Vol. 173, No. 9

JOURNAL OF BACTERIOLOGY, May 1991, p. 2993-2999

0021-9193/91/092993-07$02.00/0 Copyright © 1991, American Society for Microbiology

Expression of Regulatory nif Genes in Rhodobacter capsulatus PHILIPP HUBNER,1* JOHN C. WILLISON,2 PAULETrE M. VIGNAIS,2 AND THOMAS A. BICKLE' Department of Microbiology, Biozentrum der Universitat Basel, Klingelbergstrasse 70, CH4056 Basel, Switzerland,' and Laboratoire de Biochimie Microbienne, CNRS UA1130, Departement de Biologie Moleculaire et Structurale, Centre d'Etudes Nucleaires de Grenoble, 85X, 38041 Grenoble Cedex, France2 Received 22 October 1990/Accepted 25 February 1991

Translational fusions of the Escherichia coil lacZ gene to Rhodobacter capsulatus nif genes were constructed in order to determine the regulatory circuit of nifgene expression in R. capsulatus, a free-living photosynthetic diazotroph. The expression of nifH, nifA (copies I and II), and nifR4 was measured in different regulatory mutant strains under different physiological conditions. The expression of nifH and niR4 (the analog of ntrA in KlebsieUa pneumoniae) depends on the NIFR1/R2 system (the analog of the ntr system in K. pneumoniae), on NIFA, and on NIFR4. The expression of both copies of nifA is regulated by the NIFR1/R2 system and is modulated by the N source of the medium under anaerobic photosynthetic growth conditions. In the presence of ammonia or oxygen, moderate expression of nifA was detectable, whereas nifH and WjR4 were not expressed under these conditions. The implications for the regulatory circult of nif gene expression in R. capsulatus are discussed and compared with the situation in K. pneumoniae, another free-living diazotroph.

The photosynthetic bacterium Rhodobacter capsulatus is able to fix molecular nitrogen by using an Fe-Mo nitrogenase enzyme complex under anaerobic or microaerobic conditions (45). Several structural genes involved in the nitrogen fixation process were identified either genetically or by DNA sequence homology to known nif genes from Klebsiella pneumoniae (4, 5, 24, 47; for reviews, see references 16 and 25). The regulation of nif genes has been best studied in K. pneumoniae (for a review, see reference 15). The coordinate expression of the structural nif genes is controlled on two levels. The machinery of the first level, the ntr (nitrogen regulation) system, senses and responds to the intracellular concentration of fixed nitrogen (30). The ntr system comprises a complex signal transduction cascade (for a review, see reference 41), which finally phosphorylates the protein NTRC when the intracellular concentration of fixed nitrogen is low. The phosphorylated form of NTRC (NTRC-P) acts as a transcriptional activator of ntr-regulated operons in concert with NTRA, a specific sigma factor of the RNA polymerase (19; for reviews, see references 29 and 43). The second level of nif gene regulation is encoded by one of these ntr-regulated operons, the nifLA operon. NIFA is a nif-specific transcriptional activator (36) which, like NTRC-P, depends on the specific sigma factor NTRA. Both NTRC-P and NIFA bind to upstream activating sequences near a -24/-12 promoter sequence (for a review, see reference 43) and activate the NTRA-RNA polymerase holoenzyme by a DNA looping mechanism (46). This mechanism could involve IHF (integration host factor), which was recently reported to bind to nif promoter regions and to modulate nif gene expression (9, 20, 40). In R. capsulatus, several genes involved in the regulation of nif gene expression have been identified and characterized. Some of these genes were found to be homologous to the ntr genes of K. pneumoniae in structure but not in function. In R. capsulatus, mutations in these genes do not lead to the pleiotropic ntr phenotype, i.e., the inability to use amino acids as the N source. Since these regulatory genes *

