- Email: [email protected]
Identification and DNA sequencing of a new plasmid (pPST1) in Pseudomonas stutzeri MO-19
PLASMID
22,27 I-274 ( 1989)
Identification
MASAYAFUJITA,*
and DNA Sequencing of a New Plasmid (pPST1) in Pseudomonas stutzeri MO-l 9
MICHIOKUBOTA,~
MASAMITSUFUTAI,$
ANDAKINORIAMEMURA*"
*Department of Biotechnology, Faculty of Engineering, Fukuyama University, Higashimura-cho, Fukuyama, Iiiroshima 729-02, Japan; tllayashibara Biochemical Laboratories, Inc., 7-7 Amase-minami. Okayama 700, Japan; and *the Institule qfScie&fic and Induslrial Research, Osaka University, Ibaruki, Osaku 567, Jupun
ReceivedJuly 7, 1989; revised November 10, 1989 A cryptic plasmid, pPST1, was isolated from I’seudomonas stutzeri MO-19 and its complete nucleotide sequence was determined. This plasmid consisted of 1446 bp and could encode a putative polypeptide of 152 amino acid residues (ORFI) in an open reading frame. The putative protein contained a sequence homologous to the sequences found in DNA-binding sites. o 1989
Pseudomonas stutzeri produces an extracellular amylase that forms maltotetraose (Robyt and Ackerman, 1971). Recently, we cloned and sequenced the gene (amyf’) encoding this amylase from P. stutzeri MO-19 (Fujita et al., 1989). As the amyP gene was found to be expressed only weakly in Escherichia coli, for further studies on it, introduction of a recombinant plasmid carrying it into P. stutzeri seemed desirable. Several kinds of IncQ-based replicons have been reported. They are maintained in the majority of Gramnegative bacteria including Pseudomonasand can provide good sources for developing broad-host-range vectors (Bagdasarian et al., 198 1; Carlson et al., 1985; Mermod et al., 1986). However, their sizes are relatively large compared with those of E. coli plasmids, for example, pBR322. Small plasmids are more useful and convenient for analysis of inserted DNA. So, with a view to constructing a small useful vector for Pseudomonas,and especially for P. stutzeri, in this work we searched for new plasmids. We found a small cryptic plasmid, pPST1, in P. stutzeri MO-19 and determined its complete nucleotide sequence. ’ To whom correspondence and requests for reprints should be addressed.
271
Plasmid pPST 1 was prepared from an overnight culture of P. stutzeri MO-19 grown at 30°C in LB broth [ 1% Bacto tryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), and 1% NaCl (pH 7.0)] by the alkaline lysis procedure (Birnboim and Doly, 1979). The plasmid was linearized with SmaI, SphI, or StuI and inserted into the multiple cloning site of pUC19 (Yanisch-Perron et al., 1985). The newly constructed plasmids were then transferred into E. coli JM83 and ampicillin resistant transformants were isolated. We prepared plasmids from the transformants and analyzed the nucleotide sequence of pPST 1 inserted into pUC19 (Fig. 1). DNA sequencing of both strands was carried out by the dideoxy chain termination reaction @anger et al., 1977) by the exonuclease deletion method (Henikoff, 1984). The new Pseudomonasplasmid pPST 1 had DNA of 1446 bp in a circular form. It contained an open reading frame of 456 bp, beginning with an ATG initiation codon at nucleotide 576 and ending at nucleotide 103 1, capable of encoding a protein of 152 amino acids with a molecular weight 17,087. A sequence for a ribosome-binding site (Gold et al., 198 l), GAAAGAG, was found 9 bases up0147-619X/89
$3.00
Copyright Q 1989 by Academic Pms, Inc. All rightsof reproduction in any form reserved.
