Molec. gen. Genet. 175, 195-202 (1979)
:D by Springer-Verlag 1979
Bacillus subtilis Mutants Dependent on Streptomycin Gilda R. Quan, Kristine M. Campbell, and Glenn H. Chambliss Department of Bacteriology, Universityof Wisconsin-Madison, Madison, Wisconsin 53706, USA
Summary. Six streptomycin-dependent mutants of Bacillus subtiIis, two of which were asporogenous, were isolated. All six mutants, SD1, SD2, SD6, SD7, SD9 and SD10, contained a single mutation causing streptomycin dependence and asporogeny, but four of these mutants (SD6, SD7, SD9, SD10) contained a second mutation which phenotypically suppressed the asporogenous character of the streptomycin dependence mutation. All six mutants grew more slowly than the wild type strain BR151, but those defective in sporulation grew the slowest. The streptomycin dependence mutations of SD9 and SD10B (a sporeplus transformant from SD10 carrying both the dependence mutation and the phenotypic suppressor) lie near or possibly within the strA locus. Ribosomes from SD9, SD 10A (a spore-minus transformant from SD10 carrying only the dependence mutation), and SD10B were stimulated in vitro by concentrations of streptomycin that inhibit the activity of wild type strain BR151 ribosomes. The level of misreading as measured by poly(U)-directed isoleucine incorporation was greatly enhanced by streptomycin in wild type strain BR151 ribosomes, but misreading of mutant SD9, SD10A, and SD10B ribosomes, irrespective of the sporulation phenotype, was little affected by streptomycin. There were no apparent differences in the patterns obtained by two-dimensional polyacrylamide gel electrophoresis of the 70S ribosomal proteins of the mutants SD9, SD10A, SD10B, and wild type strain BS151.
Introduction One approach to the study of ribosome structure and function is to isolate mutants with altered responses For offprints contact: Glenn H. Chambliss
to antibiotics that affect protein synthesis. Streptomycin has been widely applied for this purpose, and streptomycin-resistant (StrR) mutants have been extensively studied. Streptomycin-dependent (StrD) mutants, on the other hand, have been less well characterized. Although StrD mutants of Staphylococcus aureus (Paine and Finland, 1948; Foley, 1950), Pseudomonas aeruginosa (Paine and Finland, 1948), Proteus morgani (Paine and Finland, 1948), Klebsiella pneumonia (Paine and Finland, 1948) and Mycobacterium ranae (Yegian and Budd, 1948; Yegian and Vanderlinde, 1949) were reported in the 1940's, they have been poorly characterized genetically. Additionally, StrD mutants of Brucella abortus (Olitzki and Szenberg, 1953), BruceIla suis (Simon and Berman, 1962), Haemophilus pertussis (Bochagova, 1959), Pasteurella pestis (Sharon, 1961), and Shigella (Sergeev et al., 1967) have been isolated, but the main emphasis of these studies was on the immunogenicity and pathogenicity of the mutants. M ore detailed genetic and biochemical studies, however, have been done on StrD mutants of Escherichia coli (Newcombe and Nyholm, 1950; Likover and Kurland, 1967; Birge and Kurland, 1969; Momose and Gorini, 1970; Sanchez-Anzaldo and Bastarrachea, 1974; Wittmann and Apirion, 1975; Polglase et al., 1978), Sahnonella o~phimurium (Goldschmidt etal., 1962), Proteus mirabilis (Hofemeister and B6hme, 1967), Pneumococcus (Mishra and Ravin, 1966), Rhizobium lupini (Garbor, 1965), Vibrio cholerae (Felsenfeld et al., 1970), Bacillus stearothermophilus (Isono, 1974), and Bacillus subtilis (Itoh et al., 1975). In some of these organisms, dependence on streptomycin can be acquired by a single mutation which often leads to pleiotropic effects on the mutant. StrD mutants of E. coli seemed to have growth factor requirements in addition to the antibiotic requirements (Newcombe and Nyholm, 1950); mutants of S. typhimurium required methionine for growth in
0026-8925/79/0175/0195/$ 01.60
196 a d d i t i o n to s t r e p t o m y c i n ( G o l d s c h m i d t et al., 1962), w h i l e m u t a n t s o f P, mirabilis w e r e c h a r a c t e r i z e d b y pleiotropic changes of the protein synthesizing appar a t u s ( H o f e m e i s t e r a n d B 6 h m e , 1967). T h e s t r e p t o m y cin d e p e n d e n c e m u t a t i o n s in E. coli ( H a s h i m o t o , 1960), Pneumococcus ( M i s h r a a n d R a v i n , 1966), R. lupini ( G a r b o r , 1965), a n d S. typhimurium ( G o l d s c h m i d t et al., 1962) lie in t h e s a m e l o c u s as t h e s t r e p t o mycin resistance mutations. Ribosomes from StrD m u t a n t s o f E. coli ( L i k o v e r a n d K u r l a n d , 1967; B i r g e a n d K u r l a n d , 1969), V. cholerae ( F e l s e n f e l d et al., 1970), B. stearothermophilus ( I s o n o , 1974), a n d B. subtills ( I t o h et al., 1975) w e r e s t u d i e d , a n d in all cases, a n a l t e r e d 30S r i b o s o m a l p r o t e i n w a s f o u n d , thus, s u g g e s t i n g a n i n v o l v e m e n t o f t h e r i b o s o m e in t h e S t r D mutations. S i n c e m a n y S t r D m u t a n t s s t u d i e d so far h a v e exh i b i t e d p l e i o t r o p i c p h e n o t y p e s , a n d since a l t e r e d r i b o s o m a l p r o t e i n s c o r r e s p o n d i n g to t h e d e p e n d e n c e m u t a t i o n h a v e b e e n f o u n d , w e c h o s e to i s o l a t e S t r D m u t a n t s o f B . subtilis t o i n v e s t i g a t e f u r t h e r t h e p o s s i b i l ity o f t h e i n v o l v e m e n t o f r i b o s o m e s in t h e r e g u l a t i o n of gene expression during sporulation. Although an a l t e r e d 30S r i b o s o m a l p r o t e i n w a s d e t e c t e d in s t r e p t o m y c i n - d e p e n d e n t s t r a i n s o f B. subtilis b y c a r b o x y m e t h y l c e l l u l o s e c h r o m a t o g r a p h y ( I t o h et al., 1975), a n d in B. stearothermophilus b y t w o - d i m e n s i o n a l gel e l e c t r o p h o r e s i s ( I s o n o , 1974), t h e i r s p o r u l a t i o n p h e n o t y p e s w e r e n o t r e p o r t e d . I n this p a p e r , we r e p o r t the i s o l a t i o n o f s t r e p t o m y c i n d e p e n d e n t m u t a n t s o f B. subtilis w h i c h s p o r u l a t e at a m u c h l o w e r f r e q u e n c y t h a n t h e w i l d t y p e strain.
