| 1. |
Serror P,
Dervyn R,
Ehrlich SD,
Maguin E,
( 2003 ) csp-like genes of Lactobacillus delbrueckii ssp. bulgaricus and their response to cold shock. PMID : 14553929 : DOI : 10.1016/S0378-1097(03)00594-9 Abstract >>
The two csp-like genes from the lactic acid bacterium Lactobacillus delbrueckii ssp. bulgaricus were characterized and designated cspA and cspB. The gene cspA has been identified using a polymerase chain reaction (PCR)-based approach with degenerated primers and further characterized using an inverse PCR strategy. cspA encodes a protein of 65 amino acid residues which displays between 81 and 77% identity with proteins CspL and CspP of Lactobacillus plantarum. cspB has been identified as a cspA ortholog using the partial sequence of the L. bulgaricus ATCC11842. cspB encodes a protein of 69 amino acids which has 42% identity with CspA. Northern blot analyses showed that cspA is transcribed as a single gene and that its transcription increased after a temperature downshift from 42 to 25 degrees C. In contrast, cspB is part of an operon transcribed at constant level irrespective of the temperature. These results indicate that cspA encodes the only Csp-like protein of L. bulgaricus induced by a downshift of temperature.
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2. |
Ishino Y,
Morgenthaler P,
Hottinger H,
Söll D,
( 1992 ) Organization and nucleotide sequence of the glutamine synthetase (glnA) gene from Lactobacillus delbrueckii subsp. bulgaricus. PMID : 1359838 : PMC : PMC183065 Abstract >>
A 3.3-kb BamHI fragment of Lactobacillus delbrueckii subsp. bulgaricus DNA was cloned and sequenced. It complements an Escherichia coli glnA deletion strain and hybridizes strongly to a DNA containing the Bacillus subtilis glnA gene. DNA sequence analysis of the L. delbrueckii subsp. bulgaricus DNA showed it to contain the glnA gene encoding class I glutamine synthetase, as judged by extensive homology with other prokaryotic glnA genes. The sequence suggests that the enzyme encoded in this gene is not controlled by adenylylation. Based on a comparison of glutamine synthetase sequences, L. delbrueckii subsp. bulgaricus is much closer to gram-positive eubacteria, especially Clostridium acetobutylicum, than to gram-negative eubacteria and archaebacteria. The fragment contains another open reading frame encoding a protein of unknown function consisting of 306 amino acids (ORF306), which is also present upstream of glnA of Bacillus cereus. In B. cereus, a repressor gene, glnR, is found between the open reading frame and glnA. Two proteins encoded by the L. delbrueckii subsp. bulgaricus gene were identified by the maxicell method; the sizes of these proteins are consistent with those of the open reading frames of ORF306 and glnA. The lack of a glnR gene in the L. delbrueckii subsp. bulgaricus DNA in this position may indicate a gene rearrangement or a different mechanism of glnA gene expression.
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3. |
Germond JE,
Lapierre L,
Delley M,
Mollet B,
Felis GE,
Dellaglio F,
( 2003 ) Evolution of the bacterial species Lactobacillus delbrueckii: a partial genomic study with reflections on prokaryotic species concept. PMID : 12519911 : DOI : 10.1093/molbev/msg012 Abstract >>
The species Lactobacillus delbrueckii consists at present of three subspecies, delbrueckii, lactis and bulgaricus, showing a high level of DNA-DNA hybridization similarity but presenting markedly different traits related to distinct ecological adaptation. The internal genetic heterogeneity of the bacterial species L. delbrueckii was analyzed. Phenotypic and several genetic traits were investigated for 61 strains belonging to this species. These included 16S rDNA sequence mutations, expression of beta-galactosidase and of the cell wall-anchored protease, the characterization of the lactose operon locus and of the sequence of lacR gene, galactose metabolism, and the distribution of insertion sequences. The high genetic heterogeneity of taxa was confirmed by every trait investigated: the lac operon was completely deleted in the subsp. delbrueckii, different mutation events in the repressor gene of the operon led to a constitutive expression of lacZ in the subsp. bulgaricus. Structural differences in the same genetic locus were probably due to the presence of different IS elements in the flanking regions. The different expression of the cell wall-anchored protease, constitutive in the subsp. bulgaricus, inducible in the subsp. lactis, and absent in the subsp. delbrueckii was also a consequence of mutations at the gene level. The galT gene for galactose metabolism was found only in the subsp. lactis, while no specific amplification product was detected in the other two subspecies. All these data, together with the absence of a specific IS element, ISL6, from the major number of strains belonging to the subsp. bulgaricus, confirmed a deep internal heterogeneity among the three subspecies. Moreover, this evidence and the directional mutations found in the 16S rDNA sequences suggested that, of the three subspecies, L. delbrueckii subsp. lactis is the taxon closer to the ancestor. Limitations of the current prokaryotic species definition were also discussed, based on presented evidences. Our results indicate the need for an accurate investigation of internal heterogeneity of bacterial species. This study has consequences on the prokaryotic species concept, since genomic flexibility of prokaryotes collides with a stable classification, necessary from a scientific and applied point of view.
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4. |
Chavagnat F,
Haueter M,
Jimeno J,
Casey MG,
( 2002 ) Comparison of partial tuf gene sequences for the identification of lactobacilli. 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.
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5. |
Lamothe GT,
Jolly L,
Mollet B,
Stingele F,
( 2002 ) Genetic and biochemical characterization of exopolysaccharide biosynthesis by Lactobacillus delbrueckii subsp. bulgaricus. PMID : 12189423 : DOI : 10.1007/s00203-002-0447-x Abstract >>
Lactobacillus delbrueckiisubsp. bulgaricus produces exopolysaccharides (EPSs), which play a role in the rheological properties of fermented food products. Lb. bulgaricus Lfi5 produces a high-molecular-weight EPS composed of galactose, glucose, and rhamnose in the molar ratio 5:1:1. An 18-kb DNA region containing 14 genes, designated epsA to epsN, was isolated by genomic DNA library screening and inverted PCR. The predicted gene products are homologous to proteins involved in the biosynthesis of other bacterial polysaccharides and the genetic organization was found to be similar to that of other eps clusters from lactic acid bacteria. Transcriptional analysis revealed that the 14 eps genes are co-ordinately expressed and transcribed as a single mRNA of 15-16 kb. The transcription start site of the promoter was mapped upstream of the first gene, epsA. Genes encoding glycosyltranferases were further studied by heterologous expression and functional assays. We showed that the epsE gene product is a phospho-glucosyltransferase initiating the biosynthesis of EPS. Heterologous expression of epsE in a Lactococcus lactis epsDmutant restored EPS production, demonstrating its role and importance in EPS biosynthesis. Functional assays of other glycosyltransferases allowed their sugar specificity to be elucidated and an overall biosynthetic pathway for EPS synthesis by Lb. bulgaricus to be proposed.
