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1. Gu  ZM, Martindale  DW, Lee  BH,     ( 1992 )

Isolation and complete sequence of the purL gene encoding FGAM synthase II in Lactobacillus casei.

Gene 119 (1)
PMID : 1398079  :   DOI  :   10.1016/0378-1119(92)90076-2    
Abstract >>
The purL gene from Lactobacillus casei, encoding phosphoribosylformylglycinamidine synthase II involved in the de novo synthesis of purines, was cloned and sequenced. The putative purL product of 741 amino acids (M(r) of 79,575) shows 25% and 53% identity to the homologous enzymes from Escherichia coli and Bacillus subtilis, respectively. In addition, partial sequences of two other pur genes (purQ and purF) and a possible third gene (purC) were obtained. All these genes are organized in an operon similar to that of B. subtilis. In contrast, the corresponding genes from E. coli and Salmonella typhimurium are scattered through the genome.
KeywordMeSH Terms
Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor
Genes, Bacterial
2. Fenster  KM, Parkin  KL, Steels  JL,     ( 2003 )

Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing, purification, and characterization.

Journal of dairy science 86 (4)
PMID : 12741535  :  
Abstract >>
An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.
KeywordMeSH Terms
3. Fenster  KM, Parkin  KL, Steele  JL,     ( 2003 )

Nucleotide sequencing, purification, and biochemical properties of an arylesterase from Lactobacillus casei LILA.

Journal of dairy science 86 (8)
PMID : 12939078  :   DOI  :   10.3168/jds.S0022-0302(03)73849-1    
Abstract >>
An esterase gene, designated estB, was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motifidentified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5 +/- 2.5 kDa, while the subunit molecular weight was 36.7 +/- 1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0,50 to 55 degrees C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10 degrees C, and 4% NaCl). Kinetic constants (KM and Vmax) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.
KeywordMeSH Terms
4. Dobson  CM, Deneer  H, Lee  S, Hemmingsen  S, Glaze  S, Ziola  B,     ( 2002 )

Phylogenetic analysis of the genus Pediococcus, including Pediococcus claussenii sp. nov., a novel lactic acid bacterium isolated from beer.

International journal of systematic and evolutionary microbiology 52 (Pt 6)
PMID : 12508860  :   DOI  :   10.1099/00207713-52-6-2003    
Abstract >>
Pediococci are found in foods and on plants and as beer-spoilage agents. The goal of the present study was to use the DNA sequences of the first three variable regions of the 165 rRNA gene, the 16S-23S rRNA internally transcribed spacer region sequence and approximately a third of the 60 kDa heat-shock protein gene to elucidate phylogenetic groupings within the genus Pediococcus. Phylogenetic trees were created with sequence data from 31 Pediococcus and three Lactobacillus isolates. Complete 16S rRNA gene sequences from selected Pediococcus isolates were also examined. The results were interpreted in relation to the currently accepted Pediococcus species. We found that, where previously done, speciation of many Pediococcus isolates is inaccurate. Also, one grouping of seven isolates did not include any currently recognized Pediococcus species type isolate. Our phylogenetic analyses support the conclusion that these seven isolates, all of brewing spoilage origin, belong to a novel species, for which the name Pediococcus claussenii sp. nov. is proposed (type strain P06(T0 = ATCC BAA-344(T) = DSM 14800(T)). Phylogenetic analysis has therefore helped to resolve problems surrounding species identification of Pediococcus isolates.
KeywordMeSH Terms
5. Chavagnat  F, Haueter  M, Jimeno  J, Casey  MG,     ( 2002 )

Comparison of partial tuf gene sequences for the identification of lactobacilli.

FEMS microbiology letters 217 (2)
PMID : 12480101  :   DOI  :   10.1111/j.1574-6968.2002.tb11472.x    
Abstract >>
Comparative analysis of partial tuf sequences was evaluated for the identification and differentiation of lactobacilli. Comparison of the amino acid sequences allowed differentiation between species and also between the subspecies of Lactobacillus delbrueckii. The nucleotide sequence comparison allowed differentiation between other subspecies and between some strains. Lactobacilli from several collections and isolates from dairy samples were clearly identified by comparison of short tuf sequences with those of the type strains. In evaluating the taxonomy of the Lactobacillus casei-related taxa, different tuf amino acid signatures are in favour of a classification into three distinct species. The type strain designation for the L. casei species is discussed.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
6. Mijakovic  I, Poncet  S, Galinier  A, Monedero  V, Fieulaine  S, Janin  J, Nessler  S, Marquez  JA, Scheffzek  K, Hasenbein  S, Hengstenberg  W, Deutscher  J,     ( 2002 )

Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: a relic of early life?

Proceedings of the National Academy of Sciences of the United States of America 99 (21)
PMID : 12359880  :   DOI  :   10.1073/pnas.212410399     PMC  :   PMC129692    
Abstract >>
In most Gram-positive bacteria, serine-46-phosphorylated HPr (P-Ser-HPr) controls the expression of numerous catabolic genes (approximately 10% of their genome) by acting as catabolite corepressor. HPr kinase/phosphorylase (HprK/P), the bifunctional sensor enzyme for catabolite repression, phosphorylates HPr, a phosphocarrier protein of the sugar-transporting phosphoenolpyruvate/glycose phosphotransferase system, in the presence of ATP and fructose-1,6-bisphosphate but dephosphorylates P-Ser-HPr when phosphate prevails over ATP and fructose-1,6-bisphosphate. We demonstrate here that P-Ser-HPr dephosphorylation leads to the formation of HPr and pyrophosphate. HprK/P, which binds phosphate at the same site as the beta phosphate of ATP, probably uses the inorganic phosphate to carry out a nucleophilic attack on the phosphoryl bond in P-Ser-HPr. HprK/P is the first enzyme known to catalyze P-protein dephosphorylation via this phospho-phosphorolysis mechanism. This reaction is reversible, and at elevated pyrophosphate concentrations, HprK/P can use pyrophosphate to phosphorylate HPr. Growth of Bacillus subtilis on glucose increased intracellular pyrophosphate to concentrations (approximately 6 mM), which in in vitro tests allowed efficient pyrophosphate-dependent HPr phosphorylation. To effectively dephosphorylate P-Ser-HPr when glucose is exhausted, the pyrophosphate concentration in the cells is lowered to 1 mM. In B. subtilis, this might be achieved by YvoE. This protein exhibits pyrophosphatase activity, and its gene is organized in an operon with hprK.
KeywordMeSH Terms
7. Fieulaine  S, Morera  S, Poncet  S, Mijakovic  I, Galinier  A, Janin  J, Deutscher  J, Nessler  S,     ( 2002 )

X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr.

Proceedings of the National Academy of Sciences of the United States of America 99 (21)
PMID : 12359875  :   DOI  :   10.1073/pnas.192368699     PMC  :   PMC129691    
Abstract >>
HPr kinase/phosphorylase (HprK/P) controls the phosphorylation state of the phosphocarrier protein HPr and regulates the utilization of carbon sources by Gram-positive bacteria. It catalyzes both the ATP-dependent phosphorylation of Ser-46 of HPr and its dephosphorylation by phosphorolysis. The latter reaction uses inorganic phosphate as substrate and produces pyrophosphate. We present here two crystal structures of a complex of the catalytic domain of Lactobacillus casei HprK/P with Bacillus subtilis HPr, both at 2.8-A resolution. One of the structures was obtained in the presence of excess pyrophosphate, reversing the phosphorolysis reaction and contains serine-phosphorylated HPr. The complex has six HPr molecules bound to the hexameric kinase. Two adjacent enzyme subunits are in contact with each HPr molecule, one through its active site and the other through its C-terminal helix. In the complex with serine-phosphorylated HPr, a phosphate ion is in a position to perform a nucleophilic attack on the phosphoserine. Although the mechanism of the phosphorylation reaction resembles that of eukaryotic protein kinases, the dephosphorylation by inorganic phosphate is unique to the HprK/P family of kinases. This study provides the structure of a protein kinase in complex with its protein substrate, giving insights into the chemistry of the phospho-transfer reactions in both directions.
KeywordMeSH Terms
8. Yebra  MJ, Pérez-Martínez  G,     ( 2002 )

Cross-talk between the L-sorbose and D-sorbitol (D-glucitol) metabolic pathways in Lactobacillus casei.

Microbiology (Reading, England) 148 (Pt 8)
PMID : 12177329  :   DOI  :   10.1099/00221287-148-8-2351    
Abstract >>
A gene encoding sorbitol-6-phosphate dehydrogenase (SorF) belonging to the sorbose operon (sorFABCDG) has been characterized in Lactobacillus casei. Inactivation of this gene revealed the presence of another sorbitol-6-phosphate dehydrogenase that was induced by D-sorbitol (D-glucitol). The gene encoding this activity (gutF) has also been isolated, sequenced and disrupted. The sorbitol-6-phosphate dehydrogenase genes (sorF, gutF) were required for growth on L-sorbose and D-sorbitol, respectively. Biochemical and transcriptional analyses of the wild-type and mutant strains demonstrated that L-sorbose and D-sorbitol induced sorF and the gene encoding the sorbose operon activator (sorR), while the expression of gutF was only activated by D-sorbitol. Furthermore, these studies indirectly suggested that a common metabolite of the L-sorbose and D-sorbitol metabolic pathways (probably D-sorbitol 6-phosphate) would act as the effector of SorR. The same effector would also be the inducer of gutF, although the two pathways seem to be subject to distinct regulatory mechanisms.
KeywordMeSH Terms
9. Felis  GE, Dellaglio  F, Mizzi  L, Torriani  S,     ( 2001 )

Comparative sequence analysis of a recA gene fragment brings new evidence for a change in the taxonomy of the Lactobacillus casei group.

International journal of systematic and evolutionary microbiology 51 (Pt 6)
PMID : 11760954  :   DOI  :   10.1099/00207713-51-6-2113     DOI  :   10.1099/00207713-51-6-2113    
Abstract >>
The taxonomic positions of species of the Lactobacillus casei group have been evaluated by sequencing and phylogenetic analysis of a 277 bp recA gene fragment. High sequence similarity between strain ATCC 393T, currently designated as the type strain of L. casei, and the type strain of Lactobacillus zeae, LMG 17315T, has been established, while L. casei ATCC 334 and Lactobacillus paracasei NCDO 151T form a single phylogenetic group. The taxonomic status of species and strains at issue is discussed.
KeywordMeSH Terms
Sequence Analysis, DNA
Sequence Analysis, DNA
10. Sun  X, Cross  JA, Bognar  AL, Baker  EN, Smith  CA,     ( 2001 )

Folate-binding triggers the activation of folylpolyglutamate synthetase.

Journal of molecular biology 310 (5)
PMID : 11501996  :   DOI  :   10.1006/jmbi.2001.4815    
Abstract >>
Folic acid is an essential vitamin for normal cell growth, primarily through its central role in one-carbon metabolism. Folate analogs (antifolates) are targeted at the same reactions and are widely used as therapeutic drugs for cancer and bacterial infections. Effective retention of folates in cells and the efficacy of antifolate drugs both depend upon the addition of a polyglutamate tail to the folate or antifolate molecule by the enzyme folylpolyglutamate synthetase (FPGS). The reaction mechanism involves the ATP-dependent activation of the free carboxylate group on the folate molecule to give an acyl phosphate intermediate, followed by attack by the incoming L-glutamate substrate. FPGS shares a number of structural and mechanistic details with the bacterial cell wall ligases MurD, MurE and MurF, and these enzymes, along with FPGS, form a subfamily of the ADP-forming amide bond ligase family. High-resolution crystallographic analyses of binary and ternary complexes of Lactobacillus casei FPGS reveal that binding of the first substrate (ATP) is not sufficient to generate an active enzyme. However, binding of folate as the second substrate triggers a large conformational change that activates FPGS and allows the enzyme to adopt a form that is then able to bind the third substrate, L-glutamate, and effect the addition of a polyglutamate tail to the folate.
KeywordMeSH Terms
11. Fieulaine  S, Morera  S, Poncet  S, Monedero  V, Gueguen-Chaignon  V, Galinier  A, Janin  J, Deutscher  J, Nessler  S,     ( 2001 )

X-ray structure of HPr kinase: a bacterial protein kinase with a P-loop nucleotide-binding domain.

The EMBO journal 20 (15)
PMID : 11483495  :   DOI  :   10.1093/emboj/20.15.3917     PMC  :   PMC149164    
Abstract >>
HPr kinase/phosphatase (HprK/P) is a key regulatory enzyme controlling carbon metabolism in Gram- positive bacteria. It catalyses the ATP-dependent phosphorylation of Ser46 in HPr, a protein of the phosphotransferase system, and also its dephosphorylation. HprK/P is unrelated to eukaryotic protein kinases, but contains the Walker motif A characteristic of nucleotide-binding proteins. We report here the X-ray structure of an active fragment of Lactobacillus casei HprK/P at 2.8 A resolution, solved by the multiwavelength anomalous dispersion method on a seleniated protein (PDB code 1jb1). The protein is a hexamer, with each subunit containing an ATP-binding domain similar to nucleoside/nucleotide kinases, and a putative HPr-binding domain unrelated to the substrate-binding domains of other kinases. The Walker motif A forms a typical P-loop which binds inorganic phosphate in the crystal. We modelled ATP binding by comparison with adenylate kinase, and designed a tentative model of the complex with HPr based on a docking simulation. The results confirm that HprK/P represents a new family of protein kinases, first identified in bacteria, but which may also have members in eukaryotes.
KeywordMeSH Terms
Bacterial Proteins
12. Kiwaki  M, Sawaki  S, Shirasawa  Y, Shimizu-Kadota  M,     ( 2000 )

Insertion of bacteriophage phiFSW into the chromosome of Lactobacillus casei strain Shirota (S-1): characterization of the attachment sites and the integrase gene.

Gene 249 (1��2��)
PMID : 10831846  :   DOI  :   10.1016/s0378-1119(00)00154-2    
Abstract >>
The integrase gene (int) on the genome of �pFSW, which is a temperate bacteriophage of Lactobacillus casei strain Shirota (formerly denoted as S-1), and the four attachment sites on the genomes of the phage and its host were characterized by sequencing. The �pFSW integrase was found to belong to the integrase family of site-specific tyrosine recombinase. The attachment sites shared a 40bp common core within which an integrative site-specific recombination occurs. The common core was flanked on one side by an additional segment of high sequence similarity. An integration plasmid, consisting of int, the phage attachment site (attP), and a selectable marker, inserted stably into the bacterial attachment site (attB) within the common core, as did the complete prophage genome at a frequency of more than 10(3)/microg of plasmid DNA. This plasmid was used as a test system for a preliminary mutational analysis of int and attP. The attB common core was located within and near the end of an open reading frame that appears to encode a homolog to glucose 6-phosphate isomerase, an enzyme of the glycolytic pathway. It is unlikely that the prophage integration inactivates this protein, since a change of only the C-terminal amino acid is predicted because of the sequence similarity between attP and attB.
KeywordMeSH Terms
Virus Integration
13. Dossonnet  V, Vadeboncoeur  C, Pérez-Martínez  G, Monedero  V, Viana  R,     ( 2000 )

Enzyme I and HPr from Lactobacillus casei: their role in sugar transport, carbon catabolite repression and inducer exclusion.

