characterisation of the klebsiella pneumoniae nitrogen-fixation regulatory proteins nifa and nifl in...

Eur. J. Biochem. 187, 353-360 (1990) FEBS 1990

Characterisation of the Klebsiella pneumoniae nitrogen-fixation regulatory proteins NIFA and NIFL in vitro Sara AUSTIN, Ncville HENDERSON and Ray DIXON

Agriculture and Food Research Council Institute of Plant Scicnce Rcsearch, Nitrogen Fixation Laboratory, University of Susscx, Brighton, England

(Received June 21:August 29, 1989) - EJB 89 0767

Transcriptional activation by the Klebsiellu pneumoniue nitrogen-fixation-specific positive control protein, NIFA, ( n f A gene product) has been demonstrated in vitro in S30 extracts from cells which overproduce this protein. The activity of NIFA was dramatically reduced in vitro in the presence of the negative regulatory protein NIFL (mfL gene product) but was not inhibited by the presence of a mutant NIFL protein, NIFL2404. Transcriptional activation from the n f H promoter by NIFA was dependent on the alternative g factor, d4, and also required the presence of an upstream activator sequence. NiFA activity was temperature-sensitive in vitro (as it is in vivo) which is due, at least in part, to the intrinsic lability of the protein itself.

The majority of overproduced NIFA and NIFL was insoluble after low-speed centrifugation and was inactive in vitro. A low level of less aggregated NiFA protein present in cell extracts was responsible for in vitro activity and this fraction was partially purified.

Transcription of the nitrogen fixation (nzj) genes in Klebsiellu pneumoniue is regulated globally by the nitrogen fixation regulatory genes ntrA, ntrB and ntrC, and specifically by the products of the n f L A operon, NIFL and NIFA, in response to changes in levels of fixed nitrogen or extracellular oxygen tension (see [l] and [2] for reviews). An alternative sigma factor, d4, the product of the rpoN (ntrA) gene, is required for recognition of nij promoters which have a pri- mary structure characterised by the conserved dinucleotides GG and GC, 24 and 12 nucleotides, respectively, upstream of the transcription start site. All known d4-dependent promoters are positively controlled by an activator protein. In the case of the n f L A operon, the activator is the product of the ntrC gene, NTRC, whereas the other niJ promoters are activated by NiFA, a product of the n f L A operon [l, 21. The activity of NTRC is modulated by the product of the ntrB gene, NTRB, which phosphorylates NTRC under conditions of nitrogen limitation and dephosphorylates it under conditions of nitrogen excess [3]. The three proteins NTKB, NTRC and cr54 have been purified and shown to be necessary, in addition to core RNA polymerase, for transcriptional activation from the nifLA promoter in vitro [4, 51.

NIFA activates nif transcription under anaerobic nitrogen-limiting conditions but becomes inactive when levels of fixed nitrogen or oxygen increase [6]. This inactivation has been attributed to the NIFL protein which appears to sense changes in the oxygen or nitrogen status of the cell [7, 81.

Correspondence to S. Austin, AFRC Institute of Plant Science Research, Nitrogen Fixation Laboratory, University of Sussex, Brighton, BNI 9RQ, England

Ahhrevicitions. UAS, upstream activator sequence; NIFL and NIFA proteins. nitrogen-fixation negative and positive regulatory proteins, respectively; IPTG, isopropyl-P-D-thiogalactopyranoside; NTRC, protein product of the ntrC gene which activates the @LA operon; NTRB, protein product of the ntrB gene which phosphorylates NTRC.

Enzyme. B-Galactosidase (EC 3.2.1.23).

NIFA binds to an upstream activator sequence (UAS) [9] which is characterised by the sequence TGTNIoACA and is typically located approximately 100 base pairs from the transcription start site, but can function when placed much further upstream [l I]. Evidence that transcriptional activation by NIFA is dependent upon the UAS being located on the correct face of the DNA helix has led to the hypothesis that the upstream-bound NIFA is brought into the vicinity of the RNA polymerase complex by formation of a DNA loop between the UAS and downstream promoter sequences [12].

The mechanism by which NIFL prevents transcriptional activation by NIFA is not yet known. The repressive effect of NIFL on NIFA activity in vivo is dependent upon the presence of ferrous or manganese ions in the medium [13]. NIFA and NIFL associate to form a complex in vivo, but it is not known whether NIFA can activate transcription while complexed to NIFL [13].

In other nitrogen-fixing organisms there does not appear to be a NIFL equivalent and in Brudyrhizobium ,juponicum NIFA itself has been shown to be directly responsive to oxygen [14], whereas K. pneumoniue NIFA is active in aerobically grown cells in the absence of NIFL [6]. Previous attempts to purify NIFA from K . pneumoniae have shown that it is very insoluble when overproduced and sediments with the cell debris after cell lysis [15]. This behaviour is presumably due to aggregation of the polypeptide chains after synthesis of the protein.

In order to study the activity of NIFA and NIFL in vitro we have developed coupled transcription-translation systems from Esrherichiu coli strains which overproduce both proteins, and examined their ability to activate transcription from the K. pneumoniue nifH promoter. The activity of both proteins in vitro corresponds with their known properties in vivo.

