You are on page 1of 4

Temperature sensitivity of a nifA-like gene in Enterobacter cloacae.

J B Zhu, Z G Li, L W Wang, S S Shen and S C Shen J. Bacteriol. 1986, 166(1):357.

Downloaded from on November 3, 2012 by guest

Updated information and services can be found at: These include:

Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more

Information about commercial reprint orders: To subscribe to to another ASM Journal go to:

JOURNAL OF BACTERIOLOGY, Apr. 1986, p. 357-359

0021-9193/86/040357-03$02.00/0 Copyright C 1986, American Society for Microbiology

Vol. 166, No. 1

Temperature Sensitivity of a nifA-Like Gene in Enterobacter cloacae

JIA-BI ZHU, ZHI-GANG LI, LI-WEN WANG, SZI-SHIH SHEN, AND SAN-CHIUN SHEN* Laboratory of Molecular Genetics, Shanghai Institute of Plant Physiology, Academia Sinica, Shanghai, People's Republic of China Received 7 August 1985/Accepted 17 November 1985
Nitrogen fixation (nif) genes of Enterobacter cloacae, a rhizosphere diazotroph of rice plants, were identified by using cloned KkebsieUa pneumoniae nf gene fragments as probes for molecular hybridization. The product of a nifA-like gene of E. cloacae appeared less temperature sensitive than the K. pneumoniae nifA gene product. This result correlates with the fact that E. cloacae can fix nitrogen at 39C, while K. pneumoniae cannot.
Downloaded from on November 3, 2012 by guest

Regulation of nif expression in Klebsiella pneumoniae involves the activity of the products of two nif genes, nifA and nifL. The nifA and nifL products are, respectively, positive and negative regulators of nifexpression (3, 5, 6,8). The nifL product exerts negative regulation of nif genes in the presence of 02, NH4', or other fixed nitrogen sources. Activation by the nifA product requires the activity of the glnF (ntrA) product (9, 11) and also the absence of high temperature (2, 18). Enterobacter cloacae, a rhizosphere diazotroph of rice plants isolated in the subtropical region of the People's Republic of China (13), was capable of growth with N2 as the sole nitrogen source at 39C, whereas nitrogen fixation in K. pneumoniae is inhibited at this temperature. In this paper we provide evidence for a nifA-like gene in E. cloacae, and we demonstrate that the E. cloacae nifA product is not as sensitive to 39C as is the nifA product of K.
pneumoniae. We investigated the effect of the product of the K. pneumoniae nifA gene on the transcription of E. cloacae nif genes by pulse-labeling the mRNA synthesized in nifderepressed E. cloacae cells. Since the nitrogenase genes of different diazotrophs are homologous (15), the radioactive mRNA obtained from a derepressed culture of E. cloacae was hybridized to the K. pneumoniae DNA fragment carrying nifHDKJ genes cleaved from plasmid pJC362 (4). The techniques used for 3H labeling and isolation of RNA were mainly those of Kaluza and Hennecke (7). Two-milliliter cultures under N2 were pulse-labeled with 100 ,uCi of [3H]uracil (specific activity, 23.6 Ci/mmol) for a period of 3 min, followed by a 5-min chase with 0.1 ml of 5-mg/ml nonradioactive uracil and then the addition of 2 ml of ice-cold minimal medium containing 20 mM sodium azide. All the samples were centrifuged immediately. RNA was extracted by the procedure described by Miller (10). The derepression experiments were carried out as follows. After being grown overnight in LB broth, the bacteria were transferred to nitrogen-free medium and then incubated anaerobically at 30C for different times as indicated in Fig. 1. For testing the ammonium repression of nif genes, 15 mM (NH4)2SO4 was added to the nitrogen-free medium. Figure 1 shows the time course of nitrogenase mRNA synthesis in anaerobically grown nitrogen-limited and NH4' excess cultures of E. cloacae. In the nitrogen-limited culture, the appearance of detectable nitrogenase mRNA preceded the appearance of acetylene-reducing activity by about 2 h (Fig. 1A). After

