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1. Sievers  M, Uermösi  C, Fehlmann  M, Krieger  S,     ( 2003 )

Cloning, sequence analysis and expression of the F1F0-ATPase beta-subunit from wine lactic acid bacteria.

Systematic and applied microbiology 26 (3)
PMID : 14529177  :  
Abstract >>
The nucleotide sequences of the genes encoding the F1F0-ATPase beta-subunit from Oenococcus oeni, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Lactobacillus brevis and Lactobacillus hilgardii were determined. Their deduced amino acid sequences showed homology values of 79-98%. Data from the alignment and ATPase tree indicated that O. oeni and L. mesenteroides subsp. mesenteroides formed a group well-separated from P. damnosus and P. parvulus and from the group comprises L. brevis and L. hilgardii. The N-terminus of the F1F0-ATPase beta-subunit of O. oeni contains a stretch of additional 38 amino acid residues. The catalytic site of the ATPase beta-subunit of the investigated strains is characterized by the two conserved motifs GGAGVGKT and GERTRE. The amplified atpD coding sequences were inserted into the pCRT7/CT-TOPO vector using TA-cloning strategy and transformed in Escherichia coli. SDS-PAGE and Western blot analyses confirmed that O. oeni has an ATPase beta-subunit protein which is larger in size than the corresponding molecules from the investigated strains.
KeywordMeSH Terms
2. Vidgrén  G, Palva  I, Pakkanen  R, Lounatmaa  K, Palva  A,     ( 1992 )

S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence.

Journal of bacteriology 174 (22)
PMID : 1429463  :   DOI  :   10.1128/jb.174.22.7419-7427.1992     PMC  :   PMC207438    
Abstract >>
The surface (S)-layer protein of Lactobacillus brevis was isolated, purified, and characterized. The S-layer protein is the major protein of the cell, with an apparent molecular mass of 46 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogold electron microscopy with polyclonal antiserum against the isolated 46-kDa protein was used to confirm the surface location of this protein. N-terminal amino acid sequences of the intact 46-kDa protein and its tryptic peptides were determined. The gene of the S-layer protein was amplified from the genome of L. brevis by polymerase chain reaction with oligonucleotides, synthesized according to the N-terminal amino acid sequences, as primers. The polymerase chain reaction fragments containing the entire S-layer gene and its regulatory regions were sequenced. Nucleic acid sequence analysis revealed one open reading frame with a capacity to encode a protein of 48,159 Da. From the regulatory region of the gene, two subsequent promoters and a ribosome binding site, showing typical features of prokaryotic consensus sequences, were found. The coding region contained a characteristic gram-positive-type signal peptide of 30 amino acids. Removal of the signal peptide results in a polypeptide of 435 amino acids, which is in excellent agreement with the size of the S-layer protein determined by SDS-PAGE. The size and the 5' end analyses of the S-layer transcripts confirmed the monocistronic nature of the S-layer operon and the functionality of the two promoters found.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
Membrane Glycoproteins
Membrane Proteins
3. Niefind  K, Müller  J, Riebel  B, Hummel  W, Schomburg  D,     ( 2003 )

The crystal structure of R-specific alcohol dehydrogenase from Lactobacillus brevis suggests the structural basis of its metal dependency.

Journal of molecular biology 327 (2)
PMID : 12628239  :   DOI  :   10.1016/s0022-2836(03)00081-0    
Abstract >>
The crystal structure of the apo-form of an R-specific alcohol dehydrogenase from Lactobacillus brevis (LB-RADH) was solved and refined to 1.8A resolution. LB-RADH is a member of the short-chain dehydrogenase/reductase (SDR) enyzme superfamily. It is a homotetramer with 251 amino acid residues per subunit and uses NADP(H) as co-enzyme. NADPH and the substrate acetophenone were modelled into the active site. The enantiospecificity of the enzyme can be explained on the basis of the resulting hypothetical ternary complex. In contrast to most other SDR enzymes, the catalytic activity of LB-RADH depends strongly on the binding of Mg(2+). Mg(2+) removal by EDTA inactivates the enzyme completely. In the crystal structure, the Mg(2+)-binding site is well defined. The ion has a perfect octahedral coordination sphere and occupies a special position concerning crystallographic and molecular point symmetry, meaning that each RADH tetramer contains two magnesium ions. The magnesium ion is no direct catalytic cofactor. However, it is structurally coupled to the putative C-terminal hinge of the substrate-binding loop and, via an extended hydrogen bonding network, to some side-chains forming the substrate binding region. Therefore, the presented structure of apo-RADH provides plausible explanations for the metal dependence of the enzyme.
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. Jakava-Viljanen  M, Avall-Jääskeläinen  S, Messner  P, Sleytr  UB, Palva  A,     ( 2002 )

Isolation of three new surface layer protein genes (slp) from Lactobacillus brevis ATCC 14869 and characterization of the change in their expression under aerated and anaerobic conditions.

Journal of bacteriology 184 (24)
PMID : 12446628  :   DOI  :   10.1128/jb.184.24.6786-6795.2002     PMC  :   PMC135479    
Abstract >>
Two new surface layer (S-layer) proteins (SlpB and SlpD) were characterized, and three slp genes (slpB, slpC, and slpD) were isolated, sequenced, and studied for their expression in Lactobacillus brevis neotype strain ATCC 14869. Under different growth conditions, L. brevis strain 14869 was found to form two colony types, smooth (S) and rough (R), and to express the S-layer proteins differently. Under aerobic conditions R-colony type cells produced SlpB and SlpD proteins, whereas under anaerobic conditions S-colony type cells synthesized essentially only SlpB. Anaerobic and aerated cultivations of ATCC 14869 cells in rich medium also resulted in S-layer protein patterns similar to those of the S- and R-colony type cells, respectively. Electron microscopy suggested the presence of only a single S-layer with an oblique structure on the cells of both colony forms. The slpB and slpC genes were located adjacent to each other, whereas the slpD gene was not closely linked to the slpB-slpC gene region. Northern analyses confirmed that both slpB and slpD formed a monocistronic transcription unit and were effectively expressed, but slpD expression was induced under aerated conditions. slpC was a silent gene under the growth conditions tested. The amino acid contents of all the L. brevis ATCC 14869 S-layer proteins were typical of S-layer proteins, whereas their sequence similarities with other S-layer proteins were negligible. The interspecies identity of the L. brevis S-layer proteins was mainly restricted to the N-terminal regions of those proteins. Furthermore, Northern analyses, expression of a PepI reporter protein under the control of the slpD promoter, and quantitative real-time PCR analysis of slpD expression under aerated and anaerobic conditions suggested that, in L. brevis ATCC 14869, the variation of S-layer protein content involves activation of transcription by a soluble factor rather than DNA rearrangements that are typical for most of the S-layer phase variation mechanisms known.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
7. Lucas  P, Lonvaud-Funel  A,     ( 2002 )

Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis IOEB 9809.

FEMS microbiology letters 211 (1)
PMID : 12052555  :   DOI  :   10.1111/j.1574-6968.2002.tb11207.x    
Abstract >>
Some lactic acid bacteria contain a tyrosine decarboxylase (TDC) which converts tyrosine to tyramine, a biogenic amine frequently encountered in fermented food and wine. Purification and microsequencing of the TDC of Lactobacillus brevis IOEB 9809 allowed us to determine a partial sequence of the TDC gene encoding 264 amino acids of the enzyme. Analysis of this protein sequence revealed typical features of pyridoxal phosphate-dependent amino acid decarboxylases while not any known decarboxylase was closely related to the TDC of L. brevis IOEB 9809. In addition, we could detect other L. brevis strains carrying a TDC gene in a rapid assay based on the polymerase chain reaction.
KeywordMeSH Terms
8. Egloff  MP, Uppenberg  J, Haalck  L, van Tilbeurgh  H,     ( 2001 )

Crystal structure of maltose phosphorylase from Lactobacillus brevis: unexpected evolutionary relationship with glucoamylases.