MATERIALS AND METHODS

Strains and plasmids

These are listed in Table 1.

354

Table 1. Bacteriul strains and plusniids Plasmids constructed in the course of this work are described in Materials and Methods

Source Strain Characteristics Reference

E. coli MC1061 uraDl3Y A(ura leu)1691 A(luc)X74, galU galK hsr strA [351

Plasmids pM M40 ApR, pKK223-3 derivative containing the l a d Q gene ~361 pNH2 5.2-kb EcaR1- Hind111 fragment carrying nifL2404 and nifA from pMF1014 ~ 3 1

pNH6 2-kb EcoRI ~ KpnI fragment carrying n f L from pNH3 in pMM40 [I 31 pNHl1 2.2-kb EcoRI ~ Hind111 fragment containing nlfA from pRT903 in pMM40 ~131 pSAl ApR, RlnA’-’lacZ translational fusion derived from pRD572 [41 pRT22 CmR, nifW’lucZ translational fusion in pACYCl84 ~ 5 1

in pMM40 pNH2 derivative substituted with 4.5-kb SmuI - Hind111 fragment from pMF1000 carrying nifL

pNH3 [I31

Plusrnid construction

The DNA templates used in the coupled transcription translation system, pRD582 and pNH4, were constructed by cloning the omega insertion element from pHP45fi into the EcoRI site of the nijH-lucZ translational fusions pSMM8 and pSMM4 respectively [lo]. The LO insertions prevent read- through transcription from the vector sequences when insert- ed upstream of the promoters [16]. pNHS was constructed by cloning the 2-kb EcoRI - Asp71 8 fragment from pNH2 containing nijL2404 into pMM40 [13]. A 1.1-kb EcoRI- Hind111 fragment from pMD189 containing a truncated K. pneumoniue nifA coding sequence which extends from the NarI site to the end of the gene (M. Drummond, unpublished results) was cloned into the expression vector pTTQl8 [17], creating pNH12, an in-frame fusion, in which the first seven codons of lucZ are fused to codons 189 to 524 of nijA. Coupled transcription-translation in S30 extracts were prepared by the method described in [18]. Overproducing strains were induced by the addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) 3 h prior to harvesting the cells. The S-30 reaction mixture was prepared as described previously [4]. 50-p1 reactions were incubated and P-galactosidase activity assayed as described previously [ 181. Supercoiled template DNA, purified on Qiagen anion-exchange resin, was added at 2 nM and purified nitrogen-regulatory proteins and ammonium sulphate fractions were added at concentrations given in the legends. S30 extracts typically contained 30 mg/ml total protein and similar levels of NIFA and NIFL2404 as judged by immunoblotting. For transcript-mapping experiments, 100-pl incubations were set up and 50-p1 aliquots were removed after 30 min incubation prior to isolation of RNA. P-Galactosidase activity was measured in the remainder of the incubations.

R N A extractions and primer extension assays

These were carried out as described in [4].

Purification of proteins

NTRB9603 and NTRC were purified as described previously [4]. For preparation of ammonium sulphate fractions containing NIFA and NIFL, 1-1 cultures of the overproducing strains were grown aerobically at 30‘C in Luria broth supplemented with the appropriate antibiotic. The cells were induced with 1 mM IPTG 3 h prior to har- vest. The cell pellet was resuspended in a buffer containing 20 mM TrisiCl, pH 7.9, 10% glycerol, 1 mM EDTA, 1 mM

dithiothreitol, 23 pg/ml lysozyme and 0.2 mM phenyl- methylsulphonylfluoride and lysed by passage through a French press. The supernatant from centrifugation at 30000 x g for 1 h was made 2% (massjvol.) in streptomycin sulphate and stirred for 20 min at 4°C. The precipitate was removed by centrifugation at 30000 x g for 1 h. To the supernatant, solid ammonium sulphate was added to 50% (mass/ vol.) saturation, stirred for 30 min at 4°C and the precipitate collected by centrifugation at 20000 x g for 45 min. The precipitate was dissolved in a buffer containing 10 mM Tris/Cl, pH 7.9, 0.2 mM EDTA, 1 mM dithiothreitol, 0.05 M NaCl and 20% glycerol, and dialysed against the same buffer for 4 h at 4°C. The dialysed ammonium sulphate fractions were stored at -80°C in small aliquots and became inactive upon repeated freezing and thawing. The fractions typically contained 30 - 60 mg/ml total protein and contained similar levels of NIFA and NIFL2404 as judged by immunoblotting.

Detection of NIFA and NIFL by Western blotting

Proteins were separated on 7.5% SDS/polyacrylamide gels and electrophoretically transferred to Immobilon polyviny- lidene difluoride membrane (Millipore). Detection was carried out using K. pneumoniue NIFA and NIFL antiserum 1131 followed by incubation in immunogold-labelled goat anti- (rabbit IgG) secondary antibody. Cross-reacting proteins were visualized by silver enhancement.