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6. |
Aubel D,
Germond JE,
Gilbert C,
Atlan D,
( 2002 ) Isolation of the patC gene encoding the cystathionine beta-lyase of Lactobacillus delbrueckii subsp. bulgaricus and molecular analysis of inter-strain variability in enzyme biosynthesis. PMID : 12101291 : DOI : 10.1099/00221287-148-7-2029 Abstract >>
The patC gene encoding the cystathionine beta-lyase (CBL) of Lactobacillus delbrueckii subsp. bulgaricus NCDO 1489 was cloned and expressed in Escherichia coli. Overexpression of CBL complemented the methionine auxotrophy of an E. coli metC mutant, demonstrating in vivo that this enzyme functions as a CBL. However, PatC is distinguishable from the MetC CBLs by a low identity in amino acid sequence, a sensitivity to iodoacetic acid, greater thermostability and a lower substrate affinity. Homologues of patC were detected in the 13 Lb. delbrueckii strains studied, but only seven of them showed CBL activity. In constrast to CBL(+) strains, all CBL-deficient strains analysed were auxotrophic for methionine. This supports the hypothesis that CBLs from lactobacilli are probably involved in methionine biosynthesis. Moreover, the results of this study suggest that post-transcriptional mechanisms account for the differences in CBL activities observed between strains of Lb. delbrueckii.
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7. |
Lapierre L,
Mollet B,
Germond JE,
( 2002 ) Regulation and adaptive evolution of lactose operon expression in Lactobacillus delbrueckii. PMID : 11807052 : DOI : 10.1128/jb.184.4.928-935.2002 PMC : PMC134810 Abstract >>
Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis are both used in the dairy industry as homofermentative lactic acid bacteria in the production of fermented milk products. After selective pressure for the fast fermentation of milk in the manufacture of yogurts, L. delbrueckii subsp. bulgaricus loses its ability to regulate lac operon expression. A series of mutations led to the constitutive expression of the lac genes. A complex of insertion sequence (IS) elements (ISL4 inside ISL5), inserted at the border of the lac promoter, induced the loss of the palindromic structure of one of the operators likely involved in the binding of regulatory factors. A lac repressor gene was discovered downstream of the beta-galactosidase gene of L. delbrueckii subsp. lactis and was shown to be inactivated by several mutations in L. delbrueckii subsp. bulgaricus. Regulatory mechanisms of the lac gene expression of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis were compared by heterologous expression in Lactococcus lactis of the two lac promoters in front of a reporter gene (beta-glucuronidase) in the presence or absence of the lac repressor gene. Insertion of the complex of IS elements in the lac promoter of L. delbrueckii subsp. bulgaricus increased the promoter's activity but did not prevent repressor binding; rather, it increased the affinity of the repressor for the promoter. Inactivation of the lac repressor by mutations was then necessary to induce the constitutive expression of the lac genes in L. delbrueckii subsp. bulgaricus.
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8. |
Razeto A,
Kochhar S,
Hottinger H,
Dauter M,
Wilson KS,
Lamzin VS,
( 2002 ) Domain closure, substrate specificity and catalysis of D-lactate dehydrogenase from Lactobacillus bulgaricus. PMID : 12054772 : DOI : 10.1016/S0022-2836(02)00086-4 Abstract >>
NAD-dependent Lactobacillus bulgaricus D-Lactate dehydrogenase (D-LDHb) catalyses the reversible conversion of pyruvate into D-lactate. Crystals of D-LDHb complexed with NADH were grown and X-ray data collected to 2.2 A. The structure of D-LDHb was solved by molecular replacement using the dimeric Lactobacillus helveticus D-LDH as a model and was refined to an R-factor of 20.7%. The two subunits of the enzyme display strong asymmetry due to different crystal environments. The opening angles of the two catalytic domains with respect to the core coenzyme binding domains differ by 16 degrees. Subunit A is in an "open" conformation typical for a dehydrogenase apo enzyme and subunit B is "closed". The NADH-binding site in subunit A is only 30% occupied, while in subunit B it is fully occupied and there is a sulphate ion in the substrate-binding pocket. A pyruvate molecule has been modelled in the active site and its orientation is in agreement with existing kinetic and structural data. On domain closure, a cluster of hydrophobic residues packs tightly around the methyl group of the modelled pyruvate molecule. At least three residues from this cluster govern the substrate specificity. Substrate binding itself contributes to the stabilisation of domain closure and activation of the enzyme. In pyruvate reduction, D-LDH can adapt another protonated residue, a lysine residue, to accomplish the role of the acid catalyst His296. Required lowering of the lysine pK(a) value is explained on the basis of the H296K mutant structure.
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9. |
Peltoniemi K,
Vesanto E,
Palva A,
( 2002 ) Genetic characterization of an oligopeptide transport system from Lactobacillus delbrueckii subsp. bulgaricus. PMID : 12029391 : DOI : 10.1007/s00203-002-0411-9 Abstract >>
The operon of the putative lactobacillar oligopeptide transport system (Opp) from Lactobacillus delbrueckii subsp. bulgaricus B14 was cloned and characterized. The opp operon was found to consist of five genes, oppD, oppF, oppB, oppC and oppA (1). In addition, an oppA (1) homolog, oppA (2), was found downstream of the operon. Sequence comparisons of the L. delbrueckii subsp. bulgaricus Opp system with other bacterial transport systems revealed the highest similarity to the oligopeptide transport system of Lactococcus lactis. Northern analyses of oppmRNAs revealed 6.1-kb and 2.1-kb transcripts, confirming that, in addition to the operon structure oppDFBCA (1), the oppA (1) gene was also expressed as a monocistronic transcript. The oppA (2) gene was expressed as a separate 2.1-kb monocistronic transcript with a low expression level. Primer-extension mapping of the 5'end of oppDFBCA (1) mRNA revealed two adjacent transcriptional start sites, and primer extension analyses of oppA (1) and oppA (2) mRNAs confirmed the location of the predicted promoters of these genes. For complementation analysis, oppA (1) alone and the operon constructs oppDFBCA (1) and oppDFBCA (2) were fused with the nisA promoter and expressed in Lactococcus lactisNZ9000Delta oppA strain. Only the L. delbrueckii subsp. bulgaricus oppDFBCA (1)genes were able to complement the L. lactis oppA mutation.
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10. |
Azcárate-Peril MA,
Raya RR,
( 2002 ) Sequence analysis of pLBB1, a cryptic plasmid from Lactobacillus delbrueckii subsp. bulgaricus. PMID : 11958563 : DOI : 10.1139/w01-137 Abstract >>
The first report of the complete nucleotide sequence of a cryptic plasmid from Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) is presented. The plasmid pLBB1 consists of 6127 bp with a GC content of 44.8%. No ssDNA was detected by hybridization experiments, which is consistent with the notion that pLBB1 does not replicate by a rolling circle mechanism. A putative replication region of pLBB1 was cloned and found to be functional in Lactobacillus johnsonii and Lactococcus lactis. Plasmid pLBB1 showed significant DNA sequence identity with plasmid pLL1212 from Lactobacillus delbrueckii subsp. lactis (Lactobacillus lactis) CRL1212 (GenBank accession No. AF109691). Four open reading frames (ORFs) larger than 100 amino acids were identified. ORFA shared similarity with a putative primase-helicase system, and ORFB and ORFC exhibited limited identity with a mobilization protein and a transposase, respectively. Curing experiments did not allowed us to assign a function to the ORFs.
