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Mutants of Staphylococcus aureus deficient in recombinational repair
279
Mutation Research, 60 (1979) 279--289
© Elsevier/North-Holland Biomedical Press
MUTANTS OF S T A P I t Y L O C O C C U S A U R E U S DEFICIENT IN RECOMBINATIONAL R E P A I R IMPROVED ISOLATION BY SELECTING F O R MUTANTS EXHIBITING C O N C U R R E N T SENSITIVITY TO U L T R A V I O L E T RADIATION AND N - M E T H Y L - N ' - N I T R O - N - N I T R O S O G U A N I D I N E
R.V. GOERING Department of Medical Microbiology, Creighton University School of Medicine, Omaha, Nebraska 68178 (U.S.A.)
(Received 28 June 1978) (Revision received 22 November 1978) (Accepted 11 December 1978)
Summary Recombination-deficient (rec) mutants of S t a p h y l o c o c c u s aureus strains 152 and Ps29 were sought b y initially screening mutagenized cultures for mutants exhibiting increased sensitivity to both ultraviolet {UV) radiation and N-methyl-N'-nitro-N-nitrosoguanidine (NG). Mutants thus isolated were analyzed for recombinational ability by transduction, and further characterized in terms of sensitivity to UV, NG, ability to repair UV-irradiated bacteriophage, and spontaneous and UV-induced DNA degradation. Mutagenesis of strain 152 yielded three isolates, one of which was rec, the second potentially lex, and the third possessing an undetermined repair deficiency. Mutagenesis of strain Ps29 resulted in the isolation of one mutant, which exhibited a rec genotype. In searching for rec mutants of S. aureus, the value of initially screening mutagenized cultures for mutants exhibiting concurrent sensitivity to UV and NG, as opposed to screening for UV sensitivity alone, is discussed. .
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Several investigators have recently reported the isolation of mutants of S t a p h y l o c o c c u s aureus deficient in the recombinational repair of DNA [8,12, 27]. Such recombination-deficient (rec) mutants are of increasing importance
to staphylococcal research including studies of DNA repair and efforts to determine the plasmid vs. chromosomal location of specific staphylococcal genes, especially those concerning antibiotic resistance [17]. In each of the previous reports, isolation of rec mutants of S. aureus followed a similar protocol: mutagenized cells were initially screened for sensitivity to ultraviolet radiation
280 (UV), with UV-sensitive isolates then examined for impaired recombination ability. The difficulty with this isolation procedure, however, is the fact that UV sensitivity is associated with a multitude of deficiencies in DNA repair (e.g., see ref. [4]). Thus, a mutagenesis procedure producing numerous UV-sensitive isolates may yield few, if any, rec mutants. Mutants deficient in post-replication repair (including rec mutants) are known to be highly susceptible to inactivation by the monofunctional alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (NG), while wild-type strains and mutants deficient in excision repair are comparatively insensitive to NG [6,9,14,16,24,25]. Accordingly, an improved procedure for the isolation of rec mutants of S. aureus is reported here, based on the initial screening of mutagenized cultures for isolates exhibiting concurrent sensitivity to UV and NG. Materials and methods Bacterial strains
For mutagenesis experiments, S. aureus strain Ps29, of the International Typing Series, and strain 152 [20] were used. Donor strains employed in transduction experiments were: S. aureus U9 (pase, tet, ol, nov) [19] which carries plasmid-linked genes for penicillinase production (pase marker) and tetracycline resistance (tet marker) [8,22], and chromosomal genes for resistance to oleandomycin (ol marker) and novobiocin (nov marker) [3,8]; and S. aureus 655CT-N which carries a plasmid-linked pase marker and a chromosomal nov marker [1,11]. Additional strains, included in DNA degradation experiments, were S. aureus 112 and 112 rec-2 [8,27] and S. aureus 8325-4 and 8325-4 his-7 recAl [27]. All cultures were maintained at 4°C on Brain Heart Infusion (BHI; Difco) agar slants. Bacteriophages and transductions
Phages 29, 80 and 83 of the International Typing Series were used for transduction and host-cell reactivation experiments. Procedures for phage maintenance, propagation, and titration were those of Pattee and Baldwin [19]. Transductions were performed by the method of Kloos and Pattee [15] with the addition of 10 -3 M CaC12 to the transduction suspension. Selective media for the antibiotic-resistance markers was prepared as described by Pattee and Baldwin [ 19,20]. Preparation o f solid m e d i u m containing N G
To directly screen mutagenized cells for NG sensitivity, a solid medium containing NG was prepared. Trypticase Soy Agar (TSA; Baltimore Biological Laboratory) was prepared at pH 6.0 by rehydrating in 0.1 M KPO4 (pH 6.0), and sterilized by autoclaving. The medium was cooled to 47°C and, immediately prior to dispensing, NG {Aldrich Chem. Co.) was freshly dissolved in 0.1 M KPO4 (pH 6.0), filter-sterilized, and added to the medium at a final concentration of 10 pg/ml. Plates of TSA containing NG (designated TSNG agar) were always used on the day of preparation. Isolation o f m u t a n t s sensitive to U V and N G S. aureus strains 152 and PS29 were mutagenized with NG (conc. = 500 pg/
281 ml) and plated on BHI agar as described previously [8]. Colonies appearing after overnight incubation at 37°C were screened by replica plating to sets of two BHI agar plates (one to receive 34 J/m s UV radiation, the other serving as an unirradiated control}, and a plate of TSNG agar. Colonies which appeared sensitive to both UV and NG (i.e., growth absent or extremely poor on UV irradiated and TSNG plates} were suspended in 0;5-ml volumes of saline (0.5% NaC1) and secondarily screened by spotting onto sets of four BHI agar plates (to receive UV dosages of 0, 8, 18 or 34 J/m 2) and a plate of TSNG agar. Only isolates exhibiting no growth on plates receiving the higher UV exposures and on TSNG agar were retained for further study.
