Mislocalization of Rieske Protein PetA Predominantly Accounts for the Aerobic Growth Defect of tat Mutants in Shewanella oneidensis

Abstract

Shewanella oneidensis exhibits a remarkable versatility in respiration, which largely relies on its various respiratory pathways. Most of these pathways are composed of secretory terminal reductases and multiple associated electron transport proteins that contain cofactors such as Fe-S, molybdopterin, and NiFe. The majority of these cofactors are inserted enzymatically in the cytoplasm, and thus are substrates of the twin-arginine translocation (Tat) protein export system, which transports fully folded proteins. Using genomic array footprinting, we discovered that loss of TatA or TatC caused a reduction in the growth rate of S. oneidensis under aerobic conditions. Mutational analysis of the predicted Tat substrates revealed that PetA, the Rieske Fe-S subunit of the ubiquinol-cytochrome c reductase, predominantly dictates the aerobic growth defect of tat mutants in S. oneidensis. In addition, evidence is presented that the signal sequence in PetA appears to be resistant to cleavage after the protein is inserted into the cytoplasmic membrane.

The Twin Arginine Translocation System Is Essential for Aerobic Growth and Full Virulence of Burkholderia thailandensis

“…The inability to export PetA would result in the failure to generate an active quinolcytochrome c oxidoreductase (QCR) complex, which is functional during aerobic growth (33). Our work shows that in B. thailandensis, it is not PetA but possibly an operon encoding two proteins of unknown function (BTH_I2176/BTH_I2175) which is required for aerobic growth.…”

“…In E. coli O157:H7 (25), Pseudomonas aeruginosa (26), Agrobacterium tumefaciens (27), and Ralstonia solanacearum (28), the Tat system is required for anaerobic growth. Conversely, in Rhizobium leguminosarum (29), Myxococcus xanthus (42), M. smegmatis (41), and Shewanella oneidensis (33), Tat mutants show reduced growth under aerobic conditions. In some bacteria, the Tat system has been suggested to be critical for aerobic growth.…”

“…Several workers have previously attributed the difficulties in constructing Tat mutants to the requirement for PetA, a Rieske iron-sulfur subunit protein of the quinol-cytochrome c oxidoreductase complex (29)(30)(31)(32)(33), and in Shewanella oneidensis, the inability to export PetA by the Tat system has been shown to result in a reduced ability to grow aerobically (33). The inability to export PetA would result in the failure to generate an active quinolcytochrome c oxidoreductase (QCR) complex, which is functional during aerobic growth (33).…”

“…Conversely, in some species, there is evidence that the Tat system plays a role in aerobic growth. The molecular basis of this is not well understood but has been suggested to reflect the inability of bacteria to translocate PetA, a Rieske iron-sulfur subunit protein of the quinol-cytochrome c oxidoreductase complex (29)(30)(31)(32)(33). We report that the Tat system is essential for aerobic growth and virulence of B. thailandensis and modulates resistance to ␤-lactam antibiotics.…”

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

“…To determine whether Shewanella nanowires contain periplasm, we expressed either GFP fused to a signal sequence that enables GFP export to the periplasm (SI Appendix, Fig. S4) after folding in the cytoplasm (35) or YFP fused to the S. oneidensis periplasmic [Fe-Fe] hydrogenase large subunit HydA (SI Appendix). We observed fluorescence along the bacterial nanowires in both constructs ( Fig.…”