Comparative sequence analysis of the tuf and recA genes and restriction fragment length polymorphism of the internal transcribed spacer region sequences supply additional tools for discriminating Bifidobacterium lactis from Bifidobacterium animalis

The relationship between Bifidobacterium lactis and Bifidobacterium animalis was examined by comparative analysis of tuf and recA gene sequences and by restriction fragment length polymorphism analysis of their internal 16S-23S transcribed spacer region sequences. The bifidobacterial strains investigated could be divided into two distinct groups within a single species based on the tuf, recA, and 16S-23S spacer region sequence analysis. Therefore, all strains of B. lactis and B. animalis could be unified as the species B. animalis and divided into two subspecies, Bifidobacterium animalis subsp. lactis and Bifidobacterium animalis subsp. animalis.     

Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rDNA-based techniques

Ribosomal DNA-based techniques including the analysis of profiles generated by ISR amplification, ISR restriction and ARDRA have been evaluated as molecular tools for identifying Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. They have been applied for the molecular characterization of 91 strains with the following identities: eight Carnobacterium including the eight type species of the genus; 61 Lactobacillus including 40 type strains out of 45 species, 13 Leuconostoc, out of them 11 are type strains and three are subspecies of Lc. mesenteroides; and nine strains representing the six species of genus Pediococcus. The genetic relationship displayed between these species by rrn-based profiles is sustained by their phylogenetic relationships and can therefore be considered useful for taxonomic purposes. Profiles obtained by ISR amplification allowed identification at genus level of Carnobacterium and Leuconostoc, and even at species level in genus Carnobacterium. Genera Lactobacillus and Pediococcus could not be distinguished from each other by applying this technique. The Lactobacillus species analysed here (45) were differentiated using ARDRA-DdeI and ISR-DdeI profiles, sequentially, and Pediococcus species by ISR-DdeI profiles. It was necessary to combine profiles generated by restriction of ISR-DdeI, ARDRA-DdeI and ARDRA-HaeIII in order to complete the identification of Leuconostoc species.     

Lactobacillus tucceti sp. nov., a new lactic acid bacterium isolated from sausage

Following the application of several molecular techniques strain R 19c, isolated from sausage by Reuter in 1970 and deposited at the DSMZ as Lactobacillus sp., has been identified as pertaining to a new species. It showed singular ISR-DdeI and ISR-HaeIII profiles that allowed its differentiation from 68 lactic acid bacteria reference strains analyzed. Phylogenetic analysis based on 16S rRNA gene sequences places this strain in the genus Lactobacillus within the Lactobacillus alimentarius group. Species L. versmoldensis is the closest phylogenetic neighbor with 96.3% sequence similarity. DNA-DNA hybridization experiments confirmed the independent status at species level of this strain. Species-specific primers for PCR detection of this new species have been developed. Phenotypically it can be distinguished from the closest relative L. versmoldensis by several traits such as the peptidoglycan type (L-Lys-Gly-D-Asp), acid production from L-rhamnose, D-mannitol and L-fucose and its inability to ferment d-galactose, d-melibiose and d-sucrose. The name Lactobacillus tucceti sp. nov. is proposed with strain R 19c(T) (=DSM 20183(T)= CECT 5920(T)) as the type strain.     

Comparison of Fructose-1,6-Bisphosphatase Gene (<Emphasis Type="Italic">fbp</Emphasis>) Sequences for the Identification of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis>

Comparative analysis of fructose-1,6-bisphosphatase gene (fbp) sequences was evaluated for the differentiation of reference and clinical strains of Lactobacillus rhamnosus. The sequences of 1,971 nucleotides of the fbp gene were determined on both DNA strands for 21 L. rhamnosus strains, representing reference, probiotic, and clinical strains. No PCR amplification of the fbp gene was observed for other species of the Lactobacillus casei complex (L. casei and L. zeae) or strains of Lactobacillus acidophilus, Streptococcus thermophilus, and Escherichia coli. Phylogenetic analysis of the fbp putative amino acid sequences of L. rhamnosus strains by the neighbor-joining method showed clear distinct positions of this species. The phylogenetic tree, derived from fbp nucleotide sequences, showed four clear divisions between strains of L. rhamnosus. From a taxonomic point of view, our results confirm for the first time that fbp gene sequences have high discriminating power for strains of L. rhamnosus that are difficult to differentiate.     

