Design and Application of Two Rapid Screening Techniques for Isolation of Mn(IV) Reduction-Deficient Mutants of Shewanella putrefaciens

Abstract

Chemical mutagenesis procedures and two newly developed rapid plate assays were used to identify two Mn(IV) reduction-deficient (Mnr) mutants of Shewanella putrefaciens. All eleven members of a set of previously isolated Fe(III) reduction-deficient (Fer) mutants displayed Mnr-positive phenotypes on the plate assays and were also capable of anaerobic growth on Mn(IV) as the sole terminal electron acceptor.

FIG. 1

Products and reactants of Mn(IV)-catalyzed oxidation of benzidine hydrochloride to benzidine blue (adapted from reference 8).

FIG. 2

Standard curve of Mn(IV) concentration as a function of absorbance at 424 nm, measured after quenching in 2 mM benzidine hydrochloride.

FIG. 3

(A and C) Plate images of Mnr mutants after application of Mnr screening techniques 1 (anaerobic) (A) and 2 (aerobic) (C) with strains oriented as follows: S. putrefaciens wild type, upper left; anaerobic respiratory mutant T121, upper right; Mnr mutant 48-4, bottom left; Mnr mutant 10-10, bottom right. (B and D) Plate images of Fer mutants after application of Mnr screening techniques 1 (anaerobic) (B) and 2 (aerobic) (D) with strains oriented as follows (from top to bottom): wild type, B29, B41, and A5 (lefthand column); T121, B31, B43, A27, and C23 (middle column); B25, B39, B45, and B101 (righthand column). Note that black color intensity corresponds to benzidine blue color intensity with each screening technique.

FIG. 4

Mn(IV) depletion (A) and anaerobic growth (B) of wild-type S. putrefaciens (•), anaerobic respiratory mutant T121 (○), and Mnr mutants 10-10 (▴) and 48-4 (▵) on Mn(IV) as the sole terminal electron acceptor. Anaerobic control experiments included incubations in which lactate (), Mn(IV) (□), or cells (⧫) were omitted. The initial cell density was (1 ± 0.2) × 10⁷ cells per ml for each culture. Mn(IV) was determined colorimetrically, and acridine orange-stained cells were counted directly. Values are means of three parallel but independent anaerobic incubations; error bars represent standard deviations. Some of the error bars cannot be seen due to the small standard deviations.

FIG. 5

Comparison of digitized colony image intensities (detected after application of screening techniques 1 and 2), final cell densities, and Mn(IV) depletion after anaerobic growth on Mn(IV) by wild-type S. putrefaciens 200, anaerobic mutant T121, Mnr mutants 48-4 and 10-10, and the 11 Fer mutants. Values of image intensities are means of pixel intensities obtained from 10 individual colonies screened on separate plates (0.40-mm² demarcated area per colony). Values for final cell densities and Mn(IV) depletion are means of three parallel but independent anaerobic growth experiments. Error bars represent standard deviations. All values were normalized to wild-type levels and expressed as percentages.