The Whole Set of Constitutive Promoters Recognized by RNA Polymerase RpoD Holoenzyme of Escherichia coli

Abstract

The promoter selectivity of Escherichia coli RNA polymerase is determined by the sigma subunit with promoter recognition activity. The model prokaryote Escherichia coli contains seven species of the sigma subunit, each recognizing a specific set of promoters. The major sigma subunit, sigma-70 encoded by rpoD, plays a major role in transcription of growth-related genes. Concomitant with the increase in detection of promoters functioning in vivo under various stressful conditions, the variation is expanding in the consensus sequence of RpoD promoters. In order to identify the canonical sequence of “constitutive promoters” that are recognized by the RNA polymerase holoenzyme containing RpoD sigma in the absence of supporting transcription factors, an in vitro mixed transcription assay was carried out using a whole set of variant promoters, each harboring one base replacement, within the model promoter with the conserved -35 and -10 sequences of RpoD promoters. The consensus sequences, TTGACA(-35) and TATAAT(-10), were identified to be ideal for the maximum level of open complex formation and the highest rate of promoter opening, respectively. For identification of the full range of constitutive promoters on the E. coli genome, a total of 2,701 RpoD holoenzyme-binding sites were identified by Genomic SELEX screening, and using the reconfirmed consensus promoter sequence, a total of maximum 669 constitutive promoters were identified, implying that the majority of hitherto identified promoters represents the TF-dependent “inducible promoters”. One unique feature of the constitutive promoters is the high level of promoter sequence conservation, about 85% carrying five-out-of-six agreements with -35 or -10 consensus sequence. The list of constitutive promoters provides the community resource toward estimation of the inducible promoters that operate under various stressful conditions in nature.

Introduction

The bacterial RNA polymerase core enzyme with the subunit structure α²ββ′ω is fully active in catalysis of RNA polymerization but is unable to initiate transcription from promoters. Transcription initiation from gene promoters requires an additional dissociable sigma subunit, which reversibly associates with the core enzyme to form the holoenzyme, and directs the core enzyme to recognize promoters for transcription initiation. Most bacteria encode multiple species of the sigma factor [1], [2]. In Escherichia coli, seven species of the sigma subunit exist, each recognizing a specific set of promoters [1], [3]. The intracellular levels of seven sigma subunits vary depending on cell growth conditions [3], [4]. Sigma replacement is a simple mechanism of switching of the pattern of genome transcription [4], [5], and the intracellular concentration of seven sigma factors is therefore a primary determinant of the pattern of genome transcription.

The sigma-70, encoded by the rpoD gene, is the primary and major sigma, which is responsible for transcription of most of the genes that are expressed in exponentially growing phase of E. coli [1], [2], [6], [7]. The holoenzyme containing RpoD sigma recognizes in vitro a pair of hexanucleotide sequence elements, TTGACA (-35) and TATAAT (-10) which are situated at 10 and 35 bp upstream, respectively, of transcription initiation sites [2, 6, 8. 9], and a spacer DNA of approximately 17 bp in length separates these two hexanucleotide sequences. This consensus sequence of RpoD promoters was originally proposed based on in vitro transcription assays of some model templates by purified RNA polymerase [10], [11]. Later the promoter sequences were determined for a variety of functioning promoters in vivo, one by one, by using ordinary molecular genetic approaches [12], [13]. More recently high-throughput experimental systems such as ChIP-chip analysis of RNA polymerase-bound DNA sequences [14], [15] and RNA-Seq analysis of whole sets of transcripts [16]–[18] have been employed for searching functioning promoters in vivo. In parallel, computational approaches have been employed to identify promoters relying on the consensus sequences predicted based on the known promoters. Up to the present time, more than 2,000 experimentally defined or computational predicted promoters have been identified, which are under the direct control of RpoD sigma (see the promoter databases such as RegulonDB [19] and EcoCyc [20]). As a result, the striking diversity appeared in the canonical promoter sequence, mainly arising from the detection of weak promoters, which are activated only in the presence of positive transcription factors (TFs).

The constitutive promoters have been defined as those that are active in vivo in all circumstances, but it is practically impossible to identify the whole set of constitutive promoters under various environmental conditions. Instead we propose in this study to define the “constitutive promoter” as the promoters that are recognized in vitro by RNA polymerase RpoD holoenzyme alone in the absence of additional supporting proteins with regulatory functions. For the identification of the whole set of constitutive promoters on the E. coli genome, we performed the Genomic SELEX screening system of genome DNA sequences recognized by the RNA polymerase holoenzyme containing RpoD sigma. The Genomic SELEX system was developed to identify DNA sequences recognized in vitro by DNA-binding transcription factors [21] and successfully applied for the identification of regulation targets of many TFs [22]. After SELEX-chip screening, a total of 2,071 sites were identified on the E. coli genome, which are recognized by the RpoD holoenzyme alone. The location of constitutive promoters within these RpoD holoenzyme-binding regions was then computationally identified using the consensus sequence, which was experimentally determined in this study using the in vitro mixed transcription assay [23]–[25]. The total number of constitutive promoters on the entire E. coli genome was thus estimated to be between minimum 492 and maximum 669. This number of constitutive promoters represents only about one-forth of the hitherto identified promoters on the E. coli genome, indicating that the rest of promoters listed in the promoter databases represents the “inducible promoters”, which are activated in the presence of supporting TFs. One unique feature of the constitutive promoters is the high-level conservation of consensus sequences, TTGACA(-35) and TATAAT(-10), each being separated by a 17-bp spacer.

In the absence of regulatory proteins with repression activity, the constitutive promoters must be always expressed. Based on the Genomic SELEX screening of the binding sites of nucleoid protein H-NS along the E. coli genome, we also propose that the H-NS plays a major role in silencing of the unnecessary expression of constitutive promoters.

Results

Genomic SELEX screening of RpoD holoenzyme-binding sequences on the E. coli genome

The constitutive promoters are transcribed in vitro by the RNA polymerase RpoD holoenzyme alone in the absence of supporting TFs. In order to identify the whole set of constitutive promoters on the entire genome of E. coli K-12 M3110, we first performed a mass-screening in vitro of the whole set of sequences that are recognized by the reconstituted RpoD holoenzyme. For this purpose, we prepared sigma-free core enzyme by passing the purified RNA polymerase three times through phosphocellulose column chromatography in the presence of 5% glycerol (note that sigma-core interaction becomes stronger in the presence of increasing glycerol concentration as used for prolonged storage of the holoenzyme [26]). The level of remaining sigma subunits was less than 0.1%, if any, as detected by both protein staining and immuno-staining with antibodies against each of all seven species of E. coli sigma subunits (RpoD, RpoN, RpoS, RpoH, RpoF, RpoE and FecI) (data not shown). The stoichiometry between core enzyme subunits was also checked by immuno-staining with antibodies against the core subunits, RpoA, RpoB, RpoB and RpoZ. The RpoD holoenzyme fully saturated with RpoD sigma was reconstituted by mixing this sigma-free core enzyme and 4-fold molar excess of purified RpoD sigma, which alone does not bind to DNA.

For the identification of DNA sequences that are recognized by RpoD holoenzyme, we employed the Genomic SELEX screening system [21], 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 [27]–[29]. The multi-copy plasmid library of 200–300 bp-long random DNA fragments was constructed from the E. coli K-12 W3110 genome [21]. The library used in this study contained 7-fold molar excess of the entire genome, and thus a single and the same sequence might be included in 7 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 RpoD RNA polymerase holoenzyme, and subjected to Genomic SELEX screening. DNA-holoenzyme complexes formed were recovered using the anti-RpoC antibody, which gave the highest level of RNA polymerase recovery among all the anti-core subunits. RNA polymerase-associated DNA was isolated from the antibody precipitates, amplified by PCR, and subjected to next cycles of SELEX. After two-cycles of SELEX screening, the final products of RpoD holoenzyme-bound DNA fragments were subjected to mapping on the genome using a DNA tilling microarray (Oxford Gene Technology, Oxford, UK) [30]–[32]. On the DNA tilling array used, the 60 b-long DNA probes are aligned at 105 bp-intervals in the order of E. coli genome sequence, 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 RpoD holoenzyme-bound DNA fragments to the tilling array.

The sequences with binding affinity to the RpoD holoenzme formed a number of peaks along the entire E. coli genome. By setting the cut-off level of 2.0% relative to the highest peak located within a spacer upstream of ssrA (SsrA smRNA) and downstream of smpB (SmpB trans-translation factor), a total of 2,701 RpoD homolenzyme-binding peaks were identified, of which 1,075 (40%) are located within 543 intergenic spacers (average 1.98 sites for each spacer) (Fig. 1A). On the other hand, a total of 1,626 (60%) peaks are located inside of 777 open reading frames (average 2.09 peaks per gene) (Fig. 1A). Since the majority of hitherto identified promoters are located within spacers, detailed search for the constitutive promoters was focused on the total of 1,075 peaks within 543 spacers.

Figure 1. Distribution and classification of the constitutive promoters.

Genomic SELEX search of RpoD holoenzyme-binding sequences was performed using the standard procedure [21]. RpoD holoenzyme-bound DNA fragments were recovered by immunoprecipitation using anti-RpoC antibody. SELEX fragments were isolated from the immuno-precipitates and subjected to mapping on the E. coli genome by using tilling DNA microarray as described previously [32], [33]. [A] Location of the constitutive promoters. A total of 2,701 RpoD holoenzyme-binding sites were identified (see Fig. 1), of which 1,075 (40%) are located within intergenic spacers. On the basis of transcription direction of flanking genes, the spacers were classified into three types: type-A between bidirectional transcription units; type-B upstream of one transcription unit but downstream of another transcription unit; and type-C, downstream of both transcription units. [B] Classification of the constitutive promoters. A total of 2,082 promoters have been identified and listed in the current versions of RegulonDB and EcoCyc databases, whereas the total number of constitutive promoters identified by Genomix SELEX screening ranges between minimum 492 and maximum 669, indicating that the majority of E. coli promoters listed in promoter database are TF-dependent inducible promoters.

Location of the constitutive promoters within the E. coli genome

The spacers containing RpoD holoenzyme-binding sites can be classified into three types (Fig. 1A): 404 peaks are located within 177 type-A spacer between bidirectional transcription units (Table 1 for the whole list; see also Table S1); 583 peaks are located within 315 type-B spacers located upstream of one transcription unit but downstream of another transcription unit (Tables 2 and 3 for the whole list; see also Table S2); and 87 peaks are located within type-C spacers downstream of both transcription units of type-C spacers. Based on the transcription direction of flanking genes, the total number of constitutive promoters was predicted to range between minimum 492 (177 A-type plus 315 B-type) and maximum 669 (177x2 A-type plus 315 B-type).

Table 1. Constitutive Promoters (Type-A Spacers).

