SEARCH

SEARCH BY CITATION

Fig. S1. Genotype of the G. metallireducens ΔfliC::Spr mutant. DNA gels showing PCR results using a primer annealing 760 bp upstream of the fliC coding sequence and a second primer annealing to the spectinomycin resistance cassette (A) and PCR results using a primer annealing 500 bp upstream of fliC and a second primer annealing within the fliC coding sequence (B) with potential mutants (lanes 1–4) and the wild-type (lane wt). The numbers on the left indicate the band sizes in kb for the NEB 1 kb ladder used as a marker (lane ladder). Genomic DNA was used as template for PCR reactions.

Fig. S2. Genotype of the G. metallireducens ΔpilA::loxP mutant. DNA gels showing PCR results using a primer annealing 600 bp upstream of pilA coding sequence and a second primer annealing in the spectinomycin resistance cassette (A) and PCR results using a primer annealing 500 bp upstream of pilA and a second primer annealing within the coding sequence of pilA (B) with potential ΔpilA::SprloxP mutants (lanes 1–5) and the wild-type (lane wt). (C) DNA gel showing PCR results using primers annealing 500 bp upstream and downstream of the pilA coding sequence with pilA mutants obtained after the introduction of the Cre recombinase expression plasmid (lanes ΔpilA::loxP 1-2), a control mutant before this treatment (lane ΔpilA::SprloxP) and the wild-type (lane wt). The numbers on the left indicate the band sizes in kb for the NEB 1 kb ladder used as a marker (lane ladder). Genomic DNA was used as template for PCR reactions.

Fig. S3. Gene deletion in G. metallireducens. Single-step gene replacement of fliC (A). A plasmid bearing a construct containing the 500 bp upstream and downstream of the coding sequence (CDS) of fliC separated by a spectinomycin resistance cassette was linearized by restriction enzyme digestion. The linearized plasmid was electroporated into G. metallireducens. Homologous recombination resulted in the replacement of the fliC wild-type allele by the mutant allele. Markerless deletion of pilA with the Cre-lox system (B). Single-step gene replacement was used to replace the pilA wild-type allele with a mutant allele in which the coding sequence of pilA was replaced by a spectinomycin resistance cassette flanked by two loxP sites. Introduction of the Cre recombinase resulted in the loss of the spectinomycin resistance cassette by recombination of the two loxP sites.

Table S1. Bacterial strains and plasmids used in this study.

Table S2. Primers used for mutant construction and genotype validation.

Appendix S1. Experimental procedures.

FilenameFormatSizeDescription
EMI4_305_sm_Appendix.doc56KSupporting info item
EMI4_305_sm_Figs.pdf108KSupporting info item
EMI4_305_sm_Tables.doc85KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.