Genome prediction of PhoB regulated promoters in Sinorhizobium meliloti and twelve proteobacteria.

In proteobacteria, genes whose expression is modulated in response to the external concentration of inorganic phosphate are often regulated by the PhoB protein which binds to a conserved motif (Pho box) within their promoter regions. Using a position weight matrix algorithm derived from known Pho box sequences, we identified 96 putative Pho regulon members whose promoter regions contained one or more Pho boxs in the Sinorhizobium meliloti genome. Expression of these genes was examined through assays of reporter gene fusions and through comparison with published microarray data. Of 96 genes, 31 were induced and 3 were repressed by Pi starvation in a PhoB dependent manner. Novel Pho regulon members included several genes of unknown function. Comparative analysis across 12 proteobacterial genomes revealed highly conserved Pho regulon members including genes involved in Pi metabolism (pstS, phnC and ppdK). Genes with no obvious association with Pi metabolism were predicted to be Pho regulon members in S.meliloti and multiple organisms. These included smc01605 and smc04317 which are annotated as substrate binding proteins of iron transporters and katA encoding catalase. This data suggests that the Pho regulon overlaps and interacts with several other control circuits, such as the oxidative stress response and iron homeostasis.

INTRODUCTION

Dissection of regulatory networks that control gene transcription is among the primary goals of the post-genomic era of biology. Whether gene expression is measured from microarrays or reporter gene fusions or other methodologies, it is generally not possible to distinguish between the direct and indirect modulation of transcription. Bioinformatic approaches to identify the regulatory networks have included the design of algorithms for genome-wide prediction of conserved regulatory DNA binding motifs (1,2). A promising approach in the delineation of transcriptional networks lies in combining genomic scanning or in silico analysis with experimental transcription data obtained from cells grown under diverse experimental conditions (1,3–12). In this report, we combine in silico prediction with experimental data obtained from reporter gene fusions and through comparisons with published microarray data. We also explore cross-species comparative genomics as a tool to identify genes whose expression is controlled by a transcriptional regulator, PhoB, in response to the phosphate starvation.

Inorganic phosphate (Pi) plays key roles in cells. In ATP, it is involved in energy metabolism, in protein phosphorylation it is responsible for regulation of transcription and many other cellular processes including chemotaxis and cell division, and perhaps most importantly, Pi is a major structural component of nucleic acids and membrane phospholipids. In many gram-negative bacteria, the transport and metabolism of Pi and phosphorous containing compounds is regulated at the transcriptional level by a two-component PhoR-PhoB signal transduction system. The Pho regulon consists of genes or operons regulated by PhoB and this has been well studied in Escherichia coli (13–15). Under Pi limiting conditions, the PhoR histidine kinase sensor undergoes autophosphorylation and subsequently donates its phosphate group to its cognate response regulator PhoB. Phosphorylated PhoB (PhoB-Pi) then modulates transcription of its targets by binding to a highly conserved 18 nt DNA sequence called the Pho box (or PhoB binding motif) which usually overlaps the −35 region of PhoB-regulated promoters (16,17). The majority of identified Pho boxes essentially comprised two 7 nt direct repeats of 5′-CTGTCAT-3′ separated by a conserved 4 nt spacer in the middle. It was postulated that the PhoB and Pho box binding complex interacts with the σ70 subunit of RNA polymerase to control transcription initiation (18–23). Over the past 30 years, about 30 Pho regulon members, which predominantly encompass an ensemble of genes involved in Pi uptake and metabolism, have been identified in E.coli as reviewed by Wanner (14).

We are studying the gram negative α-proteobacterium Sinorhizobium meliloti. This organism forms N2-fixing root-nodules on alfalfa (Medicago sativa) and its genome is unusual as in addition to a 3.4 Mb chromosome it contains two megaplasmids 1.3 and 1.7 Mb in size. Previous studies have identified several Pho regulon members in S.meliloti including the pstSCAB and phoCDET operons which encode ABC-type high affinity transport systems for Pi and in the case of phoCDET likely phosphonates (24–27). The orfA-pit operon encodes a low affinity Pi transport system whose expression is negatively regulated by PhoB (28). Other Pho regulons include the exp, phn and pta-ackA operons (16,29,30). Using DNA microarray and promoter analysis, Krol and Becker (31) identified several novel putative Pho regulon members including afuA which is annotated as an iron transport binding protein. In ongoing studies to understand the response of S.meliloti to Pi limitation, we constructed a Pho box weight matrix based on known E.coli and S.meliloti PhoB binding sites and used this matrix to predict new PhoB binding sites in the S.meliloti genome. Expression of predicted Pho regulon members then was examined through the analysis of transcriptional reporter gene fusions and through the previously reported microarray data (31). The frequency weight matrix was also employed to predict PhoB binding motifs across 12 closely related proteobacterial genomes with a goal to identifying a common set of PhoB regulated genes as might be expected from a conserved biological response to Pi-limitation.

MATERIALS AND METHODS

Construction of the Pho box weight matrix for prediction of PhoB binding sites

A total of fifteen known Pho boxes from S.meliloti and E.coli were used for weight matrix construction. Five of those Pho box sequences were collected from previously identified PhoB binding sites from S.meliloti. Of those five, four were from S.meliloti strain 1021 including one PhoB binding site upstream orfA-pit; two sites from the phoC promoter; one from the phnG promoter (24,28,32) and one Pho box was taken from the orfA-pta-ackA promoter of S.meliloti strain 104A14 (16). Ten PhoB binding sites from E.coli were phoA, phoB, phoE, phoH, phnC, pstS1, pstS2, ugpB1, ugpB2 and ugpB3 (18,33–35) (see Table 1). Following their alignment a matrix was constructed from the relative frequencies of A, T, C or G at each position of the 18 nt Pho box sequence (Table 1). This matrix was used to determine an information-based measure of potential binding sites according to the method of Schneider et al. (36). An 18 bp window was moved over the entire genome on both strands and the score (Si) at each nucleotide position (having base i) was calculated according to S = (1/18) Σ [2 + log2(F)], where F is the frequency matrix for base i at position j. This score, which ranges from −2.62 (the score of the worse match) to 1.39 (the score of the consensus sequence), is a measure of the information content of a potential binding site measured against the example set. The lowest example score, that of orfA-pit, is 0.36 and a threshold of 0.35 was used to define a ‘hit’. A scan of the entire S.meliloti genome produced about 1500 hits on each strand. These were filtered to retain only those that were between −500 to +100 bp on the coding strand from an annotated translational start site.

Table 1

Pho-box matrix

Position	#1	#2	#3	#4	#5	#6	#7	#8	#9	#10	#11	#12	#13	#14	#15	#16	#17	#18
S.meliloti orfA-pta:	T	T	G	T	C	A	A	A	C	C	G	C	C	G	T	A	A	C
S.meliloti phnG:	A	T	G	T	C	A	C	A	A	G	C	C	T	G	T	C	A	T
S.meliloti phoC1:	C	T	G	A	C	A	C	T	G	C	G	C	T	T	T	C	A	T
S.meliloti phoC2:	C	T	G	T	T	A	C	A	G	A	A	C	C	T	A	C	A	C
S.meliloti orfA-pit:	C	T	G	T	G	G	G	A	A	A	G	C	C	G	T	T	T	T
E.coli phoA	C	T	G	T	C	A	T	C	A	C	T	C	T	G	T	C	A	T
E.coli phoB	C	T	G	T	C	A	T	A	A	A	G	T	T	G	T	C	A	C
E.coli phoE	C	T	G	T	A	A	T	A	T	A	T	C	T	T	T	A	A	C
E.coli phoH	C	T	G	T	C	A	T	C	A	C	T	C	T	G	T	C	A	T
E.coli phnC	C	T	G	T	T	A	G	T	C	A	C	T	T	T	T	A	A	T
E.coli pstS1	C	T	G	T	C	A	T	A	A	A	A	C	T	G	T	C	A	T
E.coli pstS2	C	T	T	A	C	A	T	A	T	A	A	C	T	G	T	C	A	C
E.coli ugpB1	T	T	G	T	C	A	T	C	T	T	T	C	T	G	A	C	A	C
E.coli ugpB2	C	T	A	T	C	T	T	A	C	A	A	A	T	G	T	A	A	C
E.coli ugpB3	A	A	G	T	T	A	T	T	T	T	T	C	T	G	T	A	A	T
Frequencies adjusted by adding 0.03 for zero count
A	0.13	0.06	0.07	0.13	0.07	0.84	0.07	0.58	0.4	0.53	0.27	0.07	0.03	0.03	0.13	0.32	0.88	0.03
T	0.13	0.88	0.07	0.81	0.2	0.07	0.6	0.19	0.27	0.13	0.33	0.13	0.75	0.25	0.81	0.07	0.06	0.03
C	0.71	0.03	0.07	0.03	0.67	0.03	0.2	0.19	0.2	0.27	0.13	0.77	0.19	0.03	0.03	0.58	0.03	0.44
G	0.03	0.03	0.84	0.03	0.07	0.07	0.13	0.03	0.13	0.07	0.27	0.03	0.03	0.69	0.03	0.03	0.03	0.03
Consensus sequence	C	T	G	T	C	A	T	A	A	A	T	C	T	G	T	C	A	T

S.meliloti and E.coli PhoB binding sites were extracted from the following references: (16,18,24,28,32,33–35).

