Molecular Microbiology (1992) 6(19), 2805-2813

Transcriptional controi, transiation and function of the products of the five open reading frames of the Escherichia coli nir operon Nerina R. Harborne, Lesley Griffiths, Stephen J. W. Busby and Jeffrey A. Cole* School of Biochemistry, University of Birmingham, Birmingham B152TT, UK. Summary Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli n/roperon. Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC 1.6.6.4). A series of plasmids has been constructed in which each of the open reading frames has been fused in-phase with the p-galactosidase gene, lacZ. Rates of p-galactosidase synthesis during growth in different media revealed that nirB, -D, -E and -Care transcribed from the FNR-dependent promoter, p-nirB, located just upstream of the nrrB gene: expression is co-ordinately repressed by oxygen and induced during anaerobic growth. Although the nirB, -Dand-Copen reading frames are translated into protein, no translation of nirE mRNA was detected. The cysG gene product is expressed from both p-nirB and a second, FNR-independent promoter, p-cysG, located within the n/rC gene. No NADH-dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild-type bacteria. Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing. NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme.

Introduction There are two major pathways for nitrite reduction to Received 7 October, 1991; revised and accepted 15 June, 1992. *For correspondence, Tel. (021)4145400; Fax (021)4143982,

ammonia in enteric bacteria {Cole, 1988). The less active pathway in most Escherichia coii strains is the electrogenic, formate-dependent nitrite reductase which allows anaerobic growth on non-fermentable carbon sources such as glycerol (Page ef ai, 1990). The more active NADH-dependent pathway uses a cytoplasmic enzyme to remove nitrite formed as a product of nitrate reduction. As synthesis of both of these nitrite reductases is dependent on transcription activation by the FNR protein, they are active only during anaerobic growth and fulfil a dissimilatory rather than an assimilatory role (Cole, 1978). We have reported that the nir operon, which encodes the cytoplasmic, NADH-dependent nitrite reductase, contains five open reading frames (ORFs) designated nirB, nirD, nirE, nirC and cysG (Peakman et ai, 1990a). The nirB gene encodes the 90 kDa nitrite reductase apoprotein and cysG is essential for the synthesis of the sirohaem prosthetic group of both NADH-dependent nitrite reductase and NADPH-dependent sulphite reductase (Newman and Cole, 1978; Jackson et ai., 1981; Warren et al., 1990), It is not known, however, whether the other ORFs are translated into protein products and, if so, whether the polypeptides are essential for nitrite reduction or sirohaem biosynthesis. To resolve these uncertainties, segments of the nir operon have now been deleted in vitro to produce a series of in-frame fusions of each of the ORFs to the E. coli lacZ gene. Rates of expression of the resulting hybrid p-galactosidase polypeptides under different growth conditions and the effects of the deletions on nitrite reduction and cysteine synthesis have been determined.

Results and Discussion Vectors for creating protein fusions A series of plasmids was constructed which were suitable for cloning each of the ORFs of the nirB-cysG operon in phase with the £ coli [i-galactosidase gene. These plasmids were based on the protein fusion vector, pAA204 (Bingham and Busby, 1987; Fig. 1), which carries a fusion of the ga/promoter region to lacZ. First, an 8, lOor 12 bp H/ndlll linker was inserted at the BamHl site ot pAA204: the gal promoter fragment was then replaced by the

2806

N. R- Harborne, L Griffiths, S. J. W. Busby and J. A. Cole pUCIinker

1, Insert Wnd in linker at the eomHI site

8nOM2 base poir linker

Fig. 1. Construction of veciors for creating protein fusions. An 8. lOor 12bp Hind\\\ linker (Amerstiam) was first inserted at the SamHI site of pAA204 (Bmgham and Busby, 1987). The EcoRIH/rjdllHragments were then replaced by the pUC9 polylinker to generate pAA2051, -2052 and -2053. Restriction sites shown are B (BamHI), H (H/ndlll), and R (EcoRI).

2 Replace EcoR\-Hind m gal fragment with the pUC9 polylinker

pUC9 EcoRI-H/ndlll polylinker, as shown in Fig. 1, to generate a new set of vectors pAA2051, -2052 and -2053, suitable for accepting EcoRI~H/ndlll fragments to create fusions to lacZ'm any phase (Table 1).