impair only nif gene expression leading to a Nif phenotype, they were named nifRI, nifR2, and nifR4, although they have sequence homology to the K. pneumoniae genes ntrC, ntrB, and ntrA (rpoN), respectively (2, 22, 26, 27). The regulatory nifA gene in R. capsulatus is present in two copies, both of which are functional (24, 32). The DNA sequence homology between the two nifAlnijB gene regions starts 19 bp upstream of the nifA gene and ends within the stop codon of nifB (32). Furthermore, the activity of the nitrogenase enzyme complex in R. capsulatus is regulated via short-term inhibition by a covalent modification of the iron protein (23; 44). In addition, there are ntr-like R. capsulatus mutants which depend on ammonia as the N source, i.e., they are unable to use amino acids as the N source (50). These mutants contain less than 1% of the wild-type level of nitrogenase activity in intact cells, but they decrease nif gene expression only partially, implying an indirect effect of the ntr-like gene product on nif gene regulation (47). In order to further characterize the regulatory circuit of nif gene expression in R. capsulatus, we examined the expression of structural and regulatory nifgenes in different genetic backgrounds under different physiological conditions by use of newly constructed broad-host-range lacZ fusion vectors. MATERIALS AND METHODS Bacterial strains and plasmids. The R. capsulatus and Escherichia coli strains and plasmids used in this study are listed in Table 1. Media and growth conditions. E. coli was routinely grown in LB medium at 37°C (31). R. capsulatus strains were grown and maintained as described previously (48). The minimal medium was RCV (18, 45) containing DL-malate (30 mM) as the C source and (NH4)2SO4 (10 mM) as the N source. For induction of nitrogenase synthesis, strains were grown in either RCV medium containing glutamate (7 mM) as the N source (RCV-glu) or NH4-free RCV medium (RCV-N) (3). Aerobic nonphotosynthetic growth was by agitating on a roller at 30°C in 20-ml test tubes containing 2.5 ml of RCV medium with (NH4)2SO4 as the N source. Anaerobic photosynthetic growth was achieved in 12-ml stoppered screw-cap

Corresponding author. 2993

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HUBNER ET AL. TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid

Description

E. coli HB101 DH5o

recA rpsL supE hsdS

R. capsulatus B10

Wild type

SB11-7 RdcI/II AP29 Plasmids pUC19 pRK290 pRK2013 pNM480 pNM481 pNM482 pWKR61 pGA11

pGA12 pUC4-KIXX pPHU234 pPHU235 pPHU236 pPHU264 pPHU278 pPHU266 pPHU282 pPHU284 pPHU289

480dlacZAM15 A(1acZYA-argF) recA endA hsdR supE

7 GIBCO-BRL

3 27 32 48

AnifRI/R2 AnifA/nifB copy 1/11

niJR4 Apr, multiple cloning sites in lacZ' Tcr, broad-host-range vector Kmr, mobilizes pRK290 Apr, pUC-based lacZ fusion vector Apr, pUC-based lacZ fusion vector Apr, pUC-based lacZ fusion vector nifAlnijB copy I nifH nifD nifK nifR4 AnifA (copy II)

Apr aph (Km1) from TnS Broad-host-range lacZ fusion vector Broad-host-range lacZ fusion vector Broad-host-range lacZ fusion vector aph-lacZ fusion aph-lacZ fusion, no aph promoter nifH-lacZ fusion nifR4 nifAi,-lacZ fusion nifA-I1acZ fusion nifR4-lacZ fusion