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11. |
Brockmann E,
Lick S,
( 2000 ) Identification of lactobacillus delbrueckii and subspecies by hybridization probes and PCR. PMID : 10930078 : DOI : 10.1016/S0723-2020(00)80012-0 Abstract >>
Three methods addressing two different target sites were compared for identification and differentiation of the subspecies Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis/delbrueckii. A PCR method - three primer pairs that enable direct identification of the species and the two subspecies, respectively - was derived from a DNA fragment showing significant similarities to parts of the addAB genes of Bacillus sutbtilis. In addition, two oligonucleotide probes for the two subspecies were designed from that DNA region. Further, two oligonucleotide probes targeting the 16S rDNA were developed for subspecies differentiation by a one base-pair difference following identification of the species. Moreover, these probes were demonstrated to be applicable for in situ hybridization experiments. The results obtained by the different methods were in good agreement.
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12. |
Siragusa S,
De Angelis M,
Di Cagno R,
Rizzello CG,
Coda R,
Gobbetti M,
( 2007 ) Synthesis of gamma-aminobutyric acid by lactic acid bacteria isolated from a variety of Italian cheeses. PMID : 17890341 : DOI : 10.1128/AEM.01064-07 PMC : PMC2168214 Abstract >>
The concentrations of gamma-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg(-1). Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA.
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13. |
Morel F,
Atlan D,
Portalier R,
Aubel D,
( 1999 ) Characterization of a prolidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 with an unusual regulation of biosynthesis. PMID : 10075426 : DOI : 10.1099/13500872-145-2-437 Abstract >>
Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 (Lb. bulgaricus) is characterized by a high level of peptidase activities specific to proline-containing peptides. A prolidase (PepQ, EC 3.4.13.9) was purified to homogeneity and characterized as a strict dipeptidase active on X-Pro dipeptides, except Gly-Pro and Pro-Pro. The values for Km and Vmax were, respectively, 2.2 mM and 0.33 mmol min(-1) mg(-1), with Leu-Pro as the substrate. The enzyme exhibited optimal activity at 50 degrees C and pH 6.0, and required the presence of Zn2+. Size exclusion chromatographies and SDS-PAGE analysis led to the conclusion that this prolidase was a homodimer. Antibodies raised against the purified protein allowed the detection of PepQ among several Lactobacillus species but not lactococci. The pepQ gene and the upstream region were isolated and sequenced. The deduced peptide sequence showed that PepQ belongs to the M24 family of metallopeptidases. The pepR1 gene is located immediately upstream of pepQ and its product is homologous to the transcription factor CcpA, which is involved in catabolite repression of catabolic operons from Gram-positive bacteria. The pepR1-pepQ intergenic region contains a consensus catabolite-responsive element (CRE) which could be a target for PepR1 protein. Moreover, in contrast to other proline-specific enzymes from Lb. bulgaricus, PepQ biosynthesis was shown to be dependent on the composition of the culture medium, but not on the peptide concentration. A possible regulation mechanism is discussed.
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14. |
Lee JH,
Halgerson JS,
Kim JH,
O'Sullivan DJ,
( 2007 ) Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector. PMID : 17526779 : DOI : 10.1128/AEM.00099-07 PMC : PMC1932812 Abstract >>
While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three-a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria.
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15. |
Leong-Morgenthaler P,
Zwahlen MC,
Hottinger H,
( 1991 ) Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved. PMID : 1705929 : DOI : 10.1128/jb.173.6.1951-1957.1991 PMC : PMC207726 Abstract >>
The genes coding for the lactose permease and beta-galactosidase, two proteins involved in the metabolism of lactose by Lactobacillus bulgaricus, have been cloned, expressed, and found functional in Escherichia coli. The nucleotide sequences of these genes and their flanking regions have been determined, showing the presence of two contiguous open reading frames (ORFs). One of these ORFs codes for the lactose permease gene, and the other codes for the beta-galactosidase gene. The lactose permease gene is located in front of the beta-galactosidase gene, with 3 bp in the intergenic region. The two genes are probably transcribed as one operon. Primer extension studies have mapped a promoter upstream from the lactose permease gene but not the beta-galactosidase gene. This promoter is similar to those found in E. coli with general characteristics of GC-rich organisms. In addition, the sequences around the promoter contain a significantly higher number of AT base pairs (80%) than does the overall L. bulgaricus genome, which is rich in GC (GC content of 54%). The amino acid sequences obtained from translation of the ORFs are found to be highly homologous (similarity of 75%) to those from Streptococcus thermophilus. The first 460 amino acids of the lactose permease shows homology to the melibiose transport protein of E. coli. Little homology was found between the lactose permease of L. bulgaricus and E. coli, but the residues which are involved in the binding and the transport of lactose are conserved. The carboxy terminus is similar to that of the enzyme III of several phosphoenolpyruvate-dependent phosphotransferase systems.
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16. |
Mozzi F,
Vaningelgem F,
Hébert EM,
Van der Meulen R,
Foulquié Moreno MR,
Font de Valdez G,
De Vuyst L,
( 2006 ) Diversity of heteropolysaccharide-producing lactic acid bacterium strains and their biopolymers. PMID : 16751563 : DOI : 10.1128/AEM.02780-05 PMC : PMC1489642 Abstract >>
Thirty-one lactic acid bacterial strains from different species were evaluated for exopolysaccharide (EPS) production in milk. Thermophilic strains produced more EPS than mesophilic ones, but EPS yields were generally low. Ropiness or capsular polysaccharide formation was strain dependent. Six strains produced high-molecular-mass EPS. Polymers were classified into nine groups on the basis of their monomer composition. EPS from Enterococcus strains were isolated and characterized.
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17. |
van de Guchte M,
Penaud S,
Grimaldi C,
Barbe V,
Bryson K,
Nicolas P,
Robert C,
Oztas S,
Mangenot S,
Couloux A,
Loux V,
Dervyn R,
Bossy R,
Bolotin A,
Batto JM,
Walunas T,
Gibrat JF,
Bessières P,
Weissenbach J,
Ehrlich SD,
Maguin E,
( 2006 ) The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution. PMID : 16754859 : DOI : 10.1073/pnas.0603024103 PMC : PMC1482600 DOI : 10.1073/pnas.0603024103 PMC : PMC1482600 Abstract >>
Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. (i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; (ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.
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18. |
Paricharttanakul NM,
Ye S,
Menefee AL,
Javid-Majd F,
Sacchettini JC,
Reinhart GD,
( 2005 ) Kinetic and structural characterization of phosphofructokinase from Lactobacillus bulgaricus. PMID : 16285731 : DOI : 10.1021/bi051283g Abstract >>
Phosphofructokinase from Lactobacillus delbrueckii subspecies bulgaricus (LbPFK) has been reported to be a nonallosteric analogue of phosphofructokinase from Escherichia coli at pH 8.2 [Le Bras et al. (1991) Eur. J. Biochem. 198, 683-687]. A reexamination of the kinetics of this enzyme shows LbPFK to have limited binding affinity toward the allosteric ligands, MgADP and PEP, with dissociation constants of approximately 20 mM for both. Their allosteric effects are observed only at high concentrations of these ligands, with both exhibiting inhibitory effects on substrate binding. No pH dependence was observed for the binding and the influence of MgADP and PEP on the enzyme. To attempt to explain these results, the crystal structure of LbPFK was solved using molecular replacement to 1.86 A resolution. A comparative study of the LbPFK structure with that of phosphofructokinases from E. coli (EcPFK) and Bacillus stearothermophilus (BsPFK) reveals a structure with conserved fold and substrate binding site. The effector binding site, however, shows many differences that could explain the observed decreases in binding affinity for MgADP and PEP in LbPFK as compared to the other two enzymes.