Generation of gusA transcriptional gene fusions to the PCR amplified Pho box containing promoters

To construct the gusA reporter gene fusions to the Pho box containing promoters, each promoter region was PCR amplified using the primers as listed in Supplementary Table 2, and the PCR amplified promoter fragments were digested with appropriate restriction enzymes and cloned into either pFUS1 vector which is a broad host replicable vector containing promoterless gusA (uidA) gene (37) or into a suicide plasmid pTH1360 [modified pVO155 (38) by replacement of gusA coding and upstream sequences with the ones in pFUS1]. The corresponding gene fusion plasmids were verified by sequencing and subsequently introduced into S.meliloti wild-type strains RCR2011 and its derivative RmP559 (RCR2011, PhoB::TnV) strains or RmP110 and RmH852 (Rm1021, phoB) by tri-parental mating using MT616 as the helper strain as described previously (39).

β-Glucuronidase and alkaline phosphatase assays

To determine the expression of the predicted Pho box containing genes or operons in response to Pi limitation, the S.meliloti wild-type strains and its PhoB mutant harbouring the plasmid borne promoter::gusA gene fusion were inoculated in 2 ml LBmc containing 2.5 µg/ml tetracycline and grown overnight aerobically at 30°C to OD600 of ∼1.0. Luria–Bertani (LB) broth was supplemented with 2.5 mM MgSO4 and 2.5 mM CaCl2 (LBmc) (40). A total of 0.5 m of cultures were spun down in a 1.5 ml microcentrifuge tube, washed twice in 1 ml phosphate free MOPS minimal medium (P0 medium) and resuspended in 250 µl of the P0 medium. MOPS-buffered minimal medium contains 40 mM morpholinopropane sulfonic acid/20 mM potassium hydroxide; 20 mM NH4Cl; 2 mM MgSO4; 2 mM CaCl2; 100 mM NaCl; 15 mm filter-sterilized glucose as carbon source and supplied with 0.3 µg/ml biotin and 10 ng/ml CoCl2 (24,41). Ten microliter aliquots of washed cells were subcultured into 5 ml of P0 medium, or MOPS minimal medium supplied with 2 mM KH2PO4 (P2 medium). After 32 h incubation at 30°C, 2 ml cultures were spun down at 10 000 r.p.m. for 1 min and resuspended in 1 M Tris–HCl (pH 8.0) for alkaline phosphatase assays as described by Bardin et al. (24), and 3 ml cultures were left for β-glucuronidase assays according to the protocol described by Reeve et al. (37).

RESULTS AND DISCUSSION

Weight matrix prediction of potential PhoB regulated genes

A weight matrix to identify potential PhoB binding sites was generated from five S.meliloti and ten E.coli PhoB box example sequences of 18 nt length (Table 1). The nucleotide frequency matrix was used to calculate an information-based score for potential binding sites in a scan of the S.meliloti genome. Putative PhoB binding sites were defined by a score of greater than 0.35 and a location between +100 and −500 nt of the translational start codon on the transcribed strand of an annotated gene (see Materials and Methods). One hundred and three putative PhoB binding sites were found and are shown with their downstream annotated genes in Supplementary Table S1. Seven of these promoter regions contained two putative PhoB boxes, so that 96 distinct genes were found. Three out of four genes whose Pho boxes were used for matrix construction were also among those 96 genes. No orthologue of orfA-pta-ackA from S.meliloti strain 104A14 was found in Rm1021 strain.

The threshold score (0.35) used to identify putative PhoB binding sites was derived from the lowest score (that of orfA-pit) among the example sequences. With this threshold, 18 of the top 20 scores were upstream of genes found to be induced by phosphate starvation, in a PhoB-dependent manner, by gusA fusion analysis in S.meliloti (see next section). However, most putative PhoB binding sites with scores above the cut-off level did not show phosphate-dependent regulation of transcription. Possible explanations in addition to false positives are that the matrix method did not include other important features of a PhoB binding site, such as appropriately positioned −10 and −35 promoter elements. It is also possible that some genes with PhoB binding sites require interaction with additional regulatory proteins before the gene can be regulated by phosphate limitation.

Blanco et al. (23) showed that the C-terminal domain of PhoB interacts with a 22 bp region of dsDNA that consists of two direct repeats of 11 bp. Each 11 bp repeat has a conserved 7 bp region (consensus, CTGTCAT) followed by a less conserved 4 bp segment. Our weight matrix is comprised of two conserved 7 bp repeats separated by a single, less conserved 4 bp spacer, and omits the terminal 4 bp segment. However, this terminal segment is not well conserved (23) and will therefore contribute little to the weight matrix score. Furthermore, our weight matrix will reliably identify overlapping PhoB sites provided that they are separated by 4 bp ‘spacers’ and individually have component scores greater than 0.35.

Experimental validation of the predicted Pho regulon members by analysis of transcriptional gene fusions

To directly examine whether the S.meliloti genes identified by the frequency matrix were subject to phosphate-dependent regulation, we generated transcriptional reporter gene fusions to seventy-two of these candidate genes and examined their expression in defined MOPS-buffered minimal medium during growth under Pi-excess (2 mM Pi) and Pi-starvation (no Pi added) conditions (see Materials and Methods). Gene expression in a wild-type phoB+ background was compared with expression in an otherwise isogenic phoB− background (Table 2). Eighteen of the 72 promoter gene fusions were induced upon Pi-starvation in a PhoB-dependent fashion (Table 2). In addition, regardless of the media Pi concentration, gene fusions to smb20427 (putative amino acid ABC transport system), smc02886 and smc02675 (rrna) showed 3-, 2- and 10-fold more expression respectively in the wild-type background relative to the phoB− background.

