Regulation of D-xylose metabolism in Caulobacter crescentus by a LacI-type repressor

Abstract

In the oligotrophic freshwater bacterium Caulobacter crescentus, D-xylose induces expression of over 50 genes, including the xyl operon, which encodes key enzymes for xylose metabolism. The promoter (P(xylX)) controlling expression of the xyl operon is widely used as a tool for inducible heterologous gene expression in C. crescentus. We show here that P(xylX) and at least one other promoter in the xylose regulon (P(xylE)) are controlled by the CC3065 (xylR) gene product, a LacI-type repressor. Electrophoretic gel mobility shift assays showed that operator binding by XylR is greatly reduced in the presence of D-xylose. The data support the hypothesis that there is a simple regulatory mechanism in which XylR obstructs xylose-inducible promoters in the absence of the sugar; the repressor is induced to release DNA upon binding D-xylose, thereby freeing the promoter for productive interaction with RNA polymerase. XylR also has an effect on glucose metabolism, as xylR mutants exhibit reduced expression of the Entner-Doudoroff operon and their ability to utilize glucose as a sole carbon and energy source is compromised.

FIG. 1.

Caulobacter genomic loci involved in xylose metabolism and regulation. (A) The C. crescentus xyl operon (CC0823 to CC0819) and the surrounding region are shown on the top line. This region includes xylE (CC0814), which encodes a putative xylose transporter. The gene nomenclature is the nomenclature described previously (18, 24). The xylR gene (CC3065) is on the opposite side of the 4-Mb chromosome, and the approximate distance is indicated. In contrast, in the Caulobacter sp. strain K31 genome (shown below the C. crescentus operon and surrounding region), xylR is located very close to the xyl operon. Predicted coding regions in the K31 genome are based on the draft genome sequence and annotation publicly released by the U.S. Department of Energy's Joint Genome Institute (http://genome.jgi-psf.org/draft_microbes/cau_k/cau_k.home.html and http://genome.ornl.gov/microbial/caul/). Homologous genes were identified using BLAST through the Integrated Microbial Genomics web portal (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi). (B) Sequence of the C. crescentus xyl operon promoter (P_xylX) region, with the start site and putative promoter and operator motifs indicated (9, 16). The four base pairs that were altered in constitutive mutants are indicated by bold type. Predicted XylR operators found upstream of the C. crescentus xylE and Caulobacter sp. strain K31 xylX and xylE genes are aligned with the C. crescentus P_xylX operator.

FIG. 2.

Caulobacter xylose repressor, XylR. The predicted polypeptide product of C. crescentus xylR (CC3065; GenBank accession number AAK25027) is aligned with the putative polypeptide products of Caulobacter sp. strain K31 xylR (gene 1784 in the draft sequence available in September 2007) and E. coli lacI (GenBank accession number AAA24457) (21). The locations of the Tn5 insertions in the xylR1.2::Tn5 and xylR4.6::Tn5 mutants are each indicated by a line under the residue encoded by the codon in which the Tn5 inserted. HTH, helix-turn-helix.

FIG. 3.

Gel mobility shift analysis of XylR binding to the P_xylX operator. Binding reactions and electrophoresis conditions are described in Materials and Methods. (A) Lane 1, no cell extract added (negative control); lane 2, CB15 extract; lane 3, CB15 extract with 1 mM d-xylose; lane 4, CS816 (xylR1.2::Tn5) extract; lane 5, CS816 (xylR1.2::Tn5) extract with 1 mM d-xylose; lane 6, CS817 (xylR4.6::Tn5) extract; lane 7, CS817 (xylR4.6::Tn5) extract with 1 mM d-xylose. (B) Lanes 2 to 9 all contained cell extract from CB15. Lane 1 contained the negative control with no cell extract added, and lane 2 contained the positive control with CB15 cell extract and no added sugar. For the binding reactions shown in lanes 3 to 9, various sugars or amino acids were present at a concentration of 1 mM (lane 3, d-xylose; lane 4, l-xylose; lane 5, d-arabinose; lane 6, l-arabinose; lane 7, d-ribose; lane 8, d-glucose; lane 9, l-glutamate).