tubes filled with medium and incubated 1 m from a 150-W incandescent lamp in a 30°C incubation room. Under these conditions, the temperature inside the tube is around 32°C and the light intensity is between 3,000 and 6,000 lx. Antibiotics were added at the following concentrations (milligrams per liter): 25 (kanamycin), 20 (tetracycline [TC] for E. coli) and 1 (TC for R. capsulatus), and 150 (ampicillin). Conjugations with triparental matings were carried out as described previously (47). Cloning procedures. Restriction enzymes, the Klenow fragment of DNA polymerase, and the T4 DNA ligase were obtained from New England BioLabs or from Boehringer. Recombinational DNA techniques used to construct plasmids were as described previously (31). The construction of promoterless lacZ fusion vectors was carried out as follows. The insertion of an XhoI linker (dCCTCGAGG) into the SmaI site of pUC19 (49) yielded plasmid pPHU210, into which a ScaI linker (dGAGTACTC) was inserted at the HinclI site, resulting in plasmid pPHU225. The modified polylinker of pPHU225 was excised by EcoRI and HindIlI and inserted into the 8.6-kb EcoRI-HindIII fragments of plasmids pNM480, pNM481, and pNM482 (35), yielding the plasmids pPHU227, pPHU228, and pPHU229, respectively. EcoRI adaptors consisting of a 1:1 mixture of the two oligonucleotides dAATTGTCGAC and dGTCGAC, which includes a SalI site, were ligated to each of the isolated 3.9-kb DraI fragments of pPHU227, pPHU228, and pPHU229, carrying the modified polylinker in front of the promoterless lacZ gene. The DNA fragments with the ligated adaptors were digested by EcoRI, which cuts at the lacZ distal end of the modified polylinker. The resulting 3.1-kb fragments, flanked by EcoRI cohesive ends, were isolated and ligated into the EcoRI site of pRK290 (10). The

Source or reference

49 10 10 35 35 35 32 1 1 Pharmacia This study This study This study This study This study This study This study This study This study

plasmids with orientations of the lacZ gene reading away from the origin of plasmid replication (oriV) were named pPHU234, pPHU235, and pPHU236, respectively (Fig. 1). The sequences of the polylinker regions of these plasmids were confirmed by DNA sequence analysis (39). Plasmid pPHU264 (Fig. 2) has an aph-lacZ translational fusion. It carries a 0.6-kb EcoRI-PstI insertion from pUC4KIXX (Pharmacia) at the EcoRI-PstI sites of plasmid pPHU235. A promoterless aph-lacZ fusion was constructed by the insertion of the 1.0-kb BglII-ClaI fragment from pPHU264 into the BamHI-Clal sites of pPHU235 to yield pPHU278 (Fig. 2). Plasmid pPHU282 (Fig. 2) carries the nifR4 gene and a nifA11-lacZ fusion. It was constructed by insertion of a 3.0-kb Sail-BamfHI fragment from plasmid pGA12 (1) into the XhoI-BamHI sites of pPHU234. Plasmid pPHU284 (Fig. 2) carries a nifA,-lacZ fusion. It was made by cloning a 1.3-kb BamHI fragment from pPHU203 in the BamHI site of pPHU234. Plasmid pPHU203 consists of the 6.7-kb SallHindIII fragment from pWKR61 (32) inserted into SallHindIII-digested pUC19 (49). The insertion of a 1.9-kb SalI-PstI fragment from pGA12 (1) into the XhoI-PstI site of pPHU235 yielded plasmid pPHU289, which contains a niJR4-lacZ fusion (Fig. 2). Plasmid pPHU266, which expresses a nifH-lacZ fusion (Fig. 2), was constructed as follows. A 1.5-kb EcoRI-BgIII fragment from pPHU245 was ligated into the EcoRI-BamHI sites of pPHU234. Plasmid pPHU245 consists of a 2.2-kb HindIII-XhoI fragment from pPHU205 inserted at the Hindlll site of pPHU234. All these sites were filled in with the Klenow fragment of DNA polymerase. Plasmid pPHU205 originated from the insertion of a 3.5-kb EcoRI-Sall fragment from pGAll (1) into the EcoRI-SaiI site of pUC19.

1=

VOL. 173, 1991

RHODOBACTER CAPSULATUS nif GENES

2995

B

PHU234

EcoRI SacI KpnI XhoI BamHI XbaI ScaI PstI SphI HindIII 7lmAGCC= TA=CCTAGAGTCTAATCGTC=GCT GCAT GIUTGCC

IleArgAlaArqTyrProProArqGlyGlyAspProLeuGluSerSerThrArgProAlaGlyMetGlnAlaCysAspAla

PHU235 EcoRI SacI

fCGAGCTCC.

