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Overexpression of the adenylate cyclase gene cyaC facilitates current generation by Shewanella oneidensis in bioelectrochemical systems

ABSTRACT: Electrochemically active bacteria (EAB) are capable of electrochemical interactions with electrodes via extracellular electron transfer (EET) pathways and serve as essential components in bioelectrochemical systems. Previous studies have suggested that EAB, such as Shewanella oneidensis MR-1, use cyclic AMP (cAMP) receptor proteins for coordinately regulating the expression of catabolic and EET-related genes, allowing us to hypothesize that the intracellular cAMP concentration is an important factor determining electrochemical activities of EAB. The present study constructed an MR-1 mutant, cyaC-OE that overexpressed cyaC, a gene encoding a membrane-bound class III adenylate cyclase, and examined its electrochemical and transcriptomic characteristics. We show that intracellular cAMP concentration in cyaC-OE is more than double that in wild-type MR-1, and cya-OE generates approximately two-fold higher current in BES than the wild type. In addition, the expression of genes involved in EET and anaerobic carbon catabolism is up-regulated in cya-OE as compared to that in the wild type. These results suggest that enhancement of the intracellular cAMP level is a promising approach for constructing an EAB with high catabolic and electrochemical activities.

ORGANISM(S): Shewanella oneidensis MR-1 Shewanella oneidensis

PROVIDER: GSE129679 | GEO | 2019/11/01

REPOSITORIES: GEO

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Project description:Anaerobic respiration in the metal reducer Shewanella oneidensis MR-1, unlike other bacteria, is regulated by the cAMP receptor protein, CRP. 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 E. 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, DMSO, or Fe(III), whereas the 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 the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways that include DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella 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. To explore the roles of CyaC and CRP in the regulation of anaerobic respiration in Shewanella oneidensis MR-1, the genome-wide transcriptome profiles of ÎcyaC and Îcrp mutants were compared to those of the wild type. Given the growth deficiencies displayed by Îcrp and ÎcyaC under anaerobic conditions, all strains were cultured in chemostats under O2 limitation. Since both Îcrp and ÎcyaC strains are not impaired in aerobic respiration, oxygen-limited conditions provided the means to maintain equal growth rates (0.06 h-1) among the mutant and wild-type strains thus eliminating the effect of growth on the overall transcriptome profiles. At the same time, while growing aerobically, the cells decreased dissolved O2 concentration in the medium below the detection threshold (DOTâ¤1%) allowing for the expression of anaerobic respiratory genes. The latter was confirmed by the reduction of fumarate by the wild-type culture which produced stoichiometric amounts of succinate under O2-limited conditions. The optical density (OD600) of the wild type, ÎcyaC, and Îcrp chemostat cultures used for transcriptome profiling was 0.50, 0.45 and 0.40, respectively. All the experiments were carried out in duplicate.

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Overexpression of the adenylate cyclase gene cyaC facilitates current generation by Shewanella oneidensis in bioelectrochemical systems

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