G.R. Quan et al. : Streptomycin Dependence in B. subtilis a Klett-Summerson colorimeter with the No. 66 red filter in place, or with a Bausch and Lomb Spectronic 20 spectrophotometer at a wavelength of 570 nm.
Isolation of Streptomycin-Dependent Mutants Streptomycin-dependent mutants were obtained by heat mutagenesis of spores from strain BR151 (Northrup and Slepecky, 1967). BR151 was used because its amino acid requirements provided a convenient means for identification of the strain. BR151 cells were harvested from a 48 hour culture grown in liquid NSM, washed three times with double-dlstilled water, resuspended in water, and hefit shocked at 80°C for 10 min to kill vegetative cells. Samples (0.5 ml) of the heat shocked culture were dried in a vacuum oven at 50 55° C overnight. The temperature was then raised to 105°C for 2.5h, after which mutagenized spores were stored at - 2 0 ° C. Sample of the mutagenized, dry spores was then resuspended in 3 ml of A3 medium and incubated at 37° C until germination was observed mmroscoplcally. Portions (0.1 ml) of the germinated cells (containing 7 x 106 viable ceils) were plated on TBAB plates, incubated at 37° C for 3 h, and overlayed with 2.5 ml soft agar containing 5 mg streptomycin per ml. All plates were then incubated at 37° C for 2 days, and colonies that grew were screened for streptomycin dependence.
Determination of Frequeno, of Sporulation Ceils were grown in liquid NSM. Growth was monitored and shortly after the end of exponential growth, samples of the cultures were withdrawn, appropriately diluted in Davis salts, and plated on solid NSM to determine total colony forming units. Forty-eight hours after the end of exponential growth, 1 ml samples of the cultures were treated to 80° C for 10 rain, diluted appropriately in Davis salts, and plated to determine the heat-resistant colony forming units. Percentage of sporulation was defined as : heat-resistant colony forming units x 100 total colony forming units
Transformation Materials and Methods Bacterial Strains and Growth Conditions The B. subtdis strains used in this study were 168T +, prototroph, obtained from P. Schaeffer; BR151, trpC2 lys-3 metBlO, obtained from G. Wilson; Kit 1, purA16 cysA14 trpC2, a reference strain constructed by Dedonder et al. (1977); all streptomycin-dependent mutants were obtained by heat mutagenesis of BRI51 spores and were grown on Schaeffer's solid nutrient broth sporulation medium (NSM) (Schaeffer et al., 1965) containing 500 gg streptomycin sulfate per ml (Sigma). They were transferred frequently and stored at 4° C. All growth was at 37° C. Minimal medium G containing 0.12 M Trizma Base (Sigma), 0.02 M glutamic acid monopotassium salt, 3 mM sodium citrate, 2 mM MgSO4.7H20, 0.5% glucose, and pH adjusted to 7.2 with H3PO4, was supplemented with auxotrophic requirements at concentrations of 50 ~tg/ml, when needed. Antibiotic Assay Broth No. 3 (A3) and Tryptose Blood Agar Base (TBAB) were purchased from Difco. Peptone Blood Agar Base (PBAB) (Gibco) substituted for TBAB in some experiments. Davis salts used for dilution contained 0.7% K2HPO4, 0.3% KH2PO4, 0.1% (NH,)2SO4 and 0.005% MgSO4.7H20. Schaeffer's nutrient broth sporulation medium (NSM) was used for growth of cells for ribosome extractions and for sporulation studies in liquid medram. Growth was monitored by measuring culture turbidity with
Transformation procedures of BoyIan et al. (1972) were followed. DNA was extracted according to Bon and Wilson (1967), and concentrations of DNA determined by Burton's diphenylamine assay for DNA concentration (I956). Transformations were done with 0.1 ~tg of DNA per ml.
Transduction Transduction procedures of Jamet and AnagnostopouIos (1969) were followed. For the transduction experiments, a streptomycinresistant, Kit 1 strain was constructed by transformation of strain Kit 1 (purA16 cysA14 trpC2) with DNA from a spore-plus streptomycin-resistant mutant isolated by heat mutagenesis. This Kit 1StrR strain was renamed KSTP. Phage PBS-1 lysates of the StrD mutants were used to transduce KSTP. Cysteine prototrophs were selected by spreading on minimal medium G lacking cysteine. The resulting prototrophic transductants were screened on solid NSM to determine the percentage of cotransduction of the cysA mutation with the streptomycin dependence mutation, and on minimal medium G lacking adenine to determine the cotransduction of the strD mutation with the cysA and purA markers.