RpnI TA

XhoI eC

BamHI

XbaI

ScaI

Pstl

SphI Hindlll

1irGGCALCTTGGTCGAL ATRCGACCTCWn:GCATGCliRT

GCTGCC

GluPheGluLeuGlyThrProLauGluGlyGlyIleLeuEndSerArgVaILeuAspLauGlnAlaCysLysLauAlaAla PHU236 EcoRI

SacI

KpnI

XhoI

BamHI

XbaI

ScaI

PstI

SphI HindIII

WU1GAGCTCCTATGATCTGAGTc CTcGACCTCGCAT6=CCGATGCC AsnSerSerSerValProProSerArgGlyGlySerSerArgValGluTyrSerThrCysArgHisAlaSerPheAspAla

sod

FIG. 1. (A) Physical restriction map of plasmid pPHU234. Arrows indicate the direction of transcription of the genes for Tcr (tetA and tetR), for the trans replicative functions (trfA and trfB), and for the lacZ gene. The positions of the origin for plasmid DNA replication (oriV) and of the origin for the conjugative transfer of plasmid DNA (oriT) are indicated. (B) Translational phasing of the polylinker regions in plasmids pPHU234, pPHU235, and pPHU236. Restriction sites are marked by overlining. Triple asterisks indicate the eighth codon of the lacZ gene. The amino acid sequence of the translated polylinker region is given below the DNA sequence.

This 3.5-kb fragment was obtained by partial SalI digestion of the EcoRI-cleaved plasmid pGA11. Oligonucleotides. Oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer (model 380B) by using the phosphoramidite method. 3-Galactosidase assay. The P-galactosidase assay was carried out as previously described (34). Cultures of R. capsulatus cells containing a plasmid-borne nif-lacZ fusion were grown overnight in YPS medium containing 1 mg of TC per liter. RCV, RCV-glu, or RCV-N medium containing 1 mg of TC per liter in 12-ml stoppered screw-cap tubes was inoculated with 0.2 ml of freshly grown overnight cultures and incubated anaerobically in the light for 2 days as described above. Samples of 50 to 500 ,ul, depending on the expected ,-galactosidase activity, were made up to 1 ml with Z buffer (34) in 10-ml glass test tubes. After the addition of 1 drop of

0.1% sodium dodecyl sulfate (SDS) and 2 drops of chloroform, the samples were vortexed for 30 s at maximal speed in order to permeabilize the cells. After preincubation in a water bath at 28°C for 5 min, the assay was started by the addition of 200 1±l of ONPG (o-nitrophenyl-o-D-galactopyranoside; 4 mg/ml) and stopped after the development of the yellow color by the addition of 0.5 mnl of 1 M Na2CO3. The absorption of the samples was measured at X = 420 and 550 nm. In addition, A660 of the corresponding cultures was measured. ,B-Galactosidase activity was calculated by using the following formula: LacZ units = 1,000 [OD420 - k x OD550]/[°D660 x tassay (min) X Vassay(ml)I. The factor k was determined empirically and found to be 1.82 for cultures grown in RCV minimal medium. A420 and A550 were corrected by the corresponding values of a blank consisting of 1 ml of Z buffer with 200 ,ul of ONPG (4 mg/ml), 2 drops of chloroform, 1 drop of 0.1% SDS, and 0.5 ml 1 M Na2CO3. RESULTS

pPHU 264 P ES

OPbamZ

KXbhOM)

S

PSPH

pPHU 278

Sp

E So K ah) H

Sp

%g

a

B

1 >-_

9

~~pPHU2

REAOC

200 tiP

E 8

OM

rPH

S

H

A

-:

pPHU 289

FIG. 2. Partial physical map of lacZ fusions used in this study. Restriction sites: B, BamHI; G, BglII; E, EcoRI; H, Hindlll; K, KpnI; P, PstI; S, Sall; Sa, SacI; Sc, ScaI; Sm, SmaI; Sp, SphI; Xb, XbaI; Xh, XhoI. Locations and directions of known promoter (P) elements (5, 37) are shown (-). The coding part of the tested gene (_) fused to lacZ (El) is also shown (lacZ is not drawn to scale). Destroyed restriction sites are shown in parentheses.

