Molybdenum cofactor (chlorate-resistant) mutants of Klebsiella pneumoniae M5al can use hypoxanthine as the sole nitrogen source.

JOURNAL OF BACTERIOLOGY, OCt. 1992, p. 6298-6302 Vol. 174, No. 19 0021-9193/92/196298-05$02.00/0 Copyright © 1992, American Society for Microbiology Molybdenum Cofactor (Chlorate-Resistant) Mutants of Klebsiella pneumoniae M5al Can Use Hypoxanthine as the Sole Nitrogen Source ANDRES GARZ6N,1 JING LI,2 AMANDO FLORES,1 JOSEP CASADESUS,1 AND VALLEY STEWART2,3* Departamento de Gene'tica, Universidad de Sevilla, Sevilla 41071, Spain, 1 and Sections of Microbiology2 and Genetics & Development,3 Cornell University, Ithaca, New York 14853-8101 Received 23 March 1992/Accepted 3 August 1992 Selection for chlorate resistance yields mol (formerly chl) mutants with defects in molybdenum cofactor synthesis. Complementation and genetic mapping analyses indicated that the KiebsieUla pneumoniae mol genes are functionally homologous to those of Escherichia coli and occupy analogous genetic map positions. Hypoxanthine utilization in other organisms requires molybdenum cofactor as a component of xanthine dehydrogenase, and thus most chlorate-resistant mutants cannot use hypoxanthine as a sole source of nitrogen. Surprisingly, the K. pneumoniae mol mutants and the mol+ parent grew equally well with hypoxanthine as the sole nitrogen source, suggesting that K. pneumoniae has a molybdenum cofactor-independent pathway for hypoxanthine utilization. Nitrate (NO3-) serves two roles in bacterial metabolism; it can be the sole source of nitrogen for assimilation, and it can be an electron acceptor for anaerobic respiration. Klebsiella spp. will both assimilate and respire nitrate. By contrast, Escherichia coli K-12 and Salmonella typhimurium LT2 do not assimilate nitrate during aerobic growth, although both respire nitrate during anaerobic growth. Previous studies have established that nitrate assimilation and respiration are distinct processes in bacteria such as Klebsiella spp. (1, 4) and Pseudomonas aeruginosa (10). Molybdenum cofactor is a component of both respiratory and assimilatory nitrate reductase (25, 35). Chlorate (C103-) is reduced in vivo by nitrate reductase and other molybdoenzymes to yield a toxic product. Thus, mutants deficient in molybdenum cofactor biosynthesis can be selected on the basis of chlorate resistance. It is established that virtually all E. coli chlorateresistant (mol [formerly chl]) mutants have alterations in molybdenum cofactor biosynthesis or assembly (25, 35). Genetic analysis of E. coli mutants has identified five mol loci: moa (formerly chUA) and moe (formerly chlE), whose products are involved in synthesis of molybdopterin (24); mod (formerly chlD), which encodes a molybdate uptake system (11, 28); mob (formerly chlB), whose product is required for synthesis of molybdopterin guanine dinucleotide (13); and mog (formerly chlG), whose role is unknown (25, 35). The moa, mod, and moe loci each contain at least two genes (11, 14, 22). Molybdenum cofactor is required for the activities of several other enzymes, including formate dehydrogenase in bacteria and xanthine dehydrogenase in fungi and other eukaryotes (6, 21). In the ascomycete Aspergillus nidulans, molybdenum cofactor (cnx) mutants fail to grow with hypoxanthine as the sole nitrogen source, and ability to use hypoxanthine is used as one diagnostic test to determine whether a nitrate-nonassimilating strain has a mutation in molybdenum cofactor synthesis or in a nitrate reductase * Corresponding author. structural or regulatory gene (6, 10). E. coli K-12 does not use xanthine as a sole nitrogen source (33). Early genetic analyses of nitrate metabolism in K aerogenes S45 employed Chlr mol mutants (39). Subsequently, we have isolated K pneumoniae M5al mutants defective in nitrate assimilation (4) and respiration (5) by screening procedures rather than by direct selection for chlorate resistance. All