| 1. |
Ravin V,
Alatossava T,
( 2003 ) Three new insertion sequence elements ISLdl2, ISLdl3, and ISLdl4 in Lactobacillus delbrueckii: isolation, molecular characterization, and potential use for strain identification. PMID : 12749837 : Abstract >>
A group of new insertion sequence (IS) elements, ISLdl2, ISLdl3, and ISLdl4, from Lactobacillus delbrueckii subsp. lactis ATCC 15808 was isolated, characterized, and used for strain identification together with ISLdl1, recently characterized as an L. delbrueckii IS element belonging to the ISL3 family. ISLdl2 was 1367 bp in size and had a 24 bp IR and an 8 bp DR. The single ORF of ISLdl2 encoded a protein of 392 aa similar to transposases of the IS256 family. ISLdl3 had a single ORF encoding a protein of 343 aa similar to transposases of the IS30 family. Finally, ISLdl4 had a single ORF encoding a protein of 406 aa and displayed homology to the transposases of the IS110 family. ISLdl4 was only slight different from ISL4 (Accession No. AY040213). ISLdl1, ISLdl2, and ISLdl4 were present in all of the 10 L. delbrueckii subsp. lactis and subsp. delbrueckii strains tested, as well as in three of the 11 L. delbrueckii subsp. bulgaricus strains tested. ISLdl3 was present only in four closely related strains of L. delbrueckii subsp. lactis. These IS elements were not observed in Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, or Lactobacillus plantarum. A cluster of IS elements, ISLdl1, ISLdl2, ISLdl3, ISLdl4, and ISL6, was observed in L. delbrueckii subsp. lactis strain ATCC 15808. Within this cluster, ISLdl4 was inserted into ISLdl1 between the left IR and the start codon of ORF455, encoding a putative transposase. Most of the integration sites of the IS elements were strain-specific. We have observed that IS elements can migrate from one strain to another as integral parts of bacterial DNA by using phage LL-H as a vehicle. We demonstrate for the first time that inverse PCR and vectorette PCR methods with primers based on sequences of the IS elements could be used for identification of L. delbrueckii strains.
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2. |
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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3. |
Tanigawa K,
Watanabe K,
( 2011 ) Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii. PMID : 21178164 : DOI : 10.1099/mic.0.043240-0 Abstract >>
Currently, the species Lactobacillus delbrueckii is divided into four subspecies, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. indicus and L. delbrueckii subsp. lactis. These classifications were based mainly on phenotypic identification methods and few studies have used genotypic identification methods. As a result, these subspecies have not yet been reliably delineated. In this study, the four subspecies of L. delbrueckii were discriminated by phenotype and by genotypic identification [amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST)] methods. The MLST method developed here was based on the analysis of seven housekeeping genes (fusA, gyrB, hsp60, ileS, pyrG, recA and recG). The MLST method had good discriminatory ability: the 41 strains of L. delbrueckii examined were divided into 34 sequence types, with 29 sequence types represented by only a single strain. The sequence types were divided into eight groups. These groups could be discriminated as representing different subspecies. The results of the AFLP and MLST analyses were consistent. The type strain of L. delbrueckii subsp. delbrueckii, YIT 0080(T), was clearly discriminated from the other strains currently classified as members of this subspecies, which were located close to strains of L. delbrueckii subsp. lactis. The MLST scheme developed in this study should be a useful tool for the identification of strains of L. delbrueckii to the subspecies level.
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4. |
Song Y,
Sun Z,
Guo C,
Wu Y,
Liu W,
Yu J,
Menghe B,
Yang R,
Zhang H,
( 2016 ) Genetic diversity and population structure of Lactobacillus delbrueckii subspecies bulgaricus isolated from naturally fermented dairy foods. PMID : 26940047 : DOI : 10.1038/srep22704 PMC : PMC4778129 Abstract >>
Lactobacillus delbrueckii subsp. bulgaricus is one of the most widely used starter culture strains in industrial fermented dairy manufacture. It is also common in naturally fermented dairy foods made using traditional methods. The subsp. bulgaricus strains found in naturally fermented foods may be useful for improving current industrial starter cultures; however, little is known regarding its genetic diversity and population structure. Here, a collection of 298 L. delbrueckii strains from naturally fermented products in Mongolia, Russia, and West China was analyzed by multi-locus sequence typing based on eight conserved genes. The 251 confirmed subsp. bulgaricus strains produced 106 unique sequence types, the majority of which were assigned to five clonal complexes (CCs). The geographical distribution of CCs was uneven, with CC1 dominated by Mongolian and Russian isolates, and CC2-CC5 isolates exclusively from Xinjiang, China. Population structure analysis suggested six lineages, L1-L6, with various homologous recombination rates. Although L2-L5 were mainly restricted within specific regions, strains belonging to L1 and L6 were observed in diverse regions, suggesting historical transmission events. These results greatly enhance our knowledge of the population diversity of subsp. bulgaricus strains, and suggest that strains from CC1 and L4 may be useful as starter strains in industrial fermentation.
