J. ANBA,' M. BIDAUD,2 M. L. VASIL,3 AND A. LAZDUNSKI1* ..... 98 0 0 Y M T K P F S P R E V N A R V KA ..... Dayhoff, D. G. George, and W. C. Barker. 1986.
Vol. 172, No. 8
JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4685-4689
0021-9193/90/084685-05$02.00/0 Copyright © 1990, American Society for Microbiology
Nucleotide Sequence of the Pseudomonas aeruginosa phoB Gene, the Regulatory Gene for the Phosphate Regulon J. ANBA,' M. BIDAUD,2 M. L. VASIL,3 AND A. LAZDUNSKI1* Laboratoire de Chimie Bacteriennel and Laboratoire de Biochimie et de Biologie Moleculaire, C.N.R.S., 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 9, France, and Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver, Colorado 802623 Received 22 January 1990/Accepted 9 May 1990 The nucleotide sequence of Pseudomonas aeruginosa phoB was determined. The sequence data suggest that the PhoB polypeptide consists of 229 amino acid residues and has a predicted molecular weight of 25,708. In the regulatory region of the gene, a very well conserved phosphate box was found. The sequence data also predicted the presence of an open reading frame downstream of phoB, which could be phoR. The deduced amino acid sequence ofphoB was significantly homologous to that of the Escherichia coli phoB gene product and to those of several known procaryotic transcriptional regulators such as PhoP, OmpR, VirG, Dye, NtrC, and AlgR.
show that the phoR gene is adjacent to phoB, and we discuss the regulatory system of phosphate-controlled gene expression in this microorganism. The phoB gene was located on a 16-kilobase-pair DNA fragment cloned in the broad-host-range cosmid pLAFR3 (8). DNA from plasmid pPHOB3 was prepared by the method of Maniatis et al. (18). To determine the approximate location of the phoB gene, pPHOB3 DNA was digested with various restriction enzymes and fragments were subcloned in plasmid pUC19 (35). The recombinant plasmid DNAs were used to transform E. coli LEP1 (phoB23 mutant) (7), and the transformants were then tested for a wild-type phenotype (blue colonies on LP-XP plates). These plates contained Tris-glucose medium supplemented with 0.3% Proteose Peptone (Difco Laboratories) (phosphate-limiting medium) to induce Pi starvation, 50 ,ug of 5-bromo-4-chloro3-indolyl phosphate per ml, and 1% agar. For high-Pi medium (HP-XP plates), 5 mM P1 was added (7). The phoB gene was functional on a 4.6-kilobase-pair BamHI insert. This recombinant plasmid was called pUB4.6. To localize the gene more precisely, we constructed a restriction map of pUB4.6 and obtained various derivatives by deletion or subcloning (Fig. 1). Results from complementation tests on LP-XP plates suggested that phoB was located within the 1,300-base-pair (bp) PstI,-BalI2 fragment (Fig. 1, plasmid pUBL1.3). We determined the nucleotide sequence of a 1.35-kilobase-pair region that includes the phoB locus. DNA fragments (PstIh-PstI2 and PstI2-PstI3) were subcloned into the BlueScript M13KS+ vector (Stratagene). Plasmids carrying clustered deletions were then obtained from these constructs by exonuclease III and mung bean nuclease action. The helper bacteriophage M13K07 was used to obtain phage particles carrying single-stranded plasmid DNA. The nucleotide sequence was determined by the dideoxynucleotidechain termination method of Sanger et al. (24) and by using Sequenase (Genofit). The DNA sequence of the Bafl2BamH1 1.34-kilobase-pair DNA fragment is shown in Fig. 2. Computer analysis revealed that there were two open reading frames, one extending from positions 283 to 969 and the second from positions 1045 to 1341.
