Identification of Genes Involved in Cytochrome c Biogenesis in Shewanella oneidensis , Using a Modified mariner Transposon

Abstract

A modified mariner transposon, miniHimar RB1, was generated to mutagenize cells of the metal-reducing bacterium Shewanella oneidensis. The use of this transposon led to the isolation of stable mutants and allowed rapid identification of disrupted genes. Fifty-eight mutants, including BG104 and BG148 with transposon insertions in the cytochrome c maturation genes ccmC and ccmF1, respectively, were analyzed. Both mutants were deficient in anaerobic respiration and cytochrome c production.

Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms

“…GSPD, a mutant with a transposon insertion in SOO166 (gspD) was isolated by using pMiniHimar RB-1 as described (20). Mutants deficient in Fe(III) reduction were identified as described (21), and phenotypic analysis of the mutants was performed as reported (22).…”

Comparative genomics analyses on EPS biosynthesis genes required for floc formation of Zoogloea resiniphila and other activated sludge bacteria

“…The mariner transposon mutant library (pMiniHmar RB1, courtesy by Dr. Daad Saffarini) was prepared (Bouhenni et al, 2005). Transposon insertional mutants (Km r ) were screened and isolated on R2A agar plates (1.5% agar, w/v) supplemented with 50 mg/ml of kanamycin.…”

“…But the disrupted genes had not been determined due to the technical limitation at that time. Therefore, transposon mutagenesis was conducted on the Z. resiniphila MMB strain by using the mariner transposon as previously described (Bouhenni et al, 2005) and a series of floc-formation deficient mutants had been isolated (Supplementary Table S5). These mutants did not form the zoogloeal flocs that settled to the bottom of culture tubes without agitation and homogenous turbid cell cultures could be observed.…”

“…Transposon insertional mutants (Km r ) were screened and isolated on R2A agar plates (1.5% agar, w/v) supplemented with 50 mg/ml of kanamycin. The transposon insertion sites were mapped as previously described (Bouhenni et al, 2005). For in-frame deletion mutants, the two-step protocol of selection (single cross-over, antibiotic resistance) and counter-selection (double crossover, sucrose sensitivity) was conducted using the suicide vector pDS3.0 (R6K replicon, sacB, Gm R )-based constructs with a fusion of upstream and downstream sequences of the target gene as previously described (Wan et al, 2004).…”

The Role of Shewanella oneidensis MR‐1 Outer Surface Structures in Extracellular Electron Transfer

“…Growth and reduction assays were as described previously [26]. BG167, a pilD mutant of S. oneidensis MR-1, was generated using the Mini-himar RB-1 transposon [27]. Chromosomal deletions of gspG, type IV pilus biogenesis, Msh pilus biogenesis, and flagellin genes were generated using the modified suicide plasmid pER2, a derivative of pDS3 [28] using described protocols [29].…”