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, 81 (20), 7178-86

Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter Sulfurreducens

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Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter Sulfurreducens

Chi Ho Chan et al. Appl Environ Microbiol.

Abstract

Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ΔimcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. These tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.

Figures

FIG 1
FIG 1
Markerless deletion of GSU0299 from the G. sulfurreducens genome. (A) Flanking sequences of the GSU0299 transposon in the sacB vector pK18mobsacB integrate in this example upstream of GSU0299 in the presence of kanamycin. Cells plated on sucrose select for a second recombination event that can either generate the WT or the deletion allele. (B) Primers p1 to p4 depicted in panel A screen for deletion of GSU0299. (C) Full genome sequencing found 150× coverage of reads spanning the deletion junction in fgrM, verifying the transposon was removed. No reads spanned the WT junction, and no other mutations were found in the ΔGSU0299 genome. (D) The resulting ΔGSU0299, fgrM+ cells grew from the point of inoculation in 0.4% agarose, indicating motility compared to the nonmotile WT.
FIG 2
FIG 2
Slower doubling times with pBBR1 and kanamycin inhibition of biofilm growth on electrodes (A) G. sulfurreducens cells had a longer doubling time (d) in fumarate medium when carrying the high copy number plasmid pBBR1 (pSRK-Km) compared to WT and WT carrying pRK2 (pRVMCS-2; ± the standard deviations [SD]; n = 3 for each condition, 200 μg of kanamycin/ml added to plasmid-containing cultures). (B) WT G. sulfurreducens carrying pRK2-Geo2i reached a lower current density on electrodes poised at 0.24 V versus SHE at 50 h and had a longer doubling time (d) in the presence of both low (50 μg/ml) and normal (200 μg/ml) levels of kanamycin. (Representative curve from n = 6 for 5 μg/ml; n = 7 for 50 μg/ml, and n = 2 for 200 μg/ml kanamycin). (C) Promoter regions designed for vectors described in the present study. The multiple cloning site (MCS) contains the following unique restriction sites: NdeI, EcoRI, KpnI, PmlI, DraIII, SacI, AflII, BglII, SnaBI, AgeI, NheI, and PstI. (D) Sequence of the pRK2-Geo2i-inducible promoter. The GSU0800 promoter controls vanR expression. VanR binding sites on the modified acpP promoter are highlighted by boxes, and the ribosome binding site (RBS) and start codon (MET) are indicated.
FIG 3
FIG 3
Vanillate induction under Fe(III) citrate, Mn(IV) oxide, and poised electrode growth conditions. (A) Growth using Fe(III) citrate as the terminal electron acceptor in WT carrying the empty vector pRK2-Geo2i (●) and ΔimcH strain carrying pImcH16 (imcH+ in pRK2-Geo2i). ΔimcH/pImcH16 cells were induced with 50 μM vanillate at 24 h (▲) or left uninduced (◆). (Results are indicated as ± the SD; n = 3 for each condition). (B) Mn(IV) oxide reduction by WT G. sulfurreducens carrying pRK2-Geo2i (●) compared to ΔimcH carrying pImcH16 with (■) and without (◆) induction by 50 μM vanillate. (Results are indicated as ± the SD; n = 3 for each condition). (C) Current density on electrodes poised at 0.24 V versus SHE at 80 h, comparing WT carrying empty pRK2-Geo2i to ΔimcH carrying pRK2-Geo2i, and ΔimcH carrying the vanillate-inducible pImcH16 with 50 and 100 μM vanillate added at the time of inoculation. (Results are indicated as ± the SD, each point = one independent reactor; n = 27 reactors in total).
FIG 4
FIG 4
Real-time Fe(III) citrate reduction assay. Fe(III) citrate reduction by WT in 10-ml Balch tubes (○) compared to WT in the real-time 96-well assay (●). The levels of complementation of the ΔimcH strain by imcH cloned under the native constitutive promoter acpP in pImcH15 (▼), the taclac promoter in pImcH17 (▲), and the empty vanillate-inducible vector pRK2-Geo2i (■) were compared. Increasing concentrations of vanillate inducer in the culture used to grow cells increased Fe(III) citrate reduction rates in the ΔimcH strain carrying the inducible pImcH16 (◆). (Results are indicated as ± the SD; n = 3 for each condition). The rates of Fe(III) citrate reduction were normalized to the amount of cell protein.

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