Molecular microbiology 36 (3)
PMID : 10844647  :   DOI  :   10.1046/j.1365-2958.2000.01862.x    
Abstract >>
We have cloned and sequenced the Lactobacillus casei ptsH and ptsI genes, which encode enzyme I and HPr, respectively, the general components of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS). Northern blot analysis revealed that these two genes are organized in a single-transcriptional unit whose expression is partially induced. The PTS plays an important role in sugar transport in L. casei, as was confirmed by constructing enzyme I-deficient L. casei mutants, which were unable to ferment a large number of carbohydrates (fructose, mannose, mannitol, sorbose, sorbitol, amygdaline, arbutine, salicine, cellobiose, lactose, tagatose, trehalose and turanose). Phosphorylation of HPr at Ser-46 is assumed to be important for the regulation of sugar metabolism in Gram-positive bacteria. L. casei ptsH mutants were constructed in which phosphorylation of HPr at Ser-46 was either prevented or diminished (replacement of Ser-46 of HPr with Ala or Thr respectively). In a third mutant, Ile-47 of HPr was replaced with a threonine, which was assumed to reduce the affinity of P-Ser-HPr for its target protein CcpA. The ptsH mutants exhibited a less pronounced lag phase during diauxic growth in a mixture of glucose and lactose, two PTS sugars, and diauxie was abolished when cells were cultured in a mixture of glucose and the non-PTS sugars ribose or maltose. The ptsH mutants synthesizing Ser-46-Ala or Ile-47-Thr mutant HPr were partly or completely relieved from carbon catabolite repression (CCR), suggesting that the P-Ser-HPr/CcpA-mediated mechanism of CCR is common to most low G+C Gram-positive bacteria. In addition, in the three constructed ptsH mutants, glucose had lost its inhibitory effect on maltose transport, providing for the first time in vivo evidence that P-Ser-HPr participates also in inducer exclusion.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Transcription, Genetic
14. Zagorec  M, Galinier  A, Monedero  V, Dossonnet  V,     ( 2000 )

Phosphorylation of HPr by the bifunctional HPr Kinase/P-ser-HPr phosphatase from Lactobacillus casei controls catabolite repression and inducer exclusion but not inducer expulsion.

Journal of bacteriology 182 (9)
PMID : 10762262  :   DOI  :   10.1128/jb.182.9.2582-2590.2000     PMC  :   PMC111324    
Abstract >>
We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion of methyl beta-D-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative in ptsH mutants altered at Ser-46 of HPr and the hprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.
KeywordMeSH Terms
Bacterial Proteins
15. Veyrat  A, Santos  MA, Yebra  MJ,     ( 2000 )

Genetics of L-sorbose transport and metabolism in Lactobacillus casei.

Journal of bacteriology 182 (1)
PMID : 10613875  :   DOI  :   10.1128/jb.182.1.155-163.2000     PMC  :   PMC94252    
Abstract >>
Genes encoding L-sorbose metabolism of Lactobacillus casei ATCC 393 have been identified on a 6.8-kb chromosomal DNA fragment. Sequence analysis revealed seven complete genes and a partial open reading frame transcribed as two units. The deduced amino acid sequences of the first transcriptional unit (sorRE) showed high similarity to the transcriptional regulator and the L-sorbose-1-phosphate reductase of the sorbose (sor) operon from Klebsiella pneumoniae. The other genes are transcribed as one unit (sorFABCDG) in opposite direction to sorRE. The deduced peptide sequence of sorF showed homology with the D-sorbitol-6-phosphate dehydrogenase encoded in the sor operon from K. pneumoniae and sorABCD to components of the mannose phosphotransferase system (PTS) family but especially to domains EIIA, EIIB, EIIC and EIID of the phosphoenolpyruvate-dependent L-sorbose PTS from K. pneumoniae. Finally, the deduced amino acid sequence of a truncated gene (sorG) located downstream of sorD presented high similarity with ketose-1,6-bisphosphate aldolases. Results of studies on enzyme activities and transcriptional analysis revealed that the two gene clusters, sorRE and sorFABCDG, are induced by L-sorbose and subject to catabolite repression by D-glucose. Data indicating that the catabolite repression is mediated by components of the PTS elements and by CcpA, are presented. Results of sugar uptake assays in L. casei wild-type and sorBC mutant strains indicated that L-sorbose is taken up by L-sorbose-specific enzyme II and that L. casei contains an inducible D-fructose-specific PTS. Results of growth analysis of those strains and a man sorBC double mutant suggested that L-sorbose is probably also transported by the D-mannose PTS. We also present evidence, from studies on a sorR mutant, suggesting that the sorR gene encodes a positive regulator of the two sor operons. Sequence alignment of SorR, SorC (K. pneumoniae), and DeoR (Bacillus subtilis) revealed that they might constitute a new group of transcriptional regulators.
KeywordMeSH Terms
16. Guarino  DU, Bellisario  RL, Maley  GF,     ( 1979 )

The primary structure of Lactobacillus casei thymidylate synthetase. III. The use of 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine and limited tryptic peptides to establish the complete amino acid sequence of the enzyme.

The Journal of biological chemistry 254 (4)
PMID : 105005  :  
Abstract >>
N/A
KeywordMeSH Terms
Indoles
Methyltransferases
Skatole
Thymidylate Synthase
17. Polshakov  VI, Feeney  J, Gargaro  AR, Frenkiel  TA,     ( 1999 )

Structure and dynamics in solution of the complex of Lactobacillus casei dihydrofolate reductase with the new lipophilic antifolate drug trimetrexate.

Protein science : a publication of the Protein Society 8 (3)
PMID : 10091649  :   DOI  :   10.1110/ps.8.3.467     PMC  :   PMC2144292    
Abstract >>
We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase and the anticancer drug trimetrexate. Two thousand seventy distance, 345 dihedral angle, and 144 hydrogen bond restraints were obtained from analysis of multidimensional NMR spectra recorded for complexes containing 15N-labeled protein. Simulated annealing calculations produced a family of 22 structures fully consistent with the constraints. Several intermolecular protein-ligand NOEs were obtained by using a novel approach monitoring temperature effects of NOE signals resulting from dynamic processes in the bound ligand. At low temperature (5 degrees C) the trimethoxy ring of bound trimetrexate is flipping sufficiently slowly to give narrow signals in slow exchange, which give good NOE cross peaks. At higher temperature these broaden and their NOE cross peaks disappear thus allowing the signals in the lower-temperature spectrum to be identified as NOEs involving ligand protons. The binding site for trimetrexate is well defined and this was compared with the binding sites in related complexes formed with methotrexate and trimethoprim. No major conformational differences were detected between the different complexes. The 2,4-diaminopyrimidine-containing moieties in the three drugs bind essentially in the same binding pocket and the remaining parts of their molecules adapt their conformations such that they can make effective van der Waals interactions with essentially the same set of hydrophobic amino acids, the side-chain orientations and local conformations of which are not greatly changed in the different complexes (similar chi1 and chi2 values).
KeywordMeSH Terms
18. Ruas-Madiedo  P, Moreno  JA, Salazar  N, Delgado  S, Mayo  B, Margolles  A, de Los Reyes-Gavilán  CG,     ( 2007 )

Screening of exopolysaccharide-producing Lactobacillus and Bifidobacterium strains isolated from the human intestinal microbiota.

Applied and environmental microbiology 73 (13)
PMID : 17483284  :   DOI  :   10.1128/AEM.02470-06     PMC  :   PMC1932768    
Abstract >>
Using phenotypic approaches, we have detected that 17% of human intestinal Lactobacillus and Bifidobacterium strains could be exopolysaccharide (EPS) producers. However, PCR techniques showed that only 7% harbored genes related to the synthesis of heteropolysaccharides. This is the first work to screen the human intestinal ecosystem for the detection of EPS-producing strains.
KeywordMeSH Terms
19. Diancourt  L, Passet  V, Chervaux  C, Garault  P, Smokvina  T, Brisse  S,     ( 2007 )

Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination.

Applied and environmental microbiology 73 (20)
PMID : 17704267  :   DOI  :   10.1128/AEM.01095-07     PMC  :   PMC2075077    
Abstract >>
Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137(T) (= ATCC 393(T)). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (pi ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei strain diversity and evolution.
KeywordMeSH Terms
Recombination, Genetic
Sequence Analysis, DNA
20. Yebra  MJ, Zúñiga  M, Beaufils  S, Pérez-Martínez  G, Deutscher  J, Monedero  V,     ( 2007 )

Identification of a gene cluster enabling Lactobacillus casei BL23 to utilize myo-inositol.

Applied and environmental microbiology 73 (12)
PMID : 17449687  :   DOI  :   10.1128/AEM.00243-07     PMC  :   PMC1932728    
Abstract >>
Genome analysis of Lactobacillus casei BL23 revealed that, compared to L. casei ATCC 334, it carries a 12.8-kb DNA insertion containing genes involved in the catabolism of the cyclic polyol myo-inositol (MI). Indeed, L. casei ATCC 334 does not ferment MI, whereas strain BL23 is able to utilize this carbon source. The inserted DNA consists of an iolR gene encoding a DeoR family transcriptional repressor and a divergently transcribed iolTABCDG1G2EJK operon, encoding a complete MI catabolic pathway, in which the iolK gene probably codes for a malonate semialdehyde decarboxylase. The presence of iolK suggests that L. casei has two alternative pathways for the metabolism of malonic semialdehyde: (i) the classical MI catabolic pathway in which IolA (malonate semialdehyde dehydrogenase) catalyzes the formation of acetyl-coenzyme A from malonic semialdehyde and (ii) the conversion of malonic semialdehyde to acetaldehyde catalyzed by the product of iolK. The function of the iol genes was verified by the disruption of iolA, iolT, and iolD, which provided MI-negative strains. By contrast, the disruption of iolK resulted in a strain with no obvious defect in MI utilization. Transcriptional analyses conducted with different mutant strains showed that the iolTABCDG1G2EJK cluster is regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor IolR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This is the first example of an operon for MI utilization in lactic acid bacteria and illustrates the versatility of carbohydrate utilization in L. casei BL23.
KeywordMeSH Terms
Phylogeny
21. Cai  H, Rodríguez  BT, Zhang  W, Broadbent  JR, Steele  JL,     ( 2007 )

Genotypic and phenotypic characterization of Lactobacillus casei strains isolated from different ecological niches suggests frequent recombination and niche specificity.

Microbiology (Reading, England) 153 (Pt 8)
PMID : 17660430  :   DOI  :   10.1099/mic.0.2007/006452-0    
Abstract >>
Lactobacillus casei strains are lactic acid bacteria (LAB) that colonize diverse ecological niches, and have broad commercial applications. To probe their evolution and phylogeny, 40 L. casei strains were characterized; the strains included isolates from plant materials (n=9), human gastrointestinal tracts (n=7), human blood (n=1), cheeses from different geographical locations (n=22), and one strain of unknown origin. API biochemical testing identified niche-specific carbohydrate fermentation profiles. A multilocus sequence typing (MLST) scheme was developed for L. casei. Partial sequencing of six housekeeping genes (ftsZ, metRS, mutL, nrdD, pgm and polA) revealed between 11 (nrdD) and 20 (mutL) allelic types, as well as 36 sequence types. Phylogenetic analysis of MLST data by Reticulate and split decomposition analysis indicated frequent intra-species recombination. Purifying selection was detected, and is likely to have contributed to the evolution of certain L. casei genes. Pulsed-field gel electrophoresis (PFGE) using SfiI was able to discriminate all the isolates, even those not differentiated by MLST. Phylogenetic trees reconstructed based on the MLST data using minimum evolution algorithm, and the SfiI-PFGE restriction patterns using the unweighted-pair group method with arithmetic mean (UPGMA), revealed consensus clusters of strains specific to cheese and silage. Topological discrepancies between the MLST and PFGE trees were also observed, suggesting that intragenic point mutations have accumulated at a slower rate than indels and genome rearrangements in L. casei. The L. casei population analysed in this study demonstrated both a high level of phenotypic and genotypic diversity, as well as specificity to different ecological niches.
KeywordMeSH Terms
Genetic Variation
Recombination, Genetic
22. Kim  SF, Baek  SJ, Pack  MY,     ( 1991 )

Cloning and nucleotide sequence of the Lactobacillus casei lactate dehydrogenase gene.

Applied and environmental microbiology 57 (8)
PMID : 1768113  :   PMC  :   PMC183587    
Abstract >>
An allosteric L-(+)-lactate dehydrogenase gene of Lactobacillus casei ATCC 393 was cloned in Escherichia coli, and the nucleotide sequence of the gene was determined. The gene was composed of an open reading frame of 981 bp, starting with a GTG codon and ending with a TAA codon. The sequences for the promoter and ribosome binding site were identified, and a sequence for a structure resembling a rho-independent transcription terminator was also found.
KeywordMeSH Terms
23. Renouf  V, Claisse  O, Miot-Sertier  C, Lonvaud-Funel  A,     ( 2006 )

Lactic acid bacteria evolution during winemaking: use of rpoB gene as a target for PCR-DGGE analysis.

Food microbiology 23 (2)
PMID : 16942997  :   DOI  :   10.1016/j.fm.2005.01.019    
Abstract >>
Evolution of the microbial population during winemaking is crucial. Winemakers are more and more attentive to microbial aspects during fermentation. During aging, microbial stabilization is preponderant to avoid development of spoilage yeast and bacteria. Therefore, it is necessary to improve methods to study the evolution of micro-organisms and for early detection of undesirable strain. The aim of this study was to develop a culture-independent method for identifying lactic acid bacteria (LAB) and to monitoring predominant species. The benefits of PCR-DGGE for the analysis of microbial changes during winemaking were clearly demonstrated. Targeting rpoB gene allowed a reliable discrimination of each species. The primers were able to avoid the interspecies heterogeneity problem caused by the use of the 16S rRNA gene. This method was applied to study the influence of different oenological practices on LAB population and their evolution during winemaking.
KeywordMeSH Terms
24. Smith  CA, Cross  JA, Bognar  AL, Sun  X,     ( 2006 )

Mutation of Gly51 to serine in the P-loop of Lactobacillus casei folylpolyglutamate synthetase abolishes activity by altering the conformation of two adjacent loops.

