| 1. |
Turner MS,
Hafner LM,
Walsh T,
Giffard PM,
( 2003 ) Peptide surface display and secretion using two LPXTG-containing surface proteins from Lactobacillus fermentum BR11. PMID : 14532035 : DOI : 10.1128/aem.69.10.5855-5863.2003 PMC : PMC201189 Abstract >>
A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His(6)). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His(6) and CFTR peptides or His(6) peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.
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2. |
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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3. |
Pavlova SI,
Kiliç AO,
Topisirovic L,
Miladinov N,
Hatzos C,
Tao L,
( 2002 ) Characterization of a cryptic plasmid from Lactobacillus fermentum KC5b and its use for constructing a stable Lactobacillus cloning vector. PMID : 12151233 : Abstract >>
Lactobacillus fermentum KC5b, a strain originally isolated from the human vagina, contains a cryptic plasmid pKC5b. The sequence and genetic organization of the 4392-bp plasmid were determined. It contains two convergently oriented replicons, which are homologous to each other and to the stable replicon of the Enterococcus faecium plasmid pMBB1. The two replicons of pKC5b were used either individually or together to construct Lactobacillus-Escherichia coli shuttle plasmids. Only the plasmid pSP1 that carried both replicons transformed lactobacilli, suggesting a complementary function between the two replicons. Since the replicons had a high homology to those of other plasmids that replicate via a theta-like mechanism and no detectable single-stranded intermediates were found for the plasmid, it is possible that pKC5b may replicate via a theta-like mechanism. The new shuttle plasmid pSP1 has been transformed and stably maintained in several Lactobacillus strains. As an initial application, pSP1 was used to clone the S-layer protein gene (slpA) of Lactobacillus acidophilus ATCC 4356 into a heterologous vaginal Lactobacillus strain and achieved surface-bound expression of the protein.
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4. |
Vogensen FK,
Petersen A,
( 1999 ) Use of conserved randomly amplified polymorphic DNA (RAPD) fragments and RAPD pattern for characterization of Lactobacillus fermentum in Ghanaian fermented maize dough. PMID : 10388723 : PMC : PMC91476 Abstract >>
The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates.
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5. |
Glavas S,
( 1999 ) Catalytic acid/base residues of glutamate racemase. PMID : 10194325 : DOI : 10.1021/bi982663n Abstract >>
Glutamate racemase is a cofactor-independent enzyme that employs two active-site cysteine residues as acid/base catalysts during the interconversion of glutamate enantiomers. In a given reaction direction, a thiolate from one of the cysteines abstracts the alpha-proton, and the other cysteine thiol delivers a proton to the opposite face of the resulting carbanionic intermediate. This paper reports that the C73S and C184S mutants are still capable of racemizing glutamate with specificity constants about 10(3)-fold lower than those of the wild-type enzyme. A "one-base requiring" reaction, the elimination of water from N-hydroxyglutamate, has been used to deduce which thiol acts as the base for a given enantiomer. With D-N-hydroxyglutamate the C73S mutant is a much poorer catalyst than wild-type enzyme, whereas the C184S mutant is a somewhat better catalyst. This trend was reversed with L-N-hydroxyglutamate, suggesting that Cys73 is responsible for the deprotonation of D-glutamate and Cys184 is responsible for the deprotonation of L-glutamate. In addition, with C73S the Vmax/KM isotope effect on D-glutamate racemization was greater than that seen with wild-type enzyme, whereas the isotope effect with L-glutamate had decreased. The results were reversed with the C184S mutant. This is interpreted as being due to an asymmetry in the free energy profiles that is induced upon mutation, with the deprotonation step involving a serine becoming the more cleanly rate-determining of the two. These results support the above assignment and the notion that a carbanionic intermediate is formed during catalysis.
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6. |
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. 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.
