| 1. |
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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2. |
Sillanpää J,
Martínez B,
Antikainen J,
Toba T,
Kalkkinen N,
Tankka S,
Lounatmaa K,
Keränen J,
Höök M,
Westerlund-Wikström B,
Pouwels PH,
Korhonen TK,
( 2000 ) Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus. PMID : 11053389 : DOI : 10.1128/jb.182.22.6440-6450.2000 PMC : PMC94791 Abstract >>
The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.
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3. |
Hill JE,
Hemmingsen SM,
Goldade BG,
Dumonceaux TJ,
Klassen J,
Zijlstra RT,
Goh SH,
Van Kessel AG,
( 2005 ) Comparison of ileum microflora of pigs fed corn-, wheat-, or barley-based diets by chaperonin-60 sequencing and quantitative PCR. PMID : 15691942 : DOI : 10.1128/AEM.71.2.867-875.2005 PMC : PMC546709 Abstract >>
We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.
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4. |
Marcotte H,
Ferrari S,
Cesena C,
Hammarström L,
Morelli L,
Pozzi G,
Oggioni MR,
( 2004 ) The aggregation-promoting factor of Lactobacillus crispatus M247 and its genetic locus. PMID : 15357724 : DOI : 10.1111/j.1365-2672.2004.02364.x Abstract >>
Characterization of the aggregation-promoting factor (APF) of the human intestinal isolate Lactobacillus crispatus M247 and its homologous nonaggregating mutant Mu5. Western blot analysis revealed that the supernatant of both M247 and Mu5 contains a 28-kDa protein which cross reacts with the antiserum produced against the APF of Lact. gasseri 4B2. The apf genes of M247 and Mu5 strains were identical and were shown to be 672 nucleotides in length and encoding a protein of 223 amino acids with a predicted molecular weight of 24.0 kDa. Our results shows that the lost of aggregation in Mu5 is not related to a defect in secretion of the APF protein or a mutation in the apf gene. These results suggest that the mutation in Mu5 may be contained in another molecule involved in aggregation such as a possible receptor for APF.
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5. |
Ventura M,
Canchaya C,
van Sinderen D,
Fitzgerald GF,
Zink R,
( 2004 ) Bifidobacterium lactis DSM 10140: identification of the atp (atpBEFHAGDC) operon and analysis of its genetic structure, characteristics, and phylogeny. PMID : 15128574 : DOI : 10.1128/aem.70.5.3110-3121.2004 PMC : PMC404453 Abstract >>
The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species. All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases. Genes encoding the subunits of the F(1)F(0)-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of the atp operon of B. lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B. lactis DSM 10140. The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B. lactis DSM 10140 was regulated at the level of transcription and not at the enzyme assembly step.
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6. |
Strøman P,
Müller CC,
Sørensen KI,
( 2003 ) Heat shock treatment increases the frequency of loss of an erythromycin resistance-encoding transposable element from the chromosome of Lactobacillus crispatus CHCC3692. PMID : 14660363 : DOI : 10.1128/aem.69.12.7173-7180.2003 PMC : PMC309925 Abstract >>
A 3,165-bp chromosomally integrated transposon, designatedTn3692, of the gram-positive strain Lactobacillus crispatus CHCC3692 contains an erm(B) gene conferring resistance to erythromycin at concentrations of up to 250 micrograms/ml. Loss of this resistance can occur spontaneously, but the rate is substantially increased by heat shock treatment. Heat shock treatment at 60 degrees C resulted in an almost 40-fold increase in the frequency of erythromycin-sensitive cells (erythromycin MIC, 0.047 micrograms/ml). The phenotypic change was followed by a dramatic increase in transcription of the transposase gene and the concomitant loss of an approximately 2-kb DNA fragment carrying the erm(B) gene from the 3,165-bp erm transposon. In cells that were not subjected to heat shock, transcription of the transposase gene was not detectable. The upstream sequence of the transposase gene did not show any homology to known heat shock promoters in the gene data bank. Significant homology (>99%) was observed between the erythromycin resistance-encoding gene from L. crispatus CHCC3692 and the erm(B) genes from other gram-positive bacteria, such as Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium, and Lactobacillus reuteri, which strongly indicates a common origin of the erm(B) gene for these species. The transposed DNA element was not translocated to other parts of the genome of CHCC3692, as determining by Southern blotting, PCR analysis, and DNA sequencing. No other major aberrations were observed, as judged by colony morphology, growth performance of the strain, and pulsed-field gel electrophoresis. These observations suggest that heat shock treatment could be used as a tool for the removal of unwanted antibiotic resistance genes harbored in transposons flanked by insertion sequence elements or transposases in lactic acid bacteria used for animal and human food production.