Map	Promoter sequence	Right Operon	Left Gene	D	RpoD	D	Right Gene	Left Operon	Promoter sequence
0.87	TTAACG-16-AATAAT	caiTABCDE	caiT	<	HNS	>	fixA	fixABCX	GTGACA-17-TAAAGT
1.82	TTGATT-18-TGAAAT	leuLABCD	leuL	<		>	leuO*	leuO	TTCGCA-17-TATTAT
2.43	TTGTCT-18-CATAAA	coaED	coaE	<		>	guaC	guaC	TTTATA-18-GATTAT
2.99	ATGACG-18-TATAAT	gcd	gcd	<	HNS	>	hpt	hpt	TTAATA-18-TATAGG
3.06	TTTAAA-17-TATATT	can	can	<		>	yadG	yadGH
5.16	ATAACA-17-GATATT	yafV	yafV	<		>	ivy	ivy	TTGGAA-17-TATCGT
5.19	TTGCTA-17-TACAAC	fadE	fadE	<		>	lpcA	lpcA	CTGACC-17-TGTAGT
5.31	TTGTCG-17-TACAAT	dinJ-yafQ	dinJ	<	HNS	>	yafL	yafL	TTTACA-17-TATGTT
5.57	TTAAGA-17-TATATC	phoE	phoE	<	HNS	>	proB	proBA	TTGTTA-17-AATAAT
5.99	CTGCCA-16-TATGAT	insAB-2	insA2	<		>	insI-1	insI-1
6.04	TCTACA-17-TTTATT	yagA--ykgN	yagA*	<		>	yagE	yagEF	TTTACC-16-CATAGT
6.22	TTGTGA-16-AATAAT	argF	argF	<		>	ykgS	ykgS	TTGAAT-18-TGTAAT
6.25	CTGCCA-16-TATGAT	insAB-3	insA3	<		>	yagJ	yagJ
6.36	TTTATA-16-TATAGC	intF	intF	<		>	ptwF	ptwF	TTGCTA-18-TATAAA
7.07	TTGAAC-17-TTTAAT	betIBA	betI*	<	HNS	>	betT	betT	TGGACG-17-CTTAAT
7.43		yahN	yahN	<		>	yahO	yahO
8.26	TTGACA-18-TACAAT	yaiS	yaiS	<	HNS	>	tauA	tauABCD	TTGAGA-16-TACAAT
11.08		qmcA-ybbJ	qmcA	<		>	ybbL	ybbLM
12.53	TTGATT-17-TATAAC	ybcY	ybcY	<	HNS	>	tfaX	tfaX	ATGGCA-15-TTAAAT
13.05	TTGCCT-17-TACCAT	ybdK	ybdK	<		>	sokE	sokE	ATGGCA-16-AAGAAT
13.14	TTGATT-18-TATTAT	fepA	fepA	<	HNS	>	fes	fes- -entFE	AAGACA-17-AATAAT
13.37	TCGATA-17-TATCAT	fepDGC	fepD	<		>	entS	entS	ATGAAA-17-TCTTAT
13.42	ATGATA-17-TATCAT	fepB	fepB	<		>	entC	entCEBAH	ATGATA-17-TATCAT
13.73	ATGACA-16-TTTACT	dsbG	dsbG	<		>	ahpC	ahpCF	TTGCCC-17-TGTAAT
14.09	ATGACA-17-AATTAT	dcuC	dcuC	<	HNS	>	pagP	pagP	TTAAGA-17-TAAAAA
14.54	CTGACG-17-AATAAG	ybeQ	ybeQ	<	HNS	>	ybeR	ybeR-djiB
15.13	TTTACT-17-TATTTT	nagB	nagB	<		>	nagE	nagE	GTGACA-19-TTTAAT
15.69	ATGAAA-16-TGTAAT	kdpFABC	kdpF	<	HNS	>	ybfA	ybfA	TTCGCA-17-TGTAAA
16.22	TTGACA-18-TACAAA	gltA	gltA	<	HNS	>	sdhC	sdhCDAB	TTGTAA-17-TATACT
16.46	TTGAAA-18-TATTAT	mngR	mngR*	<	HNS	>	mngA	mngAB	TTAATA-19-GATAAA
17.93	CTGAAA-18-TATTGT	ybiA	ybiA	<		>	dinG	dinG	ATGCCA-16-TACAGT
18.29	TTCAAA-18-TATATC	rhtA	rhtA	<	HNS	>	ompX	ompX	TTGACT-18-TGGAAT
18.37	TTAACG-16-TATAAT	mntS	mntS	<		>	mntR*	mntR-ybiR	ATTACA-18-TATATT
19.13	TTGATA-19-TAAAAT	ybjL	ybjL	<	HNS	>	ybjM	ybjM	TTGAAA-15-GATAAT
21.47	ATGACA-17-AATAAT	ssuEADCB	ssuE	<		>	elfA	elfAD	TTTAAA-17-TATTAT
21.98	TTAACT-16-TATTCT	sulA	sulA	<	HNS	>	sxy	sxy	TTGCCC-17-TATTTT
22.59	TGGAGA-18-TACACT	gfcA	gfcA	<		>	insA4	insAB-4	CTGCCA-16-TATGAT
23.75	TTATCA-17-TAAAAT	csgDEFG	csgD*	<	HNS	>	csgB	csgBAC	CTGACA-17-TGTAGT
23.86	TGGATA-17-CAGAAT	mdoC	mdoC	<		>	mdoG	mboGH	GTGAAA-17-CTTAAT
24.03	TTAACA-16-TACATT	waaM	waaM	<		>	yceA	yceA	TTGCCG-15-AATAAT
25.16	TTTATA-17-TAAAAA	comR	comR*	<	HNS	>	bhsA	bhsA	TTCACC-17-AATAAT
25.79	TTGTAA-18-TATAAT	ymfED	ymfE	<		>	lit	lit	TTGATA-15-AATAAT
26.15	TTTGCA-17-TAAAAC	bluF	bluF*	<	HNS	>	ycgZ	ycgZ-ymgC	TTGTCA-18-TTTTAT
26.43	TTTACC-17-TGGAAT	pliG	pliG	<		>	ycgL	ycgLM	TTCAGA-18-TATAAT
26.48	ATGACA-19-TATCAT	hlyE	hlyE	<		>	umuD	umuDC	CTGGCA-16-TATAAA
26.56	CTGACC-19-CATAAA	nhaB	nhaB	<		>	fadR*	fadR	TTGATA-19-GAAAAT
26.62	TAGATA-17-TCTAAT	ycgB	ycgB	<		>	dadA	dadAX	TTATCA-17-TATTCT
26.79		ycgR	ycgR	<		>	ymgE	ymgE	TTGCCT-16-TGTAAG
26.92	TTGAAC-16-CGTAAT	dhaKLM	dhaK	<		>	dhaR*	dhaR	TTGCGA-17-CATAAG
27.10	TTGTCA-17-TAAACT	pth-ychF	pth	<		>	ychH	ychH	TTGTAA-17-CATAAC
27.37	TCGACA-16-TAAAAT	chaA	chaA	<		>	chaB	chaBC	TTGACC-18-TGTAAA
27.84	ATGAAA-17-TATCAT	hns	hns*	<		>	tdk	tdk	TTACCA-17-TATAAC
27.90	GTGACG-16-TCTAAT	adhE	adhE	<		>	ychE	ychE	TTTAAA-16-TCTAAT
29.94	ATGATA-19-TCTAAT	ycjY-ymjDC	ycjY	<		>	pgrR*	pgrR	TAGACC-18-TATCAT
31.03	TTGAAG-17-CATAGT	ldhA	ldhA	<		>	ydbH	ydbH--ydbL	TTGAAA-15-AAAAAT
31.90		azoR	azoR	<		>	hrpA	hrpA	TAGAAA-17-TATATC
32.07	TTAACA-19-TTTCAT	gapC	gapC	<	HNS	>	cybB	cybB	TTGAGA-19-GAAAAT
32.16	TTAATA-17-TATCAT	ydcI	ydcI*	<	HNS	>	ydcJ	ydcJ	CTGACA-16-TATGAT
32.48	TAAACA-18-TGTAAT	yncJ	yncJ	<		>	hicA	hicAB	TAAACA-19-TATACT
33.50	TTGCCA-15-TATAAA	bdm-sra	bdm	<	HNS	>	osmC	osmC	TTGATA-17-TATATT
34.73	TTTAAA-17-TATCTT	sad	sad	<		>	yneJ*	yneJ	TTTACT-17-AATAAT
34.84	CTGGCA-17-GATAAT	marC	marC	<		>	marR*	marRAB	TTGACT-17-TATTAT
35.08	ATGCCA-17-TTTAAG	ydfI	ydfI	<		>	ydfK	ydfK	TTCCCA-16-GATAAT
35.43		relBE-hokD	relB	<		>	ydfV	ydfV
37.33	TTGCCG-17-AATAAT	grxD	grxD	<	HNS	>	ydhO	ydhO	TTAACT-18-TAGAAT
37.44	GTGAAA-17-TAATAT	ydhB	ydhB*	<		>	ydhC	ydhC	TTCACA-17-TACACT
37.78	TTGTCT-18-TTTAAT	ydhZ	ydhZ	<	HNS	>	pykF	pykF	TTAACT-18-TATATT
38.77	TCGTCA-17-TAAAAT	thrS- -infA	thrS	<		>	arpB	arpB	ATGATG-16-TATAAC
41.11	TTGCGA-17-TATAAT	kdgR	kdgR*	<		>	yebQ	yebQ	TTATCA-17-TATAAA
41.39	TTAACA-15-TGTGAT	pphA	pphA	<		>	ryeA	ryeA	ATCACA-18-TAAAAA
41.57	TTGATT-17-TATACT	yebG	yebG	<	HNS	>	purT	purT	AAGACA-18-TATACT
41.81	ATGAAA-17-TCTCAT	znuA	znuA	<		>	znuC	znuCB	ATGAGA-17-TTTCAT
41.90	TTGTCA-15-TACAAA	yobI	yobI	<		>	yebB	yebB	CTGAAA-17-TATTAC
41.96	TTTCCA-17-TTTCAT	aspS	aspS	<		>	yecD	yecDEN	GACACA-19-TATTAT
42.61	TTGAAA-17-TATGAC	insAB-5	insA5	<		>	uspC	uspC	TTGGCA-16-TATAAG
43.11	CTAACA-19-AAAAAT	fliC	fliC	<		>	fliD	fliCST
43.23	TTGAAA-18-TTTAAT	yedD	yedD	<		>	yedE	yedEF	TTCAGA-17-TATTAT
43.32	CTGACA-16-ACTAAT	yedM	yedM	<		>	intG	intG	TTGCCC-16-CACAAT
43.89	TTGTAA-16-TATAAC	yedWV	yedW*	<		>	hluH	hluH	TAGAAA-15-CAAAAT
44.99	CTGACA-16-ACTAAT	yefM-yoeB	yefM*	<		>	hisL	hisLG--AFI	TAAACA-19-TATAAA
46.28	TTGTTA-18-TATAAC	yegI	yegI	<	HNS	>	yegJ	yegJ	ATGATA-17-TACAAT
46.68	TAAACA-18-TATATT	yegRZ	yegR	<	HNS	>	yegS	yegS	TTTAAA-17-TATTAT
48.01	TCAACA-18-CATCAT	dusC	dusC	<		>	yohJ	yohJK
48.30	ATGAAA-15-AATAAT	folE-yeiB	folE	<		>	yeiG	yeiG	CTCACA-17-TATGAT
48.42	TTAAAA-19-TATAAT	yeiE	yeiE*	<		>	yeiH	yeiH	TTACCA-15-TATAAA
49.59		napF- -ccmH	napF	<		>	yojO	yojO	TTGAGA-16-CATAAT
49.78	ATGAAA-17-TATCAT	ompC	ompC	<	HNS	>	micF	micF	TTAAGA-17-AATAAG
50.41	TTGATC-16-CATAAT	ygaL	yfaL	<	HNS	>	nrdA	nrdAB
51.81	CTGCCA-16-TATGAT	lrhA	lrhA*	<		>	alaA	alaA	TTAAGA-17-TATAAC
51.96	ATGACA-17-CATCAT	yfbV	yfbV	<		>	ackA	ackA-pta	CTGAAA-16-TAGACT
52.11	GTGACA-15-TATAGT	yfcF	yfcF	<		>	yfcG	yfcG	TCGGCA-15-TATCAA
53.46	TTGATA-17-TTTCAT	emrKV	emrK	<	HNS	>	evgA*	evgAS	TTGACA-19-TATATG
53.73	TTGCCA-17-TACTAT	yfdV	yfdY	<		>	lpxP	lpxP	TCGACA-18-AACAAT
54.02	TTTACA-17-TATTTT	glk	glk	<		>	yfeO	yfeO	CTCACA-17-CATAAC
54.09	TTGATA-17-TAGCAT	mntH	mntH	<		>	nupC	nupC	ATGACT-17-TTTAAT
54.40		yfeR	yfeR*	<		>	yfeH	yfeH	TTCTCA-17-TAGAAC
54.49	TTAATA-16-GGTAAT	zipA	zipA	<		>	cysZ	cysZK	TTAACT-19-TGAAAT
54.96	TTGGCT-17-CATAAT	ypeA-yfeZ	ypeA	<		>	amiA	amiA-hemF	TTGTCA-17-TAAAAC
55.80	TTCACC-17-TAGAAT	ypfM	ypfM	<	HNS	>	yffB	yffB- -ypfN	TTCATA-16-TTTAAT
55.97		dapA-bamC	dapA	<		>	gcvR*	gcvR	TTAAAA-18-TCTGAT
56.43	TTGCCG-16-TATAAT	upp-uraA	upp	<	HNS	>	purM	purMN	ATGATA-17-TATTTT
56.57	ATGACA-18-TATAGT	yfgF	yfgF	<	HNS	>	yfgG	yfgG	TTGACC-17-CTTAAT
56.70		guaBA	guaB	<		>	xseA	xseA	TCGACT-16-TAGAAT
57.34	TCGGCA-18-TAAGAT	trmJ	trmJ	<		>	suhB	suhB	AAGACA-17-TATACT
57.81	TTGATG-16-TATAAA	glyA	glyA	<		>	hmp	hmp	TTTACA-16-TATAAG
58.35	TAAACA-17-CATCAT	rpoE--rseC	rpoE*	<		>	nadB	nadB	TAGACT-16-TATAAG
59.01	TTGAAA-16-TATCGT	aroF-tyrA	aroF	<		>	yfiL	yfiL	TTTTCA-17-TTTTAT
59.32		ratAB	ratA	<		>	smpB	smpB
60.00	TAGATA-16-TATCAT	ileY	ileY	<	HNS	>	ygaQ	ygaQ_12	TTATCA-17-TGTAAT
60.24	TTAAGA-17-AACAAT	yqaE	yqaE	<	HNS	>	ygaV*	ygaVF	TTTAGA-17-AATACT
60.27	TTTACA-17-TTTAAT	stpA	stpA*	<	HNS	>	alaE	alaE	GTGATA-17-TCTAAT
60.83	TTGAAA-16-TTTGAT	mltB	mltB	<	HNS	>	srlA	srlA- -gutM	TTAACA-18-TATGGT
61.61	TTCACA-18-TATTTT	ygbI	ygbI*	<	HNS	>	ygbJ	ygbJK	TTCACA-16-GTTAAT
62.45	TTGACA-16-TGTGAT	ygcW	ygcW	<	HNS	>	yqcE	yqcE-ygcE	TTCTCA-18-GATAAT
62.56	TTTAAA-17-GTTAAT	queE	queE	<	HNS	>	yqcG	ygcG	TTAACA-18-GATAAT
63.94	TTGACG-18-TTTAGT	rppH-ptsP	rppH	<		>	mutH	mutH	TCGGCA-18-TTTAAT
65.80		ygfB- -visC	ygfB	<		>	zapA	zapA	TTGTCT-17-TATAGT
68.75	TAGAGA-19-TTTAAT	glgS	glgS	<	HNS	>	yqiJ	yqiJK	TTTAAA-15-TATATT
69.27	TTAACA-18-TTTTAT	yqjH	yqjH	<		>	yqjI*	yqjI	TTGCAA-16-TATAAA
69.31	TTGATC-18-TATAGT	aer	aer	<	HNS	>	patA	ygjG	TAAACA-19-TAAAAT
69.68	TTTTCA-18-TATCAT	rlmG	rlmG	<		>	ygjP	ygjP	TTGCCC-18-TATACC
70.35	TTGATA-16-TGTAAT	trcA- -G	tdcA*	<	HNS	>	tdcR	tdcR	TTTAAA-16-TATAAA
71.48	TTGACA-18-TATAAT	metY- -pnp	metY	<		>	argG	argG	ATGAAA-17-AAAAAT
71.93	TGGACT-16-TAAAAC	mlaFEDCB	mlaF	<		>	yrbG	yrbG- -lptAB	TTTACT-17-CAAAAT
72.51	TTACCA-16-CATAAA	insH-10	insH10	<		>	yhcF	yfcF	CTCACA-18-TTTAAG
72.99	ATAACA-18-TATATT	aaeXAB	aaeX	<	HNS	>	aaeR*	aaeR	TTGATA-19-TGTTAT
73.51	TTAAAA-17-TATATT	envR	envR*	<	HNS	>	acrE	acrEF	TTGAGT-19-AATAAT
73.94	CTGTCA-18-TAGAAT	smf	smf	<		>	def	def-fmt	TTGCTA-19-GATAAG
74.39	TTAATA-17-TATGAT	gspAB	gspA	<	HNS	>	gspC	gspD- -LMO	TTGATT-17-TACTAT
76.72	TTTACG-19-CATAAT	glpEGR	glpE	<		>	glpD	glpD	TTGAAA-19-TATAAC
77.61	GTGCCA-18-TGTAGT	ftsYEX	ftsY	<		>	rsmD	rsmD-yhhL	TTCACA-18-TGTTAT
78.27	TTTACA-17-GATTAT	yhiL	yhiL	<	HNS	>	yhiM	yhiM	CTGAAA-16-TATAAA
78.33	TTGCCC-17-GATAAT	yhiN	yhiN	<	HNS	>	pitA	pitA	TTCACT-18-TATAAT
78.58	ATGACG-17-TATAAA	dinQ	dinQ	<		>	arsR*	arsRBC	ATGACG-17-TATAAA
78.67	TTGTCA-18-TATAAT	insH-11	insH11	<	HNS	>	slp	slp-dctR	TTTACG-17-TAAAAT
78.93	TTGAAA-16-TATAAG	gadW	gadW*	<	HNS	>	gadY	gadY	TTCGCA-18-TATAAA
80.49	TTCACA-17-TCAAAT	bax	bax	<		>	malS	malS	TTGATA-18-TCAAAT
80.81	TTGTAA-19-TTTTAT	yiaT	yiaT	<	HNS	>	yiaU*	yiaU	TTCATA-18-TAAAAT
81.24	TATACA-18-TATAAT	yibIH	yibI	<		>	mtlA	mltADR	TCTACA-19-TACAAT
81.71	TATACA-18-TATAAT	yibB	htrL	<	HNS	>	hldD	rfaD-waaCL	TTAATA-17-CATAAA
82.01	TCTACA-17-TTTATT	waaQ- -waaK	waaQ	<	HNS	>	waaA	waaA-coaD	CTGACA-16-TTTTAT
82.28	GTCACA-16-TAAAAG	ligB	ligB	<		>	gmk	gmk	GTCACA-16-AATAAG
82.60	TTGTAA-19-TATAAT	yicJI	yicJ	<	HNS	>	selC	selC	TTATCA-18-TATAAA
83.00	TGGACA-17-GATACT	istR	istR	<	HNS	>	tisB	tisA	TTGTCC-17-TATACA
84.59	TTTATA-18-TATGAT	asnC--mnmG	asnC*	<		>	asnA	asnA	TTGATT-16-TAAAAT
85.06	TTGGCC-18-AATAAT	yifB	yifB	<	HNS	>	ilvL	ilvLXGM	TTGGCC-18-AATAAT
85.39	TTCACC-19-AATAGT	rhlB	rhlB	<		>	trxA	trxA	TTTACG-16-AATAAA
85.83	TTAACA-18-TGTAAT	aslA	aslA	<	HNS	>	glmZ	glmZ	TTGAGA-17-GATGAT
85.95	CTCACA-15-TGTAAT	hemCDKY	hemC	<		>	cyaA	cyaA	CTGACA-18-TAGGAT
87.95	TCGACA-15-TACATT	fdoG--fdhE	fdoG	<		>	fdhD	fdhD	TCGACA-15-TACATT
88.43	TTTGCA-19-TATCGT	cpxRA	cpxR*	<		>	cpxP	cpxP	ATGACG-19-TTTAAA
88.70	TGGACA-17-TACAAT	glpFKX	glpF	<		>	zapB	zapB	TGGACA-17-TACAAT
88.86	TTTGCA-18-TATGAT	priA	priA	<		>	rpmE	rpmE	CAGACA-17-TATAGC
88.93	TTGAGC-17-TAAAAT	metJ	metJ*	<		>	metB	metBL	TTGAGC-17-TAAAAT
89.48	TTCATA-17-GATACT	argE	argE	<		>	argC	argCBH	TTGACA-18-TATCAA
91.12	TTGGCT-16-TCAAAT	pepE	pepE	<		>	rluF	rluF	ATAACA-17-TATTTT
91.17	TTGACA-16-TTTATT	lysC	lysC	<		>	pgi	pgi	ATCACA-18-TACAAT
91.65		plsB	plsB	<		>	dgkA	dgkA	TTAACG-19-CATAAT
91.76	TTGATA-17-CATAAC	zur	zur*	<		>	yjbL	yjbLM	TTGTCG-18-AATAAT
91.84		qorA	qorA	<		>	dnaB	dnaB	TCGTCA-17-TAAAGT
92.01	ATGCCA-15-TTTAAT	uvrA	uvrA	<		>	ssb	ssb	TTGACC-18-TGGAAT
92.09	CTAACA-15-TATAGT	yjcB	yjcB	<	HNS	>	yjcC	yjcC	TTTTCA-16-TATAAA
94.13	GTGAAA-18-TTTCAT	yjeH	yjeH	<		>	groS	groSL	TTTTCA-17-CAGAAT
95.19	ATCACA-18-TATCAT	ulaG	ulaG	<	HNS	>	ulaA	ulaA--EF	TTAACT-15-GATAAT
95.32	TTGATT-15-GATCAT	yjfY	yjfY	<		>	rpsF	rpsF- -rplI	TTCAAA-17-TGTGAT
95.37	TTGATT-15-GATCAT	yjfZ	yjfZ	<	HNS	>	ytfA*	ytfA	TTCACA-16-AATAAA
95.51	TCGACA-15-TACATT	qorB	qorB	<		>	ytfH*	ytfH
96.37	TTGACA-17-TGTAAT	bdcA	bdcA	<		>	bdcR*	bdcR	TTGATT-17-TACAAA
96.46	TTGCAA-15-TATAAA	argI	argI	<		>	rraB	rraB	TTAAAA-16-GATTAT
96.50	TTGATA-19-TAAAAT	yjgM	yjgM	<	HNS	>	yjgN	yjgN	TTGCCA-18-TATTGT
96.99	TTAACA-17-GATAAA	insG	insG	<		>	yjhB	yjhBC	AAGACA-17-TATTGT
97.13	TTAACG-17-TAGAAT	insM	insM	<		>	yjhV	yjhV	CTGTCA-16-TATAAA
97.33	TTCTCA-17-GATAAT	fecIR	fecI**	<		>	insA7	insA-7	TTAACA-17-TATAAG
97.78	TCAACA-17-TTTAAT	nanCM	nanC	<	HNS	>	fimB	fimB	TTGGCA-16-TATATT
98.46	TTCACA-19-TTTTAT	yjiR	yjiR*	<		>	yjiS	yjiS	TTAACC-15-TAAAAG
99.15	ATGAAA-17-TTTAAT	yjjP	yjjP	<	HNS	>	yjjQ*	yjjQ-bglJ	CTGATA-17-GATAAT
99.21	TTGATA-19-GATAAT	fhuF	fhuF	<		>	yjjZ	yjjZ	TTGCAA-16-TATGAT
99.96		arcA	arcA*	<		>	yjjY	yjjY	TTGCCA-19-TACAAA
		300 genes (a)	178 (b)	K	HNS		178 (a)	291 genes (b)
		1.68 (a/b)	26 TFs		64		19 TFs	1.63 (a/b)
		63 Y-genes			36%			74 Y-genes
		20 essential						19 essential

