- Email: [email protected]
[5] Nitrogen fixation (nif) mutants of Klebsiella pneumoniae
[5]
NITROGEN FIXATION (niJ) MUTANTS OF
Klebsiella pneumoniae
47
Other Mutants Temperature-sensitive mutants of Tx20 h a v e been isolated, 8'11 but no growth factor requirements were found at the nonpermissive temperature. An interesting mutant of Tx20 which b e h a v e s like the wild-type in the light but which rapidly loses viaility and respires little in darkness has b e e n isolated and used to a t t e m p t to unravel the role(s) of e n d o g e n o u s m e t a b o l i s m in blue-green algae. 14
[5] N i t r o g e n
Fixation (nif) Mutants pneumoniae
of KlebsieHa
By K. T. SHANMUGAM and R. C. VALENTINE Mutagenesis G e n e s which code for nitrogenase in Klebsiella pneumoniae are clustered in the c h r o m o s o m e near the histidine biosynthetic operon, l'z A n u m b e r of unlinked mutations (based on generalized transduction using phage P1), which affect the production of nitrogenase have b e e n described. 3 Nitrogen fixation mutants are readily isolated using any of the various p r o c e d u r e s of mutagenesis described for isolation of auxotrophic mutants o f E. coil (see Roth4). The c o m m o n l y used chemical mutagens are E M S and N T G . P r o c e d u r e s involving the use of these mutagens for the isolation o f nitrogenase mutants are described below. The use of virulent phages for selecting spontaneously occurring deletions of nitrogenase are also presented. Use o f Ethyl Methane Sulfonate (EMS) as Mutagen. To an actively growing culture of K. pneumoniae, in sucrose minimal m e d i u m with NH4 + as the nitrogen source 5 (5 × l0 s cells/ml; 10 ml in 125-ml flask at S. Streicher, E. Gurney, and R. C. Valentine, Proc. Natl. Acad. Sci. U.S.A. 68,1174 (1971). 2 S. L. Streicher, E. Gurney, and R. C. Valentine, Nature (London) 239, 495 (1972). 3 K. T. Shanmugam, C. Morandi, and R. C. Valentine, in "Iron-Sulfur Proteins" (W. Lovenberg, ed.), Vol. 3, p. 1. Academic Press, New York, 1977. 1
4 j . R. R o t h , V o l . 17, p. 3.
5 The minimal medium contains per liter: Na2HPO4, 6.25 gm; KH2PO4, 0.75 gm; NaC1, 2.00 gm; sucrose, 15 gm; FeSO4"7H~O,0.01 gm; Na2MoO4"2H20, 0.01 gm; MgSO4"7H20, 0.20 gm; and (NH4)2SO4, 1.00; pH 7.0 [a modification of the medium by D. C. Yoch and R. M. Pengra, J. Bacteriol. 92, 618 (1966)]. Broth contains per liter: Tryptone (Difco), 10 gm; yeast extract (Difco), 5 gin; NaCI, 10 gm and sucrose, 3 gm; pH 7.0 [a modification of L broth by S. E. Luria and J. W. Burrows, J. Bacteriol. 74, 461 (1957)]. METHODS IN ENZYMOLOGY, VOL. 69
Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181969-8
48
MUTANTS
[5]
37° with shaking), EMS was added to obtain a final concentration of 1% (0.1 ml/10 ml). Incubation was continued for 1 hr with constant shaking. At the end of I hr, the cells were harvested by centrifugation (5000 g for 5 min) to remove the EMS and the carbon source. The mutagenized cells were resuspended in 10 ml of the minimal medium containing no NH4 + or sucrose (-C&N). The cell suspension in a 125-ml flask was heated in a water bath at 48° and held at that temperature for 40 min without shaking to help remove the alkylated guanine residues from the DNA. 6 Following the heat treatment, the cells were removed by centrifugation and resuspended in 10 ml of broth and incubated at 37° for 2 hr without shaking, to allow the mutagenized chromosomes to segregate. Mutants can be isolated from this culture using appropriate selection procedures described in a later section. Use o f N-Methyl-N'-nitro-N-nitrosoguanidine (NTG) as Mutagen. To obtain mutants, a culture of K. pneurnoniae was treated with NTG (Aldrich Chemical Co.), according to the procedure of Adelberg et al., 7 with modifications as