Construction of a broad-host-range lacZ fusion vector. Three translational lacZ fusion vectors were constructed by insertion of promoterless lacZ genes into the EcoRI site of the broad-host-range plasmid pRK290 (10). The lacZ genes are preceded by a modified polylinker sequence derived from pUC19 (49), which allows the insertion of foreign DNA at eight unique restriction sites (Fig. 1). The modifications which are located after the HindIII site in the parental plasmids pNM480, pNM481, and pNM482, respectively, allow the construction of translational lacZ fusions in all three possible reading frames (Fig. IB). Since the polylinker of pUC19 carries an XbaI site (TCTAGA) containing an amber stop codon, translational lacZ fusions with plasmid pPHU235 can only be obtained by choosing either the Scal, PstI, or HindIII site unless the host carries an amber suppressor mutation. Expression of aph-lacZ fusions in R. capsulatus. Since the kanamycin resistance gene (aph) from the transposon TnS can be used for insertional gene inactivation in R. capsulatus (32), aph-lacZ fusions were constructed in order to test whether the aph gene is constitutively expressed in R.

2996

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HUBNER ET AL.

TABLE 2. Expression of aph-lacZ fusions in R. capsulatus nif regulatory mutants under different physiological conditions Plasmid and straina

pPHU264 B10 RdcI/II SB11-7 AP29 pPHU278 B10

RdcI/II SB11-7 AP29

Genotype

Wild type AnifA

AnifRi nifR4

Wild type AnifA AnifRl

nifR4

,B-Galactosidase activity (U) under the following conditionsb: Dark, 02, NH4+

1,030 1,120 640 860 4.1 2.7 3.2 2.8

Light, NH4+

710 1,170 720 850 2.5 3.4 4.6 2.5

Light, Glu

600 1,010 410 490 1.9 1.9 1.1 2.1

Light, N2

690 NG

NG NG 1.1 NG NG NG

aConstruction of the plasmids is described in Materials and Methods. Relevant physical maps of the plasmids are shown in Fig. 1 and Fig. 2. b Growth conditions are described in Materials and Methods. Values represent averages of at least three independent experiments. The standard deviation was, in most cases, smaller than 50%o of the mean value. lacZ units were calculated as described in Materials and Methods. NG, no growth.

capsulatus and could thus function as a positive control throughout our experiments. Two different aph-lacZ fusions were constructed: plasmid pPHU264 carries the proposed promoter region of the aph gene (6), whereas plasmid pPHU278 does not (Fig. 2). Different R. capsulatus strains carrying either of these plasmids were tested under different physiological conditions. The results indicate that with plasmid pPHU264 the aph-lacZ fusion is well expressed in all strains under all conditions tested whereas plasmid pPHU278 lacking the promoter region of the aph gene does not express the lacZ fusion (Table 2). Since differences in the expression of the aph-lacZ fusion for a given strain or between strains are no more than twofold, we conclude that the aph gene is constitutively expressed in R. capsulatus and that the variation in the copy number of the pRK290 derivatives used in this study is smaller than twofold under the tested growth conditions. Therefore, the aph-lacZ fusion borne on plasmid pPHU264 functions as a positive control in gene expression studies in R. capsulatus. The deletion of the promoter region of the aph gene on plasmid pPHU278 reduces the expression of the aph-lacZ fusion more than 100-fold (Table 2). The residual lacZ activities are most likely due to transcription either from the tetR promoter or from the oriV region of the plasmid. Plasmid pPHU278 thus fulfills the criteria for a negative control and was included in our experiments for this purpose. Expression of nif genes in R. capsulatus. Several nif-lacZ fusions were constructed in order to study the regulatory circuit for nif gene expression in R. capsulatus. Plasmid pPHU266 carries a fusion of the nifH structural gene with lacZ, whereas plasmids pPHU282, pPHU284, and pPHU289 carry fusions with the regulatory nif genes nifA51, nifAI and nifR4, respectively (Fig. 2). Expression of the different lacZ fusions was measured under different physiological conditions which are known to influence the expression of the nif genes in R. capsulatus (3, 26). In addition, the effect of three different regulatory nif mutant strains was tested. niff. Expression of nifH (Table 3) in R. capsulatus B10 (wild type) is unmeasurable in RCV medium under aerobic conditions and under anaerobic photosynthetic conditions. Expression is strongly inducible under anaerobic conditions if the N source is either omitted (RCV-N) or changed to 7 mM glutamate (RCV-glu) (Table 3). Expression of the nifHlacZ fusion is twice as high in RCV-N medium as in RCV-glu medium. Similar ratios were found for other structural nif-lacZ fusions (nijB, nifE, nifN, and nifK; data not