Structure (London, England : 1993) 9 (8)
PMID : 11587643  :  
Abstract >>
Maltose phosphorylase (MP) is a dimeric enzyme that catalyzes the conversion of maltose and inorganic phosphate into beta-D-glucose-1-phosphate and glucose without requiring any cofactors, such as pyridoxal phosphate. The enzyme is part of operons that are involved in maltose/malto-oligosaccharide metabolism. Maltose phosphorylases have been classified in family 65 of the glycoside hydrolases. No structure is available for any member of this family. We report here the 2.15 A resolution crystal structure of the MP from Lactobacillus brevis in complex with the cosubstrate phosphate. This represents the first structure of a disaccharide phosphorylase. The structure consists of an N-terminal complex beta sandwich domain, a helical linker, an (alpha/alpha)6 barrel catalytic domain, and a C-terminal beta sheet domain. The (alpha/alpha)6 barrel has an unexpected strong structural and functional analogy with the catalytic domain of glucoamylase from Aspergillus awamori. The only conserved glutamate of MP (Glu487) superposes onto the catalytic residue Glu179 of glucoamylase and likely represents the general acid catalyst. The phosphate ion is bound in a pocket facing the carboxylate of Glu487 and is ideally positioned for nucleophilic attack of the anomeric carbon atom. This site is occupied by the catalytic base carboxylate in glucoamylase. These observations strongly suggest that maltose phosphorylase has evolved from glucoamylase. MP has probably conserved one carboxylate group for acid catalysis and has exchanged the catalytic base for a phosphate binding pocket. The relative positions of the acid catalytic group and the bound phosphate are compatible with a direct-attack mechanism of a glycosidic bond by phosphate, in accordance with inversion of configuration at the anomeric carbon as observed for this enzyme.
KeywordMeSH Terms
9. Hayashi  N, Ito  M, Horiike  S, Taguchi  H,     ( 2001 )

Molecular cloning of a putative divalent-cation transporter gene as a new genetic marker for the identification of Lactobacillus brevis strains capable of growing in beer.

Applied microbiology and biotechnology 55 (5)
PMID : 11414327  :  
Abstract >>
Random amplified polymorphic DNA (RAPD) PCR analysis of Lactobacillus brevis isolates from breweries revealed that one of the random primers could distinguish beer-spoilage strains of L. brevis from nonspoilage strains. The 1.1-kb DNA fragment amplified from all beer-spoilers included one open reading frame, termed hitA (hop-inducible cation transporter), which encodes an integral membrane protein with 11 putative trans-membrane domains and a binding protein-dependent transport signature of a non-ATP binding membrane transporter common to several prokaryotic and eukaryotic transporters. The hitA polypeptide is homologous to the natural resistance-associated macrophage protein (Nramp) family characterized as divalent-cation transport proteins in many prokaryotic and eukaryotic organisms. Northern blot analysis indicated that the hitA transcripts are expressed in cells cultivated in MRS broth supplemented with hop bitter compounds, which act as mobile-carrier ionophores, dissipating the trans-membrane pH gradient in bacteria sensitive to the hop bitter compounds by exchanging H+ for cellular divalent cations such as Mn2+. This suggests that the hitA gene products may play an important role in making the bacteria resistant to hop bitter compounds in beer by transporting metal ions such as Mn2+ into cells that no longer maintain the proton gradient.
KeywordMeSH Terms
10. Djordjevic  GM, Tchieu  JH, Saier  MH,     ( 2001 )

Genes involved in control of galactose uptake in Lactobacillus brevis and reconstitution of the regulatory system in Bacillus subtilis.

Journal of bacteriology 183 (10)
PMID : 11325952  :   DOI  :   10.1128/JB.183.10.3224-3236.2001     PMC  :   PMC95224    
Abstract >>
The heterofermentative lactic acid bacterium Lactobacillus brevis transports galactose and the nonmetabolizable galactose analogue thiomethyl-beta-galactoside (TMG) by a permease-catalyzed sugar:H(+) symport mechanism. Addition of glucose to L. brevis cells loaded with [(14)C]TMG promotes efflux and prevents accumulation of the galactoside, probably by converting the proton symporter into a uniporter. Such a process manifests itself physiologically in phenomena termed inducer expulsion and exclusion. Previous evidence suggested a direct allosteric mechanism whereby the phosphocarrier protein, HPr, phosphorylated at serine-46 [HPr(Ser-P)], binds to the galactose:H(+) symporter to uncouple sugar transport from proton symport. To elucidate the molecular mechanism of inducer control in L. brevis, we have cloned the genes encoding the HPr(Ser) kinase, HPr, enzyme I, and the galactose:H(+) symporter. The sequences of these genes were determined, and the relevant phylogenetic trees are presented. Mutant HPr derivatives in which the regulatory serine was changed to either alanine or aspartate were constructed. The cloned galP gene was integrated into the chromosome of Bacillus subtilis, and synthesis of the mutant HPr proteins in this organism was shown to promote regulation of GalP, as expected for a direct allosteric mechanism. We have thus reconstituted inducer control in an organism that does not otherwise exhibit this phenomenon. These results are consistent with the conclusion that inducer exclusion and expulsion in L. brevis operates via a multicomponent signal transduction mechanism wherein the presence of glycolytic intermediates such as fructose 1,6-bisphosphate (the intracellular effector), derived from exogenous glucose (the extracellular effector), activates HPr(Ser) kinase (the sensor) to phosphorylate HPr on Ser-46 (the messenger), which binds to the galactose:H(+) symporter (the target), resulting in uncoupling of sugar transport from proton symport (the response). This cascade allows bacteria to quickly respond to changes in external sugar concentrations. Understanding the molecular mechanism of inducer control advances our knowledge of the link between metabolic and transport processes in bacteria.
KeywordMeSH Terms
Bacterial Proteins
Gene Expression Regulation, Bacterial
11. 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
12. Scheirlinck  I, Van der Meulen  R, Van Schoor  A, Vancanneyt  M, De Vuyst  L, Vandamme  P, Huys  G,     ( 2007 )

Influence of geographical origin and flour type on diversity of lactic acid bacteria in traditional Belgian sourdoughs.

Applied and environmental microbiology 73 (19)
PMID : 17675431  :   DOI  :   10.1128/AEM.00894-07     PMC  :   PMC2075033    
Abstract >>
A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.
KeywordMeSH Terms
Bacterial Typing Techniques
Fermentation
Genetic Variation
13. Iijima  K, Suzuki  K, Ozaki  K, Yamashita  H,     ( 2006 )

horC confers beer-spoilage ability on hop-sensitive Lactobacillus brevis ABBC45cc.

