The Gene for an Exopolyphosphatase of Pseudomonas Aeruginosa
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DNA RESEARCH 6, 103-108 (1999) The Gene for an Exopolyphosphatase of Pseudomonas aeruginosa Tatsushi MIYAKE,1 Toshikazu SHIBA1'* Atsushi KAMEDA,1 Yoshiharu IHARA,1 Masanobu MUNEKATA,1 Kazuya ISHIGE,2 and Toshitada NOGUCHI2 Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan1 and Biochemicals Division, YAMASA Corporation, Choshi, Chiba 288-0056, Japan2 (Received 1 February 1999; revised 18 March 1999) Abstract In Pseudomonas aeriginosa, a gene, ppx, that encodes exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] of 506 amino acids (56,419 Da) was found downstream of the gene for polyphosphate kinase, ppk. Since ppx is located in the opposite direction of the ppk gene, they do not constitute an operon. The predicted amino acid sequence of PPX is 41% identical with Escherichia coli PPX. The gene product of ppx (paPPX) was overproduced in E. coli, and its activity was evaluated. Orthophosphate (Pi) is released from polyphosphate [poly(P)], the average chain lengths of which are 79 and 750, respectively. The amount of P; released matched the amount of poly(P) lost. Thus ppx encodes an enzyme that has exopoly(P)ase activity. Key words: Exopolyphosphatase; inorganic polyphosphate; Pseudomonas aeruginosa PAO1 1. Introduction in living organisms,1 and the characteristics of this en- 14 17 zyme have been investigated in E. yeast, " Inorganic polyphosphate [poly(P)] includes linear sponge18 and mammals.19 polymers of orthophosphate with chain lengths of up to In the course of analyzing clones of the Pseudomonas 1000 or more.1'2 Poly(P) has been found in every living 11 2 aeruginosa ppk gene, an amino acid sequence of an ad- thing, from bacteria to mammals, so far examined. Sev- jacent gene was found to be highly homologous to that eral biological functions have been suggested for cellular of exopoly(P)ase identified in E. coli. Here, we report poly(P), including a reservoir for phosphate and energy, the identification of the P. aeruginosa ppx gene (ppx), a chelator of metal ions, a buffer against alkali, a channel 2 its DNA sequence, its overexpression in E. coli, and its for DNA entry and a regulator for gene expression. In activity. Escherichia coli, poly(P) has been shown to induce the expression of the rpoS for the stationary phase specific 3 2. Materials and Methods sigma factor of RNA polymerase. Several enzymes that contribute to the metabolism 4 2.1. Materials of poly(P), including polyphosphate kinase (PPK), E. coli strain XLl-Blue (Stratagene) served as a strain exopolyphosphatase [exopoly(P)ase],112 endopolyphos- for overproducing paPPX and was also the host strain phatase,5 polyphosphate glucokinase6 and poly(P): for preparation of plasmid DNA. pTrc99A20 was from AMP phosphotransferase,7 have been identified. PPK is Pharmacia Biotech. Poly(P) with an average length an enzyme responsible for the synthesis of poly(P) poly- merizing the terminal phosphate of ATP to a poly(P) of 79 residues [Poly(P)7g] was obtained from Sigma. in a freely reversible reaction.4 The genes for PPK have A polyethyleneimine-cellulose thin layer chromatogra- been cloned from several bacteria,8"11 and sequence anal- phy plate (PEI-TLC plate) was purchased from Merck. [32P]Poly(P) was obtained by synthesis from [7-32P]ATP yses have shown that, in E. coli, the ppk gene constitutes 4 an operon with the ppx gene, and that this operon en- using purified E. coli PPK, and this material had a chain codes exopoly(P)ase.12 Exopoly(P)ase, one of the main length of approximately 750. enzymes that degrades poly(P) and release orthophos- phate (Pi) from the ends of poly(P), is widely distributed 2.2. Sequencing of DNA fragment containing a ppx ho- mologue Communicated by Satoshi Tabata The nucleotide sequence was determined by the * To whom correspondence should be addressed. Tel. & Fax. 21 +81-11-706-7816, E-mail: [email protected] dideoxy chain termination method with some specific 104 Exopolyphosphatase Gene of P. aeruginosa [Vol. 6, primers using an ABI PRISM Big-Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) and an ppx B EB B Bg ABI PRISM 310 Genetic Analyzer (Perkin Elmer). I I I 2.3. Overproduction of paPPX ppk Using two specific oligonucleotides (5'-CATGCCATG- Figure 1. Structure of the 6.8-kb insert in pPPK02Fn carrying GACTTGCAAAGCATGCCG-3' and 5'-CGGGATCCT- the ppx and ppk genes. The region that was sequenced is indi- TACCGCACGTTGAGGCTGTAG-3'), a 1.5-kb DNA cated by a dotted line with arrows. The ppx and the ppk genes fragment containing the intact open reading frame of are indicated by bold arrows. B, BamHI; Bg, Bgl II; E, EcoKl. paPPX with a Nco I site at the 5' end and a BamHI site at the 3' end was amplified by PCR. The ampli- 3. Results and Discussion fied fragment was cloned into the pTrc99A20 expression vector to give pTrcpaPPX. The ppx gene was