TABLE 1.

Phenotypes of BT3312 cells expressing the site-specific cysteine substitutions in the Aer HAMP domain

Plasmida	Protein	Succinate plate assay results	Capillary assay results	Response time (s)b		Ratio of FAD (induced/uninduced)c	Residue subtituted is Aer specificd
				O2 increase	O2 decrease
pProEX HTa	Vector	−	−	0	0	0.96 ± 0.18
pAVR2	His6-Aer2-506	+	+	173.8 ± 10.4	26.6 ± 3.0	4.98 ± 2.62
pQH30	His6-AerQ218C	+	+	>180e	26.3 ± 10.2	+	No
pQH31	His6-AerK221C	+	+	>180e	33.0 ± 5.6	+	No
pQH32	His6-AerV222C	+	+	159.3 ± 24.9	31.8 ± 5.5	+	No
pQH33	His6-AerE226C	+	+	97.2 ± 13.8	23.3 ± 5.4	+	No
pQH34	His6-AerR227C	+	+	>180e	35.3 ± 7.3	+	Yes
pQH36	His6-AerE231C	+	+	>180e	27.8 ± 2.4	+	Yes
pQH38	His6-AerR235C	−	−	0	0	1.02 ± 0.01	Yes
pQH39	His6-AerR235E	−	−	0	0	0.69 ± 0.03	Yes
pQH40	His6-AerR235K	+	+	70.0 ± 7.9	12.2 ± 1.5	+	Yes
pQH43	His6-AerD237C	+	+	103.7 ± 41.0	14.5 ± 2.2	+	No
pQH44	His6-AerL239C	+	+	174.2 ± 9.2	25.3 ± 7.4	+	No
pQH46	His6-AerT242C	+	+	>180e	23.2 ± 1.5	+	No
pQH47	His6-AerG250C	+	+	46.9 ± 17.6	25.1 ± 5.7	2.94 ± 0.62	Yes

^aAll plasmids were derived from the pProEX HTa expression vector.

^bThe data represent means ± standard deviations of results for two independent experiments, each with three trials.

^cAll Aer mutants were induced with IPTG to at least 2,500 copies per cell, which is eightfold higher than Aer expressed chromosomally (300 copies cell⁻¹). Membranes from cells with the pProEx vector alone (aer) contained ≈500 copies of FAD cell⁻¹. A 20% increase (ratio, 1.2) in total membrane FAD after IPTG induction was used to indicate FAD binding to Aer. +, aerotaxis activity was evidence that Aer was expressed and that it bound FAD in these strains.

^dYes, the residue is not conserved in HAMP domains other than Aer.

^eResponse times were variable within a range of 190 to ≈250 s.