Corresponding author.

introduction of the multicopy plasmid pST1021 (19), which carries a constitutive nifA gene of K. pneumoniae, into the same bacterial cell, the transcription of nitrogenase genes was markedly increased. The ammonia-grown culture which harbors plasmid pST1021 exhibited about the same levels of nif transcription and nitrogenase activity as did cells grown under N-limited conditions (Fig. 1B). Significantly, the synthesis of nitrogenase in E. cloacae was not affected by raising the temperature to 39C. In contrast, the effect of the K. pneumoniae nifA gene on the nif expression in E. cloacae was readily abolished at 39C (data not shown). The ni/A-like gene and nitrogenase genes of E. cloacae were identified by Southern DNA-DNA hybridization (16), with a 32P-labeled DNA fragment containing the nifA gene and portions of ni/B and nifL (niJBAL) from plasmid pST1021 and a 32P-labeled DNA fragment containing the nifHD genes and portions of ni/K and nifJ genes (niJKDHJ) from plasmid pJC362 (4) as probes. We cloned E. cloacae DNA fragments which had been partially cleaved with BamHI or HindIlI into pBR322. The resultant plasmids were then used to transform E. coli C600 (4). Using the colony hybridization procedure with 32P-labeled niJBAL or ni/KDH fragments, we selected clones that contained E. cloacae sequences homologous to those of ni/BAL and ni/KDHJ. These clones were designated pST1140 and pST1130, respectively. To further characterize the E. cloacae DNA homologous to the nif genes of K. pneumoniae, the HindIII fragments isolated from plasmids pST1140 (19.2 kilobases long) and pST1130 (7.5 kilobases) were digested with various restriction endonucleases and analyzed by Southern hybridization with 32P-labeled SalI fragments derived from plasmids pJC212, pJC276, pJC362, and pJC371 (4), which carry different K. pneumoniae nif genes. Figure 2 shows the restriction maps of the HindIII fragments of pST1140 and pST1130 and the portion of the fragments of these two plasmids hybridized by the probes is pointed out. The results indicate that the HindIII fragment from pST1140 contains sequences homologous to those of the K. pneumoniae nifBALFMSUXNEY genes carried in plasmids pJC302, pJC271, pJC391, pJC212, pJC76, and pJC151 (4) and that the HindIII fragment from pST1130 contains sequences homologous to those of the nifYKDHJ genes carried in plasmids pJC151, pJC362, and pJC371. Since the BamHI-HindIII fragment (1.4 kilobases) from the right end of the HindIII fragment of pST1140 and the BamHI-HindIII fragment (1.0 kilobase) from the left end of pST1130 were both hybridized by the sequence in pJC151, it is thus suggested that the






-j 120

7` 120



( 80




Downloaded from on November 3, 2012 by guest




FIG. 1. Synthesis of nitrogenase mRNA at 30C by E. cloacae cells which harbor the constitutive K. pneumoniae niUA gene carried by plasmid pST1021. (A) nif derepressed culture (absence of NH4+). OD6w, Optical density at 600 nm. (B) 15 mM (NH4)2SO4-supplemented medium. Symbols: 0, nitrogenase mRNA of the bacterial cells; 0, nitrogenase mRNA from the bacterial cells containing pST1021; A, nitrogenase activity as determined by the reduction of acetylene to ethylene; A, nitrogenase activity of the bacterial cells containing pST1021.





P aR




Bg B 1HgR 1 I



pJC276 pJC362


pJC212 PJC151









Bg Bg


Ii t H






pSTll30 Hind 1 fgt.

