Involvement of the multidomain regulatory protein XynR in positive control of xylanase gene expression in the ruminal anaerobe Prevotella bryantii B(1)4

Abstract

The xylanase gene cluster from the rumen anaerobe Prevotella bryantii B(1)4 was found to include a gene (xynR) that encodes a multidomain regulatory protein and is downstream from the xylanase and beta-xylosidase genes xynA and xynB. Additional genes identified upstream of xynA and xynB include xynD, which encodes an integral membrane protein that has homology with Na:solute symporters; xynE, which is related to the genes encoding acylhydrolases and arylesterases; and xynF, which has homology with the genes encoding alpha-glucuronidases. XynR includes, in a single 833-amino-acid polypeptide, a putative input domain unrelated to other database sequences, a likely transmembrane domain, histidine kinase motifs, response regulator sequences, and a C-terminal AraC-type helix-turn-helix DNA binding domain. Two transcripts (3.7 and 5.8 kb) were detected with a xynA probe, and the start site of the 3.7-kb transcript encoding xynABD was mapped to a position upstream of xynD. The DNA binding domain of XynR was purified after amplification and overexpression in Escherichia coli and was found to bind to a 141-bp DNA fragment from the region immediately upstream of xynD. In vitro transcription assays demonstrated that XynR stimulates transcription of the 3.7-kb transcript. We concluded that XynR acts as a positive regulator that activates expression of xynABD in P. bryantii B(1)4. This is the first regulatory protein that demonstrates significant homology with the two-component regulatory protein superfamily and has been shown to be involved in the regulation of polysaccharidase gene expression.

FIG. 1.

Diagram of the P. bryantii B₁4 xylan utilization operon. The available sequence of the related xylan utilization operon of B. ovatus V975 (26, 27) is shown beneath the equivalent genes in the P. bryantii B₁4 xylan utilization operon. Amino acid identities show the degrees of similarity for the three related genes. xynR (P. bryantii B₁4) and the gene indicated by the question mark (B. ovatus V975) show no sequence similarity. The recombinant protein fragment of XynR was synthesized from the PCR product obtained by using oligonucleotide primers reg-upper and reg-lower (Table 1). The probe used in Northern blotting was generated by PCR by using oligonucleotide primers FW-xynA and RV-xynB (Table 1). uORF, unidentified open reading frame.

FIG. 2.

Structure of the P. bryantii B₁4 two-component regulatory protein (XynR). The linker domain contains a characteristic membrane-spanning motif (9 amino acids; DAS transmembrane prediction server [3]). The histidine kinase domain contains the characteristic H, N, G1, F, and G2 boxes characteristic of histidine kinases of two-component regulatory proteins. The response regulator domain contains the characteristic aspartate residue that is normally phosphorylated in these domains. The effector domain contains an HTH region characteristic of the AraC family of DNA binding proteins. aa, amino acids.

FIG. 3.

Identification of a conserved sequence (boldface type and underlined) immediately upstream of P. bryantii B₁4 xylan utilization genes and other structural genes from Prevotella spp. The accession numbers for the genes are as follows: xynA and xynB, Z49241 and AJ428204; xynD, xynE, and xynF, AJ428204; ORF3, U96771; glnN, AF483911; xynC, Z79595; gdhA, U82240; cdxA, U35425; and pr23, M83379.

FIG. 4.

Northern blot identification of two mRNA transcripts from 20 μg of P. bryantii B₁4 total RNA hybridized to 1,721-bp DNA PCR probes (FW-xynA and RV-xynB) containing fragments of xynA and xynB (residues 3319 to 5040). The size of the 3.5-kb transcript corresponds to the expected size of a single polycistronic transcript comprising the xynA, xynB, and xynD genes. The size of the 5.8-kb transcript corresponds to the expected size of a single transcript comprising the xynA, xynB, xynD, and xynE genes. No hybridization of the xynA-xynB probe was observed when it was used with 20 μg of either mouse liver or Saccharomyces cerevisiae total RNA (data not shown). The sizes of the transcripts were determined by comparison with P. bryantii B₁4 16S and 23S ribosomal DNA.

FIG. 5.

Primer extension analysis with primer Trans-FITC (residues 6379 to 6398) revealed that the transcription start site (T at residue 6551) of the 3.5-kb transcript identified in Fig. 4 was located 16 bp upstream of the xynD start codon (residue 6535). Lanes A, G, C, and T contained sequencing ladders with the Trans-FITC primer. Lane S shows the transcription initiation site from P. bryantii B₁4 cells after pulse addition of 0.05% (vol/vol) WS-X.

FIG. 6.

Gel shift assay revealing binding of a 299-amino-acid recombinant protein fragment of XynR containing the HTH DNA binding domain (Fig. 2) to a ³²P-dCTP-labeled 141-bp DNA fragment (residues 6520 to 6661) containing the transcriptional start site identified for the xynABD transcript by Northern blot analysis and primer extension analysis (Fig. 4 and 5). As the concentration of the recombinant XynR polypeptide was increased, there was a concomitant decrease in the amount of free DNA. Samples were electrophoresed on 8% (wt/vol) polyacrylamide gels.

FIG. 7.

In vitro transcription assay revealing that XynR acts as a transcriptional activator of the xynABD transcript. A 780-bp PCR product containing 275 bp of sequence upstream of xynD and 505 bp of the xynD gene itself (primers IVT-F and IVT-R) was hybridized with P. bryantii B₁4 cell extract (lane 0) and different concentrations (10 and 20 μg/ml) of the recombinant XynR polypeptide (lanes 10 and 20). A nuclease protection assay revealed the presence of an undegraded 523-bp fragment of DNA that corresponds to the xynD coding sequence from the PCR product (505 bp) and the 16 bp upstream of this fragment that corresponds to the transcription initiation site (Fig. 5). Electrophoresis was carried out under denaturing conditions (1% agarose and 9% formaldehyde)

FIG. 8.

Effect of growth substrate on the expression of XynR, as determined by a nuclease protection assay. Exponentially growing cultures of P. bryantii B₁4 were pulsed with either 0.05% WS-X (XW-X), 0.05% xylose, or 0.05% glucose for 30 min, and then total RNA was extracted and 10 μg was transferred onto a Hybond-N⁺ positively charged nylon membrane. Two micrograms of S. cerevisiae total RNA (Yeast RNA) was used as a negative control. A 609-bp PCR product (obtained with primers Reg-UP and Reg-DOWN) corresponding to 398 bp of the xynR upstream sequence and 211 bp of xynR (residues 1681 to 2290) was labeled with DIG and used as a probe in the nuclease protection assay.