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7. |
Ehrmann MA,
Brandt M,
Stolz P,
Vogel RF,
Korakli M,
( 2007 ) Lactobacillus secaliphilus sp. nov., isolated from type II sourdough fermentation. PMID : 17392199 : DOI : 10.1099/ijs.0.64700-0 Abstract >>
Two strains of Gram-positive, catalase-negative, lactic acid bacteria, strains TMW 1.1309(T) and TMW 1.1313, were isolated at an interval of several years from an industrial type II sourdough. They occurred at cell numbers of 8x10(8) c.f.u. g(-1) and therefore were considered to be one of the dominant members of the microbiota in this type of fermentation. Cells of both strains grow exclusively on modified MRS containing trypsin-digested rye-bran extract. Both strains possessed identical 16S rRNA gene sequences, but could be discriminated by RAPD fingerprints. Comparative 16S rRNA and tuf gene sequence analyses positioned strain TMW 1.1309(T) as part of the Lactobacillus reuteri phylogenetic group within the genus Lactobacillus. The 16S rRNA gene sequence similarities to the closest related species, Lactobacillus coleohominis and Lactobacillus ingluviei were 97.1 and 95.4 %, respectively. The DNA G+C content of strain TMW 1.1309(T) was 48 mol%. Growth characteristics, biochemical features and DNA-DNA hybridization values below 70 % with all the nearest neighbours demonstrated that the isolates represent a novel Lactobacillus species. The name Lactobacillus secaliphilus sp. nov. is proposed for the novel isolates, with the type strain TMW 1.1309(T) (=DSM 17896(T)=CCUG 53218(T)).
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8. |
Carrera-Silva EA,
Silvestroni A,
LeBlanc JG,
Piard JC,
Savoy de Giori G,
Sesma F,
( 2006 ) A thermostable alpha-galactosidase from Lactobacillus fermentum CRL722: genetic characterization and main properties. PMID : 17048069 : DOI : 10.1007/s00284-005-0442-y Abstract >>
Alpha-galactosidase (alpha-Gal) enzyme, which is encoded by the melA gene hydrolyzes alpha-1,6 galactoside linkages found in sugars, such as raffinose and stachyose. These alpha-galacto-oligosaccharides (alpha-GOS), which are found in large quantities in vegetables, such as soy, can cause gastrointestinal disorders in sensitive individuals because monogastric animals (including humans) do not posses alpha-Gal in the gut. The use of microbial alpha-Gal is a promising alternative to eliminate alpha-GOS in soy-derived products. Using degenerate primers, the melA gene from Lactobacillus (L.) fermentum CRL722 was identified. The complete genomic sequence of melA (2223 bp), and of the genes flanking melA, were obtained using a combination of polymerase chain reaction-based techniques, and showed strong similarities with the alpha-Gal gene of thermophilic microorganisms. The alpha-Gal gene from L. fermentum CRL722 was cloned and the protein purified from cell-free extracts of the native and recombinant strains using various techniques (ion exchange chromatography, salt precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ultra-filtration); Its main biochemical properties were determined. The enzyme was active at moderately high temperatures (55 degrees C) and stable at wide ranges of temperatures and pH. The thermostable alpha-Gal from L. fermentum CRL722 could thus be used for technological applications, such as the removal of alpha-GOS found in soy products. The complete melA gene could also be inserted in other micro-organisms, that can survive in the harsh conditions of the gut to degrade alpha-GOS in situ. Both strategies would improve the overall acceptability of soy-derived products by improving their nutritional value.
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9. |
Helanto M,
Aarnikunnas J,
Palva A,
Leisola M,
Nyyssölä A,
( 2006 ) Characterization of genes involved in fructose utilization by Lactobacillus fermentum. PMID : 16741753 : DOI : 10.1007/s00203-006-0120-x Abstract >>
The genes encoding phosphoglucose isomerase (fruI) and fructokinase (fruK) of Lactobacillus fermentum NRRL-B-1932 were sequenced. They constituted an operon, which is involved in fructose metabolism of this strain by channeling intracellular fructose into the phosphoketolase pathway. A third open reading frame, unkR, upstream of the operon was identified as homologous to genes of LacI/GalR family repressors. The UnkR repressor's role in transcriptional control of the fruIK operon could, however, not be established by electrophoretic mobility shift assay (EMSA) analysis. Sequence analysis revealed two putative catabolite responsive elements (cre) in the promoter region of fruIK suggesting that the fruIK operon is under negative regulatory control by carbon catabolite repression. Expression and enzyme activity data were compatible with the assumption that the fruIK operon is repressed by glucose. No sugar specific phosphoenolpyruvate sugar transferase system activity for the transport of fructose, glucose, sucrose or mannose could be detected in L. fermentum NRRL-B-1932 cells, which suggest that fructose is taken up by a permease system.