Acta crystallographica. Section D, Biological crystallography 62 (Pt 5)
PMID : 16627949  :   DOI  :   10.1107/S0907444906009796    
Abstract >>
Based upon the three-dimensional structure of Lactobacillus casei folylpolyglutamate synthetase (FPGS), site-directed mutagenesis studies were performed on three residues associated with the ATPase site: Gly51, Ser52 and Ser73. Gly51 and Ser52 are at the end of the P-loop, which is involved in triphosphate binding. A G51S mutant enzyme and a G51S/S52T double-mutant enzyme were made in order to alter the FPGS P-loop to more closely resemble the sequences found in other ATPase and GTPase enzymes. Ser73 is on a neighboring loop (the Omega-loop) and precedes a proline residue found to be in a cis conformation. The carbonyl O atom of Ser73 is one of the protein ligands for the essential Mg(2+) ion involved in ATP binding and hydrolysis and the Omega-loop is involved in binding the folate substrate 5,10-methylenetetrahydrofolate. The serine residue was mutated to alanine and this is the only one of the three mutants which retains some FPGS activity. The structures of the G51S, G51S/S52T and S73A mutant proteins have been solved to high resolution, along with the structure of the apo wild-type FPGS. The P-loop in both the G51S and G51S/S52T mutant proteins remains unaltered, yet both structures show a large conformational rearrangement of the Omega-loop in which a cis-Pro residue has switched conformation to a trans-peptide. The structure of the Omega-loop is severely disrupted and as a consequence structural rearrangements are observed in the peptide linker joining the two domains of the enzyme. Magnesium binding in the active site is also disrupted by the presence of the serine side chain at position 51 and by the repositioning of the carbonyl O atom of Ser73 and a water molecule is bound in place of the Mg(2+) ion. The S73A mutant protein retains the cis-Pro configuration in the Omega-loop and the Mg(2+) site remains intact. The cis-Pro is also observed in the structure of the substrate-free form of FPGS (apoFPGS), maintained in the absence of Mg(2+) by a hydrogen-bonding network involving water molecules in the active site. It is only in the complete absence of water or Mg(2+) in the binding site that the cis-Pro switches to the trans conformation.
KeywordMeSH Terms
Models, Molecular
25. Yebra  MJ, Monedero  V, Zúñiga  M, Deutscher  J, Pérez-Martínez  G,     ( 2006 )

Molecular analysis of the glucose-specific phosphoenolpyruvate : sugar phosphotransferase system from Lactobacillus casei and its links with the control of sugar metabolism.

Microbiology (Reading, England) 152 (Pt 1)
PMID : 16385119  :   DOI  :   10.1099/mic.0.28293-0    
Abstract >>
Lactobacillus casei transports glucose preferentially by a mannose-class phosphoenolpyruvate : sugar phosphotransferase system (PTS). The genomic analysis of L. casei allowed the authors to find a gene cluster (manLMNO) encoding the IIAB (manL), IIC (manM) and IID (manN) proteins of a mannose-class PTS, and a putative 121 aa protein of unknown function (encoded by manO), homologues of which are also present in man clusters that encode glucose/mannose transporters in other Gram-positive bacteria. The L. casei man operon is constitutively expressed into a manLMNO messenger, but an additional manO transcript was also detected. Upstream of the man operon, two genes (upsR and upsA) were found which encode proteins resembling a transcriptional regulator and a membrane protein, respectively. Disruption of either upsR or upsA did not affect manLMNO transcription, and had no effect on glucose uptake. Cells carrying a manO deletion transported glucose at a rate similar to that of the wild-type strain. By contrast, a manM disruption resulted in cells unable to transport glucose by the PTS, thus confirming the functional role of the man genes. In addition, the manM mutant exhibited neither inducer exclusion of maltose nor glucose repression. This result confirms the need for glucose transport through the PTS to trigger these regulatory processes in L. casei.
KeywordMeSH Terms
Operon
26. Sauvageot  N, Beaufils  S, Mazé  A, Deutscher  J, Hartke  A,     ( 2006 )

Cloning and characterization of a gene encoding a cold-shock protein in Lactobacillus casei.

FEMS microbiology letters 254 (1)
PMID : 16451179  :   DOI  :   10.1111/j.1574-6968.2005.00006.x    
Abstract >>
One csp-like gene, called cspA, from the lactic acid bacterium Lactobacillus casei was identified by an inverse polymerase chain reaction approach based on degenerate primers. cspA encodes a protein of 66 amino acid residues, which displays at least 74% identity with Csp proteins of Lactobacillus genera. Northern blot analysis revealed that cspA is transcribed monocistronically and that its expression is induced after a temperature downshift from 37 degrees C to 20 degrees C. The transcriptional start site has been determined and is situated 98 bp upstream of the initiation codon. A cspA mutant strain was constructed and it showed reduced growth rate compared with the wild type at both optimal and low temperatures, demonstrating that CspA plays an important role in the physiology of L. casei.
KeywordMeSH Terms
Bacterial Proteins
Cloning, Molecular
Cold Temperature
Gene Expression Regulation, Bacterial
27. Yebra  MJ, Viana  R, Monedero  V, Deutscher  J, Pérez-Martínez  G,     ( 2004 )

An esterase gene from Lactobacillus casei cotranscribed with genes encoding a phosphoenolpyruvate:sugar phosphotransferase system and regulated by a LevR-like activator and sigma54 factor.

Journal of molecular microbiology and biotechnology 8 (2)
PMID : 15925903  :   DOI  :   10.1159/000084567    
Abstract >>
A new esterase-encoding gene was found in the draft genome sequence of Lactobacillus casei BL23 (CECT5275). It is located in an operon together with genes encoding the EIIA, EIIB, EIIC, and EIID proteins of a mannose class phosphoenolpyruvate:sugar phosphotransferase system. After overproduction in Escherichia coli and purification, the esterase could hydrolyze acetyl sugars, hence the operon was named esu for esterase-sugar uptake genes. Upstream of the genes encoding the EII components (esuABCD) and the esterase (esuE), two genes transcribed in the opposite sense were found which encode a Bacillus subtilis LevR-like transcriptional activator (esuR) and a sigma54-like transcriptional factor (rpoN). As compared with the wild-type strain, elevated fructose phosphorylation was detected in L. casei mutants constitutively expressing the esu operon. However, none of the many sugars tested could induce the esu operon. The fact that EsuE exhibits esterase activity on acetyl sugars suggests that this operon could be involved in the uptake and metabolism of esterified sugars. Expression of the esu operon is similar to that of the B. subtilis lev operon: it contains a -12,-24 consensus promoter typical of sigma54-regulated genes, and EsuR and RpoN are essential for its transcription which is negatively regulated by EIIB(Esu). The esuABCDE transcription unit represents the first sigma54-regulated operon in lactobacilli. Furthermore, replacement of His852 in the phosphoenolpyruvate:sugar phosphotransferase system regulation domain II of EsuR with Ala indicated that the transcription activator function of EsuR is inhibited by EIIB(Esu)-mediated phosphorylation at His852.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
28. Viana  R, Pérez-Martínez  G, Deutscher  J, Monedero  V,     ( 2005 )

The glycolytic genes pfk and pyk from Lactobacillus casei are induced by sugars transported by the phosphoenolpyruvate:sugar phosphotransferase system and repressed by CcpA.

Archives of microbiology 183 (6)
PMID : 16075200  :   DOI  :   10.1007/s00203-005-0003-6    
Abstract >>
In Lactobacillus casei BL23, phosphofructokinase activity was higher in cells utilizing sugars transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphofructokinase gene (pfk) was cloned from L. casei and shown to be clustered with the gene encoding pyruvate kinase (pyk). pfk and pyk genes are cotranscribed and induced upon growth on sugars transported by the PTS. Contrarily to the model proposed for Lactococcus lactis, where the global catabolite regulator protein (CcpA) is involved in PTS-induced transcription of pfk and pyk, a ccpA mutation resulted in a slight increase in pfk-pyk expression in L. casei. This weak regulation was evidenced by CcpA binding to a region of the pfk-pyk promoter which contained two cre sequences significantly deviated from the consensus. The PTS induction of pfk-pyk seems to be counteracted by the CcpA-mediated repression. Our results suggest that the need to accommodate the levels of pfk-pyk mRNA to the availability of sugars is fulfilled in L. casei by a PTS/CcpA-mediated signal transduction different from L. lactis.
KeywordMeSH Terms
Carbohydrate Metabolism
29. Dobson  CM, Chaban  B, Deneer  H, Ziola  B,     ( 2004 )

Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus zeae isolates identified by sequence signature and immunoblot phenotype.

Canadian journal of microbiology 50 (7)
PMID : 15381972  :   DOI  :   10.1139/w04-044    
Abstract >>
Species taxonomy within the Lactobacillus casei group of bacteria has been unsettled. With the goal of helping clarify the taxonomy of these bacteria, we investigated the first 3 variable regions of the 16S rRNA gene, the 16S-23S rRNA interspacer region, and one third of the chaperonin 60 gene for Lactobacillus isolates originally designated as L. casei, L. paracasei, L. rhamnosus, and L. zeae. For each genetic region, a phylogenetic tree was created and signature sequence analysis was done. As well, phenotypic analysis of the various strains was performed by immunoblotting. Both sequence signature analysis and immunoblotting gave immediate identification of L. casei, L. rhamnosus, and L. zeae isolates. These results corroborate and extend previous findings concerning these lactobacilli; therefore, we strongly endorse recent proposals for revised nomenclature. Specifically, isolate ATCC 393 is appropriately rejected as the L. casei type strain because of grouping with isolates identified as L. zeae. As well, because all other L. casei isolates, including the proposed neotype isolate ATCC 334, grouped together with isolates designated L. paracasei, we support the use of the single species L. casei and rejection of the name L. paracasei.
KeywordMeSH Terms
30. Choi  YJ, Miguez  CB, Lee  BH,     ( 2004 )

Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96.

Applied and environmental microbiology 70 (6)
PMID : 15184114  :   DOI  :   10.1128/AEM.70.6.3213-3221.2004     PMC  :   PMC427766    
Abstract >>
A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (P(mxaF)), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558. E. coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S. tag peptide. The recombinant L. casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37 degrees C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C(8)), with K(m) and k(cat) values of 14 +/- 1.08 microM and 1,245 +/- 42.3 S(-1), respectively.
KeywordMeSH Terms
31. Hensel  R, Mayr  U, Stetter  KO, Kandler  O,     ( 1977 )

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria. I. Purification and kinetics of the allosteric L-lactic acid dehydrogenase from Lactobacillus casei ssp. casei and Lactobacillus curvatus.

Archives of microbiology 112 (1)
PMID : 14601  :   DOI  :   10.1007/bf00446658    
Abstract >>
The stability, pH-dependence and kinetic properties of the Mn2+ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiency only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn2+ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.
KeywordMeSH Terms
L-Lactate Dehydrogenase
32. Ventura  M, Canchaya  C, Meylan  V, Klaenhammer  TR, Zink  R,     ( 2003 )

Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification.

Applied and environmental microbiology 69 (11)
PMID : 14602655  :   DOI  :   10.1128/aem.69.11.6908-6922.2003     PMC  :   PMC262312    
Abstract >>
We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus.
KeywordMeSH Terms
33. Bogicevic  B, Irmler  S, Portmann  R, Meile  L, Berthoud  H,     ( 2012 )

Characterization of the cysK2-ctl1-cysE2 gene cluster involved in sulfur metabolism in Lactobacillus casei.

International journal of food microbiology 152 (3)
PMID : 21745695  :   DOI  :   10.1016/j.ijfoodmicro.2011.06.015    
Abstract >>
The up- and downstream regions of ctl1 and ctl2 that encode a cystathionine lyase were analyzed in various Lactobacillus casei strains. ctl1 and ctl2 were found to be part of a gene cluster encoding two other open reading frames. One of the two open reading frames precedes ctl1 and encodes a putative cysteine synthase. The other open reading frame lies downstream of ctl1 and encodes a putative serine acetyltransferase. The gene cluster is not present in the publicly available genome sequences of L. casei ATCC 334, BL23 and Zhang. Apparently, the gene cluster was acquired by a horizontal gene transfer event and can also be found in other lactic acid bacteria such as Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. RT-PCR was used to analyze the expression of the gene cluster. Additionally, an mass spectrometry-based selected reaction monitoring method was developed for quantifying Ctl1 in a cell-free extract of lactic acid bacteria. The gene cluster cysK2-ctl1-cysE2 was expressed as single transcript, and expression was down-regulated by cysteine. In addition, cystathionine lyase activity present in cell-free extracts disappeared when L. casei was grown in the presence of cysteine. Whereas the transcript and the gene product of ctl1 protein were found in all studied ctl1(+)L. casei strains, only the transcript but not the protein or cystathionine lyase activity was detected in L. helveticus FAM2888, L. delbrueckii subsp. bulgaricus ATCC 11842 and S. thermophilus FAM17014, which actually possess a homolog of the cysK2-ctl1-cysE2 gene cluster.
KeywordMeSH Terms
Mutagenesis, Insertional
34. Panya  M, Lulitanond  V, Tangphatsornruang  S, Namwat  W, Wannasutta  R, Suebwongsa  N, Mayo  B,     ( 2012 )

Sequencing and analysis of three plasmids from Lactobacillus casei TISTR1341 and development of plasmid-derived Escherichia coli-L. casei shuttle vectors.

Applied microbiology and biotechnology 93 (1)
PMID : 21822904  :   DOI  :   10.1007/s00253-011-3503-0    
Abstract >>
Pyrosequencing followed by conventional PCR and sequencing was used to determine the complete nucleotide sequence of three plasmids (pRCEID2.9, pRCEID3.2, and pRCEID13.9) from the Lactobacillus casei strain TISTR1341. The plasmid sequences were found to be almost identical, respectively, to those of pLA106, pLA105, and pLA103 from Lactobacillus acidophilus strain TK8912, suggesting that these strains may be related. Sequence analysis and comparison indicated that pRCEID2.9 replicates by a rolling circle (RC) mechanism, while pRCEID3.2 and pRCEID13.9 probably follow a theta-type mode of replication. Replicons of pRCEID2.9 and pRCEID13.9 were used to develop Escherichia coli/L. casei compatible shuttle vectors, which were stably maintained in different genetic backgrounds. Real-time quantitative PCR analysis showed copy numbers of around 4 and 15, respectively, for the pRCEID13.9- and pRCEID2.9-derived shuttle vectors per chromosome equivalent. The functionality of vector pRCEID-LC13.9 was proved by cloning and expressing in L. casei of a green fluorescent protein gene variant from Aequorea victoria under the control of the promoter from a homologous lactate dehydrogenase gene. The new vectors might complement those currently in use for the exploitation of L. casei as a cellular factory and in other biotechnological applications.
KeywordMeSH Terms
Genetic Vectors
35. Alpert  CA, Chassy  BM,     ( 1990 )

Molecular cloning and DNA sequence of lacE, the gene encoding the lactose-specific enzyme II of the phosphotransferase system of Lactobacillus casei. Evidence that a cysteine residue is essential for sugar phosphorylation.

The Journal of biological chemistry 265 (36)
PMID : 2125053  :  
Abstract >>
The gene coding for the lactose-specific Enzyme II of the Lactobacillus casei phosphoenolpyruvate-dependent phosphotransferase system, lacE, has been isolated by molecular cloning and expressed in Escherichia coli. The DNA sequence of the lacE gene and the deduced amino acid sequence are presented. The putative translation product comprises a hydrophobic protein of 577 amino acids with a calculated molecular mass of 62,350 Da. The deduced polypeptide has a high degree of sequence similarity with the corresponding lactose-specific enzymes II of Staphylococcus aureus and Lactococcus lactis. The sequence surrounding cysteine 483 was strongly conserved in the three proteins. The identity of the lacE product as the Enzyme IIlacL.casei was demonstrated by in vitro lactose phosphorylation assays using the protein expressed in E. coli. Single replacement of each of the histidine and cysteine residues by site-directed mutagenesis pointed to cysteine 483 as an amino acid residue essential for the phosphoryl group transfer reaction.
KeywordMeSH Terms
Cysteine
Genes, Bacterial
36. Perry  KM, Fauman  EB, Finer-Moore  JS, Montfort  WR, Maley  GF, Maley  F, Stroud  RM,     ( 1990 )

Plastic adaptation toward mutations in proteins: structural comparison of thymidylate synthases.