Table 2

β-Glucuronidase activitiesa from predicted Pho-regulon gusA gene fusionsb

Genec	Wild-type 0 Pi	Wild-type 2 mM Pi	phoB− 0 Pi	phoB− 2 mM Pi	Distance	Score
SMa0041	87.4 ± 1.3	110.3 ± 5.2	70.7 ± 4.6	113.2 ± 4.5	−101, −90	0.36, 0.48
SMa0302	88 ± 4.6	123.6 ± 8.2	134.6 ± 7	187.7 ± 1.2	−21	0.45
SMa0567	139.7 ± 4.8	66 ± 2.6	116.7 ± 5.4	65.9 ± 18.5	−130	0.37
SMa0570	220.7 ± 3.5	155.9 ± 3.3	169.3 ± 33.5	128.4 ± 35.9	−41	0.38
SMa1355	275.2 ± 44.2	231.2 ± 40.2	229.9 ± 21.8	277.4 ± 24.3	−160	0.41
SMa1456	726.2 ± 14.2	763 ± 41.2	804.2 ± 56	858.5 ± 34.6	−30	0.45
SMa1836	139.1 ± 13.6	162.9 ± 9.4	122.1 ± 10.8	146.4 ± 23.8	−285	0.39
SMa2012	101.1 ± 6	100.1 ± 1.4	112.2 ± 14	102.8 ± 23.3	−381	0.39
SMa2025	132 ± 0.7	206.5 ± 21.6	121.8 ± 2.8	207.8 ± 13.6	−22	0.36
SMa2063	37.7 ± 19.5	31.4 ± 17.1	55.2 ± 12.8	75.1 ± 7.9	+349	0.55
SMb20106	592.2 ± 8.7	915 ± 52.5	460.6 ± 23.3	910.4 ± 84.1	−60	0.43
SMb20410	42.6 ± 24.7	48.1 ± 16.2	70.2 ± 13.3	87.5 ± 12.8	−6	0.44
SMb20427	1895.2 ± 78.2	1943.1 ± 78.7	671.8 ± 34.2	606 ± 43	−129	0.37
SMb20483 (crp)	95 ± 45.5	92.7 ± 24.5	92.1 ± 15.3	102.2 ± 12.7	−80, −62	0.36, 0.37
SMb20493	88 ± 14.8	131.7 ± 38.6	156.7 ± 16	216.4 ± 10.6	−41	0.63
SMb20759(phnG)*	1622.6 ± 25.9	136.6 ± 8.5	72.8 ± 4.1	115.4 ± 5.9	−66	0.40
SMb20824	1062.7 ± 54.8	1974.06 ± 229.8	996.45 ± 10.2	1709.57 ± 76.1	−154	0.38
SMb20843*	1112 ± 149	239.7 ± 11.1	179.5 ± 28.5	200.5 ± 21.8	−340	0.38
SMb20876*	2092.4 ± 134	44.5 ± 19.6	76.8 ± 13.7	69.9 ± 7.5	−98	0.43
SMb20935	722.8 ± 29.5	1182.3 ± 67.9	624.6 ± 66.3	1339.9 ± 105.2	−183	0.41
SMb20980	79.9 ± 24.1	41 ± 18.4	101.3 ± 7.4	91.4 ± 12.9	−271	0.39
SMb21144	752 ± 50	777 ± 6.7	582 ± 35.1	624.9 ± 41.9	−441	0.36
SMb21154	478.8 ± 51.4	423.4 ± 15.4	403 ± 17.3	385.5 ± 35.5	−165	0.37
SMb21177 (phoC)*	1777.4 ± 98.3	51.6 ± 5.4	65.4 ± 8.4	60.5 ± 5.3	−85, −63	0.54, 0.48
SMb21192	1231 ± 47	2424.6 ± 122.1	1165.6 ± 104.3	2752.7 ± 88.2	−356	0.42
SMb21210	258.8 ± 14.6	278 ± 6	205.2 ± 7.9	278.3 ± 13.6	−120	0.36
SMb21216	53.7 ± 13.9	78.8 ± 28.7	85.7 ± 18.1	86 ± 15.7	−355	0.37
SMb21222	123.9 ± 16.4	172.3 ± 15.4	160 ± 14.2	194.4 ± 16.1	−352	0.37
SMb21270*	1974.1 32	861.1 ± 4.1	1253.1 ± 8.0	1343.7 ± 29.4	−91, −80	0.39, 0.37
SMb21307	36.4 ± 21.7	48.6 ± 15.7	39.2 ± 7.1	63.5 ± 12.2	−424	0.42
SMb21555	296.8 ± 22.4	343.7 ± 37.4	303.9 ± 2.2	463 ± 24.5	+165	0.39
SMc00009	1829 ± 2.5.6	3616.6 ± 329.7	1631.8 ± 107.7	2613.7 ± 157.8	−302	0.37
SMc00027	61.7 ± 24.9	48.6 ± 15.9	57.8 ± 14.1	97.9 ± 8.6	−368	0.39
SMc00042	604.5 ± 3.5	1059.1 ± 76.1	631 ± 38.4	1030.7 ± 40	−62	0.43
SMc00161	619.6 ± 37.8	909.3 ± 98.8	347.6 ± 16	522.1 ± 10.6	−228, −217	0.38, 0.59
SMc00171*	1530.9 ± 19.3	124.1 ± 10.6	94 ± 1.4	128.6 ± 3.4	−52	0.47
SMc00485	5771.6 ± 682.6	5146.4 ± 238.4	3506.4 ± 90.4	3809.5 ± 254.4	−196	0.39
SMc00618 (ppk1)*	6034 ± 272	366.8 ± 29.6	266.7 ± 33.5	255.2 ± 9.7	−47	0.46
SMc00801**	673.2 ± 92.9	1824.7 ± 163	1352.5 ± 120	1719 ± 153	−57	0.87
SMc00819 (katA)*	1415.4 ± 91.2	100 ± 12.3	111.8 ± 16.5	117.2 ± 14.6	−50	0.60
SMc00978	161.6 ± 5.7	117.1 ± 7.7	158.5 ± 12.9	106.9 ± 10.1	−48	0.59
SMc00982	61.2 ± 2.3	44.6 ± 6.4	71.5 ± 25.3	65.8 ± 5.1	−359	0.39
SMc01605*	1242.6 ± 4.4	270.2 ± 0.7	95.4 ± 3.6	88.6 ± 0.4	−58	0.52
SMc01635	80.5 ± 63.6	65.1 ± 16.2	69.4 ± 7.8	78.6 ± 5.3	−207	0.36
SMc01723*	894.3 ± 3.1	314.8 ± 1.2	190.0 ± 3.6	308.2 ± 7.3	−64	0.41
SMc01849	188.5 ± 10.9	171.9 ± 4.6	175.1 ± 39.7	188.2 ± 22.7	−321	0.42
SMc01852 (pfk)*	734.3 ± 126	288.9 ± 26.5	204.6 ± 9	217.6 ± 23.7	−105	0.49
SMc01907*	955.9 ± 24.3	186.8 ± 17.5	85.9 ± 2.8	125.2 ± 4.4	−82	0.72
SMc01934	406.5 ± 22.4	706.4 ± 44.4	440.7 ± 21.7	607.6 ± 26.9	−283	0.39
SMc01952	101.7 ± 10.2	100.6 ± 13.4	123.9 ± 10.9	118.6 ± 12.8	−299	0.37
SMc02146 (pstS)*	2658.6 ± 193	100.7 ± 23.4	81.3 ± 19.8	78.7 ± 12.4	−115, −104	0.72, 0.38
SMc02315	54 ± 5.6	49.5 ± 2.6	76.4 ± 16.8	56.5 ± 8.9	−134	0.44
SMc02601**	282.4 ± 19	818.9 ± 47.3	1036.6 ± 57	1426 ± 37.9	−99	0.50
SMc02634*	2315.4 ± 146	111.4 ± 9.8	90.4 ± 0.3	114.9 ± 4.2	−77	0.56
SMc02675	342.7 ± 9.4	308.9 ± 30.5	30.7 ± 0.9	44.6 ± 1.9	−410	0.78
SMc02689	977.9 ± 54	677.5 ± 116.7	1204.5 ± 86.4	717.7 ± 4.3	−106	0.5
SMc02862 (orfA-pit)**	46.2 ± 0.18	111.3 ± 3.9	99.8 ± 6.6	123.8 ± 6.4	−83	0.37
SMc02863 (recF)	42.3 ± 40.8	36.4 ± 12	71.8 ± 5	73.6 ± 12.5	−195	0.45
SMc02886	618.4 ± 12.8	731.9 ± 45.3	290.6 ± 24.6	347.7 ± 13.3	−90	0.51
SMc02976	398.6 ± 19.2	393 ± 47	233.3 ± 19.3	307.5 ± 15.3	−73	0.36
SMc03124*	4081.4 ± 13.2	130.5 ± 1.0	126.5 ± 2.6	109.7 ± 2.6	−93	0.44
SMc03174	137.6 ± 9	196.3 ± 14.7	140.4 ± 15.1	190.7 ± 29.1	−423	0.41
SMc03243(phoA1)*	1761.4 ± 159	108.4 ± 8.3	76 ± 5.3	109.6 ± 5.1	−64, −97	0.41, 0.37
SMc03823	153.3 ± 17.8	587.6 ± 26.5	218.9 ± 8.1	728.8 ± 45.7	−206	0.46
SMc03844	260.57 ± 1.2	438.1 ± 51.2	397.9 ± 43.3	557.4 ± 23.9	−33	0.42
SMc03975	367.3 ± 25.1	544.2 ± 67.3	704.6 ± 10.8	1085 ± 29.5	+22	0.56
SMc04053*	1048 ± 80.1	149.2 ± 18	125.6 ± 29.4	164.6 ± 17.7	−142	0.39
SMc04144	235.3 ± 8	247.6 ± 24.8	173.5 ± 25	220.7 ± 13.8	−46	0.39
SMc04213	161.3 ± 6.7	170 ± 1.4	185.8 ± 11.2	168.3 ± 20.1	−169	0.36
SMc04317 (afuA)*	3284.3 ± 299	50.6 ± 23.7	76.8 ± 9.3	59.7 ± 15.7	−51	0.58
SMc04458	26.4 ± 9.1	26.7 ± 1.4	52.4 ± 6.7	54.3 ± 7.9	−230	0.37
SMc04478	1498.2 ± 100.6	3333.8 ± 118.6	1632 ± 62.7	3952.4 ± 312.8	−52	0.57

aGusA transcriptional gene fusion and expression demonstrating altered gene expression in response to Pi conditions. β-glucuronidase activities are expressed as Miller units and corresponding alkaline Phosphatase activities (data not shown) were determined as described in Materials and Methods. Each value corresponds to the mean of triplicate assays with (±) standard error. Background β-glucuronidase values from negative control (S.meliloti strains with no promoter fusions) were in the range of 42–55 Miller units. SMc02862 (orfA-pit) was a lacZ fusion.

bGene fusions integrated into the genome are shown in grey, others were generated in the plasmid pFUS1.

c*PhoB-dependent up-regulated genes, **PhoB-dependent down-regulated genes.

The Pho boxes from genes in ‘bold’ were also part of the weight matrix (Table 1).

