Nostoc Commune UTEX 584 Gene Expressing Indole Phosphate Hydrolase Activity in Escherichia Coli

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. Biotinylated RNA-DNA hybrids were visualized with a volume of isopropanol (-20°C) in the presence of 0.3 M colorimetric assay (Bethesda Research Laboratories). sodium acetate. The DNA was collected by spooling, and Construction of deletion clones. Plasmids carrying dele- the pellet was washed first in 70% (vol/vol) ethanol and then tions of the 2.9-kb EcoRI-EcoRI fragment were constructed in 90% (vol/vol) ethanol (-70°C). The pellet was dissolved in through processive deletion of pB8 and pBH6 (Table 1) with 1 mM EDTA, 10 mM Tris hydrochloride (pH 7.0) and exonuclease III, exonuclease VII, and the Klenow fragment purified further by cesium chloride density gradient ultracen- as described previously (W.-Q. Xie and M. Potts, Gene trifugation (12). Anal. Tech., in press). Purification of plasmid and phage DNAs. Plasmid DNAs Coupled in vitro transcription-translation assay. A cell-free were purified from 1-ml liquid cultures (grown overnight) by system for coupled transcription-translation of DNA was using an alkaline hydrolysis technique (GemnSeq K/RT Tech- obtained from DuPont NEN Research Products. The system nical Manual; Promega Biotec). The preparation of high-titer was supplemented with carrier-free L-[35S]methionine (1,134 liquid lysates and the purification of phage DNA were Ci mmol-1; DuPont). Conditions for the measurement of 35S achieved as described by Silhavy et al. (21). incorporation in translation products, gel electrophoresis, Southern analysis. A biotinylated RNA probe was synthe- and autoradiography were as described previously (11). sized by using the 2.9-kb N. commune UTEX 584 DNA Plasmid and phage DNAs to be used for the transcription- 710 XIE ET AL. J. BACTERIOL.

translation assays were purified through high-performance liquid chromatography. Chromatography was performed with a Gen-Pak FAX column, a DuPont Instruments series 8000 Gradient controller, and Spectro series 8000 high- performance liquid chromatography pump/detector system. A dual buffer system of 50 mM Tris hydrochloride (pH 8.1) and 1 M LiCl (in 50 mM Tris hydrochloride) was used with a gradient from 40 to 80% (vol/vol). Fractions with peaks at A260 were collected and mixed with 2 volumes of 95% (vol/vol) ethanol and then stored at -20'C. After centrifugation the pellets of DNA were washed in 70% (vol/vol) ethanol, dissolved in water, and stored at 40C until needed. Transformation. Competent cells of the various strains of E. coli were transformed with plasmid DNA in the presence of calcium chloride, rubidium chloride, and 3-[N-morpho- FIG. 1. Restriction map of the 2.9-kb DNA fragment from N. lino]propanesulfonic acid (12). commune UTEX 584 carrying iph in pMPOO5 (Table 1). The posi- tions of the phage-specific promoters SP6 and T7 are indicated. Not Detection of enzyme activities. To detect the presence of shown are multiple sites of HincII cleavage; an additional site for enzyme activities in plaques and bacterial colonies, 5- PstI is located between the two PstI sites that are indicated. Sites for bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma Chemical the following endonucleases are absent in the 2.9-kb fragment: Co.) was dissolved in dimethylformamide (50 mg ml-1), and BamHI, BgIII, HindIII, SacI, SmaI, and XbaI. Symbols: 0, AccI; 20 Ad of the solution was spread over the surface of an agar A, AvaI; 0, EcoRI; V, KpnI; *, PstI; *, Sall; O, SphI. plate (25). The plate was allowed to dry and then streaked with cells. The other substrates used to detect enzyme activities were 4-p-nitrophenyl phosphate (PNPP) (28), bis- PNPP, and 5-bromo-4-chloro-3-indolyl acetate (Sigma). Un- ATCC 23601 (Fig. 2a). A single band, corresponding to an less stated otherwise the detection of enzyme activities in EcoRI-EcoRI N. commune UTEX 584 DNA fragment of 2.9 whole cells and cell fractions, spectrophotometric assays, kb, was detected (Fig. 2b). The digests of genomic