Fusior) of the nirB-cysG oper) readmg frames to IacZ A map of the nirB-cysG operon is shown in Fig. 2A, together with the restriction sites used in this work. The starting point for generating fragments of the n/r operon to be cloned into vectors pAA2051 to -2053 was pRAR7 {Peakman et al., 1990b). This piasmid contains the r)irB promoter, the nirB, -D, -E and -C ORFs and the 5' end of cysG to position 4395, cioned as an EcoRI-H/ndlll fragment into the transcriptionai fusion vector pAA182. The pRAR7 EcoRi-H/ndlll fragment was transferred to pAA2053 to create the cysG-iacZ fusion piasmid, pRAR70 (Fig. 2B). Bai31 nuciease was then used to produce truncated derivatives of the pRAR70 fragment in which the rtir operon sequence was progressively shortened from the H/ndlll end. After Bai31 treatment, H/ndlll iinkers were added and the resulting famiiy of f c o R I H/ndlll fragments illustrated in Fig. 2B were doned into the appropriate vector to create in-phase fusions to IacZ (Table 1). These plasmids were then transformed into the lac E. coii host Ml 82 and expression of the hybrid pgalactosidase proteins during aerobic or anaerobic growth was then measured (Table 2). Only the cysG-/acZfusion protein was expressed at a significant rate during aerobic growfh. This is consistent with our previous report that the first four ORFs of the nir operon are expressed only from the FNR-dependent nir promoter, but that there is an FNR-independent promoter, p-cysG, located within the 3' end of the coding sequence for NirC. During aerobic growth, transcription of cysG is initiated at this internal promoter (Peakman etal., 1990b; see subsequent sections). During anaerobic growth, synthesis of the NirBTacZ, NirD"LacZ and NirC'LacZ fusion proteins was strongly

induced and twice as much CysGTacZ fusion protein was formed compared with the aerobic culture (Table 2). In contrast, no NirE'::'LacZ activity was found during either aerobic or anaerobic growth, indicating that the nirE message is not translated. Only background levels of pgalactosidase activity were found in bacteria transformed with any of the starting vectors, or with pRAR73 in which iacZ was fused to the nirD-nirE intergenic region (Table 2). FNR-dependent and FNR-independent expressior) of CysG Peakman et al. (1990b) proposed that the increased expression of cysG during anaerobic growth was due to transcription readthrough from the FNR-dependent nirB promoter supplementing message transcribed from the aerobically expressed p-cysG. If so, deletion of the internal cysG promoter from pRAR70 should result in FNRdependent CysG::LacZ expression only during anaerobic growth. Plasmid pRAR70D was therefore constructed in which the cysG promoter was deleted from pRAR70 (Fig. 2B, line 7). The resulting plasmid (in which nirC ORF coordinates 3492 and 4165 were joined in phase by a linker) was transformed into both £ co//M182 and the /nr derivative, JRG1728. Rates of p-galactosidase expression during aerobic and anaerobic growth were compared with the same strains which had been transformed with pRAR70 (in which both p-nirB and p-cysG are functional) and with pPMB80 which lacks the nirB promoter (Table 3). As predicted, very little CysG::LacZ polypeptide was synthesized from pRAR70D during aerobic growth, or during anaerobic growth in the fnr host. CysG'::'LacZ expression from pPMB80 was constitutive during either aerobic or anaerobic growth in both Fnr* and Fnr" hosts and, as reported by Peakman et al. (1990b), the increased expression from pRAR70 during anaerobic growth was FNR-dependent (Table 3). From this we conclude that cysG is transcribed during aerobic growth from

Genes of the Escherichia coli nir operon Table 1. Plasmids and strains used in this work. Plasmid/ Strain

Description

pAA204

pBR322-based plasmid carrying a ga/E;:/acZ fusion pAA2051, Derivatives of pAA204 allowing -2052, -2053 insertion ot promoters and fusion of foreign translation starts to (Jgalactosidase in any of the three phases (Fig. 1) pPH13 Broad-host-range plasmid with nir sequence from co-ordinates 3728 to 5613 cloned into ppRK2501; carries cysG' pPMB80 cysGii'acZ fusion lacking p-nirB. nirB. -D. -Eand-C pRAR70 Derivative of pAA2053 carrying nirB nirD nirE nirC a cysGv.lacZ fusion (Fig. 2) pRAR70D Derivative of pRAR70 from which the cysG promoter has been deleted due to an in-phase deletion in riirC pRAR71 Bal31 deletion denvative of pRAR70 resulting in an in-phase nirCv.lacZ fusion (Fig. 2) pRAR71D As pRAR71 but with two internal flg/ll fragments of nirB removed pRAR72 As pRAR71. but with a nirEwlacZ fusion pRAR73 As pRAR71 but with /acZfused to pRAR74 pRAR75 PRK2501 pR1502 pi 202

As pRAR71, but with a nirDv.lacZ fusion As pRAR71, but with a nirBwIacZ fusion Broad-host-range vector used to clone cysG mrS::/acZfusion of pRI 50 cloned into pAA2052 n/rC::/acZfusion in pAA204

Reference/Source

Bingham and Busby (1987) This work

Peakman etal. (1990b)