shown). Expression of nifH depends absolutely on nifA and on nijR4, which code for the nif-specific transcriptional activator NIFA and for the nif-specific alternative sigma factor NIFR4, respectively. Expression of nifH in a AnifRIIR2 background (strain SB11-7) in RCV-glu medium reaches only 1.3% of the wild-type level but is significantly above the background level (Table 3). Similar results were obtained for the expression of nifB, nijE, nifN, and nifK (data not shown). nfA. R. capsulatus carries a duplication of the nifA-niJB region (32) and has, therefore, two nifA genes (nifA, and nifAII). The homology between the two genes ends 19 bp upstream from nifA, so that the upstream regulatory regions of the two genes are different. Plasmids pPHU284 and pPHU282 (Fig. 2) carry nifA1- and nifA11-IacZ fusions, respectively. Both genes are induced by starvation for nitrogen in either RCV-glu or RCV-N medium (Table 3). In marked contrast to nifH, both genes are moderately expressed in the presence of 10 mM ammonia (RCV medium) under both anaerobic and aerobic growth conditions (Table 3). In the wild-type strain B10, expression of nifA1 is higher than that of nifAII under anaerobic growth conditions and, moreover, it is more responsive to nitrogen starvation. Expression of nifA, is not blocked in a nifR4 background (Table 3); the effect of NIFR4 on the expression of nifAII could not be tested because the nifA11-lacZ fusion vector also carries a copy of the niJR4 gene. On the other hand, the NIFR1INIFR2 system is needed for the expression of nifAII under all incubation conditions and for the anaerobic induction of nifAj. Expression of nifA1 under aerobic conditions and during photosynthetic, anaerobic growth in the presence of ammonia is not impaired by NIFR1/NIFR2. This is reminiscent of the regulation of the glnA operon in E. coli, where two differently regulated promoters are used (for reviews, see references 15 and 41). Since the expression of both nifA genes is higher in the nifA-nifB double deletion strain, RdcI/II, when grown on glutamate as an N source, it seems that NIFA represses its own synthesis. However, expression of the constitutive aph-lacZ fusion in this strain is also elevated to about the same level as the nifA1-lacZ

fusion. nifR4. Expression of nifR4 (Table 3) was examined by use of plasmid pPHU289 carrying a nifR4-lacZ fusion. The expression profile of nifR4 is very similar to that of nifH: it is unmeasurable during growth with oxygen or ammonia and is inducible by nitrogen starvation in either RCV-glu or

RHODOBACTER CAPSULATUS nif GENES

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2997

TABLE 3. Expression of different nif-lacZ fusions in R. capsulatus nif regulatory mutants under different physiological conditions Type of expression (ypeamd epsstrion

(plasmid) and strain'

f3-Galactosidase activity (U) under the following conditionsb:

Genotype Genotype

Dark, 02, NH4'