DNA and the use of the different substrates followed general from A. variabilis PCC 7118 and the two strains of E. coli procedures (1, 2, 4, 29). gave no hybridization signals with the RNA probe. Invitrotranscription-translation. Differentphageandphage- mid preparations (Table 1) were used in coupled transcrip- RESULTS tion-translation reactions to obtain information on the num- Isolation of a gene showing BCIP hydrolase activity. The ber and size of the gene products encoded by the 2.9-kb use of BCIP permitted the detection of a single blue plaque fragment. In parallel experiments the DNA was digested after screening -8,000 plaques in the recombinant library of with EcoRI before the transcription-translation reaction to N. commune UTEX 584 genomic DNA in phage XgtlO. The excise the 2.9-kb fragment and to uncouple any iph tran- single positive plaque was subjected to three rounds of scription from the potential control of vector-specific pro- plaque purification. DNA from the recombinant phage moters. At the completion of the transcription-translation (XgtlOPl) was purified from a 1-liter lysate of E. coli C600Hfl reactions, BCIP was added to the solutions (0.5 mM, final (Table 1); after digestion with EcoRI, the DNA insert (2.9 concentration). Indole phosphate hydrolase activity was kb) was purified through electroelution and subcloned detected in every transcription-translation reaction which through ligation in EcoRI-digested pGEM-4 and pBluescript contained iph DNA, including the reaction that contained M13+ (Table 1). Several strains of E. coli were transformed with recombinant plasmids that carried the 2.9-kb fragment in different orientations (Table 1; Fig. 1). All transformants generated bright blue colonies when they were plated on LB 1 2 3 4 5 agar plates in the presence of BCIP. Nontransformed cells plated under identical conditions gave rise to white colonies. The gene of N. commune UTEX 584 encoding the indole phosphate hydrolase (phosphatase) activity has been termed iph until such time that a definitive assignment (e.g., pho) can be given. Indole phosphate hydrolase activity in E. 1 S l 0 tF2.9kt3~~~~~~~~~~~~4-.9k coli(pMP005) was found associated with whole cells, periplasmic extracts, intact spheroplasts (after osmotic schock treatment), and sonicated spheroplasts (data not shown). Activity was not detectable in culture supernatants or in the cytoplasmic membrane-cell debris fraction, which was obtained after high-speed centrifugation of the sonicated spheroplasts. a b Southern analysis. Since endogenous phosphatases are FIG. 2. Southern hybridization analysis with biotinylated iph synthesized by the strains ofE. coli used in this study, it was riboprobe. (a) Agarose (0.8% [wt/vol]) gel electrophoresis of dif- essential to confirm that the 2.9-kb fragment originated from ferent DNA preparations each digested with EcoRI for 2 h at 37 C. Lanes: 1, genomic DNA of E. coli ATCC 23601 (10 pLg); 2, genomic N. commune UTEX 584 genomic DNA. A biotinylated RNA DNA of A. variabilis PCC 7118 (6 ,ug); 3, genomic DNA of N. probe synthesized with pB8 DNA (Table 1) was used to commune UTEX 584 (10 ,ug); 4, XgtlOP1 DNA (80 ng; EcoRI screen EcoRI digests of genomic DNA from N. commune fragments of 32.7, 10.6, and 2.9 kb (iph); 5, X HindIII size markers UTEX 584, A. variabilis PCC 7118, and E. coli C600Hfl and (0.1 ,ug). (b) Southern transfer of gel in Fig. 2a. VOL. 171, 1989 EXPRESSION OF NOSTOC iph GENE IN E. COLI 711