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10. |
Kralj S,
van Geel-Schutten GH,
Dondorff MM,
Kirsanovs S,
van der Maarel MJ,
Dijkhuizen L,
( 2004 ) Glucan synthesis in the genus Lactobacillus: isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains. PMID : 15528655 : DOI : 10.1099/mic.0.27321-0 Abstract >>
Members of the genera Streptococcus and Leuconostoc synthesize various alpha-glucans (dextran, alternan and mutan). In Lactobacillus, until now, the only glucosyltransferase (GTF) enzyme that has been characterized is gtfA of Lactobacillus reuteri 121, the first GTF enzyme synthesizing a glucan (reuteran) that contains mainly alpha-(1-->4) linkages together with alpha-(1-->6) and alpha-(1-->4,6) linkages. Recently, partial sequences of glucansucrase genes were detected in other members of the genus Lactobacillus. This paper reports, for the first time, isolation and characterization of dextransucrase and mutansucrase genes and enzymes from various Lactobacillus species and the characterization of the glucan products synthesized, which mainly have alpha-(1-->6)- and alpha-(1-->3)-glucosidic linkages. The four GTF enzymes characterized from three different Lb. reuteri strains are highly similar at the amino acid level, and consequently their protein structures are very alike. Interestingly, these four Lb. reuteri GTFs have relatively large N-terminal variable regions, containing RDV repeats, and relatively short putative glucan-binding domains with conserved and less-conserved YG-repeating units. The three other GTF enzymes, isolated from Lactobacillus sakei, Lactobacillus fermentum and Lactobacillus parabuchneri, contain smaller variable regions and larger putative glucan-binding domains compared to the Lb. reuteri GTF enzymes.
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11. |
Turner MS,
Hafner LM,
Walsh T,
Giffard PM,
( 2004 ) Identification, characterisation and specificity of a cell wall lytic enzyme from Lactobacillus fermentum BR11. PMID : 15336396 : DOI : 10.1016/j.femsle.2004.07.008 Abstract >>
Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Phiadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.
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12. |
Dellaglio F,
Torriani S,
Felis GE,
( 2004 ) Reclassification of Lactobacillus cellobiosus Rogosa et al. 1953 as a later synonym of Lactobacillus fermentum Beijerinck 1901. PMID : 15143028 : DOI : 10.1099/ijs.0.02947-0 DOI : 10.1099/ijs.0.02947-0 Abstract >>
The name Lactobacillus cellobiosus is validly published, but the species is often neglected in taxonomic studies, due to its high similarity to Lactobacillus fermentum. In the present paper, literature data concerning the two species were reviewed. Phylogenetic placement of L. cellobiosus was obtained based on 16S rDNA sequences, and genetic similarity was further investigated by comparing partial recA gene sequences for the type strains of L. cellobiosus and L. fermentum. Based on the high identity values for 16S rDNA (99 %) and recA gene (98 %) sequences, the results of DNA-DNA hybridization assays and phenotypic traits available from the literature, it is proposed that L. cellobiosus be reclassified and, as a rule of priority, renamed as L. fermentum, the first described species.
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13. |
Turner MS,
Hafner LM,
Walsh T,
Giffard PM,
( 2004 ) Identification and characterization of the novel LysM domain-containing surface protein Sep from Lactobacillus fermentum BR11 and its use as a peptide fusion partner in Lactobacillus and Lactococcus. PMID : 15184172 : DOI : 10.1128/AEM.70.6.3673-3680.2004 PMC : PMC427774 Abstract >>
Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.
