Arginine Catabolism in Pseudomonas aeruginosa

Georgia State University Research Foundation, Inc., Atlanta GA

Investigators

Abstract

The significance of arginine in the physiology of P. aeruginosa is reflected in the presence of four catabolic pathways for its utilization as a source of carbon, energy, and nitrogen as well as its ability to serve as one of the strongest chemotactic attractants for this organism. The long-term objective of this research is to elucidate the regulatory mechanisms for arginine catabolism. The first aim of this project is to determine the role of C4-dicarboxylate of the TCA cycle in reverse catabolite repression. In P. aeruginosa, the presence of acetate and the TCA cycle intermediates inhibits the utilization of sugars as well as other compounds including arginine. Studies in this laboratory have established that arginine-specific regulatory protein, ArgR, is essential for the induction of arginine catabolic operons. Preliminary data also indicated that auto-induction of ArgR in the presence of exogenous arginine was abolished in the mutants of argSU genes encoding a pair of sensor kinase/response regulator of a two-component regulatory system. In addition, the utilization of a variety of compounds including glucose, but not succinate, was severely affected in the argSU mutant. (i) Studies employing gene fusions will be conducted to test the hypothesis of C4-dicarboxylates in the TCA cycle as the signal compound of the argSU regulatory system. (ii) In vitro phosphorylation assays with either intact or His-tagged fusion proteins of ArgS and ArgU will be employed to demonstrate signal transduction in these two proteins, and effects of any of the C4-dicarboxylates on the kinetics of these biochemical reactions will also be analyzed. The second aim of this project is to identify the regulatory elements that function in reverse catabolite repression. The ArgSU system is proposed here to play a role in reverse catabolite repression. Since ArgU appears as an NtrC-like transcriptional regulator, it is expected that it could only have a direct effect on operons transcribed from s54 promoters. Its effects on operons that are transcribed from s70 promoters could be mediated by an additional regulator. (i) To identify the genomic targets of ArgU, a nickel sulfate affinity column containing His-tagged ArgU will be employed in a selection strategy to isolate its binding candidates from a cosmid genomic library. Alternatively, genomic targets of ArgU can be identified by analyses of growth suppressors of the argSU null mutant and the subsequent cloning by complementation tests. (ii) To identify the missing linking between ArgU and its affected s70 promoters, transposon mutagenesis will be employed to isolate mutants with altered expression level of b-galactosidase from an aotJ::lacZ fusion, which is known to be under the control of ArgSU. The affected gene can then be identified after sequence analysis of clones containing antibiotic-resistant genes of the transposon and its flanking regions. Alternatively, standard reverse genetics procedures will be adapted to identify this missing element. Fractions containing proteins other than ArgR that bind to the aotJ regulatory region will be identified by mobility shift assays, and subject to further purification from a DNA affinity column. Identification of the encoding gene can then be achieved after determination of the N-terminal amino acid sequence and BLAST search on the completed genomic sequence of P. aeruginosa. The enormous catabolic capability of P. aeruginosa and other related bacteria have led to a wide recognition of its potential significance in industrial and environmental biotechnology. The potential pathogenicity of P. aeruginosa in human and its related bacteria to plants is greatly enhanced by a remarkable nutritional versatility that enables this organism to survive in diverse and in harsh environments prior to infection and to efficiently adjust its metabolic activity to the existing environmental constraints following infection. These capacities have necessitated the evolution of mechanisms in control of metabolic activities that are significantly different from those found in enteric bacteria and gram-positive bacteria. Completion of this research will contribute to our knowledge of the diverse biochemical reactions and the regulatory mechanisms that is critical for long term efforts to prevent and control diseases, and to apply these microorganisms in biotechnology.