JOURNAL OF BACTERIOLOGY, Sept. 1985, p. 1285-1287 Vol. 163, No. 3 0021-9193/85/091285-03$02.00/0 Copyright © 1985, American Society for Microbiology Mol- Mutants of Klebsiella pneumoniae Requiring High Levels of Molybdate for Nitrogenase Activity JUAN IMPERIAL,t RODOLFO A. UGALDE,t VINOD K. SHAH,§ AND WINSTON J. BRILLII* Department ofBacteriology and Centerfor Studies of Nitrogen Fixation, University of Wisconsin, Madison, Wisconsin 53706 Received 8 April 1985/Accepted 20 June 1985

Mol- mutants of Klebsieila pneumoniae requiring high levels of molybdate for nitrogenase and nitrate reductase activity were characterized. The effects of mol mutations on nitrogenase activity were very similar to those caused by nifQ mutations. Mol- mutants ofK. pneumoniae appear to be equivalent to ChlD- mutants of Escherichia coli.

Molybdenum is required for the activity of a limited thesis). Strains UN5075 (mol4018::Mu) and UN5076 (mol- number of microbial enzymes (5), including nitrogenase (13) 4019: :Mu) are Mol- strains isolated from UN900 by and nitrate reductase (7). Molybdenum is found in these bacteriophage Mu mutagenesis and penicillin enrichment (J. enzymes as part of low-molecular-weight cofactors (5, 12). Zhu, unpublished results). Strain UN2458 (nifQ5031::Mu) Two different molybdenum-containing cofactors have been has been described previously (4). E. coli RK4353 (AlacU169 described previously: iron-molybdenum cofactor (FeMo- araD139 rpsL gyrA non) and its derivative RK5202 co), which is found only in nitrogenase (10, 12), and molyb- (AlacU169 araD139 rpsL gyrA non chlD202::Mu) have been denum cofactor (Mo-co), which is found in other described previously (14). Bacterial growth, derepression molybdoenzymes (5, 10). Mutants deficient in the synthesis and assay of nitrogenase, and 99Mo accumulation studies of these cofactors have been described previously (3, 11, 14). were performed by using published methods (4). Nitrate The effects of some of these mutations can be suppressed by reductase activity was assayed in whole cells (9), with the the addition of high levels of molybdate to the medium. In modification that 1-ml portion were preincubated anaerobi- ChlD- mutants of Escherichia coli, the activity of Mo-co- cally for 1 h before the assay. dependent enzymes can be restored by high molybdate The molybdate requirement for nitrogenase activity in concentrations (2, 3, 14). In Klebsiella pneumoniae, FeMo- Mol- mutants was studied by the use of acetylene reduction co-deficient mutants requiring high levels of molybdate for to follow derepression of nitrogenase under argon and by the nitrogenase activity have lesions in the gene nifQ (4). D. diazotrophic growth of cultures in molybdenum-deficient K MacNeil (Ph.D. thesis, University of Wisconsin, Madison, medium (4) supplemented with different concentrations of 1979) and J. Zhu (unpublished results) selected Nif- mutants molybdate. Figure 1 shows the dependence of acetylene of K. pneumoniae on molybdenum-deficient media. Some of reduction activity on the concentrations of molybdate added these mutants (Mol-) could grow diazotrophically on media to derepressing cultures of strains UN and UN5076. Similar containing high levels of molybdate. In this communication curves were obtained with strains UN2141 and UN5075 we report the characterization of Mol- mutants of K. pneu- (data not shown). Maximum activities for strain UN were moniae and show that they behave like NifQ- mutants with obtained at concentrations of molybdate above 100 nM, respect to nitrogenase and are equivalent to ChlD- mutants whereas 102 to 103 times more molybdate was required for of E. coli. maximum activity by Mol- strains. At