Transcriptional Response of Escherichia coli to TPEN

Abstract

DNA microarrays were used to probe the transcriptional response of Escherichia coli to N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Fifty-five transcripts were significantly up-regulated, including all of the genes that are regulated by Zur and many that are regulated by Fur. In the same TPEN-treated cells, 46 transcripts were significantly down-regulated.

The transcriptional response of Escherichia coli to elevated levels of metal ions such as Zn(II), Cd(II), Co(II), Ni(II), and Fe(II) has been probed in an effort to understand the mechanisms by which the homeostatic levels of these metal ions are maintained (10, 15, 51, 95, 96). In contrast, very few studies have probed the global response of E. coli to low levels of metal ions, presumably due to the difficulty of sufficiently depleting the growth medium of metal ions. In this study, cDNA microarrays were used to probe the transcriptional response of E. coli to stress by N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). TPEN (58, 81), a cell-permeative, divalent metal chelator, is often called “Zn(II) specific” (17, 33, 38, 48, 59, 64, 71, 79, 80, 83, 88). Our data show significant changes in the transcription of several genes in cells stressed with TPEN.

To identify genes that are differentially expressed in response to TPEN, E. coli BL21(DE3) cells were grown in minimal medium (76) until the cultures reached mid-log phase. TPEN was introduced, and the cells were cultured for 5 h; this time was chosen so that the results would correspond to previous transcriptional response studies with excess Zn(II) (51). The cultures containing 5 μM TPEN were analyzed with DNA microarrays (E. coli K12 V2 array slides from MWG-BIOTECH), and the results were compared to those from E. coli cells grown in minimal medium containing no TPEN. A minimum of six slides was used for each experiment (three slides with one combination of Cy3 and Cy5 dyes and three slides with swapped dyes). Fifty-five transcripts were significantly up-regulated (twofold) (Table 1). Four genes (ykgM, znuA, znuC, and yodA) that are regulated by Zur, the Zn(II) uptake regulator (30), exhibited significant increases in expression. The remaining Zur-regulated transcript, znuB, was also up-regulated; however, this transcript did not meet our filtering criteria: (i) P values of ≤0.5, (ii) ≥2-fold changes in expression, and (iii) consistent data in all six slides. The expression of zntA, which is regulated by ZntR and encodes the high-affinity Zn(II) exporter in E. coli (7, 14, 78), was unchanged in E. coli cultures stressed with TPEN (see the complete set of DNA array data).

TABLE 1.

