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.D-Xylose is the main constituent of xylan polymers, the major component of hemicellulose in plant cell walls. This pentose, in monomeric or polymerized form, is often present in habitats containing plant-derived biomass. The aquatic bacterium Caulobacter crescentus avidly consumes D-xylose as a carbon and energy source. D-Xylose metabolism in C. crescentus proceeds via a novel pathway initiated by the enzyme xylose dehydrogenase (XDH) (2, 24). The enzymes of this pathway are encoded in the xyl operon, whose expression is induced by D-xylose (9,16,24). In the work presented here, we identified the transcription factor controlling expression of the xyl operon and characterized the mechanism of regulation.For the past decade, the xyl operon promoter (P xylX ) has been the standard tool for engineering inducible gene expression in C. crescentus (1,10,12,15,20,25,26). This promoter is highly active when it is induced by addition of D-xylose to the medium and is virtually silent in the absence of xylose, provided that there is a single copy on the chromosome or it is in a low-copy-number plasmid vector. In its native chromosomal context, P xylX controls the expression of the five-gene xyl operon (CC0823 to CC0819) ( Fig. 1) (9). A putative operator sequence in P xylX was identified by virtue of its conservation in several xylose-regulated promoters (9). This operator overlaps the Ϫ10 region of P xylX (Fig. 1), suggesting that a DNA binding protein interacting with this site would interfere with RNA polymerase holoenzyme binding and/or transcription initiation. The simplest model for control of the xyl regulon is that a repressor ("XylR") binds to operator sites in the absence of xylose but dissociates from the operator when it is bound to xylose to allow gene expression. This model is consistent with the observation that a 4-bp mutation in the upstream half-site of the putative operator of P xylX , which did no...

Identification of a Dehydrogenase Required for Lactose Metabolism inCaulobacter crescentus

“…Second, although induction of lac expression and dehydrogenase activity is clearly observable in C. crescentus, the level of induction appears modest in comparison to other metabolic systems, both in other bacteria and in C. crescentus, including that used for xylose catabolism (the xyl system) (25,42,53). Poindexter (48) has noted that, for the majority of sugar substrates, the quantitative response of the Caulobacter cell to the sudden availability of a new substrate is not impressive and has suggested that, in dilute environments where a nutrient is likely to be available for only a short time at irregular intervals, dramatic shifts to dedicated pathways may be maladaptive; low-level maintenance of different catabolic systems at all times would be more advantageous.…”

Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing

“…Among the latter, the lac promoter of Escherichia coli, as well as its mutant and hybrid forms lacUV5 (6), Ptac (10), and Ptrc (4), have been extensively exploited in combination with its control protein, LacI. In addition, other regulatory elements have been developed and are widely used, including the arabinose promoter of E. coli (19), the xylene (xyl) promoter of Pseudomonas putida (35), and the xylose (xyl) promoter of Caulobacter crescentus (48).…”

Regulation of D-galacturonate metabolism in <i>Caulobacter crescentus</i> by HumR, a LacI-family transcriptional repressor

“…For example, transcriptome analysis of C. crescentus metabolizing xylose revealed induction of xylanases and arabinosidases, putative outer and inner-membrane transporters, and a pathway for oxidative metabolism of xylose [11,12]. The xylose regulon is under the control of XylR, a member of the LacI family of bacterial transcription factors [13]. Our results reveal an analogous galacturonate regulon in C. crescentus that is under the control of another LacI family member, HumR.…”