integrated 3.1. Identification of gene encoding paPPX downstream of the trc promoter that is inducible by iso- We previously cloned the P. aeruginosa ppk gene in propyl /3-D-thiogalactopyranoside (IPTG). E. coli XL1- a plasmid pPPK02F which carries a 6.8-kb fragment of Blue transformed by pTrcpaPPX was cultured in LB chromosomal DNA of P. aeruginosa PAOl.11 The nu- broth with 100 /ig/ml of ampicillin at 30° C until the cleotide sequencing analysis of the downstream region of mid-logarithmic growth phase. Then IPTG was added ppk showed that there is an open reading frame (ORF) to the final concentration of 1 mM and continuously cul- in the opposite direction of the ppk gene (Fig. 1). The tured for 6 hr. Cells were harvested by centrifugation, ORF is composed of 1518 bp encoding a protein of 506 and the cell pellet was suspended with 10 ml of 20 mM amino acids, and 14 bp overlapped with the 3'-terminus Tris-HCl (pH 7.5) and 2 mM MgCl2. To prepare the to- of the coding region of the ppk gene (Fig. 2A). Two tal cell extract for sodium dodecyl sulfate polyacrylamide possible initiation codons were observed at nucleotide gel electrophoresis (SDS-PAGE), a small portion of the positions 181-183 (ic-1) and 196-198 (ic-2) (Fig. 2A). cell suspension (50 fil) was removed and added to 50 fil 22 The Shine-Dalgarno sequence for ic-1 is 5 bp-matched SDS-PAGE sample buffer. The main portion of the cell with the 3'-terminus of 16S RNA of P. aeruginosa (3'- suspension was dispersed by sonication, and 20 /Ug/ml AUUCCUCUC),24 while the Shine-Dalgarno sequence DNasel and 20 //g/ml RNaseA were added. After 1 hr for ic-2 is 4 bp-matched. Therefore, ic-1 is more likely incubation at 4°C, the cell extract was centrifuged at than ic-2 to be an initiation codon, although there may be 30,000 x g for 1 hr. The resulting supernatant was used two species of paPPX that are initiated from ic-1 and ic- for exopoly(P)ase assay as the crude cell lysate. 2, respectively, in the cells. A computer-aided homology search using the FASTA program25 revealed that paPPX 2.4- Assay for exopoly(P)ase activity is 41% and 40% identical with E. coli PPX and E. colt Exopoly(P)ase activity was measured by the following GppA, respectively (Fig. 2B). E. coli gppA was first iden- tified as a mutant lacking the ability to hyrdolyze the 5'- two methods [The concentration of poly(P) is given in 26 terms of P, residues]: 1) The assay mixture (20 /xl) con- 7-phosphate of guanosine pentaphosphate (pppGpp). GppA not only has pppGpp phosphohydrolase activity taining 20 mM HEPES-KOH (pH 7.2), 4 mM MgSO4 13 (buffer A) and 0.36 mM [32P]poly(P) was incubated at but also exopoly(P)ase activity. Since E. coli PPX also 37°C for 1 hr with cell extracts (4.58 /ig protein). The P; has pppGpp phosphohydrolase avtivity, and since these three proteins are significantly homologous to each other, released from poly(P) was separated by ascending thin paPPX may also have pppGpp phosphohydrolase activ- layer chromatography on PEI-cellulose in 0.4 M LiCl ity. and 1 M formic acid and quantitated by a radio-image analyzer (BAS2000, Fujix). 2) Exopoly(P)ase activity was also assayed by directly measuring the amount of P; 3.2. Overproduction of paPPX released from poly(P). The reaction were performed at For overexpression of paPPX in E. coli, the gene was 37°C in buffer A containing 0.15 mg of cell lysate with amplified by PCR, and an expression plasmid pTrc- or without 0.3 /jmol poly(P)79 in 0.3 ml of reaction mix- paPPX was constructed, as described in Section 2.3. The ture. Pj was quantitated according to the method of plasmid pTrcpaPPX allowed IPTG-inducible production 23 Chen et al. The P; released from poly(P) was calcu- of the paPPX protein, and the translation of ppx starts lated by subtracting the Pj value of the reaction mixture from ic-1 (Fig. 2A). The total cell extract was analyzed without poly(P) from that with poly(P). by SDS-PAGE, and a protein with a molecular mass of about 60 kDa, corresponding to the calculated mass of paPPX, was overproduced (Fig. 3, lane 2). No. 2] T. Miyake et al. 105 A 91 AGTCTCTATCGTTGGTCGGCTGATTCCCGATTGATCGGCTGCCGCATTGGCGACCCTCGCCCTATCGTGGCAGGATGGCGCAGTTTTCCA 181 ^TggACTTGCAAAGCATGCCGCAAAAACCTGCCGAGGC ATTCCCGCTGATTGCCGCCCTGGACCTGGGCTCCAACAGTTTCCACCTCTCT 1MDLQSMPQKPAEAFPLIAALDLGSNSFHLC (ic-1) (ic-2) 31RILBRLGEKVQLAAGLDEERLAKANIHGEV 61NLSEEATQRGLDCLRRFAQF ISGMPQGSVR 91VVATNALREARNRSDFIRRAEEVLG 121ISGREEARLIYLGVANSMPDSGGRRLVSDI 631 151 GGGSTEFIIGQGFESELRE LQMGCVSYTQ 181RYFRDGKITPARYAQAYTAARLELMGIENS 211 LRRLGWQQAVGASGTIRAVALAIKAGGBG 901 241 GEISPDGLAWLKRKVLKLGDVEKLDLEGIK 271 PDRRTIFPAGLAILEAIFDALELEQMVBSE 301 GALREGVLYDLVGRHQHEDVRERTISSLMQ 1171 331 RYHVDPEQASRVEAKALKVLAEVGDAWEL 361 GELHRDLLSWGARVHEIGLDIAHYHYHKHG 391 AYLIEHSDLAGFSRQDQQMLSLLVRGHRR 1441 421 IPADKLAEFAEEGDKLVRLCIVLRFAILF 451 HIRGTQEMPSVRLKAEPKSLSVTFPEGWLE 16 21 481 ANPLTQADFAQEAEWLKRVGYSLNVR 1711 CGGCCAGCTTCTCCAGCAGGGTCGCCTGGG Figure 2.