A total of 1,075 RpoD holoenzyme-binding sites were identified within spacers on the entire E. coli K-12 W3110 genome. The constitutive promoters were predicted within type-A and type-B intergenic spacers (see Fig. 1A for classficiation). A total of 178 RNA polymerase RpoD holoenzyme-binding sites were identified within type-A spacers, which direct bidirectional transcription. Based on the gene orientation around these promoters, the genes and operons under the control of these promoters were estimated, that are located on either left side (left gene column) or right side (right gene column) of the respective spacers. Genes encoding transcription factors are indicated by star symbols (*) and the operons are shown in the operon columns [note that only the first and the last genes are shown for polycitronic operons]. The directions of transcription for these flanking genes are shown by arrows in column D. The map positions of left-side and right-side genes are shown in the map columns. The essential genes listed in the PEC database are underlined. The promoter sequences were predicted according to the analysis procedure described in Materials and Methods. For some spacers, multiple promoters were identified, of which the best-match promoters with the highest scores are described. The promoter sequence with complete match with the canonical promoter (see Fig. 4) is shown in bold and italic while the promoter sequence with 5-out of-6 match is shown in bold. The spacer including H-NS binding sites are shown as HNS mark in the spacer column. The numbers of hitherto identified promoters are 121 and 133 for left-ward and right-ward transcription, respectively, which correspond to 68 and 75%. Total number of genes under the control of 178 promoters were 300 for left-ward transcription, and 291 for right-ward transcription. The average numbers of genes under one promoter are 1.68 and 1.63 for left-ward and right-ward transcription, respectively. Among the total of 178 RpoD holoenyme-binding sites, 64 (36%) overlap with the H-NS-binding sites.

Table 2. Constitutive Promoters (Type-B Spacers) (Leftward transcription).