Three reporter gene fusions were found to be repressed upon Pi-starvation in a PhoB-dependent manner. These were smc00801 (transmembrane protein of unknown function), smc02601 (nadABC) and smc02862 (orfA-pit). In the wild-type background these fusions were expressed at higher levels in media containing 2 mM Pi than in Pi-starved cells. Also, in the phoB mutant background the expression level was elevated and did not alter with media Pi. We have previously reported that expression of the low-affinity Pi-transport system encoded by the orfA-pit genes is repressed by PhoB (28). The repression of smc02601- smc02602- smc02603 (nadABC) expression suggests that the observed down regulation of NAD+ synthesis in S.meliloti possibly corresponds to a slight down regulation of smc00161 expression that appears to occur upon Pi-limited growth (Table 2). The smc00161 is annotated to encode an NH3-dependent NAD+ synthetase and the promoter region of this genes was predicted to carry a promoter Pho box (Supplementary Table S1).

Comparison of Pho box predictions with DNA microarray data

Employing DNA microarrays, Krol and Becker (31) identified 98 genes (some of which were in operons) that were more than 3-fold induced in a phoB-dependent manner upon Pi limitation. An additional 50 genes showed a strong increase in expression under phosphate limitation in a partially phoB-dependent or phoB-independent manner. Krol and Becker (31) also identified potential Pho-box sequences with ≤2 mismatches from the Pho-box consensus sequence TG(A/T)CA (C/A)-NNNN-C(C/T)(G/T)TCA(C/T) defined by Summers et al. (16). Of the 19 Pho-box promoters identified by Krol and Becker (31), 14 were also identified with our weight matrix (Table 3) and data from our gene fusion experiments (Table 2) revealed that 13 of these 14 genes were regulated by media Pi in a PhoB dependent manner (Table 2). A reporter fusion to the remaining gene, sma0612 has yet to be examined. Of the five Pho-boxes not identified by our weight matrix, four were unusual (sma1809, sma1822, smc00170 (sinR) and smc00429) as they contained 3 or 5 nt in the region flanked by the 7 nt direct repeats instead of the 4 (see Table 1). The Pho-box upstream of the remaining gene, sma0045, lies on the opposite strand to sma0045 and thus would not be included in our predictions. However reporter gene fusions to these genes should be analyzed as the microarray experiments suggested these genes were induced in a phoB-dependent manner (31).

Table 3

Pho regulon members in S.meliloti as identified by this study and the microarray analysis of Krol and Becker (31).

No.	Gene	Study	Pho box motif	dDist	Score	Description	*Genomes
1	SMb20759	Known	ATGTCACAAGCCTGTCAT	−66	0.62	phnG, carbon-phosphorus lyase component	*4
2	SMb21177	Known	CTGTTACAGAACCTACAC	−85	0.54	phoC, phosphate ABC transporter ATP binding	*6
		Known	CTGACACTGCGCTTTCAT	−63	0.48
3	SMc02862	Known	CTGTGGGAAAGCCGTTTT	−83	0.37	orfA-pit, inorganic phosphate transporter	*4
4	SMa0612	Overlapa	TTGTCAGGGGCCTTTCAT	−18	0.36	FixN3, cytochrome c oxidase subunit 1
5	Smc00171	Overlapa,b	ATGTCATCTTCCTGAAAC	−52	0.47	Conserved hypothetical protein	*5
6	Smc01605	Overlapa,b	CTGTCACATAGGCTTCAT	−58	0.52	Putative ABC transporter periplasmic binding	*6
7	Smc01907	Overlapa,b	CTGTCATCCAGCTGTTAC	−82	0.72	phoA2, putative alkaline phosphatase	* 3
8	Smc02146	Overlapa,b	GTTTCACCGAACTGACAT	−104	0.38	pstS, phosphate-binding periplasmic protein	*11
		Overlapa,b	CTGTCATAAATGTTTCAC	−115	0.72
9	SMc02174	Overlapa	TTGTGAACGATCTGTCGC	−76	0.36	Hypothetical transmembrane protein	*1
10	Smc02634	Overlapa,b	TTGTCGCAGAGCTGTCAT	−77	0.56	phoA3, putative alkaline phosphatase	*2
11	Smc03124	Overlapa,b	CTGTCACGAAAGTTTCAC	−93	0.44	Putative ABC transporter periplasmic binding	*5
12	SMc03243	Overlapa,b	CTGTAAAGCCGCTGACAT	−64	0.41	phoA1, putative alkaline phosphatase	*5
		Overlapa,b	CTGTCGCCAAACCGTCCG	−97	0.37
13	SMc03961	Overlapa	TTGTCACAATCCTATCCT	−40	0.36	sqdB, sulfolipid biosynthesis protein
14	SMc04317	Overlapa,b	CTGTCACATCCCTGTAAA	−51	0.58	afuA, iron-binding periplasmic protein	*3
15	SMb20843	Gene fusionb,c	ATTTTAAATCGCCGTCAC	−340	0.38	algI, involved in acetylating surface saccharide
16	SMb21270	Gene fusionb,c	CTGGCATCTTGATGTAAT	−91	0.38	Putative transcriptional regulator
17	SMc00618	Gene fusionb	CTGTCACAGTTCCGTCGT	−47	0.46	ppk, putative polyphosphate kinase protein	*10
18	SMc00801	Gene fusionb	TTGTCATAAAACTGTCAC	−57	0.87	Hypothetical transmembrane protein
19	SMc01723	Gene fusionb	CCGTCACTACCCTGACAT	−64	0.41	Hypothetical transmembrane global homology
20	SMc01852	Gene fusionb	CTGTCATTTAATTTCCAT	−105	0.49	pfk, pyrophosphate-fructose 6-Pi 1-phosphotransferase
21	SMc02601	Gene fusionb	CTGTCACAAAACTTTCTA	−99	0.5	nadA, putative quinolinate synthetase A protein
22	SMc04053	Gene fusionb	TTGACATAAAATAGTAAT	−142	0.39	Hypothetical protein
23	SMb20876	Gene fusionb,c	AATTCGTCAATCTGTCAC	−98	0.43	Hypothetical protein	*1
24	SMc00819	Gene fusionb,c	CTGTCGTTCAGCCGTCAC	−50	0.6	katA, catalase hydroperoxidase HPII	*4
25	SMa1961	Microarrayc	CTGACAAACCGCCTTAGC	−128	0.37	Putative polyhydroxyalkanoate depolymerase
26	SMb20037	Microarrayc	ATGACGCTCACCCGTCAT	−389	0.39	aroE2, putative shikimate 5-dehydrogenase
27	SMb21317	Microarrayc	CTGTCATGCACCTGCATC	−385	0.39	expG, regulator of exopolysaccharide II synthesis	*1
28	SMc00620	Microarrayc	CTTTCACCTCGCTGTAAG	−70	0.41	Hypothetical signal peptide protein	* 2
29	SMc00772	Microarrayc	AGGTCGTCAAGGCGTCAT	−103	0.36	potH, probable putrescine transport permease	*1
30	SMc02490	Microarrayc	TTGTCATTGTTCGATCAT	−41	0.38	Hypothetical protein
31	SMc03994	Microarrayc	CTGTCAGACAGGTTCAAT	−47	0.37	suhB, putative inositol monophosphatase
32	SMc04239	Microarrayc	CTGTCGTGAAGGCTTCAT	−98	0.4	Hypothetical protein
33	SMc04280	Microarrayc	CTTTTGTAAAGATTTCAT	−81	0.37	Hypothetical signal peptide protein
34	SMc01848	Microarrayc	TCGTCATCAAAGTGTAGC	−47	0.41	Hypothetical protein (btaA-like)	* 2

aPho-box sequences detected in this study and also by Krol and Becker (31).

bPho-box sequences detected in this study and gene fusions tested for PhoB-dependent regulation.

cPho-box sequences detected only by our matrix and PhoB-dependent regulation shown by microarray data [Krol and Becker (31)]. No gene fusions were tested for #25–34.

d‘Dist’ indicates the relative distance from the annotated translational start sites.

*Indicates the conserved Pho regulon homologs having predicted Pho box in other scanned genomes (see Tables 5–8).