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14. |
Gfeller KY,
Roth M,
Meile L,
Teuber M,
( 2003 ) Sequence and genetic organization of the 19.3-kb erythromycin- and dalfopristin-resistance plasmid pLME300 from Lactobacillus fermentum ROT1. PMID : 14597008 : Abstract >>
Lactobacillus fermentum ROT1 was isolated from a raw milk dairy product. It is resistant to novobiocin, tetracycline, erythromycin and dalfopristin. A chromosomal tetracycline-resistance determinant was identified as tetM. A 19,398-bp plasmid (pLME300), present in several erythromycin-resistant strains of Lb. fermentum, was isolated from strain ROT1 and completely sequenced. Based on putative open reading frames, pLME300 contains at least four different functional regions. In region I, ORF1 shows high homologies to replication proteins of different theta-replicating plasmids. In addition, a tandem repeat of a 22-bp sequence appears 4.5 times. In region II, ORF3 may code for a methylase, and ORF4 has homologies to Mrr restriction system proteins of Deinococcus radiodurans and Escherichia coli suggesting a restriction-modification system. Region III harbours antibiotic-resistance genes, coding for a macrolide-lincosamide-streptogramin B (MLS) methylase Erm(LF) and the streptogramin A acetyltransferase Vat(E), which is identical to Vat(E) from Enterococcus faecium. Furthermore, region III shows a 91% nucleotide sequence identity to an erm-vat linkage of E. faecium. Region IV carries ORFs that appear to be involved in plasmid mobilization as characterized by a putative origin of transfer and a mobilization protein. pLME300 is the largest completely sequenced multi-resistance plasmid isolated from any Lactobacillus strain so far.
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15. |
Dan T,
Fukuda K,
Sugai-Bannai M,
Takakuwa N,
Motoshima H,
Urashima T,
( 2009 ) Characterization and expression analysis of the exopolysaccharide gene cluster in Lactobacillus fermentum TDS030603. PMID : 19966483 : DOI : 10.1271/bbb.90502 Abstract >>
Part of the exopolysaccharide gene cluster of Lactobacillus fermentum TDS030603 was characterized. It consists of 11,890 base pairs and is located in the chromosomal DNA, 13 open reading frames of which were encoded. Out of the 13 open reading frames, six were found to be involved in exopolysaccharide synthesis; however, five were similar to transposase genes of other lactobacilli, and two were functionally unrelated. Expression analysis revealed that the exopolysaccharide synthesis-related genes were expressed during cultivation. Southern analysis using specific primers for the exopolysaccharide genes indicated that duplication of the gene cluster did not occur. The plasmid-cured strain maintained its capacity for exopolysaccharide production, confirming that the exopolysaccharide gene cluster of this strain is located in the chromosomal DNA, similarly to thermophilic lactic acid bacteria. Our results indicate that this exopolysaccharide gene cluster is likely to be functional, although extensive gene rearrangement occurs.
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16. |
Macías-Rodríguez ME,
Zagorec M,
Ascencio F,
Vázquez-Juárez R,
Rojas M,
( 2009 ) Lactobacillus fermentum BCS87 expresses mucus- and mucin-binding proteins on the cell surface. PMID : 19548890 : DOI : 10.1111/j.1365-2672.2009.04368.x Abstract >>
To identify and characterize adhesion-associated proteins in the potential probiotic Lactobacillus fermentum BCS87. Protein suspensions obtained from the treatment of Lact. fermentum BCS87 with 1 mol 1(-1) LiCl were analysed by Western blotting using HRP-labelled porcine mucus and mucin. Two adhesion-associated proteins with relative molecular weight of 29 and 32 kDa were identified. The N-terminal and internal peptides of the 32 kDa protein (32-Mmubp) were sequenced, and the corresponding gene (32-mmub) was found by inverse polymerase chain reaction. The complete nucleotide sequence of 32-mmub revealed an open reading frame of 903 bp encoding a primary protein of 300 amino acids and a mature protein of 272 residues. A basic local alignment search showed 47-99% identity to solute-binding components of ATP binding cassette transporter proteins in Lactobacillus, Streptococcus and Clostridium. An OpuAC-conserved domain was identified and phylogenetic relationship analysis confirmed that 32-Mmubp belongs to the OpuAC family. Adhesion of Lact. fermentum BCS87 appeared to be mediated by two surface-associated proteins. 32-Mmubp is a component of ABC transporter system that also functions as an adhesin. Characterization of 32-Mmubp and 32-mmub will contribute to understanding the host-bacteria interactions of Lact. fermentum with the intestinal tract of pigs.