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5. |
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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6. |
Klein JR,
Dick A,
Schick J,
Matern HT,
Henrich B,
Plapp R,
( 1995 ) Molecular cloning and DNA sequence analysis of pepL, a leucyl aminopeptidase gene from Lactobacillus delbrueckii subsp. lactis DSM7290. PMID : 7737150 : Abstract >>
A genomic library of Lactobacillus delbrueckii subsp. lactis DSM7290 DNA fragments from a Sau3A partial digestion in the low-copy-number vector pLG339, was used to screen Escherichia coli for the presence of peptidases. Using the chromogenic substrate leucine-beta-naphthylamide (Leu-NH-Nap) and E. coli strain CM89 lacking the corresponding enzyme activity in an enzymic plate assay, allowed the isolation of two peptidase genes; the newly described pepL and the recently cloned and sequenced pepN. Clones could be distinguished not only by the restriction pattern of isolated plasmids but also by the rate and intensity of their colour reaction with Leu-NH-Nap. Three out of five clones were identified to express the Lactobacillus pepN gene; the others were shown to express a second aminopeptidase gene, designated pepL. This gene, together with 200 bp upstream of the proposed AUG initiation codon, was further subcloned and sequenced. The corresponding open reading frame of 897 nucleotides is predicted to encode a protein of 299 amino acids (34,541 Da). Searching the EMBL database revealed similarity to the prolinase of Lactobacillus helveticus (45.8% identity), to the iminopeptidases of Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulgaricus (25.5%), and to the Bacillus coagulans prolinase (21.5%). Minor similarities were detected for hydrolytic enzymes with serine active sites. The product encoded by the pepL gene was functional but could not be visualized on Coomassie-blue-stained polyacrylamide gels. High level expression of peptidase L in E. coli was achieved by placing the gene under the control of the T7 promoter.
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7. |
Klein JR,
Henrich B,
Plapp R,
( 1994 ) Cloning and nucleotide sequence analysis of the Lactobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC. PMID : 7851736 : DOI : 10.1111/j.1574-6968.1994.tb07299.x Abstract >>
A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-beta-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50,909 Da. The enzyme is functional and extremely overexpressed in E. coli.
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8. |
( 1994 ) NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase of Lactobacillus delbrueckii subsp. bulgaricus. Gene cloning and enzyme characterization. PMID : 7925358 : DOI : 10.1111/j.1432-1033.1994.00439.x Abstract >>
A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD(+)-dependent D-2-hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4-methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD(+)-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-oxoacid substrates except those having a branched beta-carbon.
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9. |
Germond JE,
Lapierre L,
Delley M,
Mollet B,
( 1995 ) A new mobile genetic element in Lactobacillus delbrueckii subsp. bulgaricus. PMID : 7565604 : DOI : 10.1007/bf02191640 Abstract >>
A new IS element (ISL3) was discovered in Lactobacillus delbrueckii subsp. bulgaricus during the characterization of the linkage relationships between the two genes important for milk fermentation, beta-galactosidase (lacZ) and the cell-wall associated protease (prtP). ISL3 is a 1494 bp element, flanked by 38 bp imperfect inverted repeats, and generates an 8 bp target duplication upon insertion. It contains one open reading frame, encoding a potential polypeptide of 434 amino acids, which shows significant homology (34% identity) to the transposase of the Leuconostoc mesenteroides element IS1165. Molecular analysis of spontaneous lacZ mutants revealed some strains that had sustained deletions of 7 to 30 kb in size, centered on and eliminating the copy of ISL3 next to lacZ. Other deletion endpoints were identified as located immediately adjacent to ISL3. Furthermore, genetic translocations that had occurred via transposition of ISL3 were observed fortuitously in cultures screened for deletion mutants. ISL3 can be found in one to several copies in various strains of L. delbrueckii. However, it was not present in other dairy lactic acid bacteria tested.
