FEMS Microbiology Letters 29 (1985) 5-9 5 Published by Elsevier

FEM 02159

Redox regulation of enteric nif expression is independent of the fnr gene product

(Plasmid-borne nif ; Fnr - E. coli )

Susan Hill

AFRC Unit of Nitrogen Fixation, University of Sussex, Brighton BN1 9RQ, U.K.

Received 2 May 1985 Accepted 6 May 1985

1. SUMMARY

The fnr gene product, needed for expression of anaerobic catabolism, is not needed for expression of nif in Escherichia coli.

2. INTRODUCTION

Nitrogen fixation in the facultative anaerobic Klebsiella pneumoniae is associated with anaerobic metabolism, although stimulation of nitrogen fixa- tion occurs under microaerobic conditions (30 nM 02) due to a general upgrading of the energy status [1-3]. At 0 2 concentrations near 6 tiM nitrogenase is inactivated and transcription from all nif operons, apart from nifLA, is inhibited [4-7]. At more elevated 02 concentations (60 tiM) expression from the nifL promoter is decreased [4]. The mechanism whereby 0 2 regulates the ex- pression of these genes is less well understood than is the mechanism of transcriptional control by fixed N, in which regulation at the nifL promoter by high concentrations of fixed N involves the ntr system [8 and references therein]. Regulation by low fixed N concentrations or by 0 2 is believed to involve antagonism of nifA-mediated activation by the nifL product [5-7,9]. However, the mecha-

nisms whereby either the N status or the redox status of the cell are communicated to the nifL product are unknown.

The mechanism of communication of the redox status might share common features with the regu- lation of other anaerobic metabolic processes. In E. coli, the fnr product positively controls the synthesis of anaerobic respiratory systems (e.g., fumarate and nitrate reductases) [10-12]. The fnr product has been implicated in the expression of nif because Scotnicki and Rolfe [13] failed to get expression of plasmid-borne nif in an F n r - mutant of E. coil Contrary to their finding, I report below significant nif expression from the Nif + plasmid pRD1 in that mutant and in four other F n r - strains of E. coli.

3. MATERIALS AND METHODS

3.1. Bacteria and plasmids Bacterial strains and plasmids are shown in

Table 1.

3.2. Media Nutrient broth (NB) and nutrient agar (NA)

were from Oxoid. Luria broth (LB), Luria agar (LA), minimal medium (MM), nitrogen-free

0378-1097/85/$03.30 © 1985 Federation of European Microbiological Societies

6

Table 1

Bacterial strains and plasmids

Strain Genotype or phenotype Derivation of F n r - Source and Reference or plasmid or N i rA- strain

E. coli PL2024 JRG861a JRG861b JRG861c JRG861d RK4353 RK5279

CB246 CB244

JC5466 J62-1 C l l l

K. pneumoniae U N F 686

Plasmids pRD1 pGS24 pBR322

F-, gal, trpA, trpR, rpsL F-, gal, trpA, trpR, rpsL, fnr-1 F-, gal, trpA, trpR, rpsL, fnr-8 F-, gal, trpA, trpR, rpsL~ fnr-2 F-, gal, trpA, trpR, rpsL, fnr-4 Alac U169, araD139, rpsL, gyrA, non A lac U169, araD139, rpsL, gyrA, non, fnr-250

trp trp, nirA1

trp, his, recA56, rpsE proC, his, trp, hal, lac thr, len, thi, bio, narD, rpsL

A( his-nif ), 2633 hsdR1, rpsL, recA, lacA MuhP1

K m r, Cb r, Tc r, His + , Nif + , ShiA + Tra +, IncP Cb r, Fnr + Cb', T C

- J.R. Guest [15] N G mutagenesis of PL2024 J.R. Guest [15] N G mutagenesis of PL2024 J.R. Guest [15] N G mutagenesis of PL2024 J.R. Guest [15] N G mutagenesis of PL2024 J.R. Guest [15] - V. Stewart [23] spontaneous mutation V. Stewart [23] transduced into RK4353 - J.A. Cole [19] NG mutat ion transduced J.A. Cole [19]

into CB246 N. Willetts N. Datta W.A. Venables

R.A. Dixon [4]

R.A. Dixon [20] J.R. Guest [24] Bolivar etal. [31]

medium (NFDM) are described by Cannon [14]. The Fnr + phenotypes were tested anaerobically on lactate plus nitrate (LN) and glycerol plus fumarate (GF) minimal medium of Lambden and Guest [15]. When required medium contained tryptophan (20 t~g.ml-1), kanamycin (Km, 20 /~g.ml-t), carbenicillin (Cb, 200 t~g.ml-a), streptomycin (Sm, 200/~g.m1-1) and trimethoprim (Tp, 20/~g.ml-1).

