Molecular evolution of the pathogenicity island of enterotoxigenic Bacteroides fragilis strains

Abstract

Enterotoxigenic Bacteroides fragilis (ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic B. fragilis (NTBF) strains, we found that bft and a second metalloprotease gene (mpII) are contained in an approximately 6-kb pathogenicity island (termed B. fragilis pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (bfmA and bfmB) organized in one operon and a putative origin of transfer (oriT) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (bfmC) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from Escherichia coli. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that bfmB and bfmC are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the bfmB and bfmC stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the B. fragilis chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.

FIG. 1

Partial restriction map of the region flanking bft in ETBF 86-5443-2-2. Arrow shows bft and the direction of its transcription. Probes used in the colony blot hybridizations are shown in the black bars. The results of hybridization of these probes to B. fragilis strains (Table 2) are indicated beneath each probe. The 6-kb region spanning probes G, Tox, H, and B (the striped bar) is the specific locus present only in ETBF strains (BfPAI). Only the restriction sites used for the construction of the probes are shown in the figure.

FIG. 2

Partial restriction map of the region flanking bft in ETBF 86-5443-2-2. The thicker bar (both solid and striped) shows the region sequenced, and the striped bar shows the BfPAI. (a) ORFs detected in the BfPAI and flanking regions. Arrows show the locations of the ORFs and the direction of their transcription. (b) Relative positions of the primers and the expected PCR products to determine the integration site of the BfPAI. Only the restriction sites important for the construction of the probes (Fig. 1) are shown in the figure.

FIG. 3

Putative mobilization region of the BfPAI. (a) Comparison between the putative mobilization region of BfPAI and the mobilization region of the clindamycin resistance plasmid pBFTM10. Arrows show the direction of gene transcription. (b) Nucleotide sequence (5′ to 3′) of the three IRs. (c) Nick site present in the putative oriT gene of the BfPAI.

FIG. 4

Alignment of the region surrounding the ATP/GTP-binding site motif A in BfmC, TrsK (pMRCO1), TrsK (pGO1), TraD (R100), and TraD (F). Amino acids similar to those of the BfmC sequence are in capital letters and boldface. The ATP/GTP-binding site motif A in BfmC is underlined.

FIG. 5

G+C content across the BfPAI and its flanking regions. Arrows show the location of genes and the direction of their transcription. Asterisks show the sharp declines in G+C content immediately upstream of the designated genes.

FIG. 6

PCR analysis of the left and right junctions of BfPAI. (A) Screening for the integration site by using primers P1T7 and P1T3 (see Fig. 2b). PCR yielded a product (1.6 kb) only in NTBF strains with pattern III (strain I-1345) and IV (strain LM20). Lanes: 1, 1-kb ladder molecular weight marker; 2, ETBF 86-5443-2-2 (pattern I, positive control); 3, NTBF 077225-2 (pattern II); 4, NTBF I-1345 (pattern III); 5, NTBF LM-20 (pattern IV); 6, NTBF LM-12 (pattern V). (B) Screening for the left junction by using primers P1T7 and P1T7-1 (see Fig. 2b). PCR yielded a similar-sized fragment (1.3 kb) in six ETBF strains (pattern I) and NTBF LM12 (pattern V). Lanes: 1, 1-kb ladder molecular weight marker; 2, ETBF 86-5443-2-2 (pattern I); 3, ETBF VPI 13784 (pattern I); 4, ETBF J-38-1 (pattern I); 5, ETBF DS-64 (pattern I); 6, ETBF DS-233 (pattern I); 7, ETBF DS-49 (pattern I); 8, NTBF 077225-2 (pattern II); 9, NTBF I-1345 (pattern III); 10, NTBF LM-20 (pattern IV); 11, NTBF LM-12 (pattern V). (C) Screening for the right junction by using primers P1T3-1 and P1T3 (see Fig. 2b). PCR yielded a similar-sized fragment (1.1 kb) in all six ETBF strains (pattern I). Lanes are as described for panel B.

FIG. 7

Comparison of the nucleotide-amino acid sequences of the region flanking the BfPAI of ETBF strain 86-5443-2-2 with the equivalent region of NTBF strain I-1345. Nucleotide sequences belonging to bfmB and bfmC are given in capital letters. Arrows show direction of gene transcription, and stop codons are indicated by asterisks. The 16-bp sequence lost in ETBF is given in lowercase letters.