RESULTS

Solubility of NIFA and NIFL

Previous attempts to purify NIFA have been hindered by the fact that the protein is insoluble when overproduced and sediments with cell debris after lysis [15]. In such experiments NIFL was not present, so to investigate whether the presence of NIFL would alter the solubility characteristics of NIFA we made constructs which overproduce both proteins. To avoid the repressive effect of wild-type NIFL on NIFA we used a mutant NIFL protein (NIFL2404) which allows n f A - mediated activation of nif’ transcription in the presence of fixed nitrogen and oxygen in vivo [19, 201. This mutation has been sequenced and has been shown to be a C to T transition converting Ala290 to Val 1131. Induction of expression from the tuc promoter in strains carrying the overproducing plasmid (pNH2) resulted in an accumulation of NIFA and NIFL2404 in the cells but on cell lysis and subsequent low- speed centrifugation both proteins appeared in the pellet frac-

355

tion of the cells (Fig. 1). The presence or absence of the mutant or the wild-type NIFL protein had no apparent effect on the insolubility of NIFA. NIFA and both the mutant and wild- type form of NIFL appeared to be equally insoluble when produced alone (data not shown). Western blot analysis of the cell extracts using anti-NIFA and NIFL sera revealed a small amount of NIFA and NIFL protein remaining in the extracts after low-speed centrifugation (Fig. l), although these were not visible on a Coomassie-blue-stained gel. Cenlrifuga- tion of the cell extracts at 100000 x g for 1 h resulted in sedimentation of the remaining NIFA and NIFL indicating that even this fraction of the proteins was not truly soluble (Fig. 1). Attempts to increase the solubility of the proteins with high salt or detergents such as Triton X-100, Nonidet 40, octyl glucoside and Chaps were unsuccessful. The low-speed pellet fraction, which contained the majority of the overproduced NIFL and NIFA, was only found to be soluble in 2% SDS or in high concentrations (> 3%) of Zwittergent 3 - 14. Extracts prepared anaerobically under nitrogen had the same amounts of NIFA and NIFL with the same sedimentation character-

-A -L

1 2 3 4 5 6 7

Fig. 1. Distribution of overproduced NIFA und NIFL2404 between cell pellet and supernutant after low- and high-speed centrifugation. MC1061 (pNH2) cell extracts contaming NIFA and NlFL2404 wcrc electrophoresed on 7.5% SDS/polyacrylamide gels. The proteins were detected by immunoblotting with anti-NIFA and anti-NIFL sera as described in Materials and Methods. Lanes I and 4, supernatant after ccutrifugation at 30000 x g. Lanes 2 and 5, supernatant after Centrifugation at 100000xg. Lanes 3 and 6, material pelleted at 100000 xg. Lane 7, material pelleted at 30000 xg. Lanes 1 -3, extracts prepared anaerobically. Lanes 4 - 6, extracts prepared aerobically

istics as those prepared aerobically (Fig. 1). A truncated form of NIFA lacking the N-terminal domain remained in the cell supernatant after low-speed centrifugation when overproduced, but most of the protein sedimented on centrifugation at 100000 x g for 1 h (data not shown).

Trunscriptional uctivution in vivo and in vitro

Coupled transcription-translation systems (S30s) were prepared from strain MC1061 carrying different plasmids which overproduced either wild-type NIFA alone or produced NIFA plus wild-type NIFL or NIFA plus NIFL2404. Trans- criptional activation of the nijH promoter in vivo by these strains grown aerobically in S30 medium is shown in Table 2. There was some transcriptional activation in the absence of IPTG in strains containing the pNHll and pNH2 constructs, but after addition of IPTG, NIFA activity increased in strains overproducing either wild-type NIFA alone or wild-type NTFA plus NIFL2404. There was no increase in NIFA activity upon IPTG addition in strains overproducing NIFA plus wild- type NIFL. This is in agreement with previous results which have shown that overproduction of wild-type NIFL leads to inhibition of ntf transcription, even under nitrogen-limiting conditions [I 31. Strains which overproduced the truncated NIFA lacking its N-terminal domain gave some NIFA activity but only to 30% of the wild-type level.

We initially looked to see whether we could obtain NIFA activity in vitro from strains which did not contain wild-type NIFL. The low-speed pellet fraction containing the insoluble NIFA and NIFL2404 after resuspension in S30 buffer [18] was unable to activate the nifH promoter when added to a control S30 (data not shown). We therefore investigated whether the small amount of NIFA protein present in the supernatants used to prepare the S30 extracts could activate the nifH promoter in the coupled system. We used nifH-'lacZ and g1nA'-'lacZ translational fusions as DNA templates and measured the [&galactosidase activity of the fusion proteins synthesised in vitro.

In control experiments all the S30s were able to activate expression from the glnAp2 promoter if oS4 was added (Table 3). We have shown previously that although our S30 extracts are prepared from rpo N + strains, they are apparently deficient in d4 activity [4]. Addition of purified NTRB and NTRC proteins however was not required for activity, presumably the low concentration of these proteins already present in the S30s is sufficient for activation from this promoter. The presence of either NIFA or NIFL2404 proteins in the S30 did not cause any inhibition of activation from glnAp2.