TABLE 2. Expression of iph in a cell-free coupled promoter failed to confer an Iph' phenotype in transfor- transcription-translation system mants (data not shown). Transformation of E. coli ECL8 Plasmid DNA template Vector EcoRI Lane in (phoA). DNA digest Fig. 3 activityIph isolated and purified from E. coli LE392 (rK- mK+)(pMP005) was used to transform E. coli ECL8 (phoA), a strain that None (control) - 1 lacks pGAL85 (control) pBR322 - 2 alkaline phosphatase. E. coli strains LE392(pMP005) XgtlOP1 xgt1O - 3 + and ECL8(pMP005) both hydrolyzed BCIP when plated on Xgt1OP1 XgtlO + 4 + LB agar plates, whereas E. coli strains LE392 and ECL8 2.9-kb iph fragment - 5 + gave rise to white colonies. Hydrolysis of BCIP by strains of pB8 pBluescript M13+ + 6 + E. coli transformed with pMP005 persisted when growth pB8 pBluescript M13+ - 7 + media were supplemented with 10 to 100 mM NaH2PO4- pB8 pBluescript M13+ - 8 + Na2HPO4. pB8 pBluescript M13+ + 9 + Substrate of BCIP E. coli pBH6 - specificity. Hydrolysis by pBluescript M13+ 10 + was unaffected when indole was pBH6 pBluescript M13+ + 11 + HB101(pMP005) included in assay buffers at concentrations up to 10 times the BCIP substrate concentration (40 to 200 ,uM, final concentration of indole). No color reaction was detected when 5-bromo- the 2.9-kb fragment free of any vector DNA (Table 2). 4-chloro-3-indolyl acetate was used in place of BCIP in Activity persisted when XgtlOP1, pB8, or pBH6 DNA was standard assays with whole cells or cell extracts of E. coli digested with EcoRI before use. A single polypeptide with an HB1O1(pMP005). Mr of approximately 74,000, not present in control assays, was the most obvious reaction product common to the DISCUSSION different transcription-translation assays (Fig. 3). The inten- sity of the band at Mr 74,000 was equivalent for most of the We have isolated a gene (iph) that encodes an indole assays, with the exception of the weaker signals for XgtlOP1 phosphate hydrolase from N. commune UTEX 584 as an DNA, where the molar concentration of iph DNA was early step in a long-term study of phosphatase activities of reduced relative to the input DNA (Fig. 3, lanes 3 and 4). An cyanobacteria. Southern. analysis indicated that a single additional polypeptide with an Mr of approximately 38,000 copy of iph is present within the genome of N. commune was synthesized only in the reaction supplemented with UTEX 584. In addition to the iph coding sequence, the pBH6 DNA but not when the pBH6 DNA was predigested 2.9-kb EcoRI fragment of N. commune UTEX 584 DNA with EcoRI (Fig. 3, lanes 10 and 11). carries regulatory sequences that permit the expression of Deletion analysis. Derivatives of pB8, with deletions of the iph gene both in E. coli and in a cell-free coupled approximately 1.2 kb or less at the end of the 2.9-kb insert transcription-translation system derived from E. coli. Con- distal to the lac promoter, conferred an Iph+ phenotype cerning the latter, the ability to synthesize active enzyme upon transformants of E. coli DH5-a. pB8 derivatives with from only the purified 2.9-kb fragment suggests the presence larger deletions in this region of the insert or with small of both an iph promoter sequence in the DNA and a deletions at the end of the insert proximal to the lac ribosome-binding site on the iph transcript. However, the possibility that a fortuitous E. coli-like promoter was used cannot be discounted at this time. 12 3 4 5 6 7 8 9 1011 In vitro transcription-translation analyses demonstrated that two polypeptides were synthesized from the 2.9-kb 200- fragment when it was incorporated in different DNA templates. The larger of the two polypeptides (Mr, 74,000) was .t.~ synthesized with each of the different DNA templates, including those predigested with EcoRI. Synthesis of the 97- smaller polypeptide (Mr, 38,000) occurred only in reactions with pBH6 DNA, but synthesis was prevented when pBH6 DNA was digested with EcoRI before the transcription- translation reaction. Synthesis of the smaller polypeptide 6-74 was not observed when DNA 68- pB8 (2.9-kb fragment in reverse orientation with respect to lac promoter) was used under identical conditions. Furthermore, deletions of up to 1 kb at the end of the insert distal to the lac promoter (in pB8) did not prevent synthesis of indole phosphate hydrolase. In these respects, indole phosphate hydrolase activity cannot 43- be attributed to the smaller polypeptide, which is most likely 4-38 a LacZ fusion product. Synthesis of a fusion protein of this size would be expected if, as indicated from deletion analyses, iph regulatory sequences (and the 5' end of the