Like nitrogen, phosphorus compounds are essential constituents in biological organisms. In spite of its relative abundance in nature, phosphate is a growth-limiting factor for many organisms because much of its natural supply occurs as insoluble salts. To deal with phosphate limitation, bacteria have evolved complex regulatory systems to assimilate phosphorus very efficiently. During the past several years, it has been recognized that several global regulatory networks in bacteria are controlled by transcriptional regulators which belong to a class of two-component regulatory systems that are responsive to environmental stimuli (1). More than 32 genes are expressed when Escherichia coli is grown under phosphate starvation conditions (31). The PhoB-PhoR pair is involved in the control of a large number of genes that are responsive to phosphate limitation (30). This collection of genes, all of whose promoters require PhoB, has been termed the pho regulon (30). In phoR mutants, the expression of some of these genes depends on a gene designated phoM (31), which is not part of the pho regulon. In Pseudomonas aeruginosa, phosphate limitation results in the synthesis of several protein species. These include alkaline phosphatase, an outer-membrane channel-forming protein (protein P), a binding protein for Pi and a hemolytic and a nonhemolytic phospholipase C (12, 13, 21, 22; R. M. Ostroff, A. I. Vasil, and M. L. Vasil, submitted for publication). Several other unidentified periplasmic proteins are also derepressed in low-phosphate medium (10). The mechanisms of phosphate regulation in P. aeruginosa have not yet been characterized in detail. We have previously identified and cloned two P. aeruginosa genes which can complement phoB and phoR mutations in E. coli (7). This suggests that a pho regulon system, similar to that in E. coli, may exist in P. aeruginosa, with at least two similar regulatory factors. In this report, we describe the nucleotide sequence of the P. aeruginosa phoB gene; the translated sequence of phoB was found to be highly homologous to sequences of a class of environmentally responsive regulatory genes (1). We also *
Corresponding author. 4685
4686
J. BACTERIOL.
NOTES
phoB complementation Bm
pUB 4.6
PI
P2 BD
Ni
I l
+ 4-
B2 Na I I
Ps Bs I
Bm
B4
+
pUC 19
.
pho R' i
pho B ->
ORF
pUP 2.7 pUP 1.3
pUP 4.6 A N
IL
A
pUBL 1.3
+ 1 IE
FIG. 1. Restriction map of the phoB region of P. aeruginosa DNA and the ability of recombinant plasmids to complement the phoB mutation in E. coli LEP1. Only pertinent restriction sites are indicated. Bm, BamHI; P, PstI; B, Ball; N, NruI. The locations of phoB (.) and phoR' (4m) are shown. The arrows indicate the direction of transcription. phoR' designates the part of the gene encoding the N-terminal region of PhoR. The opposite open reading frame (ORF) (") is also shown. +, Able to complement phoB mutation; -, not able to complement phoB mutation; Kb, kilobase pairs.