Preparation of Ribosomes Ribosomes were prepared according to Legault-Demare and Chambliss (1974) with the modifications described by Campbell
G.R. Quan et al.: Streptomycin Dependence in B. subtilis
197
and Chambliss (1977), with the following exceptions: the StrD mutants were grown in liquid NSM supplemented with 100 gg streptomycin per ml, and BR151 was grown without any drug; ribosomes after a 12 h wash in high salt buffer were collected by centrifugation through a 2 ml 30% sucrose cushion for 8 h at 150,000 x g.
Table 1. Growth and sporulation of streptomycin-dependent mutants in liquid NSM containing streptomycin (500 gg/ml) Strain
Growth rate (doubling time) (min)
Sporulation frequency (%)
b~ vitro Protein Synthesis
SD1 SD2 SD6 SD7 SD9 SDI0 BRI51 ~
240 201 100 105 77 100 55
0.03 0.54 49 80 100 41 80
High speed supernatant (S-150) and initiation factors (IF's) were prepared by the procedures of Legault-Demare and Chambliss (1974) with the modifications already noted (Campbell and Chambliss, 1977). The S-150 fractions used in the assays were prepared from strain 168T+ cells grown in CHT50 medium (Anagnostopoulos and Spizizen, 1961) and the IF's were prepared from strain BR151 cells. The in vitro protein synthesis assay measured the incorporation of [14C]-phenylalanine (14 cpm/pmol) into acid-insoluble material using RNA extracted from exponentially growing BR151 ceils as template. Each assay tube contained 70 gg ribosomes, 91.2 ~tg crude IF protein, 87 gg crude S-150 protein, and 300 gg BR151 RNA in a reaction volume of 0.125 ml, except where otherwise noted. The method of Lowry et al. (1951) was used to determine the protein concentrations of S-150 and IF preparations. Streptomycin was added from a stock solution made in 0.1 M Tris-HC1, pH 7.5. For the misreading experiments, procedures of Campbell and Chambliss (1977) were followed.
Two-Dimensional Polyacrylamide Gel Eleetrophoresis Two-dimensional gels were run with the buffers and gel compositions of Kaltschmidt and Wittmann (1970) as modified by Howard and Traut (1973). Ribosomal proteins were precipitated with acetone as described by Barritault et al. (1976).
Results
Isolation, Sporulation and Growth Rates of StrD Mutants S t r D m u t a n t s were i s o l a t e d by the h e a t m u t a g e n e s i s p r o c e d u r e d e s c r i b e d in Materials and Methods. F r o m 540 c o l o n i e s g r o w i n g on s t r e p t o m y c i n - c o n t a i n i n g plates, 219 c o n t a i n e d d r u g resistant cells, a n d o f this n u m b e r , 6 were f o u n d to c o n t a i n s t r e p t o m y c i n - d e p e n d e n t m u t a n t s . These m u t a n t s were n a m e d SD1, SD2, SD6, SDT, SD9 a n d SD10 respectively. T h a t each o f these m u t a n t s r e t a i n e d the a u x o t r o p h i c req u i r e m e n t s o f their p a r e n t a l strain BR151 was asc e r t a i n e d b y the fact t h a t each r e q u i r e d t r y p t o p h a n , m e t h i o n i n e , a n d lysine for g r o w t h on m i n i m a l med i u m G. This m e d i u m was devised to s u b s t i t u t e for Spizizen M i n i m a l M e d i u m (Spizizen, 1958) b e c a u s e the S t r D m u t a n t s were u n a b l e to g r o w on the latter. Beggs a n d A n d r e w s (1976) r e p o r t e d t h a t m o n o v a l e n t a n d d i v a l e n t c a t i o n s interfered with the u p t a k e o f d i h y d r o s t r e p t o m y c i n by Mycobacterium smegmatis, c o n s e q u e n t l y p r e v e n t i n g its lethal a c t i o n a g a i n s t the m i c r o o r g a n i s m . It s e e m e d p r o b a b l e t h a t the high c o n c e n t r a t i o n o f c a t i o n s in the Spizizen m i n i m a l me-
a
No streptomycin was added to this culture
d i u m i n h i b i t e d the u p t a k e o f s t r e p t o m y c i n b y the S t r D m u t a n t s , thus p r e v e n t i n g their growth. This a p p a r ently was the case since BR151, w h i c h is n o r m a l l y sensitive to s t r e p t o m y c i n , grew on Spizizen m i n i m a l m e d i u m s u p p l e m e n t e d with s t r e p t o m y c i n (500 gg/ml) but could not grow on minimal medium G containing s t r e p t o m y c i n . T h u s the r e d u c e d c a t i o n c o n c e n t r a t i o n o f m i n i m a l m e d i u m G a p p a r e n t l y a c c o u n t s for the a b i l i t y o f the d e p e n d e n t m u t a n t s to g r o w on it. Kinetics o f g r o w t h a n d f r e q u e n c y o f s p o r u l a t i o n o f SD1, SD2, SD6, SDT, SD9, SD10 a n d BR151 were d e t e r m i n e d in liquid N S M . T h e results are s h o w n on T a b l e 1. T w o o f the six m u t a n t s were quite defective in their a b i l i t y to sporulate, each s p o r u l a t i n g at less t h a n 1% the wild t y p e frequency. M u t a n t s SD6 a n d SD10 c o n s i s t e n t l y s p o r u l a t e d at a b o u t oneh a l f the f r e q u e n c y o f BR151 while m u t a n t SD9 s p o r u l a t e d slightly b e t t e r t h a n wild type. A l l m u t a n t s grew m o r e slowly t h a n the p a r e n t a l strain BR151, b u t the m u t a n t s defective in s p o r u l a t i o n grew the slowest.
