Fig. 3.

Analysis of changes in conserved residues in the predicted external loops between β-strands in the C-terminal region of HmsH. (a) Alignment of these regions of HmsH and HmsH-like proteins from: Y. pestis KIM10+ (Ype); P. fluorescens Pf-5 (Pfl); Pectobacterium atrosepticum SCR11043 (Pat; formerly Erwinia carotovora); E. coli K-12 substrain MG1655 (Eco); and X. oryzae KACC10331 (Xor). The black and grey boxes indicate residue identity and similarity, while arrows show the starts (open) and ends (filled) of the eight predicted loops between β-strands. The amino acid change line shows the amino acids individually changed to alanine. (b) Quantitative CR binding after 3 h incubation at 20 °C with CR-containing HIB medium by Y. pestis cells expressing amino acid substitutions in the predicted loops of HmsH. Controls are KIM6-2115 (in-frame ΔhmsH; HmsH⁻), KIM6+ (Hms⁺) and KIM6-2115(pNPM22) (HmsH⁺⁺). Plasmid pNPM22, which expresses hmsH from its native promoter, was also used to express HmsH with alanine substitutions. Thirteen mutants were not significantly different from positive controls (Table 2); only one of these, HmsH-D520A, along with the five mutants with significant decreases in CR binding, are shown. Data shown are the mean of two or more independent experiments with duplicate samples from each trial. Error bars, sd. For clarity, the secondary mutation (D520A) in HmsH-F651-D520A and HmsH-N655A-D520A is not shown in the labels.