The AauR-AauS Two-Component System Regulates Uptake and Metabolism of Acidic Amino Acids in Pseudomonas putida

Abstract

Pseudomonas putida KT2440 metabolizes a wide range of carbon and nitrogen sources, including many amino acids. In this study, a 54 -dependent two-component system that controls the uptake and metabolism of acidic amino acids was identified. The system (designated aau, for acidic amino acid utilization) involves a sensor histidine kinase, AauS, encoded by PP1067, and a response regulator, AauR, encoded by PP1066. aauR and aauS deletion mutants were unable to efficiently utilize aspartate (Asp), glutamate (Glu), and glutamine (Gln) as sole sources of carbon and nitrogen. Growth of the mutants was partially restored when the abovementioned amino acids were supplemented with glucose or succinate as an additional carbon source. Uptake of Gln, Asp, and asparagine (Asn) by the aauR mutant was moderately reduced, while Glu uptake was severely impaired. In the absence of glucose, the aauR mutant even secreted Glu into the medium. Furthermore, disruption of aauR affected the activities of several key enzymes of Glu and Asp metabolism, leading to the intracellular accumulation of Glu and greatly reduced survival times under conditions of nitrogen starvation. By a proteomics approach, four major proteins were identified that are downregulated during growth of the aauR mutant on Glu. Two of these were identified as periplasmic glutaminase/asparaginase and the solutebinding protein of a Glu/Asp transporter. Transcriptional analysis of lacZ fusions containing the putative promoter regions of these genes confirmed that their expression is indeed affected by the aau system. Three further periplasmic solute-binding proteins were strongly expressed during growth of the aauR deletion mutant on Glu but downregulated during cultivation on glucose/NH 4 ؉ . These systems may be involved in amino acid efflux.

Dual Involvement of CbrAB and NtrBC in the Regulation of Histidine Utilization inPseudomonas fluorescensSBW25

“…The genome of Pseudomonas contains a large number of two-component regulatory systems (.60 sensorregulator pairs per genome; Kiil et al 2005), which play a major role in detecting niche-specific signals, including nutrient availability (Monds et al 2006;Sonawane et al 2006). Previous studies on histidine utilization (as the sole source of carbon and nitrogen) showed that CbrAB was required for hut activation in P. aeruginosa (Nishijyo et al 2001) and in P. fluorescens (Zhang and Rainey 2007); more recently evidence was obtained from a suppressor analysis of a cbrAB mutant for the involvement of a second two-component system, NtrBC (Itoh et al 2007;Li and Lu 2007).…”

Characterization of a Pseudomonas putida ABC transporter (AatJMQP) required for acidic amino acid uptake: biochemical properties and regulation by the Aau two-component system

“…As judged by sequence similarity, the response regulator AauR is a DNA-binding helix-turnhelix protein belonging to the Fis family (Morett & Bork, 1998). We have further shown that expression of aat depends on s 54 , an alternative sigma factor typically involved in the transcription of genes related to nitrogen metabolism (Sonawane et al, 2006).…”

“…Genes aatP (PP1068) and aatJ (PP1071) were inactivated by a strategy described previously in detail (Sonawane et al, 2006). Briefly, internal gene fragments of about 300 bp were amplified from the KT2440 genome using primers that introduce single base-pair frameshifts and cloned into pK18, a suicide vector that cannot multiply inside P. putida unless integrated into the genome by homologous recombination.…”

“…The response regulator AauR binds to the aat promoter As the AauR-AauS two-component system has been shown to induce the expression of aatJMQP (Sonawane et al, 2006), it was of interest to delineate cis-acting elements in the aat promoter that are required for this activation. A promoter deletion series was prepared involving sequences between 283 and 2202 relative to the transcription start site (+1).…”

“…The protein content of the cells was measured by BCA assay kit (Pierce) using BSA as a standard. The kinetics of amino acid uptake from the medium was analysed by quantitative amino acid analysis, as described by Sonawane et al (2006). The binding of amino acids and other potential ligands to AatJ was studied by equilibrium dialysis.…”