followsS: cells grown overnight at 37° in 10 ml of sucrose minimal medium (with NH4 + as N source) in screwcap tubes (16 × 150 mm) were harvested by centrifugation at 12,000 g for 5 min at 4° and then washed three times with 10 ml saline to remove any traces of the medium. For mutagenesis, washed cells were incubated in 10 ml of saline containing 300 tzg/ml of NTG for 30 min at room temperature. Appropriate amount of NTG can be weighed and dissolved directly in saline. Alternatively, a stock solution of NTG can be prepared in acetone (due to poor solubility in H20) and diluted into saline to obtain the required final concentration. To remove NTG, the cells were washed by centrifugation (three times with 10-ml aliquots of saline) and finally resuspended in 1 ml of saline solution. To allow segregation of mutagenized genomes to occur, cells were grown in broth for 2 hr as described in the previous section. Since NTG is known to induce multiple, nonrandom mutations which are closely linkedfl EMS may be the mutagen of choice (especially regulatory mutants) for the isolation of mutants of nitrogenase. Selection Techniques for Mutants
nif. A mutagenized culture was appropriately diluted and plated on LB plates to obtain about 50-100 colonies per plate and incubated at 37°. 6 B. S. Strauss, J. Bacteriol. 83, 241 (1962). 7 E. A. Adelberg, M. Mandel, and G. C. C. Chen, Biochem. Biophys. Res. Commun. 18, 788 0965). a K. T. Shanmugam, I. Chan, and C. Morandi, Biochim. Biophys. Acta 408, 101 (1975). 9 N. Guerola, J. L. Ingraham, and E. Cerda-Olmedo, Nature (London), New Biol. 230, 122 (1971).
NITROGEN FIXATION (n/'/Q MUTANTS OF K l e b s i e l l a p n e u m o n i a e
[5]
49
TABLE I PROPERTIES OF nif- MUTANTS OF g . pneurnoniae" Nitrogenase components
Activity" Strain UN (parent strain U N 26 UN 106 UN 83 UN 179
CRM '~
Nitrogenase activity ~'
I
II
I
40.09
+
+
? 0.00 ? ?
+ .
+ + .
.
Cotransduction
II
MO-COfactor"
with hisD (%)
+
+
+
-
+ +
+ + .
? +
28 81 21 41
.
a Data from St. John et al. ~5 See also Elmerich et al., T M Merrick et al., t0b and Roberts el al. ~oc 0 Nitrogenase activity in crude extracts (nmoles C2H, produced per minute per milligram protein). c Based on the ability of the crude extract to complement purified nitrogenase components "'in vitro." + denotes the presence; and - denotes the absence (undetectable) of the component. d Cross-reactive material (CRM) is based on immunodiffusion experiments using Ouchterlony plate technique with antiserum prepared against purified components. e M o - C o factor is the Mo-containing, acid-stable material isolated from nitrogenase component 1.
The colonies were transferred by replica plating techniques onto sucroseminimal medium with and without NH4 ÷. These plates were incubated under N2 for 3 days at room temperature. Colonies which failed to grow in the N-free medium and grew normally in the presence of NH4 ÷ were selected. These clones were further tested for their growth and nitrogenase activity in liquid medium. About 95% of these clones were found to be defective in the production of nitrogenase. These mutations are cotransducible with his by the phage PI. Properties of some of these mutants are presented in Table I. Deletions of nif. Strains carrying deletions of the his, nif region can be isolated from phage-resistant clones by using either one of the two methods described below, a° 10 K. T. Shanmugam, A. S. Loo, and R. C. Valentine, Biochirn. Biophys. Acta 338, 545 (1974). ~0a C. Elmerich, J. H o u m a r d , L. Sibold, L. Manheimer, and N. Charpin, Molec. Gen. Genet. 165, 181 (1978). 10b M. Merrick, M. Filser, C. Kennedy, and R. Dixon, Molec. Gen. Genet. 165, 103 (1978). 10c G. P. Roberts, T. MacNeil, D. MacNeil, and W. J. Brill, J. Bacteriol. 136, 267 (1978).