The accumulation of deleterious mutations in rice genomes: a hypothesis on the cost of domestication

The extent of molecular differentiation between domesticated animals or plants and their wild relatives is postulated to be small. The availability of the complete genome sequences of two subspecies of the Asian rice, Oryza sativa (indica and japonica) and their wild relatives have provided an unprecedented opportunity to study divergence following domestication. We observed significantly more amino acid substitutions during rice domestication than can be expected from a comparison among wild species. This excess is disproportionately larger for the more radical kinds of amino acid changes (e.g. Cys<-->Tyr). We estimate that approximately a quarter of the amino acid differences between rice cultivars are deleterious, not accountable by the relaxation of selective constraints. This excess is negatively correlated with the rate of recombination, suggesting that 'hitchhiking' has occurred. We hypothesize that during domestication artificial selection increased the frequency of many deleterious mutations.     

Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region

A rapid and reliable PCR-based method for distinguishing closely related species within two groups of lactobacilli is described. Primers complementary to species-specific sequences in the 16S/23S rDNA spacer regions were designed after sequencing and sequence comparison of the spacer regions of 32 strains. The strains belong to two groups of closely related Lactobacillus species; one composed of Lactobacillus curvatus, Lactobacillus graminis and Lactobacillus sake, the other of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum. PCR assays with the designed primers and subsequent agarose gel analysis of the amplified fragments allowed the same species identification as the DNA/DNA hybridization procedure.     

Characterization of Lactobacillus sakei by the type of stereoisomers of lactic acid produced

Lactobacillus sakei strains were characterized by the shift of the type of stereoisomers of lactic acid produced in the presence of 50 mM sodium acetate in a medium. Of 27 Lactobacillus sakei strains studied, 20 strains showed high levels of DNA-DNA similarity with L. sakei NRIC 1071(T), and were confirmed as L. sakei. The three remaining strains were identified as Lactobacillus curvatus by DNA-DNA similarity, and three other strains were included in the cluster of Lactobacillus plantarum/Lactobacillus pentosus/Lactobacillus paraplantarum and one strain in the cluster of Lactobacillus paracasei on the basis of 16S rRNA gene sequences. Of the 20 L. sakei strains, 19 strains shifted the type of stereoisomers of lactic acid produced from the DL-type to the L-type in the presence of 50 mM sodium acetate. L. curvatus strains and strains included in the cluster of L. plantarum/L. pentosus/L. paraplantarum and in the cluster of L. paracasei did not shift the type of stereoisomers of lactic acid produced. The change of the type of stereoisomers of lactic acid from the DL-type to the L-type in the presence of sodium acetate was concluded to be species-specific for L. sakei and useful for identification of strains in this species.     

Divergence among genes encoding the elongation factor Tu of Yersinia Species

Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species.     

Revised classification of native probiotic strains of Lactobacillus used in Russian Federation

Thirteen strains of industrial bacterial cultures of the genus Lactobacillus (from a collection of Gabrichevsky Research Institute of Epidemiology and Microbiology) were studied. These strains were used for decades in Russian Federation for food and drug production, as ferments for lactic acid products, for production of probiotics, biologically active and veterinary preparations. Complex analysis of data on cultures obtained using microbiological and molecular-genetic methods was conducted for the first time. Biochemical characteristics of these cultures were studied and the sequence of the proximal region of 16S ribosomal RNA gene was determined. The employment of the test system API-50CHL was shown to broaden the opportunities of a more accurate biochemical identification of bacteria belonging to the genus Lactobacillus, in comparison with the set ANAEROTEST-23. According to the results obtained in a comparative analysis of nucleotide sequences of 16S rRNA gene, all strains examined show 97-99% homology of the proximal region of this gene with that of the type representatives of studied species. These data allowed taxonomic reclassification of the species position of cultures with consideration of the more advanced level of systematics. Nucleotide sequences of gene fragments of examined lactobacilli strains were recorded in NCBI database (accession numbers of deposits GU560031, GU560032, GU560033, GU560034, GU560035, GU560036, GU560037, GU560038, GU560039, GU560040, GU560041, GU560042, GU560043).     