Wild type AnifA AnifRi nifR4

0.1 0.8 0.7 0.1

Wild type AnifA AnifRI nifR4

9.2 25 6.0 6.1

Wild type A nifA AnifRI Wild type AnifA

Light,

NH4+

Light, Glu

Light, N2

590 0.1 7.5 0.1

1,330 NG NG NG

47 15 18 34

180 300 4.4 78

270 NG NG NG

17 20 1.2

26 47 2.5

74 300 1.2

57 NG NG

0.1 0.2 0.5 0.1

0.1 0.1 0.2 0.1

110 1.0 0.5 0.1

130 NG NG NG

nifH (pPHU266) B10 RdcLI/II SB11-7 AP29

nifA copy I (pPHU284) B10 RdcI/II SB11-7 AP29 nifA copy II (pPHU282) B10

RdcI/II SB11-7 nifR4 (pPHU289) B10 RdcI/II SB11-7 AP29

AnifRI nifR4

0.1 0.1 0.7 0.1

a Construction of the plasmids is described in Materials and Methods. Relevant physical maps of the plasmids are shown in Fig. 1 and 2. b Growth conditions are described in Materials and Methods. Values represent averages of at least three independent experiments. The standard deviation was, in most cases, smaller than 50% of the mean value. lacZ units were calculated as described in Materials and Methods. NG, no growth.

RCV-N medium. Expression of nijR4 reaches 10% (RCV-N) and 18% (RCV-glu) of nifHl gene expression, respectively. Expression of nifR4 depends on NIFA, NIFR1INIFR2, and interestingly on NIFR4 itself, since there is no expression in the nifR4 mutant AP29. DISCUSSION In order to examine the regulatory circuit of nif gene expression in R. capsulatus, the expression of nifHl, nifA, and nifR4 was determined in different regulatory mutant strains under different physiological conditions which are known to induce the synthesis of nitrogenase (3). Since R. capsulatus has a similar fixed nitrogen-sensing circuit (the NIFR1/NIFR2 system) to the ntr system of K. pneumoniae (28), we discuss our results with respect to the situation in K. pneumoniae (Fig. 3). It should be noted that the NIFR1/ NIFR2 system in R. capsulatus, in contrast to that in K. pneumoniae, affects only the regulation of nif genes (26) and methylammonium uptake (38), but not the use of amino acids as N sources. In this study, we show that the NIFR1/NIFR2 system in R. capsulatus is needed for the induction of the anaerobic expression of both copies of WnfA, which encodes the nifspecific transcriptional activator, NIFA. In R. capsulatus, nifA is expressed at a basal level under aerobic conditions and is inducible under anaerobic photosynthetic conditions by limiting the N source. The NIFR1INIFR2 system plays a major role in this anaerobic induction of nifA expression and a minor role in basal aerobic expression. The very low expression of nifH in R. capsulatus SB11-7 (AnifRJ/R2) under inducing conditions may be due to this low, NIFR1/ NIFR2-independent basal expression of nifA. In K. pneumoniae, nifA is also expressed under aerobic conditions, but no expression is observed in the presence of ammonia (36). In Bradyrhizobium japonicum, a dual positive control of nifA

expression was found (42). Under aerobic conditions, nifA is moderately expressed under the control of an unknown activator, whereas under microaerobic conditions, NIFA protein activates its own synthesis. In K. pneumoniae, NIFA only stimulates its own expression when present at high levels in an ntrBC background (11, 36). In R. capsulatus, no major effect of NIFA on its own synthesis was observed, but we did not test conditions under which NIFA is overexpressed. In K. pneumoniae, the expression of nifA depends on NTRA, the alternative sigma factor of RNA polymerase (36). In R. capsulatus, nifAl expression is independent of NIFR4, the nif-specific sigma factor. Since all known transcriptional activators involved in nitrogen regulation act in concert with the alternative sigma factor NTRA (for reviews, see references 29 and 43), it is very probable that nifA expression in R. capsulatus depends on an unknown sigma factor which acts together with the phosphorylated form of the transcriptional activator NIFRL. The observed differences in the expression of nifAI and nifA11 could be explained by the fact that the upstream regions of the two genes are not homologous (32). It is noteworthy that for both copies of nifA the first 200 bp of the upstream region are not sufficient for their expression (data not shown). We are currently mapping the promoter elements involved in nifA expression. In K. pneumoniae, the expression of ntrA is constitutive and is independent of NTRA, NTRB, or NTRC (33). In R. capsulatus we found that the expression of nifR4 is regulated by the N source and depends on NIFR1/NIFR2, NIFA, and NIFR4 itself. In the presence of oxygen or ammonia, no niJR4 expression was detectable. Thus the expression of nifR4 is very similar to the expression of structural nif genes such as nifH. Since the NIFR1/NIFR2 system is necessary for the expression of nifA, it is very likely that the effect of NIFR1/NIFR2 on nifR4 expression is exerted via NIFA. The expression of nifA is low, but measurable, in the presence of

