Table 1.
Bacterial strains and plasmids.
Figure 1.
Response of B. fragilis to Pi limitation.
(A) Growth curve in DMM supplemented with varying concentrations of KH2PO4. B. fragilis strain YCH46 was grown anaerobically in DMM supplemented with 6.6 mM, 0.066 mM, or 0.0066 mM of KH2PO4 at 37°C for 20 h. Optical densities of the cultures at 600 nm were measured over time. Data presented are the mean ± standard deviations of three independent cultures. (B) qPCR analysis of BF1575, BF1576, and BF2756. Total RNA was collected at mid-logarithmic phase (indicated by † in panel A). Expression levels of BF1575 (phoR homolog), BF1576 (phoB homolog), and BF2756 (pstC homolog) in wild type B. fragilis strain YCH46 were measured and normalized with transcriptional levels of rpoD. The transcriptional level of each gene under Pi limitation (0.066 mM or 0.0066 mM) is shown relative to that in DMM supplemented with 6.6 mM KH2PO4 (Pi-rich media).
Figure 2.
Effect of BF1576 deletion on the growth in Pi-limiting media.
(A) Construction of ΔBF1576 and ΔBF2185 strains. Internal deletion of each gene was confirmed by PCR using a primer pair encompassing each deletion site. W, wild type B. fragilis; M, mutant form of indicated gene; S, 100-bp ladder size markers. (B) Growth of wild type (WT), ΔBF1576 and ΔBF2185 strains under Pi-rich and Pi-limiting conditions. WT (diamonds), ΔBF1576 (squares), and ΔBF2185 (triangles) strains of B. fragilis were grown anaerobically in DMM supplemented with 6.6 mM (closed symbols) or 0.0066 mM (open symbols) of KH2PO4. Growth was measured at OD600. Data presented are the means ± standard deviations of triplicate cultures. **Significantly different from wild type (p<0.01), *Significantly different from wild type (p<0.05).
Figure 3.
Repletion of BF1576 by plasmid complementation.
B. fragilis wild type strains harboring pLYL05-Exp and the ΔBF1576 strain harboring pLYL05-Exp or pLYL1576 were grown anaerobically in a limiting amount of Pi (0.0066 mM of KH2PO4) in DMM supplemented with 50 µg/ml of cefoxitin. Growth was measured at OD600. Data were calculated from triplicate cultures and expressed as means ± standard deviations. **Significantly different from the ΔBF1576 strain harboring pLYL05-Exp (p<0.01), *Significantly different from the ΔBF1576 strain harboring pLYL05-Exp (p<0.05).
Figure 4.
Induction of pstC expression in response to Pi limitation.
Wild type, ΔBF1576, and ΔBF2185 strains of B. fragilis were cultured in DMM supplemented with varying concentrations of Pi as indicated. Total RNA was extracted from mid-logarithmic phase cultures, and pstC expression levels were compared by qPCR. The transcriptional level of pstC was normalized with that of rpoD and shown relative to that of a wild type strain in DMM supplemented with 6.6 mM KH2PO4 (Pi-rich media). Black column: wild type; gray column: ΔBF1576; white column: ΔBF2185. **Significantly different from the wild type strain (p<0.01).
Figure 5.
(A) Expression and purification of recombinant B. fragilis PhoB in E. coli. M, molecular size markers. (B) Binding of PhoB to pstC promoter. Recombinant PhoB (1.5 or 15 µM of final concentration) was mixed with the amplified promoter region of pstC or internal region of BF3397, and their interaction was evaluated by electrophoretic motility shift assay. Zero denotes that no protein was added to the reaction mixture. B: PhoB-bound probes; F: free probes.
Figure 6.
PhoB-dependent genes in B. fragilis.
(A) Classification of genes whose expression levels were altered more than 4-fold under Pi limitation (0.0066 mM of KH2PO4) in wild type and ΔphoB mutant strains, compared to levels in wild type in the presence of sufficient Pi (6.6 mM of KH2PO4). Left and right circles indicate the differentially expressed genes of wild type and ΔphoB mutant strains with expressions that were altered over 4-fold under Pi limitation. Group 1: includes 245 PhoB-dependent Pi-response genes. Group 3∶217 PhoB-dependent genes unrelated to Pi response. Group 2 contains two subgroups. Group 2a: 276 PhoB-independent Pi-response genes (fold change in expression in response to Pi-limitation was similar between wild type and ΔphoB mutant strains); Group 2b: 123 PhoB-dependent Pi response genes (the extent of change in expression in response to Pi-limitation was different >2-fold between wild type and ΔphoB mutant strains). (B) COG classification of PhoB-dependent (black bar) and PhoB-independent (white bar) genes that are differentially expressed during Pi starvation. The percentage of genes classified into each COG category is relative to the total number of genes in each group. Fifty-five percent of the PhoB-dependent (585 genes) and 60% of the PhoB-independent (276 genes) groups could not be assigned to a COG category (that data are not included in this figure).
Table 2.
Representative list of differentially expressed genes in wild type and ΔphoB B. fragilis strains in Pi-limiting conditions.
Figure 7.
qPCR verification of differentially expressed PhoB-dependent genes identified by microarray analysis.
The transcriptional levels of representative genes in wild type (closed column) and phoB deletion mutant (open column) under Pi limitation (0.0066 mM of KH2PO4) is shown in relation to those in wild type under Pi rich condition (6.6 mM of KH2PO4). Annotation of each gene is as listed in Table 2: BF1576 (PhoB), BF1575 (PhoR), BF2185 (PstC), BF1828 (UpbY, product of the first gene product in PS B), BF2585 (UpbY, product of the first gene product in PS E), BF3377 (DnaJ), BF1205 (endopeptidase Clp), BF3395 (GroEL), BF3396 (GroES), BF2645 (polyphoaphate kinase), BF1576 (alkaline phosphatase), and BF2091 (cation efflux system protein). **Significantly different from the wild type strain, *Significantly different from the wild type strain (p<0.05).
Table 3.
Peritoneal abscess formation by B. fragilis strains.
Table 4.
Viable B. fragilis cell numbers in the peritoneal abscesses a).
Figure 8.
Competitive growth assay of wild type and ΔphoB B.
fragilis strains in vitro (A) and in vivo (B). These strains were equally mixed and inoculated into GAM broth, mice intestines or the peritoneal cavity. After periodical sampling from liquid culture, feces and abscesses, samples were spread on GAM agar plates, and the population levels of wild type and ΔphoB B. fragilis strains were evaluated by discriminating colony PCR. The data were expressed as wild type/ΔphoB mutant ratios. The ratios of wild type and phoB mutant strains in the intestine and abscess were compared by Chi-square test.