In addition to the 13 Pho regulon members predicted both here and by Krol and Becker (31), both the weight matrix data and data from reporter fusion assays identified an additional 10 genes whose expression was PhoB-regulated in response to Pi limitation (Tables 2 and 3). With the exceptions of smb20843 (algI), smc00618(ppk), smc02601(nadA) and smc00801(hypothetical, global homology), these genes also showed PhoB-dependent transcription in microarray studies. The failure to detect repression of smc02601(nadA) and smc00801expression in microarray experiments is not surprising as the microarray experiments also failed to detect orfA-pit repression and this operon is known to be repressed by PhoB (25). The failure to detect induction of smb20843 (algI), smc00618 (ppk), upon Pi-starvation is more surprising as these genes appear to be highly regulated in the gene fusion experiments. Moreover expression of ppk is known to be Pi-starvation induced in many organisms. The differences between the microarray and gene fusion data could result from several factors including differences in experimental growth conditions as in microarray experiments cells were grown in 100 µM Pi source as the Pi-limitation condition. Alternatively, it is possible that the particular probes employed for ppk and smb20843 yielded low signals. Through our weight matrix scan, Pho-box sequences were also found upstream of three more genes, sma2410 (rhbF), smc01296 (rpsN) and smc01820 (putative N-carbamyl-L-amino acid aminohydrolase) (Supplementary Table S1). Promoter fusions to these three genes have not been tested yet. These genes however are shown to be repressed in a PhoB-independent manner in microarray studies (31). Further studies are required to analyze the regulation of these genes and the nature of their associated Pho-box sequences.

We note that in the case of orfA-pit, the Pho-box identified by Krol and Becker (31) lay on the opposite strand to the orfA-pit genes and is different from that identified by Bardin et al. (28). Since orfA-pit expression is negatively regulated by PhoB, it is of interest to determine the actual PhoB binding site as little is known regarding how PhoB represses transcription. In summary, of 96 genes with upstream Pho-boxes predicted by the frequency matrix genome analysis, 34 appear to be Pi and PhoB regulated as revealed from gene fusion and microarray analysis data (Table 3).

Analysis of predicted Pho regulon members across proteobacterial genomes

It is reasonable to assume that at least part of the physiological response to Pi-limitation will be conserved. As the Pho-box sequence identified by the PhoB proteins of different organisms appears to be conserved (14,16,24,28,42–44), we used the Pho-box frequency matrix described above (Table 1) to search the genomes of twelve gram negative bacteria (Table 4) for PhoB-binding sites using the same criteria as employed for S.meliloti. Genes that lay downstream of a predicted Pho-box with scores greater than 0.35 were further examined. We identified genes, such as pstSCAB, phoA, ugpA, phn and ppk that are known to be associated with phosphate metabolism (Tables 5–7). The pstS gene encodes the Pi-binding protein of the high affinity PstSCAB transport system (18,27) and expression of this system in E.coli, S.meliloti and Pseudomonas aeruginosa is known to be highly induced under Pi-limiting conditions and is PhoB dependent (27). In a number of organisms, such as Caulobacter crescentus, the pstS gene transcript is separate from the pstCAB-phoUB transcript and in these cases predicted Pho-boxes are also located upstream of the pstC gene (see Table 5).

Table 4

List of proteobacterial genomes scanned for the presence of Pho-boxes

No.	Bacterial genome	Accession no.	Number of predicted Pho regulons
1	Acinetobacter sp. ADP1	NC_005966	56
2	A.tumefaciens C58 Chromosome circular	NC_003304	51
	A.tumefaciens C58 Chromosome linear	NC_003305	35
	A.tumefaciens C58 Plasmid AT	NC_003306	7
	A.tumefaciens C58 Plasmid Ti	NC_003308	6
3	B.japonicum USDA 110	NC_004463	87
4	B.melitensis 16 M chromosome I	NC_003317	25
	B.melitensis 16 M chromosome II	NC_003318	33
5	B.suis 1330 chromosome I	NC_004310	27
	B.suis 1330 chromosome II	NC_004311	32
6	C.crescentus CB15	NC_002696	62
7	E.coli K12	NC_000913	107
8	E.coli O157:H7 Chromosome	NC_002695	96
	E.coli O157:H7 Plasmid pO157	NC_002128	5
	E.coli O157:H7 Plasmid pOSAK1	NC_002127	2
9	M.loti MAFF303099 for chromosome	NC_002678	71
	M.loti MAFF303099 plasmid pMLa	NC_002679	16
	M.loti MAFF303099 plasmid pMLb	NC_002682	7
10	P.aeruginosa PAO1	NC_002516	73
11	P.putida KT2440	NC_002947	71
12	S.meliloti 1021 chromosome	NC_003047	56
	S.meliloti 1021 plasmid pSymA	NC_003037	16
	S.meliloti 1021 plasmid pSymB	NC_003078	24
13	Rhizobium sp. NGR234 plasmid pNGR234a	NC_000914	17
	Rhizobium sp. NGR234 megaplasmid 2 contig 1	AY316747	4
	Rhizobium sp. NGR234 megaplasmid 2 contig 2	AY316746	3

