PLASMID 22,27 I-274 ( 1989)

Identification and DNA Sequencing of a New Plasmid (pPST1) in Pseudomonas stutzeri MO-l 9

MASAYAFUJITA,* 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

Received July 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 extra- cellular amylase that forms maltotetraose (Robyt and Ackerman, 1971). Recently, we cloned and sequenced the gene (amyf’) en- coding this amylase from P. stutzeri MO-19 (Fujita et al., 1989). As the amyP gene was found to be expressed only weakly in Esche- richia coli, for further studies on it, introduc- tion 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 Gram- negative bacteria including Pseudomonas and 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 plas- mid, pPST1, in P. stutzeri MO-19 and deter- mined its complete nucleotide sequence.

’ To whom correspondence and requests for reprints should be addressed.

Plasmid pPST 1 was prepared from an over- night 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 pro- cedure (Birnboim and Doly, 1979). The plas- mid 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 trans- ferred into E. coli JM83 and ampicillin resis- tant 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 Pseudomonas plasmid pPST 1 had DNA of 1446 bp in a circular form. It con- tained an open reading frame of 456 bp, be- ginning with an ATG initiation codon at nu- cleotide 576 and ending at nucleotide 103 1, capable of encoding a protein of 152 amino acids with a molecular weight 17,087. A se- quence for a ribosome-binding site (Gold et al., 198 l), GAAAGAG, was found 9 bases up-

271 0147-619X/89 $3.00 Copyright Q 1989 by Academic Pms, Inc. All rights of reproduction in any form reserved.

272 SHORT COMMUNICATIONS

Sphl 1

CCATCCCATCCACCCCCGTCAATACCTTGAGCACA 35

2

TGCTGCCTCCACCGCGTAC~CATCCA~TCTGCTCCGGGCTGGCGTAGGCGTAAACCTGCTTGGCCTTGGCGTTCTGCGGCTTGAGTTCC 125

3 4

AGTTCTGCCCCAhCGTCGTCAGGATCGCCcccGGTCTGCTTGCCCTTlTCGTAGATGCGCTTC~~TGGCTGCCGAGGTGCGCGACCCGATG 214

1 2

TAGAGCGTCCGCCCGTCTTGCTCGCGGTGAGAGTCGCCTACATGCGTGACC’rTGAGGCCGAACTTGTCTGCCGTCTCCAGACCCAGCGCG305

4 3 5

TAGAGAGAGTCCCACGCCCCCGGCTCGCAG.TAGTCGATAGTCACGTCAGCCCTGAGTAGGTTGTGCCCCCTGAACTCATCCCGAACGACA 395

GCAGCAAAOOCOGGTGCCCCGTCGCCGCTTGCAOAGGCCCATACGCGCGT~CCGACGGTGTTACCGCCCCACTGCACCCGTGCAAOCACA 485

5

GAATCGCCTCTCACGACGTTA~CCCGTTCACATTTTTCGOGGTGGTCGGCGTTACGTCCGAACGACTCGAAAGAGCCGGCCAG 575 -24 -12 SD

A Y

ATGATCAAGGAACCCGOTCGGGT~~GGCCTCGATGCTGGCGGTGTAGTAGTCGAACCTCATTTTTCACCCCCAGAGGCTCTAGAGCGCTT 665 H I KEPGRVWPR CWRCSSRTSPFI P II G S R A I.

CGCOGGGTTTTTTTGGCTCGGTGTGTAGGGGGGTTAGGTGT’~GTGACCCCCG’~AGTTACCTATGCGGGGGTTGGGCGGTCTGCAAACATG 755 R G V F 1. A II c v GGLGVV T P V V ‘r Y A 0 v G It S A N M

stul ATCCATGAGAGCGACGCAAAGGCCCGAGCATCTCGTTCATG’~GAGCCTCTCCAGACTCAAGGCC’~AGAGACCATCCCGGCCCTAGATTCA 945 1 tt E S D A K A R A S R S C E P 1. Q 1‘ Q G L E ?’ I I’ A L D S

Smal ACGTGCCAGCGCCCGGGCTTTCTGGCAGGCCCGGAGAACCCTTTGTTGGACAGGGTTAACCATAGTCAGGAACTCCGAGACAAGACCCGA 935 T C 9 II P G F L A G P EN PL L I) R V N H S Q I? I. II D K T A

6 7 7

CATCGTGACGCCCTGAACCTCAGAAAGACCCCTGATCGTGTCGTACACGTCCTGATCGAGCGTAACCGTGASTCTCGGTTTCTGTGTGGC 1025 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 A F L C 0

11 8 9 6 10 - --

CATTGGTGATGCACTCCGGGCAAAGTGATGCACCCAATCTACGCAGCCGAAGGCGAAAGGCAACCCGTGCGAATTGTTTACATGTGCGCA 1115

” :1* 12 13 12 13

- 10 14 15 ~GAGCG~ATGAGCGCATG~GATTTGTGTG~ATGTGATTT~~~ATG~ATOTG~G~ATACTGGCG~G~TA~G~TTG~~CG~~GAG~~GAGTTG 1205

14 16 9 16 17 17 CCOGCT~GTACCGGCGCTA~GCTC~~GGCC~GAT~CGGG~A~~T~GA~CCGGC~~GACGAATTTGT~~GCG~~GGTGOA~TTATTTG~~~ 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 sequences conserved in DNA-binding proteins is underlined below the amino acid sequence. For the amino acid residues underlined twice see the legend for Fig. 2. Major restriction sites are 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 sequences with the DnaA box (Fuller et al., 1984). Direct repeats are overlined and numbered.

stream of the ATG initiation codon. A poten- ouye et al., 1987) was detected in the 5’-up- tial promoter (in the -24 and -12 regions) stream region of the coding region. recognized in Pseudomonas (Dixon, 1986; In- A number of DNA-binding proteins have

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, 2 3 4 5 6 7 6 9 10 11 12 13 14 16 16 17 16 19 20

pUBI ~1 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 Asp Leu Gly Val Tyr Gin Ser Ala Ile Asn Lys Ala Ile His - - - -

ARep Gln Glu Ser Val Ala Asp Lys Met GA 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 Leu Lys - - - -

LdCR Leu Tyr Asp Val Ala Glu Tyr Ala Gly Val Ser Tyr Gln Thr Val Ser Arg Val Val Asn - - - -

Gall? Ile Lys Asp Val Ala Arg Leu Ala Gly Val Ser Val Ala Thr Val Ser Arg Val Ile Asn - - - -

PPSTl ORFl Gly Val Phe Leu Ala Arg Cys Val 9 Gly Leu Gly Val Val Thr Pro Val Val Thr Tyr - - -

Helix 3

FIG. 2. Comparison of a putative DNA-binding sequence in 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 residues in ORFI are underlined. Proposed a-helical structures and DNA-binding sequences of 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- (~3 sequences of X Cro and X repressor and the CZE-aF sequence of catabolite gene activator protein (CAP).2 These homologous sequences span 20 amino acid residues and are predicted to form a helix-turn-helix structure for DNA interactions (Pabo and Sauer, 1984). A ho- mologous sequence was 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 sequence in the pu-

tative 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 se- quence of pPST 1.

Sequences TTGAGCACA and CCATC- CACA 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

* Abbreviation used: CAP, catabolite gene activator protein.

DNA replication (Bramhill and Kornberg, 1988). Thus, these homologous sequences in 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.

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Communicated by Richard D. Kolodner