Journal of applied microbiology 100 (6)
PMID : 16696675  :   DOI  :   10.1111/j.1365-2672.2006.02869.x    
Abstract >>
To determine whether horC confers beer-spoilage ability and to evaluate the validity of horC as a trans-species genetic marker for differentiating the beer-spoilage ability of lactic acid bacteria (LAB). Hop-sensitive Lactobacillus brevis ABBC45cc was transformed with an expression plasmid, pHYchorBC, containing putative multidrug resistance gene horC and its putative regulator horB, and the transformant was designated as ABBC45cc/pHYchorBC. As a control, ABBC45cc was transformed with pHYchorB that contains horB, and the transformed strain was designated as ABBC45cc/pHYchorB. As a result of beer-spoilage assay of these transformants, ABBC45cc/pHYchorBC exhibited beer-spoilage ability, whereas ABBC45cc/pHYchorB did not. Furthermore ABBC45cc/pHYchorBC showed higher hop resistance than ABBC45cc/pHYchorB, accounting for the differences in beer-spoilage ability observed between the two transformants. ABBC45cc/pHYchorBC also exhibited higher resistance to various structurally unrelated drugs, compared with ABBC45cc/pHYchorB. horC was shown to confer hop resistance and beer-spoilage ability on ABBC45cc by presumably encoding a multidrug transporter. The finding that horC plays an important role in hop resistance and beer-spoilage ability supports the validity of horC as a trans-species genetic marker for differentiating the beer-spoilage ability of LAB.
KeywordMeSH Terms
Beer
Food Contamination
Genes, Bacterial
Industrial Microbiology
14. Park  KB, Oh  SH,     ( 2007 )

Cloning, sequencing and expression of a novel glutamate decarboxylase gene from a newly isolated lactic acid bacterium, Lactobacillus brevis OPK-3.

Bioresource technology 98 (2)
PMID : 16500100  :   DOI  :   10.1016/j.biortech.2006.01.004    
Abstract >>
Lactobacillus brevis OPK-3, having 84.292 mg/L/h of gamma-aminobutyric acid (GABA) productivity, was isolated from Kimchi, a traditional fermented food in Korea. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from the L. brevis OPK-3, using primers based on two highly conserved regions of GAD. A full-length GAD (LbGAD) clone was subsequently isolated through rapid amplification of cDNA ends (RACE) PCR. Nucleotide sequence analysis revealed that the open reading frame (ORF) consisted of 1401 bases and encoded a protein of 467 amino acid residues with a calculated molecular weight of 53.4 kDa and a pI of 5.65. The amino acid sequence deduced from LbGAD ORF showed 83%, 71%, and 60% identity to the Lactobacillus plantarum GAD, Lactococcus lactis GAD, and Listeria monocytogenes GAD sequences, respectively. The LbGAD gene was expressed in Escherichia coli strain UT481, and the extract of transformed E. coli UT481 contained an induced 53.4 kDa protein and had significantly enhanced GAD activity.
KeywordMeSH Terms
Gene Expression
15. Schlieben  NH, Niefind  K, Müller  J, Riebel  B, Hummel  W, Schomburg  D,     ( 2005 )

Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity.

Journal of molecular biology 349 (4)
PMID : 15896805  :   DOI  :   10.1016/j.jmb.2005.04.029    
Abstract >>
The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is an NADP-dependent, homotetrameric member of the extended enzyme family of short-chain dehydrogenases/reductases (SDR) with a high biotechnological application potential. Its preferred in vitro substrates are prochiral ketones like acetophenone with almost invariably a small methyl group as one substituent and a bulky (often aromatic) moiety as the other. On the basis of an atomic-resolution structure of wild-type RADH in complex with NADP and acetophenone, we designed the mutant RADH-G37D, which should possess an improved cosubstrate specificity profile for biotechnological purposes, namely, a preference for NAD rather than NADP. Comparative kinetic measurements with wild-type and mutant RADH showed that this aim was achieved. To characterize the successful mutant structurally, we determined several, partly atomic-resolution, crystal structures of RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends on an interaction between the adenosine ribose moiety of NAD and the inserted aspartate side-chain. A structural comparison between RADH-G37D as apo-enzyme and as a part of a ternary complex revealed significant rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the active site. This plasticity contributes to generate a small hydrophobic pocket for the methyl group typical for RADH substrates, and a hydrophobic coat for the second, more variable and often aromatic, substituent. Around Ser141 we even found alternative conformations in the backbone. A structural adaptability in this region, which we describe here for the first time for an SDR enzyme, is probably functionally important, because it concerns Ser142, a member of the highly conserved catalytic tetrad typical for SDR enzymes. Moreover, it affects an extended proton relay system that has been identified recently as a critical element for the catalytic mechanism in SDR enzymes.
KeywordMeSH Terms
16. Bor  YC, Moraes  C, Lee  SP, Crosby  WL, Sinskey  AJ, Batt  CA,     ( 1992 )

Cloning and sequencing the Lactobacillus brevis gene encoding xylose isomerase.

Gene 114 (1)
PMID : 1587475  :   DOI  :   10.1016/0378-1119(92)90718-5    
Abstract >>
The gene (xylA) coding for the Lactobacillus brevis xylose isomerase (Xi) has been isolated and its complete nucleotide sequence determined. L. brevis Xi was purified and the N-terminal sequence determined. All attempts to directly clone the intact xylA using a degenerative primer deduced from amino acids (aa) 10-14 were not successful. A fragment coding for the first 462 bp from the 5' end of xylA was isolated by PCR with two primers, one coding for aa M36 to W43 and the second coding for an aa sequence (WGGREG) conserved in a number of Xi's isolated from other bacteria. From the sequence of this fragment, two additional PCR primers were synthesized, which were used in an 'outward' reaction to clone a 546-bp fragment including a region upstream from the N terminus. Finally, the complete xylA gene was cloned in a 0.43-kb NlaIII-SalI fragment and a 1.9-kb SalI-EcoRI fragment. The 449-aa sequence for the L. brevis Xi shows homology with Xis isolated from other bacteria, especially within the primary catalytic domains of the enzyme.
KeywordMeSH Terms
Aldose-Ketose Isomerases
17. Stevenson  DM, Muck  RE, Shinners  KJ, Weimer  PJ,     ( 2006 )

Use of real time PCR to determine population profiles of individual species of lactic acid bacteria in alfalfa silage and stored corn stover.

Applied microbiology and biotechnology 71 (3)
PMID : 16205920  :   DOI  :   10.1007/s00253-005-0170-z    
Abstract >>
Real-time polymerase chain reaction (RT-PCR) was used to quantify seven species of lactic acid bacteria (LAB) in alfalfa silage prepared in the presence or absence of four commercial inoculants and in uninoculated corn stover harvested and stored under a variety of field conditions. Species-specific PCR primers were designed based on recA gene sequences. Commercial inoculants improved the quality of alfalfa silage, but species corresponding to those in the inoculants displayed variations in persistence over the next 96 h. Lactobacillus brevis was the most abundant LAB (12 to 32% of total sample DNA) in all of the alfalfa silages by 96 h. Modest populations (up to 10%) of Lactobacillus plantarum were also observed in inoculated silages. Pediococcus pentosaceus populations increased over time but did not exceed 2% of the total. Small populations (0.1 to 1%) of Lactobacillus buchneri and Lactococcus lactis were observed in all silages, while Lactobacillus pentosus and Enterococcus faecium were near or below detection limits. Corn stover generally displayed higher populations of L. plantarum and L. brevis and lower populations of other LAB species. The data illustrate the utility of RT-PCR for quantifying individual species of LAB in conserved forages prepared under a wide variety of conditions.
KeywordMeSH Terms
18. Fujii  T, Nakashima  K, Hayashi  N,     ( 2005 )

Random amplified polymorphic DNA-PCR based cloning of markers to identify the beer-spoilage strains of Lactobacillus brevis, Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis.