Proteins 8 (4)
PMID : 2128651  :   DOI  :   10.1002/prot.340080406    
Abstract >>
The structure of thymidylate synthase (TS) from Escherichia coli was solved from cubic crystals with a = 133 A grown under reducing conditions at pH 7.0, and refined to R = 22% at 2.1 A resolution. The structure is compared with that from Lactobacillus casei solved to R = 21% at 2.3 A resolution. The structures are compared using a difference distance matrix, which identifies a common core of residues that retains the same relationship to one another in both species. After subtraction of the effects of a 50 amino acid insert present in Lactobacillus casei, differences in position of atoms correlate with temperature factors and with distance from the nearest substituted residue. The dependence of structural difference on thermal factor is parameterized and reflects both errors in coordinates that correlate with thermal factor, and the increased width of the energy well in which atoms of high thermal factor lie. The dependence of structural difference on distance from the nearest substitution also depends on thermal factors and shows an exponential dependence with half maximal effect at 3.0 A from the substitution. This represents the plastic accommodation of the protein which is parameterized in terms of thermal B factor and distance from a mutational change.
KeywordMeSH Terms
37. Natori  Y, Kano  Y, Imamoto  F,     ( 1990 )

Nucleotide sequences and genomic constitution of five tryptophan genes of Lactobacillus casei.

Journal of biochemistry 107 (2)
PMID : 2113923  :   DOI  :   10.1093/oxfordjournals.jbchem.a123034    
Abstract >>
Five trp genes, trpD, trpC, trpF, trpB, and trpA, of Lactobacillus casei were cloned by transformation of tryptophan auxotrophic mutants of the respective trp genes in Escherichia coli. These trp genes appear to constitute an operon and are located in the above order in a segment of DNA of 6,468 base pairs. The entire nucleotide sequence of this DNA segment was determined. Five contiguous open reading frames in this segment can encode proteins consisting of 341, 260, 199, 406, and 266 amino acids, respectively, in the same direction. The amino acid sequences of these proteins exhibit 25.5-50.2% homology with the amino acid sequences of the corresponding trp enzymes of E. coli. Two trp genes, trpC and trpF, from L. casei can complement mutant alleles of the corresponding genes of E. coli. However, neither the trpA gene nor the trpB gene of L. casei can complement mutations in the E. coli trpA gene and the trpB gene, respectively, suggesting that the protein products of the L. casei and E. coli trpA and trpB genes, respectively, cannot form heterodimers of tryptophan synthetase with activity. Other features of the coding and flanking regions of the trp genes are also described.
KeywordMeSH Terms
Genomic Library
38. Toy  J, Bognar  AL,     ( 1990 )

Cloning and expression of the gene encoding Lactobacillus casei folylpoly-gamma-glutamate synthetase in Escherichia coli and determination of its primary structure.

The Journal of biological chemistry 265 (5)
PMID : 2105929  :  
Abstract >>
A genomic library of Lactobacillus casei DNA containing 10,000 individual clones was constructed in the plasmid pUC13. The gene encoding the L. casei folylpolyglutamate synthetase was isolated from the library by complementation of a folC mutant of Escherichia coli. The gene was expressed in E. coli from its own promoter and produced amplified folylpolyglutamate synthetase activity with properties identical with those of the purified L. casei enzyme. The absence of dihydrofolate synthetase activity and the preferential utilization of 5,10-methylenetetrahydrofolate, rather than 10-formyltetrahydrofolate as folate substrate, distinguishes this activity from the E. coli folylpolyglutamate synthetase-dihydrofolate synthetase. A protein of Mr = 43,000, identical with that of purified L. casei folylpolyglutamate synthetase, was expressed in maxicells containing the complementing plasmid. The nucleotide sequence of the folylpolyglutamate synthetase gene was determined. An open reading frame of 1,284 bases was found predicting a protein product of 428 amino acids with Mr = 44,169. The predicted amino acid sequence of the gene is 33% homologous to that of the E. coli folylpolyglutamate synthetase. Primer extension studies indicate that the transcription initiation site is at -59 base pairs, relative to the initiation ATG codon of the folylpolyglutamate synthetase gene, suggesting that the gene is transcribed independently of upstream genes. A second open reading frame was found downstream of the folylpolyglutamate synthetase open reading frame, overlapping the final codon by 1 base pair. This downstream gene may be co-transcribed with the folylpolyglutamate synthetase gene.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
39. Huang  CH, Lee  FL,     ( 2011 )

The dnaK gene as a molecular marker for the classification and discrimination of the Lactobacillus casei group.

Antonie van Leeuwenhoek 99 (2)
PMID : 20700765  :   DOI  :   10.1007/s10482-010-9493-6    
Abstract >>
It is hard to accurately identify specific species of the Lactobacillus casei group using phenotypic techniques alone. Some strains of this species group are considered to be probiotic and are widely applied in the food industry. In this study, we compared the use of two phylogenetic markers, the 16S rRNA and dnaK genes, for species discrimination of the members of the L. casei group using sequencing and RFLP. The results showed that L. casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus could be clearly distinguished based on the dnaK gene. The average sequence similarity for the dnaK gene (87.8%) among type strains was significantly less than that of the 16S rRNA sequence (99.1%). Therefore, the dnaK gene can be proposed as an additional molecular phylogenetic marker for L. casei that provides higher resolution than 16S rRNA. Species-specific RFLP profiles of the Lactobacillus strains were obtained with the enzyme ApoI. Our data indicate that the phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or RFLP assays.
KeywordMeSH Terms
40. Cai  H, Thompson  R, Budinich  MF, Broadbent  JR, Steele  JL,     ( 2009 )

Genome sequence and comparative genome analysis of Lactobacillus casei: insights into their niche-associated evolution.

Genome biology and evolution 1 (N/A)
PMID : 20333194  :   DOI  :   10.1093/gbe/evp019     PMC  :   PMC2817414    
Abstract >>
Lactobacillus casei is remarkably adaptable to diverse habitats and widely used in the food industry. To reveal the genomic features that contribute to its broad ecological adaptability and examine the evolution of the species, the genome sequence of L. casei ATCC 334 is analyzed and compared with other sequenced lactobacilli. This analysis reveals that ATCC 334 contains a high number of coding sequences involved in carbohydrate utilization and transcriptional regulation, reflecting its requirement for dealing with diverse environmental conditions. A comparison of the genome sequences of ATCC 334 to L. casei BL23 reveals 12 and 19 genomic islands, respectively. For a broader assessment of the genetic variability within L. casei, gene content of 21 L. casei strains isolated from various habitats (cheeses, n = 7; plant materials, n = 8; and human sources, n = 6) was examined by comparative genome hybridization with an ATCC 334-based microarray. This analysis resulted in identification of 25 hypervariable regions. One of these regions contains an overrepresentation of genes involved in carbohydrate utilization and transcriptional regulation and was thus proposed as a lifestyle adaptation island. Differences in L. casei genome inventory reveal both gene gain and gene decay. Gene gain, via acquisition of genomic islands, likely confers a fitness benefit in specific habitats. Gene decay, that is, loss of unnecessary ancestral traits, is observed in the cheese isolates and likely results in enhanced fitness in the dairy niche. This study gives the first picture of the stable versus variable regions in L. casei and provides valuable insights into evolution, lifestyle adaptation, and metabolic diversity of L. casei.
KeywordMeSH Terms
comparative genome hybridization
evolution
niche adaptation
comparative genome hybridization
evolution
niche adaptation
41. Irmler  S, Schäfer  H, Beisert  B, Rauhut  D, Berthoud  H,     ( 2009 )

Identification and characterization of a strain-dependent cystathionine beta/gamma-lyase in Lactobacillus casei potentially involved in cysteine biosynthesis.

FEMS microbiology letters 295 (1)
PMID : 19473252  :   DOI  :   10.1111/j.1574-6968.2009.01580.x    
Abstract >>
The trans-sulfuration pathways allow the interconversion of cysteine and methionine with the intermediary formation of cystathionine and homocysteine. The genome database of Lactobacillus casei ATCC 334 provides evidence that this species cannot synthesize cysteine from methionine via the trans-sulfuration pathway. However, several L. casei strains use methionine as the sole sulfur source, which implies that these strains can convert methionine to cysteine. Cystathionine synthases and lyases play a crucial role in the trans-sulfuration pathway. By applying proteomic techniques, we have identified a protein in cell-free extracts of L. casei, which showed high homology to a gene product encoded in the genome of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Lactobacillus helveticus but not in the genome of L. casei ATCC 334. The presence of the gene was only found in strains able to grow on methionine as the sole sulfur source. Moreover, two gene variants were identified. Both gene variants were cloned and expressed heterologously in Escherichia coli. The recombinant enzymes exhibited cystathionine lyase activity in vitro and also cleaved cysteine, homocysteine and methionine releasing volatile sulfur compounds.
KeywordMeSH Terms
42. Arai  K, Ishimitsu  T, Fushinobu  S, Uchikoba  H, Matsuzawa  H, Taguchi  H,     ( 2010 )

Active and inactive state structures of unliganded Lactobacillus casei allosteric L-lactate dehydrogenase.

Proteins 78 (3)
PMID : 19787773  :   DOI  :   10.1002/prot.22597    
Abstract >>
Lactobacillus casei L-lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6-bisphosphate (FBP), respectively, and exhibits unusually high pH-dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 A resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q-axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra-helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L-lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P-axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q-axis related dimer through the rigid NAD-binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH-dependent allosteric equilibrium.
KeywordMeSH Terms
43. Tsai  YK, Chen  HW, Lo  TC, Lin  TH,     ( 2009 )

Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac- to Lac+.

Microbiology (Reading, England) 155 (Pt 3)
PMID : 19246746  :   DOI  :   10.1099/mic.0.021907-0    
Abstract >>
Lactose metabolism is a changeable phenotype in strains of Lactobacillus casei. In this study, we found that L. casei ATCC 27139 was unable to utilize lactose. However, when exposed to lactose as the sole carbon source, spontaneous Lac(+) clones could be obtained. A gene cluster (lacTEGF-galKETRM) involved in the metabolism of lactose and galactose in L. casei ATCC 27139 (Lac(-)) and its Lac(+) revertant (designated strain R1) was sequenced and characterized. We found that only one nucleotide, located in the lacTEGF promoter (lacTp), of the two lac-gal gene clusters was different. The protein sequence identity between the lac-gal gene cluster and those reported previously for some L. casei (Lac(+)) strains was high; namely, 96-100 % identity was found and no premature stop codon was identified. A single point mutation located within the lacTp promoter region was also detected for each of the 41 other independently isolated Lac(+) revertants of L. casei ATCC 27139. The revertants could be divided into six classes based on the positions of the point mutations detected. Primer extension experiments conducted on transcription from lacTp revealed that the lacTp promoter of these six classes of Lac(+) revertants was functional, while that of L. casei ATCC 27139 was not. Northern blotting experiments further confirmed that the lacTEGF operon of strain R1 was induced by lactose but suppressed by glucose, whereas no blotting signal was ever detected for L. casei ATCC 27139. These results suggest that a single point mutation in the lacTp promoter was able to restore the transcription of a fully functional lacTEGF operon and cause a phenotype switch from Lac(-) to Lac(+) for L. casei ATCC 27139.
KeywordMeSH Terms
Phenotype
Point Mutation
44. Carr  MD, Birdsall  B, Frenkiel  TA, Bauer  CJ, Jimenez-Barbero  J, Polshakov  VI, McCormick  JE, Roberts  GC, Feeney  J,     ( 1991 )

Dihydrofolate reductase: sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution.

Biochemistry 30 (25)
PMID : 1905571  :   DOI  :   10.1021/bi00239a035    
Abstract >>
Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential 1H and 15N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D 15N/1H nuclear Overhauser-heteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 1H-1H through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows NOE cross peaks to be detected between NH protons even when their 1H chemical shifts are degenerate as long as the amide 15N chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+1 NOEs define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR.methotrexate.NADPH complex [Bolin et al. (1982) J. Biol. Chem. 257, 13650-13662], while the NMR-determined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-110 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of beta-sheet structure were characterized in the binary complex by strong alpha CHi-NHi+1 NOEs and by slowly exchanging amide protons. In addition, several long-range NOEs were identified linking together these stretches to form a beta-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR.methotrexate.NADPH complex, which contains an eight-stranded beta-sheet, indicating that the main body of the beta-sheet is preserved in the binary complex in solution.
KeywordMeSH Terms
45. Yasuda  E, Serata  M, Sako  T,     ( 2008 )

Suppressive effect on activation of macrophages by Lactobacillus casei strain Shirota genes determining the synthesis of cell wall-associated polysaccharides.

Applied and environmental microbiology 74 (15)
PMID : 18552190  :   DOI  :   10.1128/AEM.00412-08     PMC  :   PMC2519339    
Abstract >>
Although many Lactobacillus strains used as probiotics are believed to modulate host immune responses, the molecular natures of the components of such probiotic microorganisms directly involved in immune modulation process are largely unknown. We aimed to assess the function of polysaccharide moiety of the cell wall of Lactobacillus casei strain Shirota as a possible immune modulator which regulates cytokine production by macrophages. A gene survey of the genome sequence of L. casei Shirota hunted down a unique cluster of 10 genes, most of whose predicted amino acid sequences had similarities to various extents to known proteins involved in biosynthesis of extracellular or capsular polysaccharides from other lactic acid bacteria. Gene knockout mutants of eight genes from this cluster resulted in the loss of reactivity to L. casei Shirota-specific monoclonal antibody and extreme reduction of high-molecular-mass polysaccharides in the cell wall fraction, indicating that at least these genes are involved in biosynthesis of high-molecular-mass cell wall polysaccharides. By adding heat-killed mutant cells to mouse macrophage cell lines or to mouse spleen cells, the production of tumor necrosis factor alpha, interleukin-12 (IL-12), IL-10, and IL-6 was more stimulated than by wild-type cells. In addition, these mutants additively enhanced lipopolysaccharide-induced IL-6 production by RAW 264.7 mouse macrophage-like cells, while wild-type cells significantly suppressed the IL-6 production of RAW 264.7. Collectively, these results indicate that this cluster of genes of L. casei Shirota, which have been named cps1A, cps1B, cps1C, cps1D, cps1E, cps1F, cps1G, and cps1J, determine the synthesis of the high-molecular-mass polysaccharide moiety of the L. casei Shirota cell wall and that this polysaccharide moiety is the relevant immune modulator which may function to reduce excessive immune reactions during the activation of macrophages by L. casei Shirota.
KeywordMeSH Terms
Genes, Bacterial
46. Sheng  Y, Khanam  N, Tsaksis  Y, Shi  XM, Lu  QS, Bognar  AL,     ( 2008 )

Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site.