Table 2. Transcriptional activation of the K. pneumoniae nifH promoter in vivo by strains which overproduce NIFA Strains were grown aerobically at 30°C in S30 medium [37] to an A6nn of 0.4 and isopropyl-P-D-thiogalaetopyranosidc was then added to 1 mM where indicated. After incubation for a further 3 h, P-galactosidase activity was assayed with units expressed as described in [38]; activity is given in Miller units

Strain Characteristics 0-Galactosidase activity

- IPTG + IPTG

units

MC1061(pRT22, pMM40) MC1061(pRT22, pNH2) MC1061(pRT22, pNH3) MC1061(pRT22, pNH11) MC1061 (pRT22, pNH12)

nEfW-'lacZ, vector nzfW-'lacZ. nifL2404 nzfA n$H-'luc Z , n$L nifA nifH-'lucZ, nifA nlfH-'latZ, NAnl fA

30 30 7400 27000 700 700

4420 46470 503 13 269

356

Table 3. Expression f rom the glnA and niM promoters in S30 exlracts preparedfrom struins which overproduce NIFA and NIFL2404 /i-Galactosidasc activity is expressed as nmol o-nitrophenol formed . min- ' . kg- ' DNA at 28°C following a I-h incubation in thc coupled transcription-translation syslem. Where indicatcd, oS4 was added at 150 nM, NTRC was added at 180 nM and NTRBY603 at 30 nM. n.d., not determined

Line Strain Gene(s) IPTG Incubation Additions in vitro j?-Galactosidase activitykemplate DNA no. on plasmid in vivo temperature

in vitro d4 NTRB, NTRC glnA'-'lacZ nifH-"lucZ (PSAI)

(pRD582) (pNH4) + UAS - UAS

"C nmol. min- ' . pg-'

1 2 3 4 5 6 7 8 9

10

MC1061 MC1061 (pMM40) MC1061 (pNH11) MC1061 (pNH11) MClO6l (pNH11) MClO6l (pNH12) MC1061 (pNH2) MClO6l (pNH2) MC1061 (pNH2) MClO6l (pNH2)

nonc vector nifA nlfA nlfA NAnifA nlfL2404, nlfA n ifL2404 ,nifA nrfL2404,ni f A nrfl2404,nifA

-

+ + + + + + + +

-

32 32 32 32 31 32 32 32 37 32

+ - + - + - + - + - + - + - t - + +

- -

27.7 21.5 0.23 25.7 31.1 25.8 42.5 20.2 16.0 67.2

0.04 0.05 0.01 3.08 0.01 0.05 0.04

1 1.46 0.16 8.76

n.d. n.d. n.d. 0.07 n.d. n.d. n.d. 0.01 n.d. 3.52

Table 4. NIFA activity in ammonium sulphate,fractions from strains which overproduce NIFA and NIFL2404 d4 was added to all incubations at 150 nM. /l-Galactosidase activity is exprcsscd as nmol o-nitrophenol formed min-' . pg- ' DNA mg protein- in the ammonium sulphate fraction. n.d., not determined

Temperature of Source of ammonium Relevant proteins incubation in vitro sulphate fraction added in fractions

P-Galactosidase activity/mg protein template DNA

nif'H-'IacZ n if'H-'lacZ (pRD582) (PNH4) + UAS -UAS

"C nmol. rnin-' . pg-' . mg ' 32 MC1061 (pNH2) NIFL2404 + wild type NIFA 34.1 0.59 37 MC1061 (pNH2) NIFL2404 + wild type NIFA 2.02 n.d. 32 MC1061 (pNH11) wild-type NIFA 16.25 0.31 37 MC1061 (pNH11) wild-type NIFA 0.625 n.d. 32 MC1063 (pMM40) nonc 0.645 n.d.

Control S30s from MC1061 or from MC1061 carrying only the vector plasmid did not allow transcriptional activation from the nijH promoter (Table 3, lines 1 and 2). Trans- criptional activation of the nijH promoter could be obtained in S30s from strains overproducing either NIFA alone or NIFL2404 plus NIFA, provided that oS4 was added (Table 3, compare lines 3, 4 and 8) and there was no activation in extracts from cells in which synthesis of NIFA and NIFL2404 was not induced by IPTG (Table 3, line 7). An S30 from MC1061 (pNH12) which overproduced the truncated form of NIFA lacking its N-terminal domain could not activate the n$H promoter (Table 3, line 6). S30 extracts prepared anaerobically under nitrogen showed similar levels of NIFA activity as those prepared aerobically (data not shown).

As further evidence that the activity seen in S30s from overproducing cells was due to NIFA we constructed a DNA template (pNH4) which lacked the binding site for NIFA, the nifH UAS. No activation of this promoter was seen in any of the NIFA-containing S30s but activation was obtained in the absence of the UAS when high concentrations of purified NTRB and NTRC proteins were added (Table 3, line 10). This is in agreement with the results of Wong et al. [5] who

have also shown that NTRC can activate transcription from the n f H promoter in vitvo when added at high concentration.

The ability of NIFA to activate transcription in vivo is temperature-sensitive and is inactivated at temperatures above 34°C [6, 211. NIFA activity in vitro was also temperature-sensitive and was reduced considerably by incubation at 37 C (Table 3, compare lines 4 with 5 and 8 with 9). The optimum temperature of incubation in vitro was 32'-C (data not shown). In contrast, transcriptional activation from the o"-dependent glnAp2 promoter was not temperature- sensitive at 37'C (Table 3, compare lines 4 with 5 and 8 with 9).