Table 5

Predicted Pho-regulon orthologue groups related to Pi metabolism

Group	Gene	Pho box motif	Dist	Score	Genome source	Description-predicted function
A	Phosphate transport (high affinity)
1	SMc02146	CTGTCATAAATGTTTCAC	−115	0.72	S.meliloti	pstS, putative periplasmic phosphate-binding protein
		GTTTCACCGAACTGACAT	−104	0.38
2	ACIAD0279	CTGTCATTAAAGTTTCAT	−64	0.71	Acinetobacter sp.ADP1	phoU, transcriptional repressor for high affinity phosphate uptake
3	ACIAD1212	GCGTCATTAATTTGTCAC	−98	0.38	Acinetobactersp. ADP1	pstS, putative ABC phosphate transporter (periplasmic-binding)
		TTGTCACTGAATTGTCAT	−87	0.54
4	Atu0421	TTGACATTTCCCATTCAT	−151	0.37	A.tumefaciens	pstC, ABC transporter, membrane spanning protein
5	Atu0420	TTGTCACAAATCTTTCGT	−117	0.54	A.tumefaciens	pstS, ABC transporter, substrate binding protein [phosphate]
		CTTTCGTCAAAGTGACAT	−106	0.36
6	blr109	CTGTCATCCGACTGTCAC	−88	0.71	B.japonicum	pstS, ABC transporter phosphate-binding protein
		CTGTCACGAAACCTTCGT	−77	0.48
7	BMEI1989	GTGTCATATAAGTGTAAT	−76	0.56	B.melitensis	pstS, putative periplasmic phosphate-binding protein
		CTGTAATATTCGTGTCAT	−87	0.53
		TTGCAACAAACCTGTAAT	−98	0.37
8	BR2138	TTGCAACAAACCTGTAAT	−89	0.36	B.suis	pstS, phosphate ABC transporter, phosphate-binding protein
		CTGTAATATTAGTGTCAT	−78	0.56
		GTGTCATATGAGTGTAAT	−67	0.41
9	CC0290	TTGTCGTCAAACTGTCAT	−104	0.68	C.crescentus	pstCAB-phoUB, phosphate ABC transporter, permease protein
		CTGTCATGTAATTGTCGC	−93	0.48
10	CC1515	CTGTCGTCAAACTGTGAC	−62	0.57	C.crescentus	pstS, putative ABC transporter, periplasmic phosphate-binding
		CTGCCTTAAAACTGTCGT	−73	0.38
11	b3728	CTGTCACCTGTTTGTCCT	−56	0.36	E.coli K12	pstS, high-affinity phosphate transport phosphate-binding protein
		CTTACATATAACTGTCAC	−67	0.62
		CTGTCATATTCCTTACAT	−78	0.64
		CTGTCATAAAACTGTCAT	−89	0.97
12	ECs4664	CTGTCACCTGTTTGTCCT	−56	0.36	E.coli O157:H7	pstS, periplasmic phosphate-binding protein
		CTTACATATAACTGTCAC	−67	0.63
		CTGTCACATTCCTTACAT	−78	0.62
		GTGTCATCAAACTGTCAC	−89	0.67
		CTTTCCTTTGGGTGTCAT	−100	0.37
13	mll3723	CTGTCATGCGACTGTAAT	−79	0.58	M.loti	pstS, periplasmic phosphate-binding protein, (PBP)
		CTGACACGAAACTGTCAT	−90	0.65
14	PP5328	TTGTAATGTTTTTGTCAC	−302	0.38	P.putida	pstC, phosphate ABC transporter, permease protein
15	PP5329	TTGTCACAATGAAGTCAT	−75	0.41	P.putida	pstS, ABC transporter, periplasmic phosphate-binding protein
		CTTTCATCCAATTGTCAC	−86	0.56
16	PP2656	CTGCCATTCAATAGTCAC	−42	0.37	P.putida	pstS, phosphate ABC transporter, periplasmic phosphate-binding
		TAGTCACAAAGTTGTAAC	−31	0.49
		TTGTAACACAGCTGATGC	−20	0.36
17	PA5368	CTGTCATCTGTCTGTCAT	−277	0.74	P.aeruginosa	pstC, membrane protein component of ABC
		GCGTCATGTTGCTGTCAT	−288	0.39		phosphate transporter
18	PA5369	CTTTCATAGAGTCTTCAT	−60	0.53	P.aeruginosa	pstS-like, hypothetical protein
		CTGTCATATTCCTTTCAT	−71	0.78
		ATTTCATCCAACTGTCAT	−82	0.56
B	Phosphate transport (low affinity)
1	SMc02862	CTGTGGGAAAGCCGTTTT	−83	0.37	S.meliloti	orfA-pit, phosphate transport transmembrane protein
2	ACIAD1047	TTGTCATATAATTGTCAT	−51	0.73	Acinetobacter sp. ADP1	pit, phosphate transporter
3	bll3022	TTGTCATCCAGCCTTCAA	−49	0.53	B.japonicum	pit-like, low-affinity inorganic phosphate transporter
4	mll3637	TCGTCATACAGGGTTCAT	−56	0.36	M.loti	pit, phosphate transporter
5	PP1373	CTGTCATCTGCCTGTTAC	−67	0.56	P.putida	Low-affinity inorganic phosphate transporter
C	Phosphonate/Phosphate metabolism
1	SMb21177	CTGACACTGCGCTTTCAT	−63	0.54	S.meliloti	phoC, phosphate uptake ABC transporter ATP-binding protein
		CTGTTACAGAACCTACAC	−85	0.48
2	SMb20759	ATGTCACAAGCCTGTCAT	−66	0.4	S.meliloti	phnG, putative C-P (carbon-phosphorus lyase component protein
3	ACIAD0719	TTGATATCAAGCTTCCAT	−71	0.38	Acinetobacter sp. ADP1	phnA, putative alkylphosphonate uptake protein (PhnA)
4	Atu0181	ATGTCACACGCTTGTCAT	−67	0.47	A.tumefaciens	phnG, hypothetical protein
5	Atu0174	CTGACACTCCCGCTTCAC	−48	0.36	A.tumefaciens	phnC, ABC transporter, nucleotide binding/
		TCTTCATCAAACTGACAC	−59	0.37		ATPase [phosphonate]
		ATGTAATATTTTCTTCAT	−70	0.36
6	Atu6108	CTGTCAGAACACCCTGAT	−81	0.37	A.tumefaciens	phnA, alkylphosphonate uptake protein
7	blr7947	CTGTCATCGCTGCGTCAT	−99	0.62	B.japonicum	phoC, phosphonate metabolism protein
8	blr1221	TTGTCACGAAACAGTCAT	−12	0.47	B.japonicum	phnG, phosphonate metabolism protein
9	bll7947	ATGTCACACAAGTGTCAA	−44	0.52	B.japonicum	phoC, phosphonates transport system nucleotide binding/ATPase
10	CC0361	CCGTCACAAATCCGTTGC	−68	0.36	C.crescentus	Phosphonates ABC transporter, ATP-binding protein
11	ECs5088	CTGTTAGTCACTTTTAAT	−60	0.43	E.coli O157:H7	phnC, ATP-binding component of phosphonate transport
12	b4106	CTGTTAGTCACTTTTAAT	−60	0.42	E.coli K12	phnC, ATP-binding component of phosphonate transport
13	mll9156	TAGTCATCTCACTGTCAT	−315	0.56	M.loti	phnM homolog, phosphonate metabolism protein
14	mlr3342	CTGTCATCCACCCGTCAT	−80	0.73	M.loti	phnG, similar to phnG gene product (phosphonate metabolism)
15	PP2209	CTGACTTATAGCTGGGAT	−85	0.37	P.putida	phnW, 2-aminoethylphosphonate:pyruvate aminotransferase
16	PA3384	CTGTCATCGTCACTTCAC	−84	0.44	P.aeruginosa	phnC, ATP-binding component of ABC phosphonate transporter
		TTGTCACTCGACTGTCAT	−95	0.59
D	Pho response regulator
1	ACIAD3557	TTGCAATGATACTGTCAT	−74	0.37	Acinetobacter sp. ADP1	phoB-phoR, positive response regulator for the pho regulon PhoR
		TTGTAATAAGCCTGTCAT	−47	0.49	Acinetobacter sp. ADP1
2	Atu0419	ATGATGAATCTCTGTCAT	−58	0.36	A.tumefaciens	phoR, two component sensor kinase
		CTGTCATGAAGCTGGCCT	−47	0.38
3	blr1090	CTGTAATCTTGCTGACGT	−179	0.37	B.japonicum	phoR, phosphate regulon, two-component sensor histidine kinase
4	BMEI1624	CTGTCTTTGAACTGTCAC	−219	0.68	B.melitensis	phoR, phosphate regulon sensor protein
5	BR0298	CTGTCTTTGAACTGTCAC	−137	0.68	B.suis	phoR, putative sensor histidine kinase
6	b0399	TTTTCATAAATCTGTCAT	−64	0.41	E.coli K12	phoB, positive response regulator for pho regulon, sensor is PhoR
		CTGTCATAAATCTGACGC	−53	0.83
7	ECs0449	TTTTCATAAATCTGTCAT	−64	0.72	E.coli O157:H7	phoB-phoR operon, positive response regulator for pho regulon
		CTGTCATAAATCTGACGC	−53	0.65
8	PP5320	CTGTCACACAGCTGCAAT	−15	0.68	P.putida	phoB-phoR, DNA-binding response regulator
		CTGCAATAATTCCGTTAT	−4	0.39
9	PA5360	GTGTCACATACCTGACAC	−50	0.54	P.aeruginosa	phoB-phoR, two-component response regulator PhoB
		CTGACACAATTTCGTTAT	−39	0.42
E	Glycerol 3-phosphate transport
1	ACIAD1317	CAGTCATTGAATCTTCAT	−168	0.42	Acinetobacter sp. ADP1	gpsA, glycerol-3-phosphate dehydrogenase, biosynthetic
2	Atu5058	CTGTCATCAAAACGTCGC	−75	0.47	A.tumefaciens	ugpB, ABC transporter, substrate binding protein
		TTGCTAGACAGCCGTCAT	−29	0.37
3	Atu0305	GTGTAATAAATCTGACAC	−76	0.44	A.tumefaciens	ugpA, ABC transporter, substrate binding protein
		CTGACACGGAACTGTCAA	−87	0.47
		CTGTCAAAGAGGCGTCAT	−98	0.63
4	bll0733	CAGTCATGTGAACGTCAT	−55	0.36	B.japonicum	ABC transporter glycerol-3-phosphate-binding protein
5	blr2436	CCTTCGCCTCTCTTTCAT	−76	0.37	B.japonicum	glpD, glycerol-3-phosphate dehydrogenase
		CTTTCATCTTGCTTTCGC	−65	0.39
6	b3453	AAGTTATTTTTCTGTAAT	−75	0.37	E.coli K12	ugpB, sn-glycerol 3-phosphate transport periplasmic binding protein
		CTATCTTACAAATGTAAC	−97	0.39
		TTGTCATCTTTCTGACAC	−119	0.41
7	ECs4299	AAGTTATTTTTCTGTAAT	−75	0.37	E.coli O157:H7	ugpB, sn-glycerol 3-phosphate transport periplasmic binding protein
		CTATCTTACAAATGTAAC	−97	0.39
		CTGACACCTTACTATCTT	−108	0.41
		CCGTCACCGCCTTGTCAT	−130	0.4
8	mll3503	CTGTCACATACCTTCTCT	−61	0.37	M.loti	ugpB, sn-glycerol 3-phosphate transport system; periplasmic binding
F	Phosphatases
1	SMc03243	CTGTAAAGCCGCTGACAT	−64	0.41	S.meliloti	phoA, putative alkaline phosphatase
		CTGTCGCCAAACCGTCCG	−97	0.37
2	Smc01907	CTGTCATCCAGCTGTTAC	−82	0.72	S.meliloti	Hypothetical transmembrane protein
3	Smc02634	TTGTCGCAGAGCTGTCAT	−77	0.56	S.meliloti	Hypothetical protein, phosphatase
4	Atu1263	ATGTCATGCCACTGTCAC	−51	0.59	A.tumefaciens	Conserved hypothetical protein
5	BMEII0655	CCGTCATTCCTGTGTAAT	−53	0.38	B.melitensis	phoA, alkaline phosphatase
6	BR1200	CTGTCACACGCCTGCAAT	−80	0.44	B.suis	phoA, alkaline phosphatase
7	BRA0616	CTGTCATCGTTTCTTCGT	−75	0.39	B.suis	Hypothetical protein, phoA like gene
		CCGTCATTCCTGTGTAAT	−53	0.37
8	b0383	CTGTCATAAAGTTGTCAC	−63	0.88	E.coli K12	phoA, alkaline phosphatase
9	ECs0433	CTTTTCAACAGCTGTCAT	−5	0.39	E.coli O157:H7	phoA, alkaline phosphatase
		CTGTCATAAAGTTGTCAC	+6	0.86
10	ECs4053	TTGTCAGGTATCTGTATC	−361	0.37	E.coli O157:H7	phoA, putative alkaline phosphatase I
11	mll2704	GTGTCACATGGCCGTCAC	−45	0.46	M.loti	Probable acid phosphatase
12	mll4115	CTGTTATCAAAACTTCAT	−73	0.52	M.loti	phoA, secreted alkaline phosphatase
13	PP1044	CTGCCATCAAACTGTAAT	−45	0.66	P.putida	(uxpA) lipoprotein UxpA
		CTGTCGGATTTCTGCCAT	−56	0.41
14	PA3296	TTGTCACAAGCCCGTCAT	−82	0.53	P.aeruginosa	phoA, alkaline phosphatase
15	PA2635	CTGTCATCGTCCCGTCGC	−53	0.39	P.aeruginosa	Hypothetical protein
G	Membrane lipids
1	SMc01848	TCGTCATCAAAGTGTAGC	−47	0.41	S.meliloti	Hypothetical protein (btaA-like)
2	Atu2119	CTGTCATCAAACTGTAGC	−44	0.58	A.tumefaciens	Hypothetical protein (btaA-like)
3	mlr1574	CTGTCACCGGCCTGTCAT	+1	0.55	M.loti	Hypothetical protein (btaA-like)
H	Exopolysaccharide
1	SMb21317	CTGTCATGCACCTGCATC	−385	0.39	S.meliloti	expG, activator of exopolysaccharide II synthesis
2	SMc02851	CTTTCAAAGAGCCGCCAC	−158	0.37	S.meliloti	Putative transcription regulator protein
3	bll5036	TTGTGGCACAGGCTTAAT	−145	0.36	B.japonicum	mucS, transcriptional regulatory protein

