6ene, 86 (1990) 95-98



GENE 03373

Short Communications

Cloning and expression of draTG genes from Azospirillum lipoferum (Recombinant DNA; nitrogen fixation; covalent modification; ADP-ribosylation; P~c promoter; phage 2. library) H.-A. Fu', W.P. Fitzmaurice, G.P. Roberts b and R.H. Burris" Departments of ° Biochemistry and bBacte6olofy, and Centerfor the Study of Narogen F'tr.agon. College of Agrtculural and Life $c~ces. University of Wisconsin-Madison. Madison. WI 53706 (U.S.A.) Tel. (608) 262-2675 Received by C.R. Hutchinson: 10 August 1989 Revised: 12 September 1989 Accepted: 15 September 1989


A genomic library ofA:ospi6ilum lipoferum was constructed with phage 2.EMBIA as vector. From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), dra6, were cloned by hybridization with the heterologous probes ofRhodospi6llum rubrum. As in R. rubrum, draT is located between draO and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system). In the crude extract of Eschevichla coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity ofthe cloned genes. Southern hybridization with genomic DNA from different A:ospi~llum spp., demonstrated a correlation between observable draT6 hybridization and the biochemical demonstration of this covalent modification system.


Biological nitrogen fixation is catalyzed by the nitrogenase complex, which consists of two components" dinitrogenase (MoFe protein or component I, nifDK gene product) and dinitrogenase reductase (Fe protein or component II, nil/-/gene product) (Ludden and Burris, 1986). Correspondenceto: Dr. R.H. Burris, Department of Biochemistry,University of Wisconsin-Madison, 420 Henry Mall, Madison, Wi 53706 (U.S.A.) Tel. (608) 262.3042; Fax (908) 262-3453. " Present address: Department of Genetics, North Carolina State University, Raleigh, NC 27695 (U.S.A.) Tel. (919) 737-2287. Abbreviations: A, Azospirillum; aa, amino acid(s); Ap, ampicillin; bp, base pair(s); DRAG, dinitrogenase reductase activating glycohydrolase; DRAT, dinitrogenase reductase ADP.ribosyltransferase; IPTG, isopropyl.p.D-thiogaiactopyranoside; kb, 1000 bp; Km, kanamycin; nt, nucleotide(s); oligu, oligudeoxyribonucleotide; Ptac, trp-la¢ hybrid promoter; Rr2, dinitrogenase reductase of R. rubmm; PAGE, polyacrylamide-gel electrophoresis; SDS, sodium dodecyl sulfate; [ ], denotes plasmidcarrier state. 0378-1119/90/$03.50 O 1990ElsevierScience Publishers B.V.(BiomedicalDivision)

Because of the high energy cost of the reaction, the nitrogenase system is tightly controlled at the levels of both gene expression and enzyme activity. A. lipo/erum is a Gram - microaerophilic N2-fixingbacterium (DObereiner and Pedrosa, 1987). It is readily isolated from the surface-sterilized roots of agronomically important cereal plants, such as wheat, rice and corn. Due to its potential significance in agriculture (Okon, 1985),Azospi~llure spp. have attracted research interest. Physiological and biochemical evidence has been accumulated suggesting that A. [ipo/erum, as well as A. brasilense, possesses a posttranslational regulatory system for nitrogenase activity, apparently via ADP-ribosylation of dinitrogenase rcductase (Ludden et al., 1978; Hartmann et al., 1986; Fu et al., 1989a). The ADP-ribosylation system for nitrogenase is well studied in the photosynthetic N2-fixing bacterium R. rubrum (Ludden and Roberts, 1989). The modification of Rr2 is carried out by DRAT. NAD serves as the ADP-ribose donor with a single ADP-ribose group being attached to

96 one of the two identical subunits of the dimeric Rr2, resulting in inactivation of the enzyme. The activation of ADPribosylated Rr2 is catalyzed by DRAG, which removes the ADP-ribose group. In rive the modification reaction is favored when cells are in the dark or treated with ammonia, and activation is favored under light sufficiency and nitrogen limitation (Kanemoto and Ludden, 1984). Recently, the R. rubrum genes coding for DRAT (draT) and DRAG (draG) have been cloned and sequenced (Fitzmaurice et al., 1989), and this draT gene has been functionally expressed in the heterologous hosts Escherichia coli and Klebsieila pneumoniae (Fu et al., 198%). We report here the molecular cloning of the draTG genes of A. lipoferum by heterologous hybridization and their functional expression in E. coll.


