The Significance of Type II and PrxQ Peroxiredoxins for Antioxidative Stress Response in the Purple Bacterium Rhodobacter sphaeroides

Masahiro Wakita,Shinji Masuda,Ken Motohashi,Toru Hisabori,Hiroyuki Ohta,Ken-Ichiro Takamiya

doi:10.1074/jbc.m702855200

Abstract

Two peroxiredoxins, classified as Type II and PrxQ, were characterized in the purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides. Both recombinant proteins showed remarkable thioredoxin-dependent peroxidase activity with broad substrate specificity in vitro. Nevertheless, PrxQ of R. sphaeroides, unlike typical PrxQs studied to date, does not contain one of the two conserved catalytic Cys residues. We found that R. sphaeroides PrxQ and other PrxQ-like proteins from several organisms conserve a different second Cys residue, indicating that these proteins should be categorized into a novel PrxQ subfamily. Disruption of either the Type II or PrxQ gene in R. sphaeroides had a dramatic effect on cell viability when the cells were grown under aerobic light or oxidative stress conditions created by exogenous addition of reactive oxygen species to the medium. Growth rates of the mutants were significantly decreased compared with that of wild type under aerobic but not anaerobic conditions. These results indicate that the peroxiredoxins are crucial for antioxidative stress response in this bacterium. The gene disruptants also demonstrated reduced levels of photopigment synthesis, suggesting that the peroxiredoxins are directly or indirectly involved in regulated synthesis of the photosynthetic apparatus.

Highlights

All organisms exposed to a variety of environments must acquire adaptation systems to sustain homeostasis
We found that R. sphaeroides PrxQ and other PrxQ-like proteins from several organisms conserve a different second Cys residue, indicating that these proteins should be categorized into a novel PrxQ subfamily
We report that R. sphaeroides has two Prxs that can be classified into Type II and PrxQ Prxs and that are critical for growth under aerobic conditions

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains and Growth Conditions—R. sphaeroides strain 2.4.1 was used as a wild-type strain. For measurement of the activity of Prxs, a reaction containing 50 mM Hepes/NaOH, pH 7.3, 20 ␮M TrxA, 5 ␮M AtNTR (recombinant protein expressed in E. coli) [22], and 0.2 mM NADPH was used. The obtained plasmid was transferred into R. sphaeroides cells by conjugation with the mobilizing strain S17-1 lysogenized with ␭pir [25, 26]. Kanamycin-resistant cells on plates containing 5% sucrose were selected as double-crossover candidates, and the chromosomal insertion was confirmed by Southern hybridization. The plasmid pZJDSmpPrxQ was transferred into R. sphaeroides cells by conjugation with the mobilizing strain S17-1 lysogenized with ␭pir [25, 26]. Streptomycin/kanamycin-resistant cells were selected as double-crossover candidates, and the chromosomal insertion was confirmed by Southern hybridization. Levels of light-harvesting complexes after spectroscopic analysis of the samples were determined as described [29]

RESULTS

RsPrxT RsPrxQ RsPrxT RsPrxQ

Wild type

DISCUSSION