Skip to main content
. 2023 Jun 13;89(7):e00575-23. doi: 10.1128/aem.00575-23

FIG 1.

FIG 1

(A to C) Plasmid map of pCF702, a suicide-vector construct for deletion of the Mth60 fimbria-encoding operons (A) and the agarose gel of a corresponding PCR analysis (B), including a schematic depiction of the genomic region before (wild-type M. thermautotrophicus [w-t M.t.]) and after the deletion (Δfim) of mth58-mth61 (C). (A) The suicide-vector construct pCF702 consists of five exchangeable modules flanked by unique restriction enzyme-recognition sites, as stated in parentheses below. The origin of replication ColEI for E. coli, including a tra region for mobilization during conjugation (NotI, PmeI), the antibiotic resistance against chloramphenicol (Camr) as a selectable marker for E. coli (PmeI, SalI), and 1-kb upstream (SalI, AscI) and downstream homologous regions (FseI, NotI) for homologous recombination in M. thermautotrophicus ΔH are shown. A thermostable neomycin resistance gene (Neor) with constitutive promoter PSynth and terminator Tmcr as a selectable marker for M. thermautotrophicus ΔH is located between the homologous flanking regions (AscI, FseI). (B) PCR analysis with four primer combinations to confirm the Mth60-fimbriae operon deletion. Two primer combinations amplify a fragment inside the Mth60 fimbria-encoding operons (60, S), and two primer combinations amplify a fragment outside the Mth60 fimbria-encoding operons (M, L). These combinations result in amplified fragments of reduced lengths since the Mth60 fimbria-encoding operons (2.8 kb) were substituted with Neor (1.2 kb). (C) The schematic depiction of the genomic region before and after the deletion of mth58-61 shows the distribution of the individual genes mth58 to mth61 and the substitution with Neor after integration of the suicide-vector construct. The lines depicting primer pairs L (light blue), M (green), S (yellow), and 60 (dark red) correspond with the fragments in panel B. FW and RV represent the forward and reverse complements of the respective primer combinations. While fragments M and L appear in different lengths in both strains, S and 60 are present only in wild-type M. thermautotrophicus ΔH.