This work

A/ac./fjr'

JCB323

Chlorate-resistant Lac" strain with a chromosomal deletion of all of the genes of the /iir operon Nar' Lac" transductant of JGB323 Mac Atnr strain

JCB387 JRG1728

presence of glucose and nitrite. Whole-cell lysates were analysed by SDS-polyacrylamide gel electrophoresis (PAGE) and the protein profiles were visualized by staining with Coomassie brilliant blue (Fig. 3A). Clearly stained bands of LacZ fusion proteins were visible in tracks 3 and 4 because of the n/r6'::7acZ fusion products. No band of corresponding intensity was seen in track 2, whicfi was loaded with protein from the untransformed Lac" host bacteria. Intense bands due to NirD"LacZ and NirC'LacZ fusion proteins were also visible in tracks 5 and 8, respectively, with weaker bands of NirC'LacZ fusion proteins from pRAR7iD and p1202 in tracks 9 and 10. The CysG'lacZ band expressed from pRAR70 was very weak (frack 11), as expected from fhe correspondingly low p-galactosidase activity of the pRAR70 transformants (Tables 2 and 3). No clearly detectable hybrid LacZ polypeptides were produced in bacteria transformed with either pRAR73 (in which lacZ is fused to tfie nirD-nirE intergenic region: lane 6) or with the nirEv.laoZ fusion plasmid, pRAR72 (lane 7). Increased intensity of staining in the 90 kDa region of the gel is also apparent in tracks 5 to 8,10 and 11 due to overexpression of the nirB polypeptide encoded by plasmids pRAR70 to -74 and pi 202 (Fig. 3). In contrast, only

Table 2. Expression of different /acfusions. ^-galactosidase Expression During:

Khan efa/. (1979) Bell etal. (1990)

Plasmid

Note

aerobic growtti

Peakman et al. (1990b)

pAA2053 pRAR70 pRAR71 pRAR72 pRAR73 pRAR74 pRAR75

vector only cysG::/acZ fusion n/rC::/acZtusion n/r£::/acZ fusion intergenic /acZfusion n(/D;:/acZ fusion n//S::/acZfusion

400 60 10 20 50 50

Strain Ml 82

2807

Casadaban and Cohen (1980) Griffiths and Cole (1987) Page efa/. (1990) Spiro and Guest (1988)

the FNR-independent promoter, pcysG, but from both pcysG and the FNR-dependent nirB promoter during anaerobic growth, as proposed by Peakman et al. (1990b).

Visualization of the products of the nir operon by SDS-PAGE Sfrain JCB387 (Page etal., 1990) with a total chromosomal deletion of the nir operon was fransformed with tfie various fusion piasmids and grown anaerobically in the

anaerobic growth

10

10 800 14000 10 20

4100 2500

The Table shows p-galactosidase activities in permeabilized Ml 82 frir' cells carrying different plasmids. Activities are expressed as nmot onitrophenyl-p-D-galactoside hydrolysed min"'(mg bacterial dry mass)"'.

Table 3. FNR-dependent transcription of cysG from the rr/rSand cysG promoters. Growth Conditions

Host strain

Plasmid

Functional promoters controlling lacZ

aerobic

anaerobic

Ml 82.

pRAR70 pRAR70 pRAR70D pRAR70D pPMB80 pPMB80

p-nirB + p-cysG P'CysG p-nirB None P'CysG p-cysG

400 400 50 20 500 500

800 400 700 20 500 500

Piasmid pRAR70D was made by deleting bases 3492 to 4165, inclusive, making an in-frame deletion in nirC and inactivating p-cysG. p-galactosidase activities are as in Table 2.

2808

N. R. Harborne, L Griffiths, S. J. W. Busby and J. A. Coie

(A) Bg

D N Bt

Bg Bt

I I

'.,000

3.000

2.O00

1.000

R

r

I

5,000

PcysG

PnirB 383

29231

1*07

5613

I 3398 U2

S593

2946 3269

nirD E

nirB

cysG

nirC

(B) Bf

pRAR 70 3^.81 I- H 3350

Bt Bf

^.395

r=

1

B288 b H 3218

Bt 1

Bt

S93 ^U92

Bf

1

1

Bt

i.66

2250

A

pRAR71

(cy^ 6- iacZ) (nirC - lacZI

pRAR72

(nirE- lacZI

pRAR73

(^

pRAR7i.