50
Mux^~rs
[S]
A fresh culture of M5A1 was incubated into 10 ml of broth in a 16 × 150 mm screwcap tube and incubated at 37° without shaking until maximum cell density was reached. Incubation was continued at room temperature for about 3-5 days. For enriching deletion mutants resistant to Klebsiella phage 3 (K3), a small aliquot (0.1 ml) of this culture was inoculated into 9 ml of L broth in a 125-ml flask and grown at 37° with shaking to a cell density of about l0 s to 5 × l0 s cells/ml; this cell culture was infected with 0.1 ml of a phage stock of K3 (1.8 × 10a PFU/ml) and incubation was continued until cell lysis was complete. The lysed cell suspension was next centrifuged at 10,000 g in a Sorvall centrifuge for 5 min, and the cell pellet was collected and washed once in 10 ml of sucrose-minimal medium and resuspended in the same medium. The cell suspension was incubated for 30 min at 37° with shaking. Penicillin G (5 mg/ml, 1635 U/mg) was added, and the incubation continued for another 4 hr to enrich for histidine auxotrophs. The cells were washed in sucroseminimal medium three times and resuspended in 2 ml broth. Samples (0.1 ml) of cells escaping penicillin lysis were inoculated into 0.5 ml of L broth in a 16 × 100 mm tube and incubated at 37° until maximum density was reached. This culture was appropriately diluted for single colony formation in saline, spread onto broth plates, and incubated at 37° . These colonies were transferred by replica plating onto sucrose-minimal plates with and without histidine to score for their histidine requirement. Histidine-requiring clones were purified by restreaking and stored as stocks. A second method of selection involves the use of phage alone, omitting the penicillin-enrichment step.l° An actively growing bacterial culture (0.1 ml) was mixed with 0.1 ml of the phage stock (either K3 or K14) and 2 ml of LCTG-top agar and poured as a lawn over broth plates. After incubation overnight at 37°, about 100 phage-resistant colonies were picked and streaked onto broth plates. After 24 hr incubation, colonies were replica plated onto minimal medium plates supplemented with either histidinol or histidine, and also onto. a nitrogen-free medium supplemented with histidine. The clones which fail to grow on minimal as well as on N-free medium, putative nif deletions, were picked for further analysis. About 70% of the deletions have been reported to extend through his and into (through) the nif segment of DNA.I° Of the remaining 30%, the majority are deletions extending beyond the n/f gene cluster. Recently, phage Mu has also been utilized for generation of deletions of nif. Detailed procedures are available in the literature. 11,1z Regulatory Mutants. Most of the reported NIF regulatory mutants
11 R. N. Rao, J. Bacteriol. 128, 356 (1976). 12 M. Backhuber, W. J. Brill, and M. M. Howe, J. Bacteriol. 128, 749 (1976).