UV irradiation o f bacteria and bacteriophages Methods for the UV irradiation of bacteria and bacteriophages have been previously described [8]. Quantitative determination o f NG sensitivity The NG sensitivity of cultures was quantitatively determined in citrate solution (0.85% trisodium citrate, pH 5.0} as described previously [8]. Measurement o f post-irradiation DNA degradation Strains to be examined were grown overnight in BHI at 37°C and diluted 1 : 15 in fresh BHI containing 10 ttCi of [3H]methylthymidine/ml (spec. act. = 50 Ci/mmole) and 50 pg of deoxyadenosine/ml. After at least t w o generations of growth, cells were harvested by centrifugation, washed 2× in saline, and resuspended in BHI for an additional 1 h incubation to reduce the radioactivity in the acid-soluble pool. After an additional saline wash, cells were resuspended in saline for UV irradiation. DNA degradation was determined by diluting UVirradiated or control cultures 1 : 10 in BHI supplemented with 100 pg thymidine/ml and incubating with shaking for 3 h at 37 °C. At appropriate intervals, 0.5-ml aliquots of cell suspension were mixed with 1.0 ml of cold 10% (w/v) trichloroacetic acid (TCA) and held at 0°C for at least 2 h. Bovine serum albumin (0.1 ml of a 1.0% [w/v] solution} was then added as a carrier to each sample, followed by centrifugation at 6 0 0 0 g for 15 min. Supernatant fluid (0.5 ml) was transferred to 10 ml of liquid-scintillation cocktail (Biofluor, New England Nuclear) for counting. The radioactivity equivalent to 100% of total counts was determined by hydrolysis of 0.5-ml aliquots of cell suspension in 1.0 ml of 10% TCA at 85°C for 1 h. The samples were then chilled, centrifuged with bovine serum albumin as above, and 0.5 ml of supernatant fluid was counted in 10 ml of Biofluor. All counts were normalized by external quench correction. Isolation o f revertants resistant to NG Mutants sensitive to NG were grown overnight in BHI and ca. l 0 T to l 0 s cells were plated directly on TSNG agar. Colonies appearing after overnight incubation at 37°C were purified on TSNG agar and transferred to BHI agar slants for further study.
282 Results T r a n s d u c t i o n o f a n t i b i o t i c resistance Following mutagenesis of S. aureus strain 152, only three isolates out of
4840 colonies exhibited consistent sensitivity to both UV and NG through primary and secondary screening procedures. With S. aureus Ps29, only one isolate out of 2320 colonies was consistently sensitive to UV and NG. To assess the recombinational ability of these mutants, transduction frequencies were determined for known plasmid and chromosomal markers. Table 1 summarizes the results obtained when strain 152 and its mutants were transduced using phage 83 propagated on S. aureus 655CT-N, which is known to carry a plasmid-linked pase marker and a chromosomal n o v marker [1,11]. Of the three mutants, UVR-60 demonstrated the greatest reduction in recombinational ability as evidenced by its total inability to act as a recipient of the chromosomal nov marker. UVR-18 appeared somewhat reduced in recombinational ability {i.e., transduction frequency for the chromosomal nov marker 9% that of strain 152) but not as recombination-deficient as UVR-60. Mutant UVR-22 exhibited a transduction frequency for the nov marker indicative of wild-type recombinational ability. All three mutants were similar in poorly expressing the plasmid-linked pase marker. Table 2 indicates the results obtained when strain Ps29 and its mutant, UVR11, were transduced using phage 80 propagated on S. aureus U9 (pase, tet, ol, n o v ) which carries plasmid-linked pase and tet markers and a chromosomal nov marker [8,22]. As with the mutants of strain 152, UVR-11 poorly expressed plasmid-linked markers (pase and tet), however, the m u t a n t did exhibit a recombination deficiency as evidenced by its inability to act as a recipient of the chromosomal n o v marker. On the basis of reduced recombinational ability {Tables i and 2), mutants 152 UVR-60 and Ps29 UVR-11 were designated 152 rec-3 and Ps29 rec-4, respectively, throughout the remainder of this study. S e n s i t i v i t y to U V a n d N G
While the mutants examined in this study were all isolated on the basis of a qualitative sensitivity to UV and NG, a more quantitative measurement of TABLE 1 TRANSDUCTION FREQUENCIES OF THE nov AND pase MARKERS S T R A I N 152 A N D T H R E E M U T A N T S U S I N G P H A G E 8 3 / 6 5 5 C T - N Recipient
IN S t a p h y l o c o c c u s
aureus
Frequency of transduction a pase
nov
152 UVR-18 UVR-22
1520 (100) 280 (18) 160 (10)
1120 (100) 100 (9) 1480 (132)
UVR-60
187 (12)
0 (0)
(rec.3 )
a E x p r e s s e d as n u m b e r of transductant$ p e r 109 p l a q u e - f o r m i n g u n i t s . N u m b e r s in parentheses are relative t r a n s d u c t i o n f r e q u e n c i e s c a l c u l a t e d u s i n g t h e f r e q u e n c y o f 1 5 2 as 100%.
283 TABLE 2 T R A N S D U C T I O N F R E Q U E N C I E S OF T H E nov, tet ANDpase M A R K E R S IN S t a p l t y l o c o c c u s Ps29 A N D A M U T A N T D E R I V A T I V E U S I N G P H A G E 8 0 / U 9 (pose, tet, ol, n o v ) Recipient
aureus
Frequency of transduction a pase
no v
t et
3140 (I00) 440 (14)
1840 (I00) 0 (0)
28200 (i00) 3800 (13)
i
Ps29 UVR-I 1 (rec-4)
a E x p r e s s e d as n u m b e r o f t r a n s d u c t a n t s p e r 1 0 9 p l a q u e - f o r m i n g units. N u m b e r s in p a r e n t h e s e s are relative t r a n s d u c t i o n f r e q u e n c i e s c a l c u l a t e d u s i n g t h e f r e q u e n c y o f P s 2 9 as 100%.
these sensitivities was desired. When compared on the basis of the UV fluence required to achieve e-' inactivation, rec-3, UVR-22, and UVR-18 were, respectively, 13X, 7X, and 2X more sensitive to UV than strain 152 (Fig. 1A). A similar comparison of the NG inactivation curves of the mutants (Fig. 1B) revealed UVR-22 and rec-3 to be 18X more sensitive to NG than strain 152. Mutant UVR-18 was 7X more sensitive to NG than the parent strain. A similar analysis of the inactivation kinetics of strain PS29 and rec-4 revealed rec-4 to be 14X more sensitive to UV (Fig. 2A) and l l X more sensitive to NG (Fig. 2B) than the parent strain Ps29.
Repair of UV-irradiated phage The ability of the parent and mutant strains to repair (i.e., host-cell reactivate; HCR) UV-irradiated phage was examined. Following UV exposure, titers of viable phage did not differ significantly for phage 80 when plated on either strain 152 or its mutants (Fig. 3A), or for phage 29 when plated on either Ps29 or rec-4 (Fig. 3B).
IOC
A -
B
-
i
.J
~0
O0
-
0.00
FLUENCE(~m')
MINUTES NG EXPO~JRE
Fig. 1. Survival c u r v e s f o r S. a u r e u s strain 1 5 2 a n d t h r e e m u t a n t U V ( A ) or a 1 0 0 # g / m l c o n c e n t r a t i o n o f N G ( B ) .
derivatives w h e n e x p o s e d
either to
284 IOC
A ~29
\ \
ae 0
\
k%. \ OOI
\
\ ~LVR.I
000
,\
,\
OOCW
FLUENCE[Jim')
MINUTES NG EXPOSUI~E
Fig. 2. S u r v i v a l curves for S. a u r e u s strain Ps29 and one mutant derivative when exposed either to U V ( A ) or a 100 ~g/ml concentration of NG(B).