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19. |
Duwat P,
Ehrlich SD,
Gruss A,
( 1992 ) A general method for cloning recA genes of gram-positive bacteria by polymerase chain reaction. PMID : 1629178 : DOI : 10.1128/jb.174.15.5171-5175.1992 PMC : PMC206342 Abstract >>
An internal fragment of the recA gene from eight gram-positive organisms has been amplified by using degenerate primers in a polymerase chain reaction. The internal 348- or 360-bp recA DNA segments from Bacillus subtilis, Clostridium acetobutylicum, Lactobacillus bulgaricus, Lactobacillus helveticus, Leuconostoc mesanteroides, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus salivarus subsp. thermophilus were amplified, cloned, and sequenced. The G + C contents of the DNA from these species range from 28 to 52%. The sequences of the bacterial recA genes show strong relatedness. This method is particularly useful for the recovery of the recA genes of gram-positive bacteria and avoids the difficulties of using a genetic complementation test for cloning.
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20. |
Kochhar S,
Chuard N,
Hottinger H,
( 1992 ) Cloning and overexpression of the Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene in Escherichia coli:purification and characterization of the recombinant enzyme. PMID : 1610363 : DOI : 10.1016/0006-291x(92)91683-h Abstract >>
The Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene was amplified by the polymerase chain reaction and cloned into an Escherichia coli expression plasmid pKK223.3. Attempts to clone the full-length chromosomal DNA encoding D-lactate dehydrogenase from a partial Sau3AI lambda phage library or an enriched clone bank in E. coli were unsuccessful. The recombinant plasmid pKBULDH containing the amplified gene overexpressed D-lactate dehydrogenase (greater than 30% of total soluble protein) following induction of the tac promotor with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by two chromatographic steps with 76% recovery of enzyme activity. All the properties of the recombinant protein, e.g., optimum pH and temperature, Km and k(cat) for pyruvate as well as for other 2-oxo acids and the subunit structure were identical to the wild-type enzyme.
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21. |
Kochhar S,
Hunziker PE,
Leong-Morgenthaler P,
Hottinger H,
( 1992 ) Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site. PMID : 1569100 : Abstract >>
The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.
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22. |
Kochhar S,
Hunziker PE,
Leong-Morgenthaler P,
Hottinger H,
( 1992 ) Evolutionary relationship of NAD(+)-dependent D-lactate dehydrogenase: comparison of primary structure of 2-hydroxy acid dehydrogenases. PMID : 1567457 : DOI : 10.1016/0006-291x(92)91157-l Abstract >>
A comparison of the primary structures of NAD(+)-dependent D-lactate dehydrogenase with L-lactate dehydrogenase and L-malate dehydrogenase failed to show any sequence similarity. However, D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei, glycerate dehydrogenase from cucumber, D-3-phosphoglycerate dehydrogenase and erythronate 4-phosphate dehydrogenase from Escherichia coli showed 38%, 24%, 24% and 22% amino acid identity, respectively. The profile analysis of the aligned sequences confirmed their relatedness. The hydropathy profiles of the aligned dehydrogenases were almost identical between residues 100-300 indicating largely preserved folding patterns of their polypeptide chains. The data suggest that L- and D-specific 2-hydroxy acid dehydrogenase genes evolved from two different ancestors and thus represent two different sets of enzyme families.
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23. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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24. |
Cousin S,
Gulat-Okalla ML,
Motreff L,
Gouyette C,
Bouchier C,
Clermont D,
Bizet C,
( 2012 ) Lactobacillus gigeriorum sp. nov., isolated from chicken crop. PMID : 21421927 : DOI : 10.1099/ijs.0.028217-0 Abstract >>
In the early 1980s, a facultatively anaerobic, non-motile, short rod, designated 202(T), was isolated from a chicken crop and identified as a homofermentative lactic acid bacterium. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain was affiliated with the genus Lactobacillus, clustering within the Lactobacillus acidophilus-delbrueckii group. In this analysis, strain 202(T) appeared to be most closely related to the type strains of Lactobacillus intestinalis and Lactobacillus amylolyticus, with gene sequence similarities of 96.1 and 96.2 %, respectively. Strain 202(T) was found to differ from these two species, however, when investigated by multilocus sequence analysis, and it also differed in terms of some of its metabolic properties. On the basis of these observations, strain 202(T) is considered to represent a novel species in the genus Lactobacillus, for which the name Lactobacillus gigeriorum sp. nov. is proposed; the type strain is 202(T) (= CRBIP 24.85(T) = DSM 23908(T)).
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25. |
Tanigawa K,
Watanabe K,
( 2011 ) Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii. PMID : 21178164 : DOI : 10.1099/mic.0.043240-0 Abstract >>
Currently, the species Lactobacillus delbrueckii is divided into four subspecies, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. indicus and L. delbrueckii subsp. lactis. These classifications were based mainly on phenotypic identification methods and few studies have used genotypic identification methods. As a result, these subspecies have not yet been reliably delineated. In this study, the four subspecies of L. delbrueckii were discriminated by phenotype and by genotypic identification [amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST)] methods. The MLST method developed here was based on the analysis of seven housekeeping genes (fusA, gyrB, hsp60, ileS, pyrG, recA and recG). The MLST method had good discriminatory ability: the 41 strains of L. delbrueckii examined were divided into 34 sequence types, with 29 sequence types represented by only a single strain. The sequence types were divided into eight groups. These groups could be discriminated as representing different subspecies. The results of the AFLP and MLST analyses were consistent. The type strain of L. delbrueckii subsp. delbrueckii, YIT 0080(T), was clearly discriminated from the other strains currently classified as members of this subspecies, which were located close to strains of L. delbrueckii subsp. lactis. The MLST scheme developed in this study should be a useful tool for the identification of strains of L. delbrueckii to the subspecies level.
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26. |
Bernard N,
Ferain T,
Garmyn D,
Hols P,
Delcour J,
( 1991 ) Cloning of the D-lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli. PMID : 1915894 : DOI : 10.1016/0014-5793(91)81226-x Abstract >>
A strain of Escherichia coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LDH) was complemented with a genomic DNA library from Lactobacillus delbrueckii subsp. bulgaricus. One positive cloned showed LDH activity and production of D(-)lactate was demonstrated. The nucleotide sequence of the D-LDH gene (ldhA) revealed the spontaneous insertion of an E. coli insertion sequence IS2 upstream of the gene coding region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LDH sequences but closely related to L. casei D-hydroxyisocaproate dehydrogenase (D-HicDH).