272
SHORT COMMUNICATIONS 1 Sphl CCATCCCATCCACCCCCGTCAATACCTTGAGCACA
35
2 TGCTGCCTCCACCGCGTAC~CATCCA~TCTGCTCCGGGCTGGCGTAGGCGTAAACCTGCTTGGCCTTGGCGTTCTGCGGCTTGAGTTCC
125
4
3
AGTTCTGCCCCAhCGTCGTCAGGATCGCCcccGGTCTGCTTGCCCTTlTCGTAGATGCGCTTC~~TGGCTGCCGAGGTGCGCGACCCGATG
214
1
2
TAGAGCGTCCGCCCGTCTTGCTCGCGGTGAGAGTCGCCTACATGCGTGACC’rTGAGGCCGAACTTGTCTGCCGTCTCCAGACCCAGCGCG305 3
4
5
TAGAGAGAGTCCCACGCCCCCGGCTCGCAG.TAGTCGATAGTCACGTCAGCCCTGAGTAGGTTGTGCCCCCTGAACTCATCCCGAACGACA
395
GCAGCAAAOOCOGGTGCCCCGTCGCCGCTTGCAOAGGCCCATACGCGCGT~CCGACGGTGTTACCGCCCCACTGCACCCGTGCAAOCACA
485
5 GAATCGCCTCTCACGACGTTA~CCCGTTCACATTTTTCGOGGTGGTCGGCGTTACGTCCGAACGACTCGAAAGAGCCGGCCAG -12 -24 A Y ATGATCAAGGAACCCGOTCGGGT~~GGCCTCGATGCTGGCGGTGTAGTAGTCGAACCTCATTTTTCACCCCCAGAGGCTCTAGAGCGCTT CWRCSSRTSPFI P II KEPGRVWPR H I
575
G
S
R
A
I.
CGCOGGGTTTTTTTGGCTCGGTGTGTAGGGGGGTTAGGTGT’~GTGACCCCCG’~AGTTACCTATGCGGGGGTTGGGCGGTCTGCAAACATG ‘r Y A 0 v G GGLGVV T P V V II c v R G V F 1. A
It S
A
N
M
ATCCATGAGAGCGACGCAAAGGCCCGAGCATCTCGTTCATG’~GAGCCTCTCCAGACTCAAGGCC’~AGAGACCATCCCGGCCCTAGATTCA L E ?’ I R A S R S C E P 1. Q 1‘ Q G 1 tt E S D A K A
I’
A
L
D
S
Smal ACGTGCCAGCGCCCGGGCTTTCTGGCAGGCCCGGAGAACCCTTTGTTGGACAGGGTTAACCATAGTCAGGAACTCCGAGACAAGACCCGA S Q I? I. T C 9 II P G F L A G P EN PL L I) R V N H
II
D
K
T
A
A
F
L
C
0
SD 665
755
stul
6
7
935
7
CATCGTGACGCCCTGAACCTCAGAAAGACCCCTGATCGTGTCGTACACGTCCTGATCGAGCGTAACCGTGASTCTCGGTTTCTGTGTGGC H R D A L N L R K T P D R V V I, V I_ I II II N R D S 8 6 --9 CATTGGTGATGCACTCCGGGCAAAGTGATGCACCCAATCTACGCAGCCGAAGGCGAAAGGCAACCCGTGCGAATTGTTTACATGTGCGCA
” :1*
945
1025 10
11 1115
13
12 13 12 10 14 ~GAGCG~ATGAGCGCATG~GATTTGTGTG~ATGTGATTT~~~ATG~ATOTG~G~ATACTGGCG~G~TA~G~TTG~~CG~~GAG~~GAGTTG
15 1205
16 16 9 14 17 CCOGCT~GTACCGGCGCTA~GCTC~~GGCC~GAT~CGGG~A~~T~GA~CCGGC~~GACGAATTTGT~~GCG~~GGTGOA~TTATTTG~~~
17 1295 15
~ACACTAGCCGAGCAACGAACAGC~CGAACGA~T~AATAT~GCCG~~CAGTG~~TCCAG~TTG~GG~AATAGTTG~~GTA~TG~TTAGCC
1385
FIG. 1. Nucleotide sequence of pPST1. An open reading frame from nucleotide positions 576 to 1031 encodes protein ORFl with a possible ATG start site at position 576. Numbering begins arbitrarily at the first nucleotide of the SphI recognition sequence.A sequence homologous to the sequencesconserved in DNA-binding proteins is underlined below the amino acid sequence.For the amino acid residuesunderlined twice seethe legend for Fig. 2. Major restriction sitesare shown. The line marked SD indicates the proposed Shine-Dalgamo (ribosome-binding) sequence.The lines marked -24 and - 12 indicate a putative promoter. Horizontal arrows show homologous sequenceswith the DnaA box (Fuller et al., 1984). Direct repeats are overlined and numbered.
