Proc. Nat!. Acad. Sci. USA Vol. 89, pp. 8419-8423, September 1992

Either of two functionally redundant sensor , NarX and NarQ, is Suffcient for nitrate regulation in Esclierichia coli K-12 (two-component regulatory systems/ regulation/transmembrane signal transduction/narQ gene/nitrate respiration) Ross S. RABINt AND VALLEY STEWARTt* Sections of tMicrobiology and tGenetics and Development, Comneli University, Ithaca, NY 14853-8101 Communicated by Charles Yanofsky, June 16, 1992 (receivedfor review May 1, 1992)

ABSTRACT Nitrate acts through the response regulator regulation (4, 13), suggesting that NarX is a sensor for NarL. NarL to activate and repress anaerobic respiratory gene ex- However, NarL-dependent gene expression responds nor- pression in Escherwchw coli. The narX gene product encodes a mally to nitrate in AnarX deletion strains (14). This last cognate sensor (histidine kinase). However, previous .observation led us to hypothesize the existence of a second work discovered that NarL-mediated nitrate regulation is nitrate-responsive sensor, termed NarQ (ref. 14; Fig. 1). essentially normal in AnarX deletion mutants. In other two- In most cases examined, a single cognate sensor is essen- component regulatory systems studied,, the cognate sensor gene tial for tafget gene expression. In a few instances, however, is essential for normal regulation. We suggested that NarX- residual regulator-dependent gene expression is observed mediated signal transduction reactions are also provided by a when the sensor gene is rendered nonfunctional. At least functionally redundant nitrate sensor, NarQ. We report here three general explanations for this observation have been the identification and analysis of narQ insertion mutants. In forwarded. One idea, "cross talk" ("noise"), posits that narX I strains, a narQ::TnlO insertion had no perceptible effect inappropriate phosphorylation of a response regulator by a on nitrate regulation. However,, the same narQ::TnlO insertion noncognate sensor is responsible for low-level regulator- elmntdnitrate regulation when present in AnarX deletion dependent gene expression. A related explanation, "cross- stan.Thus, either narX+ or narQ+ was sufficient for essen- regulation," suggests that specific physiologically significant tially normal NarL-mediated nitrate regulation. The narQ gene regulation can be accomplished through regulator phosphor- mapped to 53 minutes on the E. coli genetic map, a location ylation by a specific second sensor (for review, see ref. 15). distinct from all known nitrate regulatory or target . The One example to support this latter idea is provided by the predicted NarQ sequence shares substantial similarity with regulator PhoB, which responds not only to its cognate NarX, particularly in the hsineprotein kinase region and in sensor, PhoR, but also to a different sensor, CreC. However, a region of shared similarity with the methyl-accepting chemo- phoR null alleles confer distinct phenotypes with respect to taxis proteins. Both NarQ and NarX apparently have N-ter- phosphate regulation, and creC lesions affect phoA expres- minal periplasmic domains, but the primary structures of these sion only in phoR mutants (16). Finally, it is suggested that regions are largely dissimilar in the two sequences. Analysis of low molecular weight compounds can serve as phosphory- narX* and narL mnissense alleles in narQ+ versus narQ::TnlO lation substrates for response regulators in vivo (17). backgrounds suggests that NarQ and NarX may have subtle Our evidence, reported here, suggests that the NarX/ functional differences. NarQ-NarL regulatory system represents neither "cross talk" nor "cross-regulation," but another situation in which either of two sensors is sufficient for essentially normal From studies of signal transduction pathways in prokaryotes regulation by a single physiological signal (nitrate). We have has emerged a paradigm termed two-component regulatory determined the genetic map location, nucleotide sequence,§ systems. The typical such regulatory pathway consists of a and function of the narQ gene. NarL-dependent nitrate transmembrane