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17. |
Vrancken G,
Rimaux T,
Weckx S,
De Vuyst L,
Leroy F,
( 2009 ) Environmental pH determines citrulline and ornithine release through the arginine deiminase pathway in Lactobacillus fermentum IMDO 130101. PMID : 19732985 : DOI : 10.1016/j.ijfoodmicro.2009.07.035 Abstract >>
Sourdough lactic acid bacteria (LAB) need to be adapted to a highly acidic and, therefore, challenging environment. Different mechanisms are employed to enhance competitiveness, among which conversion of arginine into ornithine through the arginine deiminase (ADI) pathway is an important one. A combined molecular and kinetic approach of the ADI pathway in Lactobacillus fermentum IMDO 130101, a highly competitive sourdough LAB strain, identified mechanisms with advantageous technological effects and quantified the impact of these effects. First, molecular analysis of the arcBCAD operon of 4.8 kb revealed the genes encoding the enzymes ornithine transcarbamoylase, carbamate kinase, arginine deiminase, and an arginine/ornithine (A/O) antiporter, respectively, with an additional A/O antiporter 702.5 kb downstream of the ADI operon. The latter could play a role in citrulline transport. Second, pH-controlled batch fermentations were carried out, generating data for the development of a mathematical model to describe the temporal evolution of the three amino acids involved in the ADI pathway (arginine, citrulline, and ornithine) as a result of the activity of these enzymes and transporter(s). Free arginine in the medium was converted completely into a mixture of citrulline and ornithine under all conditions tested. However, the ratio between these end-products and the pattern of their formation showed variation as a function of environmental pH. Under optimal pH conditions for growth, citrulline release and some further conversion into ornithine was observed. When growing under sub-optimal pH conditions, ornithine was the main product of the ADI pathway. These kinetic data suggest a role in adaptation of L. fermentum IMDO 130101 to growth under sub-optimal conditions.
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18. |
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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19. |
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. 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.
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20. |
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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21. |
Dan T,
Liu W,
Song Y,
Xu H,
Menghe B,
Zhang H,
Sun Z,
( 2016 ) Erratum: The evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST). PMID : 27004931 : DOI : 10.1186/s12866-016-0651-5 PMC : PMC4802606 Abstract >>
N/A
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22. |
Sun Z,
Harris HM,
McCann A,
Guo C,
Argimón S,
Zhang W,
Yang X,
Jeffery IB,
Cooney JC,
Kagawa TF,
Liu W,
Song Y,
Salvetti E,
Wrobel A,
Rasinkangas P,
Parkhill J,
Rea MC,
O'Sullivan O,
Ritari J,
Douillard FP,
Paul Ross R,
Yang R,
Briner AE,
Felis GE,
de Vos WM,
Barrangou R,
Klaenhammer TR,
Caufield PW,
Cui Y,
Zhang H,
O'Toole PW,
( 2015 ) Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera. PMID : 26415554 : DOI : 10.1038/ncomms9322 PMC : PMC4667430 Abstract >>
Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.