stream of the ATG initiation codon. A potential promoter (in the -24 and -12 regions) recognized in Pseudomonas(Dixon, 1986; In-
ouye et al., 1987) was detected in the 5’-upstream region of the coding region. A number of DNA-binding proteins have
273
SHORT COMMUNICATIONS ,
2
3
4
5
6
7
6
9
10
11
12
13
14
16
16
17
16
19
20
ASXI ~rp h-g b-g -Ala Met Lye His GA Ile
Gin Ser Gin Lys s
Val Ala Glu Val Ile
A cro
Gin Thr
~ys
Thr
-Ala
Lys
Val
Tyr
Gin
Ser
Ala
-Ile
Asn
Lys -Ala
ARep
Gln
Glu
Ser
Val
-Ala
Asp Lys
Met
Met
Gly
Gln
Ser
Gly
s
Gly
Ala
-Leu Phe Asn
CAP
Arg
Gin
Glu
Ile
-Gly
Gin
Ile
Val Gly Cys Ser Arg Glu Thr Val -
Gly
Arg
-Ile
LdCR
Leu Tyr
Asp Val
-Ala
Glu
Tyr
Ala
Ser
Arg -Val
pUBI
~1
Gall? PPSTl
GA
Gly -
Val
Ser
Tyr
Gln
Thr
-Val
Ile
His
Leu Lys Val
Asn
Val Ser Val Ala Thr -Val Ser Arg -Val Ile Asn -Ala Arg Leu Ala Gly Gly Val Phe Leu -Ala Arg Cys Val 9 Gly Leu Gly Val Val -Thr Pro Val -Val Thr Tyr Ile
ORFl
Asp Leu Gly -
Lys
Asp Val
Helix
3
FIG. 2. Comparison of a putative DNA-binding sequencein ORFl of pPST1, with those of other known proteins. Amino acid residues proposed to have an important role in maintaining a bihelical structure of DNA-binding sites and conserved in the sites (Pabo and Sauer, 1984)and corresponding residuesin ORFI are underlined. Proposed a-helical structures and DNA-binding sequencesof proteins other than ORFI are cited from the reviews by Pabo and Sauer (1984) and McKenzie et al. (1986).
a sequence that is homologous with the a2(~3sequencesof X Cro and X repressorand the CZE-aFsequence of catabolite gene activator protein (CAP).2 These homologous sequences span 20 amino acid residuesand are predicted to form a helix-turn-helix structure for DNA interactions (Pabo and Sauer, 1984). A homologous sequencewas also found in a highly basic protein, (Y,of plasmid PUB 110 and its possible binding to a specific DNA sequence of pUBll0 was suggested(McKenzie et al., 1986). We found a similar sequencein the putative protein (ORFl) (Fig. 2) although we have no evidence that the sequence forms a bihelical structure. Moreover, ORF 1 was highly basic (30 basic amino acids vs 14 acidic amino acids) like proteins that bind to DNA. Thus, ORFl may bind to a specific DNA sequence of pPST 1. Sequences TTGAGCACA and CCATCCACA homologous to the DnaA box, which is present in oriC of E. coli and is recognized by DnaA protein (Fuller et al., 1984) were also present in pPST1 (Fig. 1). Interaction of the DnaA protein with the DnaA box is thought to be a prerequisite for initiation of
DNA replication (Bramhill and Kornberg, 1988). Thus, these homologous sequencesin pPST 1 may form part of the replication origin and be recognized by a host factor(s) involved in DNA replication. Moreover, many direct repeats are present in pPST 1. However, the physiological significance of the direct repeats is not clear. After introduction of an appropriate genetic marker(s), plasmid pPST1 may be useful as a vector for genetic engineering of P. stutzeri.