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10. |
( 1998 ) The arbZ gene from Lactobacillus delbrueckii subsp. lactis confers to Escherichia coli the ability to utilize the beta-glucoside arbutin. PMID : 9611263 : DOI : 10.1016/s0378-1119(98)00156-5 Abstract >>
From a genomic library of the industrially used strain Lactobacillus delbrueckii subsp. lactis DSM7290, a gene designated arbZ (869bp; encoding a 33.5kDa protein) was isolated by screening E. coli transformants for the ability to utilize the beta-glucoside arbutin. Out of 9000 transformants nine were able to ferment arbutin, whereas no utilization of the beta-glucosides salicin, esculin or cellobiose could be detected. Overexpression of arbZ using the T7-polymerase-T7-promoter-system resulted in the formation of insoluble, catalytically inactive protein aggregates (inclusion bodies). Accordingly, overexpression was not accompanied by an increase in ArbZ activity. Induction of arbZ controlled by the lac promoter under conditions that reduce protein aggregation resulted in a 12-fold increase in arbutin hydrolyzing activity of intact cells and a 13-fold increase in phospho-beta-glycosidase activity in cell-free extracts of the respective transformants. Nucleotide sequence analysis revealed a second gene upstream of arbZ that was designated arbX (830bp). ArbX (32.6kDa) shared similarity with several glycosyltransferases involved in the biosynthesis of lipopolysaccharides in Gram-negative bacteria. In Lb. delbrueckii subsp. lactis DSM7290 two transcripts, one covering arbX together with arbZ and one covering arbZ alone were detected by Northern blot analysis.
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11. |
( 1998 ) An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase. PMID : 9579064 : DOI : 10.1099/00221287-144-4-905 Abstract >>
The structural genes gap, pgk and tpi encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order gap-pgk-tpi. The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, and by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed, noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gap gene, and a less abundant mRNA of 3.4 kb corresponding to the gap-pgk-tpi cluster. The absence of a visible terminator in the 3'-end of the shorter transcript and the location of this 3'-end inside the pgk gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gap gene.
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12. |
( 1997 ) Lactobacilli carry cryptic genes encoding peptidase-related proteins: characterization of a prolidase gene (pepQ) and a related cryptic gene (orfZ) from Lactobacillus delbrueckii subsp. bulgaricus. PMID : 9421914 : DOI : 10.1099/00221287-143-12-3899 Abstract >>
Two genes, pepQ and orfZ, encoding a prolidase and a prolidase-like protein, respectively, were cloned and characterized from Lactobacillus delbrueckii subsp. bulgaricus. The identity of the pepQ and orfZ genes with the Lactobacillus delbrueckii subsp. lactis prolidase gene (pepQ) was shown to be 98% and 60%, respectively. Both pepQ and orfZ were preceded by a putative promoter region. Northern analysis of pepQ mRNA revealed a 1.1 kb transcript indicating that pepQ forms a monocistronic transcriptional unit. Under the growth conditions used, no evidence was obtained that orfZ was expressed, either by mRNA size determination in Northern analysis or by primer extension analysis. With reverse transcription-PCR, however, the presence of monocistronic orfZ transcripts was established. The orfZ gene could also be overexpressed in E. coli using the vector pKK223-3. The size of the protein synthesized, 41 kDa, confirmed the molecular mass of OrfZ calculated according to DNA sequence analysis. In contrast to PepQ, which showed a substrate specificity characteristic of prolidase enzymes, no enzymic activity for the orfZ-encoded protein was found with the peptide substrates tested. These results indicate that orfZ is a cryptic gene, which is expressed at a very low level under the growth conditions used. It is noteworthy that homologues of the Lb. delbrueckii subsp. bulgaricus orfZ and pepQ genes appeared to be present in both Lb. delbrueckii subsp. lactis and Lactobacillus helveticus.
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