The first open reading frame (positions 283 to 969) encodes protein product of 229 amino acids with a predicted molecular weight of 25,708 which was subsequently identified as PhoB. This gene has a G+C content of 65%, with an intermediate codon usage bias (86% G or C) in the third position. This is typical of chromosomally encoded Pseudomonas genes (33). A homology search with the Align program (20) showed extensive homology of this protein with the PhoB proteins of E. coli (16) and Bacillus subtilis (25). Figure 3 shows their amino acids sequences. A close homology between the proteins of the three organisms was found throughout the sequence, with the 125 amino-terminal amino acids being particularly conserved (Fig. 3). The homology was greater between P. aeruginosa and E. coli PhoB proteins (59%o identical and 76% similar amino acids) than between P. aeruginosa and B. subtilis PhoB proteins (40%o identical and 59% similar amino acids). Computer analysis also revealed that the opposite strand encoded a large potential open reading fiame (extending from positions 1143 to 244) which is virtually superimposable to phoB (Figs. 1 and 2). It is well known that the E. coli phoB gene product belongs to a class of environmentally responsive regulatory proteins such as OmpR (3), VirG (34), Dye (5), NtrC (2), and AlgR (4). The deduced amino acid sequence of P. aeruginosa PhoB was compared with those of the regulatory proteins by using the Align algorithm. The P. aeruginosa PhoB Nterminal domain showed a strong similarity to the aminoterminal domains of this family of response regulators (Fig. 4). This domain has been implicated in the reception of signals transmitted through a sensor protein (1, 29), PhoR in this case. In E. coli, PhoR modulates the activator function of PhoB by phosphorylation-dephosphorylation of PhoB (14, 29). Signal transduction by phosphorylation appears to be a common mechanism for two-component regulatory systems (1, 29). It was also demonstrated that phosphorylation of PhoB enhances its binding activity to the pho boxes of pstS, one of the pho regulon genes, and activates transcription a
from this promoter (14). Pairwise comparson of protein sequences revealed a closer relationship between P. aeruginosa PhoB and E. coli OmpR than between P. aeruginosa PhoB and AlgR, another regulatory protein recently identified by P. aeruginosa (Fig. 4) (4). More specifically, in the second block of homology (positions 47 to 126), there was 61% identity (81% homology with conservative substitutions) between P. aeruginosa PhoB and E. coli OmpR, while there was only 30%o identity (60% similarity) between P. aeruginosa PhoB and P. aeruginosa AIgR. In E. coli, the two regulatory genes phoB and phoR constitute an operon, with a 61-bp intercistronic sequence between the termination codon of phoB and the initiation codon of phoR (17, 32). Both genes are induced by phosphate limitation, and the phoBR operon is positively regulated by the PhoB protein (28). In B. subtilis, the two genes also constitute an operon similar to that of E. coli (26). The second reading frame (positions 1045 to 1341) found in the presented DNA sequence (Fig. 2) could encode the