Map	Promoter sequence	Left Operon	Left Gene	D	RpoD	D	Right Gene	Right Operon
0.44	CTGCCA-16-TATGAT	insAB-1	insA-1	<		<	rpsT	rpsT
0.85		caiABCDE	caiA	<		<	caiT	caiTABCDE
3.96		yaeH	yaeH	<		<	yaeI	yaeI
5.76	TTGAAA-15-TATCAT	ykfA	ykfA	<		<	perR	perR
5.99	Internal Promoter	(insAB-afuBC)	afuB	<		<	ykgN	(yagAB-ykgN)
6.02	Internal Promoter	(yagAB-ykgN)	ykgN	<		<	yagB	(yagAB-ykgN)
6.28	TTTAGA-16-TATAAT	yagK	yagK	<	HNS	<	yagL	yagL
6.34	TTAAAA-17-TATCAT	yagN	yagN	<	HNS	<	intF	intF
6.72	TTGAAA-17-TATCTT	ykgMO	ykgM	<	HNS	<	ykgR	ykgR
6,84		ykgIB	ykgI	<	HNS	<	ykgC	ykgC
6.98	GCGACA-16-TATATT	ykgH	ykgH	<	HNS	<	betA	betA
7.77	Internal Promoter	(lacZYA)	lacA	<		<	lacY	(lacZYA)
8.14	Internal Promoter	(frmRAB)	frmA	<		<	frmR	frmRAB
8.17	TTGACA-15-TATAGT	frmRAB	frmR*	<		<	yaiO	yaiO
9.47	Internal Promoter	(xseB-ispA-dxs)	ispA	<		<	xseB	(xseB-ispA-dxs)
9.63	Internal Promoter	(cyoABCDE)	cyoD	<		<	cyoC	(cyoABCDE)
10.27	TCCACA-17-TACACT	ylaB	ylaB	<		<	ylaC	ylaC
10.30		ylaC	ylaC	<		<	maa	maa
10.33		hha	hha	<		<	tomB	tomB-hha
10.34	ATGAAA-17-TATAGT	tomB-hha	tomB	<	HNS	<	acrB	(acrAB)
14.19	ATGGCA-17-TACATT	lipA	lipA	<		<	ybeF	ybeF
14.22	TTTACA-15-TATATT	ybeF	ybeF*	<	HNS	<	lipB	lipB
15.30	TTGTAA-18-TACAAT	uof-fur	uof	<		<	fldA	fldA
15.31	TGGGCA-18-AATAAG	fldA	fldA	<		<	ybfE	ybfE
15.32	TTGGCG-18-TATTAT	ybfE	ybfE	<		<	ybfF	ybfF
16.20	TAAACA-16-TAAAAT	ybgD	ybgD	<	HNS	<	gltA	gltA
16.94	TTCAAA-17-CATATT	gpmA	gpmA	<		<	galM	(galETKM)
17.03	ATGAAA-17-TAAAAA	galETKM	galE	<	HNS	<	modF	modEF
19.38	TTGTCC-17-TAAATT	artJ	artJ	<		<	artM	artM
19.45	TTAACT-18-CATAAT	artPIQM	artP	<	HNS	<	ybjP	ybjP
19.68	TTGACG-19-TGTAAT	ybjE	ybjE	<		<	aqpZ	aqpZ
20.54	TTTACA-17-AATAAT	focA-pflB	focA	<		<	ycaO	ycaO
22.15	TTGAAA-16-TATATC	hspQ	hspQ	<		<	rlmI	rlmI
24.14	ATGACA-17-TATAAA	bssS	bssS	<	HNS	<	dinI	dinI
25.21	TTGCTA-16-GATAAT	mfd	mfd	<		<	ycfT	ycfT
25.49	Internal Promoter	(potABCD)	potB	<		<	potA	(potABCD)
26.11	TTCATA-17-CATAAT	iraM	iraM	<	HNS	<	ycgX	ycgX
26.13	TTTAAA-16-AATAAT	bluR	bluR*	<	HNS	<	bluF	bluF
27.07	Internal Promoter	( ychF )	ychF	<		<	pth	pth
28.17	TTAACA-17-TATGTT	kch	kch	<		<	yciI	yciI
28.91	TTAACA-17-TATTAT	osmB	osmB	<	HNS	<	yciT	yciT
28.92	TTGAGG-16-TATTTT	deoT	deoT	<		<	yciZ	yciZ
29.06	GTGAAA-17-GAGAAT	fabI	fabI	<		<	ycjD	ycjD
29.08	CTGACA-16-CAGAAT	ycjD	ycjD	<		<	sapF	sapDF
29.17	ATGACA-15-TTTAAT	sapABCDF	sapA	<		<	ymjA	ymjA
30.85	TGTACA-16-AATAAT	pinR	pinR	<		<	ynaE	ynaE
30.86	CTGACA-17-TACCAT	ynaE	ynaE*	<	HNS	<	uspF	uspF
31.54	CTGACA-17-AATAAC	ynbG	ynbG	<		<	insC-2	insCD-2
33.21	TTCACC-16-TCTAAT	narU	narU	<		<	yddJ	yddJ
33.25	Internal Promoter	(yddLKJ)	yddK	<		<	yddL	yddLKJ
33.42	Internal Promoter	yddM	yddM	<		<	adhP	adhP
33.58	Internal Promoter	(ddpXABCDF)	ddpB	<		<	ddpA	(ddpXABCDF)
34.03	TTGTCA-16-TATTAA	ydeN	ydeN	<		<	ydeO	ydeO
34.10	TTGAAG-16-TATATT	ydeP	ydeP	<	HNS	<	ydeQ	ydeQ
34.27	TTGACT-16-TAAAAC	hipBA	hipB	<		<	yneO	yneO
34.28	CTGACA-17-TTTAAT	yneO	yneO	<	HNS	<	lsrK	lsrRK
34.68	TTGCCG-19-TATCTT	yneF	yneF	<		<	yneG	yneG
34.71	TTTTCA-17-TAGAAA	yneHG	yneH	<		<	sad	sad
35.31	TTTATA-16-AATAAT	essQ- -rrrQ-ydfP	essQ	<	HNS	<	cspB	cspB
35.58	Internal Promoter	(rspAB)	rspB	<		<	rspA	rspAB
35.60	TTGTCA-17-TATACG	rspAB	rspA	<		<	ynfA	ynfA
36.28	TTAACG-17-AAAAAT	fumC	fumC	<		<	fumA	fumA
36.52	TTAACC-17-TATACG	uidR	uidR*	<		<	hdhA	hdhA
37.12	TTCAAA-15-TACACT	sodC	sodC	<		<	ydhF	ydhF
37.98	ATCACA-16-GATAAT	sufABCDSE	sufA	<		<	rydB	rydB
38.00	TTGTCA-16-CATATT	ydiH	ydiH	<		<	ydiI	ydiJI
38.76		infC-rpmI-rplT	infC	<		<	thrS	thrS- -rplT-pheST
39.02	ATGACT-16-AATAAT	ydjO	ydjO	<	HNS	<	cedA	cedA
39.28		ves	ves	<		<	spy	spy
39.92	TTGAAA-17-GATAAT	ydjF	ydjF*	<		<	ydjG	ydjG
40.46	TTGCCC-19-TTTTAT	yeaQ	yeaQ	<		<	yoaG	yoaG
40.65	TTAATA-18-TATCAT	fadD	fadD	<		<	yeaY	yeaY
41.07	TTGCCA-17-GATAAC	yobF-cspC	yobF	<	HNS	<	yebO	yebO
41.18		prc	prc	<		<	proQ	proQ
41.53	TTGTCC-15-CACAAT	yebE	yebE	<		<	yebF	yebF
41.62	TTCACC-17-TACACT	edd-eda	edd	<		<	zwf	zwf
41.89	TGGATA-17-TATCAT	ruvAB	ruvA	<	HNS	<	yobI	yobI
42.14	GTGACA-18-TAAAAA	torYZ	torY	<	HNS	<	cutC	cutC
42.90		pgsA	pgsA	<		<	uvrC	uvrC
42.98	TTGCAA-17-AATAAT	sdiA	sdiA*	<	HNS	<	yecC	yecC
43.07	Internal Promoter	(fliAZY)	fliZ	<		<	fliA	fliAZY
44.47	TTGAGA-16-TATATT	cobUST	cobU	<		<	insH-6	insH-6
44.49	TGTACA-17-CATGAT	yoeG	yoeG	<		<	yoeH	yoeH
44.55	TTTTCA-18-TATAAT	yoeH	yoeH	<	HNS	<	insD-3	insD-3
44.77	TTGTCA-15-AGTAAT	yeeX	yeeX	<		<	yeeA	yeeA
44.95		yeeY	yeeY	<		<	yeeZ	yeeZ
45.26	ATTACA-19-TATCAT	insH-7	insH-7	<	HNS	<	wbbK	(wbbIJK)
45.37		glf-wbbH	glf	<		<	rfbX	(rfbBDACX)
45.48	TTCACA-18-TGGAAT	rfbBDACX	rfbB	<		<	galF	galF
46.67		ogrK	ogrK	<		<	yegZ	yegZ
46.92	Internal Promoter	(gatZABCD)	gatB	<		<	gatA	(gatZABCD)
47.00		yegX	yegX	<		<	thiD	thiMD
47.63		yehS	yehS	<		<	yehT	yehT
47.89	TTGACG-18-TATGAT	pbpG	pbpG	<		<	yohC	yohC
49.05	TTGATG-17-TATCAT	yejG	yejG	<		<	bcr	bcr
49.64	TTGATG-16-TGCAAT	mqo	mqo	<	HNS	<	yojI	yojI
51.11	GTGAAA-16-GATAAT	pmrD	pmrD	<		<	menE	menE
51.61	Internal Promoter	(nuoABC- -HIJKL)	nuoH	<		<	nuoG	(nuoAB- -GHIJKL)
51.73	Internal Promoter	(nuoABC- -HIJKL)	nuoC	<		<	nuoB	(nuoAB- -GHIJKL)
51.78	TTGACA-18-TAAAAA	nuoAB- -IJKLMN	nuoA	<		<	lrhA	lrhA
52.25	TTGAAA-16-TTTAAT	hisJQMP	hisJ	<		<	argT	argT
52.87	TTGAAA-17-TATAGT	yfcV	yfcV	<	HNS	<	sixA	sixA
53.61	CTGACA-19-CATTAT	yfdV	yfdV	<		<	oxc	oxc
53.67	TTTATA-18-AATAAT	frc	frc	<	HNS	<	yfdX	yldX
57.16		sseB	sseB	<		<	pepB	pepB
57.96	TTAACT-17-TCTAAT	glmY	glmY	<		<	purL	purL
58.68	ATGATA-15-AATATT	kgtP	kgtP	<		<	rrfG	(rrsG- -rrlG-rrfG)
59.52	TAGACG-18-TGGAAT	yfjLK	yfjL	<		<	yfjM	yfjM
59.97	TTATCA-18-TTTAAT	ypjC	ypjC	<	HNS	<	ileY	ileY
61.31	Internal Promoter	(hycABCDEFGHI)	hycD	<		<	hycC	(hycABCDEFGHI)
62.01	Internal Promoter	(cusABC -ygbTF)	ygbT	<		<	casE	(cusABC- -ygbTF)
62.70	TTGATA-15-TATGAT	mazEFG	chpR	<		<	relA	relA
64.26	TTGAAA-17-TATCAT	kduI	kduI	<	HNS	<	yqeF	yqeF
64.40		yqeL	yqeL	<		<	yqeK	yqeK
65.59	GTGACG-15-TTCAAT	ygfF	ygfF	<		<	gcvP	gcvP
66.18	TTCCCA-16-TGTGAT	epd-pgk-fbaA	epd	<	HNS	<	yggC	(yggDC)
67.03	GTGACG-15-TTCAAT	yghF	yghF	<		<	yghG	yghG
67.77	TTGCCT-17-GACAAT	yghW	yghW	<		<	yghX	yghX
68.24	TTAACC-15-TAAAGT	mqsRA	mqsR	<		<	ygiV	ygiV
68.40	Internal Promoter	(nudF- -yqiA-parE)	yqiA	<		<	cpdA	(nudF- -yqiA-parE)
68.81	TTGACG-17-TAAAGT	sibD	sibD	<		<	sibE	sibE
71.46		rimP-nusA-infB	rimP	<		<	metY	metY
71.60		folP-glmM	folP	<		<	ftsH	ftsH
72.18	TTGAGG-18-CACAAT	arcB	arcB	<		<	yhcC	yhcC
72.74	TGGCCA-18-TAAAAA	sspAB	sspA	<		<	rpsI	rpsI
74.68	TTGAAA-17-TATTTT	bfd-bfr	bfd	<		<	chiA	chiA
74.87	TGGAAA-16-ATTAAT	yheO-tusDCB	yheO*	<		<	fkpA	fkpA
75.16	TTGCCA-17-CATATT	argD	argD	<		<	pabA	pabA
76.67		glpR	glpR*	<		<	glpG	glpG
77.07	TTAGCA-17-TTTAGT	gntR	gntR*	<		<	yhhW	yhhW
77.55	TTCACA-19-GATAAA	rpoH	rpoH	<		<	ftsX	ftsX
78.19	TAGACA-16-TACTAT	yhiI-rbbA-yhhJ	yhiI	<		<	yhiJ	yhiJ
78.22	TTGACG-19-TATAAT	yhiJ	yhiJ	<	HNS	<	yhiL	yhiL
78.76	TGAACA-17-TAAAAG	(hdeABD)	hdeB	<	HNS	<	hdeA	hdeABD
78.95	TTAATA-16-TGTAAT	gadX	gadX*	<	HNS	<	gadA	gadA
78.98	TTAATA-17-TATATT	gadAX	gadA	<	HNS	<	yhjA	yhjA
79.79	Internal Promoter	(dppABCDF)	dppC	<		<	dppB	(dppABCDF)
80.87	CAGACA-17-TATAAA	yiaWV	yiaW	<	HNS	<	aldB	aldB
83.00	CTGAAA-19-TGTAAA	ivbL- -uhpABC	ivbL	<		<	istR	istR
83.53		gyrB	gyrB	<		<	recF	recF
84.18		pstB-phoU	pstB	<		<	pstA	(pstCA)
85.30	CTGACA-18-GATCAT	ppiC	ppiC	<		<	yifO	yifO
86.22		yigF	yigF	<		<	yigG	yigG
87.36		glnLG	glnL	<		<	glnA	glnA
88.73	TTGCCA-18-TATACT	rraA	rraA	<		<	menA	menA-rraA
92.24	TTCCCA-15-TAAACT	yjcF	yjcF	<	HNS	<	actP	actP
92.56		yjcO	yjcO	<		<	fdhF	fdhF
93.18	TTATCA-16-TATAAA	yjdN	yjdN	<		<	yjdM	yjdM
93.46	TTTACA-17-GATACT	adiA	adiA	<	HNS	<	melR	melR
93.90	TTGAGT-19-TATAAT	cadBA	cadB	<	HNS	<	cadC	cadC
94.05	ATAACA-17-TAAAAA	dcuA	dcuA	<		<	aspA	aspA-dcuA
94.22	TTAACC-17-TAGAGT	yjeJ	yjeJ	<		<	epmB	epmB
95.44	TTTTCA-16-AAAAAT	nrdD	nrdD	<		<	treC	treC
97.36	TTAAGA-15-TTTAAT	yjhU	yjhU*	<		<	yjhF	yjhF
97.49	TTCAAA-17-TTTAAT	yjhIHG	yjhI*	<		<	sgcR	sgcR
97.61	TTTACC-17-TATCAC	sgcXBCQAER	sgcX	<		<	yjhP	yjhQP
98.20		iadA	iadA	<		<	yjiG	(yjiHG-iadA)
98.30	CTGACC-19-TACAAT	yjiK	yjiK	<		<	yjiL	yjiL
98.63	TTTACC-16-AAAAAT	mcrBC	mcrB	<		<	symE	symE
99.60		lplA	lplA	<		<	ytjB	ytjB
		290 (a)	181 (b)		HNS
		1.80 (a/b)	15 TFs		39
		69 Y-genes			24%
		14 essential