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23. |
Dan T,
Liu W,
Song Y,
Xu H,
Menghe B,
Zhang H,
Sun Z,
( 2015 ) The evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST). PMID : 25990318 : DOI : 10.1186/s12866-015-0447-z PMC : PMC4437502 Abstract >>
Lactobacillus fermentum is economically important in the production and preservation of fermented foods. A repeatable and discriminative typing method was devised to characterize L. fermentum at the molecular level. The multilocus sequence typing (MLST) scheme developed was based on analysis of the internal sequence of 11 housekeeping gene fragments (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC). MLST analysis of 203 isolates of L. fermentum from Mongolia and seven provinces/ autonomous regions in China identified 57 sequence types (ST), 27 of which were represented by only a single isolate, indicating high genetic diversity. Phylogenetic analyses based on the sequence of the 11 housekeeping gene fragments indicated that the L. fermentum isolates analyzed belonged to two major groups. A standardized index of association (I A (S)) indicated a weak clonal population structure in L. fermentum. Split decomposition analysis indicated that recombination played an important role in generating the genetic diversity observed in L. fermentum. The results from the minimum spanning tree strongly suggested that evolution of L. fermentum STs was not correlated with geography or food-type. The MLST scheme developed will be valuable for further studies on the evolution and population structure of L. fermentum isolates used in food products.
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24. |
Archer AC,
Halami PM,
( 2015 ) Probiotic attributes of Lactobacillus fermentum isolated from human feces and dairy products. PMID : 26004804 : DOI : 10.1007/s00253-015-6679-x Abstract >>
The objective of this study was to characterize native Lactobacillus fermentum isolates for their probiotic attributes. Accordingly, 12 L. fermentum isolates selected from indigenous fermented dairy products and infant fecal samples were evaluated for their probiotic properties by in vitro and PCR methods. The cultures exhibited high tolerance to acid and bile as well as survival in simulated transit fluids (above 70 %). Cell surface hydrophobicity was in the range of 0.55-57.69 % for xylene and 0.45-77.12 % for hexadecane, whereas auto-aggregation ranged between 9 and 62 %. Isolates exhibited efficient binding to mucin and fibronectin, bile salt hydrolase activity, cholesterol assimilation (49-76 %), and radical scavenging activity (37-77 %). The isolates demonstrated antibacterial activity against Listeria monocytogenes Scott A and Micrococcus luteus ATCC 9341. Molecular fingerprinting and identification of the isolates were achieved by PCR with GTG5 as well as 16S rRNA, phenylalanyl-tRNA synthetase alpha subunit (pheS), and RNA polymerase alpha subunit (rpoA) genes. This revealed the genomic diversity of the isolates from the two sources. Gene-specific amplification of probiotic marker genes was attained by PCR-based methods, and resultant products were sequenced. Multiple sequence alignment of the probiotic marker genes using bioinformatics revealed similarity to completely sequenced genomes of L. fermentum CECT 5716 and IFO 3956 with a few variations in mucin-binding protein gene sequences. Isolates designated as L. fermentum MCC 2759 and L. fermentum MCC 2760 showed the best probiotic attributes with high survival in simulated gastrointestinal fluids, in vitro adhesion, cholesterol reduction, and high antioxidative potential. Thus, these cultures could be potential probiotic candidates for application as functional foods.
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25. |
Murphree CA,
Heist EP,
Moe LA,
( 2014 ) Antibiotic resistance among cultured bacterial isolates from bioethanol fermentation facilities across the United States. 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.
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26. |
Xu Z,
Li S,
Feng X,
Zhan Y,
Xu H,
( 2014 ) Function of aspartic acid residues in optimum pH control of L-arabinose isomerase from Lactobacillus fermentum. PMID : 24220791 : DOI : 10.1007/s00253-013-5342-7 Abstract >>
L-Arabinose isomerase (L-AI) catalyzes the isomerization of L-arabinose to L-ribulose and D-galactose to D-tagatose. Most reported L-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial D-tagatose production. Lactobacillus fermentum L-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for D-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, D-galactose was isomerized into D-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-? crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other L-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of L-AIs that can be modified for desired optimum pH and better pH stability, which are useful in D-tagatose bioproduction.