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27. |
Le Bras G,
Deville-Bonne D,
Garel JR,
( 1991 ) Purification and properties of the phosphofructokinase from Lactobacillus bulgaricus. A non-allosteric analog of the enzyme from Escherichia coli. PMID : 1828763 : DOI : 10.1111/j.1432-1033.1991.tb16067.x Abstract >>
Phosphofructokinase, the enzyme which catalyzes the conversion of fructose 6-phosphate into fructose 1,6-bisphosphate in Lactobacillus bulgaricus (Lactobacillus delbrueckii, subspecies bulgaricus) has been purified to homogeneity and some of its structural and functional properties have been studied. The enzyme is a tetramer composed of four 35-kDa subunits. Its N-terminal sequence determined on 38 residues is homologous to those of the major allosteric enzymes from Escherichia coli and Bacillus stearothermophilus, suggesting that the three proteins have closely related structures. The maximum velocity of the enzyme from L. bulgaricus increases with pH according to the ionization of a group with a pK of 6.2. At all pH values, the saturation by fructose 6-phosphate is hyperbolic. At the optimum pH of 8.2, the maximum velocity and the affinities for the ATP and fructose 6-phosphate substrates are not modified by the presence of ADP or GDP nor by phosphoenolpyruvate. Partial inhibition by phosphoenolpyruvate exists at acidic pH, but is not related to an allosteric mechanism similar to that in E. coli. This inhibition results from a shift from 6.2 to 7.1 of the pK of an ionizable group which controls Vmax. Protection against thermal denaturation shows that the enzyme binds phosphoenolpyruvate and not GDP. The phosphofructokinase from L. bulgaricus appears as a structural analog of the E. coli enzyme which does not undergo an allosteric transition between two states R and T, but instead remains in a unique conformational state, intermediate between the R and T states; the active sites have an R-like conformation since they bind fructose 6-phosphate, whereas the regulatory sites have a T-like conformation since they bind phosphoenolpyruvate and not GDP.
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28. |
Nierop Groot MN,
Kleerebezem M,
( 2007 ) Mutational analysis of the Lactococcus lactis NIZO B40 exopolysaccharide (EPS) gene cluster: EPS biosynthesis correlates with unphosphorylated EpsB. PMID : 18045447 : DOI : 10.1111/j.1365-2672.2007.03516.x Abstract >>
To determine the role of the EpsA, EpsB, and EpsC proteins encoded at the 5'-end of the exopolysaccharide (EPS) gene cluster in regulation of EPS production in Lactococcus lactis. Deletion and paralog-replacement mutants of epsABCD were used to determine the function of EpsA, EpsB and EpsC in EPS production and polymer chain length determination in L. lactis. EpsA and EpsB appeared to be essential for EPS biosynthesis in L. lactis, while deletion of the phosphatase (EpsC) only had a minor effect on the EPS production level. Determination of the phosphorylation state of EpsB and analysis of a C-terminally truncated EpsB variant indicate that EPS biosynthesis in L. lactis is driven by a nonphosphorylated form of EpsB. The data presented here show that in L. lactis, EPS production is under control of a phosphoregulatory system and that EPS biosynthesis correlates with an unphosphorylated EpsB. This study provides molecular understanding of polysaccharide production in L. lactis that could eventually enable novel approaches to control EPS production by lactic acid bacteria during industrial fermentation processes.
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29. |
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. 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.
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30. |
Morovic W,
Hibberd AA,
Zabel B,
Barrangou R,
Stahl B,
( 2016 ) Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases. PMID : 27857709 : DOI : 10.3389/fmicb.2016.01747 PMC : PMC5093124 Abstract >>
Recent advances in microbiome research have brought renewed focus on beneficial bacteria, many of which are available in food and dietary supplements. Although probiotics have historically been defined as microorganisms that convey health benefits when ingested in sufficient viable amounts, this description now includes the stipulation "well defined strains," encompassing definitive taxonomy for consumer consideration and regulatory oversight. Here, we evaluated 52 commercial dietary supplements covering a range of labeled species using plate counting and targeted genotyping. Strain identities were assessed using methods recently published by the United States Pharmacopeial Convention. We also determined the relative abundance of individual bacteria by high-throughput sequencing (HTS) of the 16S rRNA sequence using paired-end 2 �� 250 bp Illumina MiSeq technology. Using these methods, we tested the hypothesis that products do contain the quantitative and qualitative list of labeled microbial species. We found that 17 samples (33%) were below label claim for CFU prior to their expiration dates. A multiplexed-PCR scheme showed that only 30/52 (58%) of the products contained a correctly labeled classification, with issues encompassing incorrect taxonomy, missing species, and un-labeled species. The HTS revealed that many blended products consisted predominantly of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis. These results highlight the need for reliable methods to determine the correct taxonomy and quantify the relative amounts of mixed microbial populations in commercial probiotic products.
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31. |
Swier LJ,
Guskov A,
Slotboom DJ,
( 2016 ) Structural insight in the toppling mechanism of an energy-coupling factor transporter. PMID : 27026363 : DOI : 10.1038/ncomms11072 PMC : PMC4820897 DOI : 10.1038/ncomms11072 PMC : PMC4820897 DOI : 10.1038/ncomms11072 PMC : PMC4820897 Abstract >>
Energy-coupling factor (ECF) transporters mediate uptake of micronutrients in prokaryotes. The transporters consist of an S-component that binds the transported substrate and an ECF module (EcfAA'T) that binds and hydrolyses ATP. The mechanism of transport is poorly understood but presumably involves an unusual step in which the membrane-embedded S-component topples over to carry the substrate across the membrane. In many ECF transporters, the S-component dissociates from the ECF module after transport. Subsequently, substrate-bound S-components out-compete the empty proteins for re-binding to the ECF module in a new round of transport. Here we present crystal structures of the folate-specific transporter ECF-FolT from Lactobacillus delbrueckii. Interaction of the ECF module with FolT stabilizes the toppled state, and simultaneously destroys the high-affinity folate-binding site, allowing substrate release into the cytosol. We hypothesize that differences in the kinetics of toppling can explain how substrate-loaded FolT out-competes apo-FolT for association with the ECF module.
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32. |
Swier LJ,
Guskov A,
Slotboom DJ,
( 2016 ) Structural insight in the toppling mechanism of an energy-coupling factor transporter. PMID : 27026363 : DOI : 10.1038/ncomms11072 PMC : PMC4820897 DOI : 10.1038/ncomms11072 PMC : PMC4820897 DOI : 10.1038/ncomms11072 PMC : PMC4820897 Abstract >>
Energy-coupling factor (ECF) transporters mediate uptake of micronutrients in prokaryotes. The transporters consist of an S-component that binds the transported substrate and an ECF module (EcfAA'T) that binds and hydrolyses ATP. The mechanism of transport is poorly understood but presumably involves an unusual step in which the membrane-embedded S-component topples over to carry the substrate across the membrane. In many ECF transporters, the S-component dissociates from the ECF module after transport. Subsequently, substrate-bound S-components out-compete the empty proteins for re-binding to the ECF module in a new round of transport. Here we present crystal structures of the folate-specific transporter ECF-FolT from Lactobacillus delbrueckii. Interaction of the ECF module with FolT stabilizes the toppled state, and simultaneously destroys the high-affinity folate-binding site, allowing substrate release into the cytosol. We hypothesize that differences in the kinetics of toppling can explain how substrate-loaded FolT out-competes apo-FolT for association with the ECF module.
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33. |
Yang L,
Chen Y,
Li Z,
Shi Y,
Li Z,
Zhao X,
( 2016 ) Complete Genome Sequence of Lactobacillus acidophilus MN-BM-F01. PMID : 26868391 : DOI : 10.1128/genomeA.01699-15 PMC : PMC4751315 Abstract >>
Lactobacillus acidophilus MN-BM-F01 was originally isolated from a traditional fermented dairy product in China. The characteristics of this bacterium are its low post-acidification ability and high acid-producing rate. Here, we report the main genome features of L. acidophilus MN-BM-F01.