Among the total of 1,075 RpoD holoenzyme-binding sites, 181 are located within type-B spacers upstream of left-side genes and downstream of right-side genes, indicating that these promoters direct leftward transcription. The genes and operons under the control of these 181 promoters were estimated, of which 16 represent putative internal promoters. Descriptions and symbols are as in Table 1. A total of 15 genes encoding transcription factors are indicated by star symbols (*). The essential genes listed in the PEC database are underlined within the operons. The promoter sequence with complete match with the canonical promoter (see Fig. 4) is shown in bold and italic while the promoter sequence with 5-out of-6 match is shown in bold. The spacers including H-NS binding sites are marked as HNS in the spacer column. Total number of genes under the control of these 181 promoters were 290 (1.80 gene per promoter). Among the total of 181 RpoD holoenyme-binding sites, 39 (24%) overlap with the H-NS-binding sites.

Table 3. Constitutive Promoters (Type-B Spacers) (Rightward transcription).

Map	Left Operon	Left Gene	D	RpoD	D	Right Gene	Right Operon	Promoter sequence
0.37	sokC	sokC	>	HNS	>	nhaA	nhaAR	TTAACC-17-TCTAAT
0.61	dapB	dapB	>		>	carA	carAB	TTGACT-17-CAGAAT
0.99	uaaU	yaaU	>		>	kefF	kefFC	TTGACT-16-TATGAC
2.27	(mraZ- -lpxC)	ftsZ	>		>	lpxC	lpxC
2.76	(pdhR- -lpd)	aceF	>		>	lpd	lpd	TTTAAA-17-TAAAAT
3.55	hrpB	hrpB	>		>	mrcB	mrcB	TTGAGA-17-TGTAAC
3.61	mrcB	mrcB	>		>	fhuA	fhuACDB	TTGCGA-18-TATTAT
3.81	clcA	clcA	>		>	erpA	erpA	TAGATA-19-TAGAAT
4.33	(bamA- -lpxA)	lpxD	>		>	fabZ	(bamA- --lpxA)	TCGCCA-15-TCTCAT
4.36	(bamA- -lpxA)	fabZ	>		>	lpxA	(bamA- -lpxA)	Internal Promoter
5.41	dinB-yafNOP	dinB	>		>	yafN	yafNOP
5.62	(proBC)	proA	>		>	thrW	thrW	TTGACG-15-TTTAAC
7.15	betT	betT	>	HNS	>	yahA	yahA	TTGATC-16-TATAAT
8.44	yaiU	yaiU	>	HNS	>	yaiV*	yaiV	TTCACT-18-TTTAAT
8.73	yaiI	yaiI	>	HNS	>	aroL	aroL-yaiA-aroM	TCGAAA-17-TATGAT
9.33	(nrdR- -pgpA)	ribD	>		>	ribE	(nrdR- -pgpA)	Internal Promoter
9.79	bloA	bolA	>		>	tig	tig	TCGACT-17-TATAAT
9.83	tig	tig	>		>	clpP	clpPX	TTGAAA-17-CATAAC
10.08	cof	cof	>		>	ybaO*	ybaO	TTGTCG-17-TAAAAT
10.70	adk	adk	>		>	hemH	hemH	TTATCA-15-GATATT
11.26	(ybbAP)	ybbP	>	HNS	>	rhsD	rhsDC-yibH	TTAATA-17-TGTAAT
11.36	(rhsD- -ylbH)	ylbH	>		>	ybbD	ybbD
11.38	ybbD	ybbD	>		>	ylbI	ylbI	TCGTCA-19-TAAAAT
12.23	(renD-emrE)	emrE	>	HNS	>	ybcK	ybcK	GTGACC-17-TAAAAA
12.24	ybcK	ybcK	>	HNS	>	ybcL	ybcLM	GTGGCA-17-TACAAT
12.29	ybcLM	ybcL	>		>	ybcM	(ybcLM)	Internal Promoter
12.55	tfaX	tfaX	>	HNS	>	appY*	appY	TTATCA-17-TTTAAT
13.06	sokE	sokE	>		>	hokE	hokE
13.56	(entcEBAH)	entH	>		>	cstA	cstA	TTTACA-15-TAAATT
14.15	pagP	pagP	>		>	cspE	cspE	TGGACA-17-TGTACT
14.57	ybeR-djiB	ybeR	>		>	djiB	(ybeR-djiB)	Internal Promoter
15.84	(ybfOC)	ybfC			>	ybfQ	ybfQ	TTTTCA-17-AATACT
16.61	(mngAB)	mngB	>	HNS	>	cydA	cydAB	TCTACA-17-TATATT
17.49	(biobFCD)	bioD	>		>	uvrB	uvrB	TTGGCA-17-TAAAAT
17.64	(moaABCDE)	moaE	>		>	ybhL	ybhL	TGCACA-17-TATCCT
17.65	ybhL	ybhL	>	HNS	>	ybhM	ybhM	ACGACA-16-TATAAA
19.21	(ybjC- -ybjN)	nfsA	>		>	rimK	rimK-ybjN
20.23	(lolA-rarA)	rarA	>		>	serS	serS	TGGCCA-17-GATAAG
20.68	(serC-aroA)	aroA	>		>	ycaL	ycaL	TTGATA-17-ATTAAT
23.37	(efeOB)	efeB	>	HNS	>	phoH	phoH	TTTATA-16-TATATT
24.80	(rpmF- -fabHDG)	fabG	>		>	acpP	acpP-fabF	TTGCAA-16-TACACT
24.84	(acpP-fabF)	fabF	>		>	pabC	pabCG- -ycfH	CTGCCA-15-GATAAG
25.12	(hinT- -ycfP)	ycfP	>	HNS	>	ndh	ndh	CTCACA-17-AACAAT
25.92	(ymfTL- -ymfS)	ymfL	>		>	ymfM	(ymfTL- -ymfS)	Internal Promoter
26.07	pinE	pinE	>		>	mcrA	mcrA	TTGTCG-17-ATTAAT
26.19	ycgZ- -ymgC	ycgZ	>		>	ymgA	(ycgZ- -ymgC)	Internal Promoter
26.20	(ycgZ- -ymgC)	ariR	>		>	ymgC	(ycgZ- -ymgC)	Internal Promoter
26.22	(ycgZ- -ymgC)	ymgC	>	HNS	>	ycgG	ycgG	TTGACG-19-TATTTT
26.27	ymgF	ymgF	>	HNS	>	ycgH	ycgHI	TTGACA-19-TATAAG
28.00	ychE	ychE	>	HNS	>	oppA	oppABCDF	TTAACA-17-AAGAAT
28.51	(yciVOQ)	yciO	>		>	yciQ	(yciVOQ)	Internal Promoter
28.71	topA	topA	>	HNS	>	cysB*	cysB	TTCACA-15-TATAAA
28.84	pgpB	pgpB	>		>	yciS	yciSM	TTGATT-18-AATCAT
31.32	(paaAB- -GHIJK)	paaB	>		>	paaC	(paaAB- -GHIJK)	Internal Promoter
31.44	(paaAB- -GHIJK)	paaJ	>		>	paaK	(paaAB- -GHIJK)	Internal Promoter
31.63	insI-2	insI-2	>		>	ydbC	ydbC	TTAACA-17-TCGAAT
32.89	yncH	yncH	>	HNS	>	rhsE	rhsE	TTGACT-17-TATTAC
34.53	(lsrACD- -tam)	lsrD	>		>	lsrB	(lsrACD- -tam)	Internal Promoter
35.16	ydfK	ydfK		HNS	>	pinQ	pinQ	TGTACA-16-AATAAT
35.51	dicF	dicF	>		>	dicB	dicB- -insD-intQ
35.98	ynfM	ynfM	>	HNS	>	asr	asr	GTCACA-18-TGTAAT
36.26	(rstAB)	rstB	>		>	tus	tus	TGGTCA-17-TATAAA
37.36	ydhO	ydhO	>		>	sodB	sodB	TTGCTA-16-AATAAG
37.49	ydhC	ydhC	>		>	cfa	cfa	CTAACA-17-TGAAAT
38.12	rprA	rprA	>		>	ydiL	ydiL	CTGATA-15-TATTGT
38.53	aroH	aroH	>	HNS	>	ydiE	ydiE	TTGATA-16-TATCAA
38.97	yniC	yniC	>		>	ydjM	ydjM	CTGAAA-17-ATTAAT
38.99	ydjM	ydjM	>		>	ydjN	ydjN	ATGACT-16-AATAAT
39.26	nadE	nadE	>		>	cho	cho	TTGTCA-15-TTTAAT
41.46	holE	holE	>		>	yobB	yobB-exoX	TATACA-16-CATAAC
43.96	(yedVZ)	yedZ	>		>	zinT	zinT	TTGTCA-18-AATAAT
44.29	amn	amn	>	HNS	>	yeeN	yeeN	TAGACG-18-TATAAT
44.60	yeeP	yeeP	>		>	isrC	isrC	TTGTCC-17-TAGAAT
44.60	isrC	isrC	>		>	flu	flu
46.62	(mdtA- -baeSR)	baeR	>		>	yegP	yegP	CTGGCA-17-CATACT
48.45	yeiH	yeiH	>		>	nfo	nfo
48.47	nfo	nfo	>		>	yeiI	yeiI
49.23	(yejLM)	yejM	>		>	proL	proL	TTGCAA-16-TAGTAT
49.61	yojO	yojO	>	HNS	>	eco	eco	GCGACA-15-TATAAA
51.29	rbn	rbn	>		>	elaD	elaD	TTAAAA-18-TGTTAT
54.54	cysZ	cysZ	>		>	cysK	cysK	ATGTCA-16-TATAGA
55.21	(yffOP)	yffP	>		>	yffQ	yffQR	CTCACA-16-TATCAC
55.23	(yffQR)	yffR	>		>	yffS	yffS
56.02	bcp	bcp	>		>	hyfA	hyfAB- -GHIJR	ATGACC-17-CAGAAT
56.02	hyfAB- -GHIJR	hyfA	>		>	hyfB	(hyfAB- -GHIJR)	Internal Promoter
57.13	sseA	sseA	>	HNS	>	ryfA	ryfA	TTGTCA-16-TATTGT
58.93	bamD	bamD	>		>	raiA	raiA	CTGTCA-18-TTTAGT
59.27	nadK	nadK	>		>	recN	recN	TTTACG-17-TATAAA
59.35	smpB	smpB	>		>	ssrA	ssrA	TGGTCA-18-TATACT
59.59	(rnlAB)	rnlB	>			yfjP	yfjPQ	TTGAAA-15-TATCAT
59.79	yfjW	yfjW	>		>	yfjX	yfjXYJZ-ypjF	TTGGCA-19-TATAAA
59.82	(yfjXYJZF)	ypjF	>		>	psaA	psaA	CTGACA-15-AACAAT
60.31	ygaM	ygaM	>		>	nrdH	nrdHIEF	TCAACA-18-TATCAT
60.46	(proVWX)	proW	>		>	proX	(proVWX)	TTATCA-16-AATAAC
60.51	(ygaXY)	ygaY	>		>	ygaZ	ygaZH	TTAAGA-17-TATAAT
60.92	(srlAEBD)	srlD	>		>	gutM*	gutM-srlRQ
61.59	mutS	mutS	>	HNS	>	pphB	pphB	TTAACG-17-TAAAAA
64.43	ygeF	ygeF	>	HNS	>	ygeG	ygeG	TTTAAA-17-TATCAA
64.49	ygeI	ygeI	>		>	pbl	pbi	TTGACC-16-GATACT
65.32	yqfG	yqfG	>		>	idi	idi	TTGTCG-18-AATCAT
65.82	zapA	zapA	>		>	ssrS	ssrS-fau
65.83	(ssrS-fau)	fau	>		>	sibC	sibC	TTGACA-15-CCTAAT
66.49	metK	metK	>	HNS	>	galP	galP	ATAACA-18-TATAAC
67.93	metC	metC	>		>	yghB	yghB	TGGACA-15-TATTGT
68.61	yqiC	yqiC	>	HNS	>	ygiL	ygiL	TCGATA-17-TATAAA
69.10	(ttdABT)	ttdB	>		>	ttdT	(ttdABT)	Internal Promoter
69.42	ebgR	ebgR	>		>	ebgA	ebgAC	TTGCCG-15-TATTTT
69.55	ygjJK	ygjJ	>		>	ygjK	(ygjJK)	Internal Promoter
69.79	aix	alx	>		>	sstT	sstT	CTGACC-17-TGTCAT
69.93	exuT	exuT	>		>	exuR*	exuR	TTTTCA-16-TAAACT
69.96	exuR	exuR	>		>	yqjA	yqjA-mzrA	TTGTCT-17-TATAAA
70.39	tdcR	tdcR	>		>	yhaB	yhaBC	TTGATA-19-GAAAAT
70.79	(agaS--agaBCDI)	agaI	>	HNS	>	yraH	yraHI	TTGATA-17-TCAAAT
71.84	ispB	ispB	>		>	sfsB*	sfsB	TTTAGA-18-TATAGT
72.93	argR	argR	>	HNS	>	yhcN	yhcN	TTGAAA-18-AATAAC
73.46	(panf-prmA)	prmA	>		>	dusB	dusB-fis	GTGCCA-18-AAAAAT
75.03	yheST	yheS	>		>	yheT	(yheST)	Internal Promoter
75.35	(nirBDC-cysG)	cysG	>	HNS	>	yhfL	yhfL	TTAACG-19-TATAAT
75.39	yhfL	yhfL	>		>	frlA	frlABCDR	CTGACA-18-TTTAAT
76.26	yhgF	yhgF	>	HNS	>	feoA	feoABC	TTATCA-15-TTTAAT
77.16	yhhZA	yhhZ	>		>	insA-6	insAB-6	TTGAAA-17-TTTAAT
77.20	(insAB-6)	insB6		HNS	>	yrhD	yrhD	TAGAGA-18-TATATT
78.71	slp-dctR	slp		HNS	>	dctR	dctR	TTAATA-17-TATTAT
80.65	yiaK	yiaK	>		>	yiaL	(yiaK- -sgbHUE)	Internal Promoter
81.13	yibA	yibA	>		>	yibJ	yibJ
81.15	yibJ	yibJ	>		>	yibG	yibG
81.20	yibV	yibV			>	yibU	yibU	TTAACT-15-GATAAT
81.79	(rfaD-waaFCL)	waaC	>		>	waaL	(rfaD-waaFCL)	Internal Promoter
82.24	yicC	yicC	>		>	dinD	dinD	GTGAGA-15-TATAAA
82.34	dinD	dinD	>		>	yicG	yicG	TTATCA-16-AAAAAT
82.48	xanP	xanP	>		>	yicH	yicH
85.10	ilvLXGMEDA	ilvL	>		>	ilvX	(ilvLXGMEDA)	Internal Promoter
85.43	trxA	trxA	>		>	rhoL	rhoL-rho	TTGACT-17-TATTAA
85.74	(rfe- -tffT- -rffM)	rffM	>		>	yifK	yifK	ATTACA-15-TTTAAT
87.25	polA	polA	>		>	spf	spf	CTGTCA-17-TAGAAA
87.47	typA	typA	>		>	yihL	yihLM
88.49	fieF	fieF	>		>	pfkA	pfkA
90.08	(rplKAJL-rpoBC)	rplL	>		>	rpoB	(rplKAJL-rpoBC)	TAGTCA-15-TGTAAG
91.98	aphA	aphA	>		>	yibQ	yibQR
92.52	(nrfABCDEFG)	nrfG	>	HNS	>	gltP	gltP	ATGCCA-18-TATTAT
94.29	ecnB	ecnB	>		>	sugE	sugE	TTGAAA-16-CAAAAT
94.89	yjeT	yjeT	>		>	purA	purA	CTGAAA-19-TTTAAG
95.01	(nsrR-rnr- -yjfIJ)	rlmB	>		>	yjfI	(nsrR-rnr- -yjfIJ)	TTAATA-17-TGGAAT
95.35	(rpsF-priB- -rplI)	rpsR	>		>	rplI	(rpsF-priB- -rplI)	Internal Promoter
97.06	(yjhBC)	yjhC	>		>	ythA	ythA
97.85	fimB	fimB	>	HNS	>	fimE	fimE	TTGTAA-17-CATAAT
98.12	(uxuAB)	uxuB	>		>	uxuR*	uxuR	GTGCCA-17-TATAGT
98.51	yjiS	yjiS	>	HNS	>	yjiT	yjiT	TTGAGA-18-TATAAA
99.35	osmY	osmY	>		>	ytjA	ytjA
99.36	ytjA	ytjA	>		>	yjjU	yjjU
99.52	(deoCABD)	deoA	>		>	deoB	(deoCABD)	Internal Promoter
99.57	(deoCABD)	deoD	>	HNS	>	yjjJ*	yjjJ	TTTTCA-18-TCTATT
99.57	yjjJ	yjjJ	>	HNS	>	yjtD	yjtD	TTGTCG-17-AATTAT
				HNS			154 (b)	271 (a)
				38			9 TFs	1.76 (a/b)
				25%			17 essential	62 Y-genes

