Development of a genetic system for Geobacter sulfurreducens

Abstract

Members of the genus Geobacter are the dominant metal-reducing microorganisms in a variety of anaerobic subsurface environments and have been shown to be involved in the bioremediation of both organic and metal contaminants. To facilitate the study of the physiology of these organisms, a genetic system was developed for Geobacter sulfurreducens. The antibiotic sensitivity of this organism was characterized, and optimal conditions for plating it at high efficiency were established. A protocol for the introduction of foreign DNA into G. sulfurreducens by electroporation was also developed. Two classes of broad-host-range vectors, IncQ and pBBR1, were found to be capable of replication in G. sulfurreducens. In particular, the IncQ plasmid pCD342 was found to be a suitable expression vector for this organism. When the information and novel methods described above were utilized, the nifD gene of G. sulfurreducens was disrupted by the single-step gene replacement method. Insertional mutagenesis of this key gene in the nitrogen fixation pathway impaired the ability of G. sulfurreducens to grow in medium lacking a source of fixed nitrogen. Expression of the nifD gene in trans complemented this phenotype. This paper constitutes the first report of genetic manipulation of a member of the Geobacter genus.

FIG. 1

Stability of IncQ and pBBR1 vectors in G. sulfurreducens. (A) Purification of IncQ plasmids from G. sulfurreducens transformants. Plasmid DNA was purified from G. sulfurreducens transformed with pJRD215 (lane 1), pJRC2 (lane 2), and pCD342 (lane 3) by alkaline lysis (26). Following purification, plasmid DNA was digested with the restriction enzymes BglII and HindIII. The sizes of the expected restriction fragments for the three plasmids are as follows: 9.1 and 1.2 kb for pJRD215; 10.4 kb for pJRC2 (23); and 8.0 and 1.7 kb for pCD342 (10). (B) Stability of pJRD215 and pBBR1MCS-2 in G. sulfurreducens under nonselective conditions. Assessment of the stability of these plasmids in the absence of antibiotic selection is described in detail in Materials and Methods. Data are means ± standard errors of four independent experiments. (C) Expression of green fluorescent protein from an IncQ expression vector in G. sulfurreducens. Green fluorescent protein expression was visualized by Nomarski microscopy using a fluorescein isothiocyanate filter. Panel i, DL1, wild-type G. sulfurreducens. Panel ii, DL1/pCD354, G. sulfurreducens transformed with pCD354 (10).

FIG. 2

Confirmation of nifD gene disruption by Southern blot analysis. (A) Restriction map of the G. sulfurreducens nifHDK operon. (B) Restriction map of pBRnif::kan. The vector pBR322 is indicated by thick dotted lines. (C) Restriction map of G. sulfurreducens nifHDK operon containing the nifD1::kan insertion mutation. (D) Southern blot of genomic DNA prepared from wild-type (lane 1) and nifD1::kan strains (lanes 2 to 5) of G. sulfurreducens. Genomic DNA was digested with the restriction enzyme EcoRV and probed with a BglII/EcoRV restriction fragment of the kanamycin resistance cassette of pBRnif::kan. Expected radiolabeled bands are as follows: lane 1, none; lanes 2 to 5, 2.3 kb. (E) Southern blot of genomic DNA prepared from wild-type (lane 1) and nifD1::kan (lanes 2 to 5) strains of G. sulfurreducens. Genomic DNA was digested with the restriction enzyme EcoRV, blotted, and then probed with a BamHI/EcoRI restriction fragment of pBRnif::kan. Expected radiolabeled bands are as follows: lane 1, 1.1 and >2.3 kb; lanes 2 to 5, 2.3 and >2.3 kb. All restriction maps were based on sequence data obtained from http://www.tigr.org.

FIG. 3

Nitrogen fixation by wild-type (DL1) and nifD1::kan (DL2D) G. sulfurreducens. FWAFC cultures of strains DL1 and DL2D were washed three times with FWAFC medium lacking both N₂ and NH₄Cl (N₂ replaced by Ar) in order to obtain cell suspensions containing as little N₂ and NH₄Cl as possible. At time zero, the washed cells were inoculated into three types of FWAFC media: (i) FWAFC, (ii) ammonium-free FWAFC, and (iii) FWAFC lacking both N₂ and NH₄Cl at a final concentration of ∼6 × 10⁶ cells/ml. Fe(II) concentration (in millimolars) and cell density (in cells/milliliter) were determined on 0.1-ml samples as previously described (18, 19). Fe(II) concentrations are means ± standard deviations of measurements obtained from three independent cultures, whereas cell densities were determined from one representative culture.

FIG. 4

Complementation of nifD1::kan phenotype by expression of nifD gene in trans. In order to select for the maintenance of pCDSnifD, strain DL2D/pCDSnifD was maintained in FWAFC medium containing 400 mg of streptomycin/ml until the beginning of the experiments. To obtain cell suspensions containing as little N₂, NH₄Cl, and streptomycin as possible, FWAFC cultures of strains DL1 (wild type) and DL2D/pCDSnifD were washed three times with FWAFC medium without both N₂ and NH₄Cl (N₂ replaced by Ar). At time zero, washed cells were inoculated into three types of FWAFC media: (i) FWAFC, (ii) ammonium-free FWAFC, and (iii) FWAFC lacking both N₂ and NH₄Cl at a final concentration of ∼3.6 × 10⁶ cells/ml. Fe(II) concentration and cell density were determined on 0.1-ml samples as previously described (18, 19). Fe(II) concentrations are the means ± standard deviations of measurements obtained from three independent cultures, whereas cell densities were determined from one representative culture.