Evidence for horizontal gene transfer in evolution of elongation factor Tu in enterococci

The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.     

Differentiation of Lactobacillus helveticus,Lactobacillus delbrueckii subsp bulgaricus,subsp lactis and subsp delbrueckii using physiological and genetic tools and reclassification of some strains from the ATCC collection

Several physiological tests of glucose metabolism and genetic tools including species specific probes and 16S rDNA sequences were used to identify strains of L. helveticus and the group of L. delbrueckii with its three subspecies lactis, bulgaricus, and delbrueckii. These species are important for the milk industry as fermenting lactic acid bacteria. The identification procedure was applied to the different strains of these species available from the ATCC collection and allowed to reclassify part of them.     

Taxonomic and strain-specific identification of the probiotic strain Lactobacillus rhamnosus 35 within the Lactobacillus casei group

Lactobacilli are lactic acid bacteria that are widespread in the environment, including the human diet and gastrointestinal tract. Some Lactobacillus strains are regarded as probiotics because they exhibit beneficial health effects on their host. In this study, the long-used probiotic strain Lactobacillus rhamnosus 35 was characterized at a molecular level and compared with seven reference strains from the Lactobacillus casei group. Analysis of rrn operon sequences confirmed that L. rhamnosus 35 indeed belongs to the L. rhamnosus species, and both temporal temperature gradient gel electrophoresis and ribotyping showed that it is closer to the probiotic strain L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG) than to the species type strain. In addition, L. casei ATCC 334 gathered in a coherent cluster with L. paracasei type strains, unlike L. casei ATCC 393, which was closer to L. zeae; this is evidence of the lack of relatedness between the two L. casei strains. Further characterization of the eight strains by pulsed-field gel electrophoresis repetitive DNA element-based PCR identified distinct patterns for each strain, whereas two isolates of L. rhamnosus 35 sampled 40 years apart could not be distinguished. By subtractive hybridization using the L. rhamnosus GG genome as a driver, we were able to isolate five L. rhamnosus 35-specific sequences, including two phage-related ones. The primer pairs designed to amplify these five regions allowed us to develop rapid and highly specific PCR-based identification methods for the probiotic strain L. rhamnosus 35.     

Identification of Lactic Acid Bacteria from Chili Bo, a Malaysian Food Ingredient

Ninety-two strains of lactic acid bacteria (LAB) were isolated from a Malaysian food ingredient, chili bo, stored for up to 25 days at 28°C with no benzoic acid (product A) or with 7,000 mg of benzoic acid kg⁻¹ (product B). The strains were divided into eight groups by traditional phenotypic tests. A total of 43 strains were selected for comparison of their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) whole-cell protein patterns with a SDS-PAGE database of LAB. Isolates from product A were identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus farciminis, Pediococcus acidilactici, Enterococcus faecalis, and Weissella confusa. Five strains belonging to clusters which could not be allocated to existing species by SDS-PAGE were further identified by 16S rRNA sequence comparison. One strain was distantly related to the Lactobacillus casei/Pediococcus group. Two strains were related to Weissella at the genus or species level. Two other strains did not belong to any previously described 16S rRNA group of LAB and occupied an intermediate position between the L. casei/Pediococcus group and the Weissella group and species of Carnobacterium. The latter two strains belong to the cluster of LAB that predominated in product B. The incidence of new species and subspecies of LAB in chili bo indicate the high probability of isolation of new LAB from certain Southeast Asian foods. None of the isolates exhibited bacteriocin activity against L. plantarum ATCC 14917 and LMG 17682.     

