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.

FIG. 1.

Map of the Bacillus thuringiensis serovar konkukian flagellin (hag) gene. The number 1 refers to the first nucleotide of the hag gene coding region. Arrowheads indicate the orientations and positions of the three primer pairs used for amplification. They are F1 and R1 at nucleotide positions 41 to 61 and 1068 to 1048, respectively; F2 and R2 (positions −13 to 7 and 1117 to 1098, respectively); and F3 and R3 (positions 16 to 35 and 1043 to 1062, respectively). C1 and C2 correspond to conserved regions 1 and 2, respectively. The shaded box corresponds to the variable, V, region between nucleotides positions 394 and 849.

FIG. 2.

Agarose gel electrophoresis of hag gene amplification products in selected B. thuringiensis serovars using the F1/R1 primer pair. Lane 1, B. thuringiensis serovar cameroun; lane 2, B. thuringiensis serovar leesis; lane 3, B. thuringiensis serovar seoulensis; lane 4, B. thuringiensis serovar oswaldocruzi; lane 5, B. thuringiensis serovar sooncheon; lane 6, B. thuringiensis serovar higo; lane 7, B. thuringiensis serovar roskildiensis; lane 8, B. thuringiensis serovar wratislaviensis; lane 9, B. thuringiensis serovar muju; lane 10, B. thuringiensis serovar navarrensis; lane 11, B. thuringiensis serovar xiaguangiensis; lane 12, B. thuringiensis serovar kim; lane 13, B. thuringiensis serovar poloniensis; lane 14, B. thuringiensis serovar palmanyolensis; lane 15, B. thuringiensis serovar rongseni; lane 16, B. thuringiensis serovar argentinensis; lane 17, B. thuringiensis serovar iberica; lane 18, B. thuringiensis serovar pingluosensis; lane 19, B. thuringiensis serovar sylvestriensis; lane 20, B. thuringiensis serovar zhaodongensis; lane 21, B. thuringiensis serovar bolivia; lane 22, B. thuringiensis serovar azorensis; lane 23, B. thuringiensis serovar pulsiensis; lane 24, B. thuringiensis serovar graciosensis; lane 25, B. thuringiensis serovar vazensis. No amplification products were detected with the F1/R1 primer pair in lanes 6, 9 and 22. Molecular weight markers are shown in lane M1, lambda DNA digested with HindIII, and lane M2, a 100-bp ladder.

FIG. 3.

Bootstrapped neighbor-joining tree of Bacillus thuringiensis (Bt) generated from the alignment of flagellin amino acid sequences. Clusters are indicated in roman numerals. Bootstrap values higher than 50% are indicated. The horizontal bar represents 10% differences in amino acids. Ba, B. anthracis; Bw, B. weihenstephanensis; Bm, B. mycoides; Bc, B. cereus.

FIG. 4.

Bootstrapped neighbor-joining tree of Bacillus thuringiensis (Bt) generated from the alignment of the variable central region (V region) flagellin amino acid sequences. Clusters are indicated in roman numerals followed by a prime sign to distinguish them from the clusters in Fig. 3. Bootstrap values higher than 50% are indicated. The horizontal bar represents 10% differences in amino acids. Bc, B. cereus; Bw, B. weihenstephanensis; Bm, B. mycoides; Ba, B. anthracis.