[5]
NITROGEN FIXATION (ni~) MUTANTS OF Klebsiella pneumoniae
51
carry defects in the assimilation of NH4+. a'13 The NH4 + produced by nitrogenase in K. pneumoniae is assimilated by the following enzymatic reactions. 14, ~5 Glutamine synthetase: G l u t a m a t e + NH4 + + A T P ~ Glutamine + A D P + Pl
Glutamate synthase: Glutamine + 2-Oxoglutarate + N A D P H ~ 2 Glutamate + N A D P ÷
NH4 ÷ is also assimilated to the level of glutamate, when the concentration of NH4 + in the medium is greater than 1 mM, by the enzyme glutamate dehydrogenase which catalyses the following reaction. 2-Oxoglutarate + NH4 ÷ + N A D P H ~ Glutamate + N A D P +
Mutations which affect the production or activity of any of these enzymes also leads to pleiotrophic defects on nitrogenase production. For example, strains lacking glutamine synthetase activity fail to produce nitrogenase activity under conditions in which the parental strain, M5A 1, produces nitrogenase. ~3 Some of the strains lacking glutamate synthase activity (Asm-), and thus are conditional glutamate auxotrophs (when the NH4 ÷ concentration is much lower than 1 mM in the medium), also produce decreased levels of nitrogenase activity. Mutant strains that induce nitrogenase even in the presence ofNH4 ÷ are found to be defective in the assimilation of NH4 + to the level of glutamate, s Isolation of these strains involve a two-step procedure, since there are two major pathways for the production of glutamate in K. pneumoniae. It is convenient to isolate Nif derepressed mutants starting with strains lacking glutamate synthase activity (Asm-). A variety of selective procedures have proved successful such as isolation of Asm- mutants lacking glutamate synthase activity and isolation of a Glu- or Gin- mutant starting with Asm- mutants as parents. One of these procedures is described below. Isolation of AsmMutants Lacking Glutamate Synthase Activity. Colonies which failed to grow under Nz as sole N source following mutagenesis are picked and checked for their growth in a medium containing NO3- as sole source of nitrogen under both aerobic and anaerobic conditions. Colonies which failed to grow under N2 as well as under NO3- (aerobic as well as anaerobic) are selected. Most of the Asm- strains are also defective in their ability to use histidine, proline or xanthine as sole source of nitrogen. Most of the colonies which fail to 13 S. L. Streicher, K. T. S h a n m u g a m , F. Ausubel, C. Morandi, and R. Goldberg, J. Bacteriol. 120, 815 (1974). 14 H. Nagatani, M. Shimizu, and R. C. Valentine, Arch. Mikrobiol. 79, 164 (1971). 15 R. T. St. John, H. M. J o h n s t o n , C. Seidman, D. Garfinkel, J. K. Gordon, V. K. Shah, and W. J. Brill, J. Bacteriol. 121,759 (1975).
52
MUTANTS
[5]
grow using N2, NO3-, histidine, proline, or xanthine as sole N source but can grow in the presence of higher concentrations of NH4 ÷ (> 1 mM) are found to be defective in the production of glutamate synthase activity. Glutamate or Glutamine-Requiring Strains Derepressed for Nitrogenase Biosynthesis. These strains lack activities of at least two of the three N H 4 ÷ assimilation enzymes and thus do not use NH4 ÷ as sole N source. These strains require glutamate as N source. Gln- strains require L-glutamine as an essential amino acid for growth. L-Glutamine can satisfy both the glutamate and glutamine requirement of the Gin- strains. A major proportion of L-glutamine can be converted into L-glutamate by glutaminases in the cell. Auxotrophic mutants in the mutagenized population can be isolated using replica plating techniques. Colonies on the LB plates are transferred to minimal medium (with NH4 + as the N source) by replica plating techniques. Clones which failed to grow in the minimal medium but are capable of growing in broth are isolated and tested for ability to grow in a glutamine (1 mg/ml) or glutamate (1 mg/ml) supplemented minimal medium. The glutamate or glutamine auxotrophs are further tested for the production and derepression of nitrogenase activity. Properties of some of these mutants are presented in Table II.
T A B L E II PROPERTIES OF NITROGENASE-DEREPRESSED MUTANTS OF g . p n e u m o n i a e a'b Nitrogenase activity
Glutamine synthetase
Glutamate synthase
Glutamate dehydrogenase
Strain
-
+NH4 +
-
+NH, +
-
+NH4 +
-
+NH4 +
M5A1 Asm-I SK-28 SK-25 SK-27
4.04 3.14 3.27 4.47 2.88
0.00 0.00 3.05 3.70 1.81
878 653 1006 0 0
229 147 1055 0 0
37 <5 <5 <5 <5
53 <5 <5 <5 <5
<5 18 <5 12 17
126 116 <5 16 15
Data from S h a n m u g a m et al. 3 b Nitrogenase activity is p.moles C2H4 produced per hour per milligram cell protein. Other e n z y m e activities are e x p r e s s e d as n m o l e s per minute per milligram protein. Strain SK-28 is a G l u - strain. Strains SK-25 and SK-27 are Gln-. The sucrose minimal m e d i u m contained L-glutamate (100 tzg/ml) in all these e x p e r i m e n t s (L-glutamine replaced L-glutamate in the e x p e r i m e n t s involving G i n - strains).