P. aeruginosa phoR gene. The deduced truncated polypeptide consists of 96 amino acid residues of the N-terminal region. When similarities were analyzed, the encoded polypeptide showed a high degree of homology to the PhoR protein of E. coli (17): 31% identical and 45% similar amino acids. Moreover, this 96-amino-acid region shows an extensive hydrophobic region, like that present in the amino-terminal portion of the E. coli PhoR protein (residues from about 18 to 60). There is a 74-bp region between the two open reading fiames, and no obvious terminator structure was found in this region. We found a sequence that may form a stemand-loop structure with a AG value of -18.7 kcal(ca. -78.2 kJ)/mol (Fig. 2); this structure is neither a typical rhoindependent terminator nor a rho-dependent terminator, since it is not followed by either CAA residues or several T residues. Such a structure, with the same AG value, was also found in the intercistronic region in E. coli, and it was postulated that it could function as a transcriptional attenuator (17). The organization of phoB and phoR in P. aerugi-
NOTES
VOL. 172, 1990 60 40 50 20 30 t0 CCTGCCATCG ACCA6CACBC CGTCCTTCTC GT6CCGGC6C CACC6TTCTG GCCA6CGC6C 6GAC6GTAGC T6GTCGTGC6 GCA6GAAGAG CAC66CCGCG GT6GCAAGAC C6STC6CGCG 100 120 80 90 1t0 70 GAAC6CC66C AGGCCAGCGC CAGACCA6CG CCGATGATTC T6TTCATC6C GTCTCTCTCC CTTGCGGCCG TCC6GTC6C6 GTCT6GTC6C 6GCTACTAAG ACAAGTAGCG CAGA6AGAG6
180 140 170 160 130 150 TGT6TCTTCA 6GTGGAGCGT TCGCCGCCC6 CAGGACGGCA CCG6AACCT6 TTGAGCATAG ACACA6AAGT CCACCTCGCA A6CGGCG66C GTCCTGCCGT GGCCTTG6AC AACTC6TATC 240 230 220 190 200 210 CTCCGCCGCG GGCA6TCG6C 6GAAACTACG CCGTT6T6TC ACATACCTGA CACAATTTCG TGTAT6GACT 66TTAAAGC CC6TCA6CC6 CCTTT6AT6C G6CAACACA6 6A66CGGC6C 300 290 260 270 280 _MetValGly LysThrIle TTATCTAATG CGGC6CAAGA CAGATC6ACC C6AGGCAAGA CCAT6GTTGG CAAGACAATC AATA6ATTAC GCCGC6TTCT GTCTAGCT6G GCTCCGTTCT 6GTACCAACC 6TTCTGTTAG
250
360 320 330 350 340 310 LeuIleValAsp AspGluAla ProIleArg GluMetIleAla ValAlaVal GluMetAla CTCATCGTTG ATGAC6AA6C ACCGATTCGC GAGATGATCG CCGTGGCCGT GGA6AT6GCC GAGTAGCAAC TACTGCTTCG TGGCTAAGC6 CTCTACTA6C 66CACCG6CA CCTCTACCGG
370
420 380 390 410 400 GlyTyrGluCys LeuGluAla GluAsnThr GInGlnAlaHis AlaValIle ValAspArg 6GCTACGAGT GCCTGGA6GC GGAAAATACC CAGCAGGCGC ACGCGGTGAT CGTCGACCGC CC6ATGCTCA CGGACCTCCG CCTTTTAT66 GTCGTCC6CG TGCGCCACTA GCAGCTG6CG 480 470 430 440 450 460 LysProHisLeu IleLeuLeu AspTrpMet LeuPro6lyThr SerGlyIle GluLeuAla AA6CCGCACC TGATCCTGCT CGACTGGATG CTCCCCGGCA CTTCCGGCAT CGAACTGGCG TTCGGCGTGG ACTAGGACGA GCTGACCTAC GAG6GGCCGT GAAGGCCGTA GCTT6ACC6C 540 490 500 510 520 530 ArgArgLeuLys ArgAsp6lu LeuThrLeu AspIleProIle IleMetLeu ThrAlaLys C6ACGCCT6A AGCGT6AC6A ACTGACCCTC GACATCCCGA TCATCATGCT CACCGCCAAG GCTGCGGACT TCGCACT6CT TGACTGGGAG CT6TAGGGCT A6TAGTACGA GTGGCGGTTC
550
560
570
580
590
600
GlyGluGluAsp AanLyslle GlnGlyLeu GluAlaGlyAla AspAspTyr IleThrLys 6GCGAGGAGG ACAACAAGAT CCAGGGCCTG GAGGCTGGCG CCGACGACTA CATCACCAAG CCGCTCCTCC TGTTGTTCTA GGTCCCGGAC CTCCGACCGC GGCTGCTGAT GTAGT6GTTC 660 610 620 630 650 640 ProPheSerPro ArgGluLeu ValAlaArg LeuLysAlaVal LeuArgArg Thr6lyPro CCGTTCTCGC CCCGCGAGCT GGTCGCCCGC CTGAAGGCCG TGCTGCGCCG CACCGGGCCT
