Molec. gen. Genet. 150, 2 0 5 - 2 0 9 (1977) © by Springer-Verlag 1977

Isolation and Characterization of Catalase Deficient Mutants of Salmonella typhimurium Steven A. Levine Dept. of Nutritional Sciences, Graduate Program in Genetics, University of California, Berkeley, California 94720, U S A

Summary. Catalase deficient mutants (kat) of Salmonella typhimurium have been isolated. The mutations katA1, katC6 and katD9 appear to map at about minute 10 on the Sahnonella chromosome. The katC6 and katD9 lesions are complemented by the E. coli F'128 (lac+ pro+) episome but the katA1 lesion is not. KatB2 maps at about minute 100. None of the mutants are oxygen sensitive; t h e y grow as well as wild type bacteria, even when aerated.

Introduction In 1893 Gottstein's publication made bacterial catalase one of the first bacterial enzymes to be described. Catalase from Micrococcus lysodeikticus was the first bacterial enzyme to be crystallized (Herbert and Pinset, 1948). The Micrococcus catalase resembles the mammalian liver catalase in having a molecular weight of about 250,000 Daltons and in containing one heine per 60,000 Daltons of protein. The catalase of Rhodopseudomonas spheroides has also been purified, and its molecular weight, catalytic activity, and spectral properties are very similar to those of the Micrococcus enzyme (Clayton, 1959). The physiological role of bacterial catalase has not been rigorously demonstrated. The enzyme is present in most aerobic bacteria, suggesting that it pi'otects the cells from oxidation by hydrogen peroxide (Deisseroth and Dounce, 1970). The recent discoveries of superoxide as the reactive intermediate formed in the autoxidation of flavins and of the correlation between the superoxide dismutase activity of bacteria and their ability to tolerate aerobic conditions suggests that superoxide dismutase may be the critical enzyme in determining the oxygen sensitivity of bacteria and that catalase serves some secondary role (McCord, Keele, and Fridovich, 1971).

To study the physiological role of catalase in the enteric bacterium Salmonella typhimurium mutants lacking catalase activity have been isolated and genetically characterized.

Materials and Methods Bacterial Strains. All strains used were derived from Salmonella typhimurium LT-2. Their characteristics are listed in Table 1. Media. Minimal glucose medium was the E m e d i u m (Vogel and Bonner, 1956) supplemented with 1% glucose. Minimal lactose and minimal glycerol media contained 1 g K2SO4, 13.5 g K 2 H P O , , 4.7 g KHEPO4, 0.1 g MgSO4-7 H20, 0.54 g NH4C1, and 10 g lactose or 16 g glycerol, respectively, per liter. Complete m e d i u m was 0.8% nutrient broth (Difco) and 0.4% NaC1. The corresponding solid media were solidified with 2% agar. All cultures were incubated at 37 C. Liquid cultures were shaken at 200 rpm an an incubator shaker (New Brunswick Sci. Co., New Brunswick, N.J.). Sodium dodecyl sulfate was purchased from General Biochemicals, Chagrin Falls, Ohio. Mutagenesis. Bacteria were grown to logarithmic phase (about 2 x 108/ml) in complete medium, collected on 0.4 la Nucleopore ® filters (Nucleopore Corp., Pleasanton, Calif.), washed, and resuspended in potassium phosphate (0.05 M, p H 7.0) buffer containing N-methyl-N'-nitro-N-nitrosoguanidine (100 ~tg/ml). The suspension was incubated at 37 C for 15 min and then filtered and washed. The cells were-then resuspended in 10 ml of complete medium and grown overnight. Isolation of Catalase Deficient Mutants. Mutagen treated cells were grown on complete medium. To identify catalase deficient mutants, an inoculating needle (Nichrome, 32 ga) was dipped into a 30% H 2 0 z solution and touched to the edge of each colony. Catalasedeficient m u t a n t s fail to evolve bubbles of oxygen gas from the HzOz. (The evolution of oxygen from the destruction of HeO z is a unique characteristic of the catalase reaction.) These mutants were restreaked on plates of complete medium and isolated colomes were retested for catalase activity. Catalase Determination. Quantitative catalase activities of cell suspensions were measured by a modification of the method of Beers and Sizer (1952). Bacterial cells were washed on 0.4 rt Nucleopore®