(nirD- lacZI

pRAR75

(nirB - IacZ)

pRAR70D

(cysG-tocZI

pRAR71D

(mrC- lucZ)

pPMB80

(cysG-IacZ)

M65i.395

1 Uu

- IacZ)

Fig. 2. A. Physical map and location of ttie five ORFs of the riirB-cysG operon located at minute 74 on the E. coli chromosome. Only restriction sites for enzymes used in this work are shown: Bg (Bg/ll), Bt (Ss(EII), C (the Hinci\ site used to construct pRAR7 and pRAR70), D (Oral), R (EcoRI). Numbers correspond to the DNA sequence presented by Peakman etai. (1990a). The m/Btranscription start at position 383 is indicated by a horizontal arrow. B. Positions of fusion junctions in the plasmids used. Plasmids pRAR71, -73, -74 and 75 were constructed by Bal31 deletion from the H/ndlll site of pRAR70. ADralsiteinmrEwasused tomakepRAR72. Plasmid pPMBSO contains the EcoRI-H/ndlll fragment of pPMB8 (Peakman etai.. 1990b) cloned into pAA2053. Plasmids pRAR70D and pRAR71 D are described in the text. The EcoRI and H/ndlll linkers upstream and downstream of each fragment are illustrated as open and tilled boxes.

a weak band of an unrelated chromosomally encoded polypeptide is seen in tracks loaded with protein from the untransformed host, or from bacteria transformed with either the n/r&i/acZ fusion plasmids or with pRAR71D. We were unable to separate the NirD, NirC or CysG polypeptides from other closely migrating proteins by one-dimensional SDS-PAGE. Note, however, the increase in staining intensity in the 12 kDa region of protein from bacteria transformed with plasmids which encode a complete NirD polypeptide (pRAR73, -72, -71, -71D, pi 202 and pRAR70 in tracks 6 to 11. respectively).

Essentiai roie of NirD in NADH-dependent nitrite reduction Only the NirB polypeptide was detected by SDS-PAGE in the small samples of active nitrite reductase purified by Jackson et ai (1981). The discovery of additional genes in the n/r operon raised the possibility that their products might be essential for NADH-dependent nitrite reductase activity, but that a low M, polypeptide might have escaped detection in our previous work. The set of fusion plasmids described in Fig. 2 enabled us to re-examine this possiblilty.

Strain JCB323 is a temperature-stabilized derivative of the n/rS::Mud1 insertion mutant, JCB303. As temperature stabilization resulted in the loss of all five genes of the n/rS-cysG operon, this strain is Nir" and requires cysteine for growth. Strain JCB323 was first transformed with pPH13, which encodes a functional cysG gene in the broad-host-range vector pRK2501 (Peakman et ai., 1990a). Purified transformants were then re-transformed with one of the fusion plasmids, pRAR70 to pRAR75. The ability of these double transformants to grow on minimal medium without cysteine or to reduce nitrite rapidly during anaerobic growth was then determined (Table 4). Plasmid pPH13, which encodes only the cysG gene, was sufficient to restore a Cys* phenotype, indicating that only the cysG^ gene and its promoter are required for sirohaem synthesis. Surprisingly, however, both the nirB'^ and the nirD' genes were essential for nitrite reduction. In contrast, neither nirE nor nirC was required for either nitrite reduction or sirohaem synthesis (Table 4). Since nirE mRNA is not translated, the lack of a requirement for a functional nirE ORF is not surprising. Any function for NirC in nitrite reduction remains to be determined, but its hydrophobicity suggests that it is a membrane-spanning integral membrane protein. Its role

Genes of the Escherichia coii nir operon

2809

B I

2

3

4

5

6

7

8

9

10

II

12 13

LocZ NirB

NirD

Fig. 3. A. Visualization by SDS-PAGE of NirB and LacZ fusion proteins produced from the n/r operon. E co/i strain JCB387 was transformed with one of the fusion plasmids, grown anaerobically in Lennox broth supplemented with 0,4% (w/v) glucose and 2.5 mM nitrite, harvested, and lysed. Proieins in the whole-cell lysate were separated by electrophoresis through a 5-25% gradient polyacrylamide gel in the presence of SDS and vrsualized by staining with Coomassie brilliant blue R250, Track 2, untransformed host bacteria. In tracks 3 to 11 bacteria were transformed with the following plasmids: 3, pRi 502 (short mrB fragment fused to lacZ].4. pRAR75 {nirB-lacZ). 5, pRAR74 (nirD-lacZ)\ 6, pRAR73 {-lacZ)\ 1. pRAR72 {nirE-lacZ);%. pRAR71 {nirC-lacZ), 9, pRAR71 D: 10, pi 202 (nirC-lacZ) and 11, pRAR70 [cysG-lacZ). Track 12: high-H marker proteins were myosin (205 kDa), |i-galactosidase (116 kDa), phosphorylase B (97 kDa), BSA (66 to 69 kDa). ovalbumin (45 kDa) and carbonic anhydrase (29-30 kDa), Track 13: low-M, marker proteins were BSA, ovalbumin, glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase, trypsinogen (24 kDa). trypsin inhibitor (20-21,5 kDa) and laotaibumin (14,2 kDa), Track 1 was loaded with a mixture of the high- and low-M, marker proteins, B, Association of the NirD polypeptide with NirB in partially purified NADH-dependent nitrite reductase. Track 1: active pool of nitrite reductase eluted from a column of DEAE Sepharose CL6B after (NH4)2S04 fractionation of soluble proteins from E, co//strain JCB323(pRAR70). Track 2 contains high- and low-M, marker proteins, as in track 1 of part 3A,