of these mutants were fully Chls, as expected, because only mutations in mol genes confer the Chlr phenotype (35). Unexpectedly, we found that all of the K pneumoniae mol mutants grew well with hypoxanthine as the sole nitrogen source. Thus, we wished to genetically characterize a representative collection of K pneumoniae mol mutants to determine whether the genetics of molybdenum cofactor biosynthesis in this organism is similar to that in E. coli. Nomenclature. Chl designates resistance (Chlr) or sensitivity (Chl') to chlorate. Nas designates the ability (Nas+) or inability (Nas-) to use nitrate as a sole nitrogen source. Nar designates the ability (Nar+) or inability (Nar-) to use nitrate as an electron acceptor for anaerobic respiration. The Nas+ and Nar+ phenotypes require expression of assimilatory or respiratory nitrate reductase and molybdenum cofactor; Chlr strains are Nas- and Nar-, but Nas- and Narstrains are not necessarily Chlr. Historically, five distinct mol (chl) loci, chlA, chlB, chlD, chlE, and chlG, have been identified through genetic analysis of E. coli mutants. Several of these loci contain more than one gene, and complete characterization of most chl loci is in progress. Recently, a new genetic nomenclature has been adopted to designate these genes (29). For example, genes in the historical chlA locus are now designated moaA, moaB, etc. Likewise, the chlB, chlD, chlE, and chlG loci are now designated mob, mod, moe, and mog, respectively. The designation mol is a collective, generic term for the moa, mob, mod, moe, and mog genes without regard to the specific locus or function. Media and culture conditions. Liquid cultures for nitrate reductase enzyme assays were grown in Yoch and Pengra nitrogen-free medium (NF medium), which was prepared as described previously (30), except that the final concentration 6298 NOTES 6299 of FeSO4 was 0.1 mM. To avoid iron precipitation, neutralized EDTA was mixed with the FeSO4 solution, and the mixture was aerated vigorously overnight (16). Nitrogen sources were added as indicated. Aerated cultures were grown in 20 ml of medium contained in 1-liter flasks, with orbital shaking at 400 rpm. Anaerobic cultures were grown in tubes sealed with rubber stobbers. The tubes were completely filled by injecting culture medium with a syringe. Liquid cultures for ,3-galactosidase assays were grown in MOPS (morpholinepropanesulfonic acid) medium as described previously (38). Plates were incubated anaerobically in Brewer jars (3). Defined media contained 0.2% glucose. Complex and indicator media for routine genetic manipulations were used as described previously (7, 20). MacConkey agar-based indicator media to test for production of formate-nitrate oxidoreductase and glycerol-fumarate oxidoreductase were prepared as previously described (38). Peptone-nitrate agar, for selecting Nas+ colonies, was prepared as described previously (37). Tests for use of alternate nitrogen sources were performed by streaking colonies on defined ammonium medium and then replica printing colonies to plates prepared with NF medium containing alternate nitrogen sources. The nitrogen sources (NH4)2SO4, NaNO3, adenine, asparagine, histidine, hypoxanthine, and urea were used at final concentrations of 0.1%; NaNO2 was used at a final concentration of 0.03%. The sulfur sources cystine and K2SO4 were used at final concentrations of 2 mM. Sodium molybdate (Na2MoO4) and sodium tungstate (Na2WO4) were added as indicated below. Chlorate agar contained 0.5% KCl03 except as indicated. Chlorate agar was made with NF medium supplemented with ammonium; with Vogel-Bonner defined medium, which contains ammonium (7); or with Luria-Bertani medium (LB medium), a complex medium (20). Chlorate sensitivity of K. pneumonuae M5al on different media. The wild-type strain K pneumoniae M5al was unable to grow on NF agar supplemented with a nitrogen source and at least 0.1% KC103. The inhibitory