CURRENT MICROBIOLOGYVol. 25

(1992), pp. 291-295

Current Microbiology O Springer-Verlag New York Inc. 1992

Cloning and Expression in Escherichia coli of an Alkaline Phosphatase (phoA) Gene from Zymomonas mobiIis T. K a r u n a k a r a n and P. G u n a s e k a r a n Department of Microbiology and Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai, India

A b s t r a c t . An alkaline p h o s p h a t a s e (phoA) gene from Z y m o m o n a s mobilis was isolated in Esche-

richia coli CC118 by use o f the plasmid Bluescript KS + . The origin o f the 6.4-kb D N A fragment in pZAP1 f r o m the c h r o m o s o m e o f Z . mobilis was confirmed by Southern blotting and hybridization studies. The Z. mobilis p h o A gene was localized at one end of the c h r o m o s o m a l insert on plasmid pZAP1. The Z. mobilis p h o A gene was e x p r e s s e d from its o w n p r o m o t e r in E. coli, and the e n z y m e was localized to the periplasmic space. Z. mobilis alkaline p h o s p h a t a s e activity in E. coli was repressed in high-phosphate media and derepressed under a phosphate-limited growth condition. T h e s e results suggest that Z. mobilis alkaline p h o s p h a t a s e is subjected to normal regulation in E. coli.

Like nitrogen, p h o s p h o r u s is an essential element in cells. Because m o s t of its natural source occurs as insoluble salts, p h o s p h a t e acts like a growth-limiting factor for m a n y organisms. Bacteria such as Escherichia coli have evolved c o m p l e x regulatory mechanisms to o v e r c o m e the p h o s p h a t e limitation and to assimilate the p h o s p h o r u s c o m p o u n d s . This system is called pho regulon [23], whose genes are scattered around the c h r o m o s o m e , and their products are found in cellular c o m p a r t m e n t s [14]. A similar m e c h a n i s m for the synthesis of e n z y m e s involved in p h o s p h a t e metabolism has also been described in diverse bacteria [7], in S. cerevisiae and in Neurospora [19]. Alkaline p h o s p h a t a s e (EC 3.1.3.1) was of prime interest to m a n y researchers because it is useful in gene fusion experiments with E. coli p h o A as a r e p o r t e r gene [16]. The gene can be utilized to study such diverse matters as protein localization and m e m b r a n e topology, as its product is active only when outside the p l a s m a m e m b r a n e . The Gram-negative, obligately fermentative b a c t e r i u m Z y m o m o n a s mobilis is well k n o w n for its tolerance to high levels of alcohol produced in the growth m e d i u m during fermentation [10, 18, 22]. Z. mobilis has been shown to possess m o r e than 70% of the fatty acids in the phospholipid fraction as cisvaccenic acid [3, 24]. It also contains a hoponoid content o f about 30 mg/g of dry cell weight, the highest concentration o b s e r v e d in bacteria so far

[9]. These specific traits allow Z. mobilis to easily survive a highly alcoholic e n v i r o n m e n t . Despite these biochemical studies, information on the envelope proteins and protein localization in Z. mobilis is still lacking. F o r elucidation of these m o l e c u l a r mechanisms, it would be appropriate to study a cell m e m b r a n e - a s s o c i a t e d e n z y m e such as alkaline phosphatase. In this study, the p h o A gene encoding alkaline p h o s p h a t a s e of Z. mobilis is cloned and its expression studied. Materials and M e t h o d s Bacteria and plasmid. Escherichia coli strains used in this study and sources are listed in Table 1. Zymomonas mobilis NRRL B-806 (obtained from Northern Regional Research Laboratory, Peoria, Illinois) was used for preparing chromosomal DNA. Z. mobilis was grown in RM medium [6] at 30~ without shaking. E. coli strains were grown in LB, M63 [6], or LP (low phosphate, [17]; glucose 2.0, Hepes 23.8, proteose peptone 5.0, NH4CI 1.1, KC11.5, MgCI20.3, g/L, pH 7.2) media with or without ampicillin (Ap 50 /xg/ml) at 37~ under aerobic conditions. The cloning vector Bluescript KS + (Stratagene, USA) was used for the construction of a genomic library. DNA manipulations. Chromosomal DNA from Z. mobilis was

prepared as described earlier [6] and partially digested with Sau3A, size-fractionated (4-8 kb), and ligated into the dephosphorylated BamHI site of Bluescript KS +. The ligation mixture was transformed into E. coli CCll8-competent cells, and the transformants were selected on LB-agar containing Ap (50/xg/ ml), IPTG (40 tzg/ml), and X-gal (20/zg/ml). The recombinant

Address reprint requests to: Dr. T. Karunakaran, Department of Periodontics, The University of Texas Health Sciences Centre, San Antonio, TX 78284-7894, USA.

292

CURRENTMICROBIOLOGYVO1.25 (1992)

Table 1. Escherichia coli strains used in this study Strains K10 CC118 U3 El5 LEP-1 C75a

Relevant g e n o t y p e wild type A phoA20

phoA14 A phoA8

phoB23 pho64

Source/reference CGSC 5023a [16] CGSC 4917a [5] CGSC 4829~ [8] CGSC 5681a [1] CGSC 5978a

a Obtained from B. Bachmann, E. coli Genetic Stock Center, Dept. of Biology, Yale University, New Haven, Connecticut.

clones were further replicated onto LB-agar plates containingAp (50 p,g/ml) and 5-bromo-4-chloro-3-indolylphosphate (Xp, 40 p,g/ ml). The positive clones were also confirmed by replica plating onto M63-agar plates containing Ap and Xp. All DNA manipulations such as rapid plasmid isolation, Southern transfer, and hybridization were performed as described in Maniatis et al. [15]. The 1.9-kb Pstl fragment from pZAP1 was biotin-ll-dUTPlabeled by the BRL random primers labeling system (BRL Life Technologies, Inc., Gaithersbnrg, Maryland) and used as a probe. The bound probe was developed by the streptavidin-alkaline phosphatase method with the BRL nonradioactive nucleic acid detection system (BRL Life Technologies, Inc.).

Enzyme assays. Alkaline phosphatase was assayed in culture su9 pernatant, toluenized cell suspensions, and in osmotic shock fluid [25], or after ultrasonic disruption [11] of cells with Sigma-104 phosphatase substrate. The alkaline phosphatase assay was performed and activity calculated according to Brickman and Beckwith [2]. Cellular extracts prepared after sonic disruption were loaded onto SDS-PAGE [12] and analyzed after Coomassie blue staining. After the cellular extract was resolved on a native PAGE, blotted onto a nitrocellulose filter, and blocked overnight with BSA, the alkaline phosphatase activity was detected with a mixture of nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3indolyl phosphate (Xp) [20]. Protein was estimated by the method of Lowry et al. [13].

Results and Discussion Cloning o f Z . mobilisphoA gene in E. coli. A genomic library of the c h r o m o s o m a l D N A of Z. mobilis N R R L B-806 was constructed by cloning Sau3A restricted and size-fractionated fragments into the BamHI site of the plasmid Bluescript KS + and then transforming into E. coli CC 118, a strain deleted for phoA gene. The library consisted of 1600 independent colonies. By screening of the library, two clones that restored alkaline phosphatase activity on L B - X p plates as an intense blue color were isolated. Plasmid D N A preparations from each of these two clones were capable of transforming E. coli strains CCl18 (AphoA20) and E l 5 (AphoAS) at high freq u e n c y to alkaline phosphatase activity. These re-

suits indicate that complementation does not result from the cloning of a suppressor gene.

Restriction mapping and localization of Z. mobilis phoA gene. Both the recombinant plasmids (pZAP1 and pZAP2) exhibited the presence of the same 6.4kb insert fragment. A restriction map was generated following digestion of pZAP1 with various restriction endonucleases (Fig. 1). To precisely localize the phoA gene in the insert fragment, a large number o f deletions were constructed and tested for the ability to complement the phoA deletion in E. coli CC 118. Removal of either 1.1-kb EcoRI (plasmid pZAP10) or 1.9-kb PstI (plasmid pZAP11) fragment from the plasmid pZAP1 resulted in the complete loss of alkaline phosphatase activity. H o w e v e r , removal of the 2.4-kb BglII fragment (pZAP12) did not affect the activity. Likewise, removal of the 3.1-kb HincIIHindIII (plasmid pZAP13) from p Z A P I resulted in complete retention of the activity. Thus, Z. mobilis phoA was localized at one end of the c h r o m o s o m a l insert following subcloning of the 2.4-kb BamHIHindIII fragment into BamHI-HindIII sites of the plasmid Bluescript KS + (plasmid pZAP14).

Southern blotting and hybridization. Southern blotting and hybridization studies were used to confirm the origin of the 6.4-kb insert in pZAP1 from Z. mobilis c h r o m o s o m e (Fig. 2). Z. mobilis N R R L B806 chromosomal D N A was digested with PstI, EcoRV, and HindIII. The restriction fragments were resolved on an agarose gel (0.7%), transferred to nitrocellulose filter, and hybridized to biotin-lldUTP-labeled 1.9-kb PstI probe, internal to Z. mobilis phoA gene, derived from the plasmid pZAP1. A positive hybridization band was obtained around 2.4-kb for Z. mobilis c h r o m o s o m a l D N A digested with PstI. Similarly, a single band o f each around 6.5 and 7.0 kb was seen for c h r o m o s o m a l D N A digested with E coR V and HindIII respectively. These results confirm that the origin of the insert in pZAP1 is from Z. mobilis chromosomal D N A . Expression and localization of Z. mobilis alkaline phosphatase in E. coli. Maximum alkaline phosphatase activity was found with E. coli carrying the plasmid pZAP1. E. coli carrying the v e c t o r alone produced just 7.3% of the activity exhibited by the recombinant plasmid pZAP1. Addition of IPTG during the growth of E. coli cells containing pZAP1 showed no effect on alkaline phosphatase activity (data not shown), indicating that the Z. mobilis phoA gene is expressed from its own promoter.

