Abstract/Details

Isolation and characterization of multicytochrome gene cluster from <i>Shewanella putrefaciens</i> MR-1 involved in iron and manganese reduction


2000 2000

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Abstract (summary)

Dissimilatory Fe(III) and Mn(IV) reduction is an anaerobic respiratory process common to many environments. A large number of bacteria that utilize these metals as terminal electron acceptors have been isolated and identified for the past decade. However, the molecular mechanisms of metal reduction remain unclear despite intensive research in this area. There are no genes or proteins identified, which are directly involved in metal reduction. The major goal of this work is to identify and isolate genes related to Fe(III) and Mn(IV) reduction as well as to determine the specific functions of their products. The organism used in this work is the facultatively anaerobic metal reducer S. putrefaciens MR-1. Transposon mutagenesis was used to generate mutants solely deficient in Fe(III) and Mn(IV) reduction. Analysis of the interrupted regions in two such mutants, SR-8 and SR-21, led to the identification of three genes, designated mtrC, mtrA and mtrB. The deduced amino acid sequence of mtrC and mtrA revealed that these genes encode deca-heme c-type cytochromes. The third gene, mtrB, was shown to encode an outer membrane protein of 679 amino acids. mRNA and Western blot analyses indicated that the three genes are organized in a single operon, mtrCAB, which is expressed constitutively. To elucidate the role of each protein in metal reduction, gene replacement was used to generate strains lacking either mtrC, mtrA or mtrB. Whole-cell suspensions of mtrCA+B+, mirC+AB+ and mtrC+A+B mutants resulted in 4.6-fold, 31-fold and 70-fold decrease in iron reduction rates, respectively. In contrast to whole cells, the levels of iron reductase activity in the crude cell extracts of the mutants were similar to that observed in the wild type. These findings strongly suggest that the products of mtrC, mtrA and mtrB play an important role in metal reduction, however, they may not constitute the terminal metal reductase complex. Analysis of the DNA region upstream of mtrCAB revealed a presence of another multicytochrome gene cluster. Although the identified genes, mtrD, mtrE, mtrF and omcA, exhibited high degree of similarity to the mtrCAB operon, there was no evidence indicating their involvement in Fe(III) and Mn(IV) reduction in S. putrefaciens.

Indexing (details)


Subject
Microbiology;
Molecular biology
Classification
0410: Microbiology
0307: Molecular biology
Identifier / keyword
Biological sciences; Iron reduction; Manganese reduction; Multicytochrome gene cluster; Schewanella putrefaciens
Title
Isolation and characterization of multicytochrome gene cluster from <i>Shewanella putrefaciens</i> MR-1 involved in iron and manganese reduction
Author
Beliaev, Alexander S.
Number of pages
92
Publication year
2000
Degree date
2000
School code
0118
Source
DAI-B 61/02, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9780599645615, 059964561X
Advisor
Saffarini, Daad A.
University/institution
University of Massachusetts Amherst
University location
United States -- Massachusetts
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
9960736
ProQuest document ID
304608608
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/304608608
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