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27. |
Kumar R,
Rajkumar H,
Kumar M,
Varikuti SR,
Athimamula R,
Shujauddin M,
Ramagoni R,
Kondapalli N,
( 2013 ) Molecular cloning, characterization and heterologous expression of bile salt hydrolase (Bsh) from Lactobacillus fermentum NCDO394. PMID : 23673477 : DOI : 10.1007/s11033-013-2607-2 Abstract >>
Bile salt hydrolase (Bsh) active probiotic strains hydrolyze bile acid amino conjugates in vivo, which triggers cholesterol consumption in liver to synthesize new bile leading to consequential cholesterol lowering. Hence, bile salt hydrolyzing potential was the criterion to select L. fermentum NCDO394 for this study and its gene encoding Bsh was identified and cloned. The resulting nucleotide sequence of bsh gene contained an open reading frame (ORF) of 978 nucleotides encoding a predicted protein of 325 amino acids with a theoretical pI of 6.39. Moreover, deduced Bsh protein had high similarity with the Bshs of L. fermentum only and also exhibited significant similarity to the Pencillin V amidases of other Lactobacillus spp. Five catalytically important amino acids were highly conserved in L. fermentum Bsh while four amino acid motifs around these active sites, were not as consistent as in other Bsh proteins. Furthermore, L. fermentum bsh gene was sub-cloned into pET-28b(+) vector, and its expression was induced with 0.05 mM isopropylthiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3). The recombinant Bsh (rBsh) was purified with homogeneity using Ni+2-NTA column and characterized for substrate specificity, pH and temperature. The rBsh hydrolyzed six major human bile salts with a slight preference towards glycine-conjugated bile salts. The optimum pH of rBsh was six, and its enzymatic activity declined below pH 5 and above pH 7. The enzyme was stable and functional even at 65 �XC while showed its maximum activity at 37 �XC. In conclusion, L. fermentum NCDO394 may be a promising candidate probiotic which may affect cholesterol metabolism in vivo.
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28. |
Thumu SC,
Halami PM,
( 2012 ) Presence of erythromycin and tetracycline resistance genes in lactic acid bacteria from fermented foods of Indian origin. PMID : 22644346 : DOI : 10.1007/s10482-012-9749-4 Abstract >>
Lactic acid bacteria (LAB) resistant to erythromycin were isolated from different food samples on selective media. The isolates were identified as Enterococcus durans, Enterococcus faecium, Enterococcus lactis, Enterococcus casseliflavus, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus fermentum, Pediococcus pentosaceus and Leuconostoc mesenteroides. Of the total 60 isolates, 88 % harbored the ermB gene. The efflux gene msrA was identified in E. faecium, E. durans, E. lactis, E. casseliflavus, P. pentosaceus and L. fermentum. Further analysis of the msrA gene by sequencing suggested its homology to msrC. Resistance to tetracycline due to the genes tetM, tetW, tetO, tetK and tetL, alone or in combination, were identified in Lactobacillus species. The tetracycline efflux genes tetK and tetL occurred in P. pentosaceus and Enterococcus species. Since it appeared that LAB had acquired these genes, fermented foods may be a source of antibiotic resistance.
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29. |
Liu GX,
Kong J,
Lu WW,
Kong WT,
Tian H,
Tian XY,
Huo GC,
( 2011 ) �]-Galactosidase with transgalactosylation activity from Lactobacillus fermentum K4. PMID : 22118071 : DOI : 10.3168/jds.2011-4479 Abstract >>
The LacLM �]-galactosidase of Lactobacillus fermentum K4 is encoded by 2 consecutive genes, lacL (large subunit) and lacM (small subunit), that share 17 overlapping nucleotides. Phylogenetic analysis revealed that this enzyme was closely related to other Lactobacillus �]-galactosidases and provided significant insight into its common and distinct characteristics. We cloned both the lacL and lacM genes of L. fermentum K4 and heterologously expressed each in Escherichia coli, although the recombinant enzyme was only functional when both were expressed on the same plasmid. We evaluated the enzymatic properties of this species-specific LacLM �]-galactosidase and discovered that it acts as both a hydrolase, bioconverting lactose into glucose and galactose, and a transgalactosylase, generating prebiotic galacto-oligosaccharides (GOS). The recombinant �]-galactosidase showed a broad pH optimum and stability around neutral pH. The optimal temperature and Michaelis constant (K(m)) for the substrates o-nitrophenyl-�]-D-galactopyranoside and lactose were, respectively, 40�XC and 45 to 50�XC and 1.31 mM and 27 mM. The enzyme activity was stimulated by some cations such as Na?, K?, and Mg??. In addition, activity was also enhanced by ethanol (15%, wt/vol). The transgalactosylation activity of L. fermentum K4 �]-galactosidase effectively and rapidly generated GOS, up to 37% of the total sugars from the reaction. Collectively, our results suggested that the �]-galactosidase from L. fermentum K4 could be exploited for the formation of GOS.
