Trans locus inhibitors limit concomitant polysaccharide synthesis in the human gut symbiont Bacteroides fragilis

Abstract

Bacteroides is an abundant genus of bacteria of the human intestinal microbiota. Bacteroides species synthesize a large number of capsular polysaccharides (PS), a biological property not shared with closely related oral species, suggesting importance for intestinal survival. Bacteroides fragilis, for example, synthesizes eight capsular polysaccharides per strain, each of which phase varies via inversion of the promoters located upstream of seven of the eight polysaccharide biosynthesis operons. In a single cell, many of these polysaccharide loci promoters can be simultaneously oriented on for transcription of the downstream biosynthesis operons. Here, we demonstrate that despite the promoter orientations, concomitant transcription of multiple polysaccharide loci within a cell is inhibited. The proteins encoded by the second gene of each of these eight loci, collectively designated the UpxZ proteins, inhibit the synthesis of heterologous polysaccharides. These unique proteins interfere with the ability of UpxY proteins encoded by other polysaccharide loci to function in transcriptional antitermination of their respective operon. The eight UpxZs have different inhibitory spectra, thus establishing a hierarchical regulatory network for polysaccharide synthesis. Limitation of concurrent polysaccharide synthesis strongly suggests that these bacteria evolved this property as an evasion-type mechanism to avoid killing by polysaccharide-targeting factors in the ecosystem.

Fig. 1.

UpxZs inhibit synthesis of subsets of polysaccharides. (A) Genetic organization of the polysaccharide biosynthesis operons demonstrating the invertible promoter (P) flanked by inverted repeats (■). upxY and upxZ are the first two genes of each operon, and are followed by 9–20 genes involved in polysaccharide synthesis. (B) Western blot analysis of synthesis of PSE and PSH from both wild type and the ΔupeZ and ΔuphZ mutants. (C) Overnight broth cultures showing the phenotypes of wild-type bacteria containing each of the upxZ genes in trans. Aggregated bacteria are visible at the bottom of many cultures. (D) Western blot analysis of the synthesis of each of the eight capsular polysaccharides from wild-type bacteria and wild type with each of the eight upxZ genes constitutively expressed from a plasmid. Cultures were mixed before analysis so that both aggregated and suspended bacteria were included. (E) Analysis of the orientations of the PSG promoter from bacteria in suspension or settled from the broth culture of the upgZ transconjugant. (F) Northern blot analysis of the PSA transcript from wild type and transconjugants containing various PSA inhibitory and noninhibitory upxZs.

Fig. 2.

Promoter orientation does not correlate with PS synthesis in B. fragilis mpi mutants due to UpxZ inhibition of heterologus PS loci transcription. (A) Schematic of the locked promoter orientations of mpiM44. PSA, PSE, and PSC promoters are locked on but the mutant expresses only PSA. (B) Western immunoblot analysis of PSA, PSE, and PSC synthesis in mpiM44, and the same mutant with successive deletions of upaZ and upeZ. (C) Northern blot analysis of PSA, PSE, and PSC transcript in mpiM44, mpiM44ΔupaZ, and mpiM44ΔupaZΔupeZ.

Fig. 3.

XylE transcriptional fusion analyses of the inhibitory action of UpaZ on PSE locus transcription. (A) Schematic of the PSE regions cloned into the promoterless xylE reporter plasmid. Each clone was amplified with the same forward primer (F) located just inside the upstream IR and contains the promoter in the on orientation and extending 44 bp (clone 8), 135 bp (clone 10), and 229 bp (clone 11) downstream of the transcriptional start site. (B) Northern blot analysis of xylE transcription when each clone is placed in the mpiM44 or mpiM44ΔupaZ background.

Fig. 4.

UpeY variants overcome UpaZ-mediated inhibition of PSE synthesis in mpiM44. (A) Schematics of the upeY and upaY wild-type and hybrid constructs added in trans to mpiM44. pLEC212 contains wild-type upeY, including its immediate 27-bp upstream region, cloned into expression vector pFD340. Transcription is driven by a vector-borne promoter (thin arrow) located 245 bp upstream of upeY. pMCL138 is the same as pLEC212 except that a strong promoter (thick arrow) is situated 51 bp upstream of upeY. pMLC55 contains wild-type upaY, including its 38-bp immediate upstream region, cloned into expression vector pFD340. pMCL74 is the same as pMCL55 except that a 51-bp region of upaY (corresponding to amino acids 62–78) was replaced with a 51-bp region of upeY (also corresponding to amino acids 62–78). This small substitution confers UpeY activity to this hybrid (7). Both pMCL116 and pMCL115 are additional hybrids of upeY and upaY and both include the 51-bp region of upeY sufficient to confer UpeY activity to UpaY. In addition, pMCL116 contains 38-bp upstream upaY through the first 183 bp of upaY with the remainder of the gene upeY, whereas pMCL115 contains 27-bp upstream upeY through the first 234 nt of upeY with the remainder of the hybrid gene derived from upaY. (B) Western blot analysis of PSE synthesis from whole cell lysates demonstrating that overexpression of UpeY overcomes inhibition of PSE synthesis in mpiM44. (C) Western blot analysis of PSE synthesis from whole cell lysates demonstrating that upaY–upeY hybrids restore PSE synthesis to mpiM44.