Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase; LiuB/LiuD) are characteristic enzymes of the catabolic pathway of acyclic terpenes (citronellol and geraniol) and of saturated methyl-branched compounds, such as leucine or isovalerate, respectively. Proteins encoded by two gene clusters (atuABCDEFGH and liuRABCDE) of Pseudomonas aeruginosa PAO1 were essential for acyclic terpene utilization (Atu) and for leucine and isovalerate utilization (Liu), respectively, as revealed by phenotype analysis of 10 insertion mutants, two-dimensional gel electrophoresis, determination of GCase and MCase activities, and Western blot analysis of wild-type and mutant strains. Analysis of the genome sequences of other pseudomonads (P. putida KT2440 and P. fluorescens Pf-5) revealed candidate genes for Liu proteins for both species and candidate genes for Atu proteins in P. fluorescens. This result concurred with the finding that P. fluorescens, but not P. putida, could grow on acyclic terpenes (citronellol and citronellate), while both species were able to utilize leucine and isovalerate. A regulatory gene, atuR, was identified upstream of atuABCDEFGH and negatively regulated expression of the atu gene cluster.

In this work, the molecular basis of aerobic citrate utilization by the gram-positive bacterium Corynebacterium glutamicum was studied. Genome analysis revealed the presence of two putative citrate transport systems. . We could subsequently show that, with 50 mM citrate as the sole carbon and energy source, the C. glutamicum wild type grew best when the minimal medium was supplemented with CaCl 2 but that MgCl 2 and SrCl 2 also supported growth. Each of the two transporters alone was sufficient for growth on citrate. The expression of citH and tctCBA was activated by citrate in the growth medium, independent of the presence or absence of glucose. This activation was dependent on the two-component signal transduction system CitAB, composed of the sensor kinase CitA and the response regulator CitB. CitAB belongs to the CitAB/DcuSR family of two-component systems, whose members control the expression of genes that are involved in the transport and catabolism of tricarboxylates or dicarboxylates. C. glutamicum CitAB is the first member of this family studied in Actinobacteria.Citrate is a ubiquitous natural compound which can be utilized as a carbon and energy source by many bacterial species. The anaerobic catabolism of citrate, which occurs, e.g., in enterobacteria like Klebsiella pneumoniae and Escherichia coli (7) and in lactic acid bacteria (14), usually involves the key enzyme citrate lyase (EC 4.1.3.6), which catalyzes the cleavage of citrate into acetate (the end product) and oxaloacetate (8,47,48). The subsequent catabolism of oxaloacetate can occur via different pathways, leading to, e.g., acetate and succinate as end products. Aerobic citrate utilization by bacteria possessing a complete tricarboxylic acid cycle usually requires only a citrate uptake system. Presently, at least five families of citrate transporters in bacteria have been characterized according to the classification system introduced by Saier (44) represented by TctABC of Salmonella enterica serovar Typhimurium (63). Whereas the transporters of the former four families consist of a single protein, the TctABC system is composed of three different subunits: two integral membrane proteins with presumably 12 (TctA) and 4 (TctB) transmembrane helices, plus a periplasmic citrate binding protein (TctC).For most citrate transporter genes studied so far with respect to regulation, expression is induced in the presence of the substrate. In many bacteria, the transcription of genes for citrate uptake and catabolism is activated by two-component signal transduction systems (TCS) consisting of a membranebound histidine kinase which controls the phosphorylation status of a soluble response regulator and thereby its activity as a transcriptional regulator. Examples are the CitA-CitB TCS of K. pneumoniae and E. coli (9, 34) and the CitS-CitT TCS of B. subtilis (64), which belong to the CitAB/DcuSR family of TCS (23). The periplasmic domains of the CitA histidine kinases from K. pneumoniae and E. coli were shown to bind citrate with high specificities and ...

SummaryExpression of the structural genes encoding the ATP-dependent proteases ClpCP and Lon in Corynebacterium glutamicum and Streptomyces lividans is activated by the transcriptional regulator ClgR in response to yet unknown environmental stimuli. As it was not known whether ClgR controls expression of additional genes we used DNA microarrays in order to comprehensively define the ClgR regulon in C.

The two-component signal transduction system consisting of the sensor kinase MtrB and the response regulator MtrA is highly conserved in corynebacteria and mycobacteria. Whereas mtrA of Mycobacterium tuberculosis was reported to be essential, we recently succeeded in creating ⌬mtrAB and ⌬mtrA deletion mutants of Corynebacterium glutamicum and provided evidence that mepA and nlpC, both encoding putative cell wall peptidases, are directly repressed by MtrA, whereas proP and betP, both encoding carriers for compatible solutes, are directly activated by MtrA. In the present study, novel MtrA target genes were identified, including mepB, encoding another putative cell wall peptidase. The repressor or activator functions of MtrA correlate with the distance between the MtrA binding site and the transcriptional start site. From the identified binding sites within 20 target promoters, a 19-bp MtrA consensus motif was derived which represents a direct repeat of 8 base pairs separated by 3 base pairs. Gene expression of a strain containing MtrA with a D53N mutation instead of wild-type MtrA resembled that of a ⌬mtrA mutant, indicating that MtrA is active in its phosphorylated form. This result was confirmed by electrophoretic mobility shift assays with phosphorylated MtrA which showed an increased binding affinity.

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