Spiro, StephenMinkoff, Susan E.2023-10-132023-10-132023-05May 2023May 2023https://hdl.handle.net/10735.1/9929Paracoccus denitrificans is a metabolically versatile Gram-negative alpha-proteobacterium that is used as a model organism for studying respiratory metabolism. One of the most notable features of P. denitrificans is its capacity to use nitrate or nitrogen oxides as terminal electron acceptors in the process of denitrification, which is the sequential conversion of nitrate (NO3-) to gaseous dinitrogen (N2). The P. denitrificans genome encodes three members of the DksA/TraR family of transcription factors, two of which appear to be bona fide DksA proteins (hence designated DksA1Pd and DksA2Pd). There is a single relA/spoT gene in the genome encoding a predicted bifunctional (p)ppGpp synthetase and hydrolase (designated RSH, for RelA/SpoT Homolog). Both DksA1Pd and DksA2Pd can rescue the amino acid auxotrophy of an Escherichia coli dksA mutant. Deletion of either dksA1 or relA/spoT eliminates the upregulation of expression of the periplasmic nitrate reductase (NAP) that is seen when cultures are grown on a reduced substrate such as butyrate. Thus, we conclude that DksA1Pd and (p)ppGpp activate nap transcription in response to growth on a reduced substrate and that these proteins therefore help to maintain redox homeostasis by activating a mechanism for the disposal of excess reductant. Moreover, we show that the redox- sensing RegAB two-component pair acts as a negative regulator of nap expression under anaerobic growth conditions. Under the same conditions, RegAB acts as a positive regulator of the expression of the membrane-associated nitrate reductase Nar, mediating reciprocal regulation of the two nitrate reductases that have distinct physiological roles. The dksA1 and relA/spoT genes are conditionally synthetically lethal; the double mutant has a null phenotype for growth on butyrate and other reduced substrates while growing normally on succinate and citrate. The flavohemoglobin gene hmp is modestly upregulated in dksA2 and relA/spoT mutants, which are also defective for biofilm formation (possibly because of enhanced scavenging of nitric oxide). In conclusion, we have successfully identified two functional DksA homologs in P. denitrificans - DksA1Pd and DksA2Pd, along with a (p)ppGpp synthetase homolog (RSH) and a two-component RegAB system. These regulators were previously unknown and found to be responsible for regulating the expression of enzymes involved in nitrate reduction and nitric oxide metabolism.application/pdfEnglishBiology, CellBiology, MolecularBiology, MicrobiologyThesis2023-10-13