these levels, the Wild-type K. pneumoniae UN and UN900 are indepen- effects of the mutation were totally suppressed. Diazotro- dent wild-type strains from cultures of strain M5al provided phic growth was dependent on molybdate in the same way by P. W. Wilson. Strain UN900 is nitrate reductase positive, (data not shown). These results can be compared with those while strain UN has a very low level of nitrate reductase obtained with NifQ- mutants, in which the same molybdate activity. Strain UN2141 (his-226 recAS6 srlA::tnt-300 mol dependence was observed (4). As with the wild-type and pTM4041) is a Mol- mutant strain unable to fix nitrogen on NifQ- mutants (4), millimolar concentrations of molybdate media containing low levels of molybdate. It was obtained inhibited both acetylene reduction activity (Fig. 1) and from a strain containing wild-type copies of the nifregulon in growth on N2 (data not shown) of Mol- mutants. Intracellu- the chromosome and pTM4041 by ethyl methanesulfonate lar molybdenum accumulation correlated with nitrogenase mutagenesis and penicillin enrichment (D. MacNeil, Ph.D. activity in Mol- mutants (Table 1). Accumulation of 99Mo in nonexchangeable pools by strain UN5076 was defective with respect to strain UN at levels of added molybdate at which * Corresponding author. low acetylene reduction activity was observed. Increasing t Present address: Departamento de Microbiologia, Facultad de the molybdate concentration in the medium resulted in Biologia, Universidad de Barcelona, 08071 Barcelona, Spain. parallel restoration of acetylene reduction activity and 99Mo t Present address: Instituto de Investigaciones Bioquimicas, accumulation. Anaerobic native gel electrophoresis of 99Mo- Fundacion Campomar, Buenos Aires, Argentina. § Present address: Department of Biochemistry, University of labeled extracts of strain UN showed that most of the 99Mo Wisconsin, Madison, WI 53706. label is present in nitrogenase component I (4). The same 11 Present address: Agracetus, 8520 University Green, Middleton, results were found with strain UN5076 (data not shown). WI 53562. Thus, the Nif phenotype of Mol- mutants, as with NifQ- 1285 1286 NOTES J. BACTERIOL.

TABLE 2. Reactivation of K. pneumoniae nitrogenase by molybdate after derepression in molybdenum-free medium .- 100 MoO42- added C2H2 reduction K. pneumoniae (I(M) activity" (%) O00 UN 0 40 %%80 40 100b UN2458 (nifQS031::Mu) 0 10 E 40 118 c 60 UN5076 (mol4019::Mu) 0 4 40 96 E a Tetracycline (50 p.g/ml) was added 5 min before the addition of molybdate bo 40 to inhibit protein synthesis (4). 0 b 100% represents 13.1 nmoL/min per unit of absorbancy at 660 nm (10 ml of culture). C

nase synthesis. In addition, the kinetics of reactivation were very similar in the different strains, with maximum levels 0* obtained after 10 min of preincubation (data not shown). 0 o2 The mol mutation also is pleiotropic on the Mo-co- 1o1i)2 iod"aIO io'x dependent enzyme activities of K. pneumoniae. Figure 2A MoO2 added (nM) shows the dependence of nitrate reductase activity on the levels of molybdate added to cultures of strains UN900 and FIG. 1. Effect of the molybdate concentration on nitrogenase UN5076 during synthesis of the enzyme. Maximum nitrate activity of K. pneumoniae. Symbols: 0, wild-type strain UN; A, reductase activities were obtained in the wild type by the strain UN5076 (mol::Mu). Assays were performed with 1-ml cul- tures for 15 min as described previously (1). mutants (4), appears to be the result of a decreased ability to incorporate molybdenum into nitrogenase component I. In 30- NifQ- mutants, the molybdenum-deficient nitrogenase com- ponent I present in whole cells