Genes up-regulated in E. coli cells stressed with TPEN

Grouping	IDa	Gene	Increase (fold)	P value	Description	Reference
Zur regulated	B0296	ykgM	23	5.5E-04	Ribosomal protein; L31 paralog	70
	B1973	yodA	21	2.8E-03	Cd(II) induced; putative Zn(II) transporter	21, 75
	B1857	znuA	7.6	1.5E-03	Periplasmic component of Zn(II)-specific ZnuABC transporter	73
	B1858	znuC	2.2	1.1E-02	ATPase of ZnuABC	73
Fur regulated	B2673	nrdH	12	1.9E-04	Glutaredoxin-like protein	39
	B2674	nrdI	12	8.8E-03	Stimulates NrdH	39
	B0595	entB	9.7	2.4E-05	2,3-Dihydro-2,3-dihydroxybenzoate synthetase	26
	B0596	entA	8.7	6.1E-03	2,3-Dihydro-2,3-dihydroxybenzoate dehydrogenase	91
	B0583	entD	7.2	1.0E-04	Enterochelin synthetase, component D	26
	B3006	exbB	5.8	4.9E-04	Uptake of enterochelin; TonB-dependent uptake of b colicins	34
	B3005	exbD	5.3	1.3E-03	Enterochelin synthetase, component d	34
	B0594	entE	5.1	1.6E-02	2,3-Dihydroxybenzoate-AMP ligase	26
	B1683	sufB	5.1	3.6E-03	Component of SufBCD cysteine desulfurase activator complex	69
	B1682	sufC	5.1	4.2E-03	ATP-binding component of SufBCD cysteine desulfurase activator complex	69
	B0151	fhuC	5.1	1.1E-03	ATP-binding component of hydroxymate-dependent iron transport	16
	B4292	fecR	4.9	2.4E-03	Regulator for fec operon, periplasmic	22
	B4293	fecI	4.8	4.5E-03	Probable RNA polymerase sigma factor	4
	B0585	fes	4.5	1.9E-02	Enterochelin esterase	13
	B1102	fhuE	4.4	3.4E-03	Outer membrane receptor for ferric iron uptake	11
	B0150	fhuA	4.3	1.9E-03	Outer membrane protein receptor for ferrichrome, colicin m	24
	B4367	fhuF	2.10	4.7E-03	Ferrioxamine B reductase	62
	B1679	sufE	2.01	1.4E-03	Cysteine desulfurase	55
	B1252	tonB	3.9	5.2E-04	TonB	45
	B1681	ynhC	3.9	3.5E-03	Component of SufBCD cysteine desulfurase activator complex	69
	B1680	sufS	3.7	6.7E-03	l-Selenocysteine lyase	72
	B2676	nrdF	3.1	8.7E-04	Subunit of ribonucleoside-diphosphate reductase II	46
	B3908	sodA	3.0	2.0E-02	Mn superoxide dismutase	36
	B4290	fecB	3.0	4.4E-02	Periplasmic component of iron dicitrate ABC transporter	31
	B2155	cirA	2.9	3.3E-03	Outer membrane receptor for colicin I receptor	12
	B0153	fhuB	2.9	5.0E-02	Component of Fe(III) hydroxamate ABC transporter	16
	B0592	fepB	2.7	2.2E-02	Fe(III) enterobactin ABC transporter	84
	B1132	ycfC	2.6	2.1E-02	Membrane protein in operon with purB	28
	B0586	entF	2.4	2.5E-02	Apo-serine activating enzyme	26
SoxRS regulated	B1611	fumC	3.8	2.6E-02	Class II fumarase	89
	B2962	yggX	3.2	6.4E-03	Protects Fe-S clusters from oxidation	67
	B2963	mltC	2.6	4.6E-05	Component of lytic murein transglycosylase C	6
	B2237	inaA	2.2	3.1E-03	pH-inducible protein involved in stress response	93
	B3924	fpr	2.1	4.8E-02	Flavodoxin NADP+ reductase	9
Other stress related	B0126	yadF	3.7	6.9E-03	Carbonic anhydrase	60
	B1053	yceE	2.5	1.3E-03	Drug resistance protein	65
	B0313	betI	2.4	5.9E-03	Transcription repressor of bet genes	49
	B1629	rsxC	2.1	2.9E-02	Component of SoxR reducing complex	47
	B4062	soxS	2.1	7.3E-03	Superoxide response regulon	52
	B1307	pspD	2.0	3.4E-02	Phage shock protein	61
Anabolic processes	B1264	trpE	5.3	3.7E-02	Anthranilate synthase component I	20
	B3161	mtr	2.9	7.6E-04	Trp transport protein	32
	B1265	trpL	2.7	3.1E-02	Trp operon leader peptide	43
	B2678	proW	2.6	2.4E-02	Pro and Gly betaine transporter	5
	B0311	betA	2.6	3.1E-03	Choline dehydrogenase	77
	B4245	pyrB	2.6	2.1E-02	Aspartate transcarbamoylase	41
	B2028	ugd	2.4	3.1E-03	UDP-glucose-6-dehydrogenase	85
	B1261	trpB	2.3	5.0E-02	Trp synthase β	44
	B3409	feoB	2.3	1.7E-02	Fe(II) transport protein	40
	B1981	shiA	2.3	4.7E-02	Shikimate transporter	92
	B0931	pncB	2.0	7.3E-03	Nicotinate phosphoribosyltransferase	94

^aID, identifier.

Twenty-nine of the up-regulated genes from TPEN-supplemented cultures are transcriptionally regulated by Fur, the iron uptake regulator (Table 1), and several are involved in Fe transport. The genes (entB, entA, entD, entE, and fes) which encode proteins involved with enterobactin synthesis and uptake (26, 29, 54) and the fec genes (fecR and fecI), which encode proteins involved in Fe import (23), were significantly up-regulated. Several other genes (fhuA, exbB, exbD, fhuD, fhuC, and fhuF) associated with ferrichrome transport in E. coli were also up-regulated (19, 56, 62).

Forty-six transcripts were significantly down-regulated in E. coli cells stressed with TPEN (Table 2). Two operons in E. coli, flgBCDEFGHIJKL and flgAMN, possess genes that encode proteins involved in flagellar biosynthesis (8). Some of these motility-related genes, namely, flgB, fliM, and motB, were previously reported as being up-regulated in E. coli cells stressed with excess Zn(II) (51). In addition, the expression of flagellar biosynthetic proteins in E. coli is affected by the concentration of copper, possibly exerting its effect via the OmpR or H-NS transcriptional regulators (42). All of the genes in the cus and cue systems, which confer copper tolerance to E. coli (68), were also down-regulated, although cusA and copA were filtered out of the data shown in Table 2. Previous studies have demonstrated that the expression levels of the cus and cue genes are dependent on aerobic/anaerobic conditions as well as on the levels of copper in the periplasm/cytoplasm (68). The expression levels of cytoplasmic ferritin (3) were also significantly down-regulated in TPEN-treated cells.

TABLE 2.

Genes down-regulated in E. coli cells stressed with TPEN

Grouping	IDb	Gene	Decrease (fold)	P value	Descriptiona	Reference
Flagellar biosynthesis	B1074	flgC	7.9	3.1E-05	Cell-proximal portion of basal-body rod	8
	B1075	flgD	7.5	3.6E-04	Initiator of hook assembly	8
	B1078	flgG	4.9	1.2E-05	Cell-distal portion of basal-body rod	8
	B1080	flgI	4.3	3.3E-04	p ring of flagellar basal body	8
	B1079	flgH	4.0	8.7E-03	Outer membrane ring protein	8
	B1077	flgF	3.9	5.4E-05	Cell-proximal portion of basal-body rod	8
	B1081	flgJ	3.9	2.8E-03	Flagellar protein	8
	B1073	flgB	3.8	3.8E-04	Cell proximal portion of basal-body rod	8
	B1072	flgA	3.4	4.7E-02	Assembly of basal-body p ring	8
	B1082	flgK	3.1	2.6E-02	Hook filament junction protein	8
	B1071	flgM	2.3	3.6E-03	Anti-flia factor	8
Sugar metabolism	B2094	gatA	6.4	5.9E-03	Enzyme IIa of PTS	66
	B2095	gatZ	5.3	5.6E-03	Putative tagatose 6-phosphate kinase	66
	B2092	gatC	4.9	2.7E-02	Enzyme IIc of PTS	66
	B2091	gatD	4.7	2.4E-03	Galactitol-1-phosphate dehydrogenase	66
	B2167	fruA	4.0	2.2E-03	Fructose specific transporter	66
	B2093	gatB	3.7	1.3E-02	Enzyme IIb of PTS	66
	B2169	fruB	3.5	3.1E-03	Fructose specific IIa component	66
	B2168	fruK	3.2	2.8E-02	Fructose 1-phosphate kinase	66
	B2096	gatY	2.6	1.7E-02	Tagatose-bisphosphate aldolase	66
	B4034	malE	2.4	8.1E-03	Maltose binding protein	66
	B3417	malP	2.3	5.0E-03	Maltodextrin phosphorylase	66
	B1198	ycgC	2.3	6.5E-03	Dihydroxyacetone kinase M	66
	B1817	manX	2.1	1.5E-03	Mannose transporter	66
Fe and Cu	B1905	ftnA	5.3	3.8E-04	Cytoplasmic ferritin	82
metabolism	B0123	yacK	2.5	2.7E-03	CueO, Cu(I) oxidase	27
	B0573	ylcC	2.5	2.7E-03	CusF, copper efflux	27
	B0572	ylcB	2.4	2.0E-02	CusC, copper efflux	27
	B0574	ylcD	2.2	1.4E-02	CusB, copper efflux	27
Stress	B1656	sodB	3.7	2.0E-03	Fe superoxide dismutase	36
	B1379	hslJ	2.3	7.4E-03	Heat shock protein	53
	B3687	ibpA	2.1	4.7E-03	Heat shock protein	50
Metabolic proteins	B3936	rpmE	3.4	2.2E-02	L31 ribosomal protein	63
	B2142	yohK	2.5	3.0E-02	Serotonin transporter	43
	B2286	nuoC	2.5	1.3E-02	Component of NADH dehydrogenase	25
	B0972	hyaA	2.5	4.8E-02	Hydrogenase small subunit	37
	B4116	adiY	2.4	2.8E-02	Transcription activator of Arg decarboxylase	86
	B0411	tsx	2.3	2.8E-2	Nucleoside channel	57
	B1380	ldhA	2.3	1.1E-02	Lactate dehydrogenase	18
	B3209	yhbL	2.3	1.8E-02	Isoprenoid biosynthesis protein	35
	B0651	ybeK	2.3	1.9E-2	Ribonucleoside hydrolase	74
	B1245	oppC	2.2	7.2E-3	ABC transporter for peptides	87
	B0872	hcr	2.2	4.0E-02	NADH oxidoreductase	90
	B1241	adhE	2.0	2.1E-03	Acetaldehyde dehydrogenase and Fe-dependent alcohol dehydrogenase	18
	B1246	oppD	2.0	3.8E-02	ABC transporter for peptides	87
	B2284	nuoF	2.0	7.0E-03	NADH dehydrogenase	25

^aPTS, phosphotransferase system.

^bID, identifier.

To validate the microarray data, two representative genes were selected for real-time PCR and assayed for the level of mRNA by a two-step, real-time PCR technique. The genes yodA and pdxH, which were up-regulated (21-fold) and exhibited no change (1.1-fold), respectively, were analyzed with real-time PCR. Real-time PCR results were as follows: yodA up-regulated, 17 ± 1; pdxH up-regulated, 1.1 ± 0.1. In order to probe whether the changes observed in cells grown in the presence of TPEN were due to the chelator, RT-PCR was used to probe for the expression levels of yodA in E. coli cells grown in minimal medium containing 5 μM TPEN and 30 μM Zn(II). There was no change in transcript levels of yodA when E. coli was cultured in this medium.

Despite the fact that TPEN is often referred to as a Zn(II)-specific chelator (17, 33, 38, 48, 59, 64, 71, 79, 80, 83, 88), the analyses of our microarray data suggest that the levels of other metal ions may have been affected by the presence of TPEN. TPEN has been reported to bind Cd(II) (K_d = 4.7 × 10⁻¹⁷), Co(II) (K_d = 2.6 × 10⁻¹⁷), Ni(II) (K_d = 2.8 × 10⁻²²), and Cu(II) (K_d = 2.9 × 10⁻²¹) more tightly than it binds Zn(II) (2). In addition, TPEN forms stable complexes with Fe(II) (K_d = 2.5 × 10⁻¹⁵) (2). Since TPEN forms much tighter complexes with Cu(II), it is likely that the down-regulation of the copper homeostasis/export transcripts, cueO, copA, cusA, cusB, cusC, and cusF, is due to low levels of intracellular copper. Twenty-nine Fur-regulated transcripts were up-regulated. Despite the fact that Fe(II) binds 1 order of magnitude less tightly to TPEN than Zn(II) (2), it is possible that TPEN lowered the intracellular concentrations of Fe(II), resulting in the transcription of Fur-regulated iron uptake proteins. The reduction in intracellular concentrations of Fe(II) could be due to direct chelation by TPEN or by oxidation of intracellular Fe(II) to Fe(III). The fact that several other SoxRS-regulated transcripts were up-regulated in cultures stressed with TPEN (Table 1) suggests oxidative stress in these cells (97). It is also possible that the reduction of intracellular Zn(II) caused by the presence of TPEN resulted in an improperly folded Fur, which requires one Zn(II) for proper structure/function (1).

Taken together, these results strongly suggest that the intracellular levels of several metal ions in E. coli can be affected by TPEN, which indicates that caution should be exercised when TPEN is used in experiments to control intracellular concentrations of Zn(II) in cells.

Microarray data accession number.

The microarray data have been loaded into the Gene Expression Omnibus (GEO) with the accession number GSE5356 (www.ncbi.nlm.nih.gov/geo).