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7. |
Ventura M,
Canchaya C,
Meylan V,
Klaenhammer TR,
Zink R,
( 2003 ) Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification. PMID : 14602655 : DOI : 10.1128/aem.69.11.6908-6922.2003 PMC : PMC262312 Abstract >>
We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus.
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8. |
Cousin S,
Gulat-Okalla ML,
Motreff L,
Gouyette C,
Bouchier C,
Clermont D,
Bizet C,
( 2012 ) Lactobacillus gigeriorum sp. nov., isolated from chicken crop. PMID : 21421927 : DOI : 10.1099/ijs.0.028217-0 Abstract >>
In the early 1980s, a facultatively anaerobic, non-motile, short rod, designated 202(T), was isolated from a chicken crop and identified as a homofermentative lactic acid bacterium. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain was affiliated with the genus Lactobacillus, clustering within the Lactobacillus acidophilus-delbrueckii group. In this analysis, strain 202(T) appeared to be most closely related to the type strains of Lactobacillus intestinalis and Lactobacillus amylolyticus, with gene sequence similarities of 96.1 and 96.2 %, respectively. Strain 202(T) was found to differ from these two species, however, when investigated by multilocus sequence analysis, and it also differed in terms of some of its metabolic properties. On the basis of these observations, strain 202(T) is considered to represent a novel species in the genus Lactobacillus, for which the name Lactobacillus gigeriorum sp. nov. is proposed; the type strain is 202(T) (= CRBIP 24.85(T) = DSM 23908(T)).
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9. |
Chen X,
Chen Y,
Li X,
Chen N,
Fang W,
( 2009 ) Characterization of surface layer proteins in Lactobacillus crispatus isolate ZJ001. PMID : 19884777 : Abstract >>
Lactobacillus crispatus (L. crispatus) ZJ001 is highly adhesive to epithelial cells and expresses S-layer proteins. In this study, S-layer genes were sequenced and expressed in E. coli to characterize the function of S-layer proteins with this particular strain. L. crispatus ZJ001 harbored two Slayer genes slpA and slpB, and only slpA gene was expressed in the bacterium, as revealed by RT-PCR and immunoassays. The mature SlpA showed 47% amino acid sequence identity to SlpB. The SlpA and SlpB of L. crispatus ZJ001 were highly homologous at the C-terminal region to other Lactobacillus S-layer proteins, but were substantially variable at N-terminal and middle regions. Electron microscopic analysis indicated that His-slpA expressed in E. coli was able to form a sheet-like structure similar to the natural S-layer, but His-slpB formed as disclike structures. In the cell binding experiments, HeLa cells were able to bind to both recombinant His-slpA and HisslpB proteins to the extent similar to the natural S-layer. The cell binding domains remain mostly in the N-terminal regions in SlpA and SlpB, as shown by high binding of truncated peptides SlpA2-228 and SlpB2-249. Our results indicated that SlpA was active and high binding to HeLa cells, and that the slpA gene could be targeted to display foreign proteins on the bacterial surface of ZJ001 as a potential mucosal vaccine vector.
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10. |
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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11. |
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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12. |
Abdelmaksoud AA,
Koparde VN,
Sheth NU,
Serrano MG,
Glascock AL,
Fettweis JM,
Strauss JF,
Buck GA,
Jefferson KK,
( 2016 ) Comparison of Lactobacillus crispatus isolates from Lactobacillus-dominated vaginal microbiomes with isolates from microbiomes containing bacterial vaginosis-associated bacteria. PMID : 26747455 : DOI : 10.1099/mic.0.000238 PMC : PMC4891990 Abstract >>
Vaginal lactobacilli can inhibit colonization by and growth of other bacteria, thereby preventing development of bacterial vaginosis (BV). Amongst the lactobacilli, Lactobacillus crispatus appears to be particularly effective at inhibiting growth of BV-associated bacteria. Nonetheless, some women who are colonized with this species can still develop clinical BV. Therefore, we sought to determine whether strains of L. crispatus that colonize women with lactobacilli-dominated vaginal microbiomes are distinct from strains that colonize women who develop BV. The genomes of L. crispatus isolates from four women with lactobacilli-dominated vaginal microbiomes (<1% 16S rRNA reads above threshold from genera other than Lactobacillus) and four women with microbiomes containing BV-associated bacteria (>12% 16S rRNA reads from bacterial taxa associated with BV) were sequenced and compared. Lactic acid production by the different strains was quantified. Phage induction in the strains was also analysed. There was considerable genetic diversity between strains, and several genes were exclusive to either the strains from Lactobacillus-dominated microbiomes or those containing BV-associated bacteria. Overall, strains from microbiomes dominated by lactobacilli did not differ from strains from microbiomes containing BV-associated bacteria with respect to lactic acid production. All of the strains contained multiple phage, but there was no clear distinction between the presence or absence of BV-associated bacteria with respect to phage-induced lysis. Genes found to be exclusive to the Lactobacillus-dominated versus BV-associated bacteria-containing microbiomes could play a role in the maintenance of vaginal health and the development of BV, respectively.