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34. |
Pang XY,
Cui WM,
Liu L,
Zhang SW,
Lv JP,
( 2014 ) Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6. PMID : 25110891 : DOI : 10.1371/journal.pone.0104829 PMC : PMC4128740 Abstract >>
Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.
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35. |
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. 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.
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36. |
Schmidt BF,
Adams RM,
Requadt C,
Power S,
Mainzer SE,
( 1989 ) Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli. PMID : 2492511 : DOI : 10.1128/jb.171.2.625-635.1989 PMC : PMC209643 Abstract >>
The Lactobacillus bulgaricus beta-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'- and 5'-flanking sequences was determined. The amino acid sequence of the beta-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) beta-galactosidase. The cloned beta-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus beta-galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these beta-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ beta-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two beta-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these beta-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic beta-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.
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37. |
Lu L,
Xu S,
Zhao R,
Zhang D,
Li Z,
Li Y,
Xiao M,
( 2012 ) Synthesis of galactooligosaccharides by CBD fusion �]-galactosidase immobilized on cellulose. PMID : 22525263 : DOI : 10.1016/j.biortech.2012.03.108 Abstract >>
The �]-galactosidase gene (bgaL3) was cloned from Lactobacillus bulgaricus L3 and fused with cellulose binding domain (CBD) using pET-35b (+) vector in Escherichia coli. The resulting fusion protein (CBD-BgaL3) was directly adsorbed onto microcrystalline cellulose with a high immobilization efficiency of 61%. A gram of cellulose was found to absorb 97.6 U of enzyme in the solution containing 100mM NaCl (pH 5.8) at room temperature for 20 min. The enzymatic and transglycosylation characteristics of the immobilized CBD-BgaL3 were similar to the free form. Using the immobilized enzyme as the catalyst, the yield of galactooligosaccharides (GOS) reached a maximum of 49% (w/w) from 400 g/L lactose (pH 7.6) at 45 �XC for 75 min, with a high productivity of 156.8 g/L/h. Reusability assay was subsequently performed under the same reaction conditions. The immobilized enzyme could retain over 85% activity after twenty batches with the GOS yields all above 40%.
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38. |
Huang CH,
Chang MT,
Huang MC,
Wang LT,
Huang L,
Lee FL,
( 2012 ) Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene. PMID : 22555934 : DOI : 10.1002/jsfa.5692 Abstract >>
To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing.
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39. |
Cui Y,
Liu W,
Qu X,
Chen Z,
Zhang X,
Liu T,
Zhang L,
( 2012 ) A two component system is involved in acid adaptation of Lactobacillus delbrueckii subsp. bulgaricus. PMID : 22177749 : DOI : 10.1016/j.micres.2011.11.003 Abstract >>
The Gram-positive bacterium Lactobacillus delbrueckii subsp. bulgaricus is of vital importance to the food industry, especially to the dairy industry. Two component systems (TCSs) are one of the most important mechanisms for environmental sensing and signal transduction in the majority of Gram-positive and Gram-negative bacteria. A typical TCS consists of a histidine protein kinase (HPK) and a cytoplasmic response regulator (RR). To investigate the functions of TCSs during acid adaptation in L. bulgaricus, we used quantitative PCR to reveal how TCSs expression changes during acid adaptation. Two TCSs (JN675228/JN675229 and JN675230/JN675231) and two HPKs (JN675236 and JN675240) were induced during acid adaptation. These TCSs were speculated to be related with the acid adaptation ability of L. bulgaricus. The mutants of JN675228/JN675229 were constructed in order to investigate the functions of JN675228/JN675229. The mutants showed reduced acid adaptation compared to that of wild type, and the complemented strains were similar to the wild-type strain. These observations suggested that JN675228 and JN675229 were involved in acid adaptation in L. bulgaricus. The interaction between JN675228 and JN675229 was identified by means of yeast two-hybrid system. The results indicated there is interaction between JN675228 and JN675229.
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40. |
( 1997 ) D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus. II. Mutagenic analysis of catalytically important residues. PMID : 9063466 : DOI : 10.1111/j.1432-1033.1997.00213.x Abstract >>
Five residues involved in catalysis and coenzyme binding have been identified in D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus by using biochemical and genetical methods. Enzyme inactivation with diethylpyrocarbonate indicated that a single histidine residue was involved in catalysis. Since H296 is the only conserved histidine in the whole family of NAD-dependent D-2-hydroxyacid dehydrogenases, we constructed the H296Q and H296S mutants and showed that their catalytic efficiencies were reduced 10(5)-fold compared with the wild-type enzyme. This low residual activity was shown to be insensitive to diethylpyrocarbonate. Taken together these data demonstrate that H296 is responsible for proton exchange in the redox reaction. Two acidic residues (D259 and E264) were candidates for maintaining H296 in the protonated state and their roles were examined by mutagenesis. The D259N and E264Q mutant enzymes both showed similar and large reductions in their Kcat/K(m) ratios (200-800-fold, depending on pH), indicating that either D259 or E264 (or both) could partner H296. The conserved R235 residue was a candidate for binding the alpha-carboxyl group of the substrate and it was changed to lysine. The R235K mutant showed a 104-fold reduced Kcat/K(m) due to both an increased K(m) and a reduced Kcat for 2-oxo-4-methylvalerate. Thus R235 plays a role in binding the substrate carboxylate similar to R171 in the L-lactate dehydrogenases. Finally, we constructed the H205Q mutant to test the role of this partially conserved histidine residue (in 10/13 enzymes of the family). This mutant enzyme displayed a 7.7-fold increased Kcat and a doubled catalytic efficiency at pH 5, was as sensitive to diethylpyrocarbonate as the wild-type but showed a sevenfold increased K(m) for NADH and a 100-fold increase in Kd for NADH together with 10-30-fold lower substrate inhibition. The transient kinetic behaviour of the H205Q mutant is as predicted from our previous study on the enzymatic mechanism of D-2-hydroxy-4-methylvalerate dehydrogenase which showed that coenzyme binding is highly pH dependent and indicated that release of the oxidised coenzyme is a significant component of the rate-limiting processes in catalysis at pH 6.5.
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41. |
( 1996 ) Asn-tRNA in Lactobacillus bulgaricus is formed by asparaginylation of tRNA and not by transamidation of Asp-tRNA. PMID : 8758990 : DOI : 10.1093/nar/24.14.2648 PMC : PMC146019 Abstract >>
In many organisms (e.g., gram-positive eubacteria) Gin-tRNA is not formed by direct glutaminylation of tRNAGln but by a specific transamidation of Glu-tRNAGln. We wondered whether a similar transamidation pathway also operates in the formation of Asn-tRNA in these organisms. Therefore we tested in S-100 preparations of Lactobacillus bulgaricus, a gram-positive eubacterium, for the conversion by an amidotransferase of [14C]Asp-tRNA to [14C]Asn-tRNA. As no transamidation was observed, we searched for genes for asparaginyl-tRNA synthetase (AsnRS). Two DNA fragments (from different locations of the L.bulgaricus chromosome) were found each containing an ORF whose sequence resembled that of the Escherichia coli asnS gene. The derived amino acid sequences of the two ORFs (432 amino acids) were the same and 41% identical with E.coli AsnRS. When one of the ORFs was expressed in E.coli, it complemented the temperature sensitivity of an E.coli asnS mutant. S-100 preparations of this transformant showed increased charging of unfractionated L.bulgaricus tRNA with asparagine. Deletion of the 3'-terminal region of the L.bulgaricus AsnRS gene led to loss of its complementation and aminoacylation properties. This indicates that L.bulgaricus contains a functional AsnRS. Thus, the transamidation pathway operates only for Gin-tRNAGln formation in this organism, and possibly in all gram-positive eubacteria.