GGCAAGAGCG GGGCGCTCGA CCAGCGGGC6 GACTTCCGGC ACGACGCGGC GTGGCCCGGA 670
680
690
700
710
720
GlyHisSerGlu AlaProIle GluValGly GlyLeuLeuLeu AspProlle SerHisArg GGCCACAGCG AGGCGCCGAT CGAGGTCGGC GGCCTGCTGC TGGACCCGAT CA6CCACCGC CCGGTGTCGC TCCGCGGCTA GCTCCAGCCG CCGGAC6AC6 ACCTGGGCTA GTCGGTGGCG 730
740
750
760
770
780
ValThrIleAsp 6lyLysPro AlaGluMet GlyProThrGlu TyrGlyLeu LeuGlnPhe GTGACCATCG ACGGCAAGCC GGCCGAGATG GGCCCCACCG A6TACGGCCT GCTGCAGTTC CACTGGTAGC TGCCGTTCGG CCG6CTCTAC CCGG6GTGGC TCATGCCG6A CGACGTCAAG 800 810 840 790 830 820 PheMetThrHis 61nGluArg AlaTyrThr ArgGly61nArg ArgAspGln ValTrpGly TTCATGACTC ACCAGGAACG CGCCTATACC CGCGGCCAAC 6TCGTGACCA G6TGTGGGGC AAGTACTGAG TGGTCCTTGC GCGGATATGG GCGCCG6TTG CAGCACTGGT CCACACCCCG
850
860
870
880
890
900
6lyAsnValTyr ValGluGlu ArgThrVal AspMetAspIle ArgArgLeu ArgLysAla GGCAACGTCT ATGTCGAGGA GCGCACCGTC 6ACATGGATA TCCGCCGCCT GC6CAAGGCG CCGTTGCAGA TACAGCTCCT CGCGTGGCAG CT6TACCTAT AGGCGGC6GA CGCGTTCCGC 910
920
930
940
950
960
LeuGlyGluVal TyrGluAsn LeuValGln ThrValArgGly ThrGlyTyr ArgPhaSer CTCGGCGAGG TCTACGAAAA TCTGGTTCAG ACTGTCCGCG GGACCGGCTA TCGTTTCTCC GAGCCGCTCC AGATGCTTTT AGACCAAGTC TGACAGGCGC CCTGGCC6AT A6CAAAGAGG 970
980
990
1000
1010
1020
, ThrLysSer*** z ACCAAGAGCT GACCCCGCTC CCG6CCGCCC AGCGGCCGAG TGGTTTCCCC TGGACGGTCC TGGTTCTC6A CTG6GGCGAG GGCC66CGGG TCGCCGGCTC ACCAAAGGGG ACCTGCCAGG 1060 1030 1080 1040 1050 1070 MetGln SerValValAsn GlnAspTrp ArgGlyAla CGTGACGGAC CGCCACTGGA CAG6ATGCAA TCCGTGGT6A ACCAAGACTG 6C6TGGAGCG SCACTGCCTG GCGGTGACCT GTCCTACGTT AGGCACCACT TGGTTCTGAC CGC&CCTCGC
4687
1110 1100 1120 t130 1140 1090 LeuIleArgHis LeuLeuLeu ValLeuAla AlaSerLeuVal LeuGlyVal ValSerGly CTGATCCGCC ACCTGCTTCT CGTGCTGGCT GCCAGCCTCG TGCTCGGCGT GGTCAGCGGC GACTAGGCGG TGGACGAAGA GCACGACCGA CGGTCGGAGC ACGAGCCGCA CCAGTCGCCG
1190 1200 1180 1160 1170 1150 HisTyrGlyTrp AlaSerAla LeuGlyLeu ArgLeuTyrLeu GlyTrpThr LeuTrpGln CATTACGGCT GGGCCTCGGC CCTCGGCCTG CGTCTCTACC TCGGCTGGAC CCTCTGGCAG GTAATGCCGA CCCGGAGCCG GGAGCCGGAC GCAGAGATGG AGCCGACCTG GGAGACCGTC 1240 1250 1260 1230 1220 1210 LeuLeuCysLeu HisGInTrp LeuArgAsn HisGlnProAsp GluProPro ProAspSer CTACTGTGCC TGCACCAGTG GCTGCGCAAC CACCA6CCAG AC6AGCCACC 6CCGGACAGC GATGACACGG ACGTGGTCAC CGACGCGTTG GTG6TCGGTC TGCTCGGTGG CGGCCTGTCG 1320 1310 1300 1290 1280 1270 TyrGlyLeuTrp GlyGluVal PheAspAsn IleTyrHlsLeu GInArgArg AanGInArg TACGGCCTCT GGG6C6AAGT CTTCGACAAT ATCTACCACC TGCAACGCCG CAACCAGCGC ATGCCGGAGA CCCC6CTTCA GAAGCTGTTA TAGATGGTGG ACGTTGCGGC GTTGGTCGCG
13!150 1340 1330 AlaArgGlyArg LeuGInArg GCCCGTGGCC GCCTGCAGCG TGGATCC CGGGCACCGG CGGACGTCGC ACCTA6G
1360
1370
1380
FIG. 2. Nucleotide sequence of the phoB region. The deduced amino acid sequences of the phoB and phoR genes are given above the DNA sequence. The termination codon ofphoB (nucleotide 972) is marked with asterisks. The putative ribosome binding site, -10 region, and Pho box are overlined. Arrows indicate the inverted repeat region whose transcript may form a stem-and-loop structure.