coding FRONT sequence) are located approximately 1 kb downstream of the lac FIG. 3. Autoradiogram of reaction products from cell-free cou- promoter (in pBH6). Autoradiographic analysis showed pled transcription-translation reactions with different iph DNA the smaller (fusion) protein to have a stronger signal than the templates (Table 2). Identical quantities of radioactivity (106 cpm) larger polypeptide (Fig. 3, lane 10). It is uncertain whether were loaded for each of the different samples. Numbers refer to this represents increased synthesis of the smaller protein or sizes of polypeptides (in kilodaltons). The major band of reference in its greater enrichment in [35S]methionine residues. Increased lane 2 (control) represents LacZ (Mr, 115,000). synthesis of the smaller protein would result from a more 712 XIE ET AL. J. BACTERIOL. efficient use of the ribosome-binding site for lacZ, as op- product may carry sequences that permit the translocation of posed to that for iph, in the E. coli-derived transcription- the protein across the cytoplasmic membrane of E. coli (7, translation system. Although the present data suggest that 14, 17). the protein with an approximate Mr of 74,000 is the gene In summary, we have cloned a nitrogen-fixing cyanobac- product of iph, final confirmation must await DNA sequence terium N. commune UTEX 584 phosphatase gene which can analysis and purification of Iph. be expressed, possibly from its own promoter, in E. coli. Indole phosphate hydrolase activity in whole cells of N. The gene product is found predominantly in the periplasm, commune UTEX 584 was regulated by the availability of where it is active, and the expression of iph both in whole phosphate. However, Pi was unable by itself to repress the cells of E. coli and in cell-free extracts can be visualized indole phosphate hydrolase activity either in whole cells or readily by hydrolysis of BCIP. in cell extracts of E. coli (Iph'). This was not unexpected, The iph gene is useful for the study of phosphatase since phosphatases are known to be subject to complex regulation in cyanobacteria and also may prove useful for the regulation. The alkaline phosphatase activity of the cyano- study of cyanobacterial promoter function and the process- bacterium Anabaena cylindrica was increased sevenfold ing of cyanobacterial membrane proteins, areas of study for after growth of the cells in phosphate-free medium, whereas which the data are limited (3, 13, 20). Coccochloris peniocytis appeared to be constitutive for this activity as no induction was observed upon starving the cells ACKNOWLEDGMENTS for phosphate (6). Regulation of alkaline phophatase in E. coli, for which there is the best understanding (24-27), is We are grateful to T. Larson for providing us with strains of E. specifically induced by phosphate starvation, as are other coli and for many helpful discussions. We also thank L. Duncan for assistance and H. Schweitzer, R. Ebel, and B. Anderson for their alkaline phosphatases (30), but there is evidence for multiple suggestions. positive regulators (28), and the scope of negative control by This study was supported by National Science Foundation grants phoR is not resolved completely (25, 27). The expression of DMB 8543002 and DCB 883068 (to M.P.). E. coli periplasmic acid phosphatase (pH optimum of 2.5) is also subject to complex regulation (2, 4). The activity of this LITERATURE CITED enzyme is influenced by phase of growth, presence or 1. Berg, P. E. 1981. Cloning and characterization of the Esche- absence of oxygen, the concentration of Pi in the medium, richia coli gene coding for alkaline phosphatase. J. Bacteriol. and the level in the cells of cyclic AMP as well as its receptor 146:660-667. protein (2). Evidence for multiple regulatory elements for the 2. Boquet, P. L., C. Manoil, and J. D. Beckwith. 1987. Use of acid phosphatase was suggested after observing that in TnphoA to detect genes for exported proteins in Escherichia standard assays with PNPP, Pi was not inhibitory (Ki, 13 coli: identification of the plasmid-encoded gene for a periplas- mM) (4). mic acid phosphatase. J. Bacteriol. 