GadE regulates fliC gene transcription and motility in Escherichia coli

Table 1 Bacterial strains and plasmids used in the study

Strain/plasmid	Description	Source
Strain
DH5a MCR	Transformation efficient strain	Gibco/BRL
MC4100	E. coli K-12 strain	Linda Kenney
EK227	E. coli K-12 strain	[53]
EF1007	gadE::Km in EK227	[54]
EF1083	gadE::Km/pPCRScript Amp gadE	[33]
NU149	Clinical isolate	[36]
NU149 gadE	ΔgadE mutation in NU149	This study
NU149 LacZ1	ΔlacZ mutation in NU149	This study
Plasmid
pUJ9	Promoterless lacZ gene, ApR	[37]
pPP2-6	Single copy plasmid, CmR	[38]
pKD4	Flp recombinase sites, KmR	[40]
pKD46	Red recombinase, ApR	[40]
pCP20	Flp recombinase, ApR	[40]
pNK2-29	fliC::lacZ on pPP2-6, ApR	This study
pPCRScript Amp gadE	gadE on pPCRScript Amp	[33]

Table 2 Effect of pH on fliC::lacZ gene transcription in Escherichia coli strain MC4100/pNK2-29 grown in buffered Luria Bertani media

pH	Gal activity1
5.5	288 ± 81.5
6	528 ± 82.5
6.5	629 ± 114
7	1111 ± 110
7.5	932 ± 190
8	738 ± 125

¹Galactosidase activity measured as Miller units.

Table 3 Effect of osmolarity on fliC::lacZ gene transcription in Escherichia coli grown in buffered pH 5.5 and 7.0 Luria Bertani media with different osmolarities

E. coli strain	NaCl (mmol/L)	Gal activity1
		pH 5.5	pH 7.0
MC4100/pNK2-29	0	308 ± 1042	1132 ± 130
MC4100/pNK2-29	100	338 ± 128	806 ± 41
MC4100/pNK2-29	200	251 ± 68.5	689 ± 173
MC4100/pNK2-29	400	62 ± 22.0	454 ± 71
NU149 LacZ1/pNK2-29	0	442 ± 72	1353 ± 98
NU149 LacZ1/pNK2-29	100	418 ± 61	976 ± 52
NU149 LacZ1/pNK2-29	200	293 ± 43	811 ± 75
NU149 LacZ1/pNK2-29	400	147 ± 39	489 ± 61

¹Galactosidase activity measured as Miller units.

²Data represents the mean ± standard deviation from three separate runs.

Table 4 Assessing a gadE and mutation and complementation on fliC::lacZ gene transcription in Escherichia coli grown in buffered pH 5.5 and 7.0 Luria Bertani media with different osmolarities

E. coli strain	Gal activity1
	pH 5.5	pH 5.52	pH 7.0	pH 7.0
EK227/pNK2-29	540 ± 513	165 ± 59	1520 ± 144	540 ± 66
EF1007/pNK2-294	1742 ± 109	470 ± 106	2196 ± 173	681 ± 135
EF1083/pNK2-29	295 ± 93	131 ± 20	794 ± 145	404 ± 41

¹Galactosidase activity measured as Miller units.

²400 mmol/L added NaCl.

³Data represents the mean ± standard deviation from three separate runs.

⁴EF1007 is gadE and EF1083 is gadE/pGadE+.

Table 5 Motility of Escherichia coli strains NU149 and EK227, their gadE mutants, and complemented gadE mutants grown in pH 5.5 Luria Bertani

Strain	Motility (mm)1
NU149	10.67 ± 1.252
NU149 gadE	57.34 ± 10.21
NU149 gadE/pPCRScript gadE	7.00 ± 0.82
EK227	8.33 ± 1.52
EF1007 (gadE)	45.00 ± 2.00
EF1083 (gadE/pPCRScript gadE)	6.67 ± 1.53

¹Spread diameter after 24 h on a motility plate measured in mm.

²Data represents the mean ± standard deviation from three separate runs.