(a) Cloning of draTG.homologous genes A genomic library of A. lipoferum was constructed by ligation of partially digested total cellular DNA (using Sau3AI) into the BamHI-treated arms of ~EMBLA (Promega Biotec, Madison, Wl). For the heterologous hybridization, an internal restriction fragment of drag of R. rubrum (Fig. IA, Fitzmaurice et al., 1989) was labeled with 32p by the oligo-labeling technique (Feinberg and Vogelstein, 1983) and used to probe the library after transfer A,

R, rut)rum 1 kid





B. A. lipoferum

nifH dmT drag





~u. m m ,,,


Insert of pilAF102:



era r



0,8 0,7 0,9 1.1 0,0 0,4 kb I I t I I I I

Fig. I. Restriction map of draTG regions. (A)The draTG region of

R. rubmm and the restriction fragments used as probes (Fitzmaurice et al., 1989; LJ. Lehman, W.P.F. and G.P.R., submitted). (B) The d~TG region of A. iipofemm indicating the region of draTG hybridization, proposed gene orientation and the region cloned on pHAFI0?. This

physicalmapis producedfroma combinationofpublishedwork(Fahsold et al., 1985) and current work. For the Southern hybridizations and washing(lowstringency)standardprocedures were followed(Maniatis et al., 1982;Fitzmanri~ et al., 1989;Fu et al., 1989a).

of the plaque's DNA to a nylon membrane (Colony/Plaque Screen, DuPont, Wilmington, DE). Phage from a plaque which hybridized to the draG probe were amplified and used for further analysis. This phage carried a 15-kb BamHI insert. The region hybridizing to the draG probe was localized to a 4.5-kb BamHI-EcoRI fragment that was subsequently cloned into pUCl9 (Yanisch-Perron et al., 1985) to yield pHAFI02 (Fig. IB). Restriction mapping of this region revealed five Sail sites. The internal draG probe hybridized to the 1. l-kb and 0.6-kb Sail fragments. As an indication of the orientation of draG transcription, a 64-fold degenerate 17-nt ofigo probe deduced from the N-terminal aa sequence of purified DRAG ofR. vubrum (Fitzmaurice et al., 1989), was labeled at the 5' end with 3zp using 1"4 polynucleotide kinase (Promega Biotec, Madison, WI), and used in Southern hybridization. This probe hybridized to the l.l-kb Sail fragment, 'suggesting that draG is transcribed toward the EcoRl site (Fig. 1B). Since the draG and draT genes in R. mbrum are contiguous, with the same orientation oftranscription, the location of the draT-homologous gene was investigated. A 32p. labeled draT probe (Fig. IA) hybridized to the 0.9-kb Sail fragment in pilAF 102, located immediately upstream from the draG-hybridizing sequence (Fig. IB). It is quite possible that the A. llpoferum draTgene extends into the 1. l-kb Sail fragment, but was not detected there since the draT probe lacks the 263 bp at the 3' end of draT.

(6) Locution of draTO relstive to nOT/ The dinitrogenase recluctase protein is the substrate for DRAG and DRAT, In R. rubrum, the nifH gene is located next to draT and is divergently transcribed (Fitzmaurice et al., 1989). Two lines of evidence suggest that the draTG region of A. I(oo[erum also is next to the nifH gene: (i)the restriction map of nifl'IDIC region from A. I#~oferum (Fahsold et al,, 1985) is identical to that of the left side of the insert in pHAFI02 (Fig, lb); (fl) 32P-labeleddraT, draG and nifH probes ofR, rubnon (Fig, la) each hybridize to the same sized fragments of genomic A. lipofemm DNA (a 6.3-kb EcoRl fragment, a 23-kb H/ndIIl fragment, and a 24-kb Xhol fragment). Digestion with BamHl separated in ntJ"and dra hybridizing regions (drag to a I5-kb BamHl fragment or a 4.$-kb BamHl-£coRI fragment and n/JH to a 4.8-kb BamHl fragment or 1.7-kb and 2.5-kb BamHIEcoRI fragments). These hybridization patterns are all consistent with the predictions of the map of Fig. IB strongly suggesting that draTG is located next to n/fH with an intervening gap of about 2.1 kb. Some hybridization was detected in this gap region to a 32P-labeled nifJ probe (3.7-kb Sail fragment ofpJC371; Collins et al., 1986) from K. pneumoniae under low stringency conditions (Fu et al., 1989a).