NITROGEN FIXATION (niJ) MUTANTS OF
Klebsiella pneumoniae
47
Other Mutants Temperature-sensitive mutants of Tx20 h a v e been isolated, 8'11 but no growth factor requirements were found at the nonpermissive temperature. An interesting mutant of Tx20 which b e h a v e s like the wild-type in the light but which rapidly loses viaility and respires little in darkness has b e e n isolated and used to a t t e m p t to unravel the role(s) of e n d o g e n o u s m e t a b o l i s m in blue-green algae. 14
[5] N i t r o g e n
Fixation (nif) Mutants pneumoniae
of KlebsieHa
By K. T. SHANMUGAM and R. C. VALENTINE Mutagenesis G e n e s which code for nitrogenase in Klebsiella pneumoniae are clustered in the c h r o m o s o m e near the histidine biosynthetic operon, l'z A n u m b e r of unlinked mutations (based on generalized transduction using phage P1), which affect the production of nitrogenase have b e e n described. 3 Nitrogen fixation mutants are readily isolated using any of the various p r o c e d u r e s of mutagenesis described for isolation of auxotrophic mutants o f E. coil (see Roth4). The c o m m o n l y used chemical mutagens are E M S and N T G . P r o c e d u r e s involving the use of these mutagens for the isolation o f nitrogenase mutants are described below. The use of virulent phages for selecting spontaneously occurring deletions of nitrogenase are also presented. Use o f Ethyl Methane Sulfonate (EMS) as Mutagen. To an actively growing culture of K. pneumoniae, in sucrose minimal m e d i u m with NH4 + as the nitrogen source 5 (5 × l0 s cells/ml; 10 ml in 125-ml flask at S. Streicher, E. Gurney, and R. C. Valentine, Proc. Natl. Acad. Sci. U.S.A. 68,1174 (1971). 2 S. L. Streicher, E. Gurney, and R. C. Valentine, Nature (London) 239, 495 (1972). 3 K. T. Shanmugam, C. Morandi, and R. C. Valentine, in "Iron-Sulfur Proteins" (W. Lovenberg, ed.), Vol. 3, p. 1. Academic Press, New York, 1977. 1
4 j . R. R o t h , V o l . 17, p. 3.
5 The minimal medium contains per liter: Na2HPO4, 6.25 gm; KH2PO4, 0.75 gm; NaC1, 2.00 gm; sucrose, 15 gm; FeSO4"7H~O,0.01 gm; Na2MoO4"2H20, 0.01 gm; MgSO4"7H20, 0.20 gm; and (NH4)2SO4, 1.00; pH 7.0 [a modification of the medium by D. C. Yoch and R. M. Pengra, J. Bacteriol. 92, 618 (1966)]. Broth contains per liter: Tryptone (Difco), 10 gm; yeast extract (Difco), 5 gin; NaCI, 10 gm and sucrose, 3 gm; pH 7.0 [a modification of L broth by S. E. Luria and J. W. Burrows, J. Bacteriol. 74, 461 (1957)]. METHODS IN ENZYMOLOGY, VOL. 69
Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181969-8
48
MUTANTS
[5]
37° with shaking), EMS was added to obtain a final concentration of 1% (0.1 ml/10 ml). Incubation was continued for 1 hr with constant shaking. At the end of I hr, the cells were harvested by centrifugation (5000 g for 5 min) to remove the EMS and the carbon source. The mutagenized cells were resuspended in 10 ml of the minimal medium containing no NH4 + or sucrose (-C&N). The cell suspension in a 125-ml flask was heated in a water bath at 48° and held at that temperature for 40 min without shaking to help remove the alkylated guanine residues from the DNA. 6 Following the heat treatment, the cells were removed by centrifugation and resuspended in 10 ml of broth and incubated at 37° for 2 hr without shaking, to allow the mutagenized chromosomes to segregate. Mutants can be isolated from this culture using appropriate selection procedures described in a later section. Use o f N-Methyl-N'-nitro-N-nitrosoguanidine (NTG) as Mutagen. To obtain mutants, a culture of K. pneurnoniae was treated with NTG (Aldrich Chemical Co.), according to the procedure of Adelberg et al., 7 with modifications as followsS: cells grown overnight at 37° in 10 ml