A family of transcriptional antitermination factors necessary for synthesis of the capsular polysaccharides of Bacteroides fragilis

Abstract

A single strain of Bacteroides fragilis synthesizes eight distinct capsular polysaccharides, designated PSA to PSH. These polysaccharides are synthesized by-products encoded by eight separate polysaccharide biosynthesis loci. The genetic architecture of each of these eight loci is similar, including the fact that the first gene of each locus is a paralog of the first gene of each of the other PS loci. These proteins are designated the UpxY family, where x is replaced by a to h, depending upon the polysaccharide locus from which it is produced. Mutational analysis of three separate upxY genes demonstrated that they are necessary and specific for transcription of their respective polysaccharide biosynthesis operon and that they function in trans. Transcriptional reporter constructs, reverse transcriptase PCR, and deletion analysis demonstrated that the UpxYs do not affect initiation of transcription, but rather prevent premature transcriptional termination within the 5' untranslated region between the promoter and the upxY gene. The UpxYs have conserved motifs that are present in NusG and NusG-like proteins. Mutation of two conserved residues within the conserved KOW motif abrogated UpaY activity, further confirming that these proteins belong to the NusG-like (NusG(SP)) family. Alignment of highly similar UpxYs led to the identification of a small region of these proteins predicted to confer specificity for their respective loci. Construction of an upaY-upeY hybrid that produced a protein in which a 17-amino-acid segment of UpaY was changed to that of UpeY altered UpaY's specificity, as it was now able to function in transcriptional antitermination of the PSE biosynthesis operon.

FIG. 1.

UpxYs are necessary for transcription of the respective PS operons. (A) Common genetic organization of the PS biosynthesis loci. The filled boxes represent IRs. Arrows represent the invertible promoter between the IRs. (B) Western immunoblots demonstrating that deletion of upaY, upeY, or uphY results in abrogation of the polysaccharide encoded by the respective locus but not of heterologous polysaccharides. When upxYs are added in trans to their respective mutant, PS synthesis is restored. (C) Northern analysis of the PSA transcript by probing with upaZ showed that UpaY is necessary for transcription of the operon. (D) Western immunoblot analysis of PSE synthesis from a mutant where the invertible PSE promoter was locked in the on orientation and its corresponding upeY deletion mutant and corresponding mutant with upeY were provided in trans. (E) Northern blot analysis of the same strains in panel C, showing that UpeY, like UpaY, is also necessary for transcription of its respective PS locus.

FIG. 2.

UpxYs require the KOW motif for activity. (A) Boxshade analysis of portions of the eight UpxY proteins, the E. coli and B. fragilis NusG proteins, and RfaH, showing the KOW motif (boxed). The G residues that were converted to A in UpaY are numbered. (B) Western immunoblot analysis of PSA expression from the wild type and ΔupaY mutant with wild-type upaY and various mutant upaY genes added in trans. Mutation of both G122 and G130 to A resulted in abrogation of UpaY activity.

FIG. 3.

UpxYs prevent premature transcriptional termination. (A) Sequence of the 5′ region of the PSA operon with demarcation of the exact nucleotides included in constructs and deleted from mutants. The downstream IR sequence is boxed. A primer 143 bp upstream of the underlined PSA promoter was used with each of the seven numbered downstream primers (arrows) to amplify regions cloned into the xylE transcriptional fusion reporter plasmid pLEC23. Each transcriptional fusion plasmid was added in trans to the wild type and the ΔupaY mutant, and the levels of XylE activity were quantified (Table 1). In addition, three areas that were deleted from the wild-type chromosome are indicated. (B) Diagram of the 5′ region of the PSA biosynthesis operon, showing primers that were used for RT-PCR analysis (arrows). Primers labeled 4, 5, and 6 correspond to the primers used for xylE constructs shown in panel A. Filled black boxes represent the inverted repeats that flank the PSA promoter. Below the map are the ethidium bromide-stained gels that show the results of each RT-PCR for the indicated regions. The four samples loaded onto each gel from left to right are the RT-PCR analysis of RNA from the wild-type (WT; RT added), ΔupaY (RT added), wild type (no RT added), and ΔupaY (no RT added). (C) Sequence of the 5′ region of the PSE operon, demarcating the exact nucleotides included in transcriptional reporter constructs. A primer 110 bp upstream of the underlined PSE promoter was used with each of the four numbered downstream primers (arrows) to amplify regions that were cloned into reporter plasmid pLEC23. Each transcriptional fusion plasmid was added in trans to the wild type and the ΔupeY mutant, and the levels of XylE activity were quantified (Table 1).

FIG. 4.

The 5′ UTR is necessary for transcription of the PSA operon. (A) Western immunoblot analysis of PSA synthesis from the wild type and the three UTR deletion mutants illustrated in Fig. 3A. (B) Northern blot analysis of the same three deletion mutants, using upaY as a probe.

FIG. 5.

Regions of UpxYs involved in specific regulation of their respective loci. (A) Primary sequence comparisons of UpaY to UpeY and UpdY to UphY. The region of UpaY that was converted to UpeY in the hybrid protein is boxed. (B) Western immunoblot analysis of the expression of PSA from the wild type, ΔupaY, and ΔupaY with upeY and the hybrid upaY-upeY (pMCL74) supplied in trans. (C) Western immunoblot analysis of the expression of PSE from the wild type, ΔupeY, and ΔupeY with upaY and the hybrid upaY-upeY (pMCL74) supplied in trans.