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42. |
( 1996 ) The genes for phosphofructokinase and pyruvate kinase of Lactobacillus delbrueckii subsp. bulgaricus constitute an operon. PMID : 8755908 : DOI : 10.1128/jb.178.15.4727-4730.1996 PMC : PMC178247 Abstract >>
In Lactobacillus delbrueckii subsp. bulgaricus, the pyk gene coding for pyruvate kinase and the pfk gene coding for phosphofructokinase formed a bicistronic operon transcribed into a 2.9-kb RNA. The nucleotide sequence of the pyk gene indicated that the encoded protein possessed an extra C-terminal domain with a potential phosphoenolpyruvate-dependent autophosphorylation site.
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43. |
( 1996 ) Lactobacillus bulgaricus asparagine synthetase and asparaginyl-tRNA synthetase: coregulation by transcription antitermination? PMID : 8636057 : DOI : 10.1128/jb.178.8.2459-2461.1996 PMC : PMC177964 Abstract >>
Genes encoding the ammonia-dependent asparagine synthetase (asnA) and asparaginyl-tRNA synthetase (asnS) have been cloned from Lactobacillus bulgaricus ATCC 11842. The nucleotide sequence suggests that asnA and asnS are organized as one operon and regulated by the tRNA-directed transcription antitermination mechanism (T. M. Henkin, Mol. Microbiol. 13:381-387, 1994).
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( 1993 ) Pyruvate kinase from Lactobacillus bulgaricus: possible regulation by competition between strong and weak effectors. PMID : 8274531 : DOI : 10.1016/0300-9084(93)90130-k Abstract >>
The pyruvate kinase from Lactobacillus bulgaricus has been purified to homogeneity. The native enzyme is composed of four probably identical subunits of relative molecular mass M(r) 72,000 +/- 4,000. The unique N-terminal amino acid sequence is homologous to those of other pyruvate kinases, especially of type I and II enzymes from Escherichia coli. The saturation of the pyruvate kinase from Lactobacillus bulgaricus is hyperbolic for ADP and cooperative for the other substrate phospho-enol-pyruvate. The enzyme is strongly activated by glucose-6-phosphate, ribose-5-phosphate, and fructose-6-phosphate, which increase the affinity for phospho-enol-pyruvate. These activators seem to stabilize the same state of the enzyme, since their maximum activations are not additive, but their partial activations can be cumulated. Pyruvate kinase is also weakly activated by AMP and inhibited by fructose-1,6-bisphosphate. However, both AMP and fructose-1,6-bisphosphate act as strong inhibitors in the presence of a strong activator, because these weak effectors suppress the activation by glucose-6-phosphate, ribose-5-phosphate, or fructose-6-phosphate. This mutual exclusion of strong and weak effectors, which appears as an original regulatory mechanism, could reflect either the binding of different effectors to different interacting sites or their competition for a unique polyvalent regulatory site in the pyruvate kinase from Lactobacillus bulgaricus.
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( 1993 ) Cloning, sequencing, and expression in Escherichia coli of the gene coding for phosphofructokinase in Lactobacillus bulgaricus. PMID : 8366023 : DOI : 10.1128/jb.175.17.5344-5349.1993 PMC : PMC206588 Abstract >>
A fragment of 1,185 bp containing the gene coding for phosphofructokinase (ATP:D-fructose-6-phosphate-1-phosphotransferase; EC 2.7.1.11) in Lactobacillus bulgaricus has been cloned, sequenced, and expressed in Escherichia coli. The amino acid sequence of this enzyme was homologous to those of the ATP-dependent phosphofructokinases from E. coli, Thermus thermophilus, Spiroplasma citri, and Bacillus stearothermophilus, suggesting that these enzymes have closely related structures despite their different regulatory properties. The recombinant protein had the same structural and functional properties as did the original enzyme. The 3' end of the 1,185-bp fragment showed the presence of an open reading frame corresponding to the N-terminal amino acid sequence of the pyruvate kinase from L. bulgaricus. This gene organization, the same as that in S. citri (C. Chevalier, C. Saillard, and J. M. Bov?, J. Bacteriol. 172:2693-2703, 1990) and B. stearothermophilus (D. Walker, W. N. Chia, and H. Muirhead, J. Mol. Biol. 228:265-276, 1992; H. Sakai and T. Ohta, Eur. J. Biochem. 311:851-859, 1993) but different from that in E. coli (H. W. Hellinga and P. R. Evans, Eur. J. Biochem. 149:363-373, 1985), indicated that the same transcription unit apparently contained the genes for phosphofructokinase and pyruvate kinase, the two key enzymes of glycolysis. The possibility that these genes could be transcribed at the same time suggested that in L. bulgaricus, the coordinated regulation of phosphofructokinase and pyruvate kinase occurs at the levels of both biosynthesis and enzymatic activity.
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( 1996 ) A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus. PMID : 8655480 : DOI : 10.1128/jb.178.11.3059-3065.1996 PMC : PMC178052 Abstract >>
Investigation of the chromosomal region downstream of the lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus revealed the presence of a gene (prtB) encoding a proteinase of 1,946 residues with a predicted molecular mass of 212 kDa. The deduced amino acid sequence showed that PrtB proteinase displays significant homology with the N termini and catalytic domains of lactococcal PrtP cell surface proteinases and is probably synthesized as a preproprotein. However, the presence of a cysteine near the histidine of the PrtB active site suggests that PrtB belongs to the subfamily of cysteine subtilisins. The C-terminal region strongly differs from those of PrtP proteinases by having a high lysine content, an imperfect duplication of 41 residues, and a degenerated sequence compared with the consensus sequence for proteins anchoring in the cell walls of gram-positive bacteria. Finally, the product of the truncated prtM-like gene located immediately upstream of the prtB gene seems too short to be involved in the maturation of PrtB.
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( 1994 ) A Lactobacillus nifS-like gene suppresses an Escherichia coli transaminase B mutation. PMID : 8031904 : DOI : 10.1016/0300-9084(94)90061-2 Abstract >>
The nifS gene was first identified in nitrogen-fixing bacteria where its protein product is essential for efficient nitrogen fixation. Here, we demonstrate that a nifS-like gene also occurs in Lactobacillus bulgaricus, an organism which does not fix nitrogen, and that the nifS gene product suppresses the leucine auxotrophy of an ilvD, ilvE Escherichia coli strain. The known nifS genes from prokaryotes and eukaryotes exhibit a high degree of sequence conservation although the genes have diverse functions, as shown by their ability to complement or suppress dissimilar mutations. It was suggested that the nifS gene products represent a group of enzymes which mediate a specific chemical reaction common to diverse metabolic pathways. The purified NifS protein from Azotobacter vinelandii was experimentally shown to be a pyridoxal phosphate-dependent cysteine desulfurase. Curiously, the NifS proteins exhibit also a remarkable sequence homology to a new class of pyridoxal phoshate-dependent aminotransferases. We show that the L bulgaricus NifS-like protein is able to replace in vivo transaminase B in E coli. This experimental observation supports the prediction that some NifS-like proteins may be aminotransferases.