nosa is reminiscent of that in E. coli and thus suggests that they may form an operon. The nucleotide sequence upstream of the coding region of phoB was examined for structures that could be relevant for the transcriptional regulation of the gene. In E. coli, an 18-nucleotide consensus sequence (phosphate [pho] box) located approximately 15 to 21 nucleotides upstream from the Pribnow box (-10 region) was found for several genes of the pho regulon, including phoB itself (16). This pho box represents the region to which the PhoB protein binds (15). The 18-bp sequence located between positions 216 and 233 closely matched with the E. coli consensus pho box sequence (14 of 18 nucleotides identical) (Fig. 2). By now, three other phosphate starvation-inducible genes in P. aeruginosa have been sequenced (23, 27; Ostroff et al., submitted). The pho boxes located upstream from the phoB and oprP genes are located 10 nucleotides from a -10 putative site, close to the E. coli consensus sequence. In the case of p1cS, no obvious homology could be found in the -35 region, but a pho box-like sequence located 201 bp from the transcription start site was found. The gene encoding the nonhemolytic phospholipase, plcN, also has a Pho box-like sequence located 170 bp upstream of the translational start site. plcN is 50-fold induced by Pi starvation, while plcS is only 6-fold induced by Pi starvation (Ostroff et al., submitted). However, these two hypothetical pho boxes are located too far upstream from the usual position to ascertain, without any mutational or protein-binding analysis, whether they are functional. One regulatory mutation, designated plcA, was isolated and located at 22 to 23 min (10 min on the recalibrated map [19]) on the P. aeruginosa chromosome (11). Strain ASON (plcA) was markedly deficient in the production of phospholipase C, alkaline phosphatase, and several unidentified extracellular proteins (9). Thus, this strain behaves like an E. coli strain which carries a mutation in phoB. To check the ability of the cloned phoB from P. aeruginosa to complement the plcA mutation, the recombinant plasmid pPHOB3 was mobilized by triparental mating from E. coli to strain ASON by using the conjugative helper plasmid pRK2013 (6).
4688
NOTES
PhoB P.a PhoB E.c PhoP 8.5
PhoB P.a PhoB E.c PhoP 8.8
J. BACTERIOL.
1 M V 6 K T I L I V D D E A P I R E N I A V A V E M A C Y E C L E A E N T OQ A H A V I VORKPHL 1 - M A R R I L V V E O E A P I R E N V C F V L EONSFOP V E A E O Y D S A V N O L N E P W P O L 1 -MNKK ILVVOO E E S I V T L L O Y N L ERS6YOV I T A S O 6 E E A L K K A E T E K P O L
50 49 49
51 I L L DWN L P 6 T S C I E L A R R L K R D E L T L D I P I I N L T AK6 E E D N K I C L E AA 100 50 I L L D W N L P 6 6 S C I O f I K H L K R E S N T R 1I P V V N L T A R C E E E D R V R 6 L E T 6 A 99 50 I V L D V N L P K L O 6 I E V C K O L RO OKLMF - -P I L N L T AKD E E F D K V L C L E L C A 97
Phob P.a 101 D D Y I T K P F S P R E L V A R L K A VLRRT---6P6H S - - - E A P I E V66LLLO 141 PhoB E.c 100 0 0 Y I T K P F S P K E L V A R I K A V N R R I - - - S P N A V --- E E V I E N 6 L SL O 140 PhoP B.s 98 0 0 Y M T K P F S P R E V N A R V K A I L R R S E I R A P S S E M K N OENE6O IV I S O L K I L 147 -
-
-
-
I
I
I
Phob P. a 142 P I S H R V T I O G K P A E N 6 P T E Y 6 L L OF F N T H Q E R A Y T R 6 O R R D a V Ia 6 N V Y V 191 PhoB E.c 141 P T S H R V N A G E E P L E N G P T E F K L L HfF N T H P E R V Y S R E I L L N H V W C T N V Y V 190 PhoP B.s 148 P D H Y E A I F K E S O L E L T P K E F E L L L Y L 6 R H K 6 R V L T R O L L L S A V M N Y O F A G 197
PhoB P.m 192 E E R T V D N D I R R L R K A L 6 E - - - - - - V Y E N LV O T V R C T 6 Y R F S T K S PhoB E.c 191 E O R T V O V H I R R L R K A L E P 6 G - - - - - H O R N V O T V R C T 6 Y R F S T R F PhoP 8.s 198 D T R I V D V H I S H L R P T K I E N N T K K P I Y - - - I K T IRC L 6 Y K L E E P K M N E
230
230 243
FIG. 3. Comparison of amino acid sequences of the P. aeruginosa PhoB, E. coli PhoB, and B. subtilis PhoP proteins. Identical residues in all or any two of the three proteins are in boldface type. Gaps have been inserted to align the three sequences for the greatest homology. Abbreviations: P.a, P. aeruginosa; E.c, E. coli; B.s, B. subtilis.