Among the total of 1,075 RpoD holoenzyme-binding sites, 154 are located within type-B spacers upstream of right-side genes and downstream of lest-side genes, indicating that these promoters direct rightward transcription. The genes and operons under the control of these 154 promoters were estimated, of which 18 represent putative internal promoters. A total of 9 genes encoding transcription factors are indicated by star symbols (*). The essential genes listed in the PEC database are underlined within the operons. The promoter sequence with complete match with the canonical promoter (see Fig. 4) is shown in bold and italic while the promoter sequence with 5-out of-6 match is shown in bold. The spacers including H-NS binding sites are marked as HNS in the spacer column. Total number of genes under the control of 154 promoters were 271 (1.76 gene per promoter). Among the total of 154 RpoD holoenyme-binding sites, 38 (25%) overlap with the H-NS-binding sites.

Type-A spacers should contain at least two promoters for bidirectional transcription. The RpoD holoenzyme-binding sites identified in a total of 177 type-A spacers should represent promoters for one or both of bidirectional transcription. Close observation of the SELEX-chip pattern indicates that two RpoD holoenzyme-binding sites can be identified if the spacer is longer than 500 bp in length (Fig. 2). For instance, two peaks of RpoD holoenzyme binding were identified within a single and the same Type-A spacer between 755 bp-long csgD-csgB and between 1458 bp-long nanC-fimB (Fig. 2). Generally this-group promoters associated with stress-response genes are located within long spacers including the binding sites of a number of TFs such as in the spacers of 755 bp-long csgD-csgB, 920 bp-long lrhA-alaA and 1456 bp-long nanC-fimB. One typical example is the promoter for the csgD gene encoding the master regulator of biofilm formation, which is under the control of more than 20 TFs [23], [33].

Figure 2. RpoD holoenzyme-binding peaks within type-A spacers.

RpoD holoenzyme-binding peaks were identified within a total of 177 type-A spacers. Some representative patterns of RpoD holoenzyme-peaks are shown, which are located between leuL-leuO (a), csgD-csgB (b), nanC-fimB (c), lrhA-alaA (d), adhE-ychE (e), waaQ-waaA (f), mdoC-mdoG (g) and ulaG-ulaA (h). Distribution of promoter -35 (indicated by orange arrows) and -10 (indicated by green arrows) signals is shown in the panel under each SELEX pattern.

The binding of RpoD holoenzyme was identified in a total of 315 type-B spacers (Fig. 1A). The binding of RpoD holoenzyme alone to type-B spacers represents the presence of at least one constitutive promoter for one direction transcription. Even for this group of constitutive promoters, more than two RpoD holoenzyme-binding peaks were identified for some spacers (Fig. 3), indicating the presence of multiple promoters for one and the same transcription units such as the cydA promoters within 847 bp spacer and the yobF promoters within 670 bp spacer. In good agreement with the presence of multiple peaks for the mngB-cydA type-B spacer, five promoters have been identified for the cydAB operon encoding cytochrome bd-1 terminal oxidase [34]–[36], of which at least two may be the constitutive promoters that function in the absence of activator TF. The collection of constitutive promoters within type-B spacers also includes a total of 40 internal promoters located within intergenic spacers of single operons (indicted by symbol “Int” in P column of Tables 2 and 3; and Table S2). These internal promoters might play physiological roles under as yet unidentified circumstances. In fact a constitutive internal promoter within the rplKAJL-rpoBC operon has been identified [37], which should contribute the expression level control between four ribosomal proteins (L11, L1, L10 and L12) and two RNA polymerase subunits (RpoB and RpoC).

Figure 3. RpoD holoenzyme-binding peaks within type-B spacers.

RpoD holoenzyme-binding peaks were identified within a total of 315 type-B spacers. Some representative patterns of RpoD holoenzyme-peaks are shown, which are located, which include the constitutive promoters for cydA (a), yfcV (b), yobF (c), ompT (d), yjeJ (e), yhcN (f), yfjL (g) and phoH (h) operons. Distribution of promoter -35 and -10 is shown below each panel.

The total number of RpoD promoters (or the transcription initiation sites) listed in the current databases (RegulonDB and EcoCyc) are as many as 2,082, indicating that the majority of known promoters represent TF-dependent “inducible promoters” that are expressed only under the support of positive regulatory proteins (Fig. 1B). Among the total of 2,082 RpoD promoters (or transcription initiation sites) listed in the current version of promoter databases, the promoter sequences are described for a total of 582, of which 434 (75%) are located within the same spacers that contain the constitutive promoters (255 in type-A spacers and 179 in type-B spacers) (shown under black background of P columns in Tables 1, 2 and 3).

Identification of the consensus sequence of constitutive promoters recognized by the RpoD holoenzyme

For identification of the constitutive promoters within type-A and type-B spacers with the binding sites of RpoD holoenzyme, we performed in silico search using the consensus sequence of constitutive promoters. The current databases of E. coli promoters include both experimentally identified and computationally predicted promoters. The prediction of promoters has been performed using the canonical promoter, TTGACA-17 bp-TATAAT, which was originally identified using the in vitro transcription studies [10], [11], [38]. We then tried to identify the consensus sequence of constitutive promoters recognized in vitro by RpoD holoenzyme alone.