of sucrose minimal medium (with NH4 + as N source) in screwcap tubes (16 × 150 mm) were harvested by centrifugation at 12,000 g for 5 min at 4° and then washed three times with 10 ml saline to remove any traces of the medium. For mutagenesis, washed cells were incubated in 10 ml of saline containing 300 tzg/ml of NTG for 30 min at room temperature. Appropriate amount of NTG can be weighed and dissolved directly in saline. Alternatively, a stock solution of NTG can be prepared in acetone (due to poor solubility in H20) and diluted into saline to obtain the required final concentration. To remove NTG, the cells were washed by centrifugation (three times with 10-ml aliquots of saline) and finally resuspended in 1 ml of saline solution. To allow segregation of mutagenized genomes to occur, cells were grown in broth for 2 hr as described in the previous section. Since NTG is known to induce multiple, nonrandom mutations which are closely linkedfl EMS may be the mutagen of choice (especially regulatory mutants) for the isolation of mutants of nitrogenase. Selection Techniques for Mutants
nif. A mutagenized culture was appropriately diluted and plated on LB plates to obtain about 50-100 colonies per plate and incubated at 37°. 6 B. S. Strauss, J. Bacteriol. 83, 241 (1962). 7 E. A. Adelberg, M. Mandel, and G. C. C. Chen, Biochem. Biophys. Res. Commun. 18, 788 0965). a K. T. Shanmugam, I. Chan, and C. Morandi, Biochim. Biophys. Acta 408, 101 (1975). 9 N. Guerola, J. L. Ingraham, and E. Cerda-Olmedo, Nature (London), New Biol. 230, 122 (1971).
NITROGEN FIXATION (n/'/Q MUTANTS OF K l e b s i e l l a p n e u m o n i a e
[5]
49
TABLE I PROPERTIES OF nif- MUTANTS OF g . pneurnoniae" Nitrogenase components
Activity" Strain UN (parent strain U N 26 UN 106 UN 83 UN 179
CRM '~
Nitrogenase activity ~'
I
II
I
40.09
+
+
? 0.00 ? ?
+ .
+ + .
.
Cotransduction
II
MO-COfactor"
with hisD (%)
+
+
+
-
+ +
+ + .
? +
28 81 21 41
.
a Data from St. John et al. ~5 See also Elmerich et al., T M Merrick et al., t0b and Roberts el al. ~oc 0 Nitrogenase activity in crude extracts (nmoles C2H, produced per minute per milligram protein). c Based on the ability of the crude extract to complement purified nitrogenase components "'in vitro." + denotes the presence; and - denotes the absence (undetectable) of the component. d Cross-reactive material (CRM) is based on immunodiffusion experiments using Ouchterlony plate technique with antiserum prepared against purified components. e M o - C o factor is the Mo-containing, acid-stable material isolated from nitrogenase component 1.
The colonies were transferred by replica plating techniques onto sucroseminimal medium with and without NH4 ÷. These plates were incubated under N2 for 3 days at room temperature. Colonies which failed to grow in the N-free medium and grew normally in the presence of NH4 ÷ were selected. These clones were further tested for their growth and nitrogenase activity in liquid medium. About 95% of these clones were found to be defective in the production of nitrogenase. These mutations are cotransducible with his by the phage PI. Properties of some of these mutants are presented in Table I. Deletions of nif. Strains carrying deletions of the his, nif region can be isolated from phage-resistant clones by using either one of the two methods described below, a° 10 K. T. Shanmugam, A. S. Loo, and R. C. Valentine, Biochirn. Biophys. Acta 338, 545 (1974). ~0a C. Elmerich, J. H o u m a r d , L. Sibold, L. Manheimer, and N. Charpin, Molec. Gen. Genet. 165, 181 (1978). 10b M. Merrick, M. Filser, C. Kennedy, and R. Dixon, Molec. Gen. Genet. 165, 103 (1978). 10c G. P. Roberts, T. MacNeil, D. MacNeil, and W. J. Brill, J. Bacteriol. 136, 267 (1978).