Enrichnzent qf' NIFA activity

We attempted to purify NIFA which remains in the crude cell extract after cell lysis and low-speed centrifugation and is active in vitvo. Crude extracts were prepared from a number of overproducing strains and subjected to streptomycin sulphate precipitation to remove ribosomes and nucleic acids. An ammonium sulphate fraction (0 - 50%) was prepared from the supernatant and the ability of this fraction to active the nifH

357

0 5 10 1 5 20 25 30

Time (min )

Fig. 2. Effect of'temnperature of incubation on stability of NIFA activity in vitro. Ammonium sulphate fractions containing NIFA and NIFL2404 were preincubated at 3 7 T , 32°C or 4°C. At the times indicated 4 pl(270 pg total protein) aliquots were removed and added to a complete S30 reaction mix containing 3 50 nM r ~ ~ ~ . The reactions were then incubated for 1 h at 32°C and the P-galactosidase activity assayed and expressed as in Table 3. (U) Preincubation at 32' C; (0) peincubation at 37'C; ( 0 ) preincubation at 4°C

promoter was assayed in an S30 from MC1061 (pMM40). Table 4 shows that NIFA activity was present in ammonium sulphate fractions containing either wild-type NIFA alone or NIFL2404 plus wild-type NIFA. A control ammonium sulphate fraction from MC1061 (pMM40) had no NIFA activity. NIFA activity was present in crude cell extracts from these strains and there was a tenfold increase in NIFA activity/ mg total protein after ammonium sulphate fractionation (data not shown). An ammonium sulphate fraction from cells containing the N-terminal truncated NIFA had a very low level of NIFA activity, although the presence of the polypeptide was confirmed by Western blotting (data not shown). The NIFA activity present in the ammonium sulphate fractions was abolished at 37°C and was also dependent upon the presence of the nifH UAS (Table 4). To see whether the loss of activity at 37'C was due to the instability of the NIFA protein itself or to one of the steps involved in transcriptional activation, we looked at the effect of preincubating an ammonium sulphate fraction containing NIFA and NIFL2404 at 32'C and 37°C before addition to the S30. Fig. 2 shows that at 37"C, NIFA activity decays more rapidly than it does at 32°C. Preincubation at 4°C did not cause any loss of NIFA activity (Fig. 2) even after several hours. There was no apparent proteolytic cleavage of NIFA or NIFL2404 at either temperature and the levels of both proteins were the same before and after a 30-min preincubation at 32°C or 37"C, as judged by immunoblotting (data not shown). This indicates that the temperature-sensitive nature of the ability of NIFA to activate transcription in vitro is at least in part due to the intrinsic lability of the protein itself.

Attempts to purify NIFA further than the ammonium sulphate fraction were not successful. Ion-exchange chromatography, affinity chromatography on heparin-agarose, hydrophobic-interaction chromatography and immunoaffin- ity chromatography were all tried. Fig. 3 shows a Western

A- L -

AM 3 4 5 6 7 8 10 12 14 16 18

A - L - -A

-L

7 8 9 u) 11 12 13 14 15 16 17 18 Fraction Nr.

Fig. 3. Heparin-agarose chromatography of a 0-50% ammonium sulphate,fiaction containing NIFA and NIFL2404. A 0 ~ 50% ammonium sulphate fraction from MC1061 (pNH2) cells was prepared as described in Materials and Methods. The fraction (10 ml) was applied to a 1.6 x 3.5 cm column of heparin-agarose in 10 mM Tris, pH 7.9, 1 mM dithiothreitol, 0.5 mM EDTA, 10 mM MgC12, 5% glycerol and 0.05 M NaCl. The column was washed with 100 ml of the same buffer and the bound protein eluted with a gradient in the range 0.05- 0.75 M NaC1. 2 0 4 aliquots were removed from selected fractions and electrophoresed on 7.5% SDS/polyacrylamide gels. The proteins were dctected by immmunoblotting using anti-NIFA and anti-NIFL sera as described in Materials and Methods. (A) Protein fractions which eluted from the column in 0.05 M NaCl. (B) Protein fractions which eluted from the column with increasing salt concentration of 0.05-0.75 M NaCl. The position of NIFA and NIFL on the blots (A and L) was located by using as a marker 0.5 pg low-speed pellet fraction from MC1061 (pNH2) cells. AM is thc 0-50% ammonium sulphate fraction which was applied to the column

blot of fractions from heparin-agarose chromatography of a 0 - 50% ammonium sulphate fraction from MC1061 (pNH2) cells. NIFA and NIFL chromatograph together indicating that they are present as a complex in the cell extracts. The appearance of both proteins in the column effluent and in most gradient fractions indicates that they exist as a heterogeneous population of molecules, possibly in different aggregation states. An ammonium sulphate fraction containing the N-terminal truncated NIFA behaved in a similar fashion on heparin agarose, implying that this protein also tends to aggregate (data not shown). There was no NIFA activity in any of the column fractions when assayed in an S30 extract, possibly because of the low concentration of NIFA present in the fractions. The heterogeneous behaviour was also apparent in extracts containing wild-type NIFA alone, indicating that it was not due to the presence of a complex with NIFL, but to some intrinsic property of the NIFA molecule itself.

