JOURNAL OF BACTERIOLOGY, Feb. 1989, p. 708-713 Vol. 171, No. 2 0021-9193/89/020708-06$02.00/0 Copyright © 1989, American Society for Microbiology Nostoc commune UTEX 584 Gene Expressing Indole Phosphate Hydrolase Activity in Escherichia coli WEN-QIN XIE,1 BRIAN A. WHITTON,2 J. WILLIAM SIMON,2 KARIN JAGERt DEBORAH REED,' AND MALCOLM POTTSl* Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061,1 and Department ofBotany, University ofDurham, Durham City DHJ 3LE, United Kingdom2 Received 18 August 1988/Accepted 9 November 1988 A gene encoding an enzyme capable of hydrolyzing indole phosphate was isolated from a recombinant gene library of Nostoc commune UTEX 584 DNA in XgtlO. The gene (designated iph) is located on a 2.9-kilobase EcoRI restriction fragment and is present in a single copy in the genome of N. commune UTEX 584. The iph gene was expressed whef the purified 2.9-kilobase DNA fragment, free of any vector sequences, was added to a cell-free coupled transcription-translation system. A polypeptide with an M, of 74,000 was synthesized when the iph gene or different iph-vector DNA templates were expressed in vitro. When carried by different multicopy plasmids and phagemids (pMP0O5, pBH6, pB8) the cyanobacterial iph gene conferred an Iph' phenotype upon various strains of Escherichia coli, including a phoA mutant. Hydrolysis of 5-bromo- 4-chloro-3-indolyl phosphate was detected in recombinant E. coli strains grown in phosphate-rich medium, and the activity persisted in assay buffers that contained phosphate. In contrast, indole phosphate hydrolase activity only developed in cells of N. commune UTEX 584, when they were partially depleted of phosphorus, and the activity associated with these cells was suppressed partially by the addition of phosphate to assay buffers. Indole phosphate hydrolase activity was detected in periplasmic extracts from E. coli (Iph') transformants, Our current understanding of the role of phosphorus in the grown in BG 11 medium under the same conditions of control of cell function in microorganisms derives largely growth. The strains of E. coli used in this study are listed in from studies with Escherichia coli (22). Four different phos- Table 1. All strains were grown in LB liquid medium (12) at phatases have been identified in the periplasm of E. col, 370C, with or without the addition of ampicillin (200 jig ml- 19 each showing hydrolytic activity with a range of substrates final concentration). In certain experiments a minimal me- that do not penetrate the cytoplasmic membrane (4, 29). The dium (8) was used; this was supplemented with different major criteria used in the characterization of these four concentrations of KH2PO4. Where necessary, liquid media phosphatases are substrate specificity and pH optimum (4, were solidified by the addition of 1.2% (wt/vol) agar. 29). Considerable data have accumulated on the genes and Recombinant DNA analyses. Unless stated otherwise, rou- enzymes involved in phosphate transport (23, 24, 26). As a tine methods were used for the manipulation of DNA (9, 12). consequence, current opinion is that the regulation of phos- Restriction endonucleases were obtained from Bethesda phate transport is complex. Research Laboratories, Inc. (Gaithersburg, Md.) and were Cyanobacteria warrant particular attention, because of the used according to the specifications of the manufacturer. key role played by the availability and turnover of phospho- The plasmid pGEM-4 and bacteriophage XgtlO were ob- rus in determining the development of water blooms or tained from Promega Biotec (Madison, Wis.). The phagemid extent of economically important nitrogen-fixing communi- pBluescript M13+ (Stratagene, La Jolla, Calif.) was a gift ties such as those in rice fields. A range of cyanobacteria from T. Larson. have been reported to show phosphatase activity (10), but Construction of recombinant DNA library. During the metabolism little is known about the regulation of phosphate isolation of rpo genes from N. commune UTEX 584 (Xie et The of a or the enzymes or genes involved. availability al., submitted), a recombinant library of N. commune UTEX recombinant gene libary of Nostoc commune UTEX 584 584 genomic DNA was constructed in the phage vector XgtlO (W.