sensor (histidine protein kinase) that detects regulation was essentially normal in strains carrying null an environmental signal and a cytoplasmic DNA-binding alleles ofeither narX or narQ but was abolished in double null protein (response regulator). The signaling mechanism in- mutants. The predicted NarQ sequence shares substantial volves autophosphorylation of the sensor on a histidine similarity with those of NarX and other membrane- residue, with subsequent phosphotransfer to an aspartate associated histidine protein kinase sensors. Thus, NarX and residue in the cognate response regulator (for reviews, see NarQ appear to be functionally redundant for nitrate signal- refs. 1-3). ing to NarL. Finally, analysis with narX* and narL missense Escherichia coli will use oxygen, nitrate, fumarate, and alleles revealed some relatively subtle differences in NarX other compounds as terminal electron acceptors for respira- and NarQ function. tion. In the absence of oxygen, nitrate induces the expression of operons encoding enzymes involved in nitrate respiration (formate dehydrogenase-N, encoded by the fdnGHI operon, MATERIALS AND METHODS and nitrate reductase, encoded by the narGHJI operon) and Strains, Plasmids, and Genetic Methods. E. coli VJS676 simultaneously represses operons encoding enzymes involved A&(argF-1ac)U169 (7) was the parent for all strains used in this in respiration of other electron acceptors (such as fumarate study. Strain constructions employed P1 kc-mediated trans- reductase, encoded by the frdABCD operon; Fig. 1). duction (18). Mutant alleles of narX and narL were trans- Nitrate activation and repression are mediated by the ferred from plasmids to the chromosome (7) via linkage to the response regulator NarL (5-10). The narL gene is immedi- adjacent marker zch-2084::fl-Cm (14), where Cm is chlor- ately downstream of the gene for a sensor, narX (7, 11, 12). amphenicol. Several alleles were used as follows: AnarX242 Certain missense mutations in narX lead to aberrant nitrate (14), narLSO4 and narLSOS (10), narX*32 (13), narX*SJJ (4),

The publication costs of this article were defrayed in part by page charge Abbreviations: Cm, chloramphenicol; Tc, tetracycline. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M94724).

8419 Downloaded by guest on September 24, 2021 8420 Genetics: Rabin and Stewart Proc. Natl. Acad Sci. USA 89 (1992)

Nitrate Reductase PO; FIG. 1. Model for nitrate regulation. Open ar- rows indicate protein-coding regions and their direc- (i ? > E tion of transcription. +, Positive regulation (activa- tion); -, negative regulation (repression), NarL is - NO3 +NO hypothesized to be a nitrate-responsive DNA- bindingprotein that, when activatedby proteinphos- phorylation, activates narGHJI, narK, andfdnGHI and represses frdABCD transcription. NarX and Sensor NarQ are involved in modulating NarL activity via FNR phosphorylation. The double-headed arrow under NarX indicates its positive (kinase) and negative Fumarate Reductase (phosphatase) roles in regulation. Uncertainty over the possible negative role for NarQ is indicated as a question mark. Fnr is required for anaerobic induc- tion of narGHJI, narK, fdnGHI, and frdABCD. FNR (Diagram is modified from ref. 4.) and narX512 (this work). Other alleles were narL25::TnlO cate, and reported values are averaged from at least two (5) and narQ251::TnlOd(Tc) (this work), where Tc is tetra- experiments. cycline. Single lysogens carrying the specialized A trans- DNA Sequencing. The entire narQ gene sequence was ducing phage Ak'(narG-lacZ)250 (19), A4(fdnG- determined on both strands by the dideoxynucleotide chain- lacZ)121ucA (20), or Ak4(frdA-1acZ)402 were isolated as terminating method, using primers complementary to the described (21). The A4(frdA-4acZ) fusion was constructed ends of bacteriophage MudK as described (20). We used 16 (21) by fusing the frd operon control region and the N-ter- MudK insertions, located at -150 base-pair intervals along minal coding region offrdA (as an 41.4-kilobase HindIII-Bgl the narQ gene, to obtain the sequence. Computer-assisted II fragment; ref. 22) in-frame with lacZ at codon 