3.58

Mapping of transcription start sites

The 5' ends of transcripts initiated from the nifHpromoter in the S30 reaction were mapped by primer extension with reverse transcriptase (Fig. 4). Transcripts originating from the nijH promoter were detected in extracts containing NIFA and NIFL2404 only when d4 was added (Fig. 4, lane 3). The 5' ends of the transcripts synthesised in these extracts corre- sponded with two G residues (on the top strand) and were identical to those obtained in vivo with RNA derived from derepressed K. pneurnoniae cells (Fig. 4, lane 1). At 37°C transcription decreased dramatically (Fig. 4, lane 4). Transcrip- tion was UAS-dependent (Fig. 4, lane 5) but the UAS-deleted promoter was activated when high concentrations of purified NTRB and NTRC proteins were also added (Fig. 4, lanes 6 and 7). Although there is a low concentration of NTRB and NTRC already present in these S30s it is obviously insufficient to activate transcription from the nifH promoter in vitro. This result also indicates that the level of NIFA present in the S30s is too low to activate the nifH promoter in the absence of the UAS. Significant levels of transcripts were detected when an ammonium sulphate fraction containing wild-type NIFA and NIFL2404 was added to a control S30 (Fig. 4, compare lanes 8 and 9). In this case, transcription was also greatly reduced at 37 'C (Fig. 4, lane 10). These results confirm the data shown in Tables 3 and 4 and indicate that the levels of P-galactosidase synthesised by the extracts are roughly proportional to transcript levels.

G A G

Ejyect qf wild-type N I F L on transcriptional activation

In the experiments described so far, activation of the nijH promoter has been studied using cell extracts containing either NIFA alone or NIFA plus the mutant protein NIFL2404, which does not inhibit NIFA activity either in vivo or in vitvo. In order to detcrminc whether wild-type NIFL could exert a repressive effect in vitvo we prepared S30 extracts from MC1061 (pNH3) cells grown aerobically containing both wild-type NIFL and NIFA. Activation of the nifH promoter in this case was reduced to 6?4 of the level seen in a control S30 containing wild-type NIFA and mutant NIFL2404 (Table 5) . In a control experiment, activation from the glnA promoter was normal in both types of S30. These results indicate that wild-type NIFL can exert an inhibitory effect on NIFA which is maintained during preparation of the extracts and subsequent assay of NIFA activity in vitro.

It has been shown that the repressive effect of NIFL on NIFA activity in vivo is dependent upon the presence of metal ions in the medium and that addition of iron chelators inhibits this and increases the level of NIFA activity [I 31. We looked at the effect of adding the iron chelators ethylenediamine- N,N'-diacetic acid and Desferal to an S30 from MC1061 (pNH3) to see whether activation from the nzfH promoter is increased. However, unlike the in vivo situation, neither had any effect on the level of NIFA activity at any of the concentrations tested (data not shown).

DISCUSSION

We have demonstrated in vitvo NIFA activity in coupled transcription-translation systems (S30s) derived from strains which overproduce this protein. S30s from strains which overproduce NIFA alone are able to activate the nifH promoter indicating that NIFL is not necessary for NIFA activity

1 2 3 4 5 6 7 8 9 1 0 Fig. 4. Prii2zc.r-pxtension analysis of' transrripts synthesised in the coupled transcv~tio?z-translation system. S30 extracts were prepared from cells containing NIFA and NIFL2404 (lanes 2-7) or from cells containing the vector plasmid only (lanes 8 - 10). Template DNA was the nifH'-'lacZ plasmid pRD582 (lanes 2-4 and 8- 10) or the UAS- deleted izifH'-'lacZ plasmid pNH4 (lanes 5 - 7) and was added at 2 nM. The reactions were incubated at 32' C unless indicated other- wise. Other additions were as follows: lane 2, nonc; lane 3, 150 nM d4; lane 4, 150 nM os4, 37°C; lanc 5, 150 nM 054; lanc 6 , 150 nM gS4, 180 nM NTRC, 30 nM NTRB9603; lane 7,150 nM d4, 360 nM NTRC, 60 nM NTRB9603; lane 8, 150 nM d4; lane 9, 150 nM d4 and 270 pg 0-50% ammonium sulphate fraction containing NlFA and NIFL2404; lanc 10 as for lane 9 but incubated at 37'C. Lane 1 shows the in vivo nifH transcription start site using RNA from K. pneumoniue grown under derepressing conditions. Size markcrs were dideoxy sequencing reactions specific for G and A residues. The 5' ends of the transcripts correspond with two G residues on the top strand previously identified as in vivo start sites by S1 mapping [39] and primer-extcnsion analysis [28]

as has been shown in vivo [20]. S30s from strains which overproduce both NIFL2404 and NlFA were generally more active than those which overproduce NlFA alone. This may

359

Table 5. Effect of wild-type NIFL an NIFA activity in vitro d4 was added to all incubations at 150 nM

Strain Relevant proteins in S30 b-Galactosidase activity/template DNA

g1nA'-'1uc.Z (pSA1) nifW-'lacZ (pRD582)

MC1061 none 16

MC1061 (pNH3) wild-type NIFL + wild-type NTFA 15 MC1061 (pNH2) NIFL2404 + wild-type NIFA 22

0.035 11.8 0.7

reflect the total levels of NIFA produced by the two constructs, since less NIFA was expressed by the pNHll con- struct compared to pNH2. However since most of the NIFA sedimented on Centrifugation after cell lysis it is difficult to quantitate differences between the two extracts and Western blot analysis showed little difference between the amounts of NIFA in the extracts. The increased activity may also be due to an interaction between NIFL2404 and NIFA since it is possible that formation of a complex between the two proteins may stabilise NIFA.