In order to experimentally confirm the consensus sequence recognized by RNA polymerase RpoD holoenzyme, we first constructed an ideal promoter with the complete set of consensus TTGACA (-35) and TATAAT (-10) sequences, separated by a spacer of 17 bp in length, starting from the lacUV5 promoter [23]. To identify the best RpoD promoter giving the highest activity and to examine the role of individual bases within two hexanucleotide sequences, we then constructed a total of 48 variant consensus promoters, each carrying one base replacement at each position of both -35 and -10 signals. For accurate measurement of the RNA product directed by each variant promoter, we employed the in vitro mixed transcription system [24], [25], in which transcription of each variant promoter was carried out in the simultaneous presence of the ideal promoter with the complete consensus sequence added as an internal reference. The test promoter directed the synthesis of 42 b-long run-off transcript while the ideal promoter directed the synthesis of 22 b-long run-off transcript [23]. The same amounts of two promoter fragments were mixed and incubated with 10-fold molar excess of RpoD holoenzyme for various time periods to allow the formation of open complexes, and then a mixture of substrates and heparin was added to allow the single-round transcription. The final level of transcripts represents the amount of RNA synthesized in 15 min reaction after the addition of substrate mixture into open complexes formed during preincubation for various times up to 30 min (referred to parameter-I in this study). Parameter-I represents the binding affinity of RpoD holoenzyme to the test promoter. On the other hand, the slope of transcript increase represents the rate of open complex formation (referred to parameter-II).

Both parameter-I and -II were determined for each variant promoter for three times and the average values are shown in Fig. 4. In each panel, the promoter activity is compared between four templates with different bases at the same position. Among the collection of -35 variants, the best promoter giving the highest activity of open complex formation (parameter-I) was identified for the consensus TTGACA sequence (Fig. 4A). This indicates that the consensus sequence of promoter -35 influences the binding affinity of RNA polymerase to the promoter in agreement with the previous estimation [23]. As to the promoter -10 signal, the best sequence giving the highest rate of open complex formation (parameter-II) was identified for the consensus TATAAT (Fig. 4B), indicating that promoter -10 influences the rate of promoter opening.

Figure 4. Determination of the consensus sequence of constitutive promoters using the in vitro mixed transcription system.

Mixtures of equal amounts of 195 bp-long template containing the ideal promoter of complete consensus sequence and 175 bp-long mutant template, each carrying one base substitution, were subjected to the in vitro mixed transcription [24], [25]. After preincubation for 0.5, 1.0, 2.5, 5.0, 7.5, 10 and 15 min, a mixture of substrates and heparin was added and RNA synthesis was allowed for 10 min. The final level of RNA synthesis represents the level of RpoD holoenzyme binding (parameter I) while the rate of open complex formation (parameter II) was determined as a reciprocal of the time required to reach the plateau level. For each set of four promoters with mutations at the same position, the promoter activities are shown as the values relative to the promoter with the highest activity.

Using this experimentally confirmed consensus sequence, TTGACA-17 bp-TATAAT, of the constitutive promoter recognized by RpoD holoenzyme alone, we search for the location of constitutive promoters within both type-A and type-B spacers including RpoD holoenzyme-binding sites.

Unique features of the RpoD constitutive promoters

After sequence analysis of the entire genome of E. coli K-12, we realized that there is no ideal sequence of RpoD promoter with perfect matching to this consensus sequence. We then analyzed whether the constitutive RpoD promoters harbor unique sequences. By setting a rather severe screening condition of the sequence matching of more than 4 out of 6 bases for both -35 and -10 signals (total score, higher than 8; the highest score, 12) and with a spacer length of 17 plus/minus 2 (score 3 for 17 bp spacer, score 2 for 16 and 18 bp spacers, and score 1 for 15 and 19 bp spacers), a total of as many as 316 promoter sequences (89%) were identified among 354 predicted constitutive promoters within type-A spacer, and a total of 226 promoter sequences (82%) were identified among 276 predicted promoters excluding the internal promoters within type-B spacer. Overall the total amount of constitutive promoters with high-level (higher than 4/6) matching with the consensus RpoD promoter for both -36 and -10 signals is more than 85% (Fig. 5A-1). This is in sharp contrast with the collection of 582 experimentally defined promoters, in which the amount of promoters containing a high-level (4/6) agreement to both -35 or -10 signals are less than 40% (Fig. 5B-1). On average, only less than half of the 12 canonical bases of the -35 and -10 boxes are conserved among the experimentally identified promoters [39]. The length of spacers between -35 and -10 signals ranges from minimum 14 to maximum 21 bp. Only 10–20% efficient promoters have been proposed to include either -35 or -10 box that resembles the consensus with five-out-of-six bases (5/6 agreement).

Figure 5. The composition of constitutive promoters.

[A] The known consensus sequences of RpoD-dependent promoter, TTGAAC (-35) and TATAAT (-10) separated by 17 plus/minus 2 bp, were searched for all type-A (177) and type-B (315) spacers (see Experimental Procedure for the analysis method). Most of the constitutive promoters carry high-levels of the consensus sequence as listed in Table 1. The composition of promoter -35 and -10 sequence was classified into 8 groups based on the conservation level of consensus sequences. About 89% of type-A promoters and 82% of type-B promoters (or 86% of A- plus B-type promoters) contain the sequence higher than 4/6 agreement with the consensus sequence at both -35 or -10 positions (A1), while only 39% of a total of 582 known promoters carries this level of consensus sequences (B1). [B] The whole set of constitutive promoter sequences were subjected to Logo analysis [73]. The Logo patterns of -35 and -10 sequences are essentially the same among the constitutive promoters within type-A and type-B spacers. The Logo pattern of the whole set of constitutive promoters was compared with the Logo pattern generated using the total of 582 experimentally identified promoters [19], [20]. The contribution of each base of the consensus -35 and -10 sequences is significantly different between the constitutive promoters and the set of known promoters.

Using a total of 669 sequences of the constitutive promoter with high-level of sequence conservation, the consensus sequence of RpoD promoter was reevaluated. The Logo plot, shown in Fig. 5A-2, indicates that: 1) the patterns of conserved bases of both -35 and -10 sequences are similar for both left and right genes with both type-A and type-B spacers (data not shown); and 2) a significant difference exists in the consensus sequence of both -35 and -10 signals between a total of 543 newly identified constitutive promoters and a total of 582 experimentally identified promoters. The Logo pattern generated from the alignment of a total of 582 experimentally identified E. coli promoters, shown in Fig. 5B-2, indicates that the level of sequence conservation is significantly higher for -10 than -35, and the conserved bases are limited, TT (positions 1 and 2) of -35 signal and TA (positions 1 and 2) and T (position 6) of -10 signal. This Logo pattern agrees well with that analyzed by Mitchell et al. [40] using the promoter set of RegulonDB [41]. In the case of constitutive promoters, -35 and -10 sequences are equally well-conserved (Fig. 5A-2). Within -35 sequence, TT (position 1 and 2) and AA (positions 4 and 6) are well-conserved but G at position 3 and C at position 5 appear less important even for the constitutive promoters. On the other hand, all six bases of TATAAT within -10 signal are equally well-conserved, indicating their equal contribution to the promoter activity. The marked difference of the consensus sequence of experimentally identified RpoD promoters from that of constitutive promoters indicates that the promoter databases include a number of inducible promoters that were active under the support of positive TFs.

Constitutive promoters carrying the binding site of silencer H-NS

The constitutive promoters direct transcription by the RpoD holoezyme alone in the absence of positive transcription factors. Expression in vivo of the constitutive promoters must be repressed under conditions where the target genes are not needed. One possible transcription factor with the silencing activity is H-NS, one of the major core nucleoid proteins with functional dichotomy [42]. H-NS is known as a universal silencer for repression of a number of xenogeneic genes [43], [44]. In parallel with the mapping of RpoD promoters, we identified a total of 987 H-NS-binding on the E. coli genome using the Genomic SELEX system [45]. Within a total of 492 spacers (177 type-A and 315 type-B) with RpoD holoenzyme-binding sites, H-NS binding sites were identified within 63 type-A (36%) (indicated as H-NS in column P of Table 1) and 77 type-B spacers (24%) (Tables 2 and 3).

The genome-silencing function of H-NS is attributable to its unique mode of DNA binding. H-NS recognizes and binds to intrinsically curved (or bent) DNA with AT-rich sequences, and such curved sequences are often located near promoters and also within transported DNA segments such as phage genomes [46], [47]. Promoter-associated DNA curvature provides H-NS with the initial contact site, leading to form DNA-H-NS filaments via cooperative protein-protein interaction [48]. Since once H-NS binds within one spacer, it could be fully covered with H-NS by spreading from the initial binding sites through high level of protein-protein cooperativity. Thus, if H-NS binds within one type-A spacer, it influences the silencing effect to promoters for both of the bidirectional transcription. Taken together we estimated that a total of 203 promoters (63x2 + 77) or 31% among a total of 669 constitutive promoters could be under the control of genome silencer H-NS, indicating that one third of the constitutive promoters are subject to silencing by H-NS.

Among the total of 203 possible targets of H-NS silencing, a total of 20 promoters are known to be under the control of H-NS. Up to the present time, however, the involvement of H-NS in promoter regulation has not analyzed seriously because this bifunctional nucleoid protein is abundant, playing both architectural and regularatory roles. The involvement of H-NS in transcription regulation of a specific gene has only been identified during the systematic search for transcription factors. For instance, the csgD (the master regulator of biofilm formation), the ndh gene (NADH:ubiqinone oxidoreductase), and the gadX gene (the stress-response regulator for acid resistance) are all under the control of more than 10 transcription factors including H-NS [4], [22]. By using the newly developed PS-TF (promoter-specific transcription factor) screening system, we have identified the involvement of H-NS for regulation of a set of genes that are all under the control of multiple transcription factors [49]. These findings altogether suggest that the involvement of H-NS in transcription silencing of a number of constitutive promoters will be established once PS-TF screening is performed.

Discussion

Definition of the constitutive promoters

The pattern of genome transcription in E. coli is determined by controlling the utilization of a limited number of RNA polymerase [4], [22]. High-throughput systems have been developed for identification of the RNA polymerase distribution within the genome. For instance, large-scale mapping of the functioning promoters in vivo on the E. coli genome has been performed by the chromatin immuno-precipitation and microarray (ChIP-chip) analysis with specific antibodies against RNA polymerase subunits [14], [15]. To prevent moving RNA polymerase along DNA, E. coli cells were treated with rifampicin, which binds to the RpoB subunit of RNA polymerase and blocks transcription initiation, thereby fixing the initiated RNA polymerase on promoters [50]. For identification of the distribution pattern of functioning RNA polymerase, RNA-Seq analysis of high throughput sequencing of transcriptomes is becoming the method of choice [16]–[18]. Concomitant with the increase in the number of promoters detected under various stressful culture conditions, the variation of promoter sequence is expanding. The fluctuation of functioning promoters is attributable to the modulation of promoter selectivity of RNA polymerase by sigma factors and transcription factors with positive and negative regulatory functions [3], [4].

The constitutive promoter has been considered to represent a set of promoters associated with the genes constitutively expressed in vivo in all circumstances. Based on this definition, however, it is practically impossible to identify the whole set of constitutive promoters of E. coli under various environmental conditions. At present, the whole sets of promoters determined by high-through put procedures (see above) are accumulating, but the experiments have been performed using different E. coli strains, under different culture conditions and using different experimental systems. In addition, even in laboratory culture conditions, it now turned clear that the steady state of cell growth does not exist and moreover, the genome expression pattern varies between individual cells within the same culture. In contrast, the whole set of constitutive promoters can be identified in vitro because in the case of E. coli, the faithful transcription can be established in vitro using purified RNA polymerase and pure DNA template under defined conditions. To avoid the complexity arisen from in vivo determination of the functioning promoters, an attempt was then made in this study to identify the whole set of the constitutive promoters using an in vitro system. Based on the results, we propose to revise the definition of the constitutive promoter as the promoter that is recognized and transcribed by the RNA polymerase holoenzyme alone in the absence of supporting TFs.

SELEX-chip search for the constitutive promoters

For this purpose, we used the improved method of Genetic SELEX screening [21], which has been successfully employed for identification of binding sites of a number of TFs on the E. coli genome [22]. As a result, the number of regulation targets markedly increased even for the TFs with known regulatory functions. For instance, the number of regulation targets increased more than 2.5 fold from 150 to 350 even for the best characterized transcription factor, CRP (cAMP receptor protein) or CAP (catabolite activator protein) [51]. This experimental system is particularly useful for short-cut estimation of the regulation targets of uncharacterized TFs including YbjK (renamed to RcdA), YcdC (renamed to RutR), YcjZ (renamed to PgrR), YdhM (renamed to NemR) and YgiP (renamed to Dan) [22]. The Genomic SELEX screening system has also been successfully employed for detection of the alteration of promoter-recognition properties of transcription factors after phosphorylation (in the case of two-component systems) or interaction with effector ligands. For instance, the selection of regulation targets of SdiA, a regulator of genes for cell division and differentiation, was found to alter differently in the presence of each of three homoserine lactone analogs, the QS signals [52].