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30. |
( 1993 ) Purification, cloning, and cofactor independence of glutamate racemase from Lactobacillus. PMID : 8385993 : DOI : 10.1021/bi00066a019 Abstract >>
Glutamate racemase has been purified more than 12,000-fold from Lactobacillus fermenti. The racemase gene has been cloned using standard hybridization techniques combined with a novel selection for in vivo glutamate racemase activity, and the racemase has been expressed in Escherichia coli as 20-25% of the total soluble cell protein. The cloned gene product is indistinguishable from that purified from Lactobacillus and is a monomer of M(r) 28,300. Both a coupled enzymatic assay and a circular dichroism assay show that the enzyme follows Michaelis-Menten kinetics, with a Km of 0.3 mM and a kcat of 70 s-1 in each reaction direction. Investigations into the cofactor dependence of glutamate racemase indicate that the enzyme employs neither pyridoxal phosphate nor a pyruvoyl group in the labilization of the proton at the stereogenic center of glutamate. Furthermore, the racemase activity is unaffected by the presence of the metal-chelating reagent EDTA. The gene sequence of the racemase is 24% identical to that of aspartate racemase from Streptococcus thermophilus and 30% identical to that of an unidentified open reading frame in the rrnB ribosomal RNA operon of E. coli. Because the two cysteine residues in glutamate racemase and their surrounding regions are well-conserved in both of these sequences, and since glutamate racemase is stabilized by the presence of reduced thiols, these residues are possible candidates for the enzymic bases that deprotonate glutamate at C-2.
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31. |
( 1993 ) Isotope effects and the identification of catalytic residues in the reaction catalyzed by glutamate racemase. PMID : 8097110 : DOI : 10.1021/bi00066a021 Abstract >>
Primary kinetic isotope effects on Vmax were observed in both reaction directions upon racemizing samples of [2-2H]glutamate with the cofactor-independent glutamate racemase from Lactobacillus. This supports a deprotonation/protonation mechanism for racemization in which the breaking of the carbon-hydrogen bond at C-2 is partially rate-determining. Substantial "overshoots" were observed when the time course of racemization of either enantiomer of glutamate was monitored using circular dichroism spectroscopy. This is consistent with a "two-base" mechanism accompanied by a kinetic isotope effect. "Competitive deuterium washout" experiments were used to measure kinetic isotope effects on Vmax/Km of 2.5 for (S)-glutamate and 3.4 for (R)-glutamate. The ratio of the notably different isotope effects was confirmed by "double competitive deuterium washout" experiments. Site-directed mutagenesis was used to generate the mutant C73A and C184A enzymes. In each case the mutant enzymes were inactive as racemases. The two mutant enzymes are, however, capable of catalyzing the elimination of HCl from opposite enantiomers of threo-3-chloroglutamic acid, a process that presumably requires only one enzymic base. This finding indicates that the active sites of the mutant enzymes are intact and that the two cysteines flank the bound substrate molecule. It appears that cysteine-73 is responsible for the abstraction of the C-2 hydrogen from (R)-glutamate and cysteine-184 abstracts the proton from (S)-glutamate in the racemization reaction of the wild-type enzyme.
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