Spontaneous and UV-induced DNA degradation The parent and mutant strains were examined for DNA degradation occurring either following UV exposure or in unirradiated control cultures. When compared to strain 152 (Fig. 4) mutants UVR-22 and rec-3 exhibited significant spontaneous DNA degradaLion (12% and 10% acid-soluble radioactivity, repectively) after 3 h incubation. Following UV exposure, UVR-22 and rec-3 exhibited DNA degradation (38% and 40%, respectively) significantly greater than that of strain 152 {11%). The UV-induced DNA degradation of UVR-18 (13%) did not differ significantly from that of 152. A comparison of strains Ps29 and rec-4 (Fig. 5) revealed 7% spontaneous
I(}C
A
B
UVR-18 UVR -22
UVR-~O
(nee-3)
~
I0 --I
UVR-H (,ec 4)
>
FLUENCE (~/m')
F i g . 3 . H o s t - c e l l r e a c t i v a t i o n o f U V - i r r a d i a t e d p h a g e 8 0 p l a t e d o n S. a u r e u s s t r a i n 1 5 2 a n d t h r e e m u t a n t d e r i v a t i v e s ( A ) o r p h a g e 2 9 P l a t e d o n S. a u r e u s s t r a i n P s 2 9 a n d o n e m u t a n t d e r i v a t i v e ( B ) .
285 152 U V R - I 8
152 80
60
40 z
o ,,,:I
,,, t~
20
0 152 UVR-60 (rec-3)
152 U V R - 2 2
z
o
80
60
40.
0 I
2
3
I
2
3
HOURS INCUBATION
Fig. 4. D N A d e g r a d a t i o n in S. a u r e u s strain 1 5 2 a n d t h r e e m u t a n t d e r i v a t i v e s w h e n e i t h e r u n i r r a d i a t e d (o) o r a f t e r 27 J / m 2 U V e x p o s u r e ( e ) .
DNA degradation for rec-4 after 3 h incubation compared to <1% spontaneous DNA degradation in strain Ps29. After UV exposure, incubation of rec-4 resulted in 36% DNA degradation compared to 6% in Ps29.
29 U V R - I I (rec-4)
29 80 1-
60 rr tU Q .¢ 40 Z 0
N
2O
I
2
13
I
2
3
HOURS INCUBATION
Fig. 5. D N A d e g r a d a t i o n in S. a u r e u s strain Ps29 a n d o n e m u t a n t d e r i v a t i v e w h e n e i t h e r u n i r r a d i a t e d (o) o r a f t e r 27 J / m 2 U V e x p o s u r e (o).
286 Of the previous reports of rec mutants in S. aureus [8,12,27] only one presents data on DNA degradation [ 12]. To allow a more thorough comparison of the mutants reported here with the rec mutants previously isolated in S. aureus, Fig. 6 indicates the spontaneous and UV-induced DNA degradation observed in the rec mutants of S. aureus (and their parent strains) previously uncharacterized in terms of DNA degradation. Strain 112 rec-2 exhibited 5% spontaneous DNA degradation. After UV exposure, DNA degradation in rec-2 increased to 34%, compared to only 5% for strain 112. Strain 8325-4 his-7 f e c A l exhibited 5% spontaneous DNA degradation. Following UV irradiation, r e c A l exhibited 55% DNA degradation compared to 9% for strain 8325-4. Characteristics o f revertants resistent to N G
To assess whether single or multiple mutational lesions were responsible for the pleiotrophic characteristics of rec-3 and rec-4, NG-resistant revertants of the mutants (termed NGR-3 and NGR-4, respectively) were selected on TSNG agar. These revertants were then compared phenotypically with their respective
112 rec-2
112 80
60
40 z 0
C- 2O rY ~D
w
A
C
o
8325-4
8325-4
recAI
his-7
z
'-' 80
60
40
20
I
2
3 HOURS
I
2
3
~NCUBATION
F i g . 6. D N A d e g r a d a t i o n i n S . a u r e u s s t r a i n 1 1 2 , 8 3 2 5 - 4 , a n d t h e i r r e c m u t a n t i r r a d i a t e d (~)) o r a f t e r 27 J / m 2 U V e x p o s u r e ( $ ) .
derivatives when either un
287 TABLE 3 C H A R A C T E R I S T I C S a OF M U T A N T S rec.3, rec-4 A N D T H E I R N G - R E S I S T A N T R E V E R T A N T S Characteristic
Strain 152 (wild-type)
rec-3
NGR-3
Ps29 (wild-type)
rec-4
NGR-4
UV sensitivity
1.0
13
1.0
1.0
14
1.0
Transduction ability
1.0
0
1.6
1.0
0
1.4
DNA d e g r a d a t i o n spontaneous
1.0
I0
1.0
1.0
14
1.0
UV-induced
J .0
1.3
1.0
3.6
6.0
1.1
a UV sensitivity of strains is e x p r e s s e d as t h e e -! f l u e n e e f o r w i l d - t y p e d i v i d e d b y t h e e -1 fluence for r e c or N G R derivatives. T r a n s d u c t i o n ability is t r a n s d u e t i o n f r e q u e n c y o f the c h r o m o s o m a l n o v m a r k e r in each strain d i v i d e d b y t h e t r a n s d u c t i o n f r e q u e n c y o f n o v in the w i l d - t y p e . D N A d e g r a d a t i o n is percent D N A d e g r a d a t i o n in each s t r a i n d i v i d e d b y p e r c e n t D N A d e g r a d a t i o n in the w i l d - t y p e .
mutant and wild-type parent strains {Table 3). With both rec-3 and rec-4, reversion to NG resistance resulted in the restoration of a wild-type phenotype, thus indicative of a single mutation responsible for the p h e n o t y p e of the mutants. Discussion Ideally, a selection procedure for isolating rec mutants should directly screen mutagenized cells for recombination deficiency. This is best accomplished in bacteria with potential for conjugation, as in the method of Clark and Margulies [5] where cells are directly examined for recombinational ability by replica plating to lawns of Hfr cells. At present, transduction and transformation are the only known means of genetic transfer in S. a u r e u s , neither of which lends itself well to the above t y p e of assay procedure. Therefore, rec mutants of S. a u r e u s have been isolated in the past initially on the basis of UV sensitivity. Primary selection of isolates solely on the basis of UV sensitivity, however, commonly results in a poor yield of rec mutants [8,27], often favoring, instread, mutants deficient in excision repair (e.g., see ref. [8] ). Excision-deficient mutants, however, do not exhibit increased sensitivity to NG [ 14,16,25], a characteristic which is associated with rec mutations [6,9,14,16,24,25]. Therefore, primary screening of mutagenized cultures for isolates exhibiting concurrent sensitivity to UV and NG reduces the population of isolates to be examined while enriching for mutants of the rec genotype. This enrichment, however, is clearly for the predominant r e c A , B and C genotypes (e.g., as in E s c h e r i c h i a coli), and not other recombination deficiencies which might be relatively less sensitive to UV and/or NG [21,23]. It is well established that sensitivity to NG is not associated exclusively with recombination deficiency (e.g. l e x mutants are also known to be sensitive to NG [25]). However, this study suggests that, at least with the S. a u r e u s strains and mutagenesis protocal employed here, rec NG-sensitive mutations occur at frequencies similar to that for rec ÷ NG-sensitive mutations, since of the four mutants reported here, two were of the rec genotype.