may be to transport nitrite across the membrane either for the uptake of external nitrite for reduction by the NADHdependent enzyme, or for the export of nitrite formed in the cytoplasm during nitrate reduction. This suggestion is consistent with the observation that NirC is homologous to the product of the ORF preceding the E. coli pfl gene which might have a function in formate transport (G. Sawers, personal communication).

Soluble extracts were prepared from anaerobic cultures of JCB323 transformed with either pRAR71D or pRAR74 and assayed spectrophotometrically for nitrite reductase activity. No activity was detected with either extract alone, but NADH was rapidly oxidized by nitrite when the two extracts were mixed on ice before being

Table 4. Essential role of NirD in NADH-dependent nitrite reduction by E, co/( strain JCB323.

Reconstitution of NADH-dependent nitrite reductase activity in vitro There are two simple explanations for the requirement of a functional nirD* gene for NADH-dependent nitrite reduction: either NirD is a subunit of the enzyme, or it catalyses the post-translational activation of NirB, for example by catalysing the insertion of sirohaem into the apoenzyme. Figure 4 illustrates an experiment designed to reconstitute NADH-dependent nitrite reductase activity by mixing inactive extracts of bacteria which synthesize only one of the two polypeptides. The nirC.JacZ fusion piasmid, pRAR71D, was used as a source of NirD polypeptide uncontaminated with NirB (line 8 of Fig. 2B). Similarly, pRAR74 encodes NirB without NirD,

pPH13

Phenotype of Transformants

(cysG')

Nir

None

•••

-

pRAR74

nirB

+

—

+

pRAR72

nirB nirD

+

+

+

pRAR71

nirB nirD nirE

+

•

pRAR70

nirB nirD nirE nirC

+

•

nir plasmid

Functional n/r genes

pRAR75

Cys

Strain JCB323 IS Nir Cys because of the deletion of all five ORFs of the nirB operon, Plasmid pPH13 carries a functional cysG gene and is compatible with the pRAR series of plasmids.

2810

N. R. Harborne, L Griffiths, S. J. W. Busby and J. A. Ccle NirD poiypeptide with NirB. The specific activity of the reconstituted nitrite reductase activity was calculated, from the slope of the linear portion of Fig. 4, to be 240nmol NADH oxidized min"^ (mg of NirB* extract)"^: this activity is similar to the specific activities of nitrite reductase in typical wild-type strains of E. ccli and in strains transformed with multicopy plasmids encoding the complete nir operon which are In the range 50 to 800 nmol NADH oxidized min"' (mg of soluble protein)"' (Macdonald and Cole, 1985). Conversely, when excess NirB"" extract was mixed en ice with different quantities of the NirD"^ extract, nitrite reductase activity again increased proportionally to a maximum value corresponding to the same equivalence point as in the preceding experiment (data not shown).

20

^0

60

BO

pl of Nir B^extract added Fig. 4. Reconstitution of NADH-dependent nitrite reductase activity tn vitro. Soluble proteins were prepared from E. co//strain JCB323 transformed with either the nirD* plasmid, pRAR71 D (which lacks a complete m/Bgene: 'NirD extract') or pRAR74 (which encodes NirB but not NirD: 'NirB extract'). Aliquots (200 \>\) of the concentrated NirD extract were mixed on ice with different volumes of the NirB extract and compensating volumes of buffer. Rates of NADH oxidation in the presence or absence of nitrite by 20 jil samples of the mixture were assayed immediately and after 5 or 10 min at 4°C. The protein concentrations of the NirB and NirD extracts were 33.0 and 29.2 mg mP', respectively.

assayed. Only very slow rates of NADH oxidation were detected with either extract alone in the absence of nitrite; these rates, which were additive when mixtures of the two extracts were assayed, were due to a background rate of NADH oxidase activity, as reported previously (Coleman etai, 1978). If NirD catalyses a post-translational modification of NirB, nitrite reductase activity should be restored slowly to a large excess of NirB by small quantities of NirD in a time- and temperature-dependent manner. Conversely, if NirD is a functional subunit of the active enzyme, stoichiometric quantities of both subunits should be required for full activity, but these subunits should be able to associate with each other rapidly to form an active enzyme even at 4°C. Aliquots of extracts of the pRAR71D transformant were therefore mixed on ice with different quantities of an extract of the pRAR74 transformant and then assayed for nitrite reductase and NADH oxidase activities (Fig. 4). The nitrite reductase activity increased in proportion to the quantity of NirB' extract added until a maximum activity was obtained, presumably because of saturation of the