effect of chlorate was exerted irrespective of the nitrogen source used (ammonium, nitrate, adenine, asparagine, histidine, or urea) and was observed even on rich media such as LB. However, chlorate sensitivity was most evident when nitrate was used as the sole nitrogen source; in this case, growth inhibition was observed at 0.01% KCl03. K pneumoniae was sensitive to chlorate both aerobically and anaerobically. Isolation of mol mutants. Spontaneous mol mutants of strain M5al were isolated by plating 0.1 ml of stationary LB cultures on NF-ammonium-chlorate or LB-chlorate agar and then incubating them aerobically at 37°C. These mutants were isolated at relatively high frequencies (approximately 10-5). Bacteriophage Mu d11734 (MudJ)-induced mol mutants were isolated by infecting strain VJSK014 with MudJ as previously described (4). Kanamycin-resistant (Kmr) colonies were then replica printed to various media to search for mutants. The moa::MudJ insertion in strain VJSK017 was isolated in a search for nitrate-nonassimilating mutants and has been previously described (4). The mol::MudJ insertions in strains VJSK152-157 were isolated by replicating Kmr colonies to Vogel-Bonner defined medium-chlorate-cystine plates, which were incubated aerobically, and those in strains VJSK158-160 were isolated by replicating Kmr colonies to MacConkey-nitrate plates, which were incubated anaerobically. This latter medium differentiates mutants defective in nitrate respiration. All putative mol::MudJ insertions were backcrossed to VJSK014 via P1 kc-mediated generalized transduction (20) to ensure that they were single insertions in a mol locus. All of the mol mutants described here were of independent origin. Complementation analysis of mol::MudJ mutants. We used the in vivo cloning method of Groisman and Casadaban (9) to clone mol+ loci from E. coli. Transductions involving pEG5005 were performed essentially as described previously (2, 9). Representative mol::Mu cts mutants (37) were transduced to Chl+ by selecting for anaerobic growth on peptone-nitrate-Km plates at 30°C. After preliminary characterization, restriction fragments from representative pEG5005-mol+ plasmids were subcloned into the generalpurpose vector pUC13 or pHG329. The mol subclones were transformed into E. coli mol::Mu cts strains, including several representative examples of each of the five mol loci moa, mob, mod, moe, and mog (37). Complementation was examined by replica printing colony-purified transformants onto LB-chlorate-glucose plates and onto MacConkey nitrate agar plates. All of these plates were incubated anaerobically. In each case, the subclones complemented only the appropriate mol::Mu cts lesions. We have not extensively characterized the inserts in these subclones. However, the preliminary restriction maps of the inserts were consistent with the maps described by Reiss et al. (26). We transformed each mol subclone into each of the K pneumoniae mol::MudJ mutants and then replica plated the transformants to chlorate agar (incubated aerobically), NF agar with nitrate as the sole nitrogen source (incubated aerobically), and MacConkey nitrate agar (incubated anaerobically). For each of the mol::MudJ mutants, one (and only one) of the mol plasmids simultaneously restored Chls, Nas+, and Nar+. These tests revealed that our collection included moa::MudJ (two), mod::MudJ (five), moe::MudJ (one), and mog::MudJ (one) mutants (Table 1). Genetic analysis of mol::MudJ mutants. The genetic maps of E. coli and K pneumoniae are probably similar, so we reasoned that homologous mol genes should have homologous genetic map positions (40). In E. coli, mod is closely linked to gal, ina is weakly linked to gal, and moe is essentially unlinked to gal. Thus, we transduced each of our VJSK014 mol::MudJ derivatives (Galr) into strain VJSK009 (Gals), selected for Kmr transductants, and scored inheritance of Galr as the nonselected marker. The mod::MudJ insertions were approximately 30% linked to gal, the moa::MudJ insertions were approximately 10% linked to gal, and the moe::MudJ and mog::MudJ insertions were less than 0.5% linked to gal. In E. coli, mog is tightly linked to thr. Thus, we also transduced all of the mol::MudJ insertions into strain VJSK207 (thr::Mu dtet). The mog::MudJ insertion was approximately 90% linked to thr::Mu dtet, while the other mol::MudJ insertions were less than 2% linked to thr::Mu dtet. Thus, the two methods employed, plasmid complementation and transductional linkage, yielded identical genetic assignments for the mol::MudJ insertions. Genetic analysis of spontaneous mol mutants. We used transduction to rapidly identify the affected mol locus in spontaneous mol mutants. Bacteriophage P1 lysates prepared on four of the spontaneous mol mutants were used to infect one representative each of moa::MudJ, mod::MudJ, moe::MudJ, and mog::MudJ, and the transduction mixtures were plated on NF-nitrate plates to select Nas+ (Chl') transductants. Three of the mutants transduced each of the recipients, except for the moa::MudJ strain, so these strains (KS10, KS11, and KS12) presumably carry lesions in the VOL. 174, 1992 6300 NOTES TABLE 1. Strains and plasmids Strain or plasmid Description Reference or source K pneumoniae strain M5al Prototroph 23a KS10 moa-102 This study KS11 moa-103 This study KS12 moa-104 This study KS13 moe-105 This study VJSK009 hsdRl Gals 4 VJSK014 Alac-2001 hsdRl Gal' Tn7 4 VJSK017 As VJSK014 but moa-106::MudJ (Lac-) This study VJSK152 As VJSK014 but mod-107::MudJ (Lac') This study VJSK154 As VJSK014 but moa-108::MudJ (Lac-) This study VJSK155 As VJSK014 but mod-109::MudJ (Lac-) This study VJSK156 As VJSK014 but mod-11O::MudJ (Lac-) This study VJSK157 As VJSK014 but mog-III::MudJ (Lac') This study VJSK158 As VJSK014 but mod-112::MudJ (Lac') This study VJSK159 As VJSK014 but mod-113::MudJ (Lac') This study VJSK160 As VJSK014 but moe-114::MudJ (Lac-) This study VJSK207 As VJSK009 but thr::Mu dtet Laboratory collection Plasmids pEG5005 Apr Kmr; Mud5005 cloning vector 9 pHG329 Apr; lacZ a polylinker 32 pUC13 Apr; lacZ a polylinker 41 pVJS501 E. coli moa'; -9-kb PstI-BglII insert in pHG329 This study pVJS502 E. coli mob'; -7-kb BamHI insert in pUC13 This study pVJS503 E. coli mod'; -10-kb PstI insert in pUC13 This study pVJS504 E. coli moe+; -4-kb SphI insert in pHG329 This study pVJS505 E. coli mog+; -7-kb EcoRI insert in pUC13 This study aCourtesy of R. A. Dixon (University of Sussex, Brighton, United Kingdom). moa locus. The other mutant tested (KS13) transduced each of the recipients, except for the moe::MudJ strain, so this mutant presumably carries a lesion in the moe locus. Control experiments in which mol::MudJ mutants were crossed with each other established the utility of this experiment for the characterization of unknown mol mutants. Summary of genetic analyses. Our combined complementation, genetic, and phenotypic analyses indicate that the K pneumoniae mol loci are homologous to those of E. coli in both function and genetic map position. Earlier work with K aerogenes S45 led to a similar conclusion (40). Thus, there seems to be nothing unusual about selection for Chlr in K pneumoniae. Our relatively small collection of mol mutants was biased toward moa and mod mutants. In E. coli, moa mutants predominate, while moe and mod mutants are also quite frequent (37). Mutations in mob and mog are much less frequently recovered in E. coli, and indeed our small collection contained no examples of mob and only one of mog. We believe that the relatively high proportion of mod::MudJ mutants recovered was due to the fact that we included cystine in the media used to isolate mol::MudJ mutants. mod mutants are phenotypically leaky, in that added molybdate can restore nearly wild-type molybdoenzyme function. Sulfur sources repress the sulfate transport system, which allows some molybdate entry into