Journal of applied microbiology 98 (5)
PMID : 15836491  :   DOI  :   10.1111/j.1365-2672.2005.02558.x    
Abstract >>
Beer-spoilage ability of lactic acid bacteria such as Lactobacillus brevis is a strain-dependent phenomenon in which the mechanism has not yet been completely clarified. In order to systematically identify genes that contribute to beer-spoilage, large-scale random amplified polymorphic DNA (RAPD)-based cloning methods was carried out. A systematic RAPD polymerase chain reaction (PCR) analysis using 600 primers was performed on beer-spoilage and on nonspoilage strains of L. brevis. Among 600 primers, three were found to amplify a single locus highly specific to beer-spoilage strains. DNA sequencing of this locus revealed a three-part operon encoding a putative glycosyl transferase, membrane protein and teichoic acid glycosylation protein. PCR analysis of typical beer-spoilage lactic acid bacteria suggested that this locus is highly specific to beer-spoilage strains. The cloned markers are highly specific to identify the beer-spoilage strains not only in L. brevis but also in Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis. This paper proves that RAPD-PCR is an efficient method for cloning the strain-specific genes from bacteria. The markers described here is one of the most useful tools to identify the beer-spoilage strains of lactic acid bacteria.
KeywordMeSH Terms
Beer
Food Contamination
Food Microbiology
19. Lucas  P, Landete  J, Coton  M, Coton  E, Lonvaud-Funel  A,     ( 2003 )

The tyrosine decarboxylase operon of Lactobacillus brevis IOEB 9809: characterization and conservation in tyramine-producing bacteria.

FEMS microbiology letters 229 (1)
PMID : 14659544  :   DOI  :   10.1016/S0378-1097(03)00787-0    
Abstract >>
Bacterial genes of tyrosine decarboxylases were recently identified. Here we continued the sequencing of the tyrosine decarboxylase locus of Lactobacillus brevis IOEB 9809 and determined a total of 7979 bp. The sequence contained four complete genes encoding a tyrosyl-tRNA synthetase, the tyrosine decarboxylase, a probable tyrosine permease and a Na+/H+ antiporter. Rapid amplification of cDNA ends (RACE) was employed to determine the 5'-end of mRNAs containing the tyrosine decarboxylase gene. It was located only 34-35 nucleotides upstream of the start codon, suggesting that the preceding tyrosyl-tRNA synthetase gene was transcribed separately. In contrast, reverse transcription-polymerase chain reactions (RT-PCRs) carried out with primers designed to amplify regions spanning gene junctions showed that some mRNAs contained the four genes. Homology searches revealed similar clusters of four genes in the genome sequences of Enterococcus faecalis and Enterococcus faecium. Phylogenetic analyses supported the hypothesis that these genes evolved all together. These data suggest that bacterial tyrosine decarboxylases are encoded in an operon containing four genes.
KeywordMeSH Terms
20. Michlmayr  H, Schümann  C, Kulbe  KD, del Hierro  AM,     ( 2011 )

Heterologously expressed family 51 alpha-L-arabinofuranosidases from Oenococcus oeni and Lactobacillus brevis.

Applied and environmental microbiology 77 (4)
PMID : 21169445  :   DOI  :   10.1128/AEM.01385-10     PMC  :   PMC3067215    
Abstract >>
Putative �\-L-arabinofuranosidases of Oenococcus oeni and Lactobacillus brevis were heterologously expressed and characterized. We report the basic functional properties of the recombinant enzymes in comparison to those of a commercial family 51 arabinosidase of Aspergillus niger.
KeywordMeSH Terms
21. Jang  SH, Yoon  BH, Chang  HI,     ( 2011 )

Complete nucleotide sequence of the temperate bacteriophage LBR48, a new member of the family Myoviridae.

Archives of virology 156 (2)
PMID : 20976608  :   DOI  :   10.1007/s00705-010-0841-7    
Abstract >>
The complete genomic sequence of LBR48, a temperate bacteriophage induced from a lysogenic strain of Lactobacillus brevis, was found to be 48,211 nucleotides long and to contain 90 putative open reading frames. Based on structural characteristics obtained from microscopic analysis and nucleic acid sequence determination, phage LBR48 can be classified as a member of the family Myoviridae. Analysis of the genome showed the conserved gene order of previously reported phages of the family Siphoviridae from lactic acid bacteria, despite low nucleotide sequence similarity. Analysis of the attachment sites revealed 15-nucleotide-long core sequences.
KeywordMeSH Terms
22. Islam  SM, Math  RK, Cho  KM, Lim  WJ, Hong  SY, Kim  JM, Yun  MG, Cho  JJ, Yun  HD,     ( 2010 )

Organophosphorus hydrolase (OpdB) of Lactobacillus brevis WCP902 from kimchi is able to degrade organophosphorus pesticides.

Journal of agricultural and food chemistry 58 (9)
PMID : 20405842  :   DOI  :   10.1021/jf903878e    
Abstract >>
Lactobacillus brevis WCP902 that is capable of biodegrading chlorpyrifos was isolated from kimchi. The opdB gene cloned from this strain revealed 825 bp, encoding 274 aa, and an enzyme molecular weight of about 27 kDa. OpdB contains the same Gly-X-Ser-X-Gly motif found in most bacterial and eukaryotic esterase, lipase, and serine hydrolases, yet it is a novel member of the GDSVG family of esterolytic enzymes. Its conserved serine residue, Ser82, is significantly involved with enzyme activity that may have application for removing some pesticides. Optimum organophosphorus hydrolase (OpdB) activity appeared at pH 6.0 and 35 degrees C and during degradation of chlorpyrifos, coumaphos, diazinon, methylparathion, and parathion.
KeywordMeSH Terms
23. Kim  HS, Kim  JY, Park  MS, Zheng  H, Ji  GE,     ( 2009 )

Cloning and expression of beta-glucuronidase from Lactobacillus brevis in E. coli and application in the bioconversion of baicalin and wogonoside.

Journal of microbiology and biotechnology 19 (12)
PMID : 20075633  :  
Abstract >>
The beta-glucuronidase (GUS) gene from Lactobacillus brevis RO1 was cloned and expressed in Escherichia coli GMS407. The GUS gene was composed of 1812 bp, encoding a 603-amino-acid protein belonging to the glycosyl hydrolase family 2 with three conserved domains. The amino acid similarity was higher than 70% with the beta-glucuronidases of various microorganisms, yet less than 58% with the beta-glucuronidase of L. gasseri ADH. Overexpression and purification of the GUS was performed in beta-glucuronidase-deficient E. coli GMS407. The purified GUS protein was 71 kDa and showed 1284 U/mg of specific activity at optimum condition of pH 5.0 and 37 degrees C. At 37 degrees C, the GUS remained stable for 80 min at pH values ranging from 5.0 to 8.0. The purified enzyme exhibited a half-life of 1 h at 60 degrees C and more than 2 h at 50 degrees C. When the purified GUS was applied to transform baicalin and wogonoside into their corresponding aglycones, 150 microM of baicalin and 125 microM of wogonoside were completely transformed into baicalein and wogonin, respectively, within 3 h.
KeywordMeSH Terms
Glucuronidase
24. Fukao  M, Tomita  H, Yakabe  T, Nomura  T, Ike  Y, Yajima  N,     ( 2009 )

Assessment of antibiotic resistance in probiotic strain Lactobacillus brevis KB290.