Biochemistry 47 (8)
PMID : 18232714  :   DOI  :   10.1021/bi701670y    
Abstract >>
The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.
KeywordMeSH Terms
Mutagenesis, Site-Directed
47. Monedero  V, Yebra  MJ, Poncet  S, Deutscher  J,     ( 2008 )

Maltose transport in Lactobacillus casei and its regulation by inducer exclusion.

Research in microbiology 159 (2)
PMID : 18096372  :   DOI  :   10.1016/j.resmic.2007.10.002    
Abstract >>
Transport of maltose in Lactobacillus casei BL23 is subject to regulation by inducer exclusion. The presence of glucose or other rapidly metabolized carbon sources blocks maltose transport by a control mechanism that depends on the phosphorylation of the HPr protein at serine residue 46. We have identified the L. casei gene cluster for maltose/maltodextrin utilization by sequence analysis and mutagenesis. It is composed of genes coding for a transcriptional regulator, oligosaccharide hydrolytic enzymes, an ABC transporter (MalEFGK2) and the enzymes for the metabolism of maltose or the degradation products of maltodextrins: maltose phosphorylase and beta-phospho-glucomutase. These genes are induced by maltose and repressed by the presence of glucose via the catabolite control protein A (CcpA). A mutant strain was constructed which expressed the hprKV267F allele and therefore formed large amounts of P-Ser-HPr even in the absence of a repressive carbon source. In this mutant, transport of maltose was severely impaired, whereas transport of sugars not subject to inducer exclusion was not changed. These results strengthen the idea that P-Ser-HPr controls inducer exclusion and make the maltose system of L. casei a suitable model for studying this process in Firmicutes.
KeywordMeSH Terms
Carbohydrate Metabolism
Gene Expression Regulation, Bacterial
48. Naser  SM, Dawyndt  P, Hoste  B, Gevers  D, Vandemeulebroecke  K, Cleenwerck  I, Vancanneyt  M, Swings  J,     ( 2007 )

Identification of lactobacilli by pheS and rpoA gene sequence analyses.

International journal of systematic and evolutionary microbiology 57 (Pt 12)
PMID : 18048724  :   DOI  :   10.1099/ijs.0.64711-0    
Abstract >>
The aim of this study was to evaluate the use of the phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) partial gene sequences for species identification of members of the genus Lactobacillus. Two hundred and one strains representing the 98 species and 17 subspecies were examined. The pheS gene sequence analysis provided an interspecies gap, which in most cases exceeded 10 % divergence, and an intraspecies variation of up to 3 %. The rpoA gene sequences revealed a somewhat lower resolution, with an interspecies gap normally exceeding 5 % and an intraspecies variation of up to 2 %. The combined use of pheS and rpoA gene sequences offers a reliable identification system for nearly all species of the genus Lactobacillus. The pheS and rpoA gene sequences provide a powerful tool for the detection of potential novel Lactobacillus species and synonymous taxa. In conclusion, the pheS and rpoA gene sequences can be used as alternative genomic markers to 16S rRNA gene sequences and have a higher discriminatory power for reliable identification of species of the genus Lactobacillus.
KeywordMeSH Terms
49. Sudhamani  M, Ismaiel  E, Geis  A, Batish  V, Heller  KJ,     ( 2008 )

Characterisation of pSMA23, a 3.5 kbp plasmid of Lactobacillus casei, and application for heterologous expression in Lactobacillus.

Plasmid 59 (1)
PMID : 17961648  :   DOI  :   10.1016/j.plasmid.2007.09.001    
Abstract >>
The complete nucleotide sequence of plasmid pSMA23 isolated from Lactobacillus casei A23 was determined. Plasmid pSMA23 is a 3497bp circular molecule with a G+C content of 38.18%. Four putative open reading frames were identified. Based on homology, two orfs were identified as genes encoding replication initiation (Rep) and mobilisation (Mob) protein, respectively. Various regulatory regions like promoters, ribosome binding site (RBS), transcriptional terminators were deduced from the sequences of rep and mob. The origin of replication (dso) was predicted. Shuttle vectors pL142 and pL157 were constructed for Escherichia coli and Lactobacillus using rep gene and ori of pSMA23 for replication in Lactobacillus, the ori of the commercial vector pBluescript SkII+ for replication in E. coli, and the erythromycin and chloramphenicol resistance genes of pE194 and pC194, respectively, as selection markers. Transformants of E. coli and Lactobacillus were obtained on media containing erythromycin and chloramphenicol, respectively, suggesting expression of the ermC and cat194 genes in both organisms. The shsp gene of plasmid pSt04 of Streptococcus thermophilus encoding a small heat shock protein and the Lactobacillus plantarum cbh gene encoding conjugated bile salts hydrolase were cloned and successfully expressed in the heterologous host Lb. casei LK1 with the aid of pSMA23-derived vectors.
KeywordMeSH Terms
Genetic Engineering
50. Irmler  S, Raboud  S, Beisert  B, Rauhut  D, Berthoud  H,     ( 2008 )

Cloning and characterization of two Lactobacillus casei genes encoding a cystathionine lyase.

Applied and environmental microbiology 74 (1)
PMID : 17993563  :   DOI  :   10.1128/AEM.00745-07     PMC  :   PMC2223195    
Abstract >>
Volatile sulfur compounds are key flavor compounds in several cheese types. To better understand the metabolism of sulfur-containing amino acids, which certainly plays a key role in the release of volatile sulfur compounds, we searched the genome database of Lactobacillus casei ATCC 334 for genes encoding putative homologs of enzymes known to degrade cysteine, cystathionine, and methionine. The search revealed that L. casei possesses two genes that putatively encode a cystathionine beta-lyase (CBL; EC 4.4.1.8). The enzyme has been implicated in the degradation of not only cystathionine but also cysteine and methionine. Recombinant CBL proteins catalyzed the degradation of L-cystathionine, O-succinyl-L-homoserine, L-cysteine, L-serine, and L-methionine to form alpha-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.
KeywordMeSH Terms
51. Blaiotta  G, Fusco  V, Ercolini  D, Aponte  M, Pepe  O, Villani  F,     ( 2008 )

Lactobacillus strain diversity based on partial hsp60 gene sequences and design of PCR-restriction fragment length polymorphism assays for species identification and differentiation.

Applied and environmental microbiology 74 (1)
PMID : 17993558  :   DOI  :   10.1128/AEM.01711-07     PMC  :   PMC2223197    
Abstract >>
A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.
KeywordMeSH Terms
Polymorphism, Restriction Fragment Length
52. Kang  J, Chung  WH, Lim  TJ, Whon  TW, Lim  S, Nam  YD,     ( 2017 )

Complete Genome Sequence of Lactobacillus casei LC5, a Potential Probiotics for Atopic Dermatitis.

Frontiers in immunology 8 (N/A)
PMID : 28439274  :   DOI  :   10.3389/fimmu.2017.00413     PMC  :   PMC5383696    
Abstract >>
N/A
KeywordMeSH Terms
Lactobacillus casei
PacBio
atopic dermatitis
genome sequence
probiotics
Lactobacillus casei
PacBio
atopic dermatitis
genome sequence
probiotics
53. Pinter  K, Davisson  VJ, Santi  DV,     ( 1988 )

Cloning, sequencing, and expression of the Lactobacillus casei thymidylate synthase gene.

DNA (Mary Ann Liebert, Inc.) 7 (4)
PMID : 2840247  :   DOI  :   10.1089/dna.1988.7.235    
Abstract >>
The thymidylate synthase (TS) gene from Lactobacillus casei has been isolated, cloned, and sequenced. The coding sequence is 948 bp and predicts a primary structure, identical to that reported by protein sequencing methods (Maley et al., 1979b). The gene has been placed in several expression systems which complement TS-deficient Escherichia coli, and express the catalytically active enzyme at levels of 10-20% of the soluble protein of E. coli. The expressed TS has kinetic and structural properties consistent with its being identical to the authentic enzyme from L. casei.
KeywordMeSH Terms
Cloning, Molecular
Genes
Genes, Bacterial
Transcription, Genetic
54. Homburg  C, Bommer  M, Wuttge  S, Hobe  C, Beck  S, Dobbek  H, Deutscher  J, Licht  A, Schneider  E,     ( 2017 )

Inducer exclusion in Firmicutes: insights into the regulation of a carbohydrate ATP binding cassette transporter from Lactobacillus casei BL23 by the signal transducing protein P-Ser46-HPr.

Molecular microbiology 105 (1)
PMID : 28370477  :   DOI  :   10.1111/mmi.13680    
Abstract >>
Catabolite repression is a mechanism that enables bacteria to control carbon utilization. As part of this global regulatory network, components of the phosphoenolpyruvate:carbohydrate phosphotransferase system inhibit the uptake of less favorable sugars when a preferred carbon source such as glucose is available. This process is termed inducer exclusion. In bacteria belonging to the phylum Firmicutes, HPr, phosphorylated at serine 46 (P-Ser46-HPr) is the key player but its mode of action is elusive. To address this question at the level of purified protein components, we have chosen a homolog of the Escherichia coli maltose/maltodextrin ATP-binding cassette transporter from Lactobacillus casei (MalE1-MalF1G1K12) as a model system. We show that the solute binding protein, MalE1, binds linear and cyclic maltodextrins but not maltose. Crystal structures of MalE1 complexed with these sugars provide a clue why maltose is not a substrate. P-Ser46-HPr inhibited MalE1/maltotetraose-stimulated ATPase activity of the transporter incorporated in proteoliposomes. Furthermore, cross-linking experiments revealed that P-Ser46-HPr contacts the nucleotide-binding subunit, MalK1, in proximity to the Walker A motif. However, P-Ser46-HPr did not block binding of ATP to MalK1. Together, our findings provide first biochemical evidence that P-Ser-HPr arrests the transport cycle by preventing ATP hydrolysis at the MalK1 subunits of the transporter.
KeywordMeSH Terms
55. Suebwongsa  N, Lulitanond  V, Mayo  B, Yotpanya  P, Panya  M,     ( 2016 )

Development of an Escherichia coli-Lactobacillus casei shuttle vector for heterologous protein expression in Lactobacillus casei.

SpringerPlus 5 (N/A)
PMID : 27026866  :   DOI  :   10.1186/s40064-016-1760-1     PMC  :   PMC4766160    
Abstract >>
There is an increasing interest to develop various lactic acid bacteria (LAB) species as mucosal delivery vehicles, for which the development of a variety of cloning and expression systems for these bacteria is of primary importance. This study reports the complete nucleotide sequence of the cryptic plasmid pRCEID7.6 derived from the chicken probiotic LAB strain Lactobacillus casei TISTR1341. Sequence analysis and comparison showed that pRCEID7.6 is composed of nine putative open reading frames. The replicon origin of pRCEID7.6 consisted of untranslated origin of replication and translated replication protein B sequences. This region was used to construct Escherichia coli/L. casei shuttle vectors carrying erythromycin and chloramphenicol resistance genes as selective markers. Segregation and structural stability of the vectors in L. casei was sufficient for most genetic applications. The feasibility of this vector for heterologous protein expression in L. casei was determined by cloning in pRCEID-LC7.6, the gene encoding the nucleocapsid protein (NP), from the influenza A virus under the control of the homologous promoter from the lactate dehydrogenase gene. L. casei carrying this recombinant plasmid was shown to successfully express the NP protein. Therefore, this shuttle vector can be used for further study in the development of mucosal delivery vehicles.
KeywordMeSH Terms
Cloning vectors
Heterologous expression
Lactobacillus casei
Oral live vaccine
Plasmid
Cloning vectors
Heterologous expression
Lactobacillus casei
Oral live vaccine
Plasmid
56. Bogicevic  B, Berthoud  H, Portmann  R, Bavan  T, Meile  L, Irmler  S,     ( 2016 )

Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

FEMS microbiology letters 363 (4)
PMID : 26790714  :   DOI  :   10.1093/femsle/fnw012     PMC  :   PMC4742844    
Abstract >>
In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE.
KeywordMeSH Terms
Lactobacillus casei
cysE
cysteine biosynthesis
serine acetyltransferase
Lactobacillus casei
cysE
cysteine biosynthesis
serine acetyltransferase
57. Bidart  GN, Rodríguez-Díaz  J, Yebra  MJ,     ( 2016 )

The Extracellular Wall-Bound �]-N-Acetylglucosaminidase from Lactobacillus casei Is Involved in the Metabolism of the Human Milk Oligosaccharide Lacto-N-Triose.

Applied and environmental microbiology 82 (2)
PMID : 26546429  :   DOI  :   10.1128/AEM.02888-15     PMC  :   PMC4711128    
Abstract >>
Human milk oligosaccharides (HMOs) are considered to play a key role in establishing and maintaining the infant gut microbiota. Lacto-N-triose forms part of both type 1 and type 2 HMOs and also of the glycan moieties of glycoproteins. Upstream of the previously characterized gene cluster involved in lacto-N-biose and galacto-N-biose metabolism from Lactobacillus casei BL23, there are two genes, bnaG and manA, encoding a �]-N-acetylglucosaminidase precursor and a mannose-6-phosphate isomerase, respectively. In this work, we show that L. casei is able to grow in the presence of lacto-N-triose as a carbon source. Inactivation of bnaG abolished the growth of L. casei on this oligosaccharide, demonstrating that BnaG is involved in its metabolism. Interestingly, whole cells of a bnaG mutant were totally devoid of �]-N-acetylglucosaminidase activity, suggesting that BnaG is an extracellular wall-attached enzyme. In addition to hydrolyzing lacto-N-triose into N-acetylglucosamine and lactose, the purified BnaG enzyme also catalyzed the hydrolysis of 3'-N-acetylglucosaminyl-mannose and 3'-N-acetylgalactosaminyl-galactose. L. casei can be cultured in the presence of 3'-N-acetylglucosaminyl-mannose as a carbon source, but, curiously, the bnaG mutant strain was not impaired in its utilization. These results indicate that the assimilation of 3'-N-acetylglucosaminyl-mannose is independent of BnaG. Enzyme activity and growth analysis with a manA-knockout mutant showed that ManA is involved in the utilization of the mannose moiety of 3'-N-acetylglucosaminyl-mannose. Here we describe the physiological role of a �]-N-acetylglucosaminidase in lactobacilli, and it supports the metabolic adaptation of L. casei to the N-acetylglucosaminide-rich gut niche.
KeywordMeSH Terms
58. Huang  CH, Chang  MT, Huang  L, Chu  WS,     ( 2015 )

The dnaJ gene as a molecular discriminator to differentiate among species and strain within the Lactobacillus casei group.