Further control experiments established that the reconstitution of nitrite reductase activity only occurred when concentrated extracts of the two transformants were mixed together before they were diluted into the assay cuvette. The nitrite reductase activities of concentrated mixtures prepared on ice did not increase during subsequent incubation at 30°C or at 4°C. Similarly, no gradual increase in activity typical of an enzyme-catalysed reaction occurred when a small quantity of one extract was mixed with a large excess of the other and incubated at 15°C or at SO^C. Thus, the stoichicmetric requirement for both NirB and NirD for NADH-dependent nitrite reduction strongly suggests that NirD is a subunit of the functional enzyme. The data are inconsistent with the alternative suggestion that NirD catalyses a post-translational modification of NirB.

c G \lnirE C=IG Gl SD.for G nirE A

T C T G C G T T A A A=T

A-T C C A= T A=T T T T T T T

nirD TAATGT

T G G = C T = A 3350

T=A

I

GCATGG^CATTT.TTTAAA, Oral

Fig. 5. Putative mRNA stem-loop structures in the nirE region. The putative Shme-Oalgarno sequence and translation star! for nirE are indicated in the upstream stem-loop structure. The Dra\ site used to construct the fusion plasmid pRAR72 is also shown.

Genes o^f/7e Escherichia coli nir operon Visualization cf NirD associated with partially purified nitrite reductase activity NADH-dependent nitrite reductase was partially purified from JCB323 transformed with pRAR70. Proteins in the resulting sample were separated by SDS-PAGE and stained with Coomassie brilliant blue (Fig. 3B). Although the most intensely stained band was the 90 kDa NirB polypeptide and several major contaminants were still present, the 12 kDa NirD polypeptide was clearly visible (lower arrow in Fig. 3B). This demonstration that the NirD polypeptide co-purifies with NirB during the initial stages in the purification of active NADH-dependent nitrite reductase complex provides further evidence that the two proteins are physically associated as subunits of the enzyme. This is an unusual structure for an NADHdependent dehydrogenase. Although binding domains for the various prosthetic groups of NirB were readily identified by comparing the derived amino acid sequence with those of other dehydrogenases (Peakman et al., 1990a), no polypeptides similar to NirD were found from searches of the data bases and, to our knowledge, no dehydrogenases with a similar, smaller subunit have been described previously. The amino acid sequence of NirD is, however, strikingly similar to the C-terminal domain of the Aspergiilus nidulans NADPH-dependent nitrite reductase (Kinghorn and Campbell, 1989). An alignment of a 76amino-acid section of NirD with the C-terminus of the NirA protein revealed 26 identities and 21 conservative changes. This similarity is sufficient to conclude that the two domains are homologous. Thus, whilst the E. celi enzyme consists of two separate polypeptides, the Aspergilius enzyme carries the NirD domain as part of a larger, single polypeptide.

Possible significance of inverted repeat sequences in the nirE region Three lines of evidence show that the nirE ORF is neither translated into a protein product nor essential for nitrite reduction. First, JCB323 transformed with the in-phase nirE3350"lacZ fusion plasmid, pRAR72, was Lac" on MacConkey-lactose-ampicillin agar and no p-galactosldase activity could be detected in this transformant under any of the growth conditions tested. Second, no Lac* clones with deletion endpoints in nirE were isolated from the Bal31 deletion experiments, although about 10 different Lac" clones were isolated with deletions terminating within nirE. Finally, attempts to generate LaC fusions to n/fE only resulted in the isolation of plasmids in which the translation termination codon for nirD was deleted with the concomitant fusion of nirE to nirD (our unpublished results). Inspection of the DNA sequence in the region of the

2811

nirE OHF revealed two inverted repeats (Fig. 5), either of which might function as a transcripion terminator to downregulate expression of nirC relative to nirB and nirD. Attempts by SI mapping to detect mRNA species which had terminated in this region were, however, consistently unsuccessful (our unpublished results). As transcription from the nirB promoter extends efficiently into the downstream nirC gene, it is more likely that the inverted repeat sequences allow the mRNA transcript to form secondary structure which protects the untranslated n/rE region from rapid degradation. However, formation of the first of these stem-loop structures in the mRNA would sequester both the Shine-Dalgarno sequence and translation start codon for nirE; this could explain why nirE is not translated (Fig. 5).