A good complementation was observed, since a wild-type phenotype was restored on LP-XP plates. We then concluded that the pIcA mutation is located in the phoB gene. The complete nucleotide sequence of the P. aeruginosa phoB gene is reported in this study. We showed that PhoB protein is highly homologous to its counterpart in E. coli and to other regulatory proteins of the same family. Arguments are given to show that the genetic organizations of the regulatory genes phoB and phoR in P. aeruginosa are similar
I N V 6 - K T I L I V I NOENYK N L V V KH V L L V 28 I NO T - PH I L I V 1I " R - 6I A W I V N V L I V 2
PhoB P.s OmpR E. c VirS A.t Oye E.c NtrC K.p A1gR P.a
Pho8 P.m
OmpR E.c VirG A.t Dye E.c NtrC K.p AlgR P.m
PhoB P.ma OmpR E.c Vlr A.t Oym E.c NtrC K.p AlgR P.a
47 48 71 47 47
K P HL I Sf HLM T V D V V 0 I N L V T PD V L
47
K P
I
- N
L P
VLOL
-
L P
9 E O N K I 9 C L E A 88 E V D R I V C L E I III E T O tKV V AL E L 87 E V O K I L L E I 87 L D A A V S A Y a
OD E A P I R E DDDNRLRA ODD V A N R H E O E L V TA N O OO S S I R W ODEPLARE
NIA- V A V E N A C Y
LLELIITLKVLERLAR
RYLT EQOF E S R L
Y I A V
L T I H A F F EA ESY L T 6 AA L 6 LD 6 Y
28 29
52 28 28 28
CTS6 I ELARRLK-RDELT LO IPI I N - L T A Ke C E 88 C E 0 6 L S I C A R L - - R S O S N P M -PII N - V T A K S E 87 110 - L 6 REOCLE I VRNLAAKS--- -D IPI I I IS60RLE - L P SKNSLLLAREL--REOA- -- NVAL N F L T 6 R D N as 86 N - P S ODCL A LL K O I KO RH- - - P N LPVI I N - T A H S D 86 N - P CLOCL OVAARL CEREA---- PP A V IF CTAHDE
LL D
V VD L N I "0 I N LSOI R V LLO I R
to those of the same genes in E. coli and B. subtilis. These two genes could constitute an operon, with phoB being promoter proximal. The presence of a very well conserved pho box in the regulatory region of phoB emphasizes the close relationships in phosphate regulation among these widely divergent organisms. We conclude, therefore, that B. subtilis, E. coli, and P. aeruginosa possess similar phosphate-regulated gene systems consisting of two components, which may be generally present among procaryotic cells.
A O O ; I ADO Y I FI A S A O YI
C C C 6 c
A
V S
A
T K P f S P R E P K P F N P R E
- - -
E A K P FS I T K P F N P R E
- - -
0L V R L K A V L R R 7 6 P 6 H S E 130 L L A R I R A V L R R O A N E L P 6 129 L AR IR V A L R V R P N V V R S L T I R A R N L L S R TN N L G T V
- - - F
152 127
Y L P K PFD I O E A V A L V R A I S H Y E O P R A P 132 6 Y L V K P V R S E D - - - L A E A L K K A S R P N R V L A A 126 FIG. 4. Similarities in the amino-terminal sequences observed in the PhoB, OmpR (2), VirG (31), Dye (4), NtrC (1), and AlgR (3) proteins. Identical amino acids found in all or any two of the six proteins are in boldface type. Abbreviations: P.a, P. aeruginosa; E.c, E. coli; A.t,
87
F
- -
A L E A
F
F V
Agrobacterium tumefaciens; K.p, Klebsiella pneumoniae.
NOTES
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