439. Inser- DNA sequence analysis employed the package of programs tions of bacteriophage MudK (Mu dII1734) into a narQ+ assembled by GCG (27). plasmid were isolated as described (23). Isolation of Mutants. The indicator strain for mutagenesis RESULTS carried 4(fdnG-acZ)108 (ref. 9) and AnarX242. Mutagene- sis was performed by insertion of TnlOd(Tc) or TnlOd(Cm) Isolation and Mapping of narQ Insertons. We previously using bacteriophage A vectors as described (24). Mutagenized found that AnarX deletion strains exhibit essentially normal cultures were plated on nutrient agar/tetrazolium/lactose NarL-dependent nitrate regulation (14). We reasoned that at medium (18) supplemented-with 40 mM NaNO3 plus Tc at 20 least one other gene (narQ) must substitute for narX gene as After aerobic incuba- function in such strains. To identify the narQ gene, we pgg/ml or Cm at 25 pg/ml required. mutagenized indicator strains with transposons TnlOd(Tc) tion, lactose-nonfermenting (red) colonies were chosen for and TnlOd(Cm), transposition-deficient derivatives of Tn1O further analysis. A variety ofphenotypic and genetic screens (24). The indicator strains carried a 4b(fdnG-LacZ) operon were used to eliminate strains defective for previously de- fusion and a large deletion of narX. These strains form Lac+ scribed genes such as fnr (5). colonies on tetrazolium/lactose/nitrate agar (18). We Cloning the narQ Gene. We used complementation analysis screened independent insertion mutants for Lac- colonies on to determine which ofthe Kohara phages (25) contains narQ. this medium. This screen yielded strains defective in four The indicator strain for complementation carried a t'(fdnG- loci: fnr, mod (chlD), narL, and narQ. Phenotypic tests lacZ) operon fusion and null alleles of narQ and narX. The allowed us to screen out the fnr and mod mutants. [Strains Kohara phage lack the cI repressor, so we used a Cm-resistant lacking mod (chlD) function are uninducible for I(fdnG- AcI+ as a helper phage. We spotted a subset of the Kohara lacZ) expression, because molybdate is poorly accumulated phages with helper phage on soft agar lawns of the indicator in such mutants (for review, see ref. 28).] strain on tryptone plates (18) supplemented with 0.2% malt- To differentiate narL and narQ mutants among the remain- ose, 10 mM MgSO4, and Cm at 20 pyg/ml. These plates were ing candidates, we performed a series of complementation replica printed to MacConkey/lactose/nitrate/Cm plates (18). tests with specialized A transducing phages (14). Strains Lac+ (red) lysogens were observed at the spot containing carrying narL insertions were phenotypically complemented phage 7A8. The narX-containing phage 13H6 was used as a by A narL+ but not by A AnarL. Conversely, strains carrying positive control. We isolated phage 7A8 DNA and cloned the narQ insertions were complemented by A narX+ but not by narQ+ gene on a lO-kilobase Pst I fragment into a plasmid A AnarX. This screen assumed that the narX- and narQ- cloning vector. The insert also contains an internal Pst I site. encoded functions are essentially interchangeable, an as- To identify the desired clone, the c1(fdnG-lacZ) narQ::TnJO sumption borne out by subsequent analysis (see below). A& narX indicator strain was again used to screen for NarQ Transductional analysis of 17 narQ: :TnlOd(Tc) and function. narQ::TnlOd(Cm) insertions revealed that all were tightly Media, Culture Conditions, and Enzyme Assays. Defined, linked (>98% cotransduction). Thus, these insertions define complex, and indicator media for routine genetic manipula- a single genetic locus, narQ. Initial Hfr-mediated conjugation tions were used as described (18, 26). Cultures for 3-galac- experiments (18) delimited the narQ locus to the 50-55 tosidase and nitrate reductase assays were grown in Mops- minute region of the E. coli genetic map. Transductional buffered minimal medium with glucose as the sole carbon analysis with known insertions (29) localized the narQ region source (6). NaNO3 (40 mM) was added as indicated. 