50
Mux^~rs
[S]
A fresh culture of M5A1 was incubated into 10 ml of broth in a 16 × 150 mm screwcap tube and incubated at 37° without shaking until maximum cell density was reached. Incubation was continued at room temperature for about 3-5 days. For enriching deletion mutants resistant to Klebsiella phage 3 (K3), a small aliquot (0.1 ml) of this culture was inoculated into 9 ml of L broth in a 125-ml flask and grown at 37° with shaking to a cell density of about l0 s to 5 × l0 s cells/ml; this cell culture was infected with 0.1 ml of a phage stock of K3 (1.8 × 10a PFU/ml) and incubation was continued until cell lysis was complete. The lysed cell suspension was next centrifuged at 10,000 g in a Sorvall centrifuge for 5 min, and the cell pellet was collected and washed once in 10 ml of sucrose-minimal medium and resuspended in the same medium. The cell suspension was incubated for 30 min at 37° with shaking. Penicillin G (5 mg/ml, 1635 U/mg) was added, and the incubation continued for another 4 hr to enrich for histidine auxotrophs. The cells were washed in sucroseminimal medium three times and resuspended in 2 ml broth. Samples (0.1 ml) of cells escaping penicillin lysis were inoculated into 0.5 ml of L broth in a 16 × 100 mm tube and incubated at 37° until maximum density was reached. This culture was appropriately diluted for single colony formation in saline, spread onto broth plates, and incubated at 37° . These colonies were transferred by replica plating onto sucrose-minimal plates with and without histidine to score for their histidine requirement. Histidine-requiring clones were purified by restreaking and stored as stocks. A second method of selection involves the use of phage alone, omitting the penicillin-enrichment step.l° An actively growing bacterial culture (0.1 ml) was mixed with 0.1 ml of the phage stock (either K3 or K14) and 2 ml of LCTG-top agar and poured as a lawn over broth plates. After incubation overnight at 37°, about 100 phage-resistant colonies were picked and streaked onto broth plates. After 24 hr incubation, colonies were replica plated onto minimal medium plates supplemented with either histidinol or histidine, and also onto. a nitrogen-free medium supplemented with histidine. The clones which fail to grow on minimal as well as on N-free medium, putative nif deletions, were picked for further analysis. About 70% of the deletions have been reported to extend through his and into (through) the nif segment of DNA.I° Of the remaining 30%, the majority are deletions extending beyond the n/f gene cluster. Recently, phage Mu has also been utilized for generation of deletions of nif. Detailed procedures are available in the literature. 11,1z Regulatory Mutants. Most of the reported NIF regulatory mutants
11 R. N. Rao, J. Bacteriol. 128, 356 (1976). 12 M. Backhuber, W. J. Brill, and M. M. Howe, J. Bacteriol. 128, 749 (1976).
