TEXT

Top Abstract Text References

Dissimilatory Mn(IV) reduction can be one of the major terminal electron-accepting processes in redox-stratified natural water systems (3, 4, 9, 20, 21). Traditional enrichment techniques (5, 11, 17, 18, 22) and 16S rRNA-targeted nucleic acid hybridization analyses (5, 7) indicate that Mn(IV)-reducing bacteria constitute a major fraction of the anaerobic microbial population that resides in organic carbon- and Mn(IV)-rich anaerobic environments. The microbially catalyzed reduction of (insoluble) Mn(IV) oxides to their (soluble) Mn(II) forms may therefore be central not only to the biogeochemical cycling of Mn but also to the oxidation of naturally occurring and contaminant organic compounds. Dissimilatory Mn(IV) reduction also greatly influences the release and subsequent distribution of inorganic phosphate and toxic trace metals that otherwise adsorb to particulate Mn(IV) surfaces (9).

The proteobacteria Shewanella and Geobacter are the most extensively studied of the Mn(IV)-reducing microorganisms (for reviews, see references 9, 10, 20, and 21). Shewanella putrefaciens is particularly well adapted to redox-stratified environments, as it is capable of growing anaerobically on a wide variety of compounds as the sole terminal electron acceptor, including oxygen (O₂), nitrate (NO₃), nitrite (NO₂), ferric iron (Fe³⁺), trimethylamine N-oxide, sulfite (SO₃²), thiosulfate (S₂O₃²), uranyl carbonate (U⁶⁺), fumarate, and Mn(IV) oxides (6, 12, 13, 17-19, 21, 23, 24). Results from a variety of biochemical studies confirm that Mn(IV) reduction by S. putrefaciens is respiratory chain linked (14, 15, 17, 19), and recent genetic analyses of S. putrefaciens mutants with multiple respiratory deficiencies indicate that electron transport to Mn(IV) proceeds through a menaquinone pool (14) and at least one c-type cytochrome (16). Despite these findings, a Mn(IV)-specific terminal reductase gene or enzyme has yet to be isolated.

The present study describes the development and application of two rapid, plate-assay-based screening techniques for identifying Mn(IV) reduction-deficient (Mnr) mutants of S. putrefaciens 200. The inability of S. putrefaciens 200 to form anaerobic colonies on Mn(IV)-supplemented solid medium [most likely due to limiting local Mn(IV) concentrations or to toxic effects associated with elevated levels of produced Mn(II) (17)] necessitated the development of alternate plate-assay-based screening methods. The Mnr mutants generated in this study will be employed in subsequent genetic complementation analyses with an S. putrefaciens 200 gene clone bank to identify Mn(IV) reduction-specific genes and gene products. A similar strategy was recently employed in the isolation of wild-type DNA fragments that restore Fe(III) reduction capability to an array of Fe(III) reduction-deficient (Fer) mutants of S. putrefaciens 200 (6).

Chemical basis of Mn(IV)-specific colorimetric assays and plate-assay-based screening techniques. Mn(IV) oxides are known to oxidize benzidine hydrochloride ([1,1'-biphenyl]-4,4'-diamine · 2HCl) to a blue-colored, meroquinoid oxidation product known as "benzidine blue." Benzidene blue consists of one molecule of benzidine in the amine form, one molecule of benzidine in the imine form, and two equivalents of a monobasic acid (8) (Fig. 1). The Mn(IV)-benzidine hydrochloride reaction was used as the chemical basis for development of a colorimetric assay for determining Mn(IV) concentrations during anaerobic growth experiments. A pronounced maximum at 424 nm was observed when Mn(IV) stock solutions (synthesized as MnO₂ according to previously described procedures [1, 2]) were quenched in a 2 mM benzidine hydrochloride solution (10% acetic acid) and analyzed spectrophotometrically (data not shown). A linear correlation ( = 15.5 cm¹) between Mn(IV) concentration and A₄₂₄ values was observed in the 0 to 100 µM Mn(IV) range (Fig. 2). The Mn(IV)-benzidine hydrochloride reaction was also used as the chemical basis for development of plate-assay-based Mnr mutant screening techniques. Benzidine blue was readily detectable on the surface of Mn(IV)-supplemented nutrient agar [5 mM Mn(IV), pH 8.5 (Difco, Detroit, Mich.)] at approximately 10 min after flooding with 2 mM benzidine hydrochloride solution (Fig. 3). It was hypothesized that S. putrefaciens colonies expressing mutant (or wild-type) Mn(IV) reduction activity could be identified by the presence (or absence) of benzidine blue beneath the colony surface.