206

S.A. Levine: Kat M u t a n t s of Salmonella typhimurium

Table 1. Bacterial strains of Salmonella typhimurium Strain

Genotype a

Sex

LT2 SA534 SA535 SA950 SB6014 TC22

Wild type, prototrophic HfrK4 serA13 HfrK5 serA!3

F Hfr Hfr FFF+

gal-50 hisD23 metC30 his-lOl4 argEll6 F'128 (argF + proB + proA + lac+)/ leu-197 trpB4 met-365 sup-49 HfrK5 trp-998 katA1 katB2 HfrK5 katC6 trp-998 HfrK5 katC9 trp-998 pro-621 his OGCBHAF644 HfrK5 trp-998 serA13

TC37 TC55 TC61 TC67 b TC70 b TR172 TR1150 a

b

Comments

clockwise transfer from minute 137 clockwise transfer from minute 66

Hfr

FHfr Hfr

Catalase-deficient Catalase-deficient Catalase-deficient Catalase-deficient

derivative derivative derivative derivative

of of of of

LT-2 LT-2 TC37 TC37

FHfr

Except for kat, these genetic markers and their properties are described by Sanderson and Demerec (1965) Stains TC67 and TC70 were obtained from independent mutagen-treated cultures of TC37

Table 2. N u m t i o n a l responses of auxotrophic derivatives of TC55 katAl and TC61 katB2

Strain

Genotype a

Substances utilized b

Substance not utilized b

Additional comments

TC56 TC57

katA1 cysE2461

Cysteine, Na2S203, CaS

NaHSO3

Interrupted mating suggests cysE

katA1 pro-1401

Proline

Ornithine

Complemented by F'128

TC58

katAI cysA2462

Cysteine, NaHSO3, CaS

NazS20 3

Confirmed by transduction and interrupted mating a

TC59

katA1 leu-1911

Leucine

Isoleucine, valine

Interrupted mating

TC60

katA1 ilvA1831

Isoleucine

Leucine, valine

Interrupted mating results are consistent with ilvA

TC62

katB2 guaB371

Guanine, xanthine

Hypoxanthine, adenine

TC63

katB2 metC1621

Methionine, homocysteine

Vit. B12, cystathionine homoserine, O-succinyl homoserine

TC64

katB2 cys-2464

Cysteine, NaHSO3, Na2S203, CaS

TC65

katB2 cysG2463

Cysteine, Na2S203, CaS

a u c

Confirmed by transduction a May be cysC, D, or H

NaHSO3

Confirmed by transduction c

The identities of the m u t a n t loci were inferred from the nutritional data summarized here and mating kinetics (data not shown) The substances utilized would permit the strains to grow on minimal glucose medium Transductions and reciprocal transductions were performed using these and a variety of other cys mutants, including cysA20 and

cysG a

Transductions and reciprocal transductions were performed between TC63 and a variety of met- strains, including TC48 (metC30)

filters and resuspended in potassium phosphate (0.05 M, p H 7.0) buffer. W h e n necessary, the cell suspensions were diluted with the same buffer. 1.9 mi of the cell suspension and 0.1 ml of a 0.5 M H 2 0 2 solution were mixed in a spectrophotometer equipped with a Beckman D U monochromator. Cell concentrations were calculated from the relationship: Cell concentration (mg dry cells/ m l ) = 0 . 4 5 x optical density of cell suspension at 650 nm. Catalase specific activities were expressed as umoles of HzO2 decomposed per min per mg of dry cells.

Introduction and Characterization of Genetic Markers. A second mutagenesis was performed to induce nutrient requirements in strain TC55 (kat-i) and TC61 (kat-2). The nutrient requirements were characterized by nutritional and genetic experiments the re-

sults o f which are summarized in Table 2. The m a p positions of the nutrient requirements were determined in interrupted mating by comparing the number of recombinants