Experimental procedures Strains, enzyme preparations and assays The E. coti K-12 strains used, listed in Table 1, were Ml 82 (Casadaban and Cohen 1980) and a derivative, JRG1728, from which the fnr gene has been deleted (Spiro and Guest, 1988), and the chlorate-resistant, Cys" strain JCB323, which carries a deletion covering all five ORFs of the n/r operon. The chlorate resistance of strain JCB323 is due to a defect in the synthesis of the molybdenum cofactor required tor nitrate reductase activity resulting in the constitutive expression of the n/r operon during anaerobic growth (Griffiths and Cole, 1987). Cultures were supplemented with 2 0 n g m r ' cysteine and 80 ng ml"^ ampicillin where appropriate. Bacteria for p-galactosidase determinations were grown either aerobically or anaerobically in the absence of nitrite and assayed as described by Jayaraman ef at. (1987), Activities are expressed as nmol o-nitrophenyl p-D-galactoside hydrolysed min"^ (mg dry cell mass)"^ and are the means of at least two independent determinations that differed by no more than 5%. The Nir phenotype of transformants was determined as described by Newman and Cole (1978). Bacteria for the preparation of cell extracts were grown ovemight in 21 conical flasks containing half-strength nutrient broth in minimal salts (Newman and Cole, 1978) supplemented with 0.8% (w/v) glucose and 2.5 mM nitrite. Bacteria were harvested by centifugation (5 minat 10 000 xg), washed in 50 mM phosphate buffer, pH 7.4, resuspended in the Tris-ascorbate-EDTA buffer of Jackson ef al. (1981) and broken in the French pressure cell. The cell extract was centifuged lor 1 h at 100 000 x gto obtain the soluble protein fraction which was used to assay NADH-dependent nitrite reductase as described by Coleman ef ai. (1978). Partially purified nitrite reductase was prepared by ammonium sulphate fractionation and fractionation on a single anion exchange column essentially as described by Jackson et ai. (1981), except that the anion exchanger was DEAE Sepharose CL6B.

Other biochemicai techniques Protein and nitrite concentrations were determined by the Folin

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N. R. Harborne, L. Griffiths, S. J. W. Busby and J. A. Cole

phenol and diazotization methods described previously (Newman and Cole, 1978; Jackson etai, 1981). For SDS-PAGE a 12 cm square slab-gel apparatus was filled with a linear gradient of polyacrylamide (either 5% or 7% to 25%, as noted in the text).

Plasmid construction and other recombinant DNA techniques The plasmids used in this work are fisted in Table 1. Ttie vectors used to clone segments of the nir operon fused in phase to the E. coli lacZ gene and the construction of plasmids with fusions to each of the ORFs of the nir operon have been described in the text. The Lac" strain of E. coli Ml 82 and the Fnr" strain JRG1728 were transformed with the resulting plasmids and plated onto MacConkey-lactose-ampicillin agar. Plasmid DNA was isolated from purified Lac* and Lac" clones and digested with Pvull to determine the approximate location of the fusion junction. The precise location of the fusion in selected clones was then determined by sequencing across the fusion junction. This procedure resulted in the isolation of a series of plasmids which encoded NirB"LaoZ, NirD"Lac2, and NirC'LacZ hybrid proteins with (i-galactosidase activity. No Lac* fusions to n/rf were'isolated by the above procedure. The nirE'iacZ fusion, pRAR72, was constructed using the Dra\ site located within n/r£ at position 3350, Plasmid R1502 contains a short fragment covering the nir promoter from bases -150 to +36 with respect to the transcription start, with an EcoRI site upstream and a Hfndlll site downstream (as in pR150 described by Bell ef ai.. 1990): this contains a n/rS7acZ fusion with only the first three amino acids of NirB. The n/rC'/acZ fusion piasmid, pi 202, was described by Peakman efa/. (1990b), Plasmid pPMBSO contains a fragment of nir operon DNA from the BsfEII site at 3729 to the H/ncll site at 4395, covering the cysG promoter region and the start of cysG as in pRAR70. An EcoRI linker is cloned upstream of position 3729 and a H/ndlll linker is located downstream of 4395: when cloned into pAA2053 this results in a cysClacZ protein fusion under the sole control of the cysG promoter (Fig. IB), Deletions that removed progressively longer sequences upstream of the cysG promoter were created by linearizing pPMB80 at the EcoRI site, using Bal31 to delete different lengths, and then restoring the EcoRI site with a linker. One of these fragments carrying a deletion to position 4165 was then ligated to the Wcol site within the nirC region of pRAR70. The resulting plasmid. pRAR70D, retains an in-phase fusion of cysG to /acZbut lacks the cysG promoter because of an in-phase deletion within the coding region of nirC, between bases 3492 and 4165. pRAR71D was made by deleting the internal nirB BglW fragments from pRAR71 (Fig. 2).