Journal of food protection 72 (9)
PMID : 19777895  :   DOI  :   10.4315/0362-028x-72.9.1923    
Abstract >>
Our purpose was to investigate the safety of the probiotic strain Lactobacillus brevis KB290. The European Qualified Presumption of Safety (QPS) evaluation approach was applied to the strain. We determined the strain's antibiotic resistance, verified it at the genetic level, and determined whether it could be transferred to intestinal microflora. Of 14 antibiotics tested, 11 showed MICs within the limits of the QPS criteria. However, the L. brevis KB290 MICs of ciprofloxacin (a fluoroquinolone), tetracycline, and vancomycin were two, four, and eight times, respectively, the breakpoint MICs suggested by the European Scientific Committee on Animal Nutrition, and the MIC of tetracycline was eight times the breakpoint MIC suggested by the European Scientific Panel on Additives and Products or Substances Used in Animal Feed. Using analysis of gapped-genome sequences, we found no known transferable determinants for tetracycline or vancomycin resistance, and we found no mutations in the quinolone resistance-determining regions of the genes encoding GyrA or ParC for ciprofloxacin resistance associated with insertion sequences, integrons, or transposons. These data were confirmed by using PCR primers specific for the respective genes. We assessed the transferability of the resistance traits in conjugation experiments with enterococci and obtained no transconjugants, strongly suggesting that the resistance traits were not transferable. This study demonstrated that the antibiotic resistance observed in L. brevis KB290 was due not to dedicated mechanisms but to intrinsic resistance. According to the QPS criteria, these results provide safety assurance for the ongoing use of L. brevis KB290 as a probiotic.
KeywordMeSH Terms
Consumer Product Safety
Probiotics
25. Coton  E, Coton  M,     ( 2009 )

Evidence of horizontal transfer as origin of strain to strain variation of the tyramine production trait in Lactobacillus brevis.

Food microbiology 26 (1)
PMID : 19028305  :   DOI  :   10.1016/j.fm.2008.07.009     DOI  :   10.1016/j.fm.2008.07.009    
Abstract >>
Lactobacillus brevis strains with the ability to decarboxylate tyrosine to tyramine have been described and the involvement of several genes constituting a tyrdc operon at the chromosomal level has been demonstrated in this species. In this study, the existence of Lb. brevis strains unable to form tyramine was observed. In order to evaluate if the tyramine-producing ability was strain-dependent or if it could be correlated to the existence of a new species or subspecies, different isolates were analysed. Analysis by M13-RAPD and sequencing of 16S rDNA, 16S-23S ISR and house-keeping gene recA confirmed that all the isolates belonged to the Lb. brevis species. Analysis of the TyrDC pathway encoding operon region in representative strains indicated the existence of a polymorphism. The genetic differences observed showed that the tyrosine decarboxylating ability is not a Lb. brevis species trait but that it is strain-dependent within this species and suggest that the genes encoding the tyramine-producing pathway constitute a genomic island.
KeywordMeSH Terms
Gene Transfer, Horizontal
Genomic Islands
Polymorphism, Genetic
Gene Transfer, Horizontal
Genomic Islands
Polymorphism, Genetic
26. Coton  E, Coton  M,     ( 2009 )

Evidence of horizontal transfer as origin of strain to strain variation of the tyramine production trait in Lactobacillus brevis.

Food microbiology 26 (1)
PMID : 19028305  :   DOI  :   10.1016/j.fm.2008.07.009     DOI  :   10.1016/j.fm.2008.07.009    
Abstract >>
Lactobacillus brevis strains with the ability to decarboxylate tyrosine to tyramine have been described and the involvement of several genes constituting a tyrdc operon at the chromosomal level has been demonstrated in this species. In this study, the existence of Lb. brevis strains unable to form tyramine was observed. In order to evaluate if the tyramine-producing ability was strain-dependent or if it could be correlated to the existence of a new species or subspecies, different isolates were analysed. Analysis by M13-RAPD and sequencing of 16S rDNA, 16S-23S ISR and house-keeping gene recA confirmed that all the isolates belonged to the Lb. brevis species. Analysis of the TyrDC pathway encoding operon region in representative strains indicated the existence of a polymorphism. The genetic differences observed showed that the tyrosine decarboxylating ability is not a Lb. brevis species trait but that it is strain-dependent within this species and suggest that the genes encoding the tyramine-producing pathway constitute a genomic island.
KeywordMeSH Terms
Gene Transfer, Horizontal
Genomic Islands
Polymorphism, Genetic
Gene Transfer, Horizontal
Genomic Islands
Polymorphism, Genetic
27. Hiraga  K, Ueno  Y, Oda  K,     ( 2008 )

Glutamate decarboxylase from Lactobacillus brevis: activation by ammonium sulfate.

Bioscience, biotechnology, and biochemistry 72 (5)
PMID : 18460820  :   DOI  :   10.1271/bbb.70782    
Abstract >>
In this study, the glutamate decarboxylase (GAD) gene from Lactobacillus brevis IFO12005 (Biosci. Biotechnol. Biochem., 61, 1168-1171 (1997)), was cloned and expressed. The deduced amino acid sequence showed 99.6% and 53.1% identity with GAD of L. brevis ATCC367 and L. lactis respectively. The His-tagged recombinant GAD showed an optimum pH of 4.5-5.0, and 54 kDa on SDS-PAGE. The GAD activity and stability was significantly dependent on the ammonium sulfate concentration, as observed in authentic GAD. Gel filtration showed that the inactive form of the GAD was a dimer. In contrast, the ammonium sulfate-activated form was a tetramer. CD spectral analyses at pH 5.5 revealed that the structures of the tetramer and the dimer were similar. Treatment of the GAD with high concentrations of ammonium sulfate and subsequent dilution with sodium glutamate was essential for tetramer formation and its activation. Thus the biochemical properties of the GAD from L. brevis IFO12005 were significantly different from those from other sources.
KeywordMeSH Terms
28. Scheirlinck  I, Van der Meulen  R, Van Schoor  A, Vancanneyt  M, De Vuyst  L, Vandamme  P, Huys  G,     ( 2008 )

Taxonomic structure and stability of the bacterial community in belgian sourdough ecosystems as assessed by culture and population fingerprinting.

Applied and environmental microbiology 74 (8)
PMID : 18310426  :   DOI  :   10.1128/AEM.02771-07     PMC  :   PMC2293155    
Abstract >>
A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.
KeywordMeSH Terms
Biodiversity
Food Microbiology
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.

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
30. 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
31. Zhu  H, Xu  G, Zhang  K, Kong  X, Han  R, Zhou  J, Ni  Y,     ( 2016 )

Crystal structure of tyrosine decarboxylase and identification of key residues involved in conformational swing and substrate binding.

Scientific reports 6 (N/A)
PMID : 27292129  :   DOI  :   10.1038/srep27779     PMC  :   PMC4904194    
Abstract >>
Tyrosine decarboxylase (TDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme and is mainly responsible for the synthesis of tyramine, an important biogenic amine. In this study, the crystal structures of the apo and holo forms of Lactobacillus brevis TDC (LbTDC) were determined. The LbTDC displays only 25% sequence identity with the only reported TDC structure. Site-directed mutagenesis of the conformationally flexible sites and catalytic center was performed to investigate the potential catalytic mechanism. It was found that H241 in the active site plays an important role in PLP binding because it has different conformations in the apo and holo structures of LbTDC. After binding to PLP, H241 rotated to the position adjacent to the PLP pyridine ring. Alanine scanning mutagenesis revealed several crucial regions that determine the substrate specificity and catalytic activity. Among the mutants, the S586A variant displayed increased catalytic efficiency and substrate affinity, which is attributed to decreased steric hindrance and increased hydrophobicity, as verified by the saturation mutagenesis at S586. Our results provide structural information about the residues important for the protein engineering of TDC to improve catalytic efficiency in the green manufacturing of tyramine.
KeywordMeSH Terms
32. Noda  M, Miyauchi  R, Danshiitsoodol  N, Higashikawa  F, Kumagai  T, Matoba  Y, Sugiyama  M,     ( 2015 )

Characterization and Mutational Analysis of a Two-Polypeptide Bacteriocin Produced by Citrus Iyo-Derived Lactobacillus brevis 174A.