Genetic Analysis of the Streptomycin-Dependent Phenotype T o d e t e r m i n e if the p h e n o t y p e s o f the S t r D m u t a n t s were due to a single m u t a t i o n , the wild t y p e strain, BR151, was t r a n s f o r m e d with D N A i s o l a t e d f r o m each m u t a n t . T r a n s f o r m a n t s , selected for abilitiy to g r o w on s t r e p t o m y c i n - c o n t a i n i n g m e d i u m , were screened for d e p e n d e n c e on the d r u g a n d for their a b i l i t y to s p o r u l a t e b y s i m p l y . o b s e r v i n g the c o l o r o f the c o l o n i e s ; colonies o f s p o r u l a t i n g cells b e c o m e b r o w n a n d o p a q u e on solid N S M , while colonies o f sporem i n u s v a r i a n t s are n o n - p i g m e n t e d a n d translucent. The suspected sporulation phenotypes were c o n f i r m e d b y p h a s e - c o n t r a s t m i c r o s c o p y . T h e results are s h o w n on T a b l e 2. Surprisingly, m o s t o f the S t r D t r a n s f o r m a n t s o f each o f the s p o r e - p l u s (Spo ÷) m u t a n t s were a s p o r o g e n o u s , suggesting t h a t each o f the
G.R. Quan et al. : Streptomycin Dependence in B. subtilis
198 2. Transformation of strain BR151 to streptomycin dependence with DNA from the streptomycin-dependentmutants
Table
Donor strain
No. of No. of No. of dependent spore-plus spore-minus transformants transformants transformants
SD1 SD2 SD6 SD7 SD9 SD10
347 86 141 165 163 443
0 0 9 20 28 12
347 86 132 145 135 431
200
I00
25
2
Time
Spo+, StrD mutants contained two mutations; one causing streptomycin dependence and asporogeny, and the second one suppressing the asporogenous phenotype.
4
6
8
I0
l houre)
Fig. 1. Growth curves of mutants SD9 (~), SD10A (©), SD10B (~), SD10 (A), and wild type strain BR151 (e). Doubling times of SD9, SD10A, SD10B, SD10 and BR151 were 77min, 135rain, 65 rain, 95 rain, and 50 rain respectively
3. Mapping of the streptomycindependence mutation. KSTP strain (purA16 cysA14 trpC2 strR) was transduced with PBS-1 lysates of SD10B and SD9, and the resultant cysteine prototrophs were screened for streptomycindependence and purine prototrophy
Table
Further Characterization of Mutants SDIO, SDIOA, SDIOB and SD9 Because of the slow growth rates of the asporogenous SD1 and SD2 mutants, a StrD Spo transformant (SD10A) of BR151 derived from SD10 was chosen for further study and characterization. A StrD Spo + transformant (SD10B) of BR151 derived from SD10 was also chosen for comparison between sporulating and non-sporulating StrD strains. Since SD9 consistently sporulated slightly better than BR151, it was also a strain of choice for further characterization. To be doubly certain that the S p o - transformants from SD 10 actually carried a single mutation causing asporogeny and streptomycin dependence and that the Spo +, StrD transformants bore two separate mutations, BR151 was transformed with D N A from SD10A and from SD10B. Out of 355 dependent transformants using SD10A D N A , 354 were spore-minus, and out of 281 dependent transformants using SD10 B D N A , 95 were spore-plus and 186 were spore-minus. This shows that S D 1 0 A does carry a single mutation, while SD10B and SD10 carry two mutations separable by transformation. Although one of the 355 dependent transformants obtained with SD 10 A D N A was spore-plus, it was apparently a mutant that had spontaneously acquired an additional mutation. This was shown by extracting its D N A and transforming BR151 strain to streptomycin dependence. The resulting transformants were predominantly spore-minus, thus, showing the same characteristics as the double mutants SD10 and SD10B. The growth curves of S D 1 0 A and SD10B are shown in Fig. 1. For comparison the growth curves of SD10, SD9 and BR15I are also included in the figure. All cultures were grown in liquid N S M sup-
Lysate
No. of Cys+
No. of Cys+ transductants that were
transductants
SD10B SD9
211 107
StrD
Ade +
Ade +StrD
i78 80
23 15
14 12
plemented with 500 ~tg streptomycin per ml, except for BR151. SD10A grows at about one half the rate of the spore-plus strains. Sporulation frequencies of these five strains at 48 hours after the onset of stationary phase were: BRI51, 80%; SD10, 41%; SD10A, 0.38%; SD10B, 51%; SD9, 100%.