97 (c) Expression of DRAG and DRAT activities in Escherickia coU

To verify the functionality of the cloned region and gain insight into gene orientation, an expression vector was constructed. The vector, pilAF210, contains the draTG sequence in pKK223-3 (Pharmacia Ltd., Piscataway, NJ) (Fig. 2), which has the inducible Pt,c promoter. The pilAF210 vector was transformed into E. coli strain CAG2041 (which carried F'/acl Q) to produce strain UQ790. To assay the DRAG and DRAT activities, E. coil cultures were grown in LB selective medium (1% tryptone/0.5• yeast extract/l ~ NaCI pH 7.5/50 #g Km per ml/100 #g Ap per ml) at 37°C. IPTG (l mM) was added to induce the expression of the Ptac controlled gene for 4 h. For the DRAG assay, crude extracts were prepared anaerobically as described earlier (Saari et al., 1983; Fu et al., 1989a). DRAG activity was estimated by coupling Hindlll Psti Sall BamH! Sinai £coR!



Ap pKK223-3


the activation of the inactive (ADP-ribosyisted) Rr2 to the acetylene reduction assay for nitrogenase activity (Burris, 1972; Saari et al., 1983). The crude extracts from £. co/i UQ790 (draT + G ÷ ) catalyzed the activation of the inactive nitmgenase complex (6.46 nmol C2H 4 formed/man/rag pro. rein). The crude extracts from the strain UQ477[pKK2233] showed no detectable activity. UQ790 grown in the absence of IPTG also had significant activity (2.7 nmol/mg/min because of leaky repression, as reported by others (Amann etal., 1983; Fu etal., 1989b). The DRAG activity in crude extracts of E. cola UQ790 is approx. one-fifth of that detected in extracts of A.//pofemm (Fu et al., 1989a). For the assay of DRAT, crude extracts were prepared aerobically by sonication (Lowery et al., 1986). DRAT activity was assayed under anaerobic conditions by determination of the incorporation of radioactivity into Rr2 with [at-32P]NAD as the donor molecule (Lowery et al., 1986). Under the conditions tested, the DRAT-specific activity in the crude extracts of UQ790 is 9.4 pmol ADP-ribose incorporated into Rr2/m8 protein/man. That the radioactivity was specifically incorporated into Rr2 was verified by autoradiography (Fu et al., 1989a); a single band appeared at the position for ADP-ribosylated subunit of Rr2. A slight decrease in activity was observed without lirrG induction of the strains. However, the crude extracts of £. coli strain with pilAF 108 which lacks Pt,© (Fig. 2) was incapable of catalyzing the 3=P-incorporation into Rr2 from [ 3=P]NAD, demonstrating that the cloned genes in pilAF210 are


Psti and Hindlll diBestien, libation

kb j

- 23.13 .9.42 - 6.56



4.32 ,2.32 " 2.02


Ap Fig. 2. Construction of the draT6 expression vector pilAF210. The Km cassette ofpUC4K (Vieira and Messing, 1982) was cut out with Sail and inserted into the Sail site between the 0.7- and 0.9-kb Sail fragments of pHAFI02 to generate pHAFI02M6, thus providing a Pstl site next to the Sail site. The Pstl-EcoRl fralpnent was cloned into pBR322 to yield pHAFI08. The H/ndlll site next to the EcoRl site in pHAFI08 is from pBR322. The 3.0-kb Pstl.Hindlil fragment of pHAFI02M6, containing draTG sequences, was inserted into the polylinker of pKK223-3 immediately after the Ptac promoter, creating pilAF210. Tc, tetracyclineresistance marker; 7', terminator.

123 Fig. 3. Hybridization ofa draTU probe ofA. lipofemm to genomic DNA. Total DNA of A. ama:onense 0ane i), A. braZen.re (lane 2) and A. Iipofemm (lane 3) was digested with EcoR! and hybridized to a sap. labelled l.l-kb Sail fragment ofpHAFI02 (Fig. IB) at 42°C overnight. Stringent conditions were usedfor washing as previously described (Fu et al., 1989a). Size markers (in kb) are shown on the right margin.


oriented transcriptionally downstream from the Ptac promoter. T~¢n together with the hybridization data, these results suggest the transcriptional organization shown in Fig. 1. (d) Correlation of "NH~-switch-off" and the p~q~nce of draTG genes Among AzospiriUum spp., A. lipoferum and A. brasilense have been demonstrated to possess a covalent modification system for dinitrogenase reductase, while no evidence for such a post-translational regulatory system has been presented for A. amazonense (Hartmann et al., 1986; Fu et al., 1989a). When the A. lipoferum draTG region was hybridized to the total cellular DNA of these three species (Fig. 3), it was found that one specific hybridization band appeared for both A. iipoferum (6.3-kb EcoRl f r ~ e n t ) and A. brasilense(12-kb EcoRl fragment), but no signoriwas seen for A. amazonense. A correlation therefore exists between the NH4+-regulation of nitrogenase activity and the presence of the draTO genes within the genus Azospirillum. (e) Conclusions The draTG genes ofA. lipoferum have been cloned, representing the first identification of this region in a non-phototroph. The draTO genes are located next to n07/in the genome. Since the enzyme activities ofdraG and draTgene products have been detected in the crude extracts of E. coil strains carrying a draTO expression vector, the identity of the cloned DNA fragment is conclusively established. The similar organization of the draTO and nl~l genes erA. llpo. ferum and R. rubrum suggests that these genes have been conserved in these two organisms during evolution.