Biological & pharmaceutical bulletin 38 (12)
PMID : 26632181  :   DOI  :   10.1248/bpb.b15-00505    
Abstract >>
In the present study, we isolated a lactic acid bacterium (LAB) from a citrus iyo fruit and identified it as Lactobacillus brevis. This plant-derived LAB strain, designated 174A, produces bacteriocin consisting of two polypeptides designated brevicin 174A-�] and 174A-�^. Although each polypeptide itself displays antibacterial activity, the ability is enhanced 100 fold by mixing both polypeptides at a 1 : 1 ratio. Significantly, brevicin 174A inhibits even the growth of several pathogenic bacteria that are more resistant to a lantibiotic bacteriocin, nisin A, which is commonly utilized as a preservative added to foodstuffs. Structural analysis of the 174A bacteriocin using a program that predicts secondary structure suggests that both component polypeptides have a positively charged N-terminal region, as well as two cysteine residues in both the N- and C-terminals. Judging from a mutational analysis of the antibacterial polypeptides, these unique amino acid sequences of 174A-�] might be important for the expression of the synergistic activity that occurs in the presence of the two polypeptides combined.
KeywordMeSH Terms
33. Wu  Q, Law  YS, Shah  NP,     ( 2015 )

Dairy Streptococcus thermophilus improves cell viability of Lactobacillus brevis NPS-QW-145 and its �^-aminobutyric acid biosynthesis ability in milk.

Scientific reports 5 (N/A)
PMID : 26245488  :   DOI  :   10.1038/srep12885     PMC  :   PMC4526857    
Abstract >>
Most high �^-aminobutyric acid (GABA) producers are Lactobacillus brevis of plant origin, which may be not able to ferment milk well due to its poor proteolytic nature as evidenced by the absence of genes encoding extracellular proteinases in its genome. In the present study, two glutamic acid decarboxylase (GAD) genes, gadA and gadB, were found in high GABA-producing L. brevis NPS-QW-145. Co-culturing of this organism with conventional dairy starters was carried out to manufacture GABA-rich fermented milk. It was observed that all the selected strains of Streptococcus thermophilus, but not Lactobacillus delbrueckii subsp. bulgaricus, improved the viability of L. brevis NPS-QW-145 in milk. Only certain strains of S. thermophilus improved the gadA mRNA level in L. brevis NPS-QW-145, thus enhanced GABA biosynthesis by the latter. These results suggest that certain S. thermophilus strains are highly recommended to co-culture with high GABA producer for manufacturing GABA-rich fermented milk.
KeywordMeSH Terms
Microbial Viability
34. Li  H, Ding  D, Cao  Y, Yu  B, Guo  L, Liu  X,     ( 2015 )

Partially overlapping primer-based PCR for genome walking.

PloS one 10 (3)
PMID : 25811779  :   DOI  :   10.1371/journal.pone.0120139     PMC  :   PMC4374871    
Abstract >>
Current genome walking methods are cumbersome to perform and can result in non-specific products. Here, we demonstrate the use of partially overlapping primer-based PCR (POP-PCR), a direct genome walking technique for the isolation of unknown flanking regions. This method exploits the partially overlapping characteristic at the 3' ends of a set of POP primers (walking primers), which guarantees that the POP primer only anneals to the POP site of the preceding PCR product at relatively low temperatures. POP primer adaptation priming at the genomic DNA/POP site occurs only once due to one low-/reduced-stringency cycle in each nested PCR, resulting in the synthesis of a pool of single-stranded DNA molecules. Of this pool, the target single-stranded DNA is replicated to the double-stranded form bound by the specific primer and the POP primer in the subsequent high-stringency cycle due to the presence of the specific primer-binding site. The non-target single stranded DNA does not become double stranded due to the absence of a binding site for any of the primers. Therefore, the POP-PCR enriches target DNA while suppressing non-target products. We successfully used POP-PCR to retrieve flanking regions bordering the gadA locus in Lactobacillus brevis NCL912, malQ in Pichia pastoris GS115, the human aldolase A gene, and hyg in rice.
KeywordMeSH Terms
Polymerase Chain Reaction
35. 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
36. Zhang  K, Ni  Y,     ( 2014 )

Tyrosine decarboxylase from Lactobacillus brevis: soluble expression and characterization.

Protein expression and purification 94 (N/A)
PMID : 24211777  :   DOI  :   10.1016/j.pep.2013.10.018    
Abstract >>
Tyrosine decarboxylase (TDC, EC 4.1.1.25) is an enzyme that catalyzes the decarboxylation of l-tyrosine to produce tyramine and CO2. In this study, a 1881-bp tdc gene from Lactobacillus brevis was cloned and heterologously expressed in Escherichia coli BL21 (DE3). Glucose was discovered to play an important role in the soluble expression of rLbTDC. After optimization, recombinant TDC (rLbTDC) was achieved in excellent solubility and a yield of 224mg rLbTDC/L broth. The C-terminal His-Tagged rLbTDC was one-step purified with 90% recovery. Based on SDS-PAGE and gel filtration analysis, rLbTDC is a dimer composed of two identical subunits of approximately 70kDa. Using l-tyrosine as substrate, the specific activity of rLbTDC was determined to be 133.5U/mg in the presence of 0.2mM pyridoxal-5'-phosphate at 40�XC and pH 5.0. The Km and Vmax values of rLbTDC were 0.59mM and 147.1�gmolmin(-1)mg(-1), respectively. In addition to l-tyrosine, rLbTDC also exhibited decarboxylase activity towards l-DOPA. This study has demonstrated, for the first time, the soluble expression of tdc gene from L. brevis in heterologous host.
KeywordMeSH Terms
Lactobacillus brevis
Pyridoxal-5′-phosphate (PLP)
Soluble expression
Tyramine
Tyrosine decarboxylase (TDC)
l-DOPA
l-Tyrosine
37. Michlmayr  H, Hell  J, Lorenz  C, Böhmdorfer  S, Rosenau  T, Kneifel  W,     ( 2013 )

Arabinoxylan oligosaccharide hydrolysis by family 43 and 51 glycosidases from Lactobacillus brevis DSM 20054.

Applied and environmental microbiology 79 (21)
PMID : 23995921  :   DOI  :   10.1128/AEM.02130-13     PMC  :   PMC3811506    
Abstract >>
Due to their potential prebiotic properties, arabinoxylan-derived oligosaccharides [(A)XOS] are of great interest as functional food and feed ingredients. While the (A)XOS metabolism of Bifidobacteriaceae has been extensively studied, information regarding lactic acid bacteria (LAB) is still limited in this context. The aim of the present study was to fill this important gap by characterizing candidate (A)XOS hydrolyzing glycoside hydrolases (GHs) identified in the genome of Lactobacillus brevis DSM 20054. Two putative GH family 43 xylosidases (XynB1 and XynB2) and a GH family 43 arabinofuranosidase (Abf3) were heterologously expressed and characterized. While the function of XynB1 remains unclear, XynB2 could efficiently hydrolyze xylooligosaccharides. Abf3 displayed high specific activity for arabinobiose but could not release arabinose from an (A)XOS preparation. However, two previously reported GH 51 arabinofuranosidases from Lb. brevis were able to specifically remove �\-1,3-linked arabinofuranosyl residues from arabino-xylooligosaccharides (AXHm3 specificity). These results imply that Lb. brevis is at least genetically equipped with functional enzymes in order to hydrolyze the depolymerization products of (arabino)xylans and arabinans. The distribution of related genes in Lactobacillales genomes indicates that GH 43 and, especially, GH 51 glycosidase genes are rare among LAB and mainly occur in obligately heterofermentative Lactobacillus spp., Pediococcus spp., members of the Leuconostoc/Weissella branch, and Enterococcus spp. Apart from the prebiotic viewpoint, this information also adds new perspectives on the carbohydrate (i.e., pentose-oligomer) metabolism of LAB species involved in the fermentation of hemicellulose-containing substrates.
KeywordMeSH Terms
38. Li  H, Li  W, Liu  X, Cao  Y,     ( 2013 )

gadA gene locus in Lactobacillus brevis NCL912 and its expression during fed-batch fermentation.