Mapping of the Spreptomycin Dependence Mutation PBS-l-mediated transduction was used to determine the genetic location of the dependence mutation carried by the mutants SD10B and SD9. A PBS-1 lysate of SD10B and SD9 was used to transduce KSTP strain (purA16 cysA14 trpC2 strR). Transductants were selected for the ability to grow in the absence of cysteine on minimal medium G containing 500 gg streptomycin per ml and screened for streptomycin dependence and purine prototrophy. Results are shown in Table 3. The cysA locus is the closest auxotrophic marker to the "ribosomal gene region" of the B. subtilis chromosome (Young and Wilson, 1972). From the results shown, the strD mutation cotransduced with the cysA marker at a frequency of approximately
G.R. Quan et al. : Streptomycin Dependence in B. subtilis
I99
300
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2 3
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:D by Springer-Verlag 1979
Bacillus subtilis Mutants Dependent on Streptomycin Gilda R. Quan, Kristine M. Campbell, and Glenn H. Chambliss Department of Bacteriology, Universityof Wisconsin-Madison, Madison, Wisconsin 53706, USA
Summary. Six streptomycin-dependent mutants of Bacillus subtiIis, two of which were asporogenous, were isolated. All six mutants, SD1, SD2, SD6, SD7, SD9 and SD10, contained a single mutation causing streptomycin dependence and asporogeny, but four of these mutants (SD6, SD7, SD9, SD10) contained a second mutation which phenotypically suppressed the asporogenous character of the streptomycin dependence mutation. All six mutants grew more slowly than the wild type strain BR151, but those defective in sporulation grew the slowest. The streptomycin dependence mutations of SD9 and SD10B (a sporeplus transformant from SD10 carrying both the dependence mutation and the phenotypic suppressor) lie near or possibly within the strA locus. Ribosomes from SD9, SD 10A (a spore-minus transformant from SD10 carrying only the dependence mutation), and SD10B were stimulated in vitro by concentrations of streptomycin that inhibit the activity of wild type strain BR151 ribosomes. The level of misreading as measured by poly(U)-directed isoleucine incorporation was greatly enhanced by streptomycin in wild type strain BR151 ribosomes, but misreading of mutant SD9, SD10A, and SD10B ribosomes, irrespective of the sporulation phenotype, was little affected by streptomycin. There were no apparent differences in the patterns obtained by two-dimensional polyacrylamide gel electrophoresis of the 70S ribosomal proteins of the mutants SD9, SD10A, SD10B, and wild type strain BS151.
Introduction One approach to the study of ribosome structure and function is to isolate mutants with altered responses For offprints contact: Glenn H. Chambliss
to antibiotics that affect protein synthesis. Streptomycin has been widely applied for this purpose, and streptomycin-resistant (StrR) mutants have been extensively studied. Streptomycin-dependent (StrD) mutants, on the other hand, have been less well characterized. Although StrD mutants of Staphylococcus aureus (Paine and Finland, 1948; Foley, 1950), Pseudomonas aeruginosa (Paine and Finland, 1948), Proteus morgani (Paine and Finland, 1948), Klebsiella pneumonia (Paine and Finland, 1948) and Mycobacterium ranae (Yegian and Budd, 1948; Yegian and Vanderlinde, 1949) were reported in the 1940's, they have been poorly characterized genetically. Additionally, StrD mutants of Brucella abortus (Olitzki and Szenberg, 1953), BruceIla suis (Simon and Berman, 1962), Haemophilus pertussis (Bochagova, 1959), Pasteurella pestis (Sharon, 1961), and Shigella (Sergeev et al., 1967) have been isolated, but the main emphasis of these studies was on the immunogenicity and pathogenicity of the mutants. M ore detailed genetic and biochemical studies, however, have been done on StrD mutants of Escherichia coli (Newcombe and Nyholm, 1950; Likover and Kurland, 1967; Birge and Kurland, 1969; Momose and Gorini, 1970; Sanchez-Anzaldo and Bastarrachea, 1974; Wittmann and Apirion, 1975; Polglase et al., 1978), Sahnonella o~phimurium (Goldschmidt etal., 1962), Proteus mirabilis (Hofemeister and B6hme, 1967), Pneumococcus (Mishra and Ravin, 1966), Rhizobium lupini (Garbor, 1965), Vibrio cholerae (Felsenfeld et al., 1970), Bacillus stearothermophilus (Isono, 1974), and Bacillus subtilis (Itoh et al., 1975). In some of these organisms, dependence on streptomycin can be acquired by a single mutation which often leads to pleiotropic effects on the mutant. StrD mutants of E. coli seemed to have growth factor requirements in addition to the antibiotic requirements (Newcombe and Nyholm, 1950); mutants of S. typhimurium required methionine for growth in
0026-8925/79/0175/0195/$ 01.60
196 a d d i t i o n to s t r e p t o m y c i n ( G o l d s c h m i d t et al., 1962), w h i l e m u t a n t s o f P, mirabilis w e r e c h a r a c t e r i z e d b y pleiotropic changes of the protein synthesizing appar a t u s ( H o f e m e i s t e r a n d B 6 h m e , 1967). T h e s t r e p t o m y cin d e p e n d e n c e m u t a t i o n s in E. coli ( H a s h i m o t o , 1960), Pneumococcus ( M i s h r a a n d R a v i n , 1966), R. lupini ( G a r b o r , 1965), a n d S. typhimurium ( G o l d s c h m i d t et al., 1962) lie in t h e s a m e l o c u s as t h e s t r e p t o mycin resistance mutations. Ribosomes from StrD m u t a n t s o f E. coli ( L i k o v e r a n d K u r l a n d , 1967; B i r g e a n d K u r l a n d , 1969), V. cholerae ( F e l s e n f e l d et al., 1970), B. stearothermophilus ( I s o n o , 1974), a n d B. subtills ( I t o h et al., 1975) w e r e s t u d i e d , a n d in all cases, a n a l t e r e d 30S r i b o s o m a l p r o t e i n w a s f o u n d , thus, s u g g e s t i n g a n i n v o l v e m e n t o f t h e r i b o s o m e in t h e S t r D mutations. S i n c e m a n y S t r D m u t a n t s s t u d i e d so far h a v e exh i b i t e d p l e i o t r o p i c p h e n o t y p e s , a n d since a l t e r e d r i b o s o m a l p r o t e i n s c o r r e s p o n d i n g to t h e d e p e n d e n c e m u t a t i o n h a v e b e e n f o u n d , w e c h o s e to i s o l a t e S t r D m u t a n t s o f B . subtilis t o i n v e s t i g a t e f u r t h e r t h e p o s s i b i l ity o f t h e i n v o l v e m e n t o f r i b o s o m e s in t h e r e g u l a t i o n of gene expression during sporulation. Although an a l t e r e d 30S r i b o s o m a l p r o t e i n w a s d e t e c t e d in s t r e p t o m y c i n - d e p e n d e n t s t r a i n s o f B. subtilis b y c a r b o x y m e t h y l c e l l u l o s e c h r o m a t o g r a p h y ( I t o h et al., 1975), a n d in B. stearothermophilus b y t w o - d i m e n s i o n a l gel e l e c t r o p h o r e s i s ( I s o n o , 1974), t h e i r s p o r u l a t i o n p h e n o t y p e s w e r e n o t r e p o r t e d . I n this p a p e r , we r e p o r t the i s o l a t i o n o f s t r e p t o m y c i n d e p e n d e n t m u t a n t s o f B. subtilis w h i c h s p o r u l a t e at a m u c h l o w e r f r e q u e n c y t h a n t h e w i l d t y p e strain.
G.R. Quan et al. : Streptomycin Dependence in B. subtilis a Klett-Summerson colorimeter with the No. 66 red filter in place, or with a Bausch and Lomb Spectronic 20 spectrophotometer at a wavelength of 570 nm.
Isolation of Streptomycin-Dependent Mutants Streptomycin-dependent mutants were obtained by heat mutagenesis of spores from strain BR151 (Northrup and Slepecky, 1967). BR151 was used because its amino acid requirements provided a convenient means for identification of the strain. BR151 cells were harvested from a 48 hour culture grown in liquid NSM, washed three times with double-dlstilled water, resuspended in water, and hefit shocked at 80°C for 10 min to kill vegetative cells. Samples (0.5 ml) of the heat shocked culture were dried in a vacuum oven at 50 55° C overnight. The temperature was then raised to 105°C for 2.5h, after which mutagenized spores were stored at - 2 0 ° C. Sample of the mutagenized, dry spores was then resuspended in 3 ml of A3 medium and incubated at 37° C until germination was observed mmroscoplcally. Portions (0.1 ml) of the germinated cells (containing 7 x 106 viable ceils) were plated on TBAB plates, incubated at 37° C for 3 h, and overlayed with 2.5 ml soft agar containing 5 mg streptomycin per ml. All plates were then incubated at 37° C for 2 days, and colonies that grew were screened for streptomycin dependence.
Determination of Frequeno, of Sporulation Ceils were grown in liquid NSM. Growth was monitored and shortly after the end of exponential growth, samples of the cultures were withdrawn, appropriately diluted in Davis salts, and plated on solid NSM to determine total colony forming units. Forty-eight hours after the end of exponential growth, 1 ml samples of the cultures were treated to 80° C for 10 rain, diluted appropriately in Davis salts, and plated to determine the heat-resistant colony forming units. Percentage of sporulation was defined as : heat-resistant colony forming units x 100 total colony forming units
Transformation Materials and Methods Bacterial Strains and Growth Conditions The B. subtdis strains used in this study were 168T +, prototroph, obtained from P. Schaeffer; BR151, trpC2 lys-3 metBlO, obtained from G. Wilson; Kit 1, purA16 cysA14 trpC2, a reference strain constructed by Dedonder et al. (1977); all streptomycin-dependent mutants were obtained by heat mutagenesis of BRI51 spores and were grown on Schaeffer's solid nutrient broth sporulation medium (NSM) (Schaeffer et al., 1965) containing 500 gg streptomycin sulfate per ml (Sigma). They were transferred frequently and stored at 4° C. All growth was at 37° C. Minimal medium G containing 0.12 M Trizma Base (Sigma), 0.02 M glutamic acid monopotassium salt, 3 mM sodium citrate, 2 mM MgSO4.7H20, 0.5% glucose, and pH adjusted to 7.2 with H3PO4, was supplemented with auxotrophic requirements at concentrations of 50 ~tg/ml, when needed. Antibiotic Assay Broth No. 3 (A3) and Tryptose Blood Agar Base (TBAB) were purchased from Difco. Peptone Blood Agar Base (PBAB) (Gibco) substituted for TBAB in some experiments. Davis salts used for dilution contained 0.7% K2HPO4, 0.3% KH2PO4, 0.1% (NH,)2SO4 and 0.005% MgSO4.7H20. Schaeffer's nutrient broth sporulation medium (NSM) was used for growth of cells for ribosome extractions and for sporulation studies in liquid medram. Growth was monitored by measuring culture turbidity with
Transformation procedures of BoyIan et al. (1972) were followed. DNA was extracted according to Bon and Wilson (1967), and concentrations of DNA determined by Burton's diphenylamine assay for DNA concentration (I956). Transformations were done with 0.1 ~tg of DNA per ml.
Transduction Transduction procedures of Jamet and AnagnostopouIos (1969) were followed. For the transduction experiments, a streptomycinresistant, Kit 1 strain was constructed by transformation of strain Kit 1 (purA16 cysA14 trpC2) with DNA from a spore-plus streptomycin-resistant mutant isolated by heat mutagenesis. This Kit 1StrR strain was renamed KSTP. Phage PBS-1 lysates of the StrD mutants were used to transduce KSTP. Cysteine prototrophs were selected by spreading on minimal medium G lacking cysteine. The resulting prototrophic transductants were screened on solid NSM to determine the percentage of cotransduction of the cysA mutation with the streptomycin dependence mutation, and on minimal medium G lacking adenine to determine the cotransduction of the strD mutation with the cysA and purA markers.
