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Dual involvement of CbrAB and NtrBC in the regulation of histidine utilization in Pseudomonas fluorescens SBW25.

ABSTRACT: Pseudomonas fluorescens SBW25 is capable of growing on histidine as a sole source of carbon and/or nitrogen. Previous work showed that the two-component regulatory system CbrAB is required for expression of the histidine utilization (hut) locus when histidine is the sole source of carbon and nitrogen. Here, using mutational analysis and transcriptional assays, we demonstrate involvement of a second two-component system, NtrBC. When histidine is the sole carbon source, transcription of the hutU operon is initiated from a sigma54-type promoter and requires CbrB (an enhancer binding protein for sigma54-recruitment). However, when histidine is the sole nitrogen source, the hutU operon is transcribed from a sigma70-type promoter and requires either CbrB or the nitrogen regulator, NtrC. No role was found for the SBW25 homolog of the nitrogen assimilation control protein (NAC). Biolog phenotypic microarray analysis of the ability of the three mutants (DeltacbrB, DeltantrC, and DeltacbrB DeltantrC) to utilize 190 carbon and 95 nitrogen substrates confirmed the central regulatory roles of CbrAB and NtrBC in cellular carbon and nitrogen catabolism: deletion of cbrB abolished growth on 20 carbon substrates; deletion of ntrC eliminated growth on 28 nitrogen substrates. A double cbrB-ntrC mutant was unable to utilize a further 14 nitrogen substrates (including histidine, proline, leucine, isoleucine, and valine). Our data show that CbrAB plays a role in regulation of both carbon and nitrogen catabolism and maintains activity of catabolic pathways under different C:N ratios.

SUBMITTER: Zhang XX

PROVIDER: S-EPMC2206070 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Dual involvement of CbrAB and NtrBC in the regulation of histidine utilization in Pseudomonas fluorescens SBW25.

Zhang Xue-Xian XX Rainey Paul B PB

Genetics 20080101 1

Pseudomonas fluorescens SBW25 is capable of growing on histidine as a sole source of carbon and/or nitrogen. Previous work showed that the two-component regulatory system CbrAB is required for expression of the histidine utilization (hut) locus when histidine is the sole source of carbon and nitrogen. Here, using mutational analysis and transcriptional assays, we demonstrate involvement of a second two-component system, NtrBC. When histidine is the sole carbon source, transcription of the hutU o ...[more]

PMID: 18202367

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Project description:HutC is known as a transcriptional repressor specific for histidine utilization (hut) genes in Gram-negative bacteria, including Pseudomonas fluorescens SBW25. However, its precise mode of protein-DNA interactions hasn't been examined with purified HutC proteins. Here, we performed electrophoretic mobility shift assay (EMSA) and DNase I footprinting using His6-tagged HutC and biotin-labeled probe of the hut promoter (PhutU). Results revealed a complex pattern of HutC oligomerization, and the specific protein-DNA interaction is disrupted by urocanate, a histidine derivative, in a concentration-dependent manner. Next, we searched for putative HutC-binding sites in the SBW25 genome. This led to the identification of 143 candidate targets with a P value less than 10-4 HutC interaction with eight selected candidate sites was subsequently confirmed by EMSA analysis, including the type IV pilus assembly protein PilZ, phospholipase C (PlcC) for phosphatidylcholine hydrolyzation, and key regulators of cellular nitrogen metabolism (NtrBC and GlnE). Finally, an isogenic hutC deletion mutant was subjected to transcriptome sequencing (RNA-seq) analysis and phenotypic characterization. When bacteria were grown on succinate and histidine, hutC deletion caused upregulation of 794 genes and downregulation of 525 genes at a P value of <0.05 with a fold change cutoff of 2.0. The hutC mutant displayed an enhanced spreading motility and pyoverdine production in laboratory media, in addition to the previously reported growth defect on the surfaces of plants. Together, our data indicate that HutC plays global regulatory roles beyond histidine catabolism through low-affinity binding with operator sites located outside the hut locus.IMPORTANCE HutC in Pseudomonas is a representative member of the GntR/HutC family of transcriptional regulators, which possess a N-terminal winged helix-turn-helix (wHTH) DNA-binding domain and a C-terminal substrate-binding domain. HutC is generally known to repress expression of histidine utilization (hut) genes through binding to the PhutU promoter with urocanate (the first intermediate of the histidine degradation pathway) as the direct inducer. Here, we first describe the detailed molecular interactions between HutC and its PhutU target site in a plant growth-promoting bacterium, P. fluorescens SBW25, and further show that HutC possesses specific DNA-binding activities with many targets in the SBW25 genome. Subsequent RNA-seq analysis and phenotypic assays revealed an unexpected global regulatory role of HutC for successful bacterial colonization in planta.

Project description: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.

Dataset Information

Dual involvement of CbrAB and NtrBC in the regulation of histidine utilization in Pseudomonas fluorescens SBW25.

Publications

Dual involvement of CbrAB and NtrBC in the regulation of histidine utilization in Pseudomonas fluorescens SBW25.

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