FEMS microbiology letters 349 (2)
PMID : 24164637  :   DOI  :   10.1111/1574-6968.12301    
Abstract >>
Normally, Lactobacillus brevis has two glutamate decarboxylase (GAD) genes; gadA and gadB. Using PCR, we cloned the gadA gene from L. brevis strain NCL912, a high yield strain for the production of gamma-aminobutyric acid (GABA). However, despite using 61 different primer pairs, including degenerate primers from conserved regions, we were unable to use PCR to clone gadB from the NCL912 strain. Furthermore, we could not clone it by genomic walking over 3000 bp downstream of the aldo-keto reductase gene, a single-copy gene that is located 1003 bp upstream of gadB in L. brevis ATCC367. Altogether, the data suggest that L. brevis NCL912 does not contain a gadB gene. By genomic walking, we cloned regions upstream and downstream of the gadA gene to obtain a 4615 bp DNA fragment that included the complete gadA locus. The locus contained the GAD gene (gadA) and the glutamate:GABA antiporter gene (gadC), which appear to be transcribed in an operon (gadCA), and a transcriptional regulator (gadR) of gadCA. During whole fed-batch fermentation, the expression of gadR, gadC and gadA was synchronized and correlated well with GABA production. The gadA locus we cloned from NCL912 has reduced homology compared with gadA loci of other L. brevis strains, and these differences might explain the ability of NCL912 to produce higher levels of GABA in culture.
KeywordMeSH Terms
Lactobacillus brevis NCL912
cloning and expression
gadA locus genes
gamma-aminobutyric acid
Fermentation
Gene Expression
Genetic Loci
39. Neveling  DP, Endo  A,     ( 2012 )

Fructophilic Lactobacillus kunkeei and Lactobacillus brevis isolated from fresh flowers, bees and bee-hives.

Current microbiology 65 (5)
PMID : 22797888  :   DOI  :   10.1007/s00284-012-0186-4    
Abstract >>
Two-hundred-and-thirty-six isolates were collected from fresh flowers, bees and bee-hives. Of these, 20 isolates preferred D-fructose as carbon source, produced lactic acid and acetic acid but trace amounts of ethanol and were classified as fructophilic. Poor growth was recorded when strains were incubated anaerobically in the presence of D-glucose as sole carbon source. Good growth was, however, recorded when D-glucose was metabolized in the presence of external electron acceptors such as fructose, pyruvate and oxygen. Nineteen of the strains were classified as Lactobacillus kunkeei and one as Lactobacillus brevis based on phenotypic characteristics, 16S rRNA sequences, recA sequences and DNA homology. This is the first description of a fructophilic strain of L. brevis.
KeywordMeSH Terms
40. Romano  A, Trip  H, Lonvaud-Funel  A, Lolkema  JS, Lucas  PM,     ( 2012 )

Evidence of two functionally distinct ornithine decarboxylation systems in lactic acid bacteria.

Applied and environmental microbiology 78 (6)
PMID : 22247134  :   DOI  :   10.1128/AEM.07161-11     PMC  :   PMC3298143    
Abstract >>
Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol.
KeywordMeSH Terms
41. Yagasaki  M, Iwata  K, Ishino  S, Azuma  M, Ozaki  A,     ( 1995 )

Cloning, purification, and properties of a cofactor-independent glutamate racemase from Lactobacillus brevis ATCC 8287.

Bioscience, biotechnology, and biochemistry 59 (4)
PMID : 7772825  :   DOI  :   10.1271/bbb.59.610    
Abstract >>
A glutamate racemase gene of Lactobacillus brevis ATCC 8287 was cloned into Escherichia coli TM93 by the phenotypic complementation of a phosphoenolpyruvate carboxylase deficiency on minimum agar medium containing D-glutamate. The gene was localized to a 1.4-kb HindIII-EcoRI DNA fragment and the total nucleotide sequence of the fragment was analyzed. The gene has typical promoter and SD sequences which appeared to function in E. coli. The deduced amino acid sequence of the enzyme had 276 amino acids and the molecular weight was calculated as 29,426. Two cysteine residues and their surrounding regions of the enzyme are homologous to those of other cofactor-independent racemases. The glutamate racemase was purified from recombinant E. coli to homogeneity and characterized. The enzyme required no cofactors for the activity, and retained its activity even in 2 M (300 g/l) L-glutamate.
KeywordMeSH Terms
42. Hermann  J, Nowotny  P, Schrader  TE, Biggel  P, Hekmat  D, Weuster-Botz  D,     ( 2018 )

Neutron and X-ray crystal structures of Lactobacillus brevis alcohol dehydrogenase reveal new insights into hydrogen-bonding pathways.

Acta crystallographica. Section F, Structural biology communications 74 (Pt 12)
PMID : 30511668  :   DOI  :   10.1107/S2053230X18015273    
Abstract >>
Lactobacillus brevis alcohol dehydrogenase (LbADH) is a well studied homotetrameric enzyme which catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. LbADH is stable and enzymatically active at elevated temperatures and accepts a broad range of substrates, making it a valuable tool in industrial biocatalysis. Here, the expression, purification and crystallization of LbADH to generate large, single crystals with a volume of up to 1 mm3 suitable for neutron diffraction studies are described. Neutron diffraction data were collected from an H/D-exchanged LbADH crystal using the BIODIFF instrument at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany to a resolution dmin of 2.15 ? in 16 days. This allowed the first neutron crystal structure of LbADH to be determined. The neutron structure revealed new details of the hydrogen-bonding network originating from the ion-binding site of LbADH and provided new insights into the reasons why divalent magnesium (Mg2+) or manganese (Mn2+) ions are necessary for its activity. X-ray diffraction data were obtained from the same crystal at the European Synchrotron Radiation Facility (ESRF), Grenoble, France to a resolution dmin of 1.48 ?. The high-resolution X-ray structure suggested partial occupancy of Mn2+ and Mg2+ at the ion-binding site. This is supported by the different binding affinity of Mn2+ and Mg2+ to the tetrameric structure calculated via free-energy molecular-dynamics simulations.
KeywordMeSH Terms
Lactobacillus brevis
alcohol dehydrogenase
hydrogen-bonding network
neutron diffraction
protein crystallization
short-chain dehydrogenases/reductases
43. Huang  J, Fang  H, Gai  ZC, Mei  JQ, Li  JN, Hu  S, Lv  CJ, Zhao  WR, Mei  LH,     ( 2018 )

Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: Crystal structure and functional analysis.

Biochemical and biophysical research communications 503 (3)
PMID : 30049439  :   DOI  :   10.1016/j.bbrc.2018.07.102    
Abstract >>
Glutamate decarboxylase (GAD), which is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme, can catalyze �\-decarboxylation of l-glutamate (L-Glu) to �^-aminobutyrate (GABA). The crystal structure of GAD in complex with PLP from Lactobacillus brevis CGMCC 1306 was successfully solved by molecular-replacement, and refined at 2.2 ? resolution to an Rwork factor of 18.76% (Rfree = 23.08%). The coenzyme pyridoxal 5-phosphate (PLP) forms a Schiff base with the active-site residue Lys279 by continuous electron density map, which is critical for catalysis by PLP-dependent decarboxylase. Gel filtration showed that the active (pH 4.8) and inactive (pH 7.0) forms of GAD are all dimer. The residues (Ser126, Ser127, Cys168, Ile211, Ser276, His278 and Ser321) play important roles in anchoring PLP cofactor inside the active site and supporting its catalytic reactivity. The mutant T215A around the putative substrate pocket displayed an 1.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme (1.227 mM-1 S-1 versus 0.777 mM-1 S-1), which was the highest activity among all variants tested. The flexible loop (Tyr308-Glu312), which is positioned near the substrate-binding site, is involved in the catalytic reaction, and the conserved residue Tyr308 plays a vital role in decarboxylation of L-Glu.
KeywordMeSH Terms
Crystal structure
Glutamate decarboxylase
Lactobacillus brevis
γ-aminobutyrate
Molecular Docking Simulation
44.     ( 2013 )

Expression and characterization of a glutamate decarboxylase from Lactobacillus brevis 877G producing �^-aminobutyric acid.

Bioscience, biotechnology, and biochemistry 77 (4)
PMID : 23563537  :   DOI  :   10.1271/bbb.120785    
Abstract >>
The glutamate decarboxylase of �^-aminobutyric acid-producing Lactobacillus brevis 877G (LbGAD) was expressed in Escherichia coli. The optimal pH and temperature for the purified LbGAD activity were respectively determined to be pH 5.2 and 45 �XC. CaCl2 was shown to be a potent activator of this LbGAD activity. The kinetic parameters for LbGAD were a Km value of 3.6 mmol/L and a Vmax value of 0.06 mmol/L/min for L-monosodium glutamate.
KeywordMeSH Terms
45. Choi  J, Lee  S, Linares-Pastén  JA, Nilsson  L,     ( 2018 )

Study on oligomerization of glutamate decarboxylase from Lactobacillus brevis using asymmetrical flow field-flow fractionation (AF4) with light scattering techniques.

Analytical and bioanalytical chemistry 410 (2)
PMID : 29167934  :   DOI  :   10.1007/s00216-017-0735-6     PMC  :   PMC5750328    
Abstract >>
In this work, asymmetrical flow field-flow fractionation (AF4) coupled with UV/Vis, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for analysis of glutamate decarboxylase (LbGadB) from Lactobacillus brevis (L. brevis). AF4 provided molecular weight (MW) (or size)-based separation of dimer, hexamer, and aggregates of LbGadB. The effect of pH on oligomerization of LbGadB was investigated, and then AF4 results were compared to those from molecular modeling. The MWs measured by AF4-UV-MALS-dRI for dimeric and hexameric forms of LbGadB were 110 and 350 kDa, respectively, which are in good agreements with those theoretically calculated (110 and 330 kDa). The molecular sizes determined by AF4-UV-MALS-dRI were also in good agreement with those obtained from molecular modeling (6 and 10 nm, respectively, for dimeric and hexameric from AF4-UV-MALS-dRI and 6.4 �� 7.6 and 7.6 �� 13.1 nm from molecular modeling). The effects of temperature, salt type, and salt concentration on oligomerization of LbGadB were also investigated using dynamic light scattering (DLS). It was found that the hexameric form of LbGadB was most stable at pH 6 and in presence of NaCl or KCl. The results indicate that AF4, in combination of various online detectors mentioned above, provides an effective tool for monitoring of oligomerization of LbGadB under different conditions, such as temperature, pH, type of salts, and salt concentrations.
KeywordMeSH Terms
Asymmetrical flow field-flow fractionation (AF4)
Glutamate decarboxylase (GAD)
Lactobacillus brevis (L. brevis)
Multi-angle light scattering (MALS)
Oligomerization
Probiotic
46.     ( 2013 )

Biosynthesis of 3-hydroxypropionic acid from glycerol in recombinant Escherichia coli expressing Lactobacillus brevis dhaB and dhaR gene clusters and E. coli K-12 aldH.

Bioresource technology 135 (N/A)
PMID : 23246300  :   DOI  :   10.1016/j.biortech.2012.11.063    
Abstract >>
3-Hydroxypropionic acid (3-HP) is a value-added chemical for polymer synthesis. For biosynthesis of 3-HP from glycerol, two dhaB and dhaR clusters encoding glycerol dehydratase and its reactivating factor, respectively, were cloned from Lactobacillus brevis KCTC33069 and expressed in Escherichia coli. Coexpression of dhaB and dhaR allowed the recombinant E. coli to convert glycerol to 3-hydroxypropionaldehyde, an intermediate of 3-HP biosynthesis. To produce 3-HP from glycerol, fed-batch fermentation with a two-step feeding strategy was designed to separate the cell growth from the 3-HP production stages. Finally, E. coli JHS00947 expressing L .brevis dhaB and dhaR, and E. coli aldH produced 14.3g/L 3-HP with 0.26 g/L-h productivity, which were 14.6 and 8.53 times higher than those of the batch culture. In conclusion, overexpression of L. brevis dhaB and dhaR clusters and E. coli aldH, and implementation of the two-step feeding strategy enabled recombinant E. coli to convert glycerol to 3-HP efficiently.
KeywordMeSH Terms
47.     ( 2012 )

RT-qPCR analysis of putative beer-spoilage gene expression during growth of Lactobacillus brevis BSO 464 and Pediococcus claussenii ATCC BAA-344(T) in beer.

Applied microbiology and biotechnology 96 (2)
PMID : 22893225  :   DOI  :   10.1007/s00253-012-4334-3    
Abstract >>
Lactic acid bacteria (LAB) contamination of beer presents a continual economic threat to brewers. Interestingly, only certain isolates of LAB can grow in the hostile beer environment (e.g., as studied here, Lactobacillus brevis BSO 464 (Lb464) and a non-ropy isolate of Pediococcus claussenii ATCC BAA-344(T) (Pc344NR)), indicating that significant genetic specialization is required. The genes hitA, horA, horB, horC, and bsrA, which have been proposed to confer beer-spoiling ability to an organism, are suspected of counteracting the antimicrobial effects of hops. However, these genes are not present in the same combination (if at all) across beer-spoiling organisms. As such, we sought to investigate the extent to which these genes participate during Lb464 and Pc344NR mid-logarithmic growth in beer through reverse transcription quantitative PCR analysis. We first determined the optimal reference gene set needed for data normalization and, for each bacterium, established that two genes were needed for accurate assessment of gene expression. Following this, we found that horA expression was induced for Pc344NR, but not for Lb464, during growth in beer. Instead, horC expression was dramatically increased in Lb464 when growing in beer, whereas no change was detected for the other putative beer-spoilage-related genes. This indicates that HorC may be one of the principle mediators enabling growth of Lb464 in beer, whereas in Pc344NR, this may be attributable to HorA. These findings not only reveal that Lb464 and Pc344NR are unique in their beer-specific genetic expression profile but also indicate that a range of genetic specialization exists among beer-spoilage bacteria.
KeywordMeSH Terms
48.     ( 1998 )

Molecular cloning and functional expression in lactobacillus plantarum 80 of xylT, encoding the D-xylose-H+ symporter of Lactobacillus brevis.

Applied and environmental microbiology 64 (12)
PMID : 9835554  :   PMC  :   PMC90914    
Abstract >>
A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to the D-xylose-H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose-H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L. brevis which is not located with the xyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D-xylulose kinase, and D-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D-xylose transport activity was reduced by sixfold, suggesting catabolite repression by D-glucose of D-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked D-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of D-xylose in L. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D-glucose. The apparent affinity constant of XylT for D-xylose was approximately 215 microM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the transport of D-xylose by XylT competitively, with a Ki of 220 microM.
KeywordMeSH Terms
Bacterial Proteins
Symporters

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