Regulation of carbon and nitrogen utilization by CbrAB and NtrBC two-component systems in Pseudomonas aeruginosa.
The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in Pseudomonas aeruginosa was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the cbrAB deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the cbr mutant were selected from minimal medium containing l-arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an ntrB allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these ntrB alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these ntrB mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the DeltacbrAB ntrB(Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in P. aeruginosa. Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed.
Li
Wei
W
Department of Biology, Georgia State University, 24 Peachtree Center Ave., Atlanta, GA 30303, USA.
Lu
Chung-Dar
CD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D002244:Carbon / Q000378:metabolism*; D004252:DNA Mutational Analysis; D017353:Gene Deletion; D005969:Glutamate Dehydrogenase / Q000032:analysis; D005970:Glutamate Synthase / Q000032:analysis; D005974:Glutamate-Ammonia Ligase / Q000032:analysis; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins / Q000235:genetics / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000502:physiology*; D017354:Point Mutation; D011073:Polyamines / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000502:physiology*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*
D001426:Bacterial Proteins; C432141:CbrA protein, Pseudomonas aeruginosa; C432142:CbrB protein, Pseudomonas aeruginosa; D051922:PII Nitrogen Regulatory Proteins; D011073:Polyamines; D014157:Transcription Factors; D006639:Histidine; D002244:Carbon; D001120:Arginine; D005970:Glutamate Synthase; D005969:Glutamate Dehydrogenase; D011494:Protein Kinases; C055699:protein kinase-phosphatase NTRB; D010749:Phosphoprotein Phosphatases; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen