Similarity and differences in the Lactobacillus acidophilus group identified by polyphasic analysis and comparative genomics

Abstract

A set of lactobacilli were investigated by polyphasic analysis. Multilocus sequence analysis, DNA typing, microarray analysis, and in silico whole-genome alignments provided a remarkably consistent pattern of similarity within the Lactobacillus acidophilus complex. On microarray analysis, 17 and 5% of the genes from Lactobacillus johnsonii strain NCC533 represented variable and strain-specific genes, respectively, when tested against four independent isolates of L. johnsonii. When projected on the NCC533 genome map, about 10 large clusters of variable genes were identified, and they were enriched around the terminus of replication. A quarter of the variable genes and two-thirds of the strain-specific genes were associated with mobile DNA. Signatures for horizontal gene transfer and modular evolution were found in prophages and in DNA from the exopolysaccharide biosynthesis cluster. On microarray hybridizations, Lactobacillus gasseri strains showed a shift to significantly lower fluorescence intensities than the L. johnsonii test strains, and only genes encoding very conserved cellular functions from L. acidophilus hybridized to the L. johnsonii array. In-silico comparative genomics showed extensive protein sequence similarity and genome synteny of L. johnsonii with L. gasseri, L. acidophilus, and Lactobacillus delbrueckii; moderate synteny with Lactobacillus casei; and scattered X-type sharing of protein sequence identity with the other sequenced lactobacilli. The observation of a stepwise decrease in similarity between the members of the L. acidophilus group suggests a strong element of vertical evolution in a natural phylogenetic group. Modern whole-genome-based techniques are thus a useful adjunct to the clarification of taxonomical relationships in problematic bacterial groups.

FIG. 1.

Polyphasic analysis of the indicated Lactobacillus isolates by MLSA (A to E), DNA typing (F and G), fermentation capacity (H), and clustering of the microarray analysis (I). In each tree, the strain is identified at the right end of the branch by a strain and an abridged genus/species identifier. La, L. acidophilus; Ld, L. delbrueckii subsp. bulgaricus; Lg, L. gasseri; Lj, L. johnsonii; Lp, L. plantarum; Lsk, L. sakei subsp. sakei; Lsl, L. salivarius subsp. salivarius. The numbers at the nodes give the bootstrap probabilities. The scale above the MLSA gives the percentage of base pair sequence identity.

FIG. 2.

Genomic diversity in the Lactobacillus acidophilus group as seen from the viewpoint of L. johnsonii strain NCC533. (Left) CGH data. Each horizontal row corresponds to an amplicon on the array, and the genes are vertically ordered according to their positions on the NCC533 genome. The columns represent the analyzed strains, and the strains are identified by their code numbers. The color code corresponding to the CGH score (BlastN-like score) is given at the bottom right of the figure; the gradient goes from black to yellow to depict the presence, divergence, or absence of a gene sequence. Some relevant gene and genetic-element positions are shown on the left side along the genome. ori, origin of replication; ter, terminus of replication. (Right inset) Signal ratio distribution of the CGH data. The reference is L. johnsonii strain NCC533. Ratios are expressed in a log₂ scale. See the text for details.

FIG. 3.

Alignment of the genetic maps of the exopolysaccharide and dTDP-rhamnose biosynthetic gene cluster in selected L. johnsonii and L. gasseri strains, identified by their code numbers on the left of the maps. Genes sharing high nucleotide sequence identity are linked by blue shading; the shading is striped when the identity is lower than 85%. The numbering of NCC533 genes follows that of the GenBank file. For ATCC 33323T, the genes 04 to 28 correspond to LGAS_1157 to LGAS_1133 of the GenBank file, respectively. The annotation is indicated by the color code explained below the figure. WZY proteins (polymerases) were tentatively classified on the basis of their similar hydrophobicity patterns.

FIG. 4.

DNA and protein sequence similarities between completely sequenced lactobacilli (identified on the y axis) as revealed by in silico genome alignments with L. johnsonii NCC533, which was used as the sequenced reference strain (MUMmer analysis). (Left) Alignments obtained with NUCmer script, highlighting the conserved regions at the DNA level. The dots represent the positions of conserved DNA sequences on the genomes. (Right) Alignments obtained with PROmer script, highlighting the conserved regions at the protein level. The dots represent the positions of conserved protein sequences on the genomes. Identities in direct or reverse orientation are indicated in blue and red, respectively. Note that the sequenced L. acidophilus strain does not correspond to the strain used in the CGH analysis.

FIG. 5.

Protein sequence similarities (PROmer) of Pediococcus pentosaceus, Oenococcus oeni, and Leuconostoc mesenteroides with L. johnsonii NCC533 as the sequenced reference strain.