288 As predicted by the selection procedure, all four mutants isolated in this study were clearly more sensitive to both UV and NG than their parent strains (Figs. 1, 2), although actual sensitivities varied somewhat among the isolates. In addition, all of the mutants were comparable to their parent strains in ability to repair UV-irradiated phage (i.e. HCR*; Fig. 3) and thus did not appear to be dificient in excision repair, which has been shown to relate to HCR ability [2,10]. On the basis of analysis by transduction, however, only two of the mutants were clearly recombination-deficient. Mutants 152 rec-3 and Ps29 rec-4 were both unable to act as recipients in the transduction of a chromosomal nov marker while expressing plasmid-linked antibiotic-resistance markers. These mutants also exhibited significant spontaneous and UV-induced DNA degradation, similar to the previously isolated rec mutants of S. aureus (Fig. 6), all suggestive of a recA genotype [9]. The restoration of a wild-type phenotype coincident with reversion to NG resistance in both rec-3 and rec-4 supports the hypothesis that the recombination-deficient phenotype of each mutant is the result of a single mutation (i.e. single site or point mutation). A similar relationship in recombination-deficient mutants of S a l m o n e l l a t y p h i m u r i u m has been described and discussed by Eisenstark et al. [6]. Mutant 152 UVR-22 demonstrated wild-type recombinational ability in transduction of the chromosomal nov marker, but exhibited elevated levels of spontaneous and UV-induced DNA degradation. This combination of characteristics suggests that UVR-22 may possess a l e x A - t y p e mutation [26], however, additional characterization of this m u t a n t (e.g., to determine whether recA protein [7,18] is produced) is required for definitive assignment of a genotype. Mutant 152 UVR-18 was a poor transduction recipient of the chromosomal nov marker. This m u t a n t was the least sensitive to UV and NG of any isolate and did not exhibit kinetics of DNA degradation significantly different from that of the parent strain, characteristics which appear similar to those of recB, C mutants in E. coli [9]. However, the current lack of information regarding DNA repair in S. aureus suggests a need for further study of this m u t a n t prior to ascribing a potential repair deficiency. Studies to further characterize all of the mutants reported here, particularly in terms of ability to produce recA protein [7,18], are currently in progress. In general, the data presented here indicate that m u t a n t selection based on concurrent sensitivity to UV and NG can be a means of significantly improving the isolation of rec mutants and other repair-deficient genotypes which are relatively u n c o m m o n , at least in S. aureus. Such mutants promise to be of considerable value in leading to a more thorough understanding of the genetics of this organism. Acknowledgements The technical assistance of Elaine Blythe is gratefully acknowledged. This investigation was supported in part by grants from Research Corporation and the Nebraska Heart Association.
289
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Margulies, I s o l a t i o n a n d c h a r a c t e r i z a t i o n o f r e c o m b i n a t i o n d e f i c i e n t m u t a n t s o f Escherichia coli K 1 2 , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 5 3 ( 1 9 6 5 ) 4 5 1 - - 4 5 9 . 6 E i s e n s t a r k , A., R. E i s e n s t a r k , J. v a n Dillewijn a n d A. R O r s c h , R a d i a t i o n - s e n s i t i v e a n d r e c o m b i n a t i o n less m u t a n t s o f S a l m o n e l l a t y p h i m u r i u m , M u t a t i o n Res., 8 ( 1 9 6 9 ) 4 9 7 - - 5 0 4 . 7 E m m e r s o n , P.T., a n d S.C. W e s t , I d e n t i f i c a t i o n o f p r o t e i n X o f Escherichia coil as t h e r e c A * / t i f ÷ g e n e p r o d u c t , Mol. G e n . G e n e t . , 1 5 5 ( 1 9 7 7 ) 7 7 - - 8 5 . 8 G o e r i n g , R . V . , a n d P.A. P a t t e e , M u t a n t s o f S t a p h y l o c o c c u s aureus w i t h i n c r e a s e d s e n s i t i v i t y to u l t r a violet r a d i a t i o n , J . B a c t e r i o l . , 1 0 6 ( 1 9 7 1 ) 1 5 7 - - 1 6 1 . 9 H o w a r d - F l a n d e r s , P., a n d R . P . B o y c e , D N A r e p a i r a n d g e n e t i c r e c o m b i n a t i o n : s t u d i e s o n m u t a n t s o f Escherichia coil d e f e c t i v e in t h e s e p r o c e s s e s , R a d i a t . Res., S u p p l . , 6 ( 1 9 6 6 ) 1 5 6 - - 1 8 4 . 1 0 t I o w a r d - F l a n d e r s , P.. R.P. B o y c e a n d L. T h e r i o t , T h r e e l o c i in Escherichia coli K - 1 2 t h a t c o n t r o l t h e excision of pyrimidine dimers and certain other mutagen products from DNA, Genetics, 53 (1966) 1119--1136. 11 Imsande, J., J.W. Zyskind and I. Mile, Regulation of staphylococcal penicilllnase synthesis, J. Bacteriol., 109 (1972) 122--123. 12 Inoue, M., H. Oahima, T. Okubo and S. Miteuhashi, Isolation of the rec mutants in Staphylococcus aureus, J. Bacteriol., 112 (1972) 1 169--1176. 13 Kada, T., K. Tutikawa and Y. Sadaie, In vitro and host-mediated "ree-assay"procedures for screening chemical mutagens; and phloxine, a mutagenic red dye detected, Mutation Res., 16 (1972) 165--174. 