derepressed with low levels of molybdate can be partially activated in the absence of protein synthesis by the addition of high levels of molybdate 20 (4). Similar results were obtained with strain UN5076 (Table 2). Nitrogenase activity was recovered to the same extent in c~~~~~~~ the wild type and in NifQ- and Mol- mutants, indicating that 10 there are no functional differences among the strains with respect to nitrogenase component I when molybdate is in excess; therefore, the mol mutation does not affect nitroge- E c A %M z~~~~~~~40- TABLE 1. Molybdenum accumulation and acetylene reduction activity' Non- radio- 99Mo C2H2 Ratio active reduc- 00 K. pneumoniae strain Na2MoO4 accumulatedb(% tio (activity/ added (% 'MO) 0 20- (pLM) Z UN900 0 98 35 0.4 0.02 100 58 0.6 1.2 11 100C 9.0 * c10~ ~ 200 0.6 69 125.0 U UN5076 (mo14019::Mu) 0 13 7 0.5 0.02 11 13 1.2 o 1.2 5 39 7.7 0 Id1 Vo13 l 1Ic&ie7lop 200 0.7 109 167.0 Mo-O4 added (nM) a The medium contained 20 ,LCi of carrier-free Na299MoO4 per ml. b Nonexchangeable counts per minute as described previously (4). 100% FIG. 2. Effect of the molybdate concentration on nitrate represents 20.4 x 106 cpm/ml of culture. reductase activity of (A) K. pneumoniae UN900 (0) and UN5076 c 100o represents 65.7 nmollmin per unit of absorbancy at 660 nm (10 ml of (mol: :Mu) (A) and of (B) E. coli RK4353 (0) and RK5202 culture). (chlD::Mu) (A). VOL. 163, 1985 NOTES 1287 addition of 10 to 100 nM molybdate, whereas 0.5 to 5 p.M This research was supported by the College of Agricultural and molybdate was required for maximum activity in the Mol- Life Sciences, University of Wisconsin, Madison, and by Public mutant. At these levels of molybdate, nitrate reductase Health Service grant GM22310 from the National Institutes of activity in the Mol- mutant was restored to wild-type levels. Health. J.I. was the recipient of a postdoctoral fellowship from the Ministry of Education of Spain. R.A.U. is a fellow of the Consejo The molybdate concentrations necessary to suppress the Nacional de Investigaciones Cientificas y Tecnicas, Argentina. effects of the mutation on nitrate reductase were lower than We thank Valley Stewart for the E. coli strains used in this study. those required to restore nitrogenase activity (Fig. 1). This might be a reflection of differences in the enzyme levels for both systems or in the affinities of both pathways for the LITERATURE CITED precursor. Figure 2B shows the same experiment performed 1. Brill, W. J., A. L. Steiner, and V. K. Shah. 1974. Effect of with E. coli RK4353 (ChlD+) and RK5202 (ChlD-). The molybdenum starvatioti and tungsten on the synthesis of nitro- curves obtained were similar to those of the wild type and genase components in Klebsiella pneumoniae. J. Bacteriol. the Mol- mutant in K. pneumoniae; however, E. coli had 118:986-989. lower levels of activity. All our Mol- mutants were resistant 2. del Campillo-Campbell, A., and A. Campbell. 1982. Molybde- to chlorate under anaerobic, molybdenum-deficient condi- num cofactor requirement for biotin sulfoxide reduction in tions (data not shown). A collection of E. coli F' plasmids Escherichia coli. J. Bacteriol. 149:469-478. spanning the whole chromosome described by Low (8) were 3. Glaser, J. H., and J. A. DeMoss. 1971. Phenotypic restoration by tested for the ability to complement the mutations in strains molybdate of nitrate reductase activity in chiD mutants of UN5076 and UN2141. F'126 complemented the mutation in Escherichia coli. J. Bacteriol. 108:854-860. 4. Imperial, J., R. A. Ugalde, V. K. Shah, and W. J. Brill. 