3.3. Conjugation and transformation Donor and recipients for conjugation were

grown in rich media (either NB or LB) for about 6 h at 30°C or 37°C. Matings were carried out either

o n NA or after washing with saline phosphate buffer [14] directly on the selective medium (MM supplemented with Km and Cb and sometimes tryptophan or Sm). Appropriate donor strains (JC5466, J62-1 or C l l l ) were chosen according to their genotype to allow selection of transcon- jugants. Transconjugants were subsequently puri- fied on the selective medium and were maintained

on this medium at room temperature with mini- mum sub-culturing.

The integrity of pRD1 in transconjugants after some C 2 H 2 reduction experiments was checked by plating diluted suspensions in saline phosphate buffer on either NA or LA. After overnight growth colonies were replicated to a lawn of UNF686 spread on the surface of NFDM Tp plates. Fol- lowing anaerobic incubation (Gas Pak) for a week at 28°C, growth in the replicated patches indicated that the his and nif markers has been transferred. In all transconjugants tested the loss of these markers was less than 10%.

Transformation of pGS24 or pBR322 was per- formed by a modification, using calcium chloride, of the freeze-and-thaw method of Dityatkin et al. [16] (M.J. Merrick, personal communication).

3.4. Preparation and analysis of recombinant plas- mid DNA

The standard techniques of plasmid preparation

using alkaline lysis a~d restriction analysis using agarose gels were those of Maniatis et al. [17].

3.5. Growth conditions, assays and analyses Growth and subsequent assay for C2H 2 reduc-

tion after N-limited growth was performed as de- scribed by Cannon [14] but with the following niodifications: LB cultures sometimes replaced NB cultures as the inoculum for the N F D M which contained the indicated sources of limiting fixed N, K m was added to cultures of transconjugants.

For nif derepression experiments strains were grown at 28°C aerobically in 10 ml of MM plus K m for 24 h. This culture was used as a 10% inoculum for 45 ml of N F D M supplemented with 15 mM (NH4)2SO 4 and K m contained in a medi- cal flat bottle. After 20 h of growth at 28°C under a slow stream of 1% CO 2 in N 2, volumes of culture (equivalent to 780 mg protein.ml-1) were harvested in plastic bijou bottles by centrifugation (at 3500 rev . /min for 15 min). The bottle was capped with a suba seal and the pellet resuspended anaerobi- cally in 3 ml of argon-sparged N F D M containing K m and, where indicated, 0.69 mM glutamine. 1 ml of C2H 2 was injected and incubation was con- tinued at 28°C. Gas samples (0.5 ml) were re- moved at intervals for gas chromatography analy- sis of C2H 4 [1].

Protein was estimated by the procedure of Lowry [18].

4. RESULTS

4.1. C2H 2 reduction by Fnr - E. coli (pRDI) trans- conjugants

Scotnicki and Rolfe [13] constructed transcon- jugants of E. coli strain JRG861a ( F n r - ) and PL2024 (wild type) carrying the Nil + plasmid FN68 and obtained significant nitrogenase activity (C :H 2 reduction) only in the wild type. In the present work the Nif ÷ plasmid pRD1 was used to construct transconjugants of 5 F n r - mutants, 1 N i r A - mutant and their respective wild-type E. coli strains. The nirA gene is considered to be synonymous with the fnr gene [19]. Plasmid pRD1 (formerly RP41; [20]) is a recombinant of RP4 and FN68, and has inherited from FN68 a segment of the K. pneumoniae (strain M5al) chromosome en- coding nif, his and several other genes [21]. The recipients and transconjugants were tested for C2H2-reducing activity in an assay procedure, of slightly different design from that used by Scot- nicki and Rolfe [13] but which has been used extensively to confirm the N i f - phenotype of K. pneumoniae mutants [14]. None of the recipients reduced acetylene (not shown). The C2H2-reduc- ing activities of the transconjugants are shown in Table 2. Activities in all the F n r - mutants com- pared to the relevant wild type were consistently much greater than that found by Scotnicki and Rolfe [13] for strain JRG861a carrying FN68 ( <

Table 2

Nif expression, measured by nitrogenase activity (C2H 2 reduction), after 18 h N-limited growth in Fnr- and Nir- strains of E. coli

Strain Relevant genotype

Nitrogenase activity N source (/~g.ml-1)

Casamino acids (50) Serine (100)

