Fig 1.
A linear representation of the pBC210 capsule locus and the function of the encoded proteins.
The original locus numbers for the genes (BCE_G9241_pBC218_00XX) are above the genes. The nomenclature that is consistent with Wzy-dependent capsules from S. pneumoniae is below each gene. The protein function for each gene product, as initially annotated or determined by homology, is listed.
Fig 2.
TS capsule locus has two potential operons.
RNA was isolated from G9241 and used to synthesize cDNA. To determine whether transcripts included multiple genes, the cDNA was used to assay for the junctions between the genes (A) by PCR (B).
Fig 3.
5’ RACE results for wchJ and wzd.
The transcriptional start sites, highlighted in gray, determined by 5’RACE are 251 bp for wchJ (A) and 172 bp for wzd from the ATG start codon, in bold. Potential -10 and -35 boxes from these start sites are underlined.
Fig 4.
Promoter activity for wchJ, wzg, and wzd.
Fragments proximal to the start codon for wchJ (160, 349, and 607 bp), wzg (871 bp), and wzd (40, 310, and 502 bp) were cloned before the promoterless lacZ in pHT304-18Z in strain ΔwchAΔhasACB. The cells were grown under capsule-inducing conditions for 4 h, and the β-galactosidase activity was measured. The bars indicate the mean of four experiments and the error bars the standard deviation. *Significantly different from empty vector control as determined by one-way ANOVA with Dunnett’s multiple comparisons test (P < 0.0001).
Fig 5.
Capsule production by capsule mutants.
Maneval stain of cells from 24 h capsule-inducing cultures. G9241 (A, B), ΔhasACB (C, D), ΔwchA (E, F), Δwzy (G, H), ΔwchJ (I, J), Δwzg (K, L), and ΔwchAΔhasACB (M) cells were either untreated (A, C, E, G, I, K, M) or treated with 200 U of hyaluronidase (B, D, F, H, J, L) prior to application of Maneval stain. Cells were visualized with oil immersion at 1,000x magnification.
Fig 6.
The HA capsule was extracted from the cell surface with chloroform from 2 ml G9241, ΔwchA, Δwzg, ΔhasACB, ΔwchAΔhasACB, and pBCXO1-/pBC210- cells at OD600 0.34–0.36. The quantity of HA capsule in the extract was determined by Stains-all assay and comparison against a standard curve of purified HA. Three strains (ΔhasACB, ΔwchAΔhasACB and pBCXO1-/pBC210-) had no detectable HA by this assay. Only ΔhasACB values are shown on the graph. The bars indicate the mean of four experiments and the error bars the standard deviation. * Significantly different (P < 0.0003) from G9241 and Δwzg as determined by one-way ANOVA with Tukey’s multiple comparisons test.
Fig 7.
Median survival of A/J and C57BL/6 mice after infection with spores.
(A) Day that A/J mice succumbed to infection after inoculation with 102 spores s.c. or 104 spores i.n. of G9241, ΔwchA, or ΔhasACB. (B) Day that C57BL/6 mice succumbed to infection after inoculation with 103 spores s.c. or 106 spores i.n. of G9241, ΔwchA, or ΔhasACB. The horizontal black line represents the median, and symbols on day 14 are mice that survived the infection. * Significantly different from G9241 spores at same dose as determined by Kruskal-Wallis with Dunn’s multiple comparisons test.
Table 1.
LD50a of spores.
Table 2.
Bacterial strains and plasmids.