3-Ga- near the purC gene at 53 minutes (-70%o linkage). lactosidase and nitrate reductase assays in permeabilized To place the narQ locus on the physical map of the E. coli cells are described (14). All cultures were assayed in dupli- chromosome, we used specialized A transducing phage from Downloaded by guest on September 24, 2021 Genetics: Rabin and Stewart Proc. Natl. Acad. Sci. USA 89 (1992) 8421 the Kohara ordered library (25) in complementation experi- pression in strains carrying various combinations ofnarQ and ments. We tested phage from the vicinity of purC for com- narX null alleles. Strains carrying either the narQ+ or the plementation of a (D(fdnG-lacZ) strain carrying both narX+ gene exhibited essentially wild-type nitrate regulation narQ::TnlO and AnarX. The Kohara phages 7A8 (nearpurC) of all three target operons (Table 1). By contrast, the AnarX and 13H6 (carrying narXI) both restored nitrate induction to narQ::TnlO double mutants were phenotypically similar to the double mutant. Subsequent alignments to the physical the narL::TnJO mutants, in that nitrate regulation was essen- map (30) revealed a gene order of dapA-purC-dapE-narQ- tially abolished. Therefore, either the narQ+ or the narX+ cysA. gene was sufficient for wild-type nitrate regulation, at least Molecular Cloning and Sequence of the narQ Gene. We under the growth conditions employed (Fig. 1). cloned the narQ gene from Kohara phage 7A8 into a plasmid Role of Conserved Histidine Residue in NarX Function. All vector. Restriction sites on the 10-kilobase cloned fragment sensors contain a histidine residue within a conserved se- matched the revised Kohara map at 2584-2594 kilobases. The quence motif. This histidine is essential for the phosphotrans- Pst I site within the narQ gene corresponds to the Pst I site fer reactions ofhistidine protein kinases (for review, see refs. at coordinate 2592 (30). The direction of narQ transcription 1-3). We used site-specific mutagenesis to change the con- is clockwise with respect to the genetic map. MudK inser- served histidine residue in NarX (His-399) to glutamine, to tions in the plasmid were tested for complementation to form allele narXSl2(H399Q). We measured (3-galactosidase delimit the narQ region. Five narQ::MudK insertions were activity as a measure of target operon expression in appro- crossed to the chromosome and genetically mapped to con- priate strain backgrounds. The phenotype conferred by the fim the identity of the narQ clone, its genetic linkage to the histidine change was indistinguishable from that conferred by previously mapped insertions, and the phenotype of con- the null deletion allele (Table 2), indicating that narX512- structed narQ::MudK mutants. (H399Q) is also a null allele, as predicted for a histidine We used several narQ::MudK insertions to determine the protein kinase. nucleotide sequence of the narQ gene, by employing primers Effects of narQ on Phenotypes Conferred by narX* Alleles. complementary to the ends of MudK. The sequence predicts Altered function alleles of the narX gene, termed narX*, that NarQ is a 63.7-kDa protein (Fig. 2). The presumed cause constitutive expression of the narGHJI and fdnGHI translation initiation region for the narQ gene is 5'-TTTG- operons in the absence of nitrate (4, 13). In diploid strains, the TGGAGAAGACGCGTGTGATT-3', where the Shine- narX* lesions are recessive to the narX+ gene, implying that Dalgarno region and presumed initiation codon are under- the NarX* proteins are deficient in a negative regulatory lined. The codon GTG is often used as an initiation codon, function (4). Our interpretation is that the NarX* proteins where it is decoded as formylmethionine (31). have reduced phosphoprotein phosphatase activity relative Nitrate Regulation in narQ::TnlO and AnarX Strains. We to histidine protein kinase activity (4). measured nitrate activation of narGHJI and fdnGHI operon Previous work with narQ+ strains showed that the expression and nitrate repression of frdABCD operon ex- narX*32(E208K) allele confers substantial constitutive (ni- trate independent) expression of and weak NARX 4(narG-4acZ) MLKRCLSPLTLVNL3UL5Z&IM& GVQAAPMRSTKRDALRMQSYR 59 11 :1 11111 1111111 constitutive expression of 4'(fdnG-4acZ). The narX*S11- NARQ mIVKRPVSA-S IM. =IT kTLLZL-qqTLRDAEAINIAGS---- LRMQSYR 55a (A224V) allele confers analogous but less-pronounced phe-