REFERENCES BAGDASARIAN, M., LURZ,R.,RUCKERT,B.,FRANKLIN, F.C.H.,BAGDASARIAN,M.M.,FREY,J.,ANDTIMMIS,
K. N. (198 1).Specific-purposeplasmid cloning vectors. II Broad host range, high copy number, RSFlOlO-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16,231-241. BIRNBOIM, H. C., AND DOLY, J. (1979). A rapid alkaline extraction procedure for screeningrecombinant plasmid DNA. Nucleic Acids Res. I, 1513- 1523. BRAMHILL, D., AND KORNBERG, A. (1988). Duplex opening by dnu4 protein at novel sequencesin initiation of replication at the origin of the Escherichia coli chro-
mosome. Cell 52,743-755. CARLSON,~. A.,STEWART,G.J.,
* Abbreviation used: CAP, catabolite gene activator protein.
AND INGRAHAM,J.L.
(1985).Thymidine salvagein Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous
274
SHORT COMMUNICATIONS
expression of Escherichia coli thymidine kinase gene. J. Bacterial. 163, 291-295. DIXON, R. (1986). The xy/ABC promoter from the Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichia coli. Mol. Gen. Genet.
MCKENZIE,T., HOSHINO,T., TANAKA, T., AND SUEOKA, N. (1986). The nucleotide sequenceof PUB 110: Some salient features in relation to replication and its regulation. Plasmid 15, 93-103. MERMOD,N., LEHRBACH,P. R., DON, R. H., AND TIMMIS. 203, 129-136. K. N. ( 1986).Gene cloning and manipulation in PseuFUJITA, M., TORIGOE,K., NAKADA, T., TSUSAKI, K., domonas. In “The Bacteria: A Treatise on Structure KUBOTA, M., SAKAI, S., AND TSUJISAKA,Y. (1989). and Function,” Vol. 10,“The Biology of Pseudomonas” Cloning and nucleotide sequence of the gene (amyP) (I. C. Gunsalus, J. R. Sokatch, and L. N. Ornston, Eds.), for maltotetraose-forming amylase from Pseudomonas pp. 325-355. Academic Press,Orlando, FL. stutzeri MO-19. J. Bacterial. 171, 1333-l 339. PABO,C. O., AND SAUER,R. T. (1984). Protein-DNA recFULLER, R. S., FUNNELL, B. E., AND KORNBERG,A. ognition. Annu. Rev. Biochem. 53, 293-32 I. (1984). The dnaA protein complex with the Escherichia ROBYT, J. F., AND ACKERMAN,R. J. (1971). Isolation, coli chromosomal replication origin (oriC) and other purification, and characterization of a maltotetraoseDNA sites. Cell 38, 889-900. producing amylase from Pseudomonasstutzeri. Arch. GOLD, L., PRIBNOW,D., SCHNEIDER,T., SHINEDLING, Biochem. Biophys. 145, 105-l 14. S.,SINGER,B. S.,ANDSTORMO,G. (1981).Translational SANGER,F., NICKLEN, S., AND COULSON, A. R. (1977). initiation in prokaryotes.Annu. Rev. Microbial. 35,365DNA sequencing with chain terminating inhibitors. 403. Proc. Natl. Acad. Sci. USA 74, 5463-5467. HENIKOFF,S. (1984). Unidirectional digestion with exoYANISCH-PERRON, C., VIEIRA, J., AND MESSING, J. (1985). nuclease III creates targeted breakpoints for DNA seImproved M 13 phage cloning vectors and host strains: quencing. Gene 28,35 l-359. Nucleotide sequences of the M 13mp18 and pUC19 INOUYE, S., NAKAZAWA, A., AND NAKAZAWA, T. (1987). vectors. Gene 33, 103-I 19. Expression of the regulatory genexylS on the TOL plasmid is positively controlled by the xylR gene product. Communicated by Richard D. Kolodner Proc. Natl. Acad. Sci. USA 84, 5 182-5 186.