‘Dist’ indicates the relative distance from the annotated translational start sites.

It was striking that multiple18 bp Pho-box sequences were predicted upstream of the pst genes in all of the genomes examined (Table 5). Multiple Pho-boxes consisted of overlapping 7 bp direct repeats separated by 4 bp spacers. The frequency matrix detected consecutive 18 bp elements and adding a terminal 4 bp spacer formed consecutive 22 bp PhoB binding sites as defined by Blanco et al. (23). The two 11 bp direct repeat sequences bind the PhoB monomers head to tail (23). The pstS promoters from E.coli K12 and O157:H7 are predicted to contain five and six of these 11 bp direct repeats, respectively. The large number of Pho-boxes in all of the pstS promoter regions presumably reflects the importance of the PstSCAB high affinity transport system in the uptake of Pi under Pi-limiting conditions. Other genes associated with phosphate metabolism for which multiple Pho-boxes sequences were detected included alkaline phosphatase-like proteins (phoA), genes involved in phosphonate uptake and metabolism (phn), in glycerol-3-phosphate uptake (ugp and glp), the regulatory genes phoB and phoR (Table 5) and genes encoding polyphosphate kinase (Table 6).

Table 6

Conserved ppk Pho box motifs in various proteobacteria

No.	Gene	Source/Pho box motif	Gene annotation/Distance from ATGa	Score
1	ACIAD1782	Acinetobacter sp. ADP1	ppk polyphosphate kinase
		TTGTAACTCGTTTGTAAC	−107	0.36
2	Atu1144	A.tumefaciens	ppk polyphosphate kinase
		CTGTCACAGTTCCGTCGT	−84	0.46
		CCGTCGTCAAACTGTTAT	−73	0.39
3	bll2813	B.japonicum	ppk2 hypothetical protein
		CTGCCATCGCCGTGTCAA	−365	0.36
4	bll4122	B.japonicum	ppk polyphosphate kinase
		ATGTCATCGAAACGTCAT	−59	0.48
5	BMEI1205	B.melitensis	ppk polyphosphate kinase,
		TTGTCATATGACAGCCAT	+10	0.37
		TTGCTATCAAATTGTCAT	−1	0.39
6	BR0748	B.suis	ppk polyphosphate kinase
		TTGTCATATGACAGCCAT	−101	0.37
		TTGCTATCAAATTGTCAT	−112	0.4
7	CC1710	C.crescentus	ppk polyphosphate kinase,
		CTGTCTTCACCGCGTCAT	+106	0.37
8	mlr8161	M.loti	ppk polyphosphate kinase
		ATGTTAGAGCACCGCCAT	+18	0.36
9	mlr8387	M.loti	ppk2 hypothetical protein
		CTGCAATAAAACCGTCAC	+74	0.51
10	PA5243	P.aeruginosa	ppk delta-aminolevulinic acid dehydratase
		TTGTTGCCAATCCGTCAT	−56	0.39
11	PA2428	P.aeruginosa	ppk2 hypothetical protein
		CCGTCACCAAACCGTCAT	−52	0.53
		TTTTCATCTAACCGTCAC	−63	0.51
12	PP5216	P.putida	ppk exopolyphosphatase
		CTGTCATATGGCCGTCAT	−119	0.73
13	PP5217	P.putida	ppk polyphosphate kinase
		GTGTAACACGGGCGTCAT	−122	0.36
14	PP1752	P.putida	ppk2 hypothetical protein
		CTGTGGGAGCGCGTTCAT	−70	0.37
15	SMc00618	S.meliloti	ppk putative polyphosphate kinase
		CTGTCACAGTTCCGTCGT	−47	0.46

aIndicates the relative distance from the annotated translational start sites.

In addition to the previously reported Pho-box in the orfA-pit promoter region of S.meliloti, Pho-box sequences were also detected in the promoter region of the orfA-pit orthologues in the α-proteobacteria, Bradyrhizobium japonicum and Mesorhizobium loti and the γ-proteobacteria Pseudomonas putida and Acinetobacter sp (Table 5). Bardin et al. (28) showed that the expression of orfA-pit in S.meliloti is repressed upon Pi-starvation, unlike in E.coli where the pit genes appear to be constitutively expressed (45) and for which no Pho-boxes were detected. The identification of putative Pho-boxes upstream of the orfA-pit genes in other bacteria suggests that these may also be repressed by Pi-starvation and that such repression may be a widespread phenomenon.

A number of predicted Pho regulon members not normally associated with Pi metabolism were identified in several genomes (Table 7). One of the genes in this category was katA encoding catalase and was recently shown to be PhoB dependent in S.meliloti and P.aeruginosa in Pi-starvation conditions (46). The detection of Pho-box elements upstream of the katA genes of C.crescentus and P.putida suggests that katA expression in these organisms is also PhoB regulated. Pho-boxes upstream of several S.meliloti ABC-class transport systems were also detected upstream of homologous clusters in other bacteria. These were smc01605, smc04317 (afuA) and smc03124 (Table 7). Both the afuABC and smc01605 gene clusters in S.meliloti are annotated as putatively involved in Fe+3 transport, however definitive evidence is lacking. Choa et al., (47) did not find either of these ABC transport systems to be up-regulated in S.meliloti when grown in iron-limiting conditions. Therefore, it appears unlikely that they are actually involved in iron transport. A third ABC system in S.meliloti, smc03124, with conserved Pho-box sequences in other proteobacteria (Table 7), is annotated as a putative peptide binding protein. The actual substrate(s) transported by this system is unknown.

Table 7

Conserved Pho regulon members either of unknown and/or not clearly associated with Pi metabolism