NARX L-LAAVPLSEKDKPLIKEMEQTAFSAELTRAAERDGQLAQLQGLQDYWRNEL-IPALMRA 117 notypes (ref. 4; Table 2). Neither allele (in single copy) affects I 11 c1(frdA-4acZ) expression in the absence of nitrate (ref. 4; NARQ LGYDLQSGSPQLNAHRQLFQQALHSPVLTNLNVWYVPEAVKTRYAHLNANWLEMONRLSK 115 Table 2). NARX QNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHR3MA3EAI.II.3TIML3RAR 177 Do NarQ functions exactly parallel those ofNarX? IfNarQ II:I I : 1I1: : I II NARQ GDLPWYQ ANINN YVNQIDLFVLALQHYAEIMTII.IaITTLRRIRHQ 175 also has phosphoprotein phosphatase activity, then the NarX* phenotype might be enhanced in a narQ::TnlO strain NARX LLQPWRQLLAMASAVSHRDFTQRANISG-RNEMAMLGTALNNMSAELAESYAVLEQRVQE 236 in the absence of nitrate. To examine this question, we l : :1 : :: 1:1111 11 NARQ WAPLNQLVTASQRIEHGQFDSPPLDTGLPNELGLLAKTFNQMSSELHKLYRSLEASVEE 235 introduced narQ::TnlO into the appropriate narX* indicator strains and determined 3-galactosidase activity as a measure NARX KTAGLEHKNQILSFLMQANRRLHSRAPLCERLSPVLNGLQNLTLLRDIELRVYDTDDEEN 296 of target operon expression. For both narX* alleles, consti- 11 I :1 : I: NARQ KTRDLHEAKRRLEVLYQCSQALNTSQIDVHCFRHILQIVRDNEAAEYLELNVGENWRISE 295 tutive 1D(narG-lacZ) and F(fdnG-lacZ) expression was el- evated in the narQ::TnlO strain background (Table 2). These NARX HQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSH 356 l: : results imply that NarQ, like NarX, may have a negative NARQ - GQPNPELPMQI------LPVTMQT-VYGELHWQMSMVS5 328 function (i.e., phosphoprotein phosphatase) in regulating NARX DQQQLVDTLVEQLTATLGLDRHQERQQQLIVMEERATIARELIgDSIAQSLSCMQVSCL 416 Table 1. Effects of narX and narQ null alleles on nar, fdn, and llIII :: : NARQ SEP-LLNSVSSMLGRGLYFNQAQKHFQQLLLMEERATIARELHDSLAQVLSYLRIQLTLL 387 frd operon expression

NARX QHQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKF 476 Enzyme specific activity : 11 IIIIIIIIIII 11 NARQ KRSIPEDNATAQSIMADFSQALNDAYRQLRELLTTFRLTLQQADLPSALREMLDTLQNQT 447 narGHJI A({fdnG-lacZ) A4{(frdA-lacZ) - - NARX GFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVT-VAQNDNQVKLTVQ 535 Genotype - NOj + NO3 NOj + NO- NO3 + NO- 11 :11111 III 11:11 :111: 1:1 I: : NARQ SAKLTLDCRLPTLALDAQMQVHLLQI IREAVLNAMICHANASEIAVSCVTAADGNHTVYIR 507 nar+ 8 530 6 710 98 11 AnarX242 17 660 10 800 97 8 NARX DNGCGVPENAIRSNHYGMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGD 595 narQ251::TnlO 9 580 6 670 100 12 111 1:1 111: 111:11 I I III ::I NARQ DNGIGIGEPKEPEGHYGLNIMRERAERLGGTLTFSQPSGGGTLVSISFRSAEGEESQLMN 566 AnarX242 narQ251::TnlO 9 8 7 5 95 120 NARX THE* 598 narL2lS::TnlO 8 8 6 26 95 140 FIG. 2. Sequence alignment of NarX and NarQ in the standard Nitrate reductase activity from the narGHJI locus was expressed single-letter code. Symbols: 1, identical residues; :, analogous resi- in Stewart and Parales units (7). (-Galactosidase activity from gene dues (Arg-Lys, Asn-Gln, Asp-Glu, Ile-Val, Leu-Met, Phe-Tyr, and fusions in attA is expressed in Miller units (18). Cultures were grown Ser-Thr); -, gaps introduced to maintain alignment. The presumed anaerobically with or without nitrate as indicated. All strains are membrane-spanning regions in NarX and NarQ and the active-site derived from strain VJS676 (Alac) and carry gene fusions on single- histidine residue in NarX are underlined. copy A specialized transducing phage. Downloaded by guest on September 24, 2021 8422 Genetics: Rabin and Stewart Proc. Nad. Acad. Sci. USA 89 (1992) Table 2. Effects of narQ+ and narQ::TnlO on NarX phenotypes The narLSOS(V88A) change confers substantial nitrate- P-Galactosidase specific activity independent constitutive narGHJI and fdnGHI operon ex- pression. This allele is dominant to narL+ (10). This consti- narQ+ narQ::TnlO tutivity is enhanced in AnarX strains, suggesting that NarX Genotype - NOj + NO3 - NO- + NOj provides a negative regulatory function (i.e., phosphoprotein (I(narG-lacZ) phosphatase; ref. 4). The phenotype of the narLSOS strains narX+ 32 2700 34 3360 was indifferent to the presence ofnarQ+ (Table 3). Analogous AnarX242 56 2840 35 26 results were