Preparation of Ribosomes Ribosomes were prepared according to Legault-Demare and Chambliss (1974) with the modifications described by Campbell
G.R. Quan et al.: Streptomycin Dependence in B. subtilis
197
and Chambliss (1977), with the following exceptions: the StrD mutants were grown in liquid NSM supplemented with 100 gg streptomycin per ml, and BR151 was grown without any drug; ribosomes after a 12 h wash in high salt buffer were collected by centrifugation through a 2 ml 30% sucrose cushion for 8 h at 150,000 x g.
Table 1. Growth and sporulation of streptomycin-dependent mutants in liquid NSM containing streptomycin (500 gg/ml) Strain
Growth rate (doubling time) (min)
Sporulation frequency (%)
b~ vitro Protein Synthesis
SD1 SD2 SD6 SD7 SD9 SDI0 BRI51 ~
240 201 100 105 77 100 55
0.03 0.54 49 80 100 41 80
High speed supernatant (S-150) and initiation factors (IF's) were prepared by the procedures of Legault-Demare and Chambliss (1974) with the modifications already noted (Campbell and Chambliss, 1977). The S-150 fractions used in the assays were prepared from strain 168T+ cells grown in CHT50 medium (Anagnostopoulos and Spizizen, 1961) and the IF's were prepared from strain BR151 cells. The in vitro protein synthesis assay measured the incorporation of [14C]-phenylalanine (14 cpm/pmol) into acid-insoluble material using RNA extracted from exponentially growing BR151 ceils as template. Each assay tube contained 70 gg ribosomes, 91.2 ~tg crude IF protein, 87 gg crude S-150 protein, and 300 gg BR151 RNA in a reaction volume of 0.125 ml, except where otherwise noted. The method of Lowry et al. (1951) was used to determine the protein concentrations of S-150 and IF preparations. Streptomycin was added from a stock solution made in 0.1 M Tris-HC1, pH 7.5. For the misreading experiments, procedures of Campbell and Chambliss (1977) were followed.
Two-Dimensional Polyacrylamide Gel Eleetrophoresis Two-dimensional gels were run with the buffers and gel compositions of Kaltschmidt and Wittmann (1970) as modified by Howard and Traut (1973). Ribosomal proteins were precipitated with acetone as described by Barritault et al. (1976).
Results
Isolation, Sporulation and Growth Rates of StrD Mutants S t r D m u t a n t s were i s o l a t e d by the h e a t m u t a g e n e s i s p r o c e d u r e d e s c r i b e d in Materials and Methods. F r o m 540 c o l o n i e s g r o w i n g on s t r e p t o m y c i n - c o n t a i n i n g plates, 219 c o n t a i n e d d r u g resistant cells, a n d o f this n u m b e r , 6 were f o u n d to c o n t a i n s t r e p t o m y c i n - d e p e n d e n t m u t a n t s . These m u t a n t s were n a m e d SD1, SD2, SD6, SDT, SD9 a n d SD10 respectively. T h a t each o f these m u t a n t s r e t a i n e d the a u x o t r o p h i c req u i r e m e n t s o f their p a r e n t a l strain BR151 was asc e r t a i n e d b y the fact t h a t each r e q u i r e d t r y p t o p h a n , m e t h i o n i n e , a n d lysine for g r o w t h on m i n i m a l med i u m G. This m e d i u m was devised to s u b s t i t u t e for Spizizen M i n i m a l M e d i u m (Spizizen, 1958) b e c a u s e the S t r D m u t a n t s were u n a b l e to g r o w on the latter. Beggs a n d A n d r e w s (1976) r e p o r t e d t h a t m o n o v a l e n t a n d d i v a l e n t c a t i o n s interfered with the u p t a k e o f d i h y d r o s t r e p t o m y c i n by Mycobacterium smegmatis, c o n s e q u e n t l y p r e v e n t i n g its lethal a c t i o n a g a i n s t the m i c r o o r g a n i s m . It s e e m e d p r o b a b l e t h a t the high c o n c e n t r a t i o n o f c a t i o n s in the Spizizen m i n i m a l me-
a
No streptomycin was added to this culture
d i u m i n h i b i t e d the u p t a k e o f s t r e p t o m y c i n b y the S t r D m u t a n t s , thus p r e v e n t i n g their growth. This a p p a r ently was the case since BR151, w h i c h is n o r m a l l y sensitive to s t r e p t o m y c i n , grew on Spizizen m i n i m a l m e d i u m s u p p l e m e n t e d with s t r e p t o m y c i n (500 gg/ml) but could not grow on minimal medium G containing s t r e p t o m y c i n . T h u s the r e d u c e d c a t i o n c o n c e n t r a t i o n o f m i n i m a l m e d i u m G a p p a r e n t l y a c c o u n t s for the a b i l i t y o f the d e p e n d e n t m u t a n t s to g r o w on it. Kinetics o f g r o w t h a n d f r e q u e n c y o f s p o r u l a t i o n o f SD1, SD2, SD6, SDT, SD9, SD10 a n d BR151 were d e t e r m i n e d in liquid N S M . T h e results are s h o w n on T a b l e 1. T w o o f the six m u t a n t s were quite defective in their a b i l i t y to sporulate, each s p o r u l a t i n g at less t h a n 1% the wild t y p e frequency. M u t a n t s SD6 a n d SD10 c o n s i s t e n t l y s p o r u l a t e d at a b o u t oneh a l f the f r e q u e n c y o f BR151 while m u t a n t SD9 s p o r u l a t e d slightly b e t t e r t h a n wild type. A l l m u t a n t s grew m o r e slowly t h a n the p a r e n t a l strain BR151, b u t the m u t a n t s defective in s p o r u l a t i o n grew the slowest.