22,27 I-274 ( 1989)
Identification
MASAYAFUJITA,*
and DNA Sequencing of a New Plasmid (pPST1) in Pseudomonas stutzeri MO-l 9
MICHIOKUBOTA,~
MASAMITSUFUTAI,$
ANDAKINORIAMEMURA*"
*Department of Biotechnology, Faculty of Engineering, Fukuyama University, Higashimura-cho, Fukuyama, Iiiroshima 729-02, Japan; tllayashibara Biochemical Laboratories, Inc., 7-7 Amase-minami. Okayama 700, Japan; and *the Institule qfScie&fic and Induslrial Research, Osaka University, Ibaruki, Osaku 567, Jupun
ReceivedJuly 7, 1989; revised November 10, 1989 A cryptic plasmid, pPST1, was isolated from I’seudomonas stutzeri MO-19 and its complete nucleotide sequence was determined. This plasmid consisted of 1446 bp and could encode a putative polypeptide of 152 amino acid residues (ORFI) in an open reading frame. The putative protein contained a sequence homologous to the sequences found in DNA-binding sites. o 1989
Pseudomonas stutzeri produces an extracellular amylase that forms maltotetraose (Robyt and Ackerman, 1971). Recently, we cloned and sequenced the gene (amyf’) encoding this amylase from P. stutzeri MO-19 (Fujita et al., 1989). As the amyP gene was found to be expressed only weakly in Escherichia coli, for further studies on it, introduction of a recombinant plasmid carrying it into P. stutzeri seemed desirable. Several kinds of IncQ-based replicons have been reported. They are maintained in the majority of Gramnegative bacteria including Pseudomonasand can provide good sources for developing broad-host-range vectors (Bagdasarian et al., 198 1; Carlson et al., 1985; Mermod et al., 1986). However, their sizes are relatively large compared with those of E. coli plasmids, for example, pBR322. Small plasmids are more useful and convenient for analysis of inserted DNA. So, with a view to constructing a small useful vector for Pseudomonas,and especially for P. stutzeri, in this work we searched for new plasmids. We found a small cryptic plasmid, pPST1, in P. stutzeri MO-19 and determined its complete nucleotide sequence. ’ To whom correspondence and requests for reprints should be addressed.
271
Plasmid pPST 1 was prepared from an overnight culture of P. stutzeri MO-19 grown at 30°C in LB broth [ 1% Bacto tryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), and 1% NaCl (pH 7.0)] by the alkaline lysis procedure (Birnboim and Doly, 1979). The plasmid was linearized with SmaI, SphI, or StuI and inserted into the multiple cloning site of pUC19 (Yanisch-Perron et al., 1985). The newly constructed plasmids were then transferred into E. coli JM83 and ampicillin resistant transformants were isolated. We prepared plasmids from the transformants and analyzed the nucleotide sequence of pPST 1 inserted into pUC19 (Fig. 1). DNA sequencing of both strands was carried out by the dideoxy chain termination reaction @anger et al., 1977) by the exonuclease deletion method (Henikoff, 1984). The new Pseudomonasplasmid pPST 1 had DNA of 1446 bp in a circular form. It contained an open reading frame of 456 bp, beginning with an ATG initiation codon at nucleotide 576 and ending at nucleotide 103 1, capable of encoding a protein of 152 amino acids with a molecular weight 17,087. A sequence for a ribosome-binding site (Gold et al., 198 l), GAAAGAG, was found 9 bases up0147-619X/89
$3.00
Copyright Q 1989 by Academic Pms, Inc. All rightsof reproduction in any form reserved.
272
SHORT COMMUNICATIONS 1 Sphl CCATCCCATCCACCCCCGTCAATACCTTGAGCACA
35
2 TGCTGCCTCCACCGCGTAC~CATCCA~TCTGCTCCGGGCTGGCGTAGGCGTAAACCTGCTTGGCCTTGGCGTTCTGCGGCTTGAGTTCC
125
4
3
AGTTCTGCCCCAhCGTCGTCAGGATCGCCcccGGTCTGCTTGCCCTTlTCGTAGATGCGCTTC~~TGGCTGCCGAGGTGCGCGACCCGATG
214
1
2
TAGAGCGTCCGCCCGTCTTGCTCGCGGTGAGAGTCGCCTACATGCGTGACC’rTGAGGCCGAACTTGTCTGCCGTCTCCAGACCCAGCGCG305 3
4
5
TAGAGAGAGTCCCACGCCCCCGGCTCGCAG.TAGTCGATAGTCACGTCAGCCCTGAGTAGGTTGTGCCCCCTGAACTCATCCCGAACGACA
395
GCAGCAAAOOCOGGTGCCCCGTCGCCGCTTGCAOAGGCCCATACGCGCGT~CCGACGGTGTTACCGCCCCACTGCACCCGTGCAAOCACA
485
5 GAATCGCCTCTCACGACGTTA~CCCGTTCACATTTTTCGOGGTGGTCGGCGTTACGTCCGAACGACTCGAAAGAGCCGGCCAG -12 -24 A Y ATGATCAAGGAACCCGOTCGGGT~~GGCCTCGATGCTGGCGGTGTAGTAGTCGAACCTCATTTTTCACCCCCAGAGGCTCTAGAGCGCTT CWRCSSRTSPFI P II KEPGRVWPR H I
575
G
S
R
A
I.
