Transcriptional and functional characterization of a putative azoreductase gene in Shewanella oneidensis MR-1

Irina Mugerfeld, Purdue University

Abstract

Shewanella oneidensis MR-1 is a facultatively anaerobic gamma-proteobacterium that can respire anaerobically by utilizing a wide variety of alternative electron acceptors including different soluble and insoluble metals. The MR-1 gene SO3585 is annotated as a putative azoreductase and was shown in previous functional genomics studies to be significantly upregulated at both the mRNA and protein levels in response to acute chromate exposure. Azoreductases are enzymes that catalyze the reductive cleavage of azo dyes to aromatic amines. Azo dyes pose a serious concern as xenobiotic pollutants that are released into the environment as industrial effluents from textile and dye manufacturing. SO3585 shares 32% amino acid sequence identity to the azoreductase from Bacillus cereus, and 28% sequence identity to Pseudomonas putida ChrR and Escherichia coli YieF, two soluble flavoproteins shown to exhibit chromate reductase activity. However, a Hidden Markov Model-based search of the TIGRFAM & Pfam database indicates that SO3585 most likely belongs to the NADPH-dependent FMN reductase family of proteins. Reverse transcription-PCR was used to show that so3585 is co-transcribed with two tightly clustered downstream genes, so3586 (encoding a predicted glyoxalase family protein) and so3587 (a hypothetical membrane-associated protein). Using 5' Rapid Amplification of cDNA Ends analysis, the transcriptional start site of the so3585-86-87 operon was localized to an adenine residue positioned 26 bp upstream of the annotated so3585 translational start codon. Phenotype characterization demonstrated that an MR-1 strain harboring a nonpolar in-frame deletion of the so3585 gene was similar to the wild type MR-1 strain in its ability to decolorize the azo dyes Orange II and Direct Blue 15 under aerobic conditions. However, growth studies showed that the mutant was hypersensitive to different transition metals, most notably Cd(II) and Cu(II). Reduction experiments showed the mutant to be initially impaired in its chromate reduction ability, although both strains eventually showed complete reduction of chromate within a similar time period. Biochemical analysis of a purified recombinant SO3585 indicated no detectable chromate or azo dye reductase activity associated with this S. oneidensis protein. In conclusion, the data suggest that the annotation of azoreductase is not correct and that the protein, in combination with SO3586 and SO3587, is likely involved in detoxification mechanisms under metal stress.

Degree

M.S.

Advisors

Thompson, Purdue University.

Subject Area

Microbiology

COinS