Aeromonas veronii strain HM21 was originally isolated by Joerg Graf from the medical leech and has been extensively characterized, including a complete genome sequence25 (link). To create the A. veronii ΔgbpA strain, a vector containing a kanamycin resistance cassette was transformed into SM10 E. coli. Conjugation between wild-type A. veronii HM21RS and the vector carrying SM10 E. coli strain was carried out, allowing the kanamycin resistance gene to replace the gbpA locus in A. veronii via allelic exchange. Candidate gbpA deletion strains were selected for loss of the plasmid and maintenance of kanamycin resistance. Insertion of the kanamycin cassette into the gbpA locus was verified in these candidates by PCR. Fluorescently marked derivatives of these strains were engineered with an established Tn7 transposon-based approach26 (link). Briefly, a cassette containing the constitutively active synthetic promoter Ptac cloned upstream of genes encoding dTomato or superfolder GFP was chromosomally inserted at the attTn7 locus to generate A. veronii attTn7::Ptac-sfGFP and A. veronii ΔgbpA attTn7::Ptac-dTomato. Joerg Graf provided the A. veronii Δt2ss mutant and isogenic complementation strain A. veronii Δt2ss+T2SS27 (link),28 (link). Ron Taylor provided V. cholerae classical O1 isolate CG842 and isogenic mutant V. cholerae ΔgbpA and complementation strain V. cholerae ΔgbpA+pGbpA17 (link).
To assay growth on minimal medium supplemented with 0.4% colloidal chitin or GlcNAc, WT and ∆gbpA Aeromonas were normalized to an OD600 of 0.005 and incubated with shaking for 24 hr at 30°C. The size and density of colloidal chitin scatters the incoming light used to monitor growth kinetics in a plate-based growth assay, leading to inconsistent readings. Thus, to determine the impact of GbpA on Aeromonas growth on colloidal chitin, each strain was grown in culture tubes and bacterial growth samples aliquoted after allowing the colloidal chitin to settle from solution. This method produced consistent and reliable measurements of bacterial growth.
To assay growth on minimal medium supplemented with 0.4% colloidal chitin or GlcNAc, WT and ∆gbpA Aeromonas were normalized to an OD600 of 0.005 and incubated with shaking for 24 hr at 30°C. The size and density of colloidal chitin scatters the incoming light used to monitor growth kinetics in a plate-based growth assay, leading to inconsistent readings. Thus, to determine the impact of GbpA on Aeromonas growth on colloidal chitin, each strain was grown in culture tubes and bacterial growth samples aliquoted after allowing the colloidal chitin to settle from solution. This method produced consistent and reliable measurements of bacterial growth.