CGCOGGGTTTTTTTGGCTCGGTGTGTAGGGGGGTTAGGTGT’~GTGACCCCCG’~AGTTACCTATGCGGGGGTTGGGCGGTCTGCAAACATG ‘r Y A 0 v G GGLGVV T P V V II c v R G V F 1. A
It S
A
N
M
ATCCATGAGAGCGACGCAAAGGCCCGAGCATCTCGTTCATG’~GAGCCTCTCCAGACTCAAGGCC’~AGAGACCATCCCGGCCCTAGATTCA L E ?’ I R A S R S C E P 1. Q 1‘ Q G 1 tt E S D A K A
I’
A
L
D
S
Smal ACGTGCCAGCGCCCGGGCTTTCTGGCAGGCCCGGAGAACCCTTTGTTGGACAGGGTTAACCATAGTCAGGAACTCCGAGACAAGACCCGA S Q I? I. T C 9 II P G F L A G P EN PL L I) R V N H
II
D
K
T
A
A
F
L
C
0
SD 665
755
stul
6
7
935
7
CATCGTGACGCCCTGAACCTCAGAAAGACCCCTGATCGTGTCGTACACGTCCTGATCGAGCGTAACCGTGASTCTCGGTTTCTGTGTGGC H R D A L N L R K T P D R V V I, V I_ I II II N R D S 8 6 --9 CATTGGTGATGCACTCCGGGCAAAGTGATGCACCCAATCTACGCAGCCGAAGGCGAAAGGCAACCCGTGCGAATTGTTTACATGTGCGCA
” :1*
945
1025 10
11 1115
13
12 13 12 10 14 ~GAGCG~ATGAGCGCATG~GATTTGTGTG~ATGTGATTT~~~ATG~ATOTG~G~ATACTGGCG~G~TA~G~TTG~~CG~~GAG~~GAGTTG
15 1205
16 16 9 14 17 CCOGCT~GTACCGGCGCTA~GCTC~~GGCC~GAT~CGGG~A~~T~GA~CCGGC~~GACGAATTTGT~~GCG~~GGTGOA~TTATTTG~~~
17 1295 15
~ACACTAGCCGAGCAACGAACAGC~CGAACGA~T~AATAT~GCCG~~CAGTG~~TCCAG~TTG~GG~AATAGTTG~~GTA~TG~TTAGCC
1385
FIG. 1. Nucleotide sequence of pPST1. An open reading frame from nucleotide positions 576 to 1031 encodes protein ORFl with a possible ATG start site at position 576. Numbering begins arbitrarily at the first nucleotide of the SphI recognition sequence.A sequence homologous to the sequencesconserved in DNA-binding proteins is underlined below the amino acid sequence.For the amino acid residuesunderlined twice seethe legend for Fig. 2. Major restriction sitesare shown. The line marked SD indicates the proposed Shine-Dalgamo (ribosome-binding) sequence.The lines marked -24 and - 12 indicate a putative promoter. Horizontal arrows show homologous sequenceswith the DnaA box (Fuller et al., 1984). Direct repeats are overlined and numbered.