Nucleotide sequence and codon usage of the elongation factor Tu(EF-Tu) gene from Mycoplasma pneumoniae

The Mycoplasma pneumoniae tuf gene, encoding the elongation factor protein Tu, was cloned and sequenced. The nucleotide sequence of the mycoplasmal gene showed about 60% homology to the sequences of tuf genes of other prokaryotes, yeast mitochondria and Euglena gracilis chloroplasts, and about 75% similarity was found when comparing the deduced amino acid sequences of the various Tu proteins. The relatively low G + C content (40%) of the M. pneumoniae DNA was reflected in a low G + C content (44.6%) of the tuf gene, and in a preferential use of adenine and uracil at the third position of codons, yet codon usage analysis revealed the presence of almost all of the codons of the genetic code in the mycoplasmal gene. Southern blot hybridization of digested DNAs of 11 Mollicutes species with the entire M. pneumoniae tuf gene and with its 5' part suggested the presence of one copy only of this gene in the representative species of the Mollicutes. In this respect, the Mollicutes resemble Gram-positive bacteria and differ from the Gram-negative bacteria, which carry two copies of the tuf gene.     

LC-MS/MS analysis of surface layer proteins as a useful method for the identification of lactobacilli from the Lactobacillus acidophilus group

For precise identification of a Lactobacillus K1 isolate, LC-MS/MS analysis of the putative surface layer protein was performed. The results obtained from LTQ-FT-ICR mass spectrometry confirmed that the analyzed protein spot is the surface layer protein originating from Lb. helveticus species. Moreover, the identified protein has the highest similarity with the surface layer protein from Lb. helveticus R0052. To evaluate the proteomic study, multilocus sequence analysis of selected housekeeping gene sequences was performed. Combination of 16S rRNA sequencing with partial sequences for the genes encoding the RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), translational elongation factor Tu (tuf), and Hsp60 chaperonins (groEL) also allowed to classify the analyzed isolate as Lb. helveticus. Further classification at the strain level was achieved by sequencing of the slp gene. This gene showed 99.8% identity with the corresponding slp gene of Lb. helveticus R0052, which is in good agreement with data obtained by nano-HPLC coupled to an LTQ-FT-ICR mass spectrometer. Finally, LC-MS/ MS analysis of surface layer proteins extracted from three other Lactobacillus strains proved that the proposed method is the appropriate molecular tool for the identification of S-layer-possessing lactobacilli at the species and even strain levels.     

M13 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp.

The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted the differentiation of species, subspecies and individual strains and the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp.     

M13 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp.

The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted the differentiation of species, subspecies and individual strains and the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp.     

Sequence diversity in S1 genes and S1 translation products of 11 serotype 3 reovirus strains.

The S1 gene nucleotide sequences of 10 type 3 (T3) reovirus strains were determined and compared with the T3 prototype Dearing strain in order to study sequence diversity in strains of a single reovirus serotype and to learn more about structure-function relationships of the two S1 translation products, sigma 1 and sigma 1s. Analysis of phylogenetic trees constructed from variation in the sigma 1-encoding S1 nucleotide sequences indicated that there is no pattern of S1 gene relatedness in these strains based on host species, geographic site, or date of isolation. This suggests that reovirus strains are transmitted rapidly between host species and that T3 strains with markedly different S1 sequences circulate simultaneously. Comparison of the deduced sigma 1 amino acid sequences of the 11 T3 strains was notable for the identification of conserved and variable regions of sequence that correlate with the proposed domain organization of sigma 1 (M.L. Nibert, T.S. Dermody, and B. N. Fields, J. Virol. 64:2976-2989, 1990). Repeat patterns of apolar residues thought to be important for sigma 1 structure were conserved in all strains examined. The deduced sigma 1s amino acid sequences of the strains were more heterogeneous than the sigma 1 sequences; however, a cluster of basic residues near the amino terminus of sigma 1s was conserved. This analysis has allowed us to investigate molecular epidemiology of T3 reovirus strains and to identify conserved and variable sequence motifs in the S1 translation products, sigma 1 or sigma 1s.