Twenty-eight divergent polysaccharide loci specifying within- and amongst-strain capsule diversity in three strains of Bacteroides fragilis

Abstract

Comparison of the complete genome sequence of Bacteroides fragilis 638R, originally isolated in the USA, was made with two previously sequenced strains isolated in the UK (NCTC 9343) and Japan (YCH46). The presence of 10 loci containing genes associated with polysaccharide (PS) biosynthesis, each including a putative Wzx flippase and Wzy polymerase, was confirmed in all three strains, despite a lack of cross-reactivity between NCTC 9343 and 638R surface PS-specific antibodies by immunolabelling and microscopy. Genomic comparisons revealed an exceptional level of PS biosynthesis locus diversity. Of the 10 divergent PS-associated loci apparent in each strain, none is similar between NCTC 9343 and 638R. YCH46 shares one locus with NCTC 9343, confirmed by mAb labelling, and a second different locus with 638R, making a total of 28 divergent PS biosynthesis loci amongst the three strains. The lack of expression of the phase-variable large capsule (LC) in strain 638R, observed in NCTC 9343, is likely to be due to a point mutation that generates a stop codon within a putative initiating glycosyltransferase, necessary for the expression of the LC in NCTC 9343. Other major sequence differences were observed to arise from different numbers and variety of inserted extra-chromosomal elements, in particular prophages. Extensive horizontal gene transfer has occurred within these strains, despite the presence of a significant number of divergent DNA restriction and modification systems that act to prevent acquisition of foreign DNA. The level of amongst-strain diversity in PS biosynthesis loci is unprecedented.

Fig. 1.

Electron micrographs of ultrathin sections of B. fragilis. (a) NCTC 9343 MC-enriched population; (b) B. fragilis 638R. Note the presence of an electron-dense layer (black arrow) outwith the outer membrane (white arrow) in both strains. Bar, 200 nm.

Fig. 2.

Four-way ACT genome comparison of within-strain variable MC-associated loci. (a) PSA/2, (b) PSB/4, (c) PSC/8, (d) PSD/5, (e) PSE/1, (f) PSF/6, (g) PSG/7, (h) PSH/3 of B. fragilis. (i) NCTC 9343, (ii) 638R, (iii)YCH46. Red or yellow coloration between sequences indicates sequence identity, no colour indicates divergent sequence, dark blue indicates an inverted promoter region. Line, conserved transcriptional regulator region. Note: sequence identity was only evident between strains NCTC 9343 and YCH46 at locus PSB/4, and strains 638R and YCH46 at locus PSC/8.

Fig. 2.

Four-way ACT genome comparison of within-strain variable MC-associated loci. (a) PSA/2, (b) PSB/4, (c) PSC/8, (d) PSD/5, (e) PSE/1, (f) PSF/6, (g) PSG/7, (h) PSH/3 of B. fragilis. (i) NCTC 9343, (ii) 638R, (iii)YCH46. Red or yellow coloration between sequences indicates sequence identity, no colour indicates divergent sequence, dark blue indicates an inverted promoter region. Line, conserved transcriptional regulator region. Note: sequence identity was only evident between strains NCTC 9343 and YCH46 at locus PSB/4, and strains 638R and YCH46 at locus PSC/8.

Fig. 3.

Immunofluorescence microscopy of B. fragilis strains. (i) NCTC 9343, (ii) 638R and (iii) YCH46 reacted with two representative mAbs, (A) and (B), raised against B. fragilis 638R, followed by anti-mouse FITC-conjugated secondary antibody, counter-stained with Evans blue. Exposure for fluorescein (green) image taken after shift of microscope slide (×100 objective). Note the within-strain variation in epitope expression in strain 638R and the lack of cross-reactivity with NCTC 9343 and YCH46.

Fig. 4.

Immunofluorescence microscopy of B. fragilis strains. (i) NCTC 9343, (ii) 638R and (iii) YCH46 reacted with mAbs raised against NCTC 9343 followed by anti-mouse FITC-conjugated secondary antibody, counter-stained with Evans blue. Exposure for fluorescein (green) image taken after shift of microscope slide (×100 objective). QUBF6 reacts with PSE/1, and QUBF19 reacts with PSB/4.

Fig. 5.

Light micrographs of eosin/carbol fuchsin negative stain (a) and immunofluorescence micrographs of bacteria reacted with anti-NCTC 9343 SC mAb followed by anti-mouse FITC-conjugated secondary antibody counter-stained with Evans blue (b). (i) B. fragilis NCTC 9343, (ii) 638R and (iii) YCH46 sampled from the 20–40 % interface of a Percoll step density gradient after centrifugation (×100 objective). (b, i) The same microscope field illustrating antibody labelling (top) and Evans blue counter stain (bottom); (b, ii) and (b, iii), combined fields. A small clear area can be observed surrounding the cells of B. fragilis NCTC 9343 and YCH46, indicating the expression of an SC. The capsule surrounding B. fragilis 638R appears more irregular. Note that 638R and YCH46 SC populations are not enriched for expression of the NCTC 9343 SC-associated epitope.

Fig. 6.

PCR products obtained from genomic DNA extracted from Percoll-purified MC, SC, LC B. fragilis NCTC 9343 populations, and mixed-capsule populations from B. fragilis 638R and YCH46, using primer pairs designed to amplify a 1.85 kb product comprising the complete BF2782 gene with its promoter in the ‘ON’ orientation (lanes 2–6) and a 1.75 kb product comprising the complete BF2782 gene with its promoter in the ‘OFF’ orientation (lanes 8–12). Lanes: 2 and 8, NCTC 9343 LC PCR; 3 and 9, NCTC 9343 SC PCR; 4 and 10, NCTC 9343 MC PCR; 5 and 11, 638R PCR; 6 and 12, YCH46 PCR; 1 and 13, 1 kb Plus DNA ladder (Invitrogen). Lane 7 is empty. Note the lack of product with the OFF orientation primers in the NCTC 9343 LC population, lane 8.

Fig. 7.

Four-way ACT genome comparison of PS-associated loci. (a) PSI/9: black arrows, inserted conjugative transposon-associated regions in YCH46; (b) PSK/11 of B. fragilis. (i) NCTC 9343, (ii) 638R, (iii) YCH46. Red or yellow coloration between sequences indicates sequence identity, no colour indicates divergent sequence.

Fig. 8.

Comparison of invertible regions R and S (arrows), active in the NCTC 9343 shotgun sequence of B. fragilis. (i) NCTC 9343, (ii) 638R, (iii) YCH46. Red coloration between sequences indicates sequence identity, no colour indicates divergent sequence, dark blue/yellow indicates inverted sequences. Note the sequence identity of inverted sequences in R and S, which suggests that these two regions form a complex intergenic shufflon.