Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

Abstract

Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.Shewanella oneidensis MR-1 is a metal reducer that uses more than 14 terminal electron acceptors for respiration. These electron acceptors include oxygen, nitrate, fumarate, dimethyl sulfoxide (DMSO), Fe(III) oxides, uranium, and chromium (21,25,26). In Escherichia coli and other bacteria, the shift from aerobic respiration to anaerobic respiration requires activation of the global transcriptional regulator FNR (11,34). FNR is an oxygen-sensing protein that is activated under anaerobic conditions by the formation of a [4Fe-4S] cluster (14). The S. oneidensis FNR homolog, EtrA, complements an E. coli FNR mutant (27) but does not appear to have the same role as the E. coli protein in S. oneidensis (17). Our previous findings demonstrated that instead of EtrA, the cyclic AMP (cAMP) receptor protein (CRP) controls anaerobic respiration in S. oneidensis MR-1 (28). crp mutants are deficient in anaerobic respiration of Fe(III), Mn(IV), fumarate, nitrate, and DMSO. Furthermore, fumarate, DMSO, and nitrate reductase activities are either severely decreased or undetectable in the crp mutants, suggesting that CRP regulates the expression of these anaerobic reductases (28).Although genetic and phenotypic data clearly have implicated CRP in the activation of anaerobic reductase systems in S. oneidensis MR-1, the mechanisms of this regulation remain unclear. CRP lacks obvious redox-sensing domains and is not expected to respond to changes in oxygen concentrations like FNR. Complementation of the S. oneidensis crp mutants with E. coli crp indicates that CRP is activa...

A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime

“…Variations in expression were regarded as not significantly altered (NS) if the fold change was below 2 or the padj value was above 0.05. The expression data is compared with a study conducted by Charania et al (2009) The heme sites within the cytochrome pool are interconnected and therefore, addition of an electron acceptor to cells where electron storage sites are fully occupied leads to a complete reoxidation of the c-type cytochrome pool. Of note, a variety of different electron acceptors leads to a similar total reoxidation of the pool.…”

“…CyaC is the major cyclic adenosine monophosphate adenylate cyclase and therefore involved in the synthesis of the effector molecule (Charania et al, 2009). Surprisingly, the cctA/fccA-deletion mutant was not affected in its transcription of essential c-type cytochromes such as mtrC, nrfA or cymA.…”

The Role of Shewanella oneidensis MR‐1 Outer Surface Structures in Extracellular Electron Transfer

“…BG167, a pilD mutant of S. oneidensis MR-1, was generated using the Mini-himar RB-1 transposon [27]. Chromosomal deletions of gspG, type IV pilus biogenesis, Msh pilus biogenesis, and flagellin genes were generated using the modified suicide plasmid pER2, a derivative of pDS3 [28] using described protocols [29]. DNA fragments for generation of deletions or complementation of mutants were amplified by PCR using Phusion polymerase (Clontech) and primers listed in Table 2.…”

Shewanella oneidensis MR-1 Sensory Box Protein Involved in Aerobic and Anoxic Growth

“…The class III adenylate cyclase was recently shown to regulate anaerobic respiration in S. oneidensis MR-1, and separate studies have shown that cyclic AMP (cAMP) levels were involved in the regulation of anaerobic respiration (8,44). Hence, SO3389 may be a sensor involved in the global regulation of different modulators for the up-and down-expression of genes related to shifts between aerobic and anoxic metabolism.…”