We thank Dr. P.W. Ludden for generous support and L.J. Lehman for enlightening discussions and technical assistance. This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, and by Department of Energy grant DE-FG02-87ERI3707, and USDA grant 87-CRCR-I-2561.

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Bun'is, R.H.: Nitrogen fixation-assay methods and techniques. Methods Enzymol. 24B (1972) 415--431. Collins, JJ., Roberts, G.P. and BriH,WJ.: Posttranscriptional control of Klebsiella pneumonias nOe mRNA stability by the n/fL product. J. Bacteriol. 168 (1986) 173-178. D6bereiner, J. and Pedrosa, F.O.: Nitrogen-fixing Bacteria in Nonleguminous Crop Plants. Science Tech. Publishers/Springer-Vedag, Madison, WI, 1987. Fahsold, R., Singh, M. and KlingmOiler, W.: Cosmid cloning of nitrogenase structure genes of Azospirillum Iipoferum. In KlingmOller, W. (Ed.). Azospirillum Ill: Genetics, Physiology, Ecology. SpringerVerlag, Berlin, 1985, pp. 30-39. Feinberg, A.P. and Vogelstein, B.: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137 (1983) 266-267. Fitzmaurice, W.P., Saari, L.L., Lowery,R.G., Ludden, P.W. and Roberts, G.P.: Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodosplrlilum rubrum. Mol. Gen. Genet. 218 (1989) 340-347. Fu, H.-A., Hartmann, A., Lowery, R.G., Fitzmaurice, W.P., Roberts, G.P. and Burris, R.H.: Posttranslational regulatory system for nitrogenase activity in Azospirillum spp. J. Bacteriol. 171 (198%) 4679-4685. Fu, H.-A.,Wirt0 H.J., Burris, R.H. and Roberts, G.P.: Functional expression of a Rhodospirlllum rubrum gene encoding dinitrogenase reductase ADP-ribosyitransferase in enteric bacteria. Gene (1989b) in press. Hartmann, A., Fu, H.-A. and Burris, R.H.: Regulation of nitrogenase activity by ammonium chloride in A=ospirlllum spp. J. Bacteriol. 165 (1986) 864-870. Kanemoto, R.H. and Ludden, P.W.: Effect of ammonia, darkness, and phenazine methosulfate on whole.cell nitrogenase activity and Fe protein modification in RhodosptrlUumrubrum. J. Bacteriol. 158 (1984) 713-720, Lowery, R.G., Saari, L.L. and Ludden, P,W,: Reversible rngulation of the nitrogenase iron protein l~om RhodospMIlum rubrum by ADPribosylation in vitro. J. Baeteriol, 166 (1986) $13-$18. Ludden, P,W. and Burris, R.H.: Nitrogenue: properties and re8ulation. in Neyra, C,A, (Ed.), Bioehemi©al Basis of Plant Breeding, Vol, !i. CRC Press, Boca Raton, FL, 1986, pp. 41-58, Ludden, P.W. and Roberts, G.P,: Regulation of nttrogenaae activity by reversible ADP-ribosylation. Curt, Topics Cell. Reg, 30 (1989) 23-33. Ludden, P,W., Okon, Y. and Burris, R.H.: The nitrogenase system of Spirlllum I!pofenon. Biochem. J. 173 (1978) 1001-1003. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Okon, Y.: A:ospirillum as a potential inoculant for agriculture, Trends Biotechnol. 3 (1985) 223-228, Saari, L,L,,Triplett, E.W. and Ludden, P.W.: Purification and properties of the activating enzyme for iron protein of nitrogenase from the photosynthetic bacterium Rhodospirlllum rubrum. J. Biol. Chem. 259 (1983) 15502-15508, Visits, J, and Messing, J.: The pUC plasmids, an Ml3mpT-derived system for insertion mutngenesis and sequencing with synthetic universal primers, Gene 19 (1982) 259-268. Yanisch-Permn, C,, Visits, J. and Messing, J.: Improved MI3 phage cloning vectors and host strains: nucleotide sequences of the Ml3mpl8 and pUCI9 vectors. Gene 33 (1985) 103-119.

Cloning and expression of draTG genes from Azospirillum lipoferum.

A genomic library of Azospirillum lipoferum was constructed with phage lambda EMBL4 as vector. From this library, the genes encoding dinitrogenase red...
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