DNA sequencing The DNA sequences across the fusion junctions of the derivatives of pRAR7 were determined by the dideoxy chain termination method using ^^S and a primer complementary to the DNA sequence 40 bases downstream of the H/ndlll site (Pharmacia). The extent of deletions towards the cysG promoter of pPMB8 was determined using the universal primer after trans-

ferring the truncated promoters as EcoRI-H/ndlll fragments to M13mp19,

Acknowledgements We are grateful to J, R. Guest for providing strain JRG1728, and to G. Sawers for permission to cite unpublished data. This research was supported by a Project Grant from the Science and Engineering Research Council,

References Bell, A., Cole, J., and Busby, S. (1990) Molecular genetic analysis of an FNR-dependent anaerobically inducible Escherichia coii promoter, Moi Microbiol 4: 1753-1763. Bingham, A., and Busby, S. (1987) Translation of ga/E and coordination of galactose operon expression in Escherichia coir, effects of insertions and deletions in the non-translated leader sequence. Mol Microbiol 1:117-124. Casadaban, M., and Cohen, S. (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol 6/0/138:179-207. Cole, J, (1978) The rapid accumulation of large quantities of ammonia during nitrite reduction by Escherichia coli. EEMS Microbiol Letts 4: 327-329. Cole, J. (1988) Assimiiatory and dissimilatory reduction of nitrite to ammonia. In Society for General Microbiology Symposium 42. The Nitrogen and Sulphur Cycles. Cole, J., and Ferguson, S. (eds). Cambridge: Cambridge University Press, pp. 281-330. Coleman, K.J. Cornish-Bowden, A., and Cole, J.A. (1978) Purification and properties of nitrite reductase from Escherichia coii. Biochem J175: 483-493. Griffiths, L., and Cole, J, (1987) Lack of redox control of the anaerobically-induced nirB gene of Escherichia coli K-12.

Arch MicrobiolU7: 364-369. Jackson, R., Cornish-Bowden, A., and Cole, J. (1981) Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coliK^2. Biochem J A93: 861-867. Jayaraman P., Peakman, T., Busby, S., Quincey, R., and Cole, J. (1987) Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the FNR protein and nitrite. J Mo/6/0/196:781-788. Khan, M., Koiter, R,, Thomas, C , Figurski, D., Meyer, R., Remaut, E., and Helinski, D. (1979) Plasmid cloning vehicles derived from plasmids ColEI, F, R6K and RK2. Meth Enzymol B8: 268-280. Kinghorn, J.R., and Campbell, E,l. (1989) Amino acid sequence relationships between bacterial, fungal and plant nitrate reductase and nitrite reductase proteins. In Molecular and Genetic Aspects of Nitrate Assimilation. Wray, J.L., and Kinghorn, J.R. (eds). Oxford: Oxford Science Publications, pp, 385-404, Macdonald, H., and Cole, J. (1985) Molecular cloning and functional analysis of the cysG and n/rSgenes of Escherichia coli K12, two closely-linked genes required for NADH-dependent nitrite reductase activity. Mol Gen Genet 200: 328-334. Newman, M., and Cole, J. (1978) The chromosomal location and pleiotropic effects of mutations in the nirA gene of Escherichia coli K12: the essential role of nirA in nitrite reduction and other anaerobic redox reactions. J Gen Microbiol •^06•.^-^ 2-

Genes of the Escherichia coii nir operon Page, L,, Griffiths. L,, and Cole, J- (1990) Different physiological roles for two independent pathways for nitrite reduction to ammonia in enteric bacteria. Arch Microbiol 154: 349354, Peakman. T-, Crouzet, J,, Mayaux, J,, Busby, S,, Mohan, S,. Harborne, N,, Wootton, J,, Nicolson, R., and Cole, J. (1990a) Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coti K-12 chromosome, Eur J Biochem 191: 315-323,

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Peakman, T,, Busby, S,, and Cole, J, (1990b) Transcriptional control of the cysG gene of Escherichia coli K-12 during aerobic and anaerobic growth, Euz-JS/ochem 191: 325-331, Spiro, S,, and Guest, J, (1988) Inactivation of the FNR protein of Escherichia coii by targeted mutagenesis in the W-terminal region, Molf^icrobiol2: 701-707. Warren. M,, Stolowich, N., Santander, P., Roessner, C , Sowa, B,, and Scott, A, (1990) Enzymatic synthesis of dihydrosirohydrochlorin (precorrin-2) and of a novel pyrrocorphin by uroporphyrinogen-lll methylase, FEfiS/-effs 261: 76-80,