14 Kimball, R.F., J.K. Setlow and M. Liu, The mutagenie and lethal effects of monofunctionalmethylating a g e n t s in s t r a i n s o f l t a e m o p h i l u s i n f l u e n z a e d e f e c t i v e i n r e p a i r p r o c e s s e s , M u t a t i o n R e s . , 12 ( 1 9 7 1 ) 21--28. 1 5 K l o o s , W.E., a n d P.A. P a t t e e , T r a n s d u c t i o n a n a l y s i s o f t h e h i s t i d i n e r e g i o n in S t a p h y l o c o c c u s aureus, J. G e n . M i c r o b i o l . , 3 9 ( 1 9 6 5 ) 1 9 5 - - 2 0 7 . 16 K u n g , A . H . C . , a n d B.T.O. Lee, T h e i s o l a t i o n a n d s u r v i v a l c h a r a c t e r i s t i c s o f r a d i a t i o n a n d c h e m i c a l - m u t a g e n sensitive m u t a n t s o f P s e u d o m o n a s aeruginosa, M u t a t i o n R e s . , 2 0 ( 1 9 7 3 ) 1 7 5 - - 1 9 0 . 17 L a c e y , R.W., A n t i b i o t i c r e s i s t a n c e plas~aids o f S t a p h y l o c o c c u s aureus a n d t h e i r clinical i m p o r t a n c e , B a c t e r i o l . Rev., 3 9 ( 1 9 7 5 ) 1 - - 3 2 . 18 M c E n t e e , K., J . E . Hesse a n d W. E p s t e i n , I d e n t i f i c a t i o n a n d r a d i o c h e m i c a l p u r i f i c a t i o n o f t h e recA p r o t e i n o f Escherichia coli K - 1 2 , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 3 ( 1 9 7 6 ) 3 9 7 9 - - 3 9 8 3 . 19 P a t t e e , P.A., a n d J . N . B a l d w i n , T r a n s d u c t i o n o f r e s i s t a n c e t o c h l o r t c t r a c y c l i n e a n d n o v o b i o c i n in S t a p h y l o c o c c u s aureus, J . B a c t e r i o l . , 8 2 ( 1 9 6 1 ) 8 7 5 - - 8 8 1 . 2 0 P a t t e e , P . A . , a n d J . N . B a l d w i n , T r a n s d u c t i o n o f r e s i s t a n c e t o s o m e m a c r o l l d e a n t i b i o t i c s in S t a p h y l o c o c c u s aureus, J . B a c t e r i o l . , 8 4 ( 1 9 6 2 ) 1 0 4 9 - - 1 0 5 5 . 21 Sin_ha, R . P . , a n d V . N . l y e r , I s o l a t i o n a n d s o m e d i s t i n c t i v e p r o p e r t i e s o f a n e w t y p e o f r e c o m b i n a t i o n d e f i c i e n t m u t a n t o f Bacillus subtilis, J. Mol. Biol., 7 2 ( 1 9 7 2 ) 7 1 1 - - 7 2 4 . 22 Sonstein, S.A., and J.N. Baldwin, Nature of the elimination of the penicilllnase plasmid from S t a p h y l o c o c c u s aureus b y s u r f a c e - a c t i v e a g e n t s , J. B a c t e r i o l . , 1 1 1 ( 1 9 7 2 ) 1 5 2 - - 1 5 6 . 2 3 S t o r m , P . K . , W.P.M. H o e k s t r a , P.G. de H a a n a n d C. V e r h o e f , G e n e t i c r e c o m b i n a t i o n i n Escherichia coli, IV. I s o l a t i o n a n d c h a r a c t e r i z a t i o n o f r e c o m b i n a t i o n - d e f i c i e n t m u t a n t s o f Escherichia coli K - 1 2 , M u t a t i o n Res., 1 3 ( 1 9 7 1 ) 9 - - 1 7 . 2 4 W i t k i n , E.M., M u t a t i o n - p r o o f a n d m u t a t i o n - p r o n e m o d e s o f survival in d e r i v a t i v e s o f Escherichia coli B d i f f e r i n g in s e n s i t i v i t y t o u l t r a v i o l e t light, B r o o k h a v e n S y m p . Biol., 2 0 ( 1 9 6 7 ) 1 7 - - 5 5 . 2 5 W i t k i n , E.M., T h e r o l e o f D N A r e p a i r a n d r e c o m b i n a t i o n in m u t a g e n e s i s , P r o c . XII Int. C o n g r . G e n e t . , 3 (1969) 225--246. 2 6 W i t k i n , E.M., U l t r a v i o l e t m u t a g e n e s l s a n d i n d u c i b l e D N A r e p a i r in Escherichia coli, B a c t e r l o l . R e v . , 4 0 (1976) 869--907. 27 W y m a n , L., R . V . G o e r i n g a n d R.P. N o v i c k , G e n e t i c c o n t r o l o f c h r o m o s o m a l a n d p l a s m i d r e c o m b i n a t i o n in S t a p h y l o c o c c u s aureus, G e n e t i c s , 7 6 ( 1 9 7 4 ) 6 8 1 - - 7 0 2 .
Mutation Research, 60 (1979) 279--289
© Elsevier/North-Holland Biomedical Press
MUTANTS OF S T A P I t Y L O C O C C U S A U R E U S DEFICIENT IN RECOMBINATIONAL R E P A I R IMPROVED ISOLATION BY SELECTING F O R MUTANTS EXHIBITING C O N C U R R E N T SENSITIVITY TO U L T R A V I O L E T RADIATION AND N - M E T H Y L - N ' - N I T R O - N - N I T R O S O G U A N I D I N E
R.V. GOERING Department of Medical Microbiology, Creighton University School of Medicine, Omaha, Nebraska 68178 (U.S.A.)
(Received 28 June 1978) (Revision received 22 November 1978) (Accepted 11 December 1978)
Summary Recombination-deficient (rec) mutants of S t a p h y l o c o c c u s aureus strains 152 and Ps29 were sought b y initially screening mutagenized cultures for mutants exhibiting increased sensitivity to both ultraviolet {UV) radiation and N-methyl-N'-nitro-N-nitrosoguanidine (NG). Mutants thus isolated were analyzed for recombinational ability by transduction, and further characterized in terms of sensitivity to UV, NG, ability to repair UV-irradiated bacteriophage, and spontaneous and UV-induced DNA degradation. Mutagenesis of strain 152 yielded three isolates, one of which was rec, the second potentially lex, and the third possessing an undetermined repair deficiency. Mutagenesis of strain Ps29 resulted in the isolation of one mutant, which exhibited a rec genotype. In searching for rec mutants of S. aureus, the value of initially screening mutagenized cultures for mutants exhibiting concurrent sensitivity to UV and NG, as opposed to screening for UV sensitivity alone, is discussed. .