-Q. Xie, K. Jager, and M. Potts, submitted for publica- (imm434 b527) and propagated in E. coli C600 (hfl) by an of a tion) provided opportunity to attempt the isolation standard methods (9). The library was constructed with N. gene the further investiga- cyanobacterial phosphatase for commune UTEX 584 genomic DNA-EcoRI restriction frag- tion metabolism in this ecologically significant, of phosphate ments (size range, 3 to 7 kilobases [kb]). The genomic DNA In the present study we nitrogen-fixing cyanobacterium. was prepared as follows. A culture was grown to a density of report the of a gene coding for an indole phosphate isolation approximately 20 g (wet weight) of cells per liter, the cells iph) from N. commune UTEX 584. hydrolase (designated were harvested by centrifugation, and the pellet was washed once of the cells) in 50 mM Tris MATERIALS AND METHODS (through suspension hydrochloride buffer (pH 8.0). The cells (40 g of wet weight) Microorganisms and growth conditions. N. commune were frozen under liquid nitrogen, ground to a powder, and UTEX 584 was grown as described previously (16) in liquid suspended in 40 ml of lysis buffer (15% [wt/vol] sucrose, 10 BG 110 medium (18). Anabaena variabilis PCC 7118 was mM EDTA, 25 mM Tris hydrochloride [pH 8.0]). This suspension was frozen (under liquid nitrogen) and thawed a * Corresponding author. total of five times. Solid lysozyme (10 mg ml-', final t Present address: Institute of Physiological Botany, University concentration) was added to the suspension, which was then of Uppsala, S-751 21 Uppsala, Sweden. incubated at 370C with gentle agitation for 4.5 h. The solution 708 VOL. 171, 1989 EXPRESSION OF NOSTOC iph GENE IN E. COLI 709 TABLE 1. Bacterial strains, plasmids, and bacteriophages Strain, plasmid, or phage Relevant characteristics Source or reference E. coli HB101 F- hsdS20 (rB- mB-) recA13 ara-14 proA2 lacYl galk2 15 rpsL20 (Str) xyl-5 mt1-i supE44 (A-) LE392 F- hsdR5l4 (rK- MK+) supE44 supF58 lacYl or A(lacIZY)6 15 galK2 galT22 metBi trpR55 (A-) ECL8 HfrC phoA8 glpD3 glpR2 relAl spoTI JhuA22 ompF627 T. Larson fadL701 pit-10 (X) C600Hf1 hflA150 [Chr::TnJO] Promega Biotec DH5-a F' endAl (rK mK+) hsdRI7 supE44 thi-I recAl gyrA96 relAl Bethesda Research Laboratories A(lacZYA-argF)U169 480dlacZAM15 (X-) ATCC 23601 Derivative of E. coli B American Type Culture Collection Plasmids pGEM-4 2.87 kb, Apr Promega Biotec pMP004 5.78 kb, Apr, iph (subclone of a 2.9-kb Nostoc genomic DNA This study EcoRI insert fragment [from XgtlOPl] in pGEM-4) pMP005 5.78 kb as pMPOO4, iph insert in reverse orientation (Fig. 1). This study pGAL85 7 kb, Apr, lacZ DuPont pB8 5.9 kb, Apr, iph (subclone of a 2.9-kb Nostoc genomic DNA This study EcoRI insert fragment [from XgtlOPl] in pBluescript M13+; orientated with unique AvaI site proximal to lacZ promoter [Fig. 1]) pBH6 5.9 kb, Apr, iph (same as pB8, insert in reverse orientation) This study Phagemid pBluescript M13+ 2.96 kb, Apr, lacZ Stratagene Inc. Bacteriophages xgtlO srI 10 b527 srI 3° imm434 (srI434+) srI 40 srl 50 Promega Biotec XgtlOPl Recombinant of XgtlO and a 2.9-kb EcoRI fragment of Nostoc This study commune UTEX 584 genomic DNA (iph) became brown and viscous within 2 h after the addition of fragment as a template (in pB8; Table 1). The synthesis was the lysozyme. The solution was then stored at 4°C overnight; performed with T7 RNA polymerase (Promega Biotec), and then 0.4 g of N-lauroylsarcosine and 0.8 mg of proteinase K precautions were observed during all manipulations of RNA. (Boehringer Mannheim Biochemicals, Indianapolis, Ind.; 20 General procedures for Southern transfer were as described Rg ml-', final concentration) were added to the solution, and previously (5). Hybridization was performed under stringent incubation was continued at 50°C for 4 h with gentle agita- conditions: the hybridization buffer contained 45% (wt/vol) tion. Approximately 1 ml of 1 M Tris hydrochloride (pH 9.0) deionized formamide, 5x SSC (lx SSC is 0.15 M sodium was added to the solution to achieve a pH of 7.0 (to chloride plus 0.015 M sodium citrate), 0.5% (wt/vol) sodium compensate for the drop in pH due to addition of N- dodecyl sulfate, 2 mnM disodium EDTA, 10 mM Tris hydro- lauroylsarcosine). The lysate was diluted with 30 ml of buffer chloride (pH 7.5), 2x Denhardt solution, and 5% (wt/vol) (50 mM EDTA, 50 mM Tris hydrochloride [pH 7.5]); then 80 polyethylene glycol (type 8000). Hybridization was per- ml of preequilibrated phenol (12) was added to the mixture, formed at 50°C for 16 h. After hybridization the filter was which was then shaken gently overnight at room tempera- washed first in lx SSC-0.1% (wt/vol) sodium dodecyl sul- ture. The aqueous phase was recovered after centrifugation fate and then in 0.2x SSC-0.1% (wt/vol) sodium dodecyl of the solution and was extracted further with equal volumes sulfate for 40 min at 50°C. Finally, the filter was washed in of phenol, phenol-chloroform (1:1), and then chloroform lx SSC-0.1% (wt/vol) sodium dodecyl sulfate. Blocking of (two extractions at room temperature). The phenol phases the filter with bovine serum albumin was performed at 60°C were extracted with distilled water (30 ml), and all the for 20 min in the presence of vanadyl-ribonucleoside com- aqueous phases were pooled before mixing with an equal plex.