stream of the ATG initiation codon. A potential promoter (in the -24 and -12 regions) recognized in Pseudomonas(Dixon, 1986; In-
ouye et al., 1987) was detected in the 5’-upstream region of the coding region. A number of DNA-binding proteins have
273
SHORT COMMUNICATIONS ,
2
3
4
5
6
7
6
9
10
11
12
13
14
16
16
17
16
19
20
ASXI ~rp h-g b-g -Ala Met Lye His GA Ile
Gin Ser Gin Lys s
Val Ala Glu Val Ile
A cro
Gin Thr
~ys
Thr
-Ala
Lys
Val
Tyr
Gin
Ser
Ala
-Ile
Asn
Lys -Ala
ARep
Gln
Glu
Ser
Val
-Ala
Asp Lys
Met
Met
Gly
Gln
Ser
Gly
s
Gly
Ala
-Leu Phe Asn
CAP
Arg
Gin
Glu
Ile
-Gly
Gin
Ile
Val Gly Cys Ser Arg Glu Thr Val -
Gly
Arg
-Ile
LdCR
Leu Tyr
Asp Val
-Ala
Glu
Tyr
Ala
Ser
Arg -Val
pUBI
~1
Gall? PPSTl
GA
Gly -
Val
Ser
Tyr
Gln
Thr
-Val
Ile
His
Leu Lys Val
Asn
Val Ser Val Ala Thr -Val Ser Arg -Val Ile Asn -Ala Arg Leu Ala Gly Gly Val Phe Leu -Ala Arg Cys Val 9 Gly Leu Gly Val Val -Thr Pro Val -Val Thr Tyr Ile
ORFl
Asp Leu Gly -
Lys
Asp Val
Helix
3
FIG. 2. Comparison of a putative DNA-binding sequencein ORFl of pPST1, with those of other known proteins. Amino acid residues proposed to have an important role in maintaining a bihelical structure of DNA-binding sites and conserved in the sites (Pabo and Sauer, 1984)and corresponding residuesin ORFI are underlined. Proposed a-helical structures and DNA-binding sequencesof proteins other than ORFI are cited from the reviews by Pabo and Sauer (1984) and McKenzie et al. (1986).
a sequence that is homologous with the a2(~3sequencesof X Cro and X repressorand the CZE-aFsequence of catabolite gene activator protein (CAP).2 These homologous sequences span 20 amino acid residuesand are predicted to form a helix-turn-helix structure for DNA interactions (Pabo and Sauer, 1984). A homologous sequencewas also found in a highly basic protein, (Y,of plasmid PUB 110 and its possible binding to a specific DNA sequence of pUBll0 was suggested(McKenzie et al., 1986). We found a similar sequencein the putative protein (ORFl) (Fig. 2) although we have no evidence that the sequence forms a bihelical structure. Moreover, ORF 1 was highly basic (30 basic amino acids vs 14 acidic amino acids) like proteins that bind to DNA. Thus, ORFl may bind to a specific DNA sequence of pPST 1. Sequences TTGAGCACA and CCATCCACA homologous to the DnaA box, which is present in oriC of E. coli and is recognized by DnaA protein (Fuller et al., 1984) were also present in pPST1 (Fig. 1). Interaction of the DnaA protein with the DnaA box is thought to be a prerequisite for initiation of
DNA replication (Bramhill and Kornberg, 1988). Thus, these homologous sequencesin pPST 1 may form part of the replication origin and be recognized by a host factor(s) involved in DNA replication. Moreover, many direct repeats are present in pPST 1. However, the physiological significance of the direct repeats is not clear. After introduction of an appropriate genetic marker(s), plasmid pPST1 may be useful as a vector for genetic engineering of P. stutzeri.
REFERENCES BAGDASARIAN, M., LURZ,R.,RUCKERT,B.,FRANKLIN, F.C.H.,BAGDASARIAN,M.M.,FREY,J.,ANDTIMMIS,
K. N. (198 1).Specific-purposeplasmid cloning vectors. II Broad host range, high copy number, RSFlOlO-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16,231-241. BIRNBOIM, H. C., AND DOLY, J. (1979). A rapid alkaline extraction procedure for screeningrecombinant plasmid DNA. Nucleic Acids Res. I, 1513- 1523. BRAMHILL, D., AND KORNBERG, A. (1988). Duplex opening by dnu4 protein at novel sequencesin initiation of replication at the origin of the Escherichia coli chro-
mosome. Cell 52,743-755. CARLSON,~. A.,STEWART,G.J.,
* Abbreviation used: CAP, catabolite gene activator protein.
AND INGRAHAM,J.L.
(1985).Thymidine salvagein Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous
274
SHORT COMMUNICATIONS
expression of Escherichia coli thymidine kinase gene. J. Bacterial. 163, 291-295. DIXON, R. (1986). The xy/ABC promoter from the Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichia coli. Mol. Gen. Genet.
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