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The copper-deprivation stimulon of Corynebacterium glutamicum comprises proteins for biogenesis of the actinobacterial cytochrome bc 1-aa 3 supercomplex.

ABSTRACT: Aerobic respiration in Corynebacterium glutamicum involves a cytochrome bc 1-aa 3 supercomplex with a diheme cytochrome c 1, which is the only c-type cytochrome in this species. This organization is considered as typical for aerobic Actinobacteria. Whereas the biogenesis of heme-copper type oxidases like cytochrome aa 3 has been studied extensively in ?-proteobacteria, yeast, and mammals, nothing is known about this process in Actinobacteria. Here, we searched for assembly proteins of the supercomplex by identifying the copper-deprivation stimulon, which might include proteins that insert copper into cytochrome aa 3 Using gene expression profiling, we found two copper starvation-induced proteins for supercomplex formation. The Cg2699 protein, named CtiP, contained 16 predicted transmembrane helices, and its sequence was similar to that of the copper importer CopD of Pseudomonas syringae in the N-terminal half and to the cytochrome oxidase maturation protein CtaG of Bacillus subtilis in its C-terminal half. CtiP deletion caused a growth defect similar to that produced by deletion of subunit I of cytochrome aa 3, increased copper tolerance, triggered expression of the copper-deprivation stimulon under copper sufficiency, and prevented co-purification of the supercomplex subunits. The secreted Cg1884 protein, named CopC, had a C-terminal transmembrane helix and contained a Cu(II)-binding motif. Its absence caused a conditional growth defect, increased copper tolerance, and also prevented co-purification of the supercomplex subunits. CtiP and CopC are conserved among aerobic Actinobacteria, and we propose a model of their functions in cytochrome aa 3 biogenesis. Furthermore, we found that the copper-deprivation response involves additional regulators besides the ECF sigma factor SigC.

SUBMITTER: Morosov X

PROVIDER: S-EPMC6177595 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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The copper-deprivation stimulon of Corynebacterium glutamicum comprises proteins for biogenesis of the actinobacterial cytochrome bc1-aa3 supercomplex.

Morosov Xenia X Davoudi Cedric-Farhad CF Baumgart Meike M Brocker Melanie M Bott Michael M

The Journal of biological chemistry 20180828 40

Aerobic respiration in Corynebacterium glutamicum involves a cytochrome bc1-aa3 supercomplex with a diheme cytochrome c1, which is the only c-type cytochrome in this species. This organization is considered as typical for aerobic Actinobacteria. Whereas the biogenesis of heme-copper type oxidases like cytochrome aa3 has been studied extensively in α-proteobacteria, yeast, and mammals, nothing is known about thi ...[more]

PMID: 30154248

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Project description:Pyruvate kinase (Pyk) catalyzes the generation of pyruvate and ATP in glycolysis and functions as a key switch in the regulation of carbon flux distribution. Both the substrates and products of Pyk are involved in the tricarboxylic acid cycle, anaplerosis and energy anabolism, which places Pyk at a primary metabolic intersection. Pyks are highly conserved in most bacteria and lower eukaryotes. Corynebacterium glutamicum is an industrial workhorse for the production of various amino acids and organic acids. Although C. glutamicum was assumed to possess only one Pyk (pyk1, NCgl2008), NCgl2809 was annotated as a pyruvate kinase with an unknown role.Here, we identified that NCgl2809 was a novel pyruvate kinase (pyk2) in C. glutamicum. Complementation of the WT?pyk1?pyk2 strain with the pyk2 gene restored its growth on D-ribose, which demonstrated that Pyk2 could substitute for Pyk1 in vivo. Pyk2 was co-dependent on Mn2+ and K+ and had a higher affinity for ADP than phosphoenolpyruvate (PEP). The catalytic activity of Pyk2 was allosterically regulated by fructose 1,6-bisphosphate (FBP) activation and ATP inhibition. Furthermore, pyk2 and ldhA, which encodes L-lactate dehydrogenase, were co-transcribed as a bicistronic mRNA under aerobic conditions and pyk2 deficiency had a slight effect on the intracellular activity of Pyk. However, the mRNA level of pyk2 in the wild-type strain under oxygen deprivation was 14.24-fold higher than that under aerobic conditions. Under oxygen deprivation, pyk1 or pyk2 deficiency decreased the generation of lactic acid, and the overexpression of either pyk1 or pyk2 increased the production of lactic acid as the activity of Pyk increased. Fed-batch fermentation of the pyk2-overexpressing WT?pyk1 strain produced 60.27?±?1.40 g/L of lactic acid, which was a 47% increase compared to the parent strain under oxygen deprivation.Pyk2 functioned as a pyruvate kinase and contributed to the increased level of Pyk activity under oxygen deprivation.

Project description:BackgroundKey mechanisms of cell division and its regulation are well understood in model bacteria such as Escherichia coli and Bacillus subtilis. In contrast, current knowledge on the regulation of cell division in Actinobacteria is rather limited. FtsZ is one of the key players in this process, but nothing is known about its transcriptional regulation in Corynebacterium glutamicum, a model organism of the Corynebacteriales.ResultsIn this study, we used DNA affinity chromatography to search for transcriptional regulators of ftsZ in C. glutamicum and identified the Cg1631 protein as candidate, which was named FtsR. Both deletion and overexpression of ftsR caused growth defects and an altered cell morphology. Plasmid-based expression of native ftsR or of homologs of the pathogenic relatives Corynebacterium diphtheriae and Mycobacterium tuberculosis in the ΔftsR mutant could at least partially reverse the mutant phenotype. Absence of ftsR caused decreased expression of ftsZ, in line with an activator function of FtsR. In vivo crosslinking followed by affinity purification of FtsR and next generation sequencing of the enriched DNA fragments confirmed the ftsZ promoter as in vivo binding site of FtsR and revealed additional potential target genes and a DNA-binding motif. Analysis of strains expressing ftsZ under control of the gluconate-inducible gntK promoter revealed that the phenotype of the ΔftsR mutant is not solely caused by reduced ftsZ expression, but involves further targets.ConclusionsIn this study, we identified and characterized FtsR as the first transcriptional regulator of FtsZ described for C. glutamicum. Both the absence and the overproduction of FtsR had severe effects on growth and cell morphology, underlining the importance of this regulatory protein. FtsR and its DNA-binding site in the promoter region of ftsZ are highly conserved in Actinobacteria, which suggests that this regulatory mechanism is also relevant for the control of cell division in related Actinobacteria.

Dataset Information

The copper-deprivation stimulon of Corynebacterium glutamicum comprises proteins for biogenesis of the actinobacterial cytochrome bc 1-aa 3 supercomplex.

Publications

The copper-deprivation stimulon of <i>Corynebacterium glutamicum</i> comprises proteins for biogenesis of the actinobacterial cytochrome <i>bc</i><sub>1</sub>-<i>aa</i><sub>3</sub> supercomplex.

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