mod strains, and therefore makes for a more stringent Chlr phenotype (15). Although the collection of mol mutants that we have genetically analyzed is small, our characterization of several dozen independent spontaneous mol mutants revealed none with phenotypes other than those reported here (17). Phenotypic characterization of mol mutants. All of the mol mutants had inhibited anaerobic growth on 0.5% chlorate, a concentration that did not cause inhibition of aerobic growth. Anaerobic growth inhibition of mol mutants caused by chlorate was merely bacteriostatic, as the mol mutants subsequently grew well upon transfer to aerobic conditions. All of the mol mutants grew well on plates with hypoxanthine as the sole nitrogen source. The growth rates and yields of the mol parent (VJSK014) and of representative moa::MudJ (VJSK017), moe::MudJ (VJSK160), and mog:: MudJ (VJSK157) strains were indistinguishable in MOPS- glucose medium with hypoxanthine (2.5 mM) as the sole nitrogen source. Each strain doubled in about 100 min and achieved a final growth yield of about 150 Klett units. In contrast, A. nidulans molybdenum cofactor mutants (cnx) fail to grow with hypoxanthine, as xanthine dehydrogenase is a molybdoenzyme in that organism (6). We previously reported that strain VJSK017 (moa-106::MudJ) was unable to grow with hypoxanthine (4). This report was in error. Our laboratory records from 1987 clearly show that this strain grew well with hypoxanthine, and that we were puzzled by this result. The other phenotypes of the K pneumoniae mol mutants were exactly as expected. All of the mol mutants were unable to grow with nitrate as the sole nitrogen source but were fully able to grow with ammonium, nitrite, adenine, asparagine, histidine, and urea. All formed small, dark-red colonies on MacConkey nitrate agar which were similar to those of E. coli mol mutants (37), while the parent M5al formed large, salmon-colored colonies indicative of efficient nitrate respiration. All mol mutants formed wild-type colonies on MacConkey glycerol-fumarate agar, indicating that none had accumulated secondary mutations inJfnr (34). All were essentially devoid of gas production, indicating severe defects in formate dehydrogenase activity. E. coli mod mutants are phenotypically suppressed by including 1 mM molybdate in the culture medium (28). J. BACTERIOL. NOTES 6301 Likewise, all of our K pneumoniae mod mutants became Chl' Nas+ Nar+ when cultured with 1 mM molybdate. Effects of tungstate on K. pneumoniae mol mutant phenotypes. Tungsten is chemically similar to molybdenum, and organisms cultured in the presence of high levels of tungstate incorporate tungsten into molybdoenzymes. Tungsten-sub- stituted enzymes are devoid of enzyme activity (25, 35). Thus, we examined the effects of 1 mM tungstate on the growth of K pneumoniae M5al and its mol derivatives. Tungstate had no effect on the growth of mol+ or mol strains cultured on NF agar supplemented with ammonium or nitrite. As expected, tungstate abolished growth of mol+ strains on NF-nitrate agar, because it inhibited assimilatory nitrate reductase activity. However, mol+ and mol strains all grew vigorously on NF-hypoxanthine agar irrespective of the presence of tungstate. This result further suggests that hypoxanthine utilization in K pneumoniae M5al does not require molybdenum cofactor. Nitrate reductase activities in mol mutants. Nitrate reductase activity was measured as nitrite produced by intact cells in exponentially growing cultures (27). After growth to an A56 of 0.5, the cultures (either aerobic or anaerobic) were treated with chloramphenicol (to avoid induction of respiratory nitrate reductase during centrifugation), harvested, washed in saline buffer, and resuspended in one-half of the original volume. The reaction mixture (0.5 M MOPS-KOH, pH 7.0, 0.4 ml; 100 mM KNO3, 0.2 ml; 1.5 mM methyl viologen, 0.2 ml; 8 mg of Na2S204 per ml, 0.2 ml; and 0.8 ml of bacterial suspension) was incubated at 37°C for 15 min, vigorously vortexed, and centrifuged. One milliliter of the supernatant was used to measure the nitrite generated during the incubation. Nitrite concentration was estimated by the method of Snell and Snell (31). Protein content was estimated by a modification of the Lowry procedure (18). Specific activity units are reported as micromoles of nitrate minute-' milligram-'. We cultured strain MSal aerobically and anaerobically to determine the activities of assimilatory and respiratory nitrate reductase, respectively. Aerated cultures grown on ammonium had very low levels of activity (0.5 mU/mg), while cultures grown with nitrate as the sole nitrogen source had substantial levels of activity (20 mU/mg). Addition of both ammonium and nitrate resulted in low levels of activity (4 mU/mg). Anaerobic cultures grown on ammonium had relatively low levels of activity (2.6 mU/mg), while nitrategrown cultures had high levels of activity (approximately 20 mU/mg) irrespective of the addition of ammonium. These results are fully consistent with previous studies which have detected nitrate-inducible, ammonium-repressible assimilatory nitrate reductase and nitrate-inducible, ammoniuminsensitive respiratory nitrate reductase in KIebsiella spp. (1, 4, 36). We also assayed nitrate reductase in several of the spontaneous mol mutants. None of the mutants had any detectable nitrate reductase activity under any growth conditions. Regulation of mod expression. Expression of genes in the E. coli mod locus is repressed by high concentrations of molybdate and is slightly induced by nitrate during anaerobic growth (19). Since three of our mod::MudJ strains were Lac', and thus presumably carried 4?(mod-lacZ) operon fusions, we examined mod expression in K pneumoniae as well. Cultures were grown anaerobically in three different media: low-molybdate (0.5 ,uM) medium, high-molybdate (100 p,M) medium, and low-molybdate medium supplemented with nitrate. These media also contained 2 mM sulfate, which represses synthesis of the sulfate transport TABLE 2. Anaerobic expression of 4D(mod-lacZ) operon fusions in response to molybdate and nitrate0 1-Galactosidase sp act' in medium containing: K pnewnoniae All strain 'ae Allele 0.50 M 10p0MpM M 0.5 pM 10 Mo0M2- + MoO42 MoO42 40 mM N03 VJSK152 mod-107::MudJ 390 35 390 VJSK158 mod-112::MudJ 290 20 290 VJSK159 mod-113::MudJ 980 85 1,040 a Strains were grown anaerobically in defined media, supplemented as indicated. I Determined as described in Materials and Methods and expressed in arbitrary units. system. Molybdate can enter cells with low efficiency through the sulfate transport system, so addition of sulfate ensures that mod strains are fully limited for molybdate (15). t-Galactosidase activity was measured in permeabilized cells as described previously (20). Specific activity units ("Miller units") are arbitrary. The results are shown in Table 2. In all three strains, expression of 4¢(mod-lacZ) was reduced approximately 10-fold by growth in high-molybdate medium. Similar results were observed with E. coli c?(modlacZ) operon fusions (19). However, in contrast to E. coli mod, the K pneumoniae 4?(mod-lacZ) fusions were not induced by nitrate (Table 2). Xanthine utilization by mol mutants. Why do K pneumoniae mol mutants retain the ability to use hypoxanthine as a sole nitrogen source? The simplest explanation is that this organism contains a molybdenum-independent pathway for hypoxanthine degradation. Unorthodox results regarding the relationship between the Chl phenotype and hypoxanthine utilization were also obtained in a study of P. aeruginosa mutants (8). Recently, it has been shown that P. aeruginosa xanthine dehydrogenase contains the Mo-molybdopterin form of the molybdenum cofactor (12), while nitrate reductase presumably contains the Mo-molybdopterin guanine dinucleotide form of the molybdenum cofactor (25). Thus, it is