( 7.5Kb)

pST1140 Hind. fgt. (19.2 Kb)

FIG. 2. Restriction maps of nif genes of E. cloacae. (A) Restriction and genetic map of nif genes of K. pneumoniae. (B) Restriction map of E. cloacae nif DNA fragments in plasmids pST1140 and pST1130. pJC Boxes (_) indicate the Sall fragments of different pJC plasmids. The regions of E. cloacae nif DNA fragments in pST1140 and pST1130 hybridizing to SalI fragments of different pJC plasmids are delineated by the vertical lines.

VOL. 166, 1986



TABLE 1. Temperature effect of the nifA-like (nifA') product of E. cloacae on the expression of the nifH-lacZ fusion


Genotype of relevant host

,B-Galactosidase units at: . Activity of relevant plasmid *3OC/390C 30opertes ratio, 30C 39C .

YMC9(pST1142 + pVSA2)a YMC9(pST1021 + pVSA2)

gln+ gln+

YMC11(pST1021 + pVSP9) YMC9(pST1142 + pVSP9) YMC9(pST1021 + pVSP9) YMC9(pVSA2) YMC9(pVSP9) YMC11(pVSA9)


YMC11(pST1142 + pVSP9)"

gln+ gln+ gln+ gln+ (glnALG)

Ec nifA' Kmr Kp nifH::lacZ, Ampr Kp nifA Cm' Kp nifH::lacZ Amp' Ec nifA' KmT Rm nifH::lacZ AmpT Kp nifA Cmr Rm nifH::lacZ Ampr Ec nifA' Km' Rm nifH::lacZ Ampr Kp nifA CmT Rm nifH::lacZ Ampr

1,773 1,721 2,145 2,339 1,102 1,311

18 169 15

1,465 660 1,638 720

1,092 1,290
22 202 15.3

Kp nifH::lacZ AmpT
Rm nifH::lacZ Ampr Rm nifH::lacZ AmpT

0.79 0.38 0.76 0.30 0.99 0.98 1.22 1.19 1.02

See reference 17. See reference 12.

Downloaded from on November 3, 2012 by guest

HindIII fragments of pST1140 and pST1130 may be linked end to end in the chromosome and that the arrangement of E. cloacae nif genes is similar to that of nif genes in K. pneumoniae. Experiments for investigation of the properties of the nifA-like gene of E. cloacae were carried out by monitoring nifA-like activity in an E. coli strain that contains the K. pneumoniae nifH promoter-lacZ fusion (pVSA2) (17) or the Rhizobium meliloti nifH-lacZ fusion (pVSP9) (12). Both the K. pneumoniae nifH and R. meliloti nifH promoters were activated by the nifA-like gene product from E. cloacae (Table 1). The notable finding is that the nifA-like product from E. cloacae was less temperature sensitive than the nifA product from K. pneumoniae. At 30 or 390C it activated the nifH promoters of K. pncamoniae and R. meliloti to almost the same level. In contrast, the activ ty of the nifA product of K. pneumoniae was decreased by 62% at 39C. Since the R. meliloti nifH promoter can be activated by either the glnG (ntrC) or nifA product of K. pneumoniae (17), expression at 39C in the wild-type Escherichia coli strain is presumably due to glnG-mediated activation. It is not surprising to find that these closely related enteric bacteria have a similar order of alignment of nif genes. E. cloacae fixes nitrogen when it is grown at 39C, whereas K. pneumoniae cannot fix nitrogen optimally at that temperature. The experiments presented in this paper indicate that the thermostability of the nifA gene product may determine the temperature limits of nitrogen fixation. In E. cloacae, which inhabits the rhizosphere of rice plants grown in subtropical regions, nitrogen fixation proceeds at a high temperature. Therefore, the E. cloacae nifA-like gene product, which tolerates a high temperature, would be favored under selective pressure.
We thank E. C. C. Lin, W. K. Maas, and W. J. Brill for their valuable suggestions and critical reading of the manuscript. We also thank Q. C. Yu for his help with molecular hybridization; W. J. Brill, J. Collins, Y. S. Qui, and D. W. Ow for providing strains and plasmids, and D. W. Ow and Q. T. Kong for stimulating discussions. This work was supported by a grant from Academia Sinica.
LITERATURE CITED 1. Backman, K., Y. M. Cehn, and B. Magasanik. 1981. Physical and genetical characterization of the glnA-glnG region of the Escherichia coli chromosome. Proc. Natl. Acad. Sci. USA 78:3737-3743. 2. Brooks, S. J., J. J. Colins, and W. J. Brill. 1984. Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature. J. Bacteriol. 157:460-464. 3. Buchanan-WoUlaston, V., M. C. Cannon, H. L. Beynon, and





