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Sequence and genetic characterization of etrA, an fnr analog that regulates anaerobic respiration in Shewanella putrefaciens MR-1.

ABSTRACT: An electron transport regulatory gene, etrA, has been isolated and characterized from the obligate respiratory bacterium Shewanella putrefaciens MR-1. The deduced amino acid sequence of etrA (EtrA) shows a high degree of identity to both the Fnr of Escherichia coli (73.6%) and the analogous protein (ANR) of Pseudomonas aeruginosa (50.8%). The four active cysteine residues of Fnr are conserved in EtrA, and the amino acid sequence of the DNA-binding domains of the two proteins are identical. Further, S. putrefaciens etrA is able to complement an fnr mutant of E. coli. In contrast to fnr, there is no recognizable Fnr box upstream of the etrA sequence. Gene replacement etrA mutants of MR-1 were deficient in growth on nitrite, thiosulfate, sulfite, trimethylamine-N-oxide, dimethyl sulfoxide, Fe(III), and fumarate, suggesting that EtrA is involved in the regulation of the corresponding reductase genes. However, the mutants were all positive for reduction of and growth on nitrate and Mn(IV), indicating that EtrA is not involved in the regulation of these two systems. Southern blots of S. putrefaciens DNA with use of etrA as a probe revealed the expected etrA bands and a second set of hybridization signals whose genetic and functional properties remain to be determined.

SUBMITTER: Saffarini DA

PROVIDER: S-EPMC206972 | biostudies-other | 1993 Dec

REPOSITORIES: biostudies-other

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Sequence and genetic characterization of etrA, an fnr analog that regulates anaerobic respiration in Shewanella putrefaciens MR-1.

Saffarini D A DA Nealson K H KH

Journal of bacteriology 19931201 24

An electron transport regulatory gene, etrA, has been isolated and characterized from the obligate respiratory bacterium Shewanella putrefaciens MR-1. The deduced amino acid sequence of etrA (EtrA) shows a high degree of identity to both the Fnr of Escherichia coli (73.6%) and the analogous protein (ANR) of Pseudomonas aeruginosa (50.8%). The four active cysteine residues of Fnr are conserved in EtrA, and the amino acid sequence of the DNA-binding domains of the two proteins are identical. Furth ...[more]

PMID: 8253682

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Project description:Anaerobically grown cells of the metal-reducing bacterium Shewanella putrefaciens MR-1 contain multiple outer membrane (OM) cytochromes. A gene replacement mutant (strain OMCB1) lacking the OM cytochrome OmcB is markedly deficient in the reduction of MnO2 and exhibits reduced rates of Fe(III) reduction. The levels of other OM cytochromes are also decreased in OMCB1. Complementation of OMCB1 with wild-type omcB did not restore any of these defects. However, a 21-kb genomic fragment from MR-1, which included omcB and 19 kb of downstream DNA, fully restored MnO2 and Fe(III) reduction and the full complement of OM cytochromes to OMCB1. A 14.7-kb DNA fragment, including omcB and 12 kb of downstream DNA, provided only a modest increase in MnO2 reduction and OM cytochrome content, but it fully restored Fe(III) citrate reduction and partially restored FeOOH reduction. While omcB mRNA was readily detected in this complement, the OmcB protein was not detected in any cellular compartment. The restoration of Fe(III) reduction despite the absence of OmcB suggests that OmcB itself is not required for Fe(III) reduction. Another OM cytochrome, OmcA, was mislocalized to the cytoplasmic membrane of OMCB1. Only the 21-kb genomic fragment was able to restore proper localization of OmcA to the OM. This 21-kb fragment does not contain omcA, but it does contain several open reading frames (ORFs) downstream from omcB. The most downstream of these ORFs (altA) encodes a putative AraC-like transcriptional regulator. However, a gene replacement mutant of altA resembled the wild type with respect to MnO2 reduction, OM cytochrome content, and the localization of OmcA and OmcB to the OM. Since OMCB1 continues to express genes immediately downstream from omcB, the lack of expression of this downstream DNA does not explain its phenotype or the need for the large complementing fragment. The results suggest that the DNA downstream of omcB must be present in cis in order to restore Fe(III) reduction, MnO2 reduction, OM cytochrome content, and the localization of OmcA and OmcB to the OM.

Dataset Information

Sequence and genetic characterization of etrA, an fnr analog that regulates anaerobic respiration in Shewanella putrefaciens MR-1.

Publications

Sequence and genetic characterization of etrA, an fnr analog that regulates anaerobic respiration in Shewanella putrefaciens MR-1.

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