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13. |
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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14. |
Stoyancheva G,
Marzotto M,
Dellaglio F,
Torriani S,
( 2014 ) Bacteriocin production and gene sequencing analysis from vaginal Lactobacillus strains. PMID : 24919535 : DOI : 10.1007/s00203-014-1003-1 Abstract >>
The human vagina is a complex and dynamic ecosystem containing an abundance of microorganisms. In women of childbearing age, this system is dominated by Lactobacillus spp. In the present work, seventeen newly isolated vaginal strains were identified by 16S rDNA sequencing and were investigated for their antimicrobial properties. Twelve of the isolated Lactobacillus strains showed activity against one or more microorganisms. Six and five of them produced substances that inhibited the growth of two different Klebsiella strains and Staphylococcus aureus, respectively. Two lactobacilli strains were active against an Escherichia coli strain, one isolate was active against an Enterococus faecalis strain and another lactobacilli strain showed antimicrobial activity against a Candida parapsilosis strain. The nature of the active compounds was additionally studied, and the presence of bacteriocin-like substances was proved. The genes related to the bacteriocin production in three of the newly isolated strains were identified and sequenced. The presence of gassericin A operon in the genome of the species Lactobacillus crispatus was described for the first time. The presence of antimicrobial activity contributes to their possible use as potential probiotic strains after further research.
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15. |
Nie C,
Liu B,
Zhang Y,
Zhao G,
Fan X,
Ning X,
Zhang W,
( 2013 ) Production and secretion of Lactobacillus crispatus �]-galactosidase in Pichia pastoris. PMID : 24012790 : DOI : 10.1016/j.pep.2013.08.019 Abstract >>
Lactobacillus �]-galactosidases are mostly heterodimeric proteins, which are encoded by the two overlapping genes, lacL and lacM, and produced in recombinant prokaryotic systems for higher yield. This is the first report on the expression of a heterodimeric �]-galactosidase from Lactobacillus crispatus B470 in Pichia pastoris. The overlapping consecutive genes, lacL and lacM, that shared 17 nucleotides were cloned from the genomic DNA of L. crispatus. A recombinant plasmid harboring both expression cassettes of lacL and lacM was constructed and transformed into P. pastoris GS115 competent cells. Two recombinant P. pastoris strains (GSLac01 and GSLac02) showed the highest �]-galactosidase activities of 24.5 and 31.0 U/ml in the culture supernatants, respectively. The recombinant �]-galactosidase (LcLacLM) from GSLac02 was purified to electrphoretic homogeneity by ion-exchange chromatography and molecular sieve chromatography. Similar to most Lactobacillus �]-galactosidases that operate at moderately thermophilic and weak acid to neutral conditions, LcLacLM showed optimal activity at 50�XC and pH 5.5-6.5. It's the first report on functional and secretory expression of LacLM-type �]-galactosidase in eukaryotic system. This strategy might be applied to the expression of other overlapping genes.
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16. |
Ramachandran P,
Lacher DW,
Pfeiler EA,
Elkins CA,
( 2013 ) Development of a tiered multilocus sequence typing scheme for members of the Lactobacillus acidophilus complex. PMID : 24038697 : DOI : 10.1128/AEM.02257-13 PMC : PMC3837765 Abstract >>
Members of the Lactobacillus acidophilus complex are associated with functional foods and dietary supplements because of purported health benefits they impart to the consumer. Many characteristics of these microorganisms are reported to be strain specific. Therefore, proper strain typing is essential for safety assessment and product labeling, and also for monitoring strain integrity for industrial production purposes. Fifty-two strains of the L. acidophilus complex (L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, and L. johnsonii) were genotyped using two established methods and compared to a novel multilocus sequence typing (MLST) scheme. PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the hsp60 gene with AluI and TaqI successfully clustered 51 of the 52 strains into the six species examined, but it lacked strain-level discrimination. Random amplified polymorphic DNA PCR (RAPD-PCR) targeting the M13 sequence resulted in highly discriminatory profiles but lacked reproducibility. In this study, an MLST scheme was developed using the conserved housekeeping genes fusA, gpmA, gyrA, gyrB, lepA, pyrG, and recA, which identified 40 sequence types that successfully clustered all of the strains into the six species. Analysis of the observed alleles suggests that nucleotide substitutions within five of the seven MLST loci have reached saturation, a finding that emphasizes the highly diverse nature of the L. acidophilus complex and our unconventional application of a typically intraspecies molecular typing tool. Our MLST results indicate that this method could be useful for characterization and strain discrimination of a multispecies complex, with the potential for taxonomic expansion to a broader collection of Lactobacillus species.