possible that the hypoxanthine-utilizing Chlr mutants of P. aeruginosa (8) have defects in the mob locus, required for synthesis of molybdopterin guanine dinucleotide (19). However, this would not explain hypoxanthine utilization by the K pneumoniae moa and moe mutants described here. Thus, the relationship between molybdenum cofactor and hypoxanthine utilization in bacteria deserves further attention. Summary. The genetic control of molybdenum cofactor synthesis in K pneumoniae appears to be analogous to that in E. coli. We identified four distinct loci, moa, mod, moe, and mog, which were functionally and genetically homologous to those of E. coli. We did not recover any K pneumoniae mob mutants; such mutants represent a minority class of E. coli mol mutants. All of the mol mutants studied failed to synthesize assimilatory and respiratory nitrate reductases and formate dehydrogenase as revealed by phenotypic tests. However, all were able to use hypoxanthine as the sole nitrogen source, indicating that this organism has a molybdenum-independent pathway for hypoxanthine utilization. We are grateful to Christina Kennedy for her interest and support and to Ray Dixon for providing strain M5al. Stephen Zinder contributed helpful discussions. We thank Lisa Collins (Cornell), VOL. 174, 1992 6302 NOTES Amy Mandel (Cornell), and M. Cruz Munioz (Universidad de Sevilla) for their help in isolating and characterizing mol mutants. This study was supported by grant 6-60-609-B from the Junta de Andalucia (awarded to J.C.) and by U.S. Department of Energy grant 91ER20027 from the Division of Energy Biosciences (awarded to V.S.). REF,ERENCES 1. Bender, R. A., and B. Friedrich. 1990. Regulation of assimilatory nitrate reductase formation in Kiebsiella aerogenes W70. J. Bacteriol. 172:7256-7259. 2. Berg, B. L., and V. Stewart. 1990. Structural genes for nitrateinduicible formate dehydrogenase in Escherichia coli K-12. Genetics 125:691-702. 3. Brewer, J. H., and D. L. Ailgeier. 1966. Safe self-contained carbon dioxide-hydrogen anaerobic system. Appl. Microbiol. 14:985-988. 4. Call, B. M., J. L. Micca, and V. Stewart. 1989. Genetic regulation of nitrate assimilation in Kiebsiella pneumoniae MSal. J. Bacteriol. 171:2666-2672. 5. Collins, L. A., and V. Stewart. Unpublished data. 6. Cove, D. J. 1979. Genetic studies of nitrate assimilation in Aspergillus nidulans. Biol. Rev. 54:291-327. 7. Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advanced bacterial genetics. A manual for genetic engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 8. Goldflam, M., and J. J. Rowe. 1983. Evidence for gene sharing in the nitrate reduction systems of Pseudomonas aeruginosa. J. Bactenrol. 1S5:1446-1449. 9. Groisman, E. A., and M. J. Casadaban. 1986. Mini-Mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing. J. Bacteriol. 168:357-364. 10. Jeter,. R M., S. R. Sias, and J. L. Ingraham. 1984. Chromosomal location and function of genes affecting Pseudomonas aeruginosa nitrate assimilation. J. Bacteriol. 157:673-677. 11. Johann, S., and S. M. Hlinton. 1987. Cloning and nucleotide sequence of the chlD locus. J. Bacteriol. 169:1911-1916. 12. Johnson, J. L., M. Chaudhury, and K V. Rajagopalan. 1991. Identification of a molybdopterin-containing molybdenum cofactor in xanthine dehydrogenase from Pseudomonas aeruginosa. BioFactors 3:103-107. 13. Johnson, J. L., L. W. Indermaur, and K V. Rijagopalan. 1991. Molybdenum cofactor biosynthesis in Escherichia co. Requirement of the chlB gene product for the formation of molybdopterin guanine dinucleotide. J. Biol. Chem. 266:12140-12145. 14. Johnson, M. E., and KL V. Rajagopalan. 1987. Involvement of chUA, E, M, and N loci in Escherichia coli molybdopterin synthesis. J. Bacteriol. 169:117-125. 15. Lee, J. H., J. C. Wendt, and K T. Shanmugam. 1990. Identification of a new gene, molR, essential for utilization of molybdate by Escherichia coli. J. Bacteriol. 172:2079-2087. 