A a B a A B

PL2024 100(2.3 + 0.1) b 100(10.5 _ 0.1) 100(1.5 + 1) N.T. JRG861a fnr-1 79 + 27 314-1 47 _+ 28 N.T. JRG861b fnr-8 N.T. 52 ___ 1 N.T. N.T. JRG861c fnr-2 261 _+ 68 N.T. 144 _+ 65 N.T. JRG861d fnr-4 88 5:31 N.T. 20 _+ 8 N.T. RK4353 100(0.2) N.T. 100(0.2) 100(0.5) RK5279 fnr-250 327 N.T. 88 208 CB246 100(2.3) 100(115:1) 100(0.3) 100(8.5 + 2) CB244 nirA1 24 9 0 40 + 3

a Values in parentheses are nmol C 2 H 4.min - 1.mg protein- 1 b Inocula (5%) grown in either LB (A) or NB (B). N.T., not tested.

0.03%, equivalent to a specific activity of < 0.01). In some Fnr- strains the activity was higher than in the relevant wild type. Differences in N source did not markedly affect activity although when inocula were grown in LB in place of NB the specific activities in mutant and wild-type strains were lower.

Further experiments were performed with the isogenic strains RK5279 (pRD1) (Fnr-) and RK4353 (pRD1) (Fnr+). Derepression of C2H 2 reduction, after 18 h anaerobic growth with excess NH~-, was followed in NFDM medium. Activity appeared after a lag of approx. 2 h and by 5 h specific activities (nmol C2H 4 produced.min-1.mg protein-I), ranged from <0.1 to 0.3 for the mutant transconjugant and from 0.3 to 0.6 for that of the wild type. The addition of glutamine (0.69 mM), which stimulates the derepression of C2H 2- reducing activity in K. pneumoniae [22], markedly enhanced derepression in RK5279 (pRD1) and RK4353 (pRD1); by 5 h the specific activity ranged from 8 to 14 and 11 to 25 respectively.

4.2. Complementation of fnr-250 by pGS24 Transductional mapping of fnr-1, -8, -2 and -4

[15], fnr-250 [23], and nirA [19], has shown a similar linkage, from 6 to 12%, to pyrF. Plasmid pGS24 carries the fnr gene, as a 1.2-kb HindlII + BamHI fragment inserted into pBR322, and com- plements mutations fnr-1, -8, -2, and -4 [24].

Confirmation that mutations fnr-250 and nirA1 reside in the fnr gene was sought by testing these mutations for complementation by pGS24. As ex- pected Cb R transformants of RK5279 (fnr-250) carrying pGS24 grew anaerobically on either GF or NF media, whereas the transformants carrying pBR322 did not. Unexpectedly transformants of CB244 (nirA1) carrying pGS24 failed to grow on these media as did tranformants carrying pBR322, whereas similar transformants of the wild-type CB246 grew.

5. DISCUSSION

• Significant nitrogenase activity was observed in transconjugants of 5 Fnr- mutants of E. coli car-

rying pRD1. The activity, although variable, was near and sometimes higher than that in the rele- vant wild-type transconjugants. Thus, in contrast to the report of Scotnicki and Rolfe [13], expres- sion of plasmid-borne nif genes showed no con- sistent requirement for the fnr gene product in E. coli.

The influence of the nirA mutation on nif ex- pression, from pRD1, was somewhat similar to that of the fnr mutations (Table 2). Analysis of" the genes nirA and fnr by co-transduction [15,19] and F' complementation [19] have indicated that they are identical. However, the failure, reported here, to complement the nirA mutation with pGS24 suggests that nirA and fnr are separate genes.

A common cause may underline the absence of nif expression from FN68, reported by Scotnicki and Rolfe [13] and the variability of expression from pRD1, reported here, in Fnr mutants of E. coli. Nitrogenase synthesis in K. pneumoniae is positively correlated with the energy status [3,25], and for activity the enzyme requires adequate sup- plies of ATP and electrons [26]. Consequently perturbations in energy status or supply of pyru- vate (the electron donor [27,28]) arising from pleiotropic effects of the fnr mutation on anaerobic catabolism could influence the timing and rate of nif derepression and magnitude of nitrogenase ac- tivity. Such perturbations probably account for the differences in the specific C2H2-reducing activities observed in parallel assays of mutant and wild type.

The finding that nif expression is independent of the fnr product cast doubt upon the possibility that the mechanisms regulating expression of nif and other anaerobic enzymes [29 and references therein], in the Enterobacteriaceae share common features. Nevertheless, there may be a common effector molecule which communicates the redox status. Circumstantial evidence relating to nif reg- ulation in K. pneumoniae suggests that such an effector may be a component of the aerobic respiratory chain, since the dissolved oxygen con- centration (100 nM) which half represses nitro- genase synthesis is near the apparent K s of the principle terminal oxidase [30].