[5]
NITROGEN FIXATION (ni~) MUTANTS OF Klebsiella pneumoniae
51
carry defects in the assimilation of NH4+. a'13 The NH4 + produced by nitrogenase in K. pneumoniae is assimilated by the following enzymatic reactions. 14, ~5 Glutamine synthetase: G l u t a m a t e + NH4 + + A T P ~ Glutamine + A D P + Pl
Glutamate synthase: Glutamine + 2-Oxoglutarate + N A D P H ~ 2 Glutamate + N A D P ÷
NH4 ÷ is also assimilated to the level of glutamate, when the concentration of NH4 + in the medium is greater than 1 mM, by the enzyme glutamate dehydrogenase which catalyses the following reaction. 2-Oxoglutarate + NH4 ÷ + N A D P H ~ Glutamate + N A D P +
Mutations which affect the production or activity of any of these enzymes also leads to pleiotrophic defects on nitrogenase production. For example, strains lacking glutamine synthetase activity fail to produce nitrogenase activity under conditions in which the parental strain, M5A 1, produces nitrogenase. ~3 Some of the strains lacking glutamate synthase activity (Asm-), and thus are conditional glutamate auxotrophs (when the NH4 ÷ concentration is much lower than 1 mM in the medium), also produce decreased levels of nitrogenase activity. Mutant strains that induce nitrogenase even in the presence ofNH4 ÷ are found to be defective in the assimilation of NH4 + to the level of glutamate, s Isolation of these strains involve a two-step procedure, since there are two major pathways for the production of glutamate in K. pneumoniae. It is convenient to isolate Nif derepressed mutants starting with strains lacking glutamate synthase activity (Asm-). A variety of selective procedures have proved successful such as isolation of Asm- mutants lacking glutamate synthase activity and isolation of a Glu- or Gin- mutant starting with Asm- mutants as parents. One of these procedures is described below. Isolation of AsmMutants Lacking Glutamate Synthase Activity. Colonies which failed to grow under Nz as sole N source following mutagenesis are picked and checked for their growth in a medium containing NO3- as sole source of nitrogen under both aerobic and anaerobic conditions. Colonies which failed to grow under N2 as well as under NO3- (aerobic as well as anaerobic) are selected. Most of the Asm- strains are also defective in their ability to use histidine, proline or xanthine as sole source of nitrogen. Most of the colonies which fail to 13 S. L. Streicher, K. T. S h a n m u g a m , F. Ausubel, C. Morandi, and R. Goldberg, J. Bacteriol. 120, 815 (1974). 14 H. Nagatani, M. Shimizu, and R. C. Valentine, Arch. Mikrobiol. 79, 164 (1971). 15 R. T. St. John, H. M. J o h n s t o n , C. Seidman, D. Garfinkel, J. K. Gordon, V. K. Shah, and W. J. Brill, J. Bacteriol. 121,759 (1975).
52
MUTANTS
[5]
grow using N2, NO3-, histidine, proline, or xanthine as sole N source but can grow in the presence of higher concentrations of NH4 ÷ (> 1 mM) are found to be defective in the production of glutamate synthase activity. Glutamate or Glutamine-Requiring Strains Derepressed for Nitrogenase Biosynthesis. These strains lack activities of at least two of the three N H 4 ÷ assimilation enzymes and thus do not use NH4 ÷ as sole N source. These strains require glutamate as N source. Gln- strains require L-glutamine as an essential amino acid for growth. L-Glutamine can satisfy both the glutamate and glutamine requirement of the Gin- strains. A major proportion of L-glutamine can be converted into L-glutamate by glutaminases in the cell. Auxotrophic mutants in the mutagenized population can be isolated using replica plating techniques. Colonies on the LB plates are transferred to minimal medium (with NH4 + as the N source) by replica plating techniques. Clones which failed to grow in the minimal medium but are capable of growing in broth are isolated and tested for ability to grow in a glutamine (1 mg/ml) or glutamate (1 mg/ml) supplemented minimal medium. The glutamate or glutamine auxotrophs are further tested for the production and derepression of nitrogenase activity. Properties of some of these mutants are presented in Table II.
T A B L E II PROPERTIES OF NITROGENASE-DEREPRESSED MUTANTS OF g . p n e u m o n i a e a'b Nitrogenase activity
Glutamine synthetase
Glutamate synthase
Glutamate dehydrogenase
Strain
-
+NH4 +
-
+NH, +
-
+NH4 +
-
+NH4 +
M5A1 Asm-I SK-28 SK-25 SK-27
4.04 3.14 3.27 4.47 2.88
0.00 0.00 3.05 3.70 1.81
878 653 1006 0 0
229 147 1055 0 0
37 <5 <5 <5 <5
53 <5 <5 <5 <5
<5 18 <5 12 17
126 116 <5 16 15
Data from S h a n m u g a m et al. 3 b Nitrogenase activity is p.moles C2H4 produced per hour per milligram cell protein. Other e n z y m e activities are e x p r e s s e d as n m o l e s per minute per milligram protein. Strain SK-28 is a G l u - strain. Strains SK-25 and SK-27 are Gln-. The sucrose minimal m e d i u m contained L-glutamate (100 tzg/ml) in all these e x p e r i m e n t s (L-glutamine replaced L-glutamate in the e x p e r i m e n t s involving G i n - strains).