SummaryThe MtrAB two-component signal transduction system is highly conserved in sequence and genomic organization in Mycobacterium and Corynebacterium species, but its function is completely unknown. Here, the role of MtrAB was studied with C. glutamicum as model organism. In contrast to M. tuberculosis , it was possible to delete the mtrAB genes in C. glutamicum . The mutant cells showed a radically different cell morphology and were more sensitive to penicillin, vancomycin and lysozyme but more resistant to ethambutol. In order to identify the molecular basis for this pleiotropic phenotype, the mRNA profiles of mutant and wild type were compared with DNA microarrays. Three genes showed a more than threefold increased RNA level in the mutant, i.e. mepA ( NCgl2411 ) encoding a putative secreted metalloprotease, ppmA ( NCgl2737 ) encoding a putative membrane-bound protease modulator, and lpqB encoding a putative lipoprotein of unknown function. Expression of plasmid-encoded mepA in Escherichia coli led to elongated cells that were hypersensitive to an osmotic downshift, supporting the idea that peptidoglycan is the target of MepA. The mRNA level of two genes was more than fivefold decreased in the mutant, i.e. betP and proP which encode transporters for the uptake of betaine and proline respectively. The microarray results were confirmed by primer extension and RNA dot blot experiments. In the latter, the transcript level of genes involved in osmoprotection was tested before and after an osmotic upshift. The mRNA level of betP , proP and lcoP was strongly reduced or undetectable in the mutant, whereas that of mscL (mechanosensitive channel) was increased. The changes in cell morphology, antibiotics susceptibility and the mRNA levels of betP , proP , lcoP , mscL and mepA could be reversed by expression of plasmid-encoded copies of mtrAB in the D D D D mtrAB mutant, confirming that these changes occurred as a consequence of the mtrAB deletion.

ABSTRACTPrevious studies have demonstrated the capability ofCorynebacterium glutamicumfor anaerobic succinate production from glucose under nongrowing conditions. In this work, we have addressed two shortfalls of this process, the formation of significant amounts of by-products and the limitation of the yield by the redox balance. To eliminate acetate formation, a derivative of the type strain ATCC 13032 (strain BOL-1), which lacked all known pathways for acetate and lactate synthesis (ΔcatΔpqoΔpta-ackAΔldhA), was constructed. Chromosomal integration of the pyruvate carboxylase genepycP458Sinto BOL-1 resulted in strain BOL-2, which catalyzed fast succinate production from glucose with a yield of 1 mol/mol and showed only little acetate formation. In order to provide additional reducing equivalents derived from the cosubstrate formate, thefdhgene fromMycobacterium vaccae, coding for an NAD+-coupled formate dehydrogenase (FDH), was chromosomally integrated into BOL-2, leading to strain BOL-3. In an anaerobic batch process with strain BOL-3, a 20% higher succinate yield from glucose was obtained in the presence of formate. A temporary metabolic blockage of strain BOL-3 was prevented by plasmid-borne overexpression of the glyceraldehyde 3-phosphate dehydrogenase genegapA. In an anaerobic fed-batch process with glucose and formate, strain BOL-3/pAN6-gapaccumulated 1,134 mM succinate in 53 h with an average succinate production rate of 1.59 mmol per g cells (dry weight) (cdw) per h. The succinate yield of 1.67 mol/mol glucose is one of the highest currently described for anaerobic succinate producers and was accompanied by a very low level of by-products (0.10 mol/mol glucose).

SummaryCorynebacterium glutamicum, an established industrial amino acid producer, has been genetically modified for efficient succinate production from the renewable carbon source glucose under fully aerobic conditions in minimal medium. The initial deletion of the succinate dehydrogenase genes (sdhCAB) led to an accumulation of 4.7 g l−1 (40 mM) succinate as well as high amounts of acetate (125 mM) as by‐product. By deleting genes for all known acetate‐producing pathways (pta‐ackA, pqo and cat) acetate production could be strongly reduced by 83% and succinate production increased up to 7.8 g l−1 (66 mM). Whereas overexpression of the glyoxylate shunt genes (aceA and aceB) or overproduction of the anaplerotic enzyme pyruvate carboxylase (PCx) had only minor effects on succinate production, simultaneous overproduction of pyruvate carboxylase and PEP carboxylase resulted in a strain that produced 9.7 g l−1 (82 mM) succinate with a specific productivity of 1.60 mmol g (cdw)−1 h−1. This value represents the highest productivity among currently described aerobic bacterial succinate producers. Optimization of the production conditions by decoupling succinate production from cell growth using the most advanced producer strain (C. glutamicumΔpqoΔpta‐ackAΔsdhCABΔcat/pAN6‐pycP458Sppc) led to an additional increase of the product yield to 0.45 mol succinate mol−1 glucose and a titre of 10.6 g l−1 (90 mM) succinate.

The response regulator HrrA of the HrrSA two-component system (previously named CgtSR11) was recently found to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Here, we provide evidence that HrrA mediates heme-dependent gene regulation in this nonpathogenic soil bacterium. Growth experiments and DNA microarray analysis revealed that C. glutamicum is able to use hemin as an alternative iron source and emphasize the involvement of the putative hemin ABC transporter HmuTUV and heme oxygenase (HmuO) in heme utilization. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of an hrrA deletion mutant and C. glutamicum wild-type strain in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase. Based on our results, it can be furthermore deduced that HrrA activates the expression of heme-containing components of the respiratory chain, namely, ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa 3 oxidase and three subunits of the cytochrome bc 1 complex. In addition, HrrA was found to repress almost all genes involved in heme biosynthesis, including those for glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Growth experiments with an hrrA deletion mutant showed that this strain is significantly impaired in heme utilization. In summary, our results provide evidence for a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum.

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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 ...

A biohybrid ring-opening olefin metathesis polymerization catalyst based on the reengineered β-barrel protein FhuA ΔCVF(tev) was chemically modified with respect to the covalently anchored Grubbs-Hoveyda type catalyst. Shortening of the spacer (1,3-propanediyl to methylene) between the N-heterocyclic carbene ligand and the cysteine site 545 increased the ROMP activity toward a water-soluble 7-oxanorbornene derivative. The cis/trans ratio of the double bond in the polymer was influenced by the hybrid catalyst.

c DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase, led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2, and IolR. Determination of the phosphoenolpyruvate carboxykinase activity of the ⌬ramA, ⌬gntR1 ⌬gntR2, and ⌬iolR deletion mutants indicated that RamA represses pck during growth on glucose about 2-fold, whereas GntR1, GntR2, and IolR activate pck expression about 2-fold irrespective of whether glucose or acetate served as the carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in a divergent orientation with respect to a iol gene cluster, encoding proteins involved in myoinositol uptake and degradation. Comparative DNA microarray analysis of the ⌬iolR mutant and the parental wild-type strain revealed strongly (>100-fold) elevated mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT1, and iolR. IolR therefore is presumably subject to negative autoregulation. A consensus DNA binding motif (5=-KGWCHTRACA-3=) which corresponds well to those of other GntR-type regulators of the HutC family was identified. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism.