The whole set of the constitutive promoters recognized by four minor sigma subunits of Escherichia coli RNA polymerase.

The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by the sigma subunit. The model prokaryote Escherichia coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. For identification of the "constitutive promoters" that are recognized by each RNAP holoenzyme alone in the absence of other supporting factors, we have performed the genomic SELEX screening in vitro for their binding sites along the E. coli K-12 W3110 genome using each of the reconstituted RNAP holoenzymes and a collection of genome DNA segments of E. coli K-12. The whole set of constitutive promoters for each RNAP holoenzyme was then estimated based on the location of RNAP-binding sites. The first successful screening of the constitutive promoters was achieved for RpoD (σ70), the principal sigma for transcription of growth-related genes. As an extension, we performed in this study the screening of constitutive promoters for four minor sigma subunits, stationary-phase specific RpoS (σ38), heat-shock specific RpoH (σ32), flagellar-chemotaxis specific RpoF (σ28) and extra-cytoplasmic stress-response RpoE (σ24). The total number of constitutive promoters were: 129~179 for RpoS; 101~142 for RpoH; 34~41 for RpoF; and 77~106 for RpoE. The list of constitutive promoters were compared with that of known promoters identified in vivo under various conditions and using varieties of E. coli strains, altogether allowing the estimation of "inducible promoters" in the presence of additional supporting factors.

Introduction

The genome of Escherichia coli K-12, the most well-characterized model prokaryote, contains a total of more than 4,500 genes, which are transcribed by a single species of the RNA polymerase (RNAP). The intracellular concentration of RNAP is, however, approximately 2,000 molecules per genome, which is less than the total number of genes or operons [1-3]. The pattern of genome expression is therefore determined by the selective distribution of a limited number of RNAP within the genome [4,5]. For adaptation to stressful environments, the pattern of genome transcription is, however, altered by modulating the promoter selectivity of RNAP through two-step interaction with two groups of the regulatory factor, i.e., 7 species of the sigma factor with promoter recognition activity at the first step [5,6] and then approximately 300 species of the transcription factor (TF) including both protein and nucleotide factors at the second step [4,5,7,8]. For understanding the genome regulation at molecular level, therefore, three kinds of the basic knowledge are absolutely needed for both all the sigma and TF factors [8,9]: (1) the whole set of regulatory target promoters, genes or operons under the control of each regulatory factor; (2) the binding affinity of the test regulatory protein to target DNA; and (3) the intracellular concentrations of the functional forms of each regulatory protein [note that the activity of TF is often controlled by effector ligands or protein modification such as phosphorylation]. Once we get these three lines of knowledge, we will be able to predict the pattern of genome transcription.

After the complete genome sequencing of E. coli K-12, its transcription pattern or transcriptome in vivo has been analyzed for various E. coli wild-type and mutant strains growing under various stress conditions, including niches within host animals, using modern technologies such as the microarray system [10,11]. The localization of RNAP and TFs on the genome was also analyzed by using ChIP-chip system [12-14]. More recently microarray was replaced by direct sequencing of RNAs [15-17] or mapping of transcription start sites [18,19]. These data are assembled in the databases such as RegulonDB [20,21] and EcoCyc [22,23]. The huge accumulation of background knowledge is absolutely needed for understanding the regulation mechanism of genome transcription as a whole in a single organism, and thus at this stage, E. coli is reassessed as the model organism. The binding sites of RNAP and TF identified in vivo using these modern techniques, however, do not represent the whole set of their binding sites because: i) their binding to regulatory sites is often interfered by other DNA-binding proteins, thereby masking their binding target sequences by antagonistic inhibitory proteins [8,9,24]; and ii) in the case of activator-dependent transcription, their binding to targets depends on the simultaneous presence of supporting factors [8,9,25]. Under the in vivo situations, therefore, it is in principle impossible to obtain the whole set of binding sites for both RNAP and TFs. In addition, the transcription-related data listed in the databases include different levels of accuracy. For instance, a number of TF-binding sites are estimated in silico relying on the consensus sequences that often include the inaccurate prediction. Another serious problem is originated from the use of various E. coli strains with different genetic background and of different culture conditions used in each experiment (for details see Discussion).

In order to avoid the problems associated with these in vivo experiments, we then decided to employ the in vitro approaches. For identification of the binding sites of RNAP and TFs, we developed the Genomic SELEX system [26] and successfully employed for search of regulatory targets for a number of TFs [8,9]. We also employed the Genomic SELEX for mapping of promoters. As described in the previous report [27], we identified a total of 2,071 sites on the E. coli K-12 genome of binding of RNAP holoenzyme containing RpoD (σ70), the major sigma for transcription of most of the growth-related genes, and mapped the location of “constitutive promoters” that are recognized by RpoD holoenzyme alone in the absence of other DNA-binding proteins [Note that the “constitutive promoter” is defined as the promoter that is recognized by RNAP alone in the absence of supporting factors while the promoters that are detected only in vivo are defined as the “inducible promoters”, supposedly under the support of accessory regulatory factors].

Besides this major house-keeping RpoD sigma (σ70), E. coli K-12 contains six alternative minor sigma factors, i.e., nitrogen-regulated gene-specific RpoN (σ54), stationary-phase nutrient-starvation specific RpoS (σ38), heat-shock response-specific RpoH (σ32), flagellar-chemotaxis specific RpoF (σ28), extra-cytoplasmic stress-response RpoE (σ24), and iron-starvation specific FecI (σ28) [4-6]. In this study, we identified the list of constitutive promoters for four minor sigma factors, RpoS, RpoH, RpoF and RpoE. Since RpoN sigma requires an additional TF such as NtrC for promoter binding, the set of promoters recognized by RpoN sigma differs depending on the species of collaborative TF. The list of promoters recognized by RpoN will be described elsewhere. On the other hand, FecI sigma is rather a unique sigma that recognizes only a specific target of the gene for fecA encoding transport of ferric citrate [28]. Thus, these two sigma factors, RpoN and FecI, are not included in this report. The list of constitutive promoters herein described provides the fundamental catalogs for the promoters recognized by the four minor sigma factors alone. The data described in this report will be deposited into TEC (Transcription Profile of Escherchia coli) database (https://shigen.nig.ac.jp/ecoli/tec/) [9]. The data of each minor sigma will be shown by ordering the sigma name (RpoS, RpoH, RpoF or RpoE) [https://shigen.nig.ac.jp/ecoli/tec/tfmap]. For details follow the instruction in TEC [9].

Results

Genomic SELEX screening for the constitutive promoters

The constitutive promoters are transcribed in vitro by the RNA polymerase holoenzyme alone in the absence of supporting factors. In order to identify the whole set of constitutive promoters on the entire genome of E. coli K-12 W3110, we performed a mass-screening in vitro of the whole set of sequences that are recognized by the reconstituted holoenzymes, each containing only one specific minor sigma factor. The sigma-free core enzyme was prepared by passing the purified RNA polymerase three times through phosphocellulose column chromatography in the presence of 5% glycerol, the stabilizer of holoenzyme complexes in the storage buffer [29]. The level of remaining sigma subunits was less than 0.1%, if any, as detected by both protein staining and immuno-staining against each of all seven species of sigma factors (RpoD, RpoN, RpoS, RpoH, RpoF, RpoE and FecI). The stoichiometry between core enzyme subunits was also checked by immune-staining with antibodies against the core subunits, RpoA, RpoB, RpoC and RpoZ. The holoenzymes fully saturated with each sigma subunit were reconstituted by mixing this sigma-free core enzyme and 4-fold molar excess of purified sigma factors, RpoS, RpoH, RpoF and RpoE. Since these sigma subunits alone are unable to bind to promoter DNA, the presence of excess sigma does not interfere with the function of RNAP holoenzymes. For the identification of DNA sequences that are recognized by each holoenzyme, we employed the Genomic SELEX screening system [26], in which a library of E. coli genome DNA fragments of 200–300 bp in length was used instead of synthetic oligonucleotides with all possible sequences used in the original SELEX method [30-32].

The multi-copy plasmid library of 200–300 bp-long random DNA fragments was constructed from the E. coli K-12 W3110 genome [26]. The library used in this study contained 6.5-fold molar excess of the entire genome, and thus a single and the same sequence might be included in 6 different overlapping segments on average, thereby increasing the resolution of mapping of SELEX fragments. In each experiment of Genomic SELEX screening, the mixture of genome DNA fragments, which was regenerated by PCR from the genome DNA library, was mixed with 2-fold molar excess of the reconstituted each RNAP holoenzyme, and subjected to Genomic SELEX screening. DNA-holoenzyme complexes formed were recovered using the anti-RpoC antibody, which gave the highest level of RNAP recovery among all the anti-core subunit antibodies. RNA polymerase-associated DNA was isolated from the antibody precipitates, amplified by PCR, and subjected to next cycles of SELEX. After repeated SELEX screening, the final products of holoenzyme-bound DNA fragments were subjected to mapping on the genome using a DNA tilling microarray (Oxford Gene Technology, Oxford, UK) [14]. The binding intensity was measured as the ratio of holoenzyme-bound DNA labeled by Cy3 against original library DNA labeled by Cy5 on an array and plotted along E. coli genome about each holoenzyme. On the DNA tilling array used, the 60 b-long probes are aligned along the E. coli genome at 105 bp-intervals, and therefore approximately 300 bp-long SELEX fragments should bind to two or more consecutive probes. This criterion was employed to avoid the background noise of non-specific binding of holoenzyme-bound DNA fragments to the tilling array [note that peaks showing hybridization to only a single probe was judged as a false-positive noise].

The binding sites were classified into two groups, one ‘within spacers’ and another ‘inside genes’. The binding sites on ‘within spacers’ were further classified into 3 types; type-A spacer located between bidirectional transcription units, type-B spacer located upstream of one transcription unit but downstream of another transcription unit, and type-C spacer located downstream of both transcription units. Based on the transcription direction of flanking genes, the total number of the constitutive promoters was predicted to range between the minimum [number of type-A spacer plus number of type-B spacer] and the maximum [number of type-A spacer x 2 plus number of type-B spacer]. The intragenic binding site was referred to type-D site. The height of binding intensity identified by SELEX-chip system is generally in good agreement with the number of clones identified by SELEX-clos (cloning-sequencing) system, indicating that these two parameters correlate with the binding affinity of test TF to DNA [4,5,8,9].

The whole set of constitutive promoters for the stationary-phase sigma RpoS

In laboratory culture of E. coli, cell growth enters into the stationary phase mainly due to the limited availability of nutrients. Upon entry into the stationary phase, the pattern of genome expression is markedly altered by turning down the growth-related genes and instead up-regulation of the stress-response genes. In switching the transcription pattern, the stationary-phase specific minor sigma RrpoS is involved [33,34]. The rpoS gene is not essential for growth under non-stress conditions, but strains carrying mutations affecting rpoS activity are extremely sensitive to environmental stresses. As in the case of other sigma factors, RpoS interacts with RNAP core enzyme and modulates its promoter recognition specificity so as to recognize a specific but large set of genes.

As noted above, the set of genes identified in vivo include a number of genes under the indirect control of RpoS. On the other hand, some target promoters of RpoS are masked in vivo due to competitive interference by other DNA-binding proteins. In order to identify the constitutive promoters directly recognized by RpoS in the absence of other DNA-binding proteins, the Genomic SELEX screening in vitro was performed using the reconstituted RNAP RpoS holoenzyme. The sequences with binding affinity to the RpoS holoenzyme formed a number of peaks along the entire E. coli genome (Fig 1). Location of peaks was aligned along the map of E. coli K-12 genome (Table 1). By setting the cut-off level of 3.0 fold-higher intensity over the background of original library DNA, a total of 218 peaks were identified, of which 125 (67%) are located within intergenic spacers and 73 (33%) are inside of open reading frames (Table 2). Since the majority of hitherto identified promoters are located within spacers and generally upstream of open reading frames, detailed search for the constitutive promoters was focused on these 125 spacer peaks. These spacers can be classified into three types: 50 peaks are located within type-A spacer between bidirectional transcription units; 79 peaks are located within type-B spacers located upstream of one transcription unit but downstream of another transcription unit; and 16 peaks are located within type-C located spacers downstream of both transcription units. Based on the transcription direction of flanking genes, the total number of RpoS constitutive promoters was predicted to range between minimum 129 (50 type-A plus 79 type-B) and maximum 179 (50x2 type-A plus 79 type-B) (Table 2). Type-A spacers should contain two promoters for bidirectional transcription, at least one of which should be RpoS-dependent promoter. The RpoS holoenzyme-binding sites identified in a total of 50 type-A spacers should represent promoters for one or both of bidirectional transcription.

Fig 1

SELEX-chip search for RNAP RpoS holoenzyme-binding sequences on the E. coli K-12 genome.

The y-axis represents the relative number of RpoS holoenzyme-bound DNA fragments whereas x-axis represents the position on the E. coli K-12 genome, in base pair. The adjacent gene on E. coli K-12 genome of peak position was indicated for high intensity peaks. The peaks located within spacer regions are shown in green color, while peaks located within open reading frames are shown in orange color. The list of RpoS holoenzyme-binding sites is described in Table 1.

Table 1

RpoS holoenzyme-binding sites on the E. coli K-12 genome.

No	Type	Map	Gene Function	Left	D	RpoS	D	Right	Gene Function	Intensity
1	D	17732		sokC	>	nhaA	>	nhaR	transcriptional activator	5.3
2	A	41946	predicted transporter	caiT	<		>	fixA	electron transfer flavoprotein	3.6
3	B	63256	RNAP-associated helicase	hepA	<		<	polB		3.8
4	D	238050		aspV	>	yafT	<	ykfM		19.0
5	B	251970		dinB	>		>	yafN	YafO-YafN toxin-antitoxin system	45.1
6	B	289870	ornithine carbamoyltransferase 2	argF	<		<	insB		25.0
7	D	296234	CP4-6 prophage protein	yagN	<	intF	>	ptwF	Xaa tRNA	70.4
8	A	296336	CP4-6 prophage integrase	intF	<		>	ptwF	Xaa tRNA	45.5
9	A	328672	transcriptional repressor	betI	<		>	betT	choline transporter	3.8
10	B	379186	frmRAB operon regulator	frmR	<		<	yaiO		50.8
11	A	383868	conserved protein	yaiS	<		>	tauA	taurine transporter	24.1
12	D	451740	cytochrome o ubiquinol oxidase	cyoA	<	ampG	<	yajG		5.9
13	B	455746		tig	>		>	clpP	ATP-dependent serine protease	7.4
14	B	467530		cof	>		>	ybaO	DNA-binding transcriptional regulator	11.5
15	D	479868	modulator of gene expression with H-NS	hha	<	tomB	<	acrB		45.1
16	B	480454	predicted protein	tomB	<		<	acrB		22.6
17	A	515034	membrane anchored protease	qmcA	<		>	ybbL	ABC superfamily transporter	70.3
18	D	532860		allA	>	allR	>	gcl	glyoxylate carboligase	3.5
19	D	562966		sfmH	>	sfmF	<	fimZ		45.3
20	D	569366		emrE	>	ybcK	>	ybcL	DLP12 prophage kinase inhibitor	6.8
21	B	570042		ybcK	>		>	ybcL	DLP12 prophage kinase inhibitor	19.7
22	A	576032	IS5 transposase and trans-activator	insH	<		>	essD	DLP12 prophage lysis protein	34.0
23	B	582672		nohB	>		>	*appY	DLP12 prophage transcriptional activator	12.9
24	D	583166		nohB	>	appY	<	ompT		11.4
25	D	606570	predicted inner membrane protein	ybdJ	<	ybdK	>	hokE	toxic polypeptide	17.3
26	A	651466	citrate lyase synthetase	citC	<		>	dpiB	CitBA TCS sensory histidine kinase	8.3
27	D	654932		dpiA	>	dcuC	>	pagP	palmitoyl transferase for Lipid A	60.7
28	D	659134		tatE	>	lipA	<	ybeF		5.9
29	B	692668	nucleoside triphosphate hydrolase	ybeZ	<		<	miaB		19.9
30	C	720060		ybfK	>		<	kdpE		3.6
31	B	770146		mngB	>		>	cydA	cytochrome d terminal oxidase, subunit I	127.5
32	D	799356		pgl	>	ybhD	>	ybhH	conserved protein	4.8
33	A	812472	hypothetical protein	ybhU	<		>	uvrB	nucleotide excision repair nuclease	12.9
34	B	837732	conserved protein	*ybiI	<		<	ybiX		19.6
35	A	849568	threonine and homoserine efflux system	rhtA	<		>	ompX	outer membrane protein	5.4
36	A	852256	ncRNA	rybA	<		>	mntR	DNA-binding transcriptional regulator	26.0
37	A	877270	SAM-dependent methyltransferase	yliG	<		>	bssR	conserved protein	5.3
38	B	891170		nfsA	>		>	rimK	ribosomal protein S6 modification protein	4.1
39	B	903170	arginine transporter	*artP	<		<	ybjP		154.2
40	A	931668	thioredoxin reductase	trxB	<		>	lrp	DNA-binding transcriptional dual regulator	5.9
41	D	943972		dmsB	>	dmsC	<	ycaC		24.3
42	B	953940	formate transporter	focA	<		<	ycaO		3.4
43	B	959450		aroA	>		>	ycaL	peptidase with chaperone function	6.6
44	B	962934		rpsA	>		>	*ihfB	integration host factor (IHF)	3.6
45	D	1028042	hemimethhylated DNA-binding protein	hspQ	<	yccW	>	yccX	predicted acylphosphatase	40.5
46	C	1049830		insB	>		<	cspH		167.2
47	B	1084156		efeB	>		>	phoH	conserved protein with NTPase domain	62.7
48	B	1120372	predicted protein	bssS	<		<	dinI		6.6
49	B	1120772	DNA damage-inducible protein I	dinI	<		<	pyrC		3.7
50	D	1144848		yceQ	>	rluC	<	yceF		23.7
51	D	1147330		rpmF	>	plsX	>	fabH	3-oxoacyl-[acyl-carrier-protein] synthase III	31.2
52	A	1168238	DNA-binding transcriptional regulator	ycfQ	<		>	bhsA	predicted protein	17.6
53	D	1187848	conserved protein	ycfD	<	phoQ	<	phoP		5.5
54	D	1211030	5-methyl-C-specific restriction nuclease	mcrA	>	elbA	<	ycgX		4.7
55	D	1212068	predicted protein	elbA	<	ycgX	<	ycgE		27.8
56	A	1214962	predicted FAD-binding phosphodiesterase	ycgF	<		>	*ycgZ	predicted protein	14.2
57	B	1215830		ariR	>		>	ymgC	predicted protein	6.7
58	C	1218960		ymgF	>		<	ymgD		15.1
59	A	1250254	dihydroxyacetone kinase	*dhaK	<		>	*dhaR	DNA-binding transcriptional regulator	15.7
60	A	1257834	peptidyl-tRNA hydrolase	pth	<		>	ychH	predicted inner membrane protein	72.5
61	D	1318064	indole-3-glycerol-P synthetase	trpC	<	trpD	<	trpE		73.6
62	B	1341438	lipoprotein	*osmB	<		<	yciT		3.4
63	D	1342872	hypothetical protein	yciZ	<	gmr	<	rnb		48.2
64	A	1359040	gamma-Glu-putrescine synthase	*puuA	<		>	*puuD	gamma-Glu-GABA hydrolase	21.8
65	D	1393856		mppA	>	ynaI	>	insH	IS5 transposase and trans-activator	4.0
66	B	1432638	DNA-binding transcriptional regulator	ynaE	<		<	uspF		10.2
67	A	1438872	pyruvate-flavodoxin oxidoreductase	ydbK	<		>	ydbJ	predicted protein	5.1
68	D	1447730		feaB	>	tynA	<	maoC		5.0
69	C	1463278		paaY	>		<	insD		6.2
70	A	1489640	ncRNA	rydC	<		>	ydcA	predicted protein	14.9
71	B	1500460		tehB	>		>	ydcL	predicted lipoprotein	7.4
72	A	1515332	hypothetical protein	yncL	<		>	ydcX	predicted inner membrane protein	28.5
73	B	1518146		yncB	>		>	mcbR	DNA-binding transcriptional regulator	4.4
74	D	1528272		yncH	>	ydcD	>	ydcC	conserved protein	9.2
75	B	1543272	predicted protein	yddJ	<		<	yddG		3.5
76	D	1553260	dehydrogenase/acetaldehyde reductase	adhP	<	maeA	<	sra		4.9
77	D	1566238	predicted diguanylate cyclase	yddV	<	yddW	<	gadC		14.5
78	B	1570272	glutamate decarboxylase B	*gadB	<		<	pqqL		14.9
79	B	1580646	conserved protein	ydeN	<		<	ydeO		8.0
80	B	1590548	DNA-binding transcriptional regulator	hipB	<		<	ydeU		94.4
81	B	1596530	predicted lipoprotein	ydeK	<		<	lsrK		12.9
82	D	1613844		yneJ	>	yneK	>	ydeA	predicted arabinose transporter	4.3
83	B	1618030		marA	>		>	marB	predicted protein	3.9
84	B	1627238		ydfH	>		>	ydfZ	conserved protein	5.5
85	A	1630740	predicted mannonate dehydrogenase	ydfI	<		>	ydfK	Qin prophage transcriptional regulator	10.8
86	A	1655452	predicted protein	ynfC	<		>	ynfD	predicted protein	32.3
87	B	1682244		rstB	>		>	tus	inhibitor of replication at Ter	77.2
88	B	1687868		manA	>		>	ydgA	conserved protein	132.4
89	B	1745130		ydhR	>		>	ydhS	protein with FAD/NAD(P)-binding domain	3.8
90	A	1753168	predicted protein	ydhZ	<		>	*pykF	pyruvate kinase I	4.2
91	D	1769372	conserved protein	ydiL	>	ydiM	>	ydiN	predicted transporter	3.2
92	C	1793764	integration host factor (IHF)	ihfA	<	pheT	<	pheS		8.1
93	D	1811050		ydjN	>	ydjO	<	cedA		29.2
94	C	1841754		gdhA	>		<	ynjI		79.5
95	B	1894766		nudL	>		>	sdaA	L-serine deaminase I	55.4
96	B	1905768	predicted protein	yobF	<		<	yebO		11.2
97	D	1921260	ncRNA	ryeA	>	ryeB	<	yebY		11.5
98	B	1927030	protease II	ptrB	<		<	yebE		6.1
99	A	1928846	conserved protein regulated by LexA	yebG	<		>	purT	P-ribosylglycinamide formyltransferase 2	63.2
100	D	1928972	conserved protein regulated by LexA	yebG	<	purT	<	eda		390.6
101	A	1944202	RuvABC resolvasome	ruvA	<		>	yebB	predicted protein	11.3
102	B	1956162	TMAO reductase III (TorYZ)	torY	<		<	cutC		17.4
103	C	1966932	CheAB TCS chemotaxis regulator	cheB	<	cheR	<	tap		8.7
104	B	1994970	DNA-binding transcriptional activator	sdiA	<		<	yecC		21.7
105	A	2023030	predicted protein	*dsrB	<		>	yodD	predicted protein	88.4
106	B	2031954		rseX	>		>	*hchA	Hsp31 molecular chaperone	7.7
107	D	2049968		asnT	>	yeeJ	>	shiA	shikimate transporter	150.8
108	D	2057732	Asn tRNA	asnW	<	yeeO	>	asnU	Asn tRNA	3.2
109	B	2061434	L,D-transpeptidase linking Lpp to murein	erfK	<		<	cobT		44.9
110	D	2103732	predicted acyl transferase	wbbJ	<	wbbI	<	rfc		4.3
111	D	2104956	conserved protein	wbbI	<	rfc	<	glf		14.6
112	D	2106934	UDP-galactopyranose mutase	glf	<	rfbX	<	rfbC		54.2
113	B	2134130	protein-tyrosine phosphatase	wzb	<		<	wza		53.9
114	B	2226932	NAD(P)-binding oxidoreductase	yohF	<		<	dusC		3.3
115	A	2247636	DNA-binding transcriptional regulator	yeiE	<		>	yeiH	conserved inner membrane protein	12.7
116	D	2255672	predicted nucleoside transporter	psuT	<	psuG	<	psuK	pseudouridine kinase	7.7
117	B	2284170		yejM	>		>	proL	Pro tRNA	9.5
118	A	2311066	outer membrane porin protein C	ompC	<		>	micF	ncRNA	16.1
119	B	2311354		micF	>		>	rcsD	RcsBC TCS phosphotransfer protein	13.8
120	D	2389234		yfbP	>	nuoN	<	nuoM		13.0
121	B	2454170	predicted fimbrial-like adhesin protein	yfcV	<		<	sixA		25.4
122	D	2467360		yfdH	>	yfdI	<	yfdK		38.3
123	C	2468764		yfdI	>		<	yfdK		26.5
124	D	2480972	predicted multidrug efflux system	emrY	<	emrK	>	*evgA	EvgAS TCS response regulator	126.6
125	A	2510860	manganese/divalent cation transporter	mntH	<		>	nupC	nucleoside (except guanosine) transporter	13.5
126	D	2532356		ptsH	>	ptsI	>	*crr	glucose-specific PTS enzyme IIA	9.2
127	B	2574144	carboxysome structural protein	eutS	<		<	maeB		11.3
128	D	2587966		narQ	>	acrD	<	ypfM		18.4
129	B	2651536		sseA	>		>	ryfA	ncRNA	40.6
130	B	2689548	ncRNA	glmY	<		<	purL		24.9
131	B	2753630		smpB	>		>	ssrA	tmRNA	60.7
132	B	2763338	CP4-57 prophage; predicted protein	yfjL	<		<	yfjM		13.8
133	B	2765760		yfjO	>		>	yfjP	CP4-57 prophage GTP-binding protein	5.2
134	D	2771468		yfjT	>	yfjW	>	yfjX	CP4-57 prophage antirestriction protein	32.6
135	D	2772262		yfjT	>	yfjW	>	yfjX	CP4-57 prophage antirestriction protein	26.4
136	B	2773166		yfjW	>		>	yfjX	CP4-57 prophage antirestriction protein	41.0
137	D	2779640		psaA	>	ypjA	<	ileY		28.8
138	B	2783272	adhesin-like autotransporter	ypjA	<		<	ileY		58.9
139	A	2784466	Ile tRNA	ileY	<		>	*csiD	predicted protein	3.7
140	A	2786358	Ile tRNA	ileY	<		>	*csiD	predicted protein	31.8
141	A	2795168	predicted membrane protein	yqaE	<		>	ygaV	DNA-binding transcriptional regulator	53.2
142	B	2882250	predicted protein	ygcL	<		<	ygcB		6.3
143	A	2903434	conserved protein	ygcF	<		>	ygcG	predicted protein	57.0
144	B	2985568		yqeG	>		>	yqeH	protein with bipartite regulator domain	32.6
145	C	2987942		yqeJ	>		<	yqeK		9.1
146	D	2990738		ygeG	>	ygeH	>	ygeI	predicted protein	6.8
147	C	2992070		ygeI	>		<	insD		6.4
148	C	2992950		ygeI	>		<	insD		6.9
149	C	2993358		ygeI	>		<	insD		13.2
150	B	3134436	phosphate transporter	pitB	<		<	gsp		12.6
151	A	3145934	predicted protein	yghW	<		>	yghZ	aldo-keto reductase	3.2
152	B	3166762	predicted cyanide hydratase	mqsR	<		<	ygiV		65.1
153	C	3181642		zupT	>		<	ribB		10.0
154	B	3183246		yqiC	>		>	ygiL	predicted fimbrial-like adhesin protein	3.7
155	D	3189250		yqiH	>	yqiI	<	glgS		68.5
156	D	3210472		rpsU	>	dnaG	>	rpoD	RNA polymerase sigma 70	21.3
157	D	3259830	L-PSP (mRNA) endoribonuclease	tdcF	<	tdcE	<	tdcD		3.6
158	D	3266232		tdcR	>	yhaB	>	yhaC	predicted protein	9.6
159	C	3326238		yhbY	>		<	greA		3.6
160	B	3335948	ABC-type organic solvent transporter	yrbC	<		<	yrbD		14.0
161	D	3345766		ptsN	>	yhbJ	>	npr	N-regulated PTS system (Npr)	75.4
162	D	3348770	ncRNA	ryhA	>	arcB	<	yhcC		5.9
163	D	3395840	ribonuclease G	rng	<	yhdE	<	mreD		4.3
164	D	3453834	general secretory pathway component	gspA	<	gspC	>	gspD	general secretory pathway component	9.3
165	B	3467930	periplasmic endochitinase	chiA	<		<	tufA		5.0
166	B	3497370		cysG	>		>	yhfL	conserved secreted peptide	13.0
167	D	3533142		pck	>	envZ	<	ompR		41.3
168	D	3542562		yhgA	>	bioH	>	gntX	gluconate periplasmic binding protein	15.9
169	B	3576742	DNA-binding transcriptional repressor	gntR	<		<	yhhW		12.2
170	B	3582734		yrhD	>		>	yrhB	predicted protein	4.3
171	A	3584864	gamma-glutamyltranspeptidase	ggt	<		>	yhhA	conserved protein	12.9
172	D	3604756		yhhN	>	zntA	<	sirA		8.8
173	B	3621930		yhhH	>		>	yhhI	predicted transposase	3.3
174	A	3632156	predicted protein	yhiJ	<		>	*yhiM	conserved inner membrane protein	32.2
175	A	3632742	predicted protein	yhiJ	<		>	*yhiM	conserved inner membrane protein	13.3
176	A	3637868	universal stress protein B	*uspB	<		>	uspA	universal stress global response regulator	22.7
177	D	3647644		arsR	>	arsB	>	arsC	arsenate reductase	31.8
178	C	3648872		arsC	>		<	insH		153.6
179	B	3656130		hdeD	>		>	gadE	DNA-binding transcriptional activator	9.1
180	B	3708672	predicted metal dependent hydrolase	eptB	<		<	yhjX		48.9
181	B	3717944		yiaG	>		>	cspA	major cold shock protein	6.1
182	B	3720058		insK	>		>	sokA	ncRNA	31.7
183	A	3749938	predicted protein	yiaT	<		>	yiaU	DNA-binding transcriptional regulator	22.4
184	B	3794944		rfaC	>		>	rfaL	O-antigen ligase	3.8
185	D	3796942		rfaL	>	waaU	<	rfaZ		46.2
186	D	3798472	lipopolysaccharide core synthesis protein	rfaZ	<	rfaY	<	rfaJ		145.8
187	D	3800958	UDP-D-glucose:LPS glucosyltransferase	rfaJ	<	rfaI	<	rfaB		15.8
188	D	3802448	UDP-galactose:LPS galactosyltransferase	rfaB	<	rfaS	<	rfaP		21.5
189	B	3834856	Sec tRNA	selC	>		>	setC	predicted sugar efflux system	22.6
190	A	3851272	ncRNA	istR	<		>	tisB	lexA-regulated toxic peptide	57.3
191	B	3886640		tnaC	>		>	tnaA	tryptophanase/L-cysteine desulfhydrase	29.5
192	D	4001054	conserved inner membrane protein	yigF	<	yigG	<	rarD		5.5
193	D	4002164	predicted inner membrane protein	yigG	<	rarD	<	yigI		13.0
194	B	4076572		yiiD	>		>	yiiE	predicted transcriptional regulator	4.5
195	D	4110740	conserved protein	yiiQ	<	yiiR	>	yiiS	conserved protein	10.8
196	A	4116232	glycerol facilitator	glpF	<		>	yiiU	conserved protein	15.6
197	B	4120330	HslUV protease	hslV	<		<	ftsN		3.6
198	D	4220334		aceK	>	arpA	<	iclR		5.4
199	B	4281230	conserved protein	yjcF	<		<	actP		83.6
200	A	4380530	fumarate reductase	frdA	<		>	poxA	predicted lysyl-tRNA synthetase	4.7
201	A	4417636	L-ascorbate 6-phosphate lactonase	ulaG	<		>	ulaA	L-ascorbate-specific PTS enzyme IIC	4.9
202	A	4432132	NAD(P)H:quinone oxidoreductase	ytfG	<		>	ytfH	predicted transcriptional regulator	90.2
203	A	4434562	2':3'-cyclic-nucleotide 2'-phosphodiesterase	cpdB	<		>	cysQ	PAPS 3'(2'),5'-bisphosphate nucleotidase	4.3
204	B	4472740		yjgJ	>		>	yjgK	conserved protein	15.2
205	C	4475268		yjgL	>		<	argI		80.9
206	A	4477770	predicted acetyltransferase	yjgM	<		>	yjgN	conserved inner membrane protein	4.1
207	A	4492648	L-idonate 5-dehydrogenase, NAD-binding	idnD	<		>	idnK	D-gluconate kinase, thermosensitive	6.0
208	B	4504448		yjhC	>		>	ythA	expressed protein	8.4
209	C	4505142		ythA	>		<	insI		5.3
210	A	4538166	N-acetylnuraminic acid OM channel protein	nanC	<		>	fimB	Tyr recombinase/fimA inversion regulator	5.5
211	A	4538758	N-acetylnuraminic acid OM channel protein	nanC	<		>	fimB	Tyr recombinase/fimA inversion regulator	127.7
212	D	4543668		fimC	>	fimD	>	fimF	minor component of type 1 fimbriae	5.0
213	C	4570642		yjiS	>		<	mcrC		41.4
214	D	4575670		yjiS	>	mcrC	<	mcrB		12.1
215	D	4576468	5-methylcytosine-specific restriction enzyme	mcrC	<	mcrB	<	symE		26.3
216	D	4588370	conserved protein	yjiX	<	*yjiY	>	tsr	methyl-accepting chemotaxis protein I	10.5
217	A	4601330	predicted inner membrane protein	yjjP	<		>	yjjQ	DNA-binding transcriptional regulator	22.9
218	B	4609336		prfC	>		>	osmY	periplasmic protein	3.7

Genomic SELEX was performed for search of the binding sites of RNAP RpoS holoenzyme. By setting the cut-off level of 3.0, a total of 218 binding sites were identified (see Fig 1 for SELEX pattern), which are aligned along the map of E. coli K12 genome. A total of 125 sites are located within intergenic spacers: 50 within type-A spacers (shown under orange background); and 79 within type-B spacers (shown under green background). The constitutive promoters of RpoS were predicted based on the adjacent genes [note that only the genes next to the RpoS holoenzyme-binding sites are shown] and the gene orientation (shown by arrows in the column of transcription direction). A total of 73 RpoS holoenzyme-binding sites are located inside open reading frames as indicated by the gene symbols shown in RpoS column.

*The genes listed in RegulonDB as the regulated targets of RpoS.

Table 2

Distribution of the binding sites of each RNAP holoenzyme.

Sigma	Total no. holoenzyme binding sites	Within Spacers			Inside Genes	Constitutive promoters
		Type-A	Type-B	Type-C		Type-A spacer	Type-B spacer	Total
RpoD	1320	177	317	49	777 (60%)	177~354	317	494~671
		543 (40%)
RpoS	218	50	79	16	73 (33%)	50~100	79	129~179
		125 (67%)
RpoH	133	41	60	6	26 (20%)	41~82	60	101~142
		107 (80%)
RpoF	105	7	27	3	68 (65%)	7~14	27	34~41
		37 (35%)
RpoE	126	29	48	7	42 (33%)	29~58	48	77~106
		84 (67%)

RNAP holoenzyme was reconstituted from the sigma-free core enzyme and 4-fold molar excess of each sigma subunit. The binding site of each holoenzyme on the genome of E. coli K-12 W3110 was determined in vitro using the improved Genomic SELEX screening system. Details of the experimental procedures are described previously [26]. The number of constitutive promoters were estimated based on the location of holoenzyme-binding sites. The number of constitutive promoters recognized by RpoD holooenzyme were described in the previous report [27].

Up to the present, two general approaches have been employed to define the RpoS regulon: the proteome analysis using two-dimensional gels of whole cell lysates [35]; and the transcriptome analysis using ChIP-chip or ChIP-Seq systems [37-39]. These studies altogether indicated that RpoS regulates, directly or indirectly, 10% (approximately 500 genes) of the E. coli genes, of which only about 140 genes were predicted to be under the direct control in vivo of RpoS [38]. The total number of RpoS promoters (or the transcription initiation sites) listed in the current RegulonDB database is as many as 164 [note that all these promoters were detected in vivo]. Of which 21 were identified by setting the cut-off level at 3.0 (Table 1, marked by asterisk), indicating that only these promoters represent the constitutive promoters and the majority of other known RpoS promoters represent the inducible promoters that are expressed only under the support of regulatory factors. Genomic SELEX analysis identified minimum 129 and maximum 179 RpoS constitutive promoters including 21 known RpoS-dependent promoters (Table 2). The highest peak (390-fold higher than the background of original library alone) was located at the 5’-proximal region of the purT gene (Fig 1), which encodes a bifunctional enzyme with both phosphoribosylglycinamide formyltransferase using formate (the third-step reaction of purine nucleotide synthesis) and acetate kinase for the synthesis of acetylphosphate (AcP). AcP might be utilized as the general phosphate donor for phosphorylation of most of the stress-response TCS (two-component system) response regulators under stressful conditions. A high-level peak (154-fold higher than the background of DNA library) was detected upstream of the artPIQM operon encoding L-arginine ABC transporter. This promoter was also identified in vivo to be RpoS dependent [36] (Table 1). High-level binding of the RpoS holoenzyme was also identified upstream of the cydAB operon encoding cytochrome oxidase for anaerobic respiration, and the hipBA operon encoding anti-toxin-toxin pair for control the persistence (Fig 1 and Table 1). RpoS-dependent constitutive promoter(s) also exists upstream of the nanCMS operon (N-acetylneuraminic acid transport and utilization) and/or the fimB gene (regulator for fimA encoding fimbrin, the major type-1 pili) (Fig 1 and Table 1). Noteworthy is that most of the RpoS-dependent promoters listed in the current databases might be those under the indirect control of RpoS [8,9,27]. Otherwise a set of RpoS-dependent promoters, designated as the inducible promoters, might be activated in the presence of additional supporting factors.

Using the newly constructed collection of E. coli promoters expressing two-fluorescent reporters, one attached to the test promoter and another to the reference promoter, we performed a systematic quantitative search in vivo for E. coli promoters that are activated in the stationary phase [39]. The activity of RpoS-dependent promoters was measured at various growth phases under various growth conditions. The results indicated that the constitutive promoters exhibited low but steady-state activity while the inducible promoters generally showed high activity during the transition from exponential growth to stationary phase.

The RpoS regulon is involved in not only cell survival in the stationary phase, but also in cross protection against various stresses, including nutrient starvation, osmotic stress, acid shock, cold shock, heat shock, and oxidative DNA damage [33,34]. Beyond entry into stationary phase, E. coli forms aggregates or biofilms that are morphologically and physiologically distinct from cells of planktonic growth. This requires coordinated production of an extracellular matrix of polysaccharide polymers and protein fibers that facilitate cell aggregation and adhesion to solid surface. The genes involved in biofilm formation and transformation into persister cells were included in the list of RpoS constitutive promoters [40,41].

The whole sets of constitutive promoters for heat-shock response sigma RpoH

When E. coli cells are exposed to higher temperature, a set of heat-shock proteins (HSPs) is markedly and transiently induced. Heat shock-induced proteins (HSPs) play major roles in controlling the structure and function of various proteins, including protein folding, assembly, transport, repair and degradation during normal growth as well as under stress conditions [42,43]. The heat-shock response is a cellular protective system for maintenance of protein homeostasis. The set of HSPs include the GroEL (HSP60) and DnaK (HSP70) chaperones and the Lon and the Clp proteases. RpoH is specifically required for expression of the genes encoding a set of HSPs as identified by proteome [44,45] and also by transcriptome analyses [46]. Genome-wide transcription profiling of the regulatory targets of RpoH was identified under the moderate induction of a plasmid-borne rpoH gene under defined, steady state growth conditions [47]. A total of 126 genes were influenced in the absence or in the over-expression of RpoH, which are organized in 85 operons. The set of genes identified in vivo by changing the level of RpoH include a large number of indirect targets, which are affected in response to the changes in the level of direct target [8,9,27]. The total number of RpoH promoters (or the transcription initiation sites) listed in the current RegulonDB database is as many as 322, but the majority of RpoH targets are predicted by the computational analysis using the consensus sequence that was predicted based on a few experimentally identified RpoH promoters.

We isolated RpoH protein for the first time and confirmed its recognition in vitro of the known HSP gene promoters [48]. Since then no serious examination in vitro has been performed to identify the RpoH function and it regulatory targets. To get insights into the regulatory role of RpoH sigma, we then performed in this study the Genomic SELEX screening using the reconstituted RNAP RpoH holoenzyme. By setting the cut-off level of 3.0 fold higher than the background of original library DNA, a total of 133 RpoH holoenzyme-binding peaks were identified (Fig 2 and Table 3), of which 107 (80%) are located within intergenic spacers and 26 (20%) are inside of open reading frames (Table 2). Since the majority of hitherto identified promoters are located within spacers, detailed search for the constitutive promoters was focused on the total of 107 peaks within spacers. The spacers containing RpoH holoenzyme-binding sites were also classified into three types (Tables 2 and 3 for the whole list): 41 peaks are located within type-A spacer; 60 peaks are located within type-B spacers; and 6 peaks are located within type-C spacers. Based on the transcription direction of flanking genes, the total number of RpoH constitutive promoters was predicted to range between minimum 101 (41 type-A plus 60 type-B) and maximum 142 (41x2 type-A plus 60 type-B) (Table 2).

Fig 2

SELEX-chip search for RNAP RpoH holoenzyme-binding sequences on the E. coli K-12 genome.

The y-axis represents the relative number of RpoH holoenzyme-bound DNA fragments whereas x-axis represents the position on the E.coli K-12 genome, in base pair. The adjacent gene on E. coli K-12 genome of peak position was indicated for high intensity peaks. The peaks located within spacer regions are shown in green color, while peaks located within open reading frames are shown in orange color. The list of RpoH holoenzyme-binding sites is described in Table 3.

Table 3

RpoH holoenzyme-binding sites on the E. coli K-12 genome.

No	Type	Map	Gene Function	Left	D	RpoH	D	Right	Gene Function	Intensity
1	A	12044	predicted protein	yaaI	<		>	*dnaK	chaperone Hsp70	3.2
2	A	142736	carbonic anhydrase	*can	<		>	yadG	ABC superfamily transporter	3.9
3	B	155442	outer membrane usher protein	htrE	<		<	ecpD		3.0
4	B	164658		hrpB	>		>	mrcB	glycosyl transferase and transpeptidase	3.8
5	B	202068		lpxD	>		>	fabZ	hydroxymyristol acyl carrier dehydratase	4.0
6	B	229134		aspU	>		>	dkgB	2,5-diketo-D-gluconate reductase B	3.0
7	C	262270		thrW	>		<	ykfI		3.6
8	B	292170	CP4-6 prophage protein	yagK	<		<	yagL		4.3
9	B	331456		betT	>		>	yahA	DNA-binding transcriptional regulator	3.5
10	D	343660		yahK	>	yahL	>	yahM	predicted protein	5.6
11	B	379186	frmRAB operon regulator	frmR	<		<	yaiO		3.0
12	B	406536		yaiA	>		>	aroM	conserved protein	3.6
13	B	477848	conserved inner membrane protein	ylaB	<		<	ylaC		3.3
14	D	543470	conserved protein	ylbA	<	allC	<	allD		10.1
15	B	557960		sfmA	>		>	sfmC	pilin chaperone	3.5
16	D	559456		sfmC	>	sfmD	>	sfmH	fimbrial-like adhesin protein	3.1
17	B	581644		nohB	>		>	appY	DLP12 transcriptional activator	3.8
18	B	592452	phage N4 receptor IM protein	nfrB	<		<	cusS		3.2
19	B	629042		ybdB	>		>	cstA	carbon starvation protein	6.0
20	D	655854	anaerobic C4-dicarboxylate transport	dcuC	<	pagP	>	cspE	DNA-binding transcriptional repressor	3.3
21	B	661936	conserved protein	*ybeD	<		<	dacA		20.1
22	D	732870		ybfA	>	*rhsC	>	ybfB	predicted inner membrane protein	3.2
23	D	747240	predicted regulator	abrB	<	ybgO	<	ybgP		3.5
24	B	784068	zinc efflux system	zitB	<		<	ybgS		3.5
25	A	784656	conserved protein	ybgS	<		>	aroG	D-arabino-heptulosonate-7P synthase	3.0
26	B	837732	conserved protein	ybiI	<		<	ybiX		3.1
27	B	913136	anaerobic terminal reductases	hcp	<		<	ybjE		3.8
28	A	918368	conserved protein	ybjX	<		>	macA	macrolide transporter	6.3
29	B	959450		aroA	>		>	ycaL	peptidase with chaperone function	3.2
30	B	985134	aspartate aminotransferase	aspC	<		<	ompF		3.4
31	A	1050632	CspA-family stress protein	*cspH	<		>	cspG	DNA-binding transcriptional regulator	3.6
32	A	1102554	DNA-binding transcriptional activator	csgD	<		>	csgB	curlin nucleator protein	4.0
33	B	1120230	predicted protein	*bssS	<		<	dinI		5.4
34	C	1195868		icd	>		<	ymfD		6.2
35	D	1198640	e14 prophage inner membrane protein	ymfE	<	lit	<	intE		3.8
36	D	1200062	e14 prophage integrase	intE	<	xisE	>	ymfI	e14 prophage; predicted protein	3.2
37	B	1218154		ycgG	>		>	ymgF	predicted protein	3.4
38	C	1219948		ymgF	>		<	ymgD		3.9
39	A	1233950	sodium:proton antiporter	nhaB	<		>	fadR	DNA-binding transcriptional regulator	3.1
40	B	1255834	predicted adhesin	ycgV	<		<	ychF		3.2
41	B	1308330	voltage-gated potassium channel	kch	<		<	yciI		3.7
42	B	1349272	enoyl-[acyl-carrier-protein] reductase	fabI	<		<	ycjD		4.3
43	A	1389946	predicted hydrolase	*ycjY	<		>	*ycjZ	DNA-binding transcriptional regulator	4.4
44	B	1432738	Rac prophage transcriptional regulator	ynaE	<		<	uspF		4.2
45	B	1486246		ydcF	>		>	aldA	aldehyde dehydrogenase A	3.7
46	B	1565470	predicted diguanylate cyclase	yddV	<		<	yddW		5.2
47	B	1568560	glutamate:aminobutyric acid antiporter	gadC	<		<	gadB		2.3
48	B	1580550	conserved protein	ydeN	<		<	ydeO		3.3
49	B	1585730	fimbrial-like adhesin protein	ydeQ	<		<	ydeR		3.7
50	B	1613766		yneJ	>		>	*yneK	predicted protein	3.0
51	A	1630638	mannonate dehydrogenase	ydfI	<		>	ydfK	Qin prophage transcriptional regulator	4.0
52	B	1639072	Qin prophage S lysis protein	essQ	<		<	cspB		3.3
53	A	1639660	Qin prophage cold shock protein	cspB	<		>	cspF	Qin prophage cold shock protein	3.7
54	B	1646444		dicA	>		>	*ydfA	Qin prophage protein	3.2
55	B	1669358		ynfM	>		>	asr	acid-shock periplasmic protein	3.7
56	B	1751846	predicted oxidoreductase	ydhV	<		<	ydhY		3.1
57	B	1762570	Fe-S cluster assembly protein	sufA	<		<	rydB		5.1
58	A	1801256	threonyl-tRNA synthetase	thrS	<		>	yniD	predicted protein	5.3
59	A	1801758	threonyl-tRNA synthetase	thrS	<		>	yniD	predicted protein	3.4
60	B	1990832	PG phosphate synthase	pgsA	<		<	uvrC		4.2
61	D	2055332		amn	>	yeeN	<	asnW		3.0
62	A	2060070	DNA-binding transcriptionall regulator	nac	<		>	asnV	Asn tRNA	3.2
63	B	2096360	LPS O-antigen length regulator	*cld	<		<	ugd		3.2
64	D	2104956	conserved protein	wbbI	<	*rfc	<	glf		3.4
65	A	2261530	fructose-specific PTS enzyme IIA	fruB	<		>	setB	lactose/glucose efflux system	3.3
66	B	2276452	predicted protein	yejG	<		<	bcr		3.1
67	A	2342846	hypothetical protein	ypaB	<		>	nrdA	ribonucleoside diphosphate reductase	3.4
68	D	2355640		glpC	>	yfaD	>	ypaA	predicted protein	3.4
69	D	2363146		nudI	>	ais	>	arnB	uridine aminotransferase	3.8
70	B	2403570	NADH:ubiquinone oxidoreductase	nuoA	<		<	lrhA		5.1
71	A	2405036	DNA-binding transcriptional regulator	lrhA	<		>	yfbQ	predicted aminotransferase	3.2
72	D	2476546	DNA-binding transcriptional regulator	dsdC	<	dsdX	>	dsdA	D-serine ammonia-lyase	3.6
73	D	2489940	predicted transporter	yfdV	<	oxc	<	frc		3.6
74	D	2490262	predicted oxalyl-CoA decarboxylase	oxc	<	frc	<	yfdX		3.6
75	A	2492430	predicted protein	yfdX	<		>	ypdI	lipoprotein for colanic acid biosynthesis	3.0
76	A	2493362	predicted inner membrane protein	yfdY	<		>	lpxP	palmitoleoyl-ACP acyltransferase	3.6
77	D	2532356		ptsH	>	ptsI	>	crr	glucose-specific PTS enzyme IIA	3.2
78	A	2597838	dihydrodipicolinate synthase	dapA	<		>	gcvR	DNA-binding transcriptional repressor	3.1
79	B	2599140		bcp	>		>	hyfA	hydrogenase 4, 4Fe-4S subunit	3.1
80	D	2626032		ppx	>	yfgF	>	yfgG	predicted protein	3.6
81	A	2696642	conserved protein	yfhB	<		>	yfhH	DNA-binding transcriptional regulator	3.3
82	A	2714742	pyruvate formate lyase subunit	yfiD	<		>	ung	uracil-DNA-glycosylase	4.8
83	A	2739338	D-arabino-heptulosonate-7P synthase	aroF	<		>	yfiL	predicted protein	3.5
84	A	2823870	lytic murein transglycosylase B	mltB	<		>	srlA	glucitol/sorbitol-specific PTS IIC	3.1
85	A	2837534	DNA-binding transcriptional repressor	ascG	<		>	ascF	cellobiose/arbutin/salicin PTS IIB-IIC	3.5
86	D	2884946	predicted protein	ygcL	<	ygcB	<	cysH		3.4
87	A	2898372	deoxygluconate dehydrogenase	ygcW	<		>	yqcE	predicted transporter	4.6
88	A	2932264	L-fuculose-1-phosphate aldolase	fucA	<		>	fucP	L-fucose transporter	3.3
89	B	2967056	nucleotide hydrolase	rppH	<		<	ygdT		4.5
90	A	2976930	diaminopimelate decarboxylase	lysA	<		>	lysR	DNA-binding transcriptional regulator	3.4
91	B	2985164		yqeG	>		>	yqeH	protein with bipartite regulator domain	3.3
92	D	2985970		yqeG	>	yqeH	>	yqeI	transcriptional regulator	3.2
93	D	2991152		ygeG	>	ygeH	>	ygeI	predicted protein	3.7
94	C	2991858		ygeI	>		<	insD		3.5
95	B	3067930	mechanosensitive channel	mscS	<		<	fbaA		3.0
96	B	3166268	DNA-binding transcriptional regulator	ygiT	<		<	mqsR		3.0
97	B	3237654		alx	>		>	sstT	sodium:serine/threonine symporter	6.9
98	D	3266066		tdcR	>	yhaB	>	yhaC	predicted protein	3.0
99	A	3276944	DNA-binding transcriptional regulator	agaR	<		>	kbaZ	tagatose 6-phosphate aldolase 1	3.6
100	B	3319952	predicted hydrolase, inner membrane	yhbX	<		<	leuU		3.2
101	A	3325832	23S rRNA methyltransferase	rrmJ	<		>	yhbY	predicted RNA-binding protein	14.1
102	D	3360232		gltF	>	*yhcA	>	yhcD	predicted outer membrane protein	3.2
103	B	3375554	stringent starvation protein A	sspA	<		<	rpsI		3.7
104	B	3387142	p-hydroxybenzoic acid efflux system	*aaeA	<		<	aaeX		3.1
105	A	3559934	thiosulfate:cyanide sulfurtransferase	glpE	<		>	glpD	sn-glycerol-3-phosphate dehydrogenase	3.5
106	D	3629568	predicted HlyD family secretion protein	yhiI	<	yhiJ	>	yhiM	conserved inner membrane protein	3.6
107	C	3634072		yhiM	>		<	yhiN		3.8
108	B	3655654		hdeD	>		>	gadE	DNA-binding transcriptional activator	4.1
109	C	3661646		mdtF	>		<	gadW		3.7
110	B	3681552	C4-dicarboxylic acid citrate transporter	dctA	<		<	yhjK		3.0
111	A	3717248	conserved protein	yiaF	<		>	yiaG	transcriptional regulator	3.0
112	B	3717858		yiaG	>		>	cspA	major cold shock protein	3.0
113	B	3720058		insK	>		>	sokA	ncRNA	3.0
114	A	3826772	glutamate transporter	gltS	<		>	yicE	predicted transporter	4.4
115	B	3834954		selC	>		>	setC	predicted sugar efflux system	3.4
116	A	3865668	heat shock chaperone	*ibpA	<		>	yidQ	conserved outer membrane protein	4.6
117	A	3939432	predicted transcriptional regulator	yieP	<		>	*rrsC	16S ribosomal RNA of rrnC operon	3.6
118	A	4002730	conserved protein	yigI	<		>	*pldA	outer membrane phospholipase A	3.7
119	B	4068432	predicted aldose-1-epimerase	yihR	<		<	yihS		7.5
120	B	4360430	DNA-binding transcriptional activator	cadC	<		<	pheU		4.0
121	B	4364770	C4-dicarboxylate antiporter	dcuA	<		<	aspA		4.6
122	A	4366568	aspartate ammonia-lyase	aspA	<		>	*fxsA	inner membrane protein	19.2
123	D	4371850		yjeI	>	*yjeJ	<	yjeK		4.9
124	D	4486746		yjgQ	>	yjgR	<	idnR		4.1
125	B	4494638		leuX	>		>	insC	KpLE2 phage IS2 element repressor	3.1
126	B	4518430	KpLE2 transcriptional regulator	yjhU	<		<	yjhF		3.1
127	B	4530030	KpLE2 phage endoglucanase	sgcX	<		<	yjhP		3.3
128	A	4538050	N-acetylnuraminic acid channel protein	nanC	<		>	fimB	tyrosine recombinase, fimA regulator	3.5
129	A	4538964	N-acetylnuraminic acid channel protein	nanC	<		>	fimB	tyrosine recombinase, fimA regulator	3.4
130	B	4540968		fimE	>		>	fimA	major type 1 subunit fimbrin (pilin)	3.4
131	A	4549330	fructuronate transporter	gntP	<		>	uxuA	mannonate hydrolase	5.0
132	D	4576468	5-methylcytosine restriction enzyme	mcrC	<	mcrB	<	symE		3.5
133	A	4633370	DNA-binding transcriptional activator	rob	<		>	*creA	conserved protein	3.3

Genomic SELEX was performed for search of the binding sites of RNAP RpoH holoenzyme. By setting the cut-off level of 3.0, a total of 133 binding sites were identified (see Fig 2 for SELEX pattern), which are aligned along the map of E. coli K12 genome. A total of 107 sites are located within intergenic spacers: 41 wihin type-A spacers (shown under orange background); and 60 within type-B spacers (shown under green background). The constitutive promoters of RpoH were predicted based on the adjacent genes [note that only the genes next to the RpoH holoenzyme-binding sites are shown] and the gene orientation (shown by arrows in the column of transcription direction). A total of 26 RpoH holoenzyme-binding sites are located inside open reading frames as indicated by the gene symbols shown in RpoH column.

* The genes listed in RegulonDB as the regulated targets of RpoH.

Among a total of 322 RpoH promoters (or the transcription initiation sites) listed in RegulonDB database, 20 were identified setting the cut-off level at 3.0 (Table 3, marked by asterisk). The majority of RpoH promoters in the database were suggested to belong to the inducible promoters that are expressed only under the support of other positive regulatory factors. Otherwise these RpoH promoters might represent the inaccurate prediction as note above. Genomic SELEX analysis identified minimum 100 and maximum 140 RpoH constitutive promoters including 18 known RpoH dependent promoters (Table 2). The highest peak was 20-fold intensity that was detected on promoter region of the ybeD gene, which encodes a conserved protein of unknown function under regulation of RpoH (Fig 2) [49], followed by high-level peaks at the aspA-fxsA and the rlmJ-yhbY intergenic regions. The fxsA gene encodes an inner membrane protein, which is involved in sensitivity control to bacteriophage T7 [49]. The rlmE gene encodes 23S rRNA 2’-O-ribose U2552 metyltransferase, and has been proposed to carry RpoH-dependent promoter [50]. The regulatory target of RpoH sigma identified by Genomic SELEX expands to a set of genes related to varieties of stress-response genes beyond the HSP genes. In fact, the genes for response to environmental insults such as ethanol, alkaline pH, and hyperosmotic shock and the genes for proteolysis and cell division have been indicated under the control of RpoH. The set of RpoH-regulon genes thus identified in vivo, however, vary depending on the culture conditions.

The whole sets of constitutive promoters for the flagella-chemotaxis sigma RpoF

The bacterial flagellum is a complex organelle consisting of three distinctive structural parts, the basal body, the hook and the filament [51]. The synthesis, assembly and function of the flagellar and chemotaxis system require the expression of more than 50 genes, which are divided into three temporally regulated transcriptional classes based on the hierarchy of expression order: class-I (early), class-II (middle), and class-III (late) [52,53]. The class-1 (early) consists of a single operon including two genes, flhD and flhC, each encoding transcription factor FlhD and FlhC, respectively, which together form a complex, FlhD2-FlhC2 or FlhD4-FlhC2, that activates transcription of a set of class-2 (middle) genes, including both the rpoF sigma gene (renamed fliA) and the flgM gene encoding the anti-RpoF factor [51,52]. RpoF is the sigma factor for flagellar chemotaxis, which recognizes the promoters of motility and flagellar synthesis genes. The regulatory target of RpoF in Salmonella was identified to include a set of genes that were classified into the class-3 operons of the flagella regulon [54,55]. More than 30 genes have been proposed to carry promoters that are under the control of RpoF sigma, including a set of the structural genes for flagella formation, and the chemotaxis genes encoding sensor of environmental signals affecting the motility control [54,56]. With use the combination of ChIP-chip, ChIP-seq and RNA-seq systems, a more comprehensive screening was recently performed for identification of the regulatory targets of RpoF sigma in E. coli [57]. A total of 52 RpoF-binding sites were identified in vivo on the genome of exponentially growing E. coli K-12 MG1655 cells in a rich LB medium, with a considerable level of over-lapping with the hitherto identified target genes of the RpoF regulon. The total number of RpoF promoters (or the transcription initiation sites) listed in the current RegulonDB database is as many as 144, which have been identified in vivo using ChIP-chip and ChIP-RNA Seq analyses. Most of the targets predicted by the in vivo data, however, represent those indirectly affected upon knock-out of the rpoF gene or over-production of RpoF.

We then performed the Genomic SELEX screening in vitro for search of the direct target promoters, genes and operons under the control of RpoF using the reconstituted RNAP RpoF holoenzyme. By setting the cut-off level of 4.0 fold higher than the background of original library DNA, a total of 105 RpoF holoenzyme-binding peaks were identified (Fig 3 and Table 4), of which 37 (35%) are located within intergenic spacers and 68 (65%) are inside of genes (Table 2). One unique feature of RpoF holoenzyme is its high-level (65%) binding to inside of open reading frames of a number of genes. A high-level (60%) of RNAP binding was also identified for RpoD holoenzyme [27]. The identification of the promoter-like sequences inside these genes awaits further analysis. The spacers containing RpoF holoenzyme-binding sites were also classified into three types (Tables 2 and 4 for the whole list): 7 peaks are located within type-A spacer; 27 peaks are located within type-B spacers; and 3 peaks are located within type-C spacers. Based on the transcription direction of flanking genes, the total number of RpoF constitutive promoters was predicted to range between minimum 34 (7 type-A plus 27 type-B) and maximum 41 (7x2 type-A plus 27 type-B) (Table 2). The total number of RpoF promoters (or the transcription initiation sites) listed in the current RegulonDB database is as many as 144. Of which 14 were identified setting cut-off level at 4.0 (Table 4, marked by asterisk), indicating that these promoters are constitutive promoters and the majority of known promoters represent the inducible promoters that are expressed only under the support of positive regulatory factors.

Fig 3

SELEX-chip search for RNAP RpoF holoenzyme-binding sequences on the E. coli K-12 genome.

The y-axis represents the relative number of RpoF holoenzyme-bound DNA fragments whereas x-axis represents the position on the E. coli K-12 genome, in base pair. The adjacent gene on E. coli K-12 genome of peak position was indicated for high intensity peaks. The peaks located within spacer regions are shown in green color, while peaks located within open reading frames are shown in orange color. The list of RpoF holoenzyme-binding sites is described in Table 4.

Table 4

RpoF holoenzyme-binding sites on the E. coli K-12 genome.

No	Type	Map	Gene Function	Left	D	RpoF	D	Right	Gene Function	Intensity
1	D	25672		ileS	>	lspA	>	fkpB	peptidyl-prolyl cis-trans isomerase	8.1
2	B	68342	L-arabinose isomerase	araA	<		<	araB		4.3
3	D	109556		secM	>	secA	>	mutT	NTP pyrophosphohydrolase	5.5
4	A	131366	predicted protein	yacH	<		>	acnB	aconitate hydratase	4.6
5	B	164658		hrpB	>		>	mrcB	glycosyl transferase and transpeptidase	6.4
6	D	198960		rseP	>	bamA	>	skp	periplasmic chaperone	4.7
7	B	202068		lpxD	>		>	fabZ	hydroxymyristol acyl carrier dehydratase	7.1
8	D	298660	conserved protein	yagQ	<	yagR	<	yagS		4.4
9	D	343660		yahK	>	yahL	>	yahM	predicted protein	27.1
10	D	353230		prpD	>	prpE	>	codB	cytosine transporter	4.7
11	B	463170		ppiD	>		>	ybaV	conserved protein	5.4
12	D	472938		glnK	>	amtB	<	tesB		4.0
13	D	477460		ybaA	>	ylaB	<	ylaC		7.8
14	D	482072	predicted protein	tomB	<	acrB	<	acrA		4.5
15	D	543470	conserved protein	ylbA	<	allC	<	allD		12.3
16	D	633930	predicted oxidoreductase	ybdH	<	ybdL	<	ybdM		5.4
17	D	652632	citrate lyase synthetase	citC	<	dpiB	>	dpiA	CitAB TCS response regulator	5.0
18	D	747240	predicted regulator	abrB	<	ybgO	<	ybgP		13.1
19	D	761630		sucA	>	sucB	>	sucC	succinyl-CoA synthetase	4.7
20	B	794336		ybhT	>		>	*modA	molybdate transporter subunit	9.2
21	B	822944	cardiolipin synthase 2	ybhO	<		<	ybhP		4.9
22	B	837732	conserved protein	ybiI	<		<	ybiX		5.7
23	D	844530		rlmF	>	ybiO	<	glnQ		9.0
24	B	874568		yliE	>		>	yliF	predicted diguanylate cyclase	4.3
25	D	878444		bssR	>	yliI	<	yliJ		5.3
26	D	893136		ybjN	>	potF	>	potG	ABC superfamily putrescine transporter	4.1
27	B	956734		ycaP	>		>	serC	3-phosphoserine aminotransferase	6.2
28	D	1039338		appC	>	appB	>	yccB	hypothetical protein	5.0
29	D	1048250	conserved protein	gfcC	<	gfcB	<	gfcA		4.0
30	B	1062060	modulator of CbpA co-chaperone	cbpM	<		<	cbpA		5.2
31	B	1129438	anti-sigma factor for FliA (sigma 28)	*flgM	<		<	flgA		15.0
32	B	1133868		flgF	>		>	flgG	flagellar component	6.7
33	B	1137530		flgJ	>		>	*flgK	flagellar hook-filament junction protein 1	27.2
34	D	1158658		ptsG	>	fhuE	>	hinT	purine nucleoside phosphoramidase	6.5
35	B	1193064	tRNA methyltransferase	mnmA	<		<	nudJ		5.3
36	D	1236068		fadR	>	ycgB	>	dadA	D-amino acid dehydrogenase	6.9
37	A	1243852	protein involved in flagellar function	*ycgR	<		>	ymgE	predicted inner membrane protein	4.6
38	B	1349272	enoyl-[acyl-carrier-protein] reductase	fabI	<		<	ycjD		5.6
39	D	1356550	predicted protein	ymjA	<	puuP	<	puuA		5.0
40	D	1392064		ycjZ	>	mppA	<	ynaI		6.5
41	A	1434934	outer membrane pore protein N	ompN	<		>	micC	ncRNA	5.9
42	B	1488236		aldA	>		>	cybB	cytochrome b561	13.9
43	D	1504230	predicted benzoate transporter	ydcO	<	ydcN	>	ydcP	predicted peptidase	16.1
44	B	1644248		ydfV	>		>	*flxA	Qin prophage; predicted protein	23.2
45	D	1661452		ynfF	>	ynfG	>	ynfH	oxidoreductase, membrane subunit	5.5
46	D	1734472		sodB	>	ydhP	<	ynhF		6.1
47	D	1794968	integration host factor (IHF)	ihfA	<	pheT	<	pheS		12.3
48	A	1801758	threonyl-tRNA synthetase	thrS	<		>	yniD	predicted protein	5.3
49	B	1815172	conserved protein	*chbG	<		<	chbF		4.2
50	D	1822736		cho	>	*ves	<	spy		10.8
51	D	1861032	methionine sulfoxide reductase B	msrB	<	gapA	>	yeaD	conserved protein	4.2
52	B	1887966	acyl-CoA synthetase	fadD	<		<	yeaY		9.9
53	A	1906860	predicted protein	mgrB	<		>	yobH	predicted protein	8.3
54	D	1914836	methionine-(R)-sulfoxide reductase	yebR	<	yebS	>	yebT	conserved protein	14.4
55	D	1928332	conserved protein	yebE	<	yebF	<	yebG		4.3
56	D	1938762	myristoyl-ACP-dependent acyltransferase	lpxM	<	yebA	<	znuA		11.9
57	D	1971862	purine-binding chemotaxis protein	cheW	<	cheA	<	motB		4.3
58	D	1988066	predicted protein	*yecH	<	tyrP	<	yecA		7.9
59	B	1999848	RNA polymerase sigma 28	*fliA	<		<	fliC		46.2
60	D	2233032		yeiS	>	yeiT	>	yeiA	Dihydropyrimidine dehydrogenase	4.8
61	D	2262454	fructose-specific PTS enzymes IIA	fruB	<	setB	<	yeiW		6.2
62	D	2379830	N-acyltransferase	elaA	<	rbn	>	elaD	predicted enzyme	12.5
63	B	2403570	NADH:ubiquinone oxidoreductase	nuoA	<		<	lrhA		3.5
64	D	2412454	conserved inner membrane protein	yfbV	<	ackA	>	pta	phosphate acetyltransferase	6.2
65	D	2456468	phosphohistidine phosphatase	sixA	<	fadJ	<	fadI		4.5
66	D	2609062		hyfH	>	hyfI	>	hyfJ	processing element hydrogenase 4	7.1
67	D	2684772	serine hydroxymethyltransferase	glyA	<	hmp	<	glnB		5.8
68	D	2745566	30S ribosomal subunit protein S16	rpsP	<	ffh	>	ypjD	predicted inner membrane protein	5.9
69	D	2868142	L-isoaspartate carboxymethyltransfeerase	pcm	<	surE	<	truD		5.8
70	D	2916840		barA	>	gudD	<	gudX		5.4
71	D	2952958	exonuclease V (RecBCD complex),	recD	<	recB	<	ptrA		8.0
72	D	2965066	diacylglyceryl transferase	lgt	<	ptsP	<	rppH		6.7
73	D	3087058		metK	>	galP	>	yggI	conserved protein	6.4
74	D	3124360	glycolate oxidase iron-sulfur subunit	glcF	<	glcE	<	glcD		4.0
75	C	3146946		yghZ	>		<	yqhA		5.2
76	B	3197666	deadenylyltransferase/adenylyltransferase	glnE	<		<	ygiF		8.8
77	B	3237654		alx	>		>	sstT	sodium:serine/threonine symporter	9.9
78	D	3338440	ABC superfamily toluene transporter	yrbF	<	yrbG	>	kdsD	D-arabinose 5-phosphate isomerase	6.2
79	D	3344772		hpf	>	ptsN	>	yhbJ	protein with NTP hydrolase domain	5.9
80	C	3388568		aaeR	>		<	tldD		4.7
81	D	3452166	type II secretion divergon	gspB	<	gspA	>	gspC	general secretory pathway component	11.8
82	D	3454168	general secretory pathway component	gspA	<	gspC	>	gspD	general secretory pathway component	14.7
83	D	3478072	predicted protein	yheV	<	kefB	<	kefG		6.1
84	D	3515848	predicted protein	damX	<	aroB	<	aroK		4.5
85	C	3528652		hslO	>		<	yhgE		4.0
86	D	3534454	TCS sensory histidine kinase	envZ	<	ompR	>	*greB	transcription elongation factor	4.3
87	D	3585866		yhhA	>	ugpQ	<	ugpC		18.1
88	D	3633746	predicted protein	yhiJ	<	*yhiM	<	yhiN		4.8
89	D	3689064	endo-1,4-D-glucanase	bcsZ	<	bcsB	<	bcsA		4.6
90	B	3779166		lldD	>		>	yibK	predicted rRNA methylase	9.2
91	D	3789732	threonine 3-dehydrogenase	tdh	<	kbl	<	htrL		4.9
92	A	3826672	glutamate transporter	gltS	<		>	yicE	predicted transporter	5.4
93	D	3829430		yicE	>	yicH	<	yicI		4.3
94	B	3906432	phosphate transporter	pstB	<		<	pstA		4.1
95	D	3945232		trpT	>	*hdfR	>	yifE	conserved protein	4.7
96	B	3951430		ilvE	>		>	ilvD	dihydroxyacid dehydratase	8.6
97	D	4003446	conserved protein	yigI	<	pldA	>	recQ	ATP-dependent DNA helicase	4.5
98	D	4032158		yigZ	>	trkH	>	hemG	protoporphyrin oxidase, flavoprotein	6.0
99	B	4068432	predicted aldose-1-epimerase	yihR	<		<	yihS		9.6
100	D	4178758		rplJ	>	rplL	>	rpoB	RNA polymerase, beta subunit	6.7
101	D	4286472	acetyl-CoA synthetase	acs	<	nrfA	>	nrfB	nitrite reductase	5.8
102	D	4327160	conserved protein	yjdM	<	*yjdA	>	yjcZ	conserved protein	12.4
103	D	4338862	biodegradative arginine decarboxylase	adiA	<	melR	>	melA	alpha-galactosidase, NAD(P)-binding	4.5
104	D	4535430	conserved protein	yjhX	<	yjhS	<	nanM		4.7
105	A	4589632	predicted inner membrane protein	yjiY	<		>	*tsr	methyl-accepting chemotaxis protein I	5.3

Genomic SELEX was performed for search of the binding sites of RNAP RpoF holoenzyme. By setting the cut-off level of 3.0, a total of 105 binding sites were identified (see Fig 3 for SELEX pattern), which are aligned along the map of E. coli K12 genome. A total of 37 sites are located within intergenic spacers: 7 wihin type-A spacers (shown under orange background); and 27 within type-B spacers (shown under green background). The constitutive promoters of RpoF were predicted based on the adjacent genes [note that only the genes next to the RpoF holoenzyme-binding sites are shown] and the gene orientation (shown by arrows in the column of transcription direction). A total of as many as 68 RpoF holoenzyme-binding sites are located inside open reading frames as indicated by the gene symbols shown in RpoF column.

*The genes listed in RegulonDB as the regulated targets of RpoF.

The highest peak was 46-fold intensity detected on promoter region of rpoF itself (Fig 3), indicating the autoregulation as already suggested [58]. A high-level peak was also identified upstream of the flgK gene, which encodes flagellar hook-filament junction protein that connects the filament to the hook, and its transcription has been shown in vitro under the direct control of RpoF [59]. The flgM gene encodes the anti-sigma factor for RpoF [55]. FlgM forms a complex with RpoF, thereby inactivating its sigma function but protects its degradation by the Lon protease for preservation [60].

The whole set of constitutive promoters for extra-cytoplasmic stress response sigma RpoE

The bacterial cell envelope is a dynamic compartment, changing its structure and function in response to environmental conditions. Accordingly, the integrity of envelope is maintained through frequent modulation of its composition and components. The minor sigma factor RpoE plays a central role in this process, by controlling the selective expression of envelope components [61]. The regulatory targets have been estimated after proteome and transcriptome analyses in vivo [62-64]. The activity of RpoE is negatively regulated by a membrane-bound anti-sigma factor RseA, which sequesters RpoE under unstressed conditions. Within membrane, RseA is associated at its C-terminal domain with a periplasmic protein RseB, which senses misfolded proteins for release and activation of RpoE from RpoE-RseA complexes [62,65]. The total number of RpoE promoters (or the transcription initiation sites) listed in the current RegulonDB database is as many as 518, of which most are identified by computational analyses based on the consensus sequence of RpoE promoters without experimental analysis. After SELEX screening as noted below, most of these RpoE promoters must be inaccurate estimation due to the error in the consensus sequence.

The Genomic SELEX screening system was employed as a short-cut approach for identification of the RpoE regulon. Previously we purified RpoE and examined its promoter selectivity using an in vitro transcription system [66]. Using this purified RpoE, we performed SELEX screening. By setting the cut-off level of 4.0 fold against original library DNA, a total of 126 RpoE holoenzyme-binding peaks were identified (Fig 4 and Table 5), of which 84 (67%) are located within intergenic spacers and 42 (33%) are inside of open reading frames (Tables 2 and 5 for the whole set). Since the majority of hitherto identified promoters are located within spacers, detailed search for the constitutive promoters was focused on the total of 84 peaks within spacers. The spacers containing RpoE holoenzyme-binding sites were also classified into three types (Table 2): 29 peaks are located within type-A spacer; 48 peaks are located within type-B spacers; and 7 peaks are located within type-C spacers. Based on the transcription direction of flanking genes, the total number of RpoE constitutive promoters was predicted to range between minimum 77 (29 type-A plus 48 type-B) and maximum 106 (29x2 type-A plus 48 type-B) (Table 2). Within the set of constitutive promoters identified by setting the cut-off level at 4.0, a total of 19 known RpoE promoters were identified (Table 5, marked by asterisk). The majority of known promoters represent the inducible promoters that are expressed only under the support of positive regulatory factors.

Fig 4

SELEX-chip search for RNAP RpoE holoenzyme-binding sequences on the E. coli K-12 genome.

The y-axis represents the relative number of RpoE holoenzyme-bound DNA fragments whereas x-axis represents the position on the E.coli genome, in base pair. The adjacent gene on E. coli K-12 genome of peak position was indicated for high intensity peaks. The peaks located within spacer regions are shown in green color, while peaks located within open reading frames are shown in orange color. The list of RpoE holoenzyme-binding sites is described in Table 5.

Table 5

RpoE holoenzyme-binding sites on the E. coli K-12 genome.

No.	Type	Map	Left Gene Function	Left	D	RpoE	D	Right	Right Gene Function	Intensity
1	D	154236	fimbrial-like adhesin protein	yadM	<	htrE	<	ecpD		5.0
2	B	164658		hrpB	>		>	mrcB	glycosyl transferase and transpeptidase	4.3
3	B	202068		lpxD	>		>	fabZ	hydroxymyristol acyl carrier dehydratase	30.9
4	C	262270		thrW	>		<	ykfI		4.6
5	D	270562	CP4-6 prophage transcriptional regulator	perR	<	insI	<	insH		4.6
6	B	292358	CP4-6 prophage; conserved protein	yagK	<		<	yagL		4.7
7	D	302738	xanthine dehydrogenase 2Fe-2S subunit	yagT	<	yagU	<	ykgJ		5.3
8	A	319448	oxidoreductase with FAD-binding domain	*ykgC	<		>	ykgD	DNA-binding transcriptional regulator	4.0
9	C	323832		ykgG	>		<	ykgH		5.6
10	B	331456		betT	>		>	yahA	DNA-binding transcriptional regulator	4.9
11	d	343660		yahK	>	yahL	>	*yahM	predicted protein	17.8
12	A	345542	neutral amino-acid efflux system	yahN	<		>	yahO	predicted protein	4.0
13	B	477848	conserved inner membrane protein	ylaB	<		<	ylaC		4.5
14	D	543560	conserved protein	*ylbA	<	allC	<	allD		9.6
15	B	581644		nohB	>		>	appY	DLP12 DNA-binding transcriptional activator	11.5
16	B	629042		ybdB	>		>	cstA	carbon starvation protein	4.3
17	B	728638		ybfA	>		>	rhsC	rhsC element core protein RshC	5.6
18	D	732870		ybfA	>	rhsC	>	ybfB	predicted inner membrane protein	4.1
19	D	747240	predicted regulator	abrB	<	*ybgO	<	ybgP		9.3
20	A	753948	citrate synthase	gltA	<		>	sdhC	succinate dehydrogenase	4.7
21	B	773834		ybgE	>		>	ybgC	predicted acyl-CoA thioesterase	4.2
22	B	837732	conserved protein	ybiI	<		<	ybiX		4.1
23	D	838958	conserved protein	ybiX	<	fiu	<	mcbA		4.3
24	D	844530		rlmF	>	ybiO	<	glnQ		6.0
25	A	862768	pyruvate formate lyase activating enzyme	ybiY	<		>	fsaA	fructose-6-phosphate aldolase 1	4.4
26	B	887366	ncRNA	*rybB	<		<	ybjL		55.5
27	B	986332	outer membrane porin 1a (Ia;b;F)	ompF	<		<	asnS		5.0
28	D	992744		pepN	>	ssuB	<	ssuC		5.3
29	D	1045134	exopolysaccharide export protein	gfcE	<	gfcD	<	gfcC		5.1
30	C	1049830		insB	>		<	cspH		6.3
31	A	1050632	CspA-family stress protein	*cspH	<		>	cspG	DNA-binding transcriptional regulator	4.7
32	D	1088072	predicted glycosyl transferase	pgaC	<	pgaB	<	pgaA		4.8
33	B	1166772		ndh	>		>	ycfJ	predicted protein	4.0
34	C	1195868		icd	>		<	ymfD		6.4
35	B	1218154		ycgG	>		>	*ymgF	predicted protein	5.4
36	D	1259856		ychH	>	ychM	<	prs		5.3
37	D	1342036	lipoprotein	osmB	<	*yciT	<	yciZ		4.3
38	B	1349272	enoyl-[acyl-carrier-protein] reductaset	fabI	<		<	ycjD		5.6
39	B	1384744		ycjF	>		>	tyrR	DNA-binding transcriptional regulator	5.0
40	A	1389946	predicted hydrolase	ycjY	<		>	ycjZ	DNA-binding transcriptional regulator	17.2
41	B	1432738	Rac prophage transcriptional regulator	ynaE	<		<	uspF		5.6
42	D	1468142	KpLE2 phage-like IS repressor InsA	insC	<	insI	>	ydbC	NAD(P)-binding oxidoreductase	5.4
43	D	1500658		tehB	>	*ydcL	>	ydcM	predicted transposase	4.8
44	B	1518232		yncB	>		>	mcbR	DNA-binding transcriptional regulator	4.5
45	B	1542352	nitrate/nitrite transporter	narU	<		<	yddJ		4.9
46	B	1585730	fimbrial-like adhesin protein	ydeQ	<		<	ydeR		4.4
47	B	1588350	fimbrial-like adhesin protein	ydeS	<		<	hipA		4.5
48	A	1630638	predicted mannonate dehydrogenase	ydfI	<		>	*ydfK	Qin prophage transcriptional regulator	5.1
49	B	1638930	Qin prophage; predicted S lysis protein	essQ	<		<	cspB		7.0
50	A	1639660	Qin prophage; cold shock protein	cspB	<		>	*cspF	Qin prophage; cold shock protein	7.3
51	A	1676254	pyridine nucleotide transhydrogenase	pntA	<		>	ydgH	predicted protein	5.0
52	B	1751846	predicted oxidoreductase	ydhV	<		<	ydhY		6.6
53	A	1801758	threonyl-tRNA synthetase	thrS	<		>	yniD	predicted protein	10.1
54	D	1852370	predicted transporter	*ydjE	<	*ydjF	<	ydjG		4.4
55	B	1887966	acyl-CoA synthetase	fadD	<		<	yeaY		4.5
56	A	1923044	conserved protein	yobA	<		>	holE	DNA polymerase III	4.5
57	D	1938762	myristoyl-acyl carrier ACP acyltransferase	lpxM	<	*yebA	<	znuA		7.1
58	A	2060070	DNA-binding transcriptional dual regulator	nac	<		>	asnV	Asn tRNA	4.4
59	D	2103368	predicted acyl transferase	wbbJ	<	wbbI	<	rfc		4.2
60	D	2104870	conserved protein	wbbI	<	rfc	<	glf		5.7
61	D	2144358	uridine/cytidine kinase	udk	<	yegE	<	alkA		5.8
62	B	2196338		metG	>		>	yehI	conserved protein	8.6
63	D	2210570	conserved protein	yehS	<	yehT	<	yehU		5.2
64	D	2214150		yohO	>	yehW	<	yehX		4.0
65	D	2302258		yojO	>	eco	<	mqo		7.2
66	D	2369270		arnD	>	arnT	>	arnE	conserved protein	5.9
67	A	2405036	DNA-binding transcriptional repressor	lrhA	<		>	yfbQ	predicted aminotransferase	4.0
68	D	2441138	3-oxoacyl-[acyl-carrier-protein] synthase I	fabB	<	mnmC	<	yfcL		5.7
69	A	2459264	conserved protein	yfcZ	<		>	fadL	long-chain fatty acid OM transporter	5.3
70	D	2484966		evgA	>	evgS	<	yfdE		5.2
71	D	2490262	predicted oxalyl-CoA decarboxylase	*oxc	<	frc	<	yfdX		4.9
72	D	2492854	predicted protein	yfdX	<	*ypdI	<	yfdY		4.1
73	A	2507446	glucokinase	glk	<		>	yfeO	predicted ion channel protein	4.3
74	A	2535342	pyridoxal-pyridoxamine kinase	pdxK	<		>	*yfeK	predicted protein	7.5
75	D	2649866	fused transglycosylase/transpeptidase	pbpC	<	yfhM	>	sseA	3-mercaptopyruvate sulfurtransferase	6.1
76	A	2696642	conserved protein	yfhB	<		>	yfhH	DNA-binding transcriptional regulator	4.7
77	A	2708134	RNA polymerase sigma 24	rpoE	<		>	nadB	quinolinate synthase	6.3
78	B	2761570	CP4-57 prophage protein	yfjK	<		<	yfjL		5.6
79	B	2763272	CP4-57 prophage protein	yfjL	<		<	yfjM		3.2
80	D	2764730	CP4-57 prophage protein	yfjM	<	rnlA	>	yfjO	CP4-57 prophage protein	6.1
81	B	2773454		yfjW	>		>	yfjX	CP4-57 prophage antirestriction protein	6.1
82	C	2796040		ygaP	>		<	stpA		4.3
83	A	2812736	S-ribosylhomocysteinase	luxS	<		>	*micA	ncRNA	40.9
84	A	2874372	sulfate adenylyltransferase	cysD	<		>	*iap	aminopeptidase	4.2
85	D	2881556	predicted protein	ygcK	<	ygcL	<	ygcB		10.9
86	D	2894072	predicted flavoprotein	ygcQ	<	ygcR	<	ygcS		4.0
87	D	2903952	conserved protein	ygcF	<	*ygcG	<	eno		4.5
88	B	2985164		yqeG	>		>	yqeH	protein with bipartite regulator domain	4.0
89	A	3004270	DNA-binding transcriptional regulator	ygeV	<		>	*ygeW	conserved protein	4.5
90	D	3036652	ssDNA exonuclease	recJ	<	*dsbC	<	xerD		5.1
91	B	3237758		alx	>		>	sstT	sodium:serine/threonine symporter	23.4
92	D	3246540		yqjA	>	yqjB	>	yqjC	conserved protein	4.4
93	A	3276944	DNA-binding transcriptional dual regulator	agaR	<		>	kbaZ	tagatose 6-phosphate aldolase 1	4.2
94	B	3375554	stringent starvation protein A	sspA	<		<	rpsI		4.7
95	B	3387142	p-hydroxybenzoic acid efflux system	aaeA	<		<	aaeX		4.9
96	C	3528652		hslO	>		<	yhgE		4.1
97	A	3559934	thiosulfate:cyanide sulfurtransferase	glpE	<		>	glpD	sn-glycerol-3-phosphate dehydrogenase	4.0
98	B	3576850	DNA-binding transcriptional repressor	gntR	<		<	yhhW		4.9
99	A	3579162	ncRNA	ryhB	<		>	yhhY	predicted acetyltransferase	6.5
100	B	3599044	RNA polymerase sigma 32	*rpoH	<		<	ftsX		37.4
101	C	3661646		mdtF	>		<	gadW		6.7
102	D	3666862	glutamate decarboxylase A	gadA	<	yhjA	>	treF	cytoplasmic trehalase	6.6
103	B	3720058		insK	>		>	sokA	ncRNA	7.3
104	B	3739660	4Fe-4S ferredoxin-type hydrogenase	ysaA	<		<	yiaJ		4.2
105	D	3764054	predicted glutathione S-transferase	yibF	<	rhsA	>	yibA	lyase containing HEAT-repeat	4.0
106	B	3779166		lldD	>		>	yibK	predicted rRNA methylase	10.8
107	B	3828470		yicE	>		>	yicH	conserved protein	4.2
108	D	3829430		yicE	>	yicH	<	yicI		8.2
109	D	3862372		yidL	>	yidP	<	yidE		4.3
110	B	3906432	phosphate transporter subunit	pstB	<		<	pstA		5.2
111	D	3955270		ilvA	>	ilvY	>	ilvC	ketol-acid reductoisomerase	4.1
112	A	3958552	peptidyl-prolyl cis-trans isomerase C	ppiC	<		>	rep	DNA helicase/ssDNA-dependent ATPase	4.8
113	B	3978772		rffM	>		>	yifK	predicted transporter	4.0
114	B	4068538	predicted aldose-1-epimerase	yihR	<		<	yihS		10.7
115	B	4173964		thrT	>		>	tufB	protein chain elongation factor EF-Tu	5.7
116	B	4187734		rpoC	>		>	yjaZ	heat shock protein	4.0
117	A	4212172	predicted acetyltransferase	yjaB	<		>	metA	homoserine O-transsuccinylase	4.3
118	B	4249366		malM	>		>	ubiC	chorismate pyruvate lyase	5.7
119	B	4364770	C4-dicarboxylate antiporter	dcuA	<		<	aspA		5.2
120	A	4368630	predicted transporter	yjeH	<		>	groS	Cpn10 chaperonin GroES	4.3
121	A	4380530	anaerobic fumarate reductase	frdA	<		>	poxA	predicted lysyl-tRNA synthetase	4.0
122	B	4427854		fklB	>		>	cycA	D-alanine/D-serine/glycine transporter	8.0
123	D	4486746		yjgQ	>	yjgR	<	idnR		9.4
124	B	4530030	KpLE2 phage endoglucanase	sgcX	<		<	yjhP		5.2
125	B	4533162	conserved protein	yjhX	<		<	yjhS		4.0
126	A	4538964	N-acetylnuraminic acid outer membrane channel protein	nanC	<		>	fimB	tyrosine recombinase/fimA regulator	6.4
127	B	4638610		yjjY	>		>	*yjtD	predicted rRNA methyltransferase	4.6

Genomic SELEX was performed for search of the binding sites of RNAP RpoE holoenzyme. By setting the cut-off level of 4.0, a total of 126 binding sites were identified (see Fig 4 for SELEX pattern), which are aligned along the map of E. coli K12 genome. A total of 84 sites are located within intergenic spacers: 29 wihin type-A spacers (shown under orange background); and 48 within type-B spacers (shown under green background). The constitutive promoters of RpoE were predicted based on the adjacent genes [note that only the genes next to the RpoE holoenzyme-binding sites are shown] and the gene orientation (shown by arrows in the column of transcription direction). A total of 42 RpoE holoenzyme-binding sites are located inside open reading frames as indicated by the gene symbols shown in RpoE column.

*The genes listed in RegulonDB as the regulated targets of RpoE.

Genomic SELEX analysis identified minimum 77 and maximum 106 for the RpoE constitutive promoters. The highest peak (55-fold intensity) was detected in the promoter region of rybB, which encodes a small regulatory RNA for expression control of some outer membrane proteins. The rybB promoter is known to be regulated by RpoE (Fig 4) [67]. The second highest peak was located on the luxS-micA intergenic region. The micA gene again encodes a small regulatory RNA that regulates expression of many genes including outer membrane proteins [68,69]. The micA promoter is also established under the control of RpoE. These sRNAs control the repair of damages in the outer membrane that took place in response to envelope stress [70,71]. High-intensity peaks were detected on some other known RpoE-dependent promoters such as rpoH, pgrR and ycjY [72,73]. Among the total of 136 binding sites of RpoE holoenzyme, 36 overlaps with that of RpoH holoenzyme. Most of these overlapping sites are related to the genes that are expressed under envelope stresss or heat-shock stress.

The intracellular levels of all seven sigma factors in E. coli K-12 W3110

In this study, we determined the constitutive promoters for the four minor sigma factors, RpoS, RpoH, RpoF and RpoE, from E. coli K-12 W3110. These promoters are recognized by the RNAP holoenzyme containing each sigma in the absence of other supporting factors. Using the mixed reconstitution in vitro of RNAP holoenzyme in the presence of all seven sigma factors, we estimated the binding affinity of each sigma to the common core enzyme, the order being RpoD (highest) > RpoN > RpoF > RpoH > FecI > RpoE > RpoS (lowest) [74]. Once we get the knowledge of intracellular concentrations of these sigma factors, it should be possible to predict the expression levels of the regulatory target genes and operons under the control of the constitutive promoters of each sigma factor. Including these four minor sigma factors, we then determined the intracellular concentrations of all seven sigma subunits. For this purpose, antibodies were made against each of the purified sigma factors that were also used for SELEX screening.

E. coli K-12 W3110 type-A was cultivated with shaking at 37°C in LB medium, and the whole cell lysates were prepared in both exponential growing phase and the stationary phase. By using the quantitative immuno-blotting method and the purified sigma proteins as the reference controls, we measured the concentrations of all seven sigma subunits. The measurement was carried out for two independent cultures, and the immuno-blot analysis was repeated for all the samples. The intracellular concentration of RpoD sigma is maintained at a constant level (on average, 160 fmol/μg total protein) throughout the transition from the exponential growth phase to the stationary phase (Fig 5) in good agreement with the previous estimation [1,75]. Based on the relative level of RNAP core enzyme subunits, the number of RpoD sigma was estimated to be 800 molecules per genome [note that the number of RNAP core enzyme is 2,000 molecules per genome]. The level of RpoD sigma in E. coli K-12 MG1655 was about half the level of W3110 (data not shown). The second abundant sigma was RpoF (70 fmol/μg protein at log phase and 80 fmol/μg protein at stationary phase) (Fig 5), but the unneccesary RpoF is stored in an inactive form by forming complexes with the anti-RpoF sigma, FlgM [76]. RpoN is also always present at a constant level (approximately 40 fmol/μg protein) in both log and stationary phases (Fig 5). The levels of other four sigma, RpoS, RpoH, RpoE and FecI, are very low during the steady-state growth (Fig 5A), but upon entry into the stationary phase, the level of RpoS increased markedly up to the level (about 100 fmol/μg protein) higher than other five minor sigma factors (Fig 5B). Taking the intracellular concentrations and the binding affinity of sigma to RNAP core enzyme as noted above, we are now able to estimate the level of each RNAP holoenzyme. Noteworthy is that the total number of all seven sigma factors is approximately as many as that of the core enzyme, but the RNAP involved in the transcription cycle or the elongation of RNA chains is considered to lack sigma subunit, the RNAP not involved in transcription should be stored as the holoenzyme forms.

Fig 5

Intracellular concentrations of seven sigma factors in E. coli K-12 W3110 type-A strain.

E. coli K-12 W3110 type-A strain was grown in LB medium at 37°C with shaking. Cells were harvested at various times and cell lysates were subjected to the quantitative immuno-blot analysis of all seven sigma factors as described in Materials and Methods. [A] The sigma levels at exponential growth phase; [B] the sigma levels in the stationary phase.

Discussion

Seven species of the sigma subunit exist in E. coli K-12, the widely used model E. coli strain. Here we identified the whole set of constitutive promoters for four minor sigma factors, RpoS, RpoH, RpoF and RpoE, by using the Genomic SELEX system. Up to the present time, the binding sites of RNAP and TF have been identified in vivo using the high-throughput systems such as ChIP-chip, ChIP-seq and RNA-seq systems. Even using these modern techniques, however, it is in principle impossible to obtain the whole set of binding sites for both RNAP and TFs because of the competition with other DNA-binding proteins in binding to DNA targets [8,9,24] [note that E. coli contains more than 500 DNA-binding proteins [77], and because the binding of RNAP and TFs often depends on the supporting factors for binding to targets [8,9,25].

The computational approaches in silico have also been employed to identify the target binding sequences of RNAP and TFs, relying on the consensus sequences predicted based on the known target sequences listed in the databases such as RegulonDB [20,21] and EcoCyc [22,23] (Table 2) (Details of the promoter list and the evidence are in Supplemental Information: S1 Table for RpoS; S2 Table for RpoH; S3 Table for RpoF; and S4 Table for RpoE). In particular, more than 80% of RpoE-dependent promoters were predicted in silico (Table 5; and S4 Table). The consensus sequences, however, often include the inaccurate non-target sequences due to the lack of experiments for confirmation or some regulators recognize wide-varieties of the binding sequences [8,9,27,78]. Another serious problem associated with in vivo approaches is the difference in genetic background of E. coli strains used. Up to the present, the complete genome sequence has been determined for more than 1,000 different E. coli strains, allowing the prediction of about 3,000 core genes for all strains but at least one third of the total genes on the E. coli genome are different among the hitherto sequenced E. coli genome [79]. The difference in genetic background exists even in the RNAP and TF genes and between not only different strains but also different stocks of the same E. coli strain. For instance, the difference in the gene encoding the stationary-phase sigma RpoS was first identified between laboratory stocks of a single and the same E. coli K-12 W3110 strain [80]. The widely used databases such as RegulonDB [20,21] and EcoCyc [22,23] include huge collections of useful data of transcription in vivo, but care should be taken to use these data for theoretical prediction of transcription regulation, in particular, as to the bacterial strains and culture conditions used in each experiment.

In this study, we performed the SELEX screening for the constitutive promoters that are recognized in vitro by four minor sigma factors, RpoS, RpoH, RpoF and RpoE, but in the absence of repressors, activators and other DNA-binding proteins. It should be noted that all the proteins and promoters used in this study are prepared from a single and the same E. coli K-12 W3110. Here we also determined the intracellular concentrations of all seven sigma factors in both growing and stationary-phase cells of E. coli K-12 W3110. These data altogether will be used for our ultimate purpose of the prediction of genome expression under a given condition. The list of constitutive promoters for the minor sigma factors will be deposited into TEC database (Transcriptional profile of Escherichia coli database: https://shigen.nig.ac.jp/ecoli/tec/top/) [9].

Materials and methods

Bacterial strains and plasmids

E. coli K12 W3350 type-A containing the full-set of seven sigma factors [80] was used for purification of RNA polymerase and the template DNA for Genomic SELEX screening of RpoS, RpoH, RpoF and RpoE promoters. E. coli BL21(DE3) was used for the expression and purification of sigma and core enzyme subunit proteins. Expression plasmids for the core enzyme subunits (pRpoA, pRpoB and pRpoC) and all seven sigma subunits (pRpoD, pRpoN, pRpoS, pRpoH, pRpoF, pRpoE and pFecI) were constructed by ligating the respective coding sequences, which were prepared by PCR amplification of the E. coli K12 W3350 type-A genome DNA as template, into pET21 expression vector essentially according to the standard procedure used for expression of all sigma and all transcription factors in this laboratory [74,81].

Purification of core RNA polymerase

RNAP was purified from log-phase cells of E. coli K-12 W3350 by the standard procedure [29]. Separation of the native core from holoenzymes was performed by passing the purified RNAP through P11-phosphocellulose column in the presence of 50% glycerol. To remove trace amounts of the core enzyme-associated sigma factors, the purified RNAP in the storage buffer containing 50% glycerol was dialyzed against the same buffer but containing 5% glycerol and fractionated by P11-phosphocellulose column chromatography in the presence of 5% glycerol. The level of remaining sigma factors was less than 0.1%, if any, as checked of SDS-PAGE gels by both protein-staining with a silver reagent and immuno-staining with antibodies against each of seven sigma factors.

Purification of core and sigma subunits

The core enzyme subunits (RpoA, RpoB, RpoC and RpoZ) were expressed using the respective expression plasmids and purified by two cycles of column chromatography through DEAE (DE52) and P11-phosphocellulose [29]. Sigma subunits were expressed and purified by ion-exchange column chromatography through DE52 and P11 followed by Sephacryl S-300 gel filtration column. The purified sigma and core subunit proteins were more than 99% pure as judged by both protein-staining and immuno-staining of SDS-PAGE gels.

Preparation of antibodies

Antibodies against sigma factors and core enzyme subunits were produced in rabbits by injecting purified sigma proteins [75,76]. Antibodies against each RNA polymerase protein were produced in two rabbits, and after examination of antibody activity using immune-blot analysis, the batch of higher activity was used in this study. Anti-RpoD, anti-RpoS, anti-RpoN, anti-RpoH, anti-RpoF, anti-RpoE, anti-FecI and anti-RpoC used in this study did not cross-react with each other. These antibodies were produced in the Nippon Institute for Biological Science (One, Tokyo) and the Animal Laboratory of Mitsubishi Chemical Medience Co. (Uto, Kumamoto, Japan).

Genomic SELEX screening of RNA polymerase holoenzyme binding sequences

The Genomic SELEX screening was carried out under the standard procedure [26,27]. This method was developed by improvement of the original SELEX methods [30-32]. A mixture of DNA fragments of the E. coli K-12 W3110 genome was prepared after sonication of purified genome DNA, and cloned into a multi-copy plasmid pBR322 at EcoRV site. In each SELEX screening, the DNA mixture was regenerated by PCR using a pair of primers with the flanking sequences of pBR322 EcoRV. For SELEX screening, 5 pmol of the mixture of DNA fragments and 10 pmol of RNA polymerase holoenzyme were mixed in a binding buffer (10 mM Tris-HCl, pH 7.8 at 4°C, 3 mM magnesium acetate, 150 mM NaCl, and 1.25 mg/ml bovine serum albumin) and incubated for 30 min at 37°C. The DNA-RNA polymerase mixture was treated with anti-RpoC antibody and DNA fragments recovered from the complexes were PCR-amplified and subjected to next cycle of SELEX for enrichment of RNA polymerase-bound DNA fragments.

For SELEX-chip analysis, DNA samples were isolated from the DNA-protein complexes at the final state of SELEX, PCR-amplified and labeled with Cy5 while the original DNA library was labeled with Cy3. The fluorescent labeled DNA mixtures were hybridized to a DNA microarray consisting of 43,450 species of 60 b-long DNA probe, which are designed to cover the entire E. coli K-12 MG1655 genome at 105 bp interval (Oxford Gene Technology, Oxford, UK) [14]. The fluorescent intensity of test sample at each probe was normalized with that of the corresponding peak of original library. After normalization of each pattern, the Cy5/Cy3 ratio was measured and plotted along the E. coli K-12 MG1655 genome. The gene organization is almost identical between two well-characterized E. coli K-12 strains except for a long-range inversion between the rrnD and rrnE operons.

Immuno-blot analysis for determination of sigma levels

For the measurement of sigma factors in E. coli K-12 W3110, a quantitative Western blot analysis was employed with the anti-sigma antibodies as employed in the previous studies [66,75,76]. In brief, cell lysates were treated with a SDS (sodium dodecyl sulfate) sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 1% 2-mercaptoethanol, 10% glycerol, and 0.025% bromophenol blue) and separated on SDS–7.5 or 10% polyacrylamide gels. Proteins in gels were directly electro-blotted onto polyvinylidene difluoride membranes (Nippon Genetics). Blots were blocked overnight at 48C in 3% BSA in PBS (phosphate-buffered saline), probed with the polyclonal antibodies against each sigma factor, washed with 0.5% Tween 20 in PBS, and incubated with goat anti-rabbit immunoglobulin G conjugated with hydroxyperoxidase (Cappel). The blots were developed with 3,3’-diaminobenzidine tetrahydrochloride (Dojindo). Staining intensity was measured with a PDI image analyzer system equipped with a white light scanner. The standard curve for the calculation of each sigma level was prepared from the immuno-blot patterns of increasing concentrations of each sigma factor. Under the standard Western-blot conditions herein employed, the linearity was detected over a 10-fold range at least between 2 and 20 ng sigma proteins. The determination of test sigma proteins subunits was first performed using several different volumes of the cell lysates. Using the optimum volumes of cell lysates to give the sigma concentrations within the linear range of standard curves, we finally repeated the determination of individual sigma factors.

RpoS-dependent promoters listed in RegulonDB.

Promoters listed in RegulonDB are classified into those not identified as the constitutive promoters (A) and the constitutive promoters identified by SELEX screening (B). Evidence for each promoter are as described in RegulonDB (see Table 5): (Group-A) Promoters were experimentally identified by using HTTIM (high-throughput transcription initiation mapping), TIM (transcription initiation mapping), FP (footprinting), or IDA (inferred by direct promoter assay; (Class-B) Promoters were predicted based on AIPP (automated inference of promoter position), ICWHO (inferred computationally without human oversight), HIPP (human inference of promoter position), NTAS (non-traceable author statement), TASES (traceable author statement to experimental support), TAS (traceable author statement), IMP (inferred from mutant) or IEP (inferred from expression pattern).

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RpoH-dependent promoters listed in RegulonDB.

Promoters listed in RegulonDB are classified into those not identified as the constitutive promoters (A) and the constitutive promoters identified by SELEX screening (B). Evidence for each promoter are as described in S1 Table.

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RpoF-dependent promoters listed in RegulonDB.

Promoters listed in RegulonDB are classified into those not identified as the constitutive promoters (A) and the constitutive promoters identified by SELEX screening (B). Evidence for each promoter are as described in S1 Table.

(PDF)

Click here for additional data file.

Promoters listed in RegulonDB are classified into those not identified as the constitutive promoters (A) and the constitutive promoters identified by SELEX screening (B). Evidence for each promoter are as described in S1 Table.

(PDF)

Click here for additional data file.