observed with D(frdA-4acZ) expression (data narXSl2(H399Q) 40 3140 33 32 not shown). This suggests that the negative regulatory role of narX*511(A224V) 610 3530 940 3090 NarQ functions differently from that of NarX. narX*32(E208K) 1250 2840 2010 2010 (WdnG-lacZ) DISCUSSION narX+ 5 1000 5 1040 AnarX242 8 1130 4 8 NarQ and NarX Are Homologous But Not Identical. The narXSl2(H399Q) 5 1210 4 9 primary structures of NarQ and NarX share a similar overall narX*SJJ(A224V) 19 1050 33 970 architecture (Figs. 2 and 3). The N-terminal portion of each narX*32(E208K) 71 753 110 370 appears to contain two transmembrane-spanning regions (ID(frdA-lacZ) flanking a periplasmic-exposed region. Both contain a linker narX+ 92 10 90 14 region analogous to those in the methyl-accepting chemotaxis AnarX242 93 9 90 100 proteins (4). The C-terminal regions, while similar to those of narXSl2(H399Q) 104 7 96 110 histidine protein kinases (1), are unconventional and place narX*SJJ(A224V) 93 7 100 8 NarX and NarQ in a small subfamily of sensors along with narX*32(E208K) 95 35 89 65 DegS and UhpB (11). The region around the conserved histidine residue that is found in all histidine protein kinases Specific activity was expressed in Miller units (18). Cultures were grown anaerobically with or without nitrate as indicated. All strains (His-399 in NarX and His-370 in NarQ) is distinctive in this are derived from strain VJS676 (Alac) and carry gene fusions on NarX subfamily (11). The NarQ and NarX sequences are single-copy A specialized transducing phage. identical over a stretch of 14 residues in this area (Figs. 2 and 3). Evidence that this conserved histidine residue is func- target operon expression. However, the overall effect was tionally important for NarX activity is presented in Table 2 small. Furthermore, cF(frdA-4acZ) expression in the absence (see Results). of nitrate was indifferent to NarQ (Table 2). Preliminary computer analyses indicate that NarQ is more In the presence of nitrate, narX*32 strains are somewhat closely related to NarX than either protein is to any other deficient in CF(narG-lacZ) and I)(fdnG-lacZ) activation and sensor. Despite these architectural similarities, the two se- in 4'(frdA-lacZ) repression (refs. 4 and 13; Table 2). Intro- quences are only -28% identical. The periplasmic domains of duction ofa narQ: :TnlO lesion further reduced this activation the two proteins appear unrelated with the exception of an and repression (Table 2), suggesting that NarQ provides 8-residue stretch of identity just distal to the first transmem- brane region (Figs. 2 and 3). This observation was quite substantial NarL kinase activity in narX*32 strains. On the if nitrate binds to the domains of other hand, even with a functional narQ+ gene, the narX*32 surprising; periplasmic in nitrate NarX and NarQ, then one might expect to find considerable strains are somewhat defective regulation of the sequence similarity in this periplasmic region. There is also three operons. This suggests that the narQ gene product a large stretch of sequence dissimilarity in the region between cannot provide full-level positive function (i.e., protein ki- the linker and the beginning of the histidine protein kinase nase) in a narX*32 strain. (Recall that the narQ+ gene homologies (Figs. 2 and 3). product appears fully functional in narX null allele strain NarX and NarQ Share Simbir Positive Regulatory Func- backgrounds; Table 1.) tions. Is NarQ functionally interchangeable with NarX, or are Effects of narQ on Phenotypes Conferred by narL Missense there subtle or pronounced differences between the two Alleles. Two missense alleles of narL have been described proteins, as might be suggested by the substantial sequence (10). Strains carrying the narL504(D11OK) change require the dissimilarity? In otherwise wild-type strains, either narX+ or narX+ gene for full nitrate activation of narGHJI operon narQ+ is sufficient for full activation and repression of target expression. This allele is recessive to the narL+ gene (10). operon expression. This suggests that the positive regulatory 4'(narG-lacZ) activation in the narL504 narX+ strains was