As an extension of screening of the whole set of RpoD promoters, we have successfully performed the screening of promoters recognized by RNA holoenzymes containing minor sigma factors, RpoS, RpoH, RpoF, RpoE and FecI. In the case of RpoN sigma factor, it requires enhancer-binding proteins such as NtrC and for formation of stable RNA polymerase-promoter complexes. Results will be described elsewhere.

Physiological roles of the constitutive promoters

The genes under the control of constitutive promoters are supposed to be expressed constitutively although unnecessary genes are subject to repression by silencers (see the H-NS chapter). One short-cut interpretation is that the essential genes are under the control of constitutive promoters. After systematic deletion of the genome, the total number of essential genes in the E. coli genome has been minimized to generate the minimal genome consisting of 302 essential genes ([53]; also see PEC database). Among 302 essential genes, 70 (23%) were identified to be under the control of constitutive promoters. Noteworthy is that the expression of some essential genes should be under the control of constitutively expressed positive transcription factors. In fact, we have identified the presence of approximately 100 species of E. coli transcription factor throughout cell growth.

Marked variation of the promoter sequences listed in databases must have been arisen from several different factors: 1) the list of RpoD-dependent promoters include those recognized by RpoS (and possibly other minor sigma factors), of which the promoter recognition properties overlaps with RpoD [54], [55]; 2) the list also includes promoters that are functional in vivo only under the support of positive TFs [56]–[58]; and 3) a variety of promoter-like sequences have been identified in silico to be promoters. In fact approximately 60% of the RpoD holoenzyme-binding sites are located inside open reading frames (see Fig. 1A). Binding of RNA polymerase on some open reading frames has been recognized [59]. The unexpected high number of RpoD holoenzyme-binding sites inside open reading frames raises a possibility of an as yet unidentified functional role(s) for the RpoD holoenzyme. These promoter-like sequences may contribute transcription initiation from internal promoters and/or blocking the migration of elongation complexes. Clustering of promoter-like sequences within the E. coli genome were predicted by Collado-Vides and colleagues [60] while Ozoline and colleagues proposed the presence of as many as 78 ‘Promoter Islands’ [59], [61]. These promoter-like sequences could form transcriptionally inactive complexes with RNA polymerase but might contribute to increase local concentrations of RNA polymerase on the genome.

Some of the constitutive promoters with high-level of conservation of the consensus sequence are located within long spacers with long UTR sequences but lacking protein-coding sequences of reasonable sizes. In these cases, it would be worthwhile to test as yet unidentified regulatory sRNAs [62]. Noteworthy is that such long spacers including the constitutive promoters often correspond to the Promoter Islands.

The consensus sequence of constitutive promoters

The canonical model of RpoD promoters consisting of two hexanucleotide sequences, TTGACA -35 signal and TATAAT -10 signal, each being separated by 17 bp linker was originally identified using in vitro transcription of some model templates by purified RNA polymerase [10], [11]. Here we identified the consensus sequence of as many as 669 constitutive promoters. The most significant feature of constitutive promoters is the high-level conservation of canonical TTGACA(-35)-17bp-TATAAT(-10) sequence. We also identified the roles and -35 and -10 signals and the conservation level of each base within these two hexanucleotide sequences. The promoter -35 TTGACA signal plays a key role in binding the RNA polymerase (see Fig. 4) but G at position 3 and C at position 5 are relatively less important for the constitutive promoters, suggesting that both play roles in TF-depending inducible promoters. The promoter -10 TATAAT signal plays a major role in promoter opening (see Fig. 4) in agreement with the previous proposals [63], [64], but the novel finding is that all six bases of TATAAT are equally important for this -10 signal function, supporting the hypothesis that the cooperativity of energy threshold, but not interaction of individual bases with RpoD, are important factors guiding the dynamics and selectivity of promoter open complex formation [65].

Regulation of the conserved promoter

The constitutive promoters must be repressed in cases when the genes under their control are not necessary. A group of silencing proteins play roles in preventing the potentially harmful effects of uncontrolled expression of the constitutive promoters. In E. coli, the H-NS family proteins are the major players of anti-silencing [44], [45], [66]. The high-level of overlapping was observed in the distribution between the constitutive promoters (see Tables 1, 2 and 3) and the binding sites of silencer H-NS [45]. A total of 203 (30%) of the constitutive promoters were predicted to be under the control of H-NS silencer, but silencing proteins for other 70% promoters remain unidentified. The spectrum of silencing targets by H-NS should be modulated by interaction with the members of Hha/YdgT family of small-sized co-regulators [67], [68]. Possible involvement of other growth condition-specific nucleoid proteins in silencing such as Dps (DNA-binding protein in starved cells) [69] and Dan (DNA-binding protein under anaerobic conditions) [32] awaits further studies.

It should be noted that the constitutive promoters are also subject to activation by positive TFs for enhanced expression of the target genes. For instance, the csgD gene encoding the master regulator of biofilm formation carries one of the Type-A constitutive promoters, which is controlled by as many as 20 positive and negative TFs [22], [33]. The level of constitutive csgD promoter is higher than that of csgB promoters directing transcription toward opposite orientation (see Fig. 2A[b]), but once the regulator CsgD is produced, it antagonizes H-NS silencer and the expression of the csgBAC operon is markedly enhanced, leading to production of curli fimbriae for biofilm formation.

In conclusion, we classified E. coli promoters into the constitutive promoters recognized by RNA polymerase holoenzyme alone and the transcription factor-assisted inducible promoters. Using the information of RpoD holoenzyme-binding sites identified by Genomic SELEX screening system, we predicted a total of 669 constitutive promoters with high-level conservation of the promoter consensus sequences. This finding indicates that the majority of hitherto identified promoters represent the TF-dependent inducible promoters.

Materials and Methods

Bacterial strains and plasmids

E. coli K12 W3350 type-A [70] was used for purification of RNA polymerase and the template DNA for Genomic SELEX screening of RpoD 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 (pRpoD, pRpoA, pRpoB and pRpoC) and all seven sigma subunits (pRpoD, pRpoS, pRpoN, pRpoH, pRpoF, pRpoE and pFec) 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 seven sigma subunits and all 300 transcription factors in this laboratory [71], [72].

Purification of core RNA polymerase

RNA polymerase was purified from log-phase cells of E. coli K-12 W3350 by the standard procedure [26]. Separation of the core and holoenzymes was performed by passing the purified RNA polymerase through P11 phosphocellulose column in the presence of 50% glycerol [26]. To remove trace amounts of the core enzyme-associated sigma factors, the purified RNA polymerase in the storage buffer containing 50% glycerol was dialyzed against the same buffer but containing 5% glycerol and fractionated by phosphocellulose column chromatography in the presence of 5% glycerol [26]. The level of remaining sigma factors was less than 0.1%, if any, as checked by 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 [26]. Sigma subunits were expressed and purified by ion-exchange column chromatography through DE52 and P11 followed by Sephacryl S-300 gel filtration column [26]. The purified sigma and core subunit proteins were more than 99% pure as judged by protein staining of SDS-PAGE gels.

Preparation of antibodies

Antibodies against RpoD sigma and core enzyme subunits were produced in rabbits by injecting purified sigma proteins. The protocol for antibody production was raised following the Ethical Guidelines proposed by the Science Council of Japan and the Japanese Government, and approved by the Committee on the Ethics of Animal Experiments in the Animal Laboratory of Mitsubishi Chemical Medience Co. (Uto, Kumamoto, Japan). Antibodies against each RNA polymerase proteins 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-RpoC used in this study did not cross-react with each other.

Genomic SELEX screening of RpoD holoenzyme-binding sequences

The genomic SELEX method was carried out as previously described [21]. 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. In each SELEX screening, the DNA mixture was regenerated by PCR. For SELEX screening, 5 pmol of the mixture of DNA fragments and 10 pmol RNA polymerase RpoD 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 genome at 105 bp interval (Oxford Gene Technology, Oxford, UK) [27]–[29]. 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 genome.

Mixed transcription assay in vitro

The promoter sequence of 195 bp-long lacUV5 template and its 3′ truncated 175 bp-long DNA was modified to the consensus sequence, TTGACA(-35)-17 bp-TATAAT(-10), each producing run-off transcripts of 42 and 22 nucleotides in length, respectively. Starting from these DNA fragments with the ideal promoter, the complete set of variant consensus promoters, each containing one base substitution at all positions of the hexanucleotide sequences of promoter -35 and -10, was prepared as described previously [23]. The test promoters of variant consensus promoters directed the synthesis of 42 b-long RNA while the reference template containing the original consensus promoter directed the synthesis of 22 b-long RNA. The in vitro mixed transcription was performed under the standard single-round reaction conditions in the presence of [³²P]UTP as a labeled substrate [23]–[25]. In brief, a mixture of these two templates was preincubated with RpoD holoenzyme for various times (0.5, 1.0, 2.5, 5.0, 10, 15 and 30 min) at 37°C in the standard transcription assay buffer, and then a mixture of substrates and heparin was added to allow a single-round transcription for 15 min. Heparin inactivates RNA polymerase that was not involved in open complex formation. RNA products were separated by electrophoresis on 10% PAGE containing 8.3 M urea. The amount of RNA was determined by measuring the intensity of ³²P radioactivity and corrected for the U content of each transcript.

Promoter sequence analysis

The complete genome sequence of E. coli K-12 MG1655 (U00096.2) was used for the promoter sequence analysis. The perl scripts for finding the candidate sequences of promoters include the following functions: 1) extraction of every 6 bases in the genome sequences with sliding 1 bp; 2) comparison of the 6 base sequences with TTGACA (-35) and calculation of the score by setting one match scoring 1 point, one mis-match scoring 0 point, and without gap; 3) comparison of the 6 bases with TATAAT (-10) under the same conditions; and 4) extraction of a pair of -35 and -10 hexanucleotide sequences by setting the best-match score of 12 (6 at -35 and at -10 signal); 6) extraction of a pair of -35 and -10 signals with a spacer length of 17 plus/minus 2, giving the score of +3 for 17 bp, +2 for 16 and 18 bp; and +1 for 15 and 19 bp, respectively. Thus, the maximum score for the best-match promoter in both -35 and -10 sequences separated by 17 bp spacer is 15.

The Logo pattern analysis of promoter sequences was performed using the established sequence logo generator [73].

Supporting Information

Table S1

RNA polymerase RpoD holoenzyme-binding sites on the E. coli genome. A total of 1,075 RpoD holoenzyme-binding sites were identified within spacers on the entire E. coli K-12 W3110 genome. The binding sites identified within intergenic spacers were classified into Type-A, Type-B and Type-C (see Fig. 2 and Table 1). The constitutive promoters were predicted based on the location of the RpoD holoenzyme-binding sites. [S1A] Promoters within type-A spacers. The genes under the constitutive promoters on left- and right-sides of type-A intergenic spacers and their functions are described. The genes shown under filled background encodes DNA-binding transcription factors (TFs), each regulating a set of target genes. The spacers under grey background contain two distinct peaks of holoenzyme binding. The positions of these genes on the genome are described on the left-end column while the levels of SELEX-peaks relative to the highest peak are described on the right-end column. [S1B] Promoters within type-B spacers. The genes under the constitutive promoters on either left- or right-sides of type-B intergenic spacers and their functions are described. The positions of these genes on the genome are described on the left-end column while the levels of SELEX-peaks relative to the highest peak are described on the right-end column. The genes shown under black background encodes DNA-binding TFs. The spacers under grey background contain two distinct peaks of holoenzyme binding.

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Table S2

Operons under the control of the constitutive promoters. Operons under the control of constitutive promoters are listed on the operon columns. Promoters shown by shaded background represent those listed in RegulonDB and EcoCyc databases. Spacers containing H-NS binding sites are indicated by HNS. [S2A] Operons under the control of constitutive promoters within type-A spacers. The constitutive promoters identified within type-A spacers direct bidirectional transcription of the operons indicated on left- or right-operon columns. The map positions of the promoter-proximal genes are indicated on the left- and right-end columns. [S2B] Operons under the control of constitutive promoters within type-B spacers. The constitutive promoters identified within type-B spacers direct transcription toward one direction.

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Funding Statement

This study was supported by Grants-in-Aid for Scientific Research from MEXT (Ministry of Education, Culture, Sports, Science and Technology of Japan): (A) (21241047) to AI, (B) (18310133) to AI, (C) (25430173) to AI, and Young Scientists (B) (24710214) to TS. This study was performed under the MEXT-Supported Program for the Strategic Research Foundation at Private Universities 208-2012 (S0801037). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.