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Sequence diversity of the Bacillus thuringiensis and B. cereus sensu lato flagellin (H antigen) protein: comparison with H serotype diversity.

ABSTRACT: We set out to analyze the sequence diversity of the Bacillus thuringiensis flagellin (H antigen [Hag]) protein and compare it with H serotype diversity. Some other Bacillus cereus sensu lato species and strains were added for comparison. The internal sequences of the flagellin (hag) alleles from 80 Bacillus thuringiensis strains and 16 strains from the B. cereus sensu lato group were amplified and cloned, and their nucleotide sequences were determined and translated into amino acids. The flagellin allele nucleotide sequences for 10 additional strains were retrieved from GenBank for a total of 106 Bacillus species and strains used in this study. These included 82 B. thuringiensis strains from 67 H serotypes, 5 B. cereus strains, 3 Bacillus anthracis strains, 3 Bacillus mycoides strains, 11 Bacillus weihenstephanensis strains, 1 Bacillus halodurans strain, and 1 Bacillus subtilis strain. The first 111 and the last 66 amino acids were conserved. They were referred to as the C1 and C2 regions, respectively. The central region, however, was highly variable and is referred to as the V region. Two bootstrapped neighbor-joining trees were generated: a first one from the alignment of the translated amino acid sequences of the amplified internal sequences of the hag alleles and a second one from the alignment of the V region amino acid sequences, respectively. Of the eight clusters revealed in the tree inferred from the entire C1-V-C2 region amino acid sequences, seven were present in corresponding clusters in the tree inferred from the V region amino acid sequences. With regard to B. thuringiensis, in most cases, different serovars had different flagellin amino acid sequences, as might have been expected. Surprisingly, however, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. Likewise, serovars from the same H serotypes were most often found clustered together, with exceptions. Indeed, some serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. Species-wise, B. halodurans, B. subtilis, and B. anthracis formed specific branches, whereas the other four species, all in the B. cereus sensu lato group, B. mycoides, B. weihenstephanensis, B. cereus, and B. thuringiensis, did not form four specific clusters as might have been expected. Rather, strains from any of these four species were placed side by side with strains from the other species. In the B. cereus sensu lato group, B. anthracis excepted, the distribution of strains was not species specific.

SUBMITTER: Xu D

PROVIDER: S-EPMC1489342 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Sequence diversity of the Bacillus thuringiensis and B. cereus sensu lato flagellin (H antigen) protein: comparison with H serotype diversity.

Xu Dong D Côté Jean-Charles JC

Applied and environmental microbiology 20060701 7

We set out to analyze the sequence diversity of the Bacillus thuringiensis flagellin (H antigen [Hag]) protein and compare it with H serotype diversity. Some other Bacillus cereus sensu lato species and strains were added for comparison. The internal sequences of the flagellin (hag) alleles from 80 Bacillus thuringiensis strains and 16 strains from the B. cereus sensu lato group were amplified and cloned, and their nucleotide sequences were determined and translated into amino acids. The flagell ...[more]

PMID: 16820457

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Project description:In Bacillus thuringiensis, the hag gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Specific flagellin amino acid sequences have been correlated to specific B. thuringiensis H serotypes, H1 to H67. Ten H serotypes, however, contain three or more antigenic subfactors, labeled a, b, c, d, or e, and have been subdivided into 23 serovars. In the present study, we set out to analyze the sequence diversity of flagellins among serovars from the same H serotypes. We studied the hag genes in 39 B. thuringiensis strains representing the 23 serovars from the 10 H serotypes mentioned above. A serovar and a biovar from an 11th H serotype were also included. The hag genes were amplified and cloned and their nucleotide sequences were determined and translated into amino acid sequences, or the sequences were retrieved directly from GenBank when available. Strains of the H3 serotype contained two or three copies of the fla gene, an ortholog of the hag gene. Strains of the H6 serotype contained three copies. Strains of all other H serotypes each contained a single copy of the hag gene. Alignments of amino acid sequences from all copies in all strains of the H3 serotype revealed short signature sequences, GGAG and SGG, GPDPDDAVKNLT, and DITTTK, that appeared to be specific to the H3c, H3d, and H3e antigenic subfactors, respectively. Similar short signature sequences, GDIT, AFIK, TSAGKA, and SAPSKG, were revealed for H8b, H8c, H20b, and H20c, respectively. Amino acid sequences in the flagellin central variable region were highly conserved among serovars of the H3, H5, H11, and H20 serotypes and much more divergent among serovars of the H4, H10, H18, H24, and H28 serotypes. Two bootstrapped neighbor-joining trees were respectively generated from the alignments of the amino acid sequences translated from all copies of the hag genes in the B. thuringiensis strains of the H3 and H6 serotypes. Sequence identities and relationships were revealed. A third bootstrapped neighbor-joining tree was generated, this one from the alignment of the flagellin amino acid sequences from all the B. thuringiensis strains in the study. Eight clusters, I to VIII, were revealed. Although most clusters contained strains and serovars from the same H serotype, clusters VII and VIII contained serovars from different H serotypes.