T. Karunakaran and P. Gunasekaran: Z. mobilis Alkaline Phosphatase

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2

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~

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Fig. 1. Subcloning and deletion analysis of the recombinant plasmid pZAP1. The line represents Z. mobilis chromosomal insert, and the broken line represents the deleted portion of the insert. The ability to complement E. coli phoA mutation was correlated with the physical map to localize the coding region of phoA (marked by an arrow) on the recombinant plasmids, and the alkaline phosphatase activity is indicated in the column to the right. E. coli CC118 carrying only the vector (pBluescript KS +) exhibited just 7.3% of the relative activity. HIII, HindIII; EV, EcoRV; EI, EcoRI; P, PstI; BII, BglII; B, BamHI; HII, HincII; BI, BglI; X, XbaI; S, S a d .

Table 2. Localization of Z. mobilis alkaline phosphatase activity in E. colia Secreted into the medium

Released in shock fluid

Remaining in cells

11.35 -+ 4.40

84.18 + 4.18

4.47 -+ 4.34

a Enzyme activity is expressed as a percentage of total activity.

Fig. 2. Southern transfer and hybridization of Z. mobil& NRRL B-806 chromosomal DNA with biotin-11-dUTP-labeled, 1.9-kb PstI fragment derived from the plasmid pZAP1. Lane a: plasmid Bluescript KS + ; lanes b-d: Z. mobilis chromosomal DNA digested with various enzymes; lane b, PstI; lane c, EcoRV; lane d, HindIII.

In many Gram-negative bacteria alkaline phosphatase is a periplasmic enzyme [4]. The localization ofZ. mobilis alkaline phosphatase activity was done by submitting E. coli El5 carrying pZAP1 to osmotic shock as described by Willis et al. [25]. It was found that Z. mobilis alkaline phosphatase was cell associated in E. coli. About 84% of Z. mobilis alkaline phosphatase was released into the osmotic fluid (Table 2). Under similar conditions, wild-type E. coli (K 10) released about 91% of its alkaline phosphatase activity into the osmotic fluid. These results indicate that Z, mobilis alkaline phosphatase activity is localized in E. coli periplasmic space. E. coli cells carrying various recombinant plasmids mentioned in Fig. 1 or pBluescript KS + were

294

CURRENTMICROBIOLOGYVol. 25 (1992) Table 3. Alkaline phosphatase levels in E. coli strains carrying the plasmid pZAP1 Alkaline phosphatase levela (mU/A600) E. coli strains CCll8 E15 U3

(AphoA20) (AphoA8) (phoA14)

HP mediumb

LP medium

0.027 0.031 0.039

0.742 0.587 0.673

a Results represent the average of three independent experiments. b LP medium supplemented with 10 mM phosphate.

Fig. 3. SDS-PAGE analysis of the polypeptides encoded by the recombinant plasmids carrying Z. mobilis phoA gene. Lane a, pZAPI3; lane b, pZAP12; lane c, pZAPll; lane d, pBluescript KS +; lane e, molecular weight markers; lane f, pZAP1.

grown overnight in LB-broth containing Ap (100/xg/ ml) and sonicated. When the cellular extract was subjected to S D S - P A G E (Fig. 3) after Coomassie blue staining, a major polypeptide of about 60 kDa was observed in E. coli cells carrying the Z. mobilis p h o A gene. H o w e v e r , this polypeptide was not detected in extracts of E. coli cells carrying pZAP11 or pBluescript KS + . The molecular weight of Z. mobilis P h o A was also confirmed by active staining as described in Materials and Methods. Various E. coli strains defective in p h o A gene were complemented to alkaline phosphatase activity with pZAP1. They were grown in low- and highphosphate media containing Ap (100 /xg/ml), and alkaline phosphatase activity was measured with their cell extract. Activity in all three strains was repressed in high-phosphate medium and was derepressed under phosphate-limited growth conditions. The derepression of alkaline phosphatase in these strains ranged from 17- to 27-fold (Table 3). These results suggest that Z. mobilis p h o A gene is normally regulated by phosphate, and Z. mobilis p h o A is expressed from its own promoter. Earlier, Michel and Baratti [17] reported an alkaline phosphatase in Z. mobilis that is not phosphate repressible. The results presented here are contrary to their finding, as the p h o A gene is subjected to normal regulation by phosphate in E. coli. H o w e v e r , the molecular weight of Z. mobilis P h o A is within the range of the molecular weight suggested by Michel and Baratti [17]. It is possible that the same e n z y m e is regulated differently in E. coli and Z. mobilis. Usually in E. coli,

the control of p h o A gene expression in response to phosphate limitation requires the function of both the positive (phoB) and negative (phoR) regulatory genes [21, 23]. The plasmid pZAP1 failed to complement E. coli strains LEP-1 or C75a, indicating that the plasmid pZAP1 contains neither of the E. coli analogous genes.

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