Lipase Expression in Pseudomonas alcaligenes Is Under the Control of a Two-Component Regulatory System

Abstract

Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set up a search method for genes controlling lipase expression by use of a cosmid library containing fragments of P. alcaligenes genomic DNA. A screen for lipase hyperproduction resulted in the selection of multiple transformants, of which the best-producing strains comprised cosmids that shared an overlapping genomic fragment. Within this fragment, two previously unidentified genes were found and named lipQ and lipR. Their encoded proteins belong to the NtrBC family of regulators that regulate gene expression via binding to a specific upstream activator sequence (UAS). Such an NtrC-like UAS was identified in a previous study in the P. alcaligenes lipase promoter, strongly suggesting that LipR acts as a positive regulator of lipase expression. The regulating role could be confirmed by down-regulated lipase expression in a strain with an inactivated lipR gene and a threefold increase in lipase yield in a large-scale fermentation when expressing the lipQR operon from the multicopy plasmid pLAFR3. Finally, cell extracts of a LipR-overexpressing strain caused a retardation of the lipase promoter fragment in a band shift assay. Our results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system.

Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa

“…In P. alcaligenes, the expression of the lipA gene coding for secreted lipase is positively controlled by RpoN and by the LipQ/LipR twocomponent system. LipQ and LipR are homologues of P. aeruginosa CbrA and CbrB, respectively (9,18). At Ϫ130 to Ϫ112 of the lipA promoter, a UAS has been proposed as a LipR binding site (7).…”

“…AERUGINOSA CbrB ACTIVATES crcZ AND lipA PROMOTERS 2789 (18). All of the substrates mentioned sustain the growth of pseudomonads to high cell population densities but are energetically less favorable than C 4 dicarboxylates, glutamate, and aspartate, which are preferred substrates of pseudomonads (30,33).…”

“…The CbrB protein is a transcriptional activator for 54 RNA polymerase and belongs to the NtrC family of response regulators (30). Genetic evidence indicates that CbrB positively controls the expression of the crcZ sRNA gene in P. aeruginosa (36), the lipA (lipase) gene in Pseudomonas alcaligenes (7,18), and the hutU (histidine utilization) operon of P. aeruginosa and P. fluorescens (16,40). The fact that cbrB and crcZ mutants of P. aeruginosa have similar, but not identical, phenotypes suggests that most, but not all, activities of the CbrA/CbrB system are mediated by the sRNA CrcZ (36).…”

The Sensor Kinase CbrA Is a Global Regulator That Modulates Metabolism, Virulence, and Antibiotic Resistance in Pseudomonas aeruginosa

“…A two-component system, LipQR, was discovered in Pseudomonas alcaligenes and demonstrated to be involved in the regulation of lipase expression (20). The LipQR system exhibits significant homology to the CbrAB system in P. aeruginosa (20).…”

“…A two-component system, LipQR, was discovered in Pseudomonas alcaligenes and demonstrated to be involved in the regulation of lipase expression (20). The LipQR system exhibits significant homology to the CbrAB system in P. aeruginosa (20). Analysis of the transcriptome profile of P. aeruginosa exposed to sublethal concentrations of tobramycin revealed downregulation of the cbrA gene (K. N. Kindrachuk, L. Fernandez, M. Bains, and R. E. W. Hancock, submitted for publication).…”