A C K N O W L E D G E M E N T S

I t hank Prof . J .R . Pos tga t e and Dr . M.J . M e r -

r ick for c o n s t r u c t i v e c r i t i c i sm of the m a n u s c r i p t ,

Mis s M e l i t a F a r r u g i a for p r e p a r a t i o n of the type-

scr ip t and Prof . J .R. G u e s t and Drs . J .A. C o l e and

V. S t ewar t for bac t e r i a l s t ra ins a n d p lasmids .

R E F E R E N C E S

[1] Hill, S. (1976) J. Gen Microbiol. 93, 335-345. [2] Hill, S., Turner, G.L. and Bergersen, F.J. (1984) J. Gen.

Microbiol. 130, 1061-1067. [3] Hill, S., Turner, G.L. and Bergersen, F.J. (1984) in

Advances in Nitrogen Fixation Research (Veeger, C. and Newton, W.E., Eds.), pp. 260, Martinus Nijhoff/Junk, The Hague.

[4] Dixon, R.A., Eady, R.R., Espin, G., Hill, S., laccarino, M., Kahn, D. and Merrick, M. (1980) Nature 286, 128-132.

[5] Hill, S., Kennedy, C., Kavanagh, E., Goldberg, R.B. and Hanau, R. (1981) Nature 290, 424-426.

[6] Merrick, M., Hill, S., Hennecke, H., Hahn, M., Dixon, R. and Kennedy, C. (1982) Mol. Gen. Genet. 185, 75-81.

[7] Cannon, M., Hill, S., Kavanagh, E. and Cannon, F. (1985) Mol. Gen. Genet. 198, 198-206.

[8] Dixon, R.A. (1984) J. Gen. Microbiol. 130, 2745-2755. [9] Filser, M., Merrick, M. and Cannon, F. (1983) Mol. Gen.

Genet. 191, 485-491. [10] Shaw, D.J., Rice, D.W. and Guest, J.R. (1983) J. Mol.

Biol. 186, 241-247. [11] Unden, G. and Guest, J.R. (1984) FEBS Lett. 170,

321-325. [12] Unden, G. and Guest, J.R. (1985) Eur. J. Biochem. 146,

193-199.

[13] Scomicki, M.L. and Rolfe, B.G. (1979) Aust. J. Biol. Sci. 32, 637-649.

[14] Cannon, F.C. (1970) in Methods for Evaluating Biological Nitrogen Fixation (Bergersen, F.J., Ed.), pp. 367-413, Wiley, Chichester.

[15] Lambden, P.R. and Guest, J.R. (1976) J. Gen. Microbiol. 97, 145-160.

[16] Dityatkin, S.Y., Lisovskaya, K.V., Panzhava, N.N. and Ilinshenko, B.M. (1972) Biochim. Biophys. Acta 281, 319-323.

[17] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molec- ular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[18] Lowry, O.J., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275.

[19] Newman, B.M. and Cole, J.A. (1978) J. Gen. Microbiol. 106, 1-12.

[20] Dixon, R.A., Cannon, F. and Kondorosi, A. (1976) Nature 260, 268-271.

[21] MacNeil, D., Supiano, M.A. and Brill, W.J. (1979) J. Bacteriol. 138, 1041-1045.

[22] Nair, M.B. and Eady, R.R. (1984) J. Gen. Microbiol. 130, 3063-3069.

[23] Stewart, V. (1982) J. Bacteriol. 151, 1320-1325. [24] Shaw, D.J. and Guest, J.R. (1982) J. Gen. Microbiol. 128,

2221-2228. [25] Jensen, J.S. and Kennedy, C. (1982) EMBO J. 1,197-204. [26] Eady, R.R. and Postgate, J.R. (1974) Nature 249, 805-810. [27] Hill, S. and Kavanagh, E.P. (1980) J. Bacteriol. 141,

470-475. [28] Shah, V.K., Stacey, G. and Brill, W.J. (1983) J. Biol.

Chem. 258, 12066-12068. [29] Gottesman, S. (1984) Annu. Rev. Genet. 18, 415-441. [30] Bergersen, F.J., Kennedy, C. and Hill, S. (1982) J. Gen.

Microbiol. 128, 909-915. [31] Bolivar, F., Rodriguez, R., Greene, P.J., Betlach, M.,

Heyneker, J.K., Boyer, H.W., Crosa, J. and Falkow, S. (1977) Gene 2, 95-113.