Genetic Analysis of the Streptomycin-Dependent Phenotype T o d e t e r m i n e if the p h e n o t y p e s o f the S t r D m u t a n t s were due to a single m u t a t i o n , the wild t y p e strain, BR151, was t r a n s f o r m e d with D N A i s o l a t e d f r o m each m u t a n t . T r a n s f o r m a n t s , selected for abilitiy to g r o w on s t r e p t o m y c i n - c o n t a i n i n g m e d i u m , were screened for d e p e n d e n c e on the d r u g a n d for their a b i l i t y to s p o r u l a t e b y s i m p l y . o b s e r v i n g the c o l o r o f the c o l o n i e s ; colonies o f s p o r u l a t i n g cells b e c o m e b r o w n a n d o p a q u e on solid N S M , while colonies o f sporem i n u s v a r i a n t s are n o n - p i g m e n t e d a n d translucent. The suspected sporulation phenotypes were c o n f i r m e d b y p h a s e - c o n t r a s t m i c r o s c o p y . T h e results are s h o w n on T a b l e 2. Surprisingly, m o s t o f the S t r D t r a n s f o r m a n t s o f each o f the s p o r e - p l u s (Spo ÷) m u t a n t s were a s p o r o g e n o u s , suggesting t h a t each o f the
G.R. Quan et al. : Streptomycin Dependence in B. subtilis
198 2. Transformation of strain BR151 to streptomycin dependence with DNA from the streptomycin-dependentmutants
Table
Donor strain
No. of No. of No. of dependent spore-plus spore-minus transformants transformants transformants
SD1 SD2 SD6 SD7 SD9 SD10
347 86 141 165 163 443
0 0 9 20 28 12
347 86 132 145 135 431
200
I00
25
2
Time
Spo+, StrD mutants contained two mutations; one causing streptomycin dependence and asporogeny, and the second one suppressing the asporogenous phenotype.
4
6
8
I0
l houre)
Fig. 1. Growth curves of mutants SD9 (~), SD10A (©), SD10B (~), SD10 (A), and wild type strain BR151 (e). Doubling times of SD9, SD10A, SD10B, SD10 and BR151 were 77min, 135rain, 65 rain, 95 rain, and 50 rain respectively
3. Mapping of the streptomycindependence mutation. KSTP strain (purA16 cysA14 trpC2 strR) was transduced with PBS-1 lysates of SD10B and SD9, and the resultant cysteine prototrophs were screened for streptomycindependence and purine prototrophy
Table
Further Characterization of Mutants SDIO, SDIOA, SDIOB and SD9 Because of the slow growth rates of the asporogenous SD1 and SD2 mutants, a StrD Spo transformant (SD10A) of BR151 derived from SD10 was chosen for further study and characterization. A StrD Spo + transformant (SD10B) of BR151 derived from SD10 was also chosen for comparison between sporulating and non-sporulating StrD strains. Since SD9 consistently sporulated slightly better than BR151, it was also a strain of choice for further characterization. To be doubly certain that the S p o - transformants from SD 10 actually carried a single mutation causing asporogeny and streptomycin dependence and that the Spo +, StrD transformants bore two separate mutations, BR151 was transformed with D N A from SD10A and from SD10B. Out of 355 dependent transformants using SD10A D N A , 354 were spore-minus, and out of 281 dependent transformants using SD10 B D N A , 95 were spore-plus and 186 were spore-minus. This shows that S D 1 0 A does carry a single mutation, while SD10B and SD10 carry two mutations separable by transformation. Although one of the 355 dependent transformants obtained with SD 10 A D N A was spore-plus, it was apparently a mutant that had spontaneously acquired an additional mutation. This was shown by extracting its D N A and transforming BR151 strain to streptomycin dependence. The resulting transformants were predominantly spore-minus, thus, showing the same characteristics as the double mutants SD10 and SD10B. The growth curves of S D 1 0 A and SD10B are shown in Fig. 1. For comparison the growth curves of SD10, SD9 and BR15I are also included in the figure. All cultures were grown in liquid N S M sup-
Lysate
No. of Cys+
No. of Cys+ transductants that were
transductants
SD10B SD9
211 107
StrD
Ade +
Ade +StrD
i78 80
23 15
14 12
plemented with 500 ~tg streptomycin per ml, except for BR151. SD10A grows at about one half the rate of the spore-plus strains. Sporulation frequencies of these five strains at 48 hours after the onset of stationary phase were: BRI51, 80%; SD10, 41%; SD10A, 0.38%; SD10B, 51%; SD9, 100%.
Mapping of the Spreptomycin Dependence Mutation PBS-l-mediated transduction was used to determine the genetic location of the dependence mutation carried by the mutants SD10B and SD9. A PBS-1 lysate of SD10B and SD9 was used to transduce KSTP strain (purA16 cysA14 trpC2 strR). Transductants were selected for the ability to grow in the absence of cysteine on minimal medium G containing 500 gg streptomycin per ml and screened for streptomycin dependence and purine prototrophy. Results are shown in Table 3. The cysA locus is the closest auxotrophic marker to the "ribosomal gene region" of the B. subtilis chromosome (Young and Wilson, 1972). From the results shown, the strD mutation cotransduced with the cysA marker at a frequency of approximately
G.R. Quan et al. : Streptomycin Dependence in B. subtilis
I99
300
~oo
2 3
200 _> u