View larger version (7K): [in this window] [in a new window]   FIG. 1.   Products and reactants of Mn(IV)-catalyzed oxidation of benzidine hydrochloride to benzidine blue (adapted from reference 8).

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Standard curve of Mn(IV) concentration as a function of absorbance at 424 nm, measured after quenching in 2 mM benzidine hydrochloride.

View larger version (86K): [in this window] [in a new window]   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.

Identification of putative Mnr mutants. Screening technique 1 consisted of transferring mutagenized colonies (arising from ethyl methane sulfonate-treated cells, as described previously [6]) to Mn(IV)-supplemented nutrient agar and incubating them anaerobically for 24 h. Putative Mnr mutant phenotypes could be differentiated from the wild type at 10 min after flooding of the agar surface with benzidine hydrochloride; Mn(IV) was visually detectable as benzidine blue beneath putative Mnr mutant colonies (and anaerobic respiratory mutant T121) yet was not detectable beneath wild-type colonies (Fig. 3A). Approximately 1,000 mutagenized colonies were visually scored for the Mnr mutant phenotype, and four putative Mnr mutants were identified (Table 1). Three of the four putative Mnr mutants were deficient in the ability to grow anaerobically on a broad spectrum of compounds as the sole terminal electron acceptor and were not studied further (synthetic growth medium, terminal electron acceptor concentrations, and anaerobic growth conditions were identical to those described previously [6]). Cell numbers were determined via direct counts of acridine orange-stained cells according to previously described procedures (11). Particulate Mn(IV) was reduced to soluble Mn(II) prior to counting via addition of sodium dithionite (17). The fourth putative Mnr mutant (10-10) was proficient in anaerobic growth on all terminal electron acceptors except Mn(IV). During anaerobic growth on Mn(IV), strain 10-10 exhibited a lag phase nearly identical to that of the wild-type strain yet reached a final cell density that was only 20% of that reached by the wild-type strain (Fig. 4). In addition, strain 10-10 depleted Mn(IV) to levels that were only 39% of that depleted by the wild-type strain [as detected via the Mn(IV)-specific colorimetric assay described above]. Anaerobic respiratory mutant T121 (capable only of aerobic growth [25]) was unable to grow anaerobically [or to deplete Mn(IV)] under these experimental conditions. The wild-type strain was unable to grow anaerobically [or to deplete Mn(IV)] in anaerobic control experiments in which Mn(IV) (or lactate) was omitted. Mn(IV) was not depleted in abiotic control experiments (Fig. 4).

View larger version (20K): [in this window] [in a new window]   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.

View larger version (50K): [in this window] [in a new window]   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.

Analysis of digitized images of the Mnr and Fer mutant strains. Images displayed by colonies after application of the two screening techniques were recorded as 8-bit TIFF files with the UVP 7500 Gel Documentation System (UVP, Inc., Upland, Calif.). Image intensity (reported as mean pixel value in Table 2) was measured with NIH Image software, version 1.58 (anonymous FTP [zippy.nimh.nih.gov]; National Institutes of Health, Bethesda, Md.). Colony boundaries were demarcated with the circular selection tool, and image intensity was noted as the mean pixel value within each selected area. Errors associated with mean pixel differences within each colony (i.e., as a function of the demarcated area), between colonies of identical strains on the same plate, or between colonies of identical strains on different plates were minimal (Table 2). Analysis of digitized images of the 11 Fer mutants (Fig. 5) and the 20 randomly selected Mnr-positive control strains (Table 2) revealed that these strains produced images whose intensities were nearly identical to that of the wild-type strain. The near-wild-type images produced by these strains correlated with their ability to grow anaerobically on Mn(IV) as the sole terminal electron acceptor (Fig. 5). Analysis of digitized images of Mnr mutants 10-10 and 48-4 (as well as anaerobic respiratory mutant T121) after application of each screening technique revealed that our visual inspection methods were discerning Mnr mutants with image intensities of <14% of the wild-type strain (Fig. 3; Table 2). It is possible that Mnr mutants which display intensities of >14% exist and are therefore not detected by our visual inspection methods. Current work in the development of Mnr plate-assay-based screening techniques centers on automation of Mnr mutant detection methods via computer-assisted analysis of digitized colony images.