A cytochrome c encoded by the nar operon is required for the synthesis of active respiratory nitrate reductase in Thermus thermophilus

Abstract

A cytochrome c (NarC) is encoded as the ¢rst gene of the operon for nitrate respiration in Thermus thermophilus. NarC is required for anaerobic growth and for the synthesis of active nitrate reductase (NR). The K K and N N subunits (NarG, NarJ) of the NR were constitutively expressed in narC: :kat mutants, but NarG appeared in the soluble fraction instead of associated with the membranes. Our data demonstrate for NarC an essential role in the synthesis of active enzyme and for the attachment to the membrane of the respiratory NR from T. thermophilus. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Membrane-Associated Maturation of the Heterotetrameric Nitrate Reductase ofThermus thermophilus

“…Digestion was stopped by boiling in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, and the proteins were separated by SDS-PAGE (14). NarG and NarJ were identified by Western blot with specific rabbit antisera (29) and detected by chemiluminescence with the ECL detection kit (Amersham).…”

“…Isolation of insertion mutants. The isolation of the narC::kat mutant has already been described (29). In this mutant, the kat cassette interrupted narC at nucleotide position 125 from its ATG translation start codon.…”

“…Upstream and downstream DNA sequences were added in both cases to provide appropriate arms for homologous recombination. Trans- formation and mutant selection were carried out as described previously (29). Mutants were checked by PCR and Southern blot.…”

“…The first gene of the operon is also specific to T. thermophilus and codes for a membrane-bound di-heme c-type cytochrome (NarC). We have shown that in the absence of NarC, the NarG protein remains soluble and inactive in nitrate reduction (29). Consequently, we proposed that NarC was required for the attachment of the NarGH complex to the membrane and that this binding was necessary for a subsequent "maturation" step.…”

Two Putative c -Type Multiheme Cytochromes Required for the Expression of OmcB, an Outer Membrane Protein Essential for Optimal Fe(III) Reduction in Geobacter sulfurreducens

“…Unlike the case for the low-molecular-weight cytochrome OmcF, which was found to be required either for transcriptional activation of the omcB gene or for the stability its transcripts (14), deletion of the omcG and omcH genes did not affect levels of omcB mRNA. In the case of the c-type cytochrome nitrate reductases of Wolinella succinogenes and Thermus thermophilus, deletion of a second cytochrome which served as a membrane anchor led to accumulation of the nitrate reductase in the soluble rather than the membrane fraction (28,33). However, failure to detect OmcB in the outer membrane of the triple mutant did not appear to be due to mistargeting or failure to incorporate heme, since OmcB could not be detected by Western blotting in either the inner membrane-enriched or soluble fractions of the triple mutant (data not shown).…”