1984. both strains (data not shown). This plasmid contains a Role of the n(fQ gene product in the incorporation of molybde- wild-type copy of the E. coli chiD gene. When a plasmid num into nitrogenase in Klebsiella pneumoniae. J. Bacteriol. containing the nif regulon from K. pneumoniae was intro- 158:187-194. duced into a ChlD- E. coli strain, nitrogenase activity was 5. Johnson, J. 1980. The molybdenum cofactor common to nitrate affected and, although only to a limited extent, could be reductase, xanthine dehydrogenase and sulphite oxidase, p. restored by high levels of molybdate (6). The combined 345-384. In M. Coughlan (ed.), Molybdenum and molybdenum- evidence suggests that MolP mutants in K. pneumoniae containing enzymes. Pergamon Press, Ltd., Oxford. probably are genetically equivalent to ChlD- mutants in E. 6. Kennedy, C., and J, R. Postgate. 1977. Expression of Klebsiella coli. However, we favor the use of the Mol nomenclature pneumoniae nitrogen fixation genes in nitrate reductase mutants of Escherichia coli. J. Gen. Microbiol. 98:551-557. because of the pleiotropic niature of the phenotype. 7. Lester, R. L., and J. A. DeMoss. 1971. Effects of molybdate and The effects of mol and nifQ tnutations on the nitrogenase selenite on formate and nitrate metabolism in Escherichia coli. system of K. pneumoniae were indistinguishable by the J. Bacteriol. 105:1006-1014. criteria described above. However, the nifQ gene product 8. Low, B. 1973. Rapid mapping of conditional and auxotrophic was implicated in the biosynthesis of FeMo-co (4), whereas mutations in Escherichia coli K-12. J. Bacteriol. 113:798-812. the mol gene product is required for synthesis of both 9. Newman, B. M., and J. A. Cole. 1978. The chromosomal FeMo-co and Mo-co. Thus, mol must affect an early step in location and pleiotropic effects of mutations of the nirA+ gene of the processing of molybdenum before the branching of the Escherichia coli K12: the essential role of nirA+ in nitrite FeMo-co and Mo-co pathways (13) but later than the trans- reduction and in other anaerobic redox reactions. J. Gen. was not Microbiol. 106:1-12. port of molybdate into the cell, since 99Mo uptake 10. Pienkos, P. T., V. K. Shah, and W. J. Brill. 1977. Molybdenum impaired in E. coli ChlD- (3) and K. pneumoniae Mol cofactors from molybdoenzymes and in vitro reconstitution of strains (data similar to NifQ- mutants; see Fig. 3 in refer- nitrogenase and nitrate reductase. Proc. Natl. Acad. Sci. USA ence 4). The ability of molybdate to suppress the effects of 74:5468-5471. nifQ and mol mutations appears to be a general feature ofthe 11. Roberts, G. P., T. MacNeil, D. MacNeil, and W. J. Brill. 1978. early steps in the molybdenum processing pathways, since Regulation and characterization of protein products coded by ChlG- mutants also can be partially cured by molybdate (2, the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J. 14). Perhaps this is due to reactions which may occur Bacteriol. 136:267-279. nonenzymatically at high substrate concentrations. In this 12. Shah, V. K., and W. J. Brill. 1977. Isolation of an iron- molybdenum cofactor from nitrogenase. Proc. Natl. Acad. Sci. case, NifQ- mutants which lack an activity in a late step in USA 74:3249-3253. the FeMo-co biosynthetic pathway should be cured more 13. Shah, V. K., R. A. Ugalde, J. Imperial, and W. J. Brili. 1984. easily than Mol- mutants. This appears to be the case, since Molybdenum in nitrogenase. Annu. Rev. Biochem. 53:231-257. Mol- mutants are "tighter" in low-nmolybdenum media and 14. Stewart, V., and C. H. MacGregor. 1982. Nitrate reductase in most of the Nif-, Mo-dependent mutants are Mol- rather Escherichia coli K-12: involvement of chIC, chlE, and chiG loci. than NifQ- (unpublished results). J. Bacteriol. 151:788-799.