Table 2.

Effect of amino acid substitutions in HmsH on biofilm phenotypes and protein level

Amino acid substitutions in HmsH	CR plate	CV staining (%)*	CR binding (%)*	Protein level†
Substitution in loop 1‡
HmsH-D520A	Red colonies	80 %	100 %	RL
Substitution in loop 2
HmsH-F549A	Red colonies	85 %	100 %	RL
Substitutions in loop 4
HmsH-P615A	Red colonies	95 %	83 %	RL
HmsH-R617A	Red colonies	89 %	100 %	RL
Substitutions in loop 5
HmsH-F651A-D520A§	Red colonies	105 %	63 %	<WT
HmsH-D653A	Pink colonies	83 %	46 %	<WT
HmsH-N655A-D520A§	Small pink colonies	98 %	60 %	<WT
HmsH-R657A	Red colonies	89 %	60 %	≥WT <RL
Substitutions in loop 6
HmsH-P697A	Red colonies	97 %	90 %	≥WT <RL
HmsH-D691A	Red colonies	80 %	100 %	RL
HmsH-Y694A	Red colonies	95 %	53 %	RL
Substitution in loop 7
HmsH-Y737A	Red colonies	101 %	80 %	RL
Substitutions in loop 8
HmsH-P769A	Red colonies	93 %	83 %	≥WT <RL
HmsH-E775A	Red colonies	97 %	116 %	RL
HmsH-D771A	Red colonies	85 %	93 %	RL
HmsH-R774A	Red colonies	85 %	90 %	RL
Substitutions in the N-terminal region
HmsH-D263A	White-to-pink, then red colonies	58 %	100 %	>WT
HmsH-E345A-D520A§	Red colonies	95 %	118 %	>WT
HmsH-E347A	Red colonies	103 %	88 %	>WT
HmsH-D397A-D520A§	Red colonies	86 %	103 %	>WT
HmsH-R433A-D520A§	Red colonies	100 %	107 %	>WT
HmsH-R479A	White-to-pink, then red colonies	46 %	100 %	>WT
HmsH-R491A-D691A||	Red colonies	99 %	122 %	>WT

*CR-binding values are represented as a percentage of the hmsHFRS⁺ strain (100 %=0.30 A₅₀₀ units). CV-staining values are represented as a percentage of the ΔhmsH(pNPM22) strain (100 %=1.72 A₅₇₀ units).

†WT, level of HmsH expression from the chromosome; RL, level of HmsH expression from pNPM22.

‡External loops between β-strands are numbered starting at the N terminus of HmsH.

§D520A secondary mutation identified by sequencing.

||D691A secondary mutation identified by sequencing.