169:1663-1669. Hydrolysis of PNPP by the indole phosphate hydrolase in 3. Bullerjahn, G. S., and L. A. Sherman. 1986. Identification of a carotenoid-binding protein in the cytoplasmic membrane from the E. coli transformants was obscured by a much higher the heterotrophic cyanobacterium Synechocystis sp. strain background level of PNPP hydrolysis due to host periplas- PCC6714. J. Bacteriol. 167:396-399. mic phosphatases (29). The high PNPP background hydrol- 4. Dassa, E., M. Cahu, B. Desjoyaux-Cherel, and P. L. Boquet. ysis was observed for all the strains tested, the apparent pH 1982. The acid phosphatase with optimum pH of 2.5 of Esche- optima were between 5.0 to 6.0, and the activity persisted in richia coli. Physiological and biochemical study. J. Biol. Chem. the presence of phosphate. It appears that these host acid 257:6669-6676. phosphatases have less affinity for BCIP than does the 5. Defrancesco, N., and M. Potts. 1988. Cloning of nifHD from cyanobacterial indole phosphate hydrolase (or the host alka- Nostoc commune UTEX 584 and of a flanking region homolo- line phosphatase). This explains how it was possible to gous to part of the Azotobacter vinelandii nifU gene. J. Bacte- of on the lawn riol. 170:3297-3300. detect the single blue positive plaque XgtlOP1 6. Doonan, B. B., and T. E. Jensen. 1980. Ultrastructural localiza- of E. coli C600 (hfl). tion of alkaline phosphatase in the cyanobacterium Coccochlo- The indole phosphate hydrolase does not cleave 5-bromo- ris peniocytis and Anabaena cylindrica. Protoplasma 102:189- 4-chloro-3-indolyl acetate, suggesting that activity is not due 197. to an esterase, and the hydrolysis of BCIP was unaffected by 7. Ferenci, T., and T. J. Silhavy. 1987. Sequence information excess indole. The latter result confirms that BCIP hydrol- required for protein translocation from the cytoplasm. J. Bac- ysis is not an artifact of some recognition of the cloned gene teriol. 169:5339-5342. product for the indole ring system of BCIP. 8. Garin, A., and C. Levinthal. 1960. A fine structure genetic and The cellular localization of phosphatases, even those with chemical study of the enzyme alkaline phosphatase. Biochim. rather similar properties, varies with the organism. For the Biophys. Acta 38:470-483. 9. Glover, D. M. (ed.). 1985. DNA cloning, vol. 1., a practical prokaryotes E. coli and Micrococcus sodonensis the alkaline approach. IRL Press, Oxford. phosphatases are located in the periplasm and extracellu- 10. Healey, F. P. 1982. Phosphate, p. 105-124. In N. G. Carr, and larly, respectively, whereas for the yeast Neurospora crassa B. A. Whitton (ed.), The biology of cyanobacteria. Blackwell the enzyme is intracellular (30). The phytoflagellate Scientific Publications, Oxford. Ochromonas danica secretes an acid phosphatase that 11. Jager, K., and M. Potts. 1988. In vitro translation of mRNA shows properties different from those of the intracellular from Nostoc commune (Cyanobacteria). Arch. Microbiol. 149: enzyme (15). Different intra- and extracellular acid phos- 225-231. phatases have also been described for fungi (19). Phos- 12. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular phatase activity in A. cylindrica was found to be associated cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. with the cell wall (6), in contrast to C. peniocytis, where 13. Olie, J. J., and M. Potts. 1986. Purification and biochemical phosphatase was secreted. Indole phosphate hydrolase ac- analysis of the cytoplasmic membrane from the desiccation- tivity in E. coli transformants was found exclusively external tolerant cyanobacterium Nostoc commune UTEX 584. Appl. to the cytoplasmic membrane and was associated predomi- Environ. Microbiol. 52:706-710. nantly with the periplasm. This suggests that the iph gene 14. Oliver, D. 1985. Protein secretion in Escherichia coli. Annu. VOL. 171, 1989 EXPRESSION OF NOSTOC iph GENE IN E. COLI 713

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