functions of NarX and NarQ are similar. A subtle difference indifferent to the presence of narQ+ (Table 3). However, the in positive function was revealed by analysis of the narLS04 residual 4D(narG-lacZ) activation in the AnarX strains was allele. Nitrate activation in narLS04 strains was essentially fully dependent upon narQ+ (Table 3). Analogous results normal with narX+ narQ::TnlO but was reduced with AnarX were observed with 4(frdA-lacZ) expression (data not narQ+ (Table 3). Apparently, NarX and NarQ interacted shown). These results indicate that the interactions of NarX somewhat differently with the NarL504 protein; perhaps this and NarQ with the NarL504 protein, at least, are not iden- hints at subtle differences in their interactions with wild-type tical. NarL. Table 3. Effects of narX and narQ on F(narG-lacZ) expression in strains carrying narL missense alleles P-Galactosidase specific activity narX+ narQ+ narX+ narQ::TnJO AnarX narQ+ AnarX narQ::TnlO Genotype - NO + NO3 - NO- + NO7 - NO- + NO- - NO3 + NO3 narL+ 29 2750 28 3300 49 2880 28 29 narL504(D11OK) 35 2950 37 3300 28 1460 27 29 narL505(V88A) 1280 3010 1290 3340 3920 3040 4290 3250 Specific activity was expressed in Miller units (18). Cultures were grown anaerobically with or without nitrate as indicated. All strains are derived from strain VJS676 (Alac) and carry gene fusions on single-copy A specialized transducing phage. Downloaded by guest on September 24, 2021 Genetics: Rabin and Stewart Proc. Natl. Acad. Sci. USA 89 (1992) 8423

TM-1 A TM-2 Tsr HPK-I B HPK-I1 HPK-III NARX H = ~~~~3=fl~~c NARO ala} N - izrn umu~~~~~~ 50 residues

FIG. 3. Conserved features in NarX and NarQ. TM-1 and TM-2, presumed transmembrane regions; A, an 8-residue identity between the two sequences (the periplasmic regions are otherwise dissimilar); Tsr, similarity shared with the linker regions of methyl-accepting chemotaxis proteins such as Tsr; HPK-I (histidine protein kinase), a 14-residue identity between the two sequences in the region of the active-site histidine residue, shown as "H"; HPK-II and HPK-Ill, other regions that are well conserved in the NarX subfamily of sensor proteins; B, an 11-residue identity between the two sequences (residues 444-454 in NarX).

Do NarX and NarQ Share Similar Negative Regulatory We are indebted to Lisa Collins for her excellent technical assis- Functions? Our genetic analyses of narL and narX (4, 10) tance. Carol Gross, Nancy Kleckner, and Robert Simons were suggest that NarX plays both positive and negative roles in generous in providing many useful strains. The Kohara mini-set was nitrate regulation (i.e., protein kinase and provided by Kenneth Rudd via Joseph Calvo. We thank Joseph phosphoprotein Calvo and the members ofour laboratory for their helpful advice and phosphatase). Do NarX and NarQ, therefore, also share interest. This study was supported by Public Health Service Grant similar negative regulatory functions? Analysis of narL and GM36877 from the National Institute of General Medical Sciences narX* missense alleles suggests that the two proteins may (V.S.) and by a National Science Foundation Graduate Fellowship have differences in negative regulation. (R.S.R.). (i) The constitutive phenotype conferred by the narL505 1. Stock, J. B., Ninfa, A. J. & Stock, A. M. (1989) Microbiol. Rev. 53, allele is greatly enhanced after introduction of AnarX (10), 450-490. suggesting that NarX has a negative effect on the phenotype 2. Igo, M. M., Slauch, J. M. & Silhavy, T. J. (1990) New Biol. 2, 5-9. conferred by narL505. This constitutivity was only slightly 3. Bourret, R. B., Borkovich, K. A. & Simon, M. I. (1991)Annu. Rev. enhanced by introduction of narQ::TnlO (Table 3). Thus, Biochem. 60, 401-441. NarQ had only a small effect on narLSOS-mediated constitu- 4. Collins, L. A., Egan, S. M. & Stewart, V. (1992) J. Bacteriol. 174, tivity. (ii) The constitutive phenotype conferred by narX* 3667-3675. 5. Stewart, V. (1982) J. Bacteriol. 151, 1325-1330. alleles was only slightly enhanced by introducing narQ: :TnlO 6. Iuchi, S. & Lin, E. C. C. (1987) Proc. Nati. Acad. Sci. USA 84, (Table 2). We previously concluded, based on complemen- 3901-3905. tation analysis, that the narX* changes affect the negative 7. Stewart, V. & Parales, J., Jr. (1988) J. Bacteriol. 