Abstract
Shewanella oneidensis, a metal reducer and facultative anaerobe, expresses a large number of c-type cytochromes, many of which function as anaerobic reductases. All of these proteins contain the typical heme-binding motif CXXCH and require the Ccm proteins for maturation. Two c-type cytochrome reductases also possess atypical heme-binding sites, the NrfA nitrite reductase (CXXCK) and the SirA sulfite reductase (CX12NKGCH). S. oneidensis MR-1 encodes two cytochrome c synthetases (CcmF and SirE) and two apocytochrome c chaperones (CcmI and SirG). SirE located in the sir gene cluster is required for the maturation of SirA, but not NrfA. Here we show that maturation of SirA requires the combined function of the two apocytochrome c chaperones CcmI and SirG. Loss of either protein resulted in decreased sulfite reductase. Furthermore, SirA was not detected in a mutant that lacked both chaperones, perhaps due to misfolding or instability. These results suggest that CcmI interacts with SirEFG during SirA maturation, and with CcmF during maturation of NrfA. Additionally, we show that CRP regulates expression of sirA via the newly identified transcriptional regulatory protein, SirR.
Highlights
Shewanella species are facultative anaerobes that are abundant in freshwater and marine environments and are best known for their ability to use metals as electron acceptors for anaerobic respiration
To determine if SirR regulates genes required for anaerobic growth, a sirR (∆sirR) mutant was generated and tested for anerobic respiration with diverse electron acceptors utilized by the wild type S. oneidensis MR-1
Expression of the sirA operon was eliminated in both the ∆sirR and ∆crp mutants (Fig. 2b). These results indicated that both SirR and cyclic AMP receptor protein (CRP) were required for sulfite reduction, yet it was unknown whether these proteins regulated sirA expression independently or as part of a regulatory cascade
Summary
Shewanella species are facultative anaerobes that are abundant in freshwater and marine environments and are best known for their ability to use metals as electron acceptors for anaerobic respiration (see Fredrickson et al for review[1]). Similar to other Gram-negative bacteria, maturation of c-type cytochromes in S. oneidensis requires proteins encoded by genes in the ccmABCDE and ccmFGH operons. SO_0265 was identified as ccmI, and was initially determined to have a nonessential role in cytochrome c maturation in S. oneidensis[28], but subsequently found to have a role in maturation of canonical heme binding motifs and to be required for maturation of the nitrite reductase, NrfA29. It remains unknown if or what role CcmI plays in maturation of the sulfite reductase. We show that CRP controls expression of SirR, which in turn regulates expression of the sulfite reductase operon
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