Project description:BackgroundBacillus cereus sensu lato (s. l.) is an ecologically diverse bacterial group of medical and agricultural significance. In this study, I use publicly available genomes and novel bioinformatic workflows to characterize the B. cereus s. l. pan-genome and perform the largest phylogenetic and population genetic analyses of this group to date in terms of the number of genes and taxa included. With these fundamental data in hand, I identify genes associated with particular phenotypic traits (i.e., "pan-GWAS" analysis), and quantify the degree to which taxa sharing common attributes are phylogenetically clustered.MethodsA rapid k-mer based approach (Mash) was used to create reduced representations of selected Bacillus genomes, and a fast distance-based phylogenetic analysis of this data (FastME) was performed to determine which species should be included in B. cereus s. l. The complete genomes of eight B. cereus s. l. species were annotated de novo with Prokka, and these annotations were used by Roary to produce the B. cereus s. l. pan-genome. Scoary was used to associate gene presence and absence patterns with various phenotypes. The orthologous protein sequence clusters produced by Roary were filtered and used to build HaMStR databases of gene models that were used in turn to construct phylogenetic data matrices. Phylogenetic analyses used RAxML, DendroPy, ClonalFrameML, PAUP*, and SplitsTree. Bayesian model-based population genetic analysis assigned taxa to clusters using hierBAPS. The genealogical sorting index was used to quantify the phylogenetic clustering of taxa sharing common attributes.ResultsThe B. cereus s. l. pan-genome currently consists of ≈60,000 genes, ≈600 of which are "core" (common to at least 99% of taxa sampled). Pan-GWAS analysis revealed genes associated with phenotypes such as isolation source, oxygen requirement, and ability to cause diseases such as anthrax or food poisoning. Extensive phylogenetic analyses using an unprecedented amount of data produced phylogenies that were largely concordant with each other and with previous studies. Phylogenetic support as measured by bootstrap probabilities increased markedly when all suitable pan-genome data was included in phylogenetic analyses, as opposed to when only core genes were used. Bayesian population genetic analysis recommended subdividing the three major clades of B. cereus s. l. into nine clusters. Taxa sharing common traits and species designations exhibited varying degrees of phylogenetic clustering.ConclusionsAll phylogenetic analyses recapitulated two previously used classification systems, and taxa were consistently assigned to the same major clade and group. By including accessory genes from the pan-genome in the phylogenetic analyses, I produced an exceptionally well-supported phylogeny of 114 complete B. cereus s. l. genomes. The best-performing methods were used to produce a phylogeny of all 498 publicly available B. cereus s. l. genomes, which was in turn used to compare three different classification systems and to test the monophyly status of various B. cereus s. l. species. The majority of the methodology used in this study is generic and could be leveraged to produce pan-genome estimates and similarly robust phylogenetic hypotheses for other bacterial groups.

Dataset Information

Sequence diversity of the Bacillus thuringiensis and B. cereus sensu lato flagellin (H antigen) protein: comparison with H serotype diversity.

Publications

Sequence diversity of the Bacillus thuringiensis and B. cereus sensu lato flagellin (H antigen) protein: comparison with H serotype diversity.

Similar Datasets

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