The NIFA protein has some similarities to a family of transcriptional activator proteins of which NTRC is a member [22]. NIFA and NTRC are very similar in their central domains and also show homology in their C-terminal domains, whereas the N-terminal domains are different. NTRC is a soluble protein when overproduced and it seemed likely that the N-terminal domain of NIFA was responsible for its solubility characteristics. A truncated NIFA protein lacking the N-terminal domain was overproduced and although it remained in the cell supernatant after centrifugation at 30000 x g , it tended to sediment at I00000 xg, indicating that the deleted protein still aggregates. The truncated NIFA had a reduced level of NIFA activity in vivo and little or no activity in vitro. The NIFA proteins from Rhizohium meliloti and R. juponicum have been reported to be fully active as transcriptional activators in vivo when their N-termini are deleted [23, 241. However as nothing is known about the solubility or stability of these truncated proteins compared to the wild- type, the results could simply reflect differences in these characteristics.

We have demonstrated that the ability of NTFA to activate transcription is temperature-sensitive in vitro as it is in vivo and that the reason for this is due partly to the intrinsic lability of the NIFA protein itself. One or more of the steps involved in transcriptional activation, i.e. binding at the UAS, DNA- loop formation or formation of the open complex, may also be temperature-sensitive. NIFA activity in vitro is dependent upon the presence of the UAS since the concentration of NIFA in the extracts used is apparently too low to allow transcriptional activation in its absence. Some activation of promoters lacking UASs is observed in vivo with NIFA from K. pneumoniae, R. meliloti and B. juponicum [25 - 271. Nucleo- tides in the - 15 to - 17 region have been shown to be involved in determining the degree of activation by NTFA which is unable to bind to the UAS [28]. Binding of NIFA at the UAS is thought to increase the local concentration of NIFA and to orientate the activator appropriately to allow interaction with aS4 RNA polymerase bound at the -24 to - 12 region.

It has only been possible to purify NIFA and NIFL partially due to their insoluble nature when overproduced. It would appear that the tendency to aggregate is an intrinsic

property of these proteins. The insolubility of NIFA has been reported previously using constructs which overproduce NIFA alone [15]. However, overproducing NIFL and NIFA together from the same plasmid had no effect on the solubility of either protein and both wild-type NIFL and NIFL2404 were equally insoluble whether synthesised alone or with NIFA. There is no apparent structure in the amino acid sequences of these proteins to indicate why they are insoluble. They do not have strongly hydrophobic regions nor do they appear to be integral or peripheral membrane proteins. Many proteins overproduced in E. coli are insoluble and accumulate in inclusion bodies which pellet with cell debris. This is thought to be due to incorrect folding of the proteins after synthesis and formation of intermolecular disulphide link- ages. Various strategies to refold proteins correctly have been devised, mostly involving denaturing the insoluble protein in strong denaturants such as guanidinium chloride or urea and then allowing the protein to refold correctly by gradually removing the denaturant [29]. Attempts to solubilise NIFA and NlFL from cell pellets in this manner were unsuccessful as the renatured protein had no activity in vitro and tended to reprecipitate as soon as the denaturant was removed.

Purification of NIFA from cell extracts which had in vitro activity and contained small amounts of soluble protein was possible as far as ammonium sulphate fractionation. However the heterogeneous behaviour of NIFA and NIFL on conven- tional column chromatography together with loss of NIFA activity in vitro precluded any further purification of the proteins by these methods. Further attempts to purify NIFA and NlFL by other methods are in progress.

The presence of wild-type NIFL in S30 extracts almost completely abolished NIFA activity. This repressive effect of NlFL on NIFA does not appear to be dependent on the presence of metal ions, as it is in vivo, as addition of iron chelators to the S30s do not restore NIFA activity. This suggests that inactivation of NIFA by NIFL in vivo is not reversible in vitro and may indicate that metal ions are required for a component of the signal-transduction pathway rather than for NIFL activity itself. In contrast, metal-dependent regulation of the aerobactin operon by the Fur protein has been demonstrated in vitro [30]. Previous results have indicated that NIFA and NIFL are expressed in stoichio- metric amounts and form a protein complex [13]. This suggests that repression of NIFA by NIFL may involve a protein- protein interaction. This is in contrast to the situation in the family of two-component regulatory systems where phosphorylation is required for signal transduction at least in the NTRB-NTRC and CHEA-CHEY protein pairs [31, 321. One member of another pair ENVZ has also been shown to be phosphorylated in vitro but the physiological relevance of this has not been demonstrated [33]. NIFL and NIFA do not fit the conserved domain pattern seen in this family of proteins

although there are some homologous regions [34]. It seems therefore possible that NIFL modulates NIFA by a mechanism other than protein phosphorylation.