Molecular and cellular probes 29 (6)
PMID : 26050941  :   DOI  :   10.1016/j.mcp.2015.05.016    
Abstract >>
Identifying Lactobacillus casei and its closely related taxa at the species and strain level using only phenotypic and genotypic (16S rDNA sequence homology analysis) techniques often yields inaccurate results. In this study, the dnaJ chaperone gene was investigated as a molecular target for inter- and intraspecies discrimination within the Lb. casei group as well as for the development of specific primers for species identification. The results showed that most of the examined strains could be clearly distinguished from closely related species based on the sequenced fragments. At the interspecies level, the dnaJ sequence similarities were 81.7%-85.5%. However, at the intraspecies level, the dnaJ sequence similarities were 96.2-100% and could be assigned to different haplotypes in Lactobacillus paracasei and Lactobacillus rhamnosus, respectively. Compared to the 16S rRNA gene, the dnaJ sequence showed greater variation at both the species and strain level. Thus, the dnaJ gene can be proposed as an alternative marker for the Lb. casei group that provides higher discriminatory power than the 16S rRNA gene. In addition, species-specific primers were developed and subsequently employed in two-plex minisequencing analysis and shown to be specific for Lb. paracasei and Lb. rhamnosus. Our data indicate that phylogenetic relationships in the Lb. casei group can be resolved using comparative sequence analysis of the dnaJ gene and that the Lb. paracasei and Lb. rhamnosus species can be simultaneously identified using a novel species-specific minisequencing assay.
KeywordMeSH Terms
Inter- and intraspecies discrimination
Lactobacillus casei group
Species-specific minisequencing
dnaJ gene
Inter- and intraspecies discrimination
Lactobacillus casei group
Species-specific minisequencing
dnaJ gene
Inter- and intraspecies discrimination
Lactobacillus casei group
Species-specific minisequencing
dnaJ gene
Inter- and intraspecies discrimination
Lactobacillus casei group
Species-specific minisequencing
dnaJ gene
Inter- and intraspecies discrimination
Lactobacillus casei group
Species-specific minisequencing
dnaJ gene
Inter- and intraspecies discrimination
Lactobacillus casei group
Species-specific minisequencing
dnaJ gene
59. Kim  MS, Kim  JE, Yoon  YS, Kim  TH, Seo  JG, Chung  MJ, Yum  DY,     ( 2015 )

Improvement of atopic dermatitis-like skin lesions by IL-4 inhibition of P14 protein isolated from Lactobacillus casei in NC/Nga mice.

Applied microbiology and biotechnology 99 (17)
PMID : 25687448  :   DOI  :   10.1007/s00253-015-6455-y    
Abstract >>
Atopic dermatitis (AD) is a chronic inflammatory skin disease, with a complex etiology encompassing immunologic responses. AD is frequently associated with elevated serum immunoglobulin (Ig) E levels and is exacerbated by a variety of environmental factors, which contribute to its pathogenesis. However, the etiology of AD remains unknown. Recently, reports have documented the role of lactic acid bacteria (LAB) in the treatment and prevention of AD in humans and mice. The LAB, Lactobacillus casei (LC), is frequently used in the treatment of AD. To identify the active component of LC, we screened fractions obtained from the ion exchange chromatography of LC extracts. Using this approach, we identified the candidate protein, P14. We examined whether the P14 protein has anti-atopic properties, using both in vitro and in vivo models. Our results showed that the P14 protein selectively downregulated serum IgE and interleukin-4 cytokine levels, as well as the AD index and scratching score in AD-like NC/Nga mice. In addition, histological examination was also effective in mice. These results suggest that the P14 protein has potential therapeutic effects and that it may also serve as an effective immunomodulatory agent for treating patients with AD.
KeywordMeSH Terms
60. Sun  Z, Harris  HM, McCann  A, Guo  C, Argimón  S, Zhang  W, Yang  X, Jeffery  IB, Cooney  JC, Kagawa  TF, Liu  W, Song  Y, Salvetti  E, Wrobel  A, Rasinkangas  P, Parkhill  J, Rea  MC, O'Sullivan  O, Ritari  J, Douillard  FP, Paul Ross  R, Yang  R, Briner  AE, Felis  GE, de Vos  WM, Barrangou  R, Klaenhammer  TR, Caufield  PW, Cui  Y, Zhang  H, O'Toole  PW,     ( 2015 )

Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera.

Nature communications 6 (N/A)
PMID : 26415554  :   DOI  :   10.1038/ncomms9322     PMC  :   PMC4667430     DOI  :   10.1038/ncomms9322     PMC  :   PMC4667430    
Abstract >>
Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.
KeywordMeSH Terms
Phylogeny
Phylogeny
61. Murphree  CA, Heist  EP, Moe  LA,     ( 2014 )

Antibiotic resistance among cultured bacterial isolates from bioethanol fermentation facilities across the United States.

Current microbiology 69 (3)
PMID : 24748439  :   DOI  :   10.1007/s00284-014-0583-y    
Abstract >>
Bacterial contamination of fuel ethanol fermentations by lactic acid bacteria (LAB) can have crippling effects on bioethanol production. Producers have had success controlling bacterial growth through prophylactic addition of antibiotics to fermentors, yet concerns have arisen about antibiotic resistance among the LAB. Here, we report on mechanisms used by 32 LAB isolates from eight different US bioethanol facilities to persist under conditions of antibiotic stress. Minimum inhibitory concentration assays with penicillin, erythromycin, and virginiamycin revealed broad resistance to each of the antibiotics as well as high levels of resistance to individual antibiotics. Phenotypic assays revealed that antibiotic inactivation mechanisms contributed to the high levels of individual resistances among the isolates, especially to erythromycin and virginiamycin, yet none of the isolates appeared to use a �]-lactamase. Biofilm formation was noted among the majority of the isolates and may contribute to persistence under low levels of antibiotics. Nearly all of the isolates carried at least one canonical antibiotic resistance gene and many carried more than one. The erythromycin ribosomal methyltransferase (erm) gene class was found in 19 of 32 isolates, yet a number of these isolates exhibit little to no resistance to erythromycin. The erm genes were present in 15 isolates that encoded more than one antibiotic resistance mechanism, suggestive of potential genetic linkages.
KeywordMeSH Terms
Drug Resistance, Bacterial
Industrial Microbiology
62. Chen  Z, Lin  J, Ma  C, Zhao  S, She  Q, Liang  Y,     ( 2014 )

Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon.

Applied microbiology and biotechnology 98 (13)
PMID : 24652065  :   DOI  :   10.1007/s00253-014-5649-z    
Abstract >>
Many lactic acid bacteria carry different plasmids, particularly those that replicate via a theta mechanism. Here we describe Lactobacillus casei MCJ(CCTCC AB20130356), a new isolate that contains pMC11, carrying two distinct theta-type replicons. Each replicon contained an iteron in the origin of replication (oriV1 or oriV2) and a gene coding for the replicase (RepA_1 or RepB_1), both of which are essential for plasmid replication. Escherichia coli/Lactobacillus shuttle vectors were constructed with each replicon, yielding pEL5.7 and pEL5.6 that are based on oriV2 and oriV1 replicons, respectively. These plasmids showed distinct properties: pEL5.7 was capable of replicating in L. casei MCJ�G1 and Lactobacillus delbrueckii subsp. lactic LBCH-1 but failed to do so in two other tested lactobacilli strains whereas pEL5.6 replicated in three different strains, including L. casei MCJ�G1, L. casei NJ, Lactobacillus paracasei LPC-37 and L. delbrueckii subsp. lactic LBCH-1. Plasmid stability was studied: pEL5.6 and pEL5.7 were very stably maintained in L. casei, as the loss rate was lower than 1 % per generation. pEL5.7 was also stable in L. delbrueckii subsp. lactic LBCH-1 with the loss rate estimated to be 3 %. These vectors were employed to express a green fluorescent protein (GFP) using the promoter of S-layer protein SlpA from Lactobacillus acidophilus. And a growth-phase regulated expression of GFP was observed in different strains. In conclusion, these shuttle vectors provide efficient genetic tools for DNA cloning and heterologous gene expression in lactobacilli.
KeywordMeSH Terms
Genetic Vectors
Replication Origin
63. Zhang  H, You  C, Ren  J, Xu  D, Han  M, Liao  W,     ( 2014 )

A simple one-step PCR walking method and its application of bacterial rRNA for sequencing identification.

Current microbiology 68 (4)
PMID : 24322403  :   DOI  :   10.1007/s00284-013-0488-1    
Abstract >>
There are many PCR walking methods applied currently, and they all have examples of successful application in organisms which are more complex than bacteria. However, to a certain extent, it will be more convenient for researchers if the complicated operation and poor specificity for bacteria can be improved. Here, we introduced an improved one-step PCR walking method of bacteria. Using a specific primer of the known sequence together with a universal semirandom primer, the unknown sequence adjacent to a known sequence can be obtained easily by just one ordinary round PCR. The products can be gel purified and directly sequenced. Specific primers were designed according to the gene sequence of bacterial rRNA, and the variable and adjacent gene sequences were obtained by this method. The sequence analysis of the product showed that it can improve the resolution of bacterial identification to the species level.
KeywordMeSH Terms
64. von Schillde  MA, Hörmannsperger  G, Weiher  M, Alpert  CA, Hahne  H, Bäuerl  C, van Huynegem  K, Steidler  L, Hrncir  T, Pérez-Martínez  G, Kuster  B, Haller  D,     ( 2012 )

Lactocepin secreted by Lactobacillus exerts anti-inflammatory effects by selectively degrading proinflammatory chemokines.

Cell host & microbe 11 (4)
PMID : 22520466  :   DOI  :   10.1016/j.chom.2012.02.006    
Abstract >>
The intestinal microbiota has been linked to inflammatory bowel diseases (IBD), and oral treatment with specific bacteria can ameliorate IBD. One bacterial mixture, VSL#3, containing Lactobacillus, Bifidobacterium, and Streptococcus, was clinically shown to reduce inflammation in IBD patients and normalize intestinal levels of IP-10, a lymphocyte-recruiting chemokine, in a murine colitis model. We identified Lactobacillus paracasei prtP-encoded lactocepin as a protease that selectively degrades secreted, cell-associated, and tissue-distributed IP-10, resulting in significantly reduced lymphocyte recruitment after intraperitoneal injection in an ileitis model. A human Lactobacillus casei isolate was also found to encode lactocepin and degrade IP-10. L. casei feeding studies in a murine colitis model (T cell transferred Rag2(-/-) mice) revealed that a prtP-disruption mutant was significantly less potent in reducing IP-10 levels, T cell infiltration and inflammation in cecal tissue compared to the isogenic wild-type strain. Thus, lactocepin-based therapies may be effective treatments for chemokine-mediated diseases like IBD.
KeywordMeSH Terms
65. Serata  M, Iino  T, Yasuda  E, Sako  T,     ( 2012 )

Roles of thioredoxin and thioredoxin reductase in the resistance to oxidative stress in Lactobacillus casei.

Microbiology (Reading, England) 158 (Pt 4)
PMID : 22301908  :   DOI  :   10.1099/mic.0.053942-0    
Abstract >>
The Lactobacillus casei strain Shirota used in this study has in the genome four putative thioredoxin genes designated trxA1, trxA2, trxA3 and trxA4, and one putative thioredoxin reductase gene designated trxB. To elucidate the roles of the thioredoxins and the thioredoxin reductase against oxidative stress in L. casei, we constructed gene disruption mutants, in which each of the genes trxA1, trxA2 and trxB, or both trxA1 and trxA2 were disrupted, and we characterized their growth and response to oxidative stresses. In aerobic conditions, the trxA1 (MS108) and the trxA2 (MS109) mutants had moderate growth defects, and the trxA1 trxA2 double mutant (MS110) had a severe growth defect, which was characterized by elongation of doubling time and a lower final turbidity level. Furthermore, the trxB mutant (MS111), which is defective in thioredoxin reductase, lost the ability to grow under aerobic conditions, although it grew partially under anaerobic conditions. The growth of these mutants, however, could be substantially restored by the addition of dithiothreitol or reduced glutathione. In addition, MS110 and MS111 were more sensitive to hydrogen peroxide and disulfide stress than the wild-type. In particular, the stress sensitivity of MS111 was significantly increased. On the other hand, transcription of all these genes was only weakly affected by these oxidative stresses. Taken together, these results suggest that the thioredoxin-thioredoxin reductase system is the major thiol/disulfide redox system and is essential to allow the facultative anaerobe L. casei to grow under aerobic conditions.
KeywordMeSH Terms
Oxidative Stress
66. Bogicevic  B, Berthoud  H, Portmann  R, Meile  L, Irmler  S,     ( 2012 )

CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity.

Applied microbiology and biotechnology 94 (5)
PMID : 22113557  :   DOI  :   10.1007/s00253-011-3677-5    
Abstract >>
A gene encoding an O-acetyl-L-serine sulfhydrylase (cysK) was cloned from Lactobacillus casei FAM18110 and expressed in Escherichia coli. The purified recombinant enzyme synthesized cysteine from sulfide and O-acetyl-L-serine at pH 5.5 and pH 7.4. At pH 7.4, the apparent K(M) for O-acetyl-L-serine (OAS) and sulfide were 0.6 and 6.7 mM, respectively. Furthermore, the enzyme showed cysteine desulfurization activity in the presence of dithiothreitol at pH 7.5, but not at pH 5.5. The apparent K(M) for L-cysteine was 0.7 mM. The synthesis of cystathionine from homocysteine and serine or OAS was not observed. When expressed in a cysMK mutant of Escherichia coli, the cloned gene complemented the cysteine auxotrophy of the mutant. These findings suggested that the gene product is mainly involved in cysteine biosynthesis in L. casei. Quantitative real-time PCR and a mass spectrometric assay based on selected reaction monitoring demonstrated that L. casei FAM18110 is constitutively overexpressing cysK.
KeywordMeSH Terms
67.     ( 1997 )

Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA.

Journal of bacteriology 179 (21)
PMID : 9352913  :   DOI  :   10.1128/jb.179.21.6657-6664.1997     PMC  :   PMC179592    
Abstract >>
The chromosomal ccpA gene from Lactobacillus casei ATCC 393 has been cloned and sequenced. It encodes the CcpA protein, a central catabolite regulator belonging to the LacI-GalR family of bacterial repressors, and shows 54% identity with CcpA proteins from Bacillus subtilis and Bacillus megaterium. The L. casei ccpA gene was able to complement a B. subtilis ccpA mutant. An L. casei ccpA mutant showed increased doubling times and a relief of the catabolite repression of some enzymatic activities, such as N-acetylglucosaminidase and phospho-beta-galactosidase. Detailed analysis of CcpA activity was performed by using the promoter region of the L. casei chromosomal lacTEGF operon which is subject to catabolite repression and contains a catabolite responsive element (cre) consensus sequence. Deletion of this cre site or the presence of the ccpA mutation abolished the catabolite repression of a lacp::gusA fusion. These data support the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria.
KeywordMeSH Terms
Bacterial Proteins
Gene Expression Regulation, Bacterial
Gene Expression Regulation, Enzymologic
Glycoside Hydrolases
68.     ( 1995 )

Role of the conserved tryptophan 82 of Lactobacillus casei thymidylate synthase.

