Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria

Abstract

Tunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of the Pseudomonas putida F1 cym/cmt system. The resulting promoter, termed P Q5 , responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in other Alphaproteobacteria, such as the model organisms Caulobacter crescentus, Paracoccus denitrificans, and Methylobacterium extorquens. In the noninduced state, expression from P Q5 is low enough to allow gene depletion analysis, as demonstrated with the essential gene phyP of Sphingomonas sp. strain Fr1. A set of P Q5 -based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins. R egulated gene expression systems are a powerful tool to study physiology, allowing, for example, dosage-effect studies, conditional expression of toxic alleles, and depletion analysis of essential genes; accordingly, they are well developed for model organisms (1-7) but are missing for many non-model organisms. Most systems rely on a transcriptional repressor that tightly binds to operator sites in the promoter region of target genes in the absence of an inducer, thereby preventing transcription; when an inducer is present, it allosterically binds to and inactivates the transcriptional regulator, leading to derepression of promoters. Despite this simple concept, identification of inducible promoters, control elements, and inducing conditions in a particular organism is not always a trivial task. On the other hand, it is often difficult to exploit a particular system for use in organisms other than the original host because of the need for dedicated transporters for the inducer or a different promoter specificity of the RNA polymerase holoenzyme or the requirement of a coactivator for full promoter activity (8). Some of these obstacles can be circumvented by engineering artificial inducible promoters by placing a constitutive minimal promoter known to be active in a particular organism with operator sequences and the repressor of a heterologous system (2, 9, 10). We here describe such a system for sphingomonads, a phylogenetically diverse group of environmentally abundant bacteria comprising the genera Sphingomonas, Sphingobium, Novosphingobium, and Sphingopyxis (11). Members of this group are well known for their unusual ability to degrade a wide range of compounds, including pesticides, herbicides, xenobiotics, and many other aromatics. Due to this property, they are prospective candidates for bioremediation; in addition, several str...

Complex two-component signaling regulates the general stress response in Alphaproteobacteria

“…Also in agreement with a role of Paks in the GSR, we were able to delete phyP in the sextuple or septuple kinase mutant backgrounds. Because the PhyR phosphatase PhyP is essential in a WT background preventing lethal hyperphosphorylation of PhyR (10,24), the successful deletion of phyP suggested that a major source of PhyR phosphorylation is absent in these genetic backgrounds. However, because the GSR activity in the sextuple or septuple kinase mutants was still substantially higher than in a phyR mutant ( Fig.…”

“…To identify factors positively regulating the PhyR-NepR-σ EcfG cascade, we conducted a genetic screen, performing transposon mutagenesis on a strain expressing the dominant streptomycin-sensitive rpsL1 allele (25) under control of the σ EcfG -dependent ecfG2 promoter (10,24). This construct would allow selection for mutants with an impaired PhyR-NepR-σ EcfG cascade on medium containing streptomycin (details are provided in SI Materials and Methods).…”

“…2D), it was not evident whether all kinases or only some were involved in the GSR. To address this question, we individually overexpressed kinases from the cumate-inducible promoter P Q5 (24) in the WT background carrying the nhaA2p-lacZ + fusion; to follow protein levels by Western blotting, kinases were fused C-terminally to SYFP2, a GFP derivative. Overexpression of all pak genes (i.e., pakA to pakG) activated the GSR threefold to fivefold, whereas overproduction of the remaining HRXXN kinase (Sphme2-DRAFT_2368), an unrelated NtrB-like kinase from S. melonis Fr1 (Sphme2DRAFT_3203), or SYFP2 alone failed to do so (Fig.…”

“…Among other evidence, this conclusion is based on the findings that phyP is essential unless in a ΔphyR or ΔecfG background (10), and that PhyP depletion leads to lethal overactivation of the GSR in a manner that requires the phosphorylatable Asp residue (Asp194) of PhyR (24). Assuming that PhyP acts solely as a phosphatase, this notion implies that alternative phosphodonors for PhyR must exist.…”

Two-Tiered Histidine Kinase Pathway Involved in Heat Shock and Salt Sensing in the General Stress Response of Sphingomonas melonis Fr1

“…In Sphingomonas melonis Fr1, seven HRXXN kinases, called PhyR-activating histidine kinases (Paks; PakA to PakG), are thought to activate the GSR by directly phosphorylating PhyR (7), while another HRXXN kinase, PhyP, seems to be a phosphatase of PhyR, presumably preventing deleterious overactivation of the GSR under nonstress conditions (6,11). Paks also phosphorylate the single-domain response regulator (SDRR) SdrG, which acts genetically upstream of phyR and plays an important role in the GSR, through a currently unknown mechanism (7).…”