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Several investigators have recently reported the isolation of mutants of S t a p h y l o c o c c u s aureus deficient in the recombinational repair of DNA [8,12, 27]. Such recombination-deficient (rec) mutants are of increasing importance
to staphylococcal research including studies of DNA repair and efforts to determine the plasmid vs. chromosomal location of specific staphylococcal genes, especially those concerning antibiotic resistance [17]. In each of the previous reports, isolation of rec mutants of S. aureus followed a similar protocol: mutagenized cells were initially screened for sensitivity to ultraviolet radiation
280 (UV), with UV-sensitive isolates then examined for impaired recombination ability. The difficulty with this isolation procedure, however, is the fact that UV sensitivity is associated with a multitude of deficiencies in DNA repair (e.g., see ref. [4]). Thus, a mutagenesis procedure producing numerous UV-sensitive isolates may yield few, if any, rec mutants. Mutants deficient in post-replication repair (including rec mutants) are known to be highly susceptible to inactivation by the monofunctional alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (NG), while wild-type strains and mutants deficient in excision repair are comparatively insensitive to NG [6,9,14,16,24,25]. Accordingly, an improved procedure for the isolation of rec mutants of S. aureus is reported here, based on the initial screening of mutagenized cultures for isolates exhibiting concurrent sensitivity to UV and NG. Materials and methods Bacterial strains
For mutagenesis experiments, S. aureus strain Ps29, of the International Typing Series, and strain 152 [20] were used. Donor strains employed in transduction experiments were: S. aureus U9 (pase, tet, ol, nov) [19] which carries plasmid-linked genes for penicillinase production (pase marker) and tetracycline resistance (tet marker) [8,22], and chromosomal genes for resistance to oleandomycin (ol marker) and novobiocin (nov marker) [3,8]; and S. aureus 655CT-N which carries a plasmid-linked pase marker and a chromosomal nov marker [1,11]. Additional strains, included in DNA degradation experiments, were S. aureus 112 and 112 rec-2 [8,27] and S. aureus 8325-4 and 8325-4 his-7 recAl [27]. All cultures were maintained at 4°C on Brain Heart Infusion (BHI; Difco) agar slants. Bacteriophages and transductions
Phages 29, 80 and 83 of the International Typing Series were used for transduction and host-cell reactivation experiments. Procedures for phage maintenance, propagation, and titration were those of Pattee and Baldwin [19]. Transductions were performed by the method of Kloos and Pattee [15] with the addition of 10 -3 M CaC12 to the transduction suspension. Selective media for the antibiotic-resistance markers was prepared as described by Pattee and Baldwin [ 19,20]. Preparation o f solid m e d i u m containing N G
To directly screen mutagenized cells for NG sensitivity, a solid medium containing NG was prepared. Trypticase Soy Agar (TSA; Baltimore Biological Laboratory) was prepared at pH 6.0 by rehydrating in 0.1 M KPO4 (pH 6.0), and sterilized by autoclaving. The medium was cooled to 47°C and, immediately prior to dispensing, NG {Aldrich Chem. Co.) was freshly dissolved in 0.1 M KPO4 (pH 6.0), filter-sterilized, and added to the medium at a final concentration of 10 pg/ml. Plates of TSA containing NG (designated TSNG agar) were always used on the day of preparation. Isolation o f m u t a n t s sensitive to U V and N G S. aureus strains 152 and PS29 were mutagenized with NG (conc. = 500 pg/
281 ml) and plated on BHI agar as described previously [8]. Colonies appearing after overnight incubation at 37°C were screened by replica plating to sets of two BHI agar plates (one to receive 34 J/m s UV radiation, the other serving as an unirradiated control}, and a plate of TSNG agar. Colonies which appeared sensitive to both UV and NG (i.e., growth absent or extremely poor on UV irradiated and TSNG plates} were suspended in 0;5-ml volumes of saline (0.5% NaC1) and secondarily screened by spotting onto sets of four BHI agar plates (to receive UV dosages of 0, 8, 18 or 34 J/m 2) and a plate of TSNG agar. Only isolates exhibiting no growth on plates receiving the higher UV exposures and on TSNG agar were retained for further study.
UV irradiation o f bacteria and bacteriophages Methods for the UV irradiation of bacteria and bacteriophages have been previously described [8]. Quantitative determination o f NG sensitivity The NG sensitivity of cultures was quantitatively determined in citrate solution (0.85% trisodium citrate, pH 5.0} as described previously [8]. Measurement o f post-irradiation DNA degradation Strains to be examined were grown overnight in BHI at 37°C and diluted 1 : 15 in fresh BHI containing 10 ttCi of [3H]methylthymidine/ml (spec. act. = 50 Ci/mmole) and 50 pg of deoxyadenosine/ml. After at least t w o generations of growth, cells were harvested by centrifugation, washed 2× in saline, and resuspended in BHI for an additional 1 h incubation to reduce the radioactivity in the acid-soluble pool. After an additional saline wash, cells were resuspended in saline for UV irradiation. DNA degradation was determined by diluting UVirradiated or control cultures 1 : 10 in BHI supplemented with 100 pg thymidine/ml and incubating with shaking for 3 h at 37 °C. At appropriate intervals, 0.5-ml aliquots of cell suspension were mixed with 1.0 ml of cold 10% (w/v) trichloroacetic acid (TCA) and held at 0°C for at least 2 h. Bovine serum albumin (0.1 ml of a 1.0% [w/v] solution} was then added as a carrier to each sample, followed by centrifugation at 6 0 0 0 g for 15 min. Supernatant fluid (0.5 ml) was transferred to 10 ml of liquid-scintillation cocktail (Biofluor, New England Nuclear) for counting. The radioactivity equivalent to 100% of total counts was determined by hydrolysis of 0.5-ml aliquots of cell suspension in 1.0 ml of 10% TCA at 85°C for 1 h. The samples were then chilled, centrifuged with bovine serum albumin as above, and 0.5 ml of supernatant fluid was counted in 10 ml of Biofluor. All counts were normalized by external quench correction. Isolation o f revertants resistant to NG Mutants sensitive to NG were grown overnight in BHI and ca. l 0 T to l 0 s cells were plated directly on TSNG agar. Colonies appearing after overnight incubation at 37°C were purified on TSNG agar and transferred to BHI agar slants for further study.
282 Results T r a n s d u c t i o n o f a n t i b i o t i c resistance Following mutagenesis of S. aureus strain 152, only three isolates out of
4840 colonies exhibited consistent sensitivity to both UV and NG through primary and secondary screening procedures. With S. aureus Ps29, only one isolate out of 2320 colonies was consistently sensitive to UV and NG. To assess the recombinational ability of these mutants, transduction frequencies were determined for known plasmid and chromosomal markers. Table 1 summarizes the results obtained when strain 152 and its mutants were transduced using phage 83 propagated on S. aureus 655CT-N, which is known to carry a plasmid-linked pase marker and a chromosomal n o v marker [1,11]. Of the three mutants, UVR-60 demonstrated the greatest reduction in recombinational ability as evidenced by its total inability to act as a recipient of the chromosomal nov marker. UVR-18 appeared somewhat reduced in recombinational ability {i.e., transduction frequency for the chromosomal nov marker 9% that of strain 152) but not as recombination-deficient as UVR-60. Mutant UVR-22 exhibited a transduction frequency for the nov marker indicative of wild-type recombinational ability. All three mutants were similar in poorly expressing the plasmid-linked pase marker. Table 2 indicates the results obtained when strain Ps29 and its mutant, UVR11, were transduced using phage 80 propagated on S. aureus U9 (pase, tet, ol, n o v ) which carries plasmid-linked pase and tet markers and a chromosomal nov marker [8,22]. As with the mutants of strain 152, UVR-11 poorly expressed plasmid-linked markers (pase and tet), however, the m u t a n t did exhibit a recombination deficiency as evidenced by its inability to act as a recipient of the chromosomal n o v marker. On the basis of reduced recombinational ability {Tables i and 2), mutants 152 UVR-60 and Ps29 UVR-11 were designated 152 rec-3 and Ps29 rec-4, respectively, throughout the remainder of this study. S e n s i t i v i t y to U V a n d N G
While the mutants examined in this study were all isolated on the basis of a qualitative sensitivity to UV and NG, a more quantitative measurement of TABLE 1 TRANSDUCTION FREQUENCIES OF THE nov AND pase MARKERS S T R A I N 152 A N D T H R E E M U T A N T S U S I N G P H A G E 8 3 / 6 5 5 C T - N Recipient
IN S t a p h y l o c o c c u s
aureus
Frequency of transduction a pase
nov
152 UVR-18 UVR-22
1520 (100) 280 (18) 160 (10)
1120 (100) 100 (9) 1480 (132)
UVR-60
187 (12)
0 (0)
(rec.3 )
a E x p r e s s e d as n u m b e r of transductant$ p e r 109 p l a q u e - f o r m i n g u n i t s . N u m b e r s in parentheses are relative t r a n s d u c t i o n f r e q u e n c i e s c a l c u l a t e d u s i n g t h e f r e q u e n c y o f 1 5 2 as 100%.