Chemistry & biology 2 (9)
PMID : 9383465  :  
Abstract >>
Thymidylate synthase (TS; EC 2.1.1.45) catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) by 5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate) to produce 2'-deoxythymidine-5'-monophosphate (dTMP) and 7,8-dihydrofolate (H2folate). Major advances in the understanding of the mechanism of TS have been made by studying site-specific mutants of the enzyme. Trp82 is completely conserved in all of the 20 TS sequences known. It forms part of the CH2H4folate binding pocket, is reported to be a component of a catalytically important H-bond network, and is suspected to be the source of an unusual absorbance change at 330 nm when TS forms a ternary complex with 5-fluoro-dTMP and CH2H4folate. We therefore prepared and characterized a set of 12 mutants at position 82 of Lactobacillus casei TS. Eight Trp82 mutants were active enough for us to determine their kinetic constants for dTMP production, while four were inactive. The active mutants had higher Km values for dUMP (2- to 10-fold) and CH2H4folate (2- to 27-fold), and lower kcat values (12- to 250-fold) than wild-type TS. The most active mutants were those containing the aromatic side chains Phe and His at position 82. All of the Trp82 mutants catalyzed the debromination of 5-bromo-dUMP with kinetic parameters similar to those of wild-type TS, and all formed ternary complexes with 5-fluoro-dUMP and CH2H4folate. The absence of Trp82 did not prevent the absorbance change at 330 nm on ternary complex formation. Trp82, a completely conserved residue that was shown by X-ray crystallography to interact directly with CH2H4folate and indirectly with dUMP, does not appear to be essential for binding or catalysis. We do, however, find a preference for an aromatic side chain at position 82. Trp82 does not contribute to the unique spectral change at 330 nm that accompanies TS ternary complex formation.
KeywordMeSH Terms
69.     ( 1997 )

Establishing a model to study the regulation of the lactose operon in Lactobacillus casei.

FEMS microbiology letters 148 (1)
PMID : 9066115  :   DOI  :   10.1111/j.1574-6968.1997.tb10271.x    
Abstract >>
The chromosomally encoded lactose-specific phosphoenol pyruvate-dependent phosphotransferase system (PTS) has been investigated in Lactobacillus casei ATCC 393 [pLZ15-] and it was considered an excellent system to study the regulation of the lactose operon. This chromosomal operon has been cloned and sequenced, being 99% homologous to that encoded on the plasmid pLZ64. Expression of the lactose operon in different mutants of L. casei ATCC 393 [pLZ15-] and primer extension analysis revealed that it is subject to a dual regulation: (i) glucose repression possibly mediated by CcpA and PTS elements, and (ii) induction by lactose through transcriptional antitermination.
KeywordMeSH Terms
70.     ( 1997 )

The lac operon of Lactobacillus casei contains lacT, a gene coding for a protein of the Bg1G family of transcriptional antiterminators.

Journal of bacteriology 179 (5)
PMID : 9045813  :   DOI  :   10.1128/jb.179.5.1555-1562.1997     PMC  :   PMC178866    
Abstract >>
The 5' region of the lac operon of Lactobacillus casei has been investigated. An open reading frame of 293 codons, designated lacT, was identified upstream of lacE. The gene product encoded by lacT is related to the family of transcriptional antiterminator proteins, which includes BglG from Escherichia coli, ArbG from Erwinia chrysanthemi, SacT, SacY, and LicT from Bacillus subtilis, and BglR from Lactococcus lactis. Amino acid sequence identities range from 35 to 24%, while similarities range from 56 to 47%. The transcriptional start site of the lac operon was identified upstream of lacT. The corresponding mRNA would contain in the 5' region a sequence with high similarity to the consensus RNA binding site of transcriptional antiterminators overlapping a sequence capable of folding into a structure that resembles a rho-independent terminator. LacT was shown to be active as an antiterminator in a B. subtilis test system using the sacB target sequence. lacT directly precedes lacEGF, the genes coding for enzyme IICB, phospho-beta-galactosidase, and enzyme IIA, and these genes are followed by a sequence that appears to encode a second rho-independent transcription terminator-like structure. Northern hybridizations with probes against lacT, lacE, and lacF revealed transcripts of similar sizes for the lac mRNAs of several L. casei strains. Since the length of the lac mRNA is just sufficient to contain lacTEGF, we conclude that the lac operon of L. casei does not contain the genes of the accessory tagatose-6-phosphate pathway as occurs in the lac operons of Lactococcus lactis, Streptococcus mutans, or Staphylococcus aureus.
KeywordMeSH Terms
Lac Operon
Transcription, Genetic
71.     ( 1993 )

Refined structures of substrate-bound and phosphate-bound thymidylate synthase from Lactobacillus casei.

Journal of molecular biology 232 (4)
PMID : 8371269  :   DOI  :   10.1006/jmbi.1993.1463    
Abstract >>
Crystal structures of two crystal forms of the complex of Lactobacillus casei (TS) with its substrate dUMP have been solved and refined at 2.55 A resolution. The two crystal forms differ by approximately 5% in the c-axis length. The TS-dUMP complexes are symmetric dimers with dUMP bound equivalently in both active sites. dUMP is non-covalently bound in the same conformation as in ternary complexes of TS with dUMP and cofactor or cofactor analogs. The same hydrogen bonds are made between TS and substrate in the binary and ternary complexes. We have also determined the 2.36 A crystal structure of phosphate-bound L. casei TS. This structure has been refined to an R-factor of 19.3% with highly constrained geometry. Refinement has revealed the locations of all residues in the protein, including the disordered residues 90 to 119, which are part of an insert found only in the L. casei and Staphylococcus aureus transposon Tn4003 TS sequences. The 2.9 A multiple isomorphous replacement (MIR) structure of L. casei TS in a complex with its substrate dUMP has been refined to a crystallographic R-factor of 15.5%. Reducing agents were withheld from crystallization solutions during MIR structure determination to allow heavy-metal labeling of the cysteine residues. Therefore, the active-site cysteine residue in this structure is oxidized and the dUMP is found at half-occupancy in the active site. No significant conformational difference was found between the phosphate-bound and dUMP-bound structures. The TS-dUMP structures were better ordered than the phosphate-bound TS or the oxidized TS-dUMP, particularly Arg23, which is clearly hydrogen-bonded to the phosphate group of dUMP. A large and a small P6(1)22 crystal form are observed for both phosphate-bound and dUMP-bound L. casei TS. The small cell forms of the phosphate-bound and dUMP-bound enzyme are isomorphous, whereas the cell constants of the larger cell form change slightly when dUMP is bound (c = 240 A versus c = 243 A). For both liganded and unliganded enzyme, conversion from the small to the large crystal form sometimes occurs spontaneously, and the crystal packing changes at a single interface. Conversion may be the result of a small change in pH in the mother liquor surrounding the crystal. A single intermolecular contact between symmetry-related Asp287 residues is disrupted on going from the small to the large crystal form.
KeywordMeSH Terms
72.     ( 1994 )

Role of the D-alanyl carrier protein in the biosynthesis of D-alanyl-lipoteichoic acid.

Journal of bacteriology 176 (3)
PMID : 8300523  :   DOI  :   10.1128/jb.176.3.681-690.1994     PMC  :   PMC205105    
Abstract >>
D-Alanyl-lipoteichoic acid (D-alanyl-LTA) is a widespread macroamphiphile which plays a vital role in the growth and development of gram-positive organisms. The biosynthesis of this polymer requires the enzymic activation of D-alanine for its transfer to the membrane-associated LTA (mLTA). A small, heat-stable, and acidic protein that is required for this transfer was purified to greater than 98% homogeneity from Lactobacillus casei ATCC 7469. This protein, previously named the D-alanine-membrane acceptor ligase (V. M. Reusch, Jr., and F. C. Neuhaus, J. Biol. Chem. 246:6136-6143, 1971), functions as the D-alanyl carrier protein (Dcp). The amino acid composition, beta-alanine content, and N-terminal sequence of this protein are similar to those of the acyl carrier proteins (ACPs) of fatty acid biosynthesis. The isolation of Dcp and its derivative, D-alanyl approximately Dcp, has allowed the characterization of two novel reactions in the pathway for D-alanyl-mLTA biosynthesis: (i) the ligation of Dcp with D-alanine and (ii) the transfer of D-alanine from D-alanyl approximately Dcp to a membrane acceptor. It has not been established whether the membrane acceptor is mLTA or another intermediate in the pathway for D-alanyl-mLTA biosynthesis. Since the D-alanine-activating enzyme (EC 6.1.1.13) catalyzes the ligation reaction, this enzyme functions as the D-alanine-Dcp ligase (Dcl). Dcl also ligated the ACPs from Escherichia coli, Vibrio harveyi, and Saccharopolyspora erythraea with D-alanine. In contrast to the relaxed specificity of Dcl in the ligation reaction, the transfer of D-alanine to the membrane acceptor was highly specific for Dcp and did not occur with other ACPs. This transfer was observed by using only D-[14C]alanyl approximately Dcp and purified L. casei membranes. Thus, D-alanyl approximately Dcp is an essential intermediate in the transfer of D-alanine from Dcl to the membrane acceptor. The formation of D-alanine esters of mLTA provides a mechanism for modulating the net anionic charge in the cell wall.
KeywordMeSH Terms
73.     ( 1993 )

Isolation and complete sequence of the purL gene encoding FGAM synthase II in Lactobacillus casei.

Gene 133 (1)
PMID : 8224889  :   DOI  :   10.1016/0378-1119(93)90240-4    
Abstract >>
N/A
KeywordMeSH Terms
Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor
74.     ( 1993 )

Lactobacillus casei contains a member of the CRP-FNR family.

Nucleic acids research 21 (3)
PMID : 8441692  :   DOI  :   10.1093/nar/21.3.753     PMC  :   PMC309187    
Abstract >>
N/A
KeywordMeSH Terms
Escherichia coli Proteins
Iron-Sulfur Proteins
Multigene Family
75.     ( 1993 )

Cloning and sequence determination of the valS gene, encoding valyl-tRNA synthetase in Lactobacillus casei.

Journal of bacteriology 175 (8)
PMID : 8468307  :   DOI  :   10.1128/jb.175.8.2475-2478.1993     PMC  :   PMC204542    
Abstract >>
The DNA sequence of the valS gene from Lactobacillus casei and the predicted amino acid sequence of its valyl-tRNA synthetase product have been determined. An open reading frame coding for a protein of 901 amino acids was found. A clone containing the intact L. casei valS gene functionally complemented the temperature-sensitive growth of the valS mutant strain 236c of Escherichia coli. The valS gene and the downstream folylpolyglutamate synthetase gene are transcribed in the same direction but are separated by a putative transcription terminator.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
76. Taguchi  H, Ohta  T,     ( 1995 )

Role of histidine 188 in fructose 1,6-bisphosphate- and divalent cation-regulated L-lactate dehydrogenase of Lactobacillus casei.

Bioscience, biotechnology, and biochemistry 59 (3)
PMID : 7766183  :   DOI  :   10.1271/bbb.59.451    
Abstract >>
A fructose 1,6-bisphosphate [Fru(1,6)P2] and divalent cation-regulated allosteric L-lactate dehydrogenase (L-LDH) (EC 1.1.1.27) of Lactobacillus casei was highly produced in Escherichia coli cells, together with its mutant enzyme, in which His-188 was replaced by Asp. Under acidic conditions, the mutant enzyme showed positive allosteric regulations by the substrate pyruvate and its analogues, like the wild-type enzyme, but not by Fru(1,6)P2, which even inhibited the stimulative effects of the alternative activation factors. In addition, Mn2+ ions also showed greatly reduced inhibitory effects on the mutant enzyme. Under neutral conditions, on the other hand, the reaction of the mutant enzyme was slightly enhanced by Fru(1,6)P2, but not further stimulated by additional Mn2+ ions, unlike the case of the wild-type enzyme. These results indicate that His-188 is, though not essential for the regulation by the alternative factors, essential for the cooperative regulation by Fru(1,6)P2 and divalent cations in L. casei L-LDH.
KeywordMeSH Terms
77. Batley  KE, Morris  HR,     ( 1977 )

Dihydrofolate reductase from Lactobacillus casei: N-terminal sequence and comparison with the substrate binding region of other reductases.

Biochemical and biophysical research communications 75 (4)
PMID : 405008  :   DOI  :   10.1016/0006-291x(77)91482-6    
Abstract >>
N/A
KeywordMeSH Terms
Tetrahydrofolate Dehydrogenase
78. Andrews  J, Clore  GM, Davies  RW, Gronenborn  AM, Gronenborn  B, Kalderon  D, Papadopoulos  PC, Schäfer  S, Sims  PF, Stancombe  R,     ( 1985 )

Nucleotide sequence of the dihydrofolate reductase gene of methotrexate-resistant Lactobacillus casei.

Gene 35 (1��2��)
PMID : 3928445  :   DOI  :   10.1016/0378-1119(85)90174-x    
Abstract >>
The nucleotide sequence of the dihydrofolate reductase (DHFR) gene of a methotrexate-resistant strain of Lactobacillus casei, which is the source of DHFR for nuclear magnetic resonance (NMR) studies, has been determined. The derived amino acid sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at position 8 and proline instead of leucine at position 90. The nucleotide sequences of 320-bp 5' and 335-bp 3' flanking regions of this gene have also been determined.
KeywordMeSH Terms
Genes, Bacterial
79. Bognar  AL, Shane  B,     ( 1983 )

Purification and properties of Lactobacillus casei folylpoly-gamma-glutamate synthetase.

The Journal of biological chemistry 258 (20)
PMID : 6138353  :  
Abstract >>
Folylpolyglutamate synthetase was purified 200,000-fold from extracts of Lactobacillus casei. The homogeneous protein was a monomer of Mr = 43,000. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to 5,10-methylene-tetrahydropteroylmono-, di-, and triglutamate substrates and metabolized (6R)-5,10-methylene-tetrahydro[3H]folate to the tetraglutamate derivative. Other folate derivatives were poor substrates or lacked activity. The specificity of the nucleotide site was wide. The magnesium salts of dATP, GTP, ITP, and UTP were effective alternate substrates for the reaction. The specificity of the glutamate binding site was very narrow. Of a wide variety of analogs tested, only L-homocysteate and 4-fluoroglutamate demonstrated affinity for the enzyme. Kinetic studies were consistent with an ordered Ter Ter mechanism with MgATP binding first to the enzyme, folate second, and glutamate last. The order of product dissociation from the enzyme was ADP, folate product, and Pi. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. The Michaelis constants for (6R)-5,10-methylene-tetrahydropteroyldiglutamate, the most effective folate substrate, MgATP, and L-glutamate were 2.3 microM, 5.6 mM, and 423 microM, respectively. Adenosine 5'-(3-thio)triphosphate and beta, gamma-methylene-ATP were inhibitors of the reaction and had higher affinities for the enzyme than ATP.
KeywordMeSH Terms
80. Morgan  WD, Birdsall  B, Polshakov  VI, Sali  D, Kompis  I, Feeney  J,     ( 1995 )

Solution structure of a brodimoprim analogue in its complex with Lactobacillus casei dihydrofolate reductase.