16. Luque, F., E. Santero, J. R. Medina, and M. Tortolero. 1987. Mutants of Azotobacter vimelandii altered in the regulation of nitrate assimilation. Arch. Microbiol. 148:231-235. 17. Mandel, A. F., and V. Stewart. Unpublished data. 18. Markwell, M. A. K, S. M. Haas, J. L. Bieber, and N. E. Tolbert. 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87:206-210. 19. Miller, J. B., D. B. Scott, and N. K. Amy. 1987. Molybdenumsensitive transcriptional regulation of the chlD locus of Eschenichia coli. J. Bacteriol. 169:1853-1860. 20. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 21. Nason, A., K.-Y. Lee, S.-S. Pan, P. A. Ketchum, A. Lamberti, and J. DeVries. 1971. In vitro formation of assimilatory reduced nicotinamide adenine phosphate:nitrate reductase from a Neurospora crassa mutant and a component of molybdenum enzymes. Proc. Natl. Acad. Sci. USA 68:3242-3246. 22. Nohno, T., Y. Kasai, and T. Salto. 1988. Cloning and sequencing of the Escherichia coli chlEN operon involved in molybdopterin synthesis. J. Bacteriol. 170:4097-4102. 23. Pengra, R. M., and P. W. Wilson. 1958. Physiology of nitrogen fixation by Aerobacter aerogenes. J. Bacteriol. 75:21-25. 24. Pitterle, D. M., and K. V. Rajagopalan. 1989. Two proteins encoded at the chUA locus constitute the converting factor of Escherichia coli chUIA. J. Bacteriol. 171:3373-3378. 25. Rajagopalan, K. V., and J. L. Johnson. 1992. The pterin molybdenum cofactors. J. Biol. Chem. 267:10199-10202. 26. Reiss, J., A. Kleinhofs, and W. Klingmfiller. 1987. Cloning of seven differently complementing DNA fragments with chl functions from Escherichia coli K-12. Mol. Gen. Genet. 206:352- 355. 27. Santero, E., F. Luque, J. R. Medina, and M. Tortolero. 1986. Isolation of ntrA-like mutants of Azotobacter vinelandii. J. Bacteriol. 166:541-544. 28. Scott, D., and N. K. Amy. 1989. Molybdenum accumulation in chlD mutants of Eschenichia coli. J. Bacteriol. 171:1284-1287. 29. Shanmupm, K. T., D. H. Boxer, J. A. Cole, M. Chippaux, J. A. DeMoss, G. Giordano, R. P. Gunsalus, E. C. C. iUn, K. V. Rajagopalan, and V. Stewart. Proposed nomenclature for the genes involved in molybdenum metabolism in Escherichia coli and Salmonella typhimurium. Submitted for publication. 30. Smibert, R. M., and N. R Krieg. 1981. General characterization, p. 437. In P. Gerhardt (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. 31. Snell, F. D., and C. T. Snell. 1949. Colorimetric methods of analysis, vol. 3, p. 804-805. Van Nostrand, New York. 32. Stewart, G. S. A. B., S. Lubinsky-Mink, C. G. Jackson, A. Kassel, and J. Kuhn. 1986. pHG165: a pBR322 copy number derivative of pUC8 for cloning and expression. Plasmid 15:172- 181. 33. Stewart, V. Unpublished data. 34. Stewart, V. 1982. Requirement of Fnr and NarL functions for nitrate reductase expression in Escherichia coli K-12. J. Bacteriol. 151:1325-1330. 35. Stewart, V. 1988. Nitrate respiration in relation to facultative metabolism in enterobacteria. Microbiol. Rev. 52:190-232. 36. Stewart, V., and B. M. Cali. 1990. Genetic evidence that NarL function is not required for nitrate regulation of nitrate assimilation in Klebsiella pneumoniae M5al. J. Bacteriol. 172:4482- 4488. 37. Stewart, V., and C. H. MacGregor. 1982. Nitrate reductase in Escherichia coli K-12: involvement of chiC, chlE, and chlG loci. J. Bacteriol. 151:788-799. 38. Stewart, V., and J. Parales, Jr. 1988. Identification and expression of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12. J. Bacteriol. 170:1589-1597. 39. Stouthamer, A. H., C. W. Bettenhausen, and J. van Hartingsveldt. 1967. Nitrate reduction in Aerobacter aerogenes. III. Nitrate reduction, chlorate resistance and formate metabolism in mutant strains. Arch. Mikrobiol. 58:228-247. 40. Stouthamer, A. H., and K. Pietersma. 1969. Deletion mapping of resistance against chlorate in Klebsiella aerogenes. Mol. Gen. Genet. 106:174-179. 41. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103-119. J. BACTERIOL.