Group	Gene	Pho box motif	Dist	Score	Genome source	Description:(predicted) function
A
1	SMc01605	CTGTCACATAGGCTTCAT	−58	0.52	S.meliloti	ABC transporter, periplasmic substrate-binding Fe+3
2	Atu2147	TTGCAATCCAACCGTCAC	−63	0.39	A.tumefaciens	ABC transporter, substrate binding protein
3	BMEII1120	CTGTCATCCAACCGTCAT	−96	0.73	B.melitensis	Iron(III)-binding periplasmic protein
4	BRA0115	CTGTCATCCAACCGTCAT	−79	0.77	B.suis	ABC transporter, periplasmic substrate-binding
5	mll3069	CTTTCATCTGGCTGTCAC	−58	0.55	M.loti	ABC transporter, periplasmic binding protein
6	PA3250	CTGACATGAAACCGTCAT	−124	0.58	P.aeruginosa	Hypothetical protein (smc01605 homolog)
7	PP1726	CCTACATCAAACTGTCAC	−119	0.39	P.putida	ABC transporter, periplasmic binding protein
B
1	SMc04317	CTGTCACATCCCTGTAAA	−51	0.58	S.meliloti	afuA, iron-binding periplasmic protein
2	Atu0202	CTGGCATCCATCCGATAT	−66	0.37	A.tumefaciens	ABC transporter, substrate binding protein (iron)
3	afuA2	CTGTCACATACATGTTAT	−46	0.54	A.tumefaciens	Atu2014 ABC transporter, substrate binding
4	mll3626	CTGTCATCCACCAGTCAT	−79	0.62	M.loti	ABC transporter, periplasmic substrate-binding (iron)
5	y4fP	ATGTAATTAAACTGACAT	+24	0.53	Rhizobium sp. NGR234	Probable ABC transporter periplasmic binding
C
1	SMb20876	AATTCGTCAATCTGTCAC	−98	0.43	S.meliloti	Hypothetical protein
2	Atu2329	CGGTCACACGCCTGTCAT	−161	0.51	A.tumefaciens	Conserved hypothetical protein
D
1	Smc00171	ATGTCATCTTCCTGAAAC	−52	0.47	S.meliloti	Conserved hypothetical protein
2	Atu1649	CTGTCATGTTGCTGAAAC	−64	0.51	A.tumefaciens	Conserved hypothetical protein
3	bll5904	CGGTCATCCCCCTGTCAC	−24	0.52	B.japonicum	Hypothetical protein
4	CC3344	CTGTCAGAAAGTCCGCAT	−152	0.38	C.crescentus	Hypothetical protein
5	mll0806	CTGACATCGCGATTTCAC	−27	0.44	M.loti	Hypothetical protein
7	PP4510	TTGCCATGGCGCTGTCAT	−43	0.37	P.putida	Conserved hypothetical protein
E
1	SMc00819	CTGTCGTTCAGCCGTCAC	−50	0.6	S.meliloti	katA, monofunctional catalase HPII
2	Atu4642	TAGTCATCTTCATGACAG	−133	0.37	A.tumefaciens	katA, bifunctional catalase/peroxidase HPI
3	CC3043	CGGTCGGTAAGGTGTCAC	−60	0.36	C.crescentus	Catalase/peroxidase
4	PA4236	CTGTCATTCATCCTTAAC	−157	0.67	P.aeruginosa	katA, monofunctional catalase HPII
5	PP3668	CTGAAAGAACACTGGCAT	−41	0.41	P.putida	Catalase/peroxidase HPI
F
1	SMc03124	CTGTCACGAAAGTTTCAC	−93	0.44	S.meliloti	Putative periplasmic binding ABC transporter
2	Atu6138	TTGCAACAAAACTGTCAC	−77	0.43	A.tumefaciens	accA, ABC transporter, substrate binding protein
		CTGTCACCAAACTTTCAT	−66	0.76
3	blr0308	CTGTCGGCTCACTGACAC	−445	0.45	B.japonicum	ABC transporter peptide-binding protein
4	BMEI1934	ATGTCATATTTCTGAAAT	−418	0.56	B.melitensis	Putative periplasmic binding ABC transporter
5	BMEII0505	CTGACACATGGCTGGAAC	−23	0.37	B.melitensis	Oligopeptide transport system permease protein
6	BRA0786	CTGACACATGGCTGGAAC	−23	0.37	B.suis	Peptide ABC transporter, substrate binding protein
7	mlr9269	CCCTCATAAAACCGTCAT	−85	0.47	M.loti	Extracellular solute-binding protein
8	mll9149	TTGTCATTCGACTGTCAT	−89	0.62	M.loti	Oligopeptide ABC transporter oligopeptide-binding
G
	SMc00620	CTTTCACCTCGCTGTAAG	−70	0.41	S.meliloti	Hypothetical signal peptide protein
	blr7070	CTTTCACAAAACTGAAAC	−44	0.57	B.japonicum	Hypothetical protein
	mlr1518	CTGTCACGCAAGCATCAT	−47	0.38	M.loti	Hypothetical protein
H
	SMc00772	AGGTCGTCAAGGCGTCAT	−103	0.36	S.meliloti	potH, putrescine transport system permease
	BR1610	TTGTCATCGCAGTGCCAT	−51	0.39	B.suis	Spermidine/putrescine ABC transporter, permease
I
	SMc02174	TTGTGAACGATCTGTCGC	−76	0.36	S.meliloti	Hypothetical transmembrane protein
	mll8170	CTGTGAACAATGTGTCGC	−72	0.36	M.loti	Hypothetical protein

‘Dist’ indicates the relative distance from the annotated translational start sites.

We identified a putative Pho-box upstream of smc00772 (potH)- gene clusters well as orthologues in M.loti and Brucella suis (Table 7). Although fusion data for smc00772 is unavailable, the potFGHI ABC-class, putative putrescine transporter cluster was identified as upregulated by Pi-limitation in the microarray analysis (31), although no Pho-box was identified by them. The putative Pho-box upstream of smc00772 (potH) lies within the coding region of potG (smc00771), instead of upstream of the regulator (potF). The fact that Pho-box-like sequences were identified upstream of genes similar to S.meliloti potH in M.loti and B.suis suggest that putrescine transport may be PhoB-regulated across a range of organisms and should be further investigated.

In response to Pi-starvation, S.meliloti replaces phospholipids with other non-Pi-containing lipids sulphoquinovosyl diacylglycerols (SL), ornithine-containing lipids (OL) and diacylglyceryl-N,N,N-trimethylhomoserines (DGTS) (48,49). In Rhodobacter sphaeroides it was demonstrated that the smc01848 homolog btaA is directly involved in DGTS biosynthesis (50) and recently Lopez-Lara et al. (51) established that smc01848 and smc01849 (btaAB) are required for DGTS synthesis. A Pho-box is predicted 64 nt from the smc01848 start codon and orthologs of smc01848 in M.loti (mlr1574) and Agrobacterium tumefaciens (atu2119) also have predicted Pho boxes in the corresponding promoter regions (Table 5). These data strongly suggest that DGTS synthesis induced upon Pi limitation is mediated directly via PhoR-PhoB system.

Pi starvation and polyphosphate metabolism

Inorganic polyphosphates (polyPi) are linear polymers of orthophosphate residues linked by high-energy phosphoanhydride bonds. These polymers can vary in size from 3 to over 1000 phosphate residues. PolyPi is ubiquitous and the enzyme primarily responsible for polyPi synthesis in E.coli is polyP kinase (PPK), which uses the gamma phosphate of ATP to make the polymer. PolyPi can also be hydrolyzed to Pi either by exopolyhosphatases (PPX) or by endopolyphosphatases (PPN). The identification and assignment of Pho-boxes was sometimes complicated by differences in genome annotation, as in the case of genes encoding polyphosphate kinase (ppk) (Table 6). Here Pho boxes were predicted in the ppk promoter regions of 10 of the 12 genomes examined. However, the predicted Pho-box from both M.loti (52) and C.crescentus (53) were located within the annotated gene coding regions. Alignment of the Ppk amino acid sequence suggests that the actual start codons of the ppk genes in M.loti and C.crescentus are downstream of the annotated start codons (data not shown). Our reporter gene fusion data showed that the S.meliloti ppk gene was strongly induced member of the Pho regulon. However, most strikingly, the weight matrix did not detect a ppk Pho box either from the E.coli K12 genome or the E.coli O157 genome, even at very low cut-off (0.18). In E.coli there is genetic evidence demonstrated that polyphosphates accumulate upon Pi starvation and depend on PhoB, although the E.coli ppk promoter has never been mapped. Therefore, it is likely that E.coli PhoB regulates ppk indirectly as suggested elsewhere (54).

CONCLUSION

Several complementary approaches were integrated to investigate the cellular response to Pi starvation. As a first step, computational identification of PhoB binding motifs predicted 96 potential Pho regulon members from the entire S.meliloti genome. These were subsequently investigated by genetic screening of transcriptional reporter gene fusions and through comparisons with recently available microarray data (31). It was found that 34 out of the 96 in silico predicted Pho regulon members were regulated by Pi concentration in a PhoB dependent manner (Table 3). These 34 Pho regulon members were analyzed in silico for conservation or co-occurrence across 12 genomes scanned (Tables 5 and 7). Nineteen of these 34 candidates were also predicted as having upstream Pho-boxes in at least one of the other genomes scanned in this study. The in silico analysis provided evidence for the conservation of a core Pho regulon in bacteria and suggests that these organisms share a common response to Pi limitation. Such a conservation is not surprising as for example in both plants and yeast one of the major responses to Pi-limitation is the induction of a high affinity Pi transport system and the induction of scavenging enzymes, such as alkaline phosphatases.

Extending the Pho-box analysis to many more genomes should define the core group of genes that respond to Pi-starvation. Further it will allow the identification of subgroups of genes, such as katA, whose expression is regulated by PhoB in some organisms but not in others. Analysis of the distribution of such data may lead to the recognition of associations between particular regulatory patterns and other phenotypic properties of the organisms.

SUPPLEMENTARY DATA

Supplemental Data are available at NAR online.