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HUBNER ET AL. low fixed nitrogen

Rhodobacter capsulatus

(GLNB-UMP)~+

low

rati:

a-ketoglutarate

}Binase NTRBLeCG(N

2 }=kinase

\+ATP

(NiiR'P

{UflkfoWfl sigma factor ?)

V =NTRC-P /

active

Klebsiella pneumoniae

/+ATP

tAnsciptional NTRC-Pactive

NTRA activatorssig ! \ , / factor

NIFA NIFR4 * I t /RA

~~~NIFA

m specific I naf specific

nif specific

transUiptional activatox

t

siga factor I

other nifgenes (eg. nifHIDK)

transcriptional activator

~~~~~~~~~~~~~~~~~~I I

other nif genes (eg. nifHDK)

FIG. 3. Comparison of nitrogen fixation regulatory models for R. capsulatus and K. pneumoniae. The model is based in part on the work presented here and in part on reviews (15, 25). UTase, uridyltransferase (encoded by ginD). The relevant differences between the regulatory circuits of R. capsulatus and K. pneumoniae are discussed in the text.

oxygen or ammonia. Thus the question arises why nifR4 is not expressed under these conditions. One possible explanation is that the nifR4 promoter cannot compete with all the other nif promoters for NIFA protein under conditions where NIFA is limiting. On the other hand, it was shown that in B. japonicum NIFA activity is oxygen sensitive, and four essential cysteine residues which could be involved in binding an oxygen-sensitive metal ion were identified (1214). These four cysteine residues lie within the central domain of NIFA which, in the case of Rhizobium meliloti, is sufficient for the transcriptional activation of nif genes (21). Moreover, the four cysteine residues are conserved between the NIFA of rhizobia and R. capsulatus but are not found in K. pneumoniae (12, 32). In K. pneumoniae, another protein, NIFL, is responsible for the inactivation of NIFA in the presence of oxygen or ammonia (8, 17). In rhizobia and R. capsulatus, evidence for a nifL-like gene is lacking. Thus it is likely that the observed oxygen sensitivity of nifR4 expression as well as the expression of other structural nif genes in R. capsulatus is a consequence of the postulated oxygen sensitivity of the transcriptional activator NIFA. This is in line with the finding that, in R. capsulatus glnB mutants, nif gene expression is not regulated by ammonia but is still repressed under aerobic conditions (28). The need of NIFR4 for its own expression confirms that NIFA acts in concert with the nif-specific sigma factor NIFR4. Therefore, expression of nijR4 is autoregulated by a positive feedback mechanism. The effect of ammonia on nifR4 expression is presumably exerted via the NIFR1/NIFR2 system, as also appears to be the case with nifA expression. We conclude that there are two levels of controls involved in nif gene expression in R. capsulatus (Fig. 3). The NIFR1/ NIFR2 system senses the intracellular concentration of fixed nitrogen and responds by controlling the expression of nifA. The transcriptional activator NIFR1 and, most likely, an unknown alternative sigma factor are involved in this step. On the second level of control, the nif-specific transcriptional activator NIFA induces, in concert with the nifspecific sigma factor NIFR4, the structural nif genes.

ACKNOWLEDGMENTS We are grateful to R. G. Kranz and W. Klipp for generously providing strains and plasmids. We thank P. Richaud for synthesis of the oligonucleotides and B. Cauvin, A. Alias, Y. Jouanneau, E. Schatt, C. Duport, and J. Pierrard for discussions and friendly cooperation all along during this work. The research was supported by grants from the Swiss National Science Foundation to T.A.B. and by a fellowship from the Swiss National Science Foundation to P.H.

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