TABLE 1.

Cys replacements of residues in loop VIII–IX, TMS IX, and TMS XI of NhaA For characterization of the mutations, EP432 cells were transformed with the plasmids carrying the indicated mutations. The expression level was expressed as a percentage of the control cells (EP432/pCl-HAH4) encoding CL-NhaA. Growth experiments were conducted on agar plates at 37 °C with high Na⁺ (0.6 m) at the indicated pH values. + + +, number and size of the colonies after 48 h of incubation at neutral pH; + +, number as above but smaller sized colonies after 48 h of incubation at alkaline pH; +, both number and size of colonies were reduced; –, no growth. The apparent K_m for NaCl was determined at pH 8.5 as described under “Experimental Procedures.” ND, not determined.

Mutation	Expression	Growth, 0.6 m NaCl		Apparent Km, Na+
		pH 7	pH 8.3
	%			mm
E241Ca	120	+ + +	+ +	ND
K242C	48	+	+	1.9
H243C	116	+ + +	+ +	1.2
G244C	82	+ + +	+ +	0.9
R245C	87	+ + +	+ +	4.8
S246C	98	+ + +	+ +	1.7
P247C	161	+ + +	+ +	1.2
A248C	84	+ + +	+ +	1.3
K249C	80	+ + +	+ +	2.6
R250C	89	+ + +	+	1.9
L251C	72	+ + +	+ +	2
E252C	83	+ + +	+ +	10
H253C	37	+ + +	+	1.5
V254Ca	110	+ + +	+ +	ND
L255C	47	+ + +	+ +	0.8
H256C	80	+ + +	+ +	3.5
P257C	50	+ + +	+ +	0.6
W258C	81	+ + +	+ +	0.4
V259C	50	+ + +	+ +	1.4
L262C	78	+ + +	+ +	0.6
L264C	83	+ + +	+ +	0.9
F267C	95	+ + +	–	0.8
F344C	40	+ + +	+	0.16
F267C/F344C	37	–	–
CL-NhaA	100	+ + +	+ +	0.5
pBR322		–	–

^aThe data were obtained from Ref. 25