283 TABLE 2 T R A N S D U C T I O N F R E Q U E N C I E S OF T H E nov, tet ANDpase M A R K E R S IN S t a p l t y l o c o c c u s Ps29 A N D A M U T A N T D E R I V A T I V E U S I N G P H A G E 8 0 / U 9 (pose, tet, ol, n o v ) Recipient
aureus
Frequency of transduction a pase
no v
t et
3140 (I00) 440 (14)
1840 (I00) 0 (0)
28200 (i00) 3800 (13)
i
Ps29 UVR-I 1 (rec-4)
a E x p r e s s e d as n u m b e r o f t r a n s d u c t a n t s p e r 1 0 9 p l a q u e - f o r m i n g units. N u m b e r s in p a r e n t h e s e s are relative t r a n s d u c t i o n f r e q u e n c i e s c a l c u l a t e d u s i n g t h e f r e q u e n c y o f P s 2 9 as 100%.
these sensitivities was desired. When compared on the basis of the UV fluence required to achieve e-' inactivation, rec-3, UVR-22, and UVR-18 were, respectively, 13X, 7X, and 2X more sensitive to UV than strain 152 (Fig. 1A). A similar comparison of the NG inactivation curves of the mutants (Fig. 1B) revealed UVR-22 and rec-3 to be 18X more sensitive to NG than strain 152. Mutant UVR-18 was 7X more sensitive to NG than the parent strain. A similar analysis of the inactivation kinetics of strain PS29 and rec-4 revealed rec-4 to be 14X more sensitive to UV (Fig. 2A) and l l X more sensitive to NG (Fig. 2B) than the parent strain Ps29.
Repair of UV-irradiated phage The ability of the parent and mutant strains to repair (i.e., host-cell reactivate; HCR) UV-irradiated phage was examined. Following UV exposure, titers of viable phage did not differ significantly for phage 80 when plated on either strain 152 or its mutants (Fig. 3A), or for phage 29 when plated on either Ps29 or rec-4 (Fig. 3B).
IOC
A -
B
-
i
.J
~0
O0
-
0.00
FLUENCE(~m')
MINUTES NG EXPO~JRE
Fig. 1. Survival c u r v e s f o r S. a u r e u s strain 1 5 2 a n d t h r e e m u t a n t U V ( A ) or a 1 0 0 # g / m l c o n c e n t r a t i o n o f N G ( B ) .
derivatives w h e n e x p o s e d
either to
284 IOC
A ~29
\ \
ae 0
\
k%. \ OOI
\
\ ~LVR.I
000
,\
,\
OOCW
FLUENCE[Jim')
MINUTES NG EXPOSUI~E
Fig. 2. S u r v i v a l curves for S. a u r e u s strain Ps29 and one mutant derivative when exposed either to U V ( A ) or a 100 ~g/ml concentration of NG(B).
Spontaneous and UV-induced DNA degradation The parent and mutant strains were examined for DNA degradation occurring either following UV exposure or in unirradiated control cultures. When compared to strain 152 (Fig. 4) mutants UVR-22 and rec-3 exhibited significant spontaneous DNA degradaLion (12% and 10% acid-soluble radioactivity, repectively) after 3 h incubation. Following UV exposure, UVR-22 and rec-3 exhibited DNA degradation (38% and 40%, respectively) significantly greater than that of strain 152 {11%). The UV-induced DNA degradation of UVR-18 (13%) did not differ significantly from that of 152. A comparison of strains Ps29 and rec-4 (Fig. 5) revealed 7% spontaneous
I(}C
A
B
UVR-18 UVR -22
UVR-~O
(nee-3)
~
I0 --I
UVR-H (,ec 4)
>
FLUENCE (~/m')
F i g . 3 . H o s t - c e l l r e a c t i v a t i o n o f U V - i r r a d i a t e d p h a g e 8 0 p l a t e d o n S. a u r e u s s t r a i n 1 5 2 a n d t h r e e m u t a n t d e r i v a t i v e s ( A ) o r p h a g e 2 9 P l a t e d o n S. a u r e u s s t r a i n P s 2 9 a n d o n e m u t a n t d e r i v a t i v e ( B ) .
285 152 U V R - I 8
152 80
60
40 z
o ,,,:I
,,, t~
20
0 152 UVR-60 (rec-3)
152 U V R - 2 2
z
o
80
60
40.
0 I
2
3
I
2
3
HOURS INCUBATION
Fig. 4. D N A d e g r a d a t i o n in S. a u r e u s strain 1 5 2 a n d t h r e e m u t a n t d e r i v a t i v e s w h e n e i t h e r u n i r r a d i a t e d (o) o r a f t e r 27 J / m 2 U V e x p o s u r e ( e ) .
DNA degradation for rec-4 after 3 h incubation compared to <1% spontaneous DNA degradation in strain Ps29. After UV exposure, incubation of rec-4 resulted in 36% DNA degradation compared to 6% in Ps29.
29 U V R - I I (rec-4)
29 80 1-
60 rr tU Q .¢ 40 Z 0
N
2O
I
2
13
I
2
3
HOURS INCUBATION
Fig. 5. D N A d e g r a d a t i o n in S. a u r e u s strain Ps29 a n d o n e m u t a n t d e r i v a t i v e w h e n e i t h e r u n i r r a d i a t e d (o) o r a f t e r 27 J / m 2 U V e x p o s u r e (o).