Wc wish to thank Martin Drummond for providing plasmid pMD189. We also wish to thank Martin Buck, Mike Merrick, and Barry Smith for their comments on the manuscript, and Beryl Scutt for typing it.

REFERENCES 1. Dixon, R. (1988) in The nirrogen and sulphur cycles (Cole, J.

A. & Ferguson, S. J., eds) pp. 417-438, Cambridge University Press, Cambridge.

2. Gussin, G. N., Ronson, C. W. & Ausubel, F. M. (1986) Annu. Rev. Gen. 20, 567 - 591.

3. Ninfa, A. & Magasanik, B. (1986) Proc. Nut1 Acad. Sci. USA 83, 5909-5913.

4. Austin, S., Hcnderson, N . & Dixon, R. (1987) Mol. Microhiol. I ,

5 . Wong, P.-K., Popham, D., Keener, J. & Kustu, S. (1987) J .

6. Buchanan-Wollaston, V., Cannon, M. C., Beynon, J. L. & Can-

7. Merrick, M., Hill, S., Hennecke, H., Hahn, M., Dixon, R. &

8. Hill, S., Kennedy, C., Kavanagh, E., Goldberg, R. B. & Hanau,

9. Morett, E. & Buck, M. (1988) Proc. Nutl Acad. Sci. U S A 85,

10. Buck, M., Miller, S., Drummond, M. & Dixon, R. (1986) Nature

11. Buck, M., Woodcock, J., Cannon, W., Mitchenall, L. & Drummond, M. (1987) Mol. Gen. Genet. 210, 140-144.

12. Buck, M., Cannon, W. & Woodcock, J . (1987) Mol. Microbiol. I , 243 - 249.

13. Henderson, N., Austin, S. & Dixon, R. A. (1989) Mol. Gen. Genet.

14. Fischer, H.-M. & Hennecke, 11. (1987) Mol. Gen. Genet. 209,

92-100.

Bacterial. 169, 2876- 2880.

non, F. C. (1981) Nature 294,176-778.

Kennedy, C . (1982) Mol. Gen. Genet. 185, 75-81.

R. (1981) Nature 290, 424-426.

9401 - 9405.

320, 374-378.

216,484 - 49 1.

621 - 626.

15. Tuli, R. & Merrick, M. (1988) J . Gen. Microhiol. 134, 425-432. 16. Prentki, P. & Krisch, H. (1984) Gene 29, 303-313. 17. Stark, M. (1987) Gene51, 255-267. 18. Hawkes, T., Merrick, M. & Dixon, R. (1985) Mol. Gen. Genet.

19. Filser, M., Merrick, M. & Cannon, F. (1983) Mol. Gen. Genet.

20. Arnott, M., Sidoti, C., Hill, S. & Merrick, M. (1989) Arch.

21. Brooks, S. J., Collins, J . J . & Brill, W. J. (1984) J . Bacferiol. 157,

22. Drummond, M., Whitty. P. & Wootton, J. (1986) EMBO 1. 5,

23. Beynon, J. L., Williams, M. K. & Cannon, F. C. (1988) EMBO

24. Fischer, H.-M., Bruderer, T. & Hennecke, H. (1988) Nucleic Acids

25. Morett, E., Cannon, W. & Buck, M. (1988) Nucleic Acids Res.

26. Better, M., Ditta, G. & Hclinski, D. (1985) EMBO J . 4 , 2419-

27. Gubler, M. (1989) Mol. Microhiol. 3, 149-159. 28. Buck, M. & Cannon, W. (1989) Nucleic Acids Rex 17, 2597-

29. Marston, F. (1986) Biochem. J . 240, I - 12. 30. Bagg, A. & Neilands, T. (1987) Biochem. J . 26, 5471 -5477. 31. Ninfa, A,, Ninfa, E., Lupas, A., Stock, A,, Magasanik, B. &

32. Hess, J . F., Oosawa, K., Matsumura, P. & Simon, M. I. (1987)

33. Igo, M. & Silhavy, T. J. (1988) 1. Bacteriol. 170, 5971 -5973. 34. Drummond, M. & Wootton, J. (1987) Mol. Microbiol. 1, 37-44. 35. Casadaban, M., Chou, J . & Cohen, S. (1980) J . Bucteriol. 143,

36. Kleiner, D., Paul, W. & Merrick, M. (1988) J . Gen. Microhiol.

37. Collins, J. (1979) Gene 6, 29 -47. 38. Minchin, S., Austin, S. & Dixon, R. A. (1988) Mol. Microbiol. 2,

39. Sundaresan, V., Jones, J. D. G., Ow, D. W. & Ausubel, F. M.

201,492-498.

191,485-491.

Microhiol. 151, 180-182.

460 - 464.

441 -447.

J . 7, 7-14.

R ~ s . 16, 2207 - 2247.

16, 11 469 - 1 1488.

2424.

261 2.

Stock, J. (1988) Proc. Nut1 Acad. Sci. USA 85, 5492-5496.

Proc. Nutl Acad. Sci. USA 84, 7609 - 761 3.

971 -980.

134, 1779 - 1784.

433 -442.

(1983) Nature 301, 728-132.

characterisation of the klebsiella pneumoniae nitrogen-fixation regulatory proteins nifa and nifl in...

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