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17. |
Huang CH,
Chang MT,
Huang MC,
Wang LT,
Huang L,
Lee FL,
( 2012 ) Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene. PMID : 22555934 : DOI : 10.1002/jsfa.5692 Abstract >>
To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing.
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18. |
Sun Z,
Kong J,
Hu S,
Kong W,
Lu W,
Liu W,
( 2013 ) Characterization of a S-layer protein from Lactobacillus crispatus K313 and the domains responsible for binding to cell wall and adherence to collagen. PMID : 22526799 : DOI : 10.1007/s00253-012-4044-x Abstract >>
It was previously shown that the surface (S)-layer proteins covering the cell surface of Lactobacillus crispatus K313 were involved in the adherence of this strain to human intestinal cell line HT-29. To further elucidate the structures and functions of S-layers, three putative S-layer protein genes (slpA, slpB, and slpC) of L. crispatus K313 were amplified by PCR, sequenced, and characterized in detail. Quantitative real-time PCR analysis reveals that slpA was silent under the tested conditions; whereas slpB and slpC, the putative amino acid sequences which exhibited minor similarities to the previously reported S-layer proteins in L. crispatus, were actively expressed. slpB, which was predominantly expressed in L. crispatus K313, was further investigated for its functional domains. Genetic truncation of the untranslated leader sequence (UTLS) of slpB results in a reduction in protein production, indicating that the UTLS contributed to the efficient S-layer protein expression. By producing a set of N- and C-terminally truncated recombinant SlpB proteins in Escherichia coli, the cell wall-binding region was mapped to the C terminus, where rSlpB(380-501) was sufficient for binding to isolated cell wall fragments. Moreover, the binding ability of the C terminus was variable among the Lactobacillus species (S-layer- and non-S-layer-producing strains), and teichoic acid may be acting as the receptor of SlpB. To determine the adhesion region of SlpB to extracellular matrix proteins, ELISA was performed. Binding to immobilized types I and IV collagen was observed with the His-SlpB(1-379) peptides, suggesting that the extracellular matrix protein-binding domain was located in the N terminus.
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19. |
Hu S,
Kong J,
Sun Z,
Han L,
Kong W,
Yang P,
( 2011 ) Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein. PMID : 22035337 : DOI : 10.1186/1475-2859-10-86 PMC : PMC3215925 Abstract >>
Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB). In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Multiple sequence alignment revealed that the C-terminal region (LcsB) of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP) or beta-galactosidase (Gal) was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological application.
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Sun Z,
Wang X,
Zhang X,
Wu H,
Zou Y,
Li P,
Sun C,
Xu W,
Liu F,
Wang D,
( 2018 ) Class III bacteriocin Helveticin-M causes sublethal damage on target cells through impairment of cell wall and membrane. PMID : 29349568 : DOI : 10.1007/s10295-018-2008-6 Abstract >>
Helveticin-M, a novel Class III bacteriocin produced by Lactobacillus crispatus exhibited an antimicrobial activity against Staphylococcus aureus, S. saprophyticus, and Enterobacter cloacae. To understand how Helveticin-M injured target cells, Helveticin-M was cloned and heterologously expressed in Escherichia coli. Subsequently, the cell wall organization and cell membrane integrity of target cells were determined. The mechanism of cellular damage differed according to bacterial species. Based on morphology analysis, Helveticin-M disrupted the cell wall of Gram-positive bacteria and disorganized the outer membrane of Gram-negative bacteria, therefore, altering surface structure. Helveticin-M also disrupted the inner membrane, as confirmed by leakage of intracellular ATP from cells and depolarization of membrane potential of target bacteria. Based on cell population analysis, Helveticin-M treatment caused the increase of cell membrane permeability, but the cytosolic enzymes were not influenced, indicating that it was the sublethal injury. Therefore, the mode of Helveticin-M action is bacteriostatic rather than bactericidal.
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