8. 9. 10. 11. 12.


15. 16.

18. 19.

F. C. Cannon. 1981. Role of the nifA gene product in the regulation of nif expression in Klebsiella pneumoniae. Nature (London) 294:776-778. Collins, J. J., and W. J. Brill. 1985. Control of Klebsiella pneumoniae nif mRNA synthesis. J. Bacteriol. 162:1186-1190. Dixon, R., R. R. Eady, G. Espin, S. Hill, M. laccarino, D. Kahn, and M. Merrick. 1980. Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions. Nature (London) 286:128-132. Hill, S., C. Kennedy, E. Kavanaugh, R. B. Goldberg, and R. Hanau. 1981. Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in Klebsiella pneumoniae. Nature (London) 290:424426. Kaluza, K., and H. Hennecke. 1981. Regulation of nitrogenase messenger RNA synthesis and stability in Klebsiella Arch. Microbiol. 130:38-43. Merrick, M., S. Hill, H. Hennecke, M. Hahn, R. Dixon, and C. Kennedy. 1982. Repressor properties of the nifL gene product in Klebsiella pneumoniae. Mol. Gen. Genet. 185:75-81. Merrick, M. J. 1983. Nitrogen control of nif regulation in Klebsiella pneumoniae: involvement of the ntrA gene and analogies between ntrC and nifA. EMBO J. 2:39-44. Miller, J. 1972. Experiments in molecular genetics, p. 338-343, 352-355. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Ow, D. W., and F. M. Ausubel. 1983. Regulation of nitrogen metabolism genes by nifA gene product in Klebsiella pneumoniae. Nature (London) 301:307-313. Ow, D. W., Y. Xiong, Q. Gu, and S.-C. Shen. 1985. Mutational analysis of the Klebsiella pneumoniae nitrogenase promoter: sequences essential for positive control by nifA and ntrC (glnG) products. J. Bacteriol. 161:868-874. Qiu, Y. S., S. P. Zhou, X. Z. Mo, D. S. Wang, and J. H. Hong. 1981. Study of nitrogen fixing bacteria associated with rice root. Acta Microbiol. Sin. 21:473-476. Roberts, G. P., T. MacNeil, D. MacNeil, and W. J. Brill. 1978. Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J. Bacteriol. 136:267-279. Ruvkun, G. B., and F. M. Ausubel. 1980. Interspecies homology of nitrogenase genes. Proc. Natl. Acad. Sci. USA 77:191-195. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Sundaresan, V., D. W. Ow, and F. M. Ausubel. 1983. Activation of Klebsiella pneumoniae and Rhizobium meliloti nitrogenase promoters by gln (ntr) regulatory proteins. Proc. Natl. Acad. Sci. USA 80:4030-4034. Zhu, J., and W. J. Brill. 1981. Temperature sensitivity of the regulation of nitrogenase synthesis by Klebsiella pneumoniae. J. Bacteriol. 145:1116-1118. Zhu, J. B., G. Yu, Q. Jiang, L. Wang, and S. Shen. 1983. Effect of nifA product on suppression of Nif phenotype of gln mutation and constitutive synthesis of nitrogenase in Klebsiella pneumoniae. Sci. Sin. 26:1258-1268.