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( 1993 ) Prediction of structurally conserved regions of D-specific hydroxy acid dehydrogenases by multiple alignment with formate dehydrogenase. PMID : 8476420 : DOI : 10.1006/bbrc.1993.1398 Abstract >>
We propose a multiple alignment of the sequence of formate dehydrogenase with the D-specific 2-hydroxy acid dehydrogenases family. Structurally conserved regions are predicted for those sequences corresponding to important regions of the catalytic and the coenzyme binding domains defined from the known three-dimensional structure of the formate dehydrogenase, namely the nicotinamide binding site (beta D to beta F) and the beta A-loop-alpha B region containing the typical glycine pattern of the adenosine binding site, the catalytic histidine/aspartic acid pair and an arginine probably involved in the interaction with the carboxyl group of the substrate.
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( 1994 ) Proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397: purification and characterization. PMID : 8012576 : DOI : 10.1099/00221287-140-3-537 Abstract >>
Proline iminopeptidase (PepIP) is a major peptidase in Lactobacillus delbrueckii subsp. bularicus CNRZ397, encoded by the pepIP gene. Amplification and expression of this gene in Escherichia coli K12 resulted in a very high level of enzyme production. Moreover, export into the E. coli periplasm of 45% of PepIP activity allowed us to purify the enzyme easily by a single ion-exchange chromatography step. PepIP is a trimer of Mr 100000 , composed of three identical subunits. In the presence of 0.1% BSA, PepIP activity was optimal at pH 6-7 and stable at temperatures below 40 degrees C. The enzyme was strongly inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, by bestatin and by heavy metal ions. It was also inactivated by p-chloromercuribenzoate, but was reactivated by adding dithiothreitol. PepIP is characterized by a high specificity towards di- or tripeptides with proline at the NH2-terminal position, but is not able to hydrolyse longer peptides, or peptides with hydroxyproline at the NH2-end. The NH2-terminal amino acid sequence of the purified PepIP corresponds to the amino acid sequence deduced from the nucleotide sequence of the pepIP gene.
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( 1994 ) Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397. PMID : 8012575 : DOI : 10.1099/00221287-140-3-527 Abstract >>
The proline iminopeptidase (PepIP) of Lactobacillus delbrueckii subsp. bulgaricus is a major peptidase located in the cell envelope. Its structural gene (pepIP) has been cloned into pUC18 and expressed at a very high level in Escherichia coli to give a PepIP activity 15,000-fold higher than that found in L. delbrueckii subsp. bulgaricus. The nucleotide sequence of the pepIP gene revealed an open reading frame of 295 codons encoding a protein with a predicted M(r) of 33,006, which is consistent with the apparent size of the gene product. The amino acid sequence of PepIP shows significant homology with those of other hydrolases involved in the degradation of cyclic compounds. In particular, there is a region which includes an identified catalytic site containing a serine residue and a motif specific for the active sites of prolyloligopeptidases (Gly-X-Ser-X-Gly-Gly). The PepIP opens a new way for supplying cells with proline using the peptides resulting from the proteolytic degradation of caseins.
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51. |
Santos JA,
Rempel S,
Mous ST,
Pereira CT,
Ter Beek J,
de Gier JW,
Guskov A,
Slotboom DJ,
( 2018 ) Functional and structural characterization of an ECF-type ABC transporter for vitamin B12. PMID : 29809140 : DOI : 10.7554/eLife.35828 PMC : PMC5997447 DOI : 10.7554/eLife.35828 PMC : PMC5997447 Abstract >>
Vitamin B12 (cobalamin) is the most complex B-type vitamin and is synthetized exclusively in a limited number of prokaryotes. Its biologically active variants contain rare organometallic bonds, which are used by enzymes in a variety of central metabolic pathways such as L-methionine synthesis and ribonucleotide reduction. Although its biosynthesis and role as co-factor are well understood, knowledge about uptake of cobalamin by prokaryotic auxotrophs is scarce. Here, we characterize a cobalamin-specific ECF-type ABC transporter from Lactobacillus delbrueckii, ECF-CbrT, and demonstrate that it mediates the specific, ATP-dependent uptake of cobalamin. We solved the crystal structure of ECF-CbrT in an apo conformation to 3.4 ? resolution. Comparison with the ECF transporter for folate (ECF-FolT2) from the same organism, reveals how the identical ECF module adjusts to interact with the different substrate binding proteins FolT2 and CbrT. ECF-CbrT is unrelated to the well-characterized B12 transporter BtuCDF, but their biochemical features indicate functional convergence.
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( 2013 ) Structural characterization of a D-isomer specific 2-hydroxyacid dehydrogenase from Lactobacillus delbrueckii ssp. bulgaricus. PMID : 23110853 : DOI : 10.1016/j.jsb.2012.10.009 Abstract >>
Hydroxyacid dehydrogenases, responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids in lactic acid producing bacteria, have a range of biotechnology applications including antibiotic synthesis, flavor development in dairy products and the production of valuable synthons. The genome of Lactobacillus delbrueckii ssp. bulgaricus, a member of the heterogeneous group of lactic acid bacteria, encodes multiple hydroxyacid dehydrogenases whose structural and functional properties remain poorly characterized. Here, we report the apo and coenzyme NAD? complexed crystal structures of the L. bulgaricusD-isomer specific 2-hydroxyacid dehydrogenase, D2-HDH. Comparison with closely related members of the NAD-dependent dehydrogenase family reveals that whilst the D2-HDH core fold is structurally conserved, the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme.
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( 1999 ) The prolyl aminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus belongs to the alpha/beta hydrolase fold family. PMID : 9989236 : DOI : 10.1016/s0167-4838(98)00264-7 Abstract >>
Prolyl aminopeptidase (PepIP) of Lactobacillus delbrueckii subsp. bulgaricus displays the Gly-x-Ser-x-Gly-Gly consensus motif surrounding the catalytic serine of the prolyl oligopeptidases family. Sequence comparison revealed that this motif and two other domains appear well conserved among bacterial PepIPs and members of the alpha/beta hydrolase fold family. Secondary structural predictions of PepIP were performed from amino acid sequence and corroborated by circular dichroism analysis. These predictions well matched the core structure of alpha/beta hydrolases organised in eight beta-sheets connected by alpha-helices. We obtained 26 mutants of PepIP by chemical or site-directed mutagenesis. Most substitutions associated with stable and inactive mutant proteins were mainly located in the three conserved boxes (including the catalytic serine motif). Taken together, our results strongly suggest that PepIP belongs to the alpha/beta hydrolase fold family and that Ser107, Asp246 and His273 constitute the catalytic triad of the enzyme.
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( 1998 ) An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase. PMID : 9579064 : DOI : 10.1099/00221287-144-4-905 Abstract >>
The structural genes gap, pgk and tpi encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order gap-pgk-tpi. The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, and by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed, noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gap gene, and a less abundant mRNA of 3.4 kb corresponding to the gap-pgk-tpi cluster. The absence of a visible terminator in the 3'-end of the shorter transcript and the location of this 3'-end inside the pgk gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gap gene.
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