170, 1589-1597. regulatory function of NarX (4). If NarQ provides a similar 8. Kalman, L. V. & Gunsalus, R. P. (1988) J. Bacteriol. 170, 623-629. 9. Berg, B. L. & Stewart, V. (1990Y Genetics 125, 691-702. negative role, then one would expect the NarX* phenotype 10. Egan, S. M. & Stewart, V. (1991) J. Bacteriol. 173, 4424-4432. to be much more pronounced when NarQ is absent. Of 11. Stewart, V., Parales, J., Jr., & Merkel, S. M. (1989) J. Bacteriol. course, these presumed differences in negative functions are 171, 2229-2234. so far revealed only indirectly, through analysis of narL and 12. Nohno, T., Noji, S., Taniguchi, S. & Saito, T. (1989) Nucleic Acids narX* missense alleles. Res. 17, 2947-2957. 13. Kalman, L. V. & Gunsalus, R. P. (1990) J. Bacteriol. 172, 7049- Effect of the narX*32(E208K) Change on NarX Function. 7056. Kalman and Gunsalus (13) isolated narX*32 in a screen for 14. Egan, S. M. & Stewart, V. (1990) J. Bacteriol. 172, 5020-5029. mutants with constitutively repressed 4D(frdA-1acZ) expres- 15. Wanner, B. L. (1992) J. Bacteriol. 174, 2053-2058. sion. This allele also causes nitrate-independent activation of 16. Wanner, B. L. (1987) J. Bacteriol. 169, 900-903. In anal- 17. Lukat, G. S., McCleary, W. R., Stock, A. M. & Stock, J. B. (1992) 41(narG-lacZ) and c1(fdnG-lacZ) expression. diploid Proc. Natd. Acad. Sci. USA 89, 718-722. ysis, the constitutivity conferred by narX*32 is recessive to 18. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring narXI (4). This latter observation led us to conclude that the Harbor Lab., Cold Spring Harbor, NY). NarX*32 protein was deficient in its negative function rela- 19. Rabin, R. S., Collins, L. A. & Stewart, V. (1992) Proc. Nati. Acad. tive to its positive function (4). Sci. USA 89, in press. 20. Berg, B. L., Li, J., Heider, J. & Stewart, V. (1991) J. Biol. Chem. The narX*32 strains are also slightly defective in nitrate 266, 22380-22385. activation and repression of target operon expression, and 21. Simons, R. W., Houman, F. & Kleckner, N. (1987) Gene 53, 85-96. this defect is codominant with narX+ (4). Introduction of 22. Jones, H. M. & Gunsalus, R. P. (1987) J. Bacteriol. 169, 3340-3349. narQ::TnlO exacerbated this defect (Table 2). This latter 23. Castilho, B. A., Olfson, P. & Casadaban, M. J. (1984) J. Bacteriol. result suggests that NarX*32 is rather seriously impaired for 158, 488-495. 24. Kleckner, N., Bender, J. & Gottesman, S. (1991) Methods Enzymol. nitrate-mediated activation and repression. Because the 204, 139-180. narX*32 defect is apparent in narQ+ strains and because it is 25. Kohara, Y., Akiyama, K. & Isono, K. (1987) Cell 50, 495-508. codominant with narX+, it is possible that NarX*32 inter- 26. Davis, R. W., Botstein, D. & Roth, J. R. (1980) AdvancedBacterial feres with the positive functions of NarX and NarQ. Genetics: A Manualfor Genetic Engineering (Cold Spring Harbor Why Does Nitrate Regulation Involve Two Sensors? Exam- Lab., Cold Spring Harbor, NY). 27. Devereux, J., Haeberli, P. & Smithies, 0. (1984) NucleicAcids Res. ples of truly redundant prokaryotic regulatory functions are 12, 387-395. rare. Our initial observations, summarized above, suggest 28. Stewart, V. (1988) Microbiol. Rev. 52, 190-232. that the NarX and NarQ regulatory functions are interchang- 29. Singer, M., Baker, T. A., Schnitzler, G., Deischel, S. M., Goel, M., able in otherwise wild-type strains. Perhaps NarX and NarQ Dove, W., Jaacks, K. J., Grossman, A. D., Erickson, J. W. & have differential abilities to sense nitrate under physiological Gross, C. A. (1989) Microbiol. Rev. 53, 1-24. 30. Rudd, K. E. (1992)A Short Course inBacterial Genetics Handbook, conditions other than balanced growth in defined medium. ed. Miller, J. H. (Cold Spring Harbor Lab., Cold Spring Harbor, Clues to the answer(s) for this question will come as we learn NY), 2.3-2.43. more about narX and narQ gene expression and function. 31. Gold, L. (1988) Annu. Rev. Biochem. 57, 199-233. Downloaded by guest on September 24, 2021