286 Of the previous reports of rec mutants in S. aureus [8,12,27] only one presents data on DNA degradation [ 12]. To allow a more thorough comparison of the mutants reported here with the rec mutants previously isolated in S. aureus, Fig. 6 indicates the spontaneous and UV-induced DNA degradation observed in the rec mutants of S. aureus (and their parent strains) previously uncharacterized in terms of DNA degradation. Strain 112 rec-2 exhibited 5% spontaneous DNA degradation. After UV exposure, DNA degradation in rec-2 increased to 34%, compared to only 5% for strain 112. Strain 8325-4 his-7 f e c A l exhibited 5% spontaneous DNA degradation. Following UV irradiation, r e c A l exhibited 55% DNA degradation compared to 9% for strain 8325-4. Characteristics o f revertants resistent to N G
To assess whether single or multiple mutational lesions were responsible for the pleiotrophic characteristics of rec-3 and rec-4, NG-resistant revertants of the mutants (termed NGR-3 and NGR-4, respectively) were selected on TSNG agar. These revertants were then compared phenotypically with their respective
112 rec-2
112 80
60
40 z 0
C- 2O rY ~D
w
A
C
o
8325-4
8325-4
recAI
his-7
z
'-' 80
60
40
20
I
2
3 HOURS
I
2
3
~NCUBATION
F i g . 6. D N A d e g r a d a t i o n i n S . a u r e u s s t r a i n 1 1 2 , 8 3 2 5 - 4 , a n d t h e i r r e c m u t a n t i r r a d i a t e d (~)) o r a f t e r 27 J / m 2 U V e x p o s u r e ( $ ) .
derivatives when either un
287 TABLE 3 C H A R A C T E R I S T I C S a OF M U T A N T S rec.3, rec-4 A N D T H E I R N G - R E S I S T A N T R E V E R T A N T S Characteristic
Strain 152 (wild-type)
rec-3
NGR-3
Ps29 (wild-type)
rec-4
NGR-4
UV sensitivity
1.0
13
1.0
1.0
14
1.0
Transduction ability
1.0
0
1.6
1.0
0
1.4
DNA d e g r a d a t i o n spontaneous
1.0
I0
1.0
1.0
14
1.0
UV-induced
J .0
1.3
1.0
3.6
6.0
1.1
a UV sensitivity of strains is e x p r e s s e d as t h e e -! f l u e n e e f o r w i l d - t y p e d i v i d e d b y t h e e -1 fluence for r e c or N G R derivatives. T r a n s d u c t i o n ability is t r a n s d u e t i o n f r e q u e n c y o f the c h r o m o s o m a l n o v m a r k e r in each strain d i v i d e d b y t h e t r a n s d u c t i o n f r e q u e n c y o f n o v in the w i l d - t y p e . D N A d e g r a d a t i o n is percent D N A d e g r a d a t i o n in each s t r a i n d i v i d e d b y p e r c e n t D N A d e g r a d a t i o n in the w i l d - t y p e .
mutant and wild-type parent strains {Table 3). With both rec-3 and rec-4, reversion to NG resistance resulted in the restoration of a wild-type phenotype, thus indicative of a single mutation responsible for the p h e n o t y p e of the mutants. Discussion Ideally, a selection procedure for isolating rec mutants should directly screen mutagenized cells for recombination deficiency. This is best accomplished in bacteria with potential for conjugation, as in the method of Clark and Margulies [5] where cells are directly examined for recombinational ability by replica plating to lawns of Hfr cells. At present, transduction and transformation are the only known means of genetic transfer in S. a u r e u s , neither of which lends itself well to the above t y p e of assay procedure. Therefore, rec mutants of S. a u r e u s have been isolated in the past initially on the basis of UV sensitivity. Primary selection of isolates solely on the basis of UV sensitivity, however, commonly results in a poor yield of rec mutants [8,27], often favoring, instread, mutants deficient in excision repair (e.g., see ref. [8] ). Excision-deficient mutants, however, do not exhibit increased sensitivity to NG [ 14,16,25], a characteristic which is associated with rec mutations [6,9,14,16,24,25]. Therefore, primary screening of mutagenized cultures for isolates exhibiting concurrent sensitivity to UV and NG reduces the population of isolates to be examined while enriching for mutants of the rec genotype. This enrichment, however, is clearly for the predominant r e c A , B and C genotypes (e.g., as in E s c h e r i c h i a coli), and not other recombination deficiencies which might be relatively less sensitive to UV and/or NG [21,23]. It is well established that sensitivity to NG is not associated exclusively with recombination deficiency (e.g. l e x mutants are also known to be sensitive to NG [25]). However, this study suggests that, at least with the S. a u r e u s strains and mutagenesis protocal employed here, rec NG-sensitive mutations occur at frequencies similar to that for rec ÷ NG-sensitive mutations, since of the four mutants reported here, two were of the rec genotype.
288 As predicted by the selection procedure, all four mutants isolated in this study were clearly more sensitive to both UV and NG than their parent strains (Figs. 1, 2), although actual sensitivities varied somewhat among the isolates. In addition, all of the mutants were comparable to their parent strains in ability to repair UV-irradiated phage (i.e. HCR*; Fig. 3) and thus did not appear to be dificient in excision repair, which has been shown to relate to HCR ability [2,10]. On the basis of analysis by transduction, however, only two of the mutants were clearly recombination-deficient. Mutants 152 rec-3 and Ps29 rec-4 were both unable to act as recipients in the transduction of a chromosomal nov marker while expressing plasmid-linked antibiotic-resistance markers. These mutants also exhibited significant spontaneous and UV-induced DNA degradation, similar to the previously isolated rec mutants of S. aureus (Fig. 6), all suggestive of a recA genotype [9]. The restoration of a wild-type phenotype coincident with reversion to NG resistance in both rec-3 and rec-4 supports the hypothesis that the recombination-deficient phenotype of each mutant is the result of a single mutation (i.e. single site or point mutation). A similar relationship in recombination-deficient mutants of S a l m o n e l l a t y p h i m u r i u m has been described and discussed by Eisenstark et al. [6]. Mutant 152 UVR-22 demonstrated wild-type recombinational ability in transduction of the chromosomal nov marker, but exhibited elevated levels of spontaneous and UV-induced DNA degradation. This combination of characteristics suggests that UVR-22 may possess a l e x A - t y p e mutation [26], however, additional characterization of this m u t a n t (e.g., to determine whether recA protein [7,18] is produced) is required for definitive assignment of a genotype. Mutant 152 UVR-18 was a poor transduction recipient of the chromosomal nov marker. This m u t a n t was the least sensitive to UV and NG of any isolate and did not exhibit kinetics of DNA degradation significantly different from that of the parent strain, characteristics which appear similar to those of recB, C mutants in E. coli [9]. However, the current lack of information regarding DNA repair in S. aureus suggests a need for further study of this m u t a n t prior to ascribing a potential repair deficiency. Studies to further characterize all of the mutants reported here, particularly in terms of ability to produce recA protein [7,18], are currently in progress. In general, the data presented here indicate that m u t a n t selection based on concurrent sensitivity to UV and NG can be a means of significantly improving the isolation of rec mutants and other repair-deficient genotypes which are relatively u n c o m m o n , at least in S. aureus. Such mutants promise to be of considerable value in leading to a more thorough understanding of the genetics of this organism. Acknowledgements The technical assistance of Elaine Blythe is gratefully acknowledged. This investigation was supported in part by grants from Research Corporation and the Nebraska Heart Association.
289
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