Biochemistry 34 (37)
PMID : 7547901  :   DOI  :   10.1021/bi00037a006    
Abstract >>
Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating-frame NOESY (ROESY) spectra were used to assign essentially all the protons in a 1:1 complex of Lactobacillus casei dihydrofolate reductase formed with an analogue of the antibacterial drug brodimoprim [2,4-diamino-5-(3',5'-dimethoxy-4'-bromobenzyl)pyrimidine]. The analogue has a 4,6-dicarboxylic acid side chain substituted on the 3'-O position designed to interact with the Arg 57 and His 28 residues in L. casei dihydrofolate reductase; it binds a factor of 10(3) more tightly to the enzyme than does the parent compound. Thirty-eight intermolecular and 11 intramolecular NOEs were measured involving the bound brodimoprim-4,6-dicarboxylic acid analogue. These provided the distance constraints used in conjunction with an energy minimization and simulated annealing protocol (using Discover from Biosym Ltd.) to dock the brodimoprim analogue into dihydrofolate reductase. In calculations where side chains and backbone fragments for binding-site residues were allowed flexibility, 90% of the 40 calculated structures had reasonable covalent geometry and none of them had NOE distance violations of greater than 0.36 A. The conformations of the aromatic rings in the bound ligand were well-defined in all the structures, with torsion angles tau 1 = -153 degrees +/- 4 degrees (C4-C5-C7-C1') and tau 2 = 53 degrees +/- 4 degrees (C5-C7-C1'-C2'): the aromatic rings of the ligand occupied essentially the same space in all the calculated structures (root mean square deviation value 1.83 A). Inclusion of the electrostatic interactions into the energy minimizations indicated that structures in which the 4,6-dicarboxylate group of the ligand interacts with the side chains of Arg 57 and His 28 are of low energy. Significant differences in side-chain and backbone conformations were detected between binding-site residues in the enzyme complexes with the brodimorpim analogue and methotrexate.
KeywordMeSH Terms
81. Hensel  R, Mayr  U, Yang  CY,     ( 1983 )

The complete primary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei.

European journal of biochemistry 134 (3)
PMID : 6411465  :   DOI  :   10.1111/j.1432-1033.1983.tb07595.x    
Abstract >>
The polypeptide chain of the allosteric L-lactate dehydrogenase (EC 1.1.1.27) of Lactobacillus casei consists of 325 amino acid residues. Despite the strikingly different enzymatic characteristics of the allosteric L-lactate dehydrogenase of L. casei and of the non-allosteric vertebrate enzymes, the sequence of the allosteric enzyme shows a distinct homology with that of the non-allosteric vertebrate enzymes (average identity: 37%). An especially high sequence homology can be identified within the active center (average identity: 70%). A clear deviation of the L. casei enzyme from the vertebrate enzyme is the lack of the first 12 amino acid residues at the N terminus and an additional 7 amino acid residues at the C terminus. The localization of the binding site of the allosteric effector D-fructose 1,6-bisphosphate and pH and effector-induced changes of the spectroscopic properties are discussed on the basis of the primary structure.
KeywordMeSH Terms
82. Filman  DJ, Bolin  JT, Matthews  DA, Kraut  J,     ( 1982 )

Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. II. Environment of bound NADPH and implications for catalysis.

The Journal of biological chemistry 257 (22)
PMID : 6815179  :  
Abstract >>
New details of NADPH binding to Lactobacillus casei dihydrofolate reductase have become visible as a result of crystallographic refinement to an R factor of 0.152 at 1.7 A resolution. Conformational torsion angles for bound NADPH have been extensively revised and specific interatomic contacts responsible for cofactor binding have been identified. In addition, several structurally conserved water molecules are seen to mediate the protein-ligand interaction. In the nicotinamide binding site three oxygen atoms of the enzyme lie in the plane of the pyridine ring and close to ring carbons 2, 4, and 6. The placement of these polar groups suggests that the enzyme stabilizes a C4-carbonium electronic isomer of oxidized nicotinamide in the transition state. Pyramidalization of ring nitrogen N1 in the transition state might be promoted by a fixed water molecule positioned to donate a hydrogen bond to the N1 lone pair orbital. Pyramidalization could also relieve an unfavorable steric contact due to the observed rotation of the nicotinamide's carboxamide group by 180 degrees from its most stable conformation.
KeywordMeSH Terms
83. Alpert  CA, Chassy  BM,     ( 1988 )

Molecular cloning and nucleotide sequence of the factor IIIlac gene of Lactobacillus casei.

Gene 62 (2)
PMID : 3130296  :   DOI  :   10.1016/0378-1119(88)90565-3    
Abstract >>
The lactose-specific factor III (FIIIlac of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Lactobacillus casei and purified to homogeneity by conventional protein purification methods. Its apparent native Mr, estimated from steric exclusion chromatography (approx. 39 kDa), and subunit Mr, estimated from sodium dodecyl sulfate-polyacrylamide gels, indicated that it exists as a trimer of identical subunits of 13 kDa. The gene for FIII L. casei lac was cloned into Escherichia coli using the vector pUC18. The coding sequences were contained on an 860-bp BglII-HindIII DNA fragment of the L. casei lactose plasmid, pLZ64. A protein identical in properties to FIII L. casei lac was isolated from clones of E. coli carrying this DNA insert. The nucleotide sequence of the FIII L. casei lac gene was determined by the dideoxy chain-termination technique. The 336-bp open reading frame for FIII L. casei lac was followed by a stem-loop structure, analogous to a Rho-independent terminator. We concluded that the FIII L. casei lac was the terminal gene in what appears to be an operon comprised of the lactose-PTS-P-beta Gal-coding genes. Comparison of the deduced amino acid sequence of FIII L. caseilac with the amino acid sequence of FIII S. aureus lac (derived from peptide sequencing) demonstrated a high degree of homology (49 identical residues and 21 conservative exchanges out of 103 total aa residues). The FIII L. casei lac lacked his82, previously identified as the phosphorylation site of FIII S. aureus. lac His80 was proposed to be the site of histidyl phosphorylation of FIII L. casei lac.
KeywordMeSH Terms
Genes, Bacterial
84. Porter  EV, Chassy  BM,     ( 1988 )

Nucleotide sequence of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei: comparison to analogous pbg genes of other gram-positive organisms.

Gene 62 (2)
PMID : 3130295  :   DOI  :   10.1016/0378-1119(88)90564-1    
Abstract >>
Lactose metabolism in Lactobacillus casei occurs via phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and subsequent cleavage of lactose-6-phosphate by beta-D-phosphogalactoside galactohydrolase (P-beta Gal). The genes for lactose uptake and P-beta Gal have been shown to be plasmid-associated in L. casei 64H [Chassy et al., Curr. Microbiol. 1 (1978) 141-144]. The cloned P-beta Gal-coding gene (pbg) previously described [Lee et al., J. Bacteriol. 152 (1982) 1138-1146] was subcloned on a 2.9-kb KpnI-Bg/II fragment isolated from pLZ605. Sequence analysis of this fragment revealed an open reading frame of 1422 bp capable of coding for a protein product containing 474 amino acids and having an Mr of 53,989. The L. casei protein showed a high degree of homology to the proteins whose sequence was deduced from the nucleotide sequence of the pbg genes of Staphylococcus aureus and Streptococcus lactis. Because of the significant homologies observed, as reflected in amino acid content as well as predicted structural characteristics of the three proteins, we suggest a common origin for the P-beta Gals of these three organisms.
KeywordMeSH Terms
Genes, Bacterial
Glycoside Hydrolases
85. Shimizu-Kadota  M, Kiwaki  M, Hirokawa  H, Tsuchida  N,     ( 1985 )

ISL1: a new transposable element in Lactobacillus casei.

Molecular & general genetics : MGG 200 (2)
PMID : 2993817  :   DOI  :   10.1007/bf00425423    
Abstract >>
The genome structures of a temperate Lactobacillus phage, phi FSW, and its virulent mutants, phi FSVs, were examined by restriction, heteroduplex and nucleotide-sequence analyses. The results showed that two out of three phi FSVs had the same 1.3 kbp insertion (designated as ISL1) at different positions in the phi FSW sequence. ISL1 was 1,256 bp long and contained at least two long open reading frames of 279 and 822 bases on one strand. Inverted repeats were found at the termini of the ISL1 which was bracketed by 3 bp direct repeats of the phi FSW sequence. From this evidence, we concluded that ISL1 was a transposable element in Lactobacillus casei.
KeywordMeSH Terms
DNA Transposable Elements
Genes, Bacterial
Genes, Viral
86.     ( 1978 )

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Sequences of the cyanogen bromide peptides and complete sequences of the enzyme.

The Journal of biological chemistry 253 (18)
PMID : 98527  :  
Abstract >>
The complete amino acid sequence of dihydrofolate reductase from an amethopterin-resistant strain of Lactobacillus casei has been determined by sequence analysis of peptides produced by cleavage with cyanogen bromide, trypsin, staphylococcal protease, and myxobacter protease. Comparison of this sequence with those of reductases from other bacterial sources shows that the enzymes are homologous. The Lactobacillus casei reductase sequences shows a 29% sequence identity with that of the Escherichia coli enzyme and a 34% identity with the sequence of the enzyme from Streptococcus faecium. The NH2-terminal 68 residues of the L. casei reductase show a 54% sequence identity with that of the enzyme from S. faecium.
KeywordMeSH Terms
Tetrahydrofolate Dehydrogenase
87.     ( 1998 )

Suppression of the ptsH mutation in Escherichia coli and Salmonella typhimurium by a DNA fragment from Lactobacillus casei.

Journal of bacteriology 180 (19)
PMID : 9748463  :   PMC  :   PMC107566    
Abstract >>
A DNA fragment from Lactobacillus casei that restores growth to Escherichia coli and Salmonella typhimurium ptsH mutants on glucose and other substrates of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been isolated. These mutants lack the HPr protein, a general component of the PTS. Sequencing of the cloned fragment revealed the absence of ptsH homologues. Instead, the complementation ability was located in a 120-bp fragment that contained a sequence homologue to the binding site of the Cra regulator from enteric bacteria. Experiments indicated that the reversion of the ptsH phenotype was due to a titration of the Cra protein, which allowed the constitutive expression of the fructose operon.
KeywordMeSH Terms
Escherichia coli Proteins
Suppression, Genetic
88.     ( 1998 )

The gal genes for the Leloir pathway of Lactobacillus casei 64H.

Applied and environmental microbiology 64 (6)
PMID : 9603808  :   PMC  :   PMC106272    
Abstract >>
The gal genes from the chromosome of Lactobacillus casei 64H were cloned by complementation of the galK2 mutation of Escherichia coli HB101. The pUC19 derivative pKBL1 in one complementation-positive clone contained a 5.8-kb DNA HindIII fragment. Detailed studies with other E. coli K-12 strains indicated that plasmid pKBL1 contains the genes coding for a galactokinase (GalK), a galactose 1-phosphate-uridyltransferase (GalT), and a UDP-galactose 4-epimerase (GalE). In vitro assays demonstrated that the three enzymatic activities are expressed from pKBL1. Sequence analysis revealed that pKBL1 contained two additional genes, one coding for a repressor protein of the LacI-GalR-family and the other coding for an aldose 1-epimerase (mutarotase). The gene order of the L. casei gal operon is galKETRM. Because parts of the gene for the mutarotase as well as the promoter region upstream of galK were not cloned on pKBL1, the regions flanking the HindIII fragment of pKBL1 were amplified by inverse PCR. Northern blot analysis showed that the gal genes constitute an operon that is transcribed from two promoters. The galKp promoter is inducible by galactose in the medium, while galEp constitutes a semiconstitutive promoter located in galK.
KeywordMeSH Terms
Genes, Bacterial
89.     ( 1997 )

Detection and speciation of bacteria through PCR using universal major cold-shock protein primer oligomers.

Journal of industrial microbiology & biotechnology 19 (4)
PMID : 9439003  :  
Abstract >>
The detection of bacteria using PCR is a well-established diagnostic technique. However, conventional PCR requires the use of DNA primer oligomers that are specific to the target organism and, as a consequence, a sample can only be tested for the presence of that specific target. A significant advantage would be to probe a sample for the presence of any bacteria, followed by identification. To achieve this it is necessary to identify a DNA sequence common to all bacteria. Here we demonstrate that such a sequence may be that encoding the major cold-shock proteins. Using two universal PCR primer oligomers from conserved regions of these gene homologues, we have amplified a 200 base-pair DNA sequence from more than 30 diverse Gram-positive and Gram-negative bacteria, including representatives from the genera Aeromonas, Bacillus, Citrobacter, Enterobacter, Enterococcus, Escherichia, Klebsiella, Lactobacillus, Lactococcus, Listeria, Pediococcus, Photobacterium, Proteus, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia. Sequence analysis of the amplified products confirmed a high level of DNA homology. Significantly, however, there are sufficient nucleotide variations to allow the unique allocation of each amplified sequence to its parental bacterium.
KeywordMeSH Terms
DNA Primers
90.     ( 1998 )

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase.

Proceedings of the National Academy of Sciences of the United States of America 95 (12)
PMID : 9618466  :   DOI  :   10.1073/pnas.95.12.6647     PMC  :   PMC22582    
Abstract >>
Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-A resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Omega loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.
KeywordMeSH Terms
Protein Conformation
91.     ( 1998 )

The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate.

Journal of molecular biology 277 (1)
PMID : 9514736  :   DOI  :   10.1006/jmbi.1997.1560    
Abstract >>
We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase (18.3 kDa, 162 amino acid residues) formed with the anticancer drug methotrexate using 2531 distance, 361 dihedral angle and 48 hydrogen bond restraints obtained from analysis of multidimensional NMR spectra. Simulated annealing calculations produced a family of 21 structures fully consistent with the constraints. The structure has four alpha-helices and eight beta-strands with two other regions, comprising residues 11 to 14 and 126 to 127, also interacting with each other in a beta-sheet manner. The methotrexate binding site is very well defined and the structure around its glutamate moiety was improved by including restraints reflecting the previously determined specific interactions between the glutamate alpha-carboxylate group with Arg57 and the gamma-carboxylate group with His28. The overall fold of the binary complex in solution is very similar to that observed in the X-ray studies of the ternary complex of L. casei dihydrofolate reductase formed with methotrexate and NADPH (the structures of the binary and ternary complexes have a root-mean-square difference over the backbone atoms of 0.97 A). Thus no major conformational change takes place when NADPH binds to the binary complex. In the binary complex, the loop comprising residues 9 to 23 which forms part of the active site has been shown to be in the "closed" conformation as defined by M. R. Sawaya & J. Kraut, who considered the corresponding loops in crystal structures of complexes of dihydrofolate reductases from several organisms. Thus the absence of the NADPH does not result in the "occluded" form of the loop as seen in crystal studies of some other dihydrofolate reductases in the absence of coenzyme. Some regions of the structure in the binary complex which form interaction sites for NADPH are less well defined than other regions. However, in general terms, the NADPH binding site appears to be essentially pre-formed in the binary complex. This may contribute to the tighter binding of coenzyme in the presence of methotrexate.
KeywordMeSH Terms

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