Genetic and biochemical analysis of the serine/threonine protein kinases PknA, PknB, PknG and PknL of Corynebacterium glutamicum: evidence for non-essentiality and for phosphorylation of OdhI and FtsZ by multiple kinases.
We previously showed that the 2-oxoglutarate dehydrogenase inhibitor protein OdhI of Corynebacterium glutamicum is phosphorylated by PknG at Thr14, but that also additional serine/threonine protein kinases (STPKs) can phosphorylate OdhI. To identify these, a set of three single (DeltapknA, DeltapknB, DeltapknL), five double (DeltapknAG, DeltapknAL, DeltapknBG, DeltapknBL, DeltapknLG) and two triple deletion mutants (DeltapknALG, DeltapknBLG) were constructed. The existence of these mutants shows that PknA, PknB, PknG and PknL are not essential in C. glutamicum. Analysis of the OdhI phosphorylation status in the mutant strains revealed that all four STPKs can contribute to OdhI phosphorylation, with PknG being the most important one. Only mutants in which pknG was deleted showed a strong growth inhibition on agar plates containing glutamine as carbon and nitrogen source. Thr14 and Thr15 of OdhI were shown to be phosphorylated in vivo, either individually or simultaneously, and evidence for up to two additional phosphorylation sites was obtained. Dephosphorylation of OdhI was shown to be catalysed by the phospho-Ser/Thr protein phosphatase Ppp. Besides OdhI, the cell division protein FtsZ was identified as substrate of PknA, PknB and PknL and of the phosphatase Ppp, suggesting a role of these proteins in cell division.
Institut für Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich, Germany.
Christian
C
Schultz
Axel
A
Niebisch
Astrid
A
Schwaiger
Ulrike
U
Viets
Sabine
S
Metzger
Marc
M
Bramkamp
Michael
M
Bott
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D007655:Ketoglutarate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism; D007656:Ketoglutaric Acids; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D004269:DNA, Bacterial; C071525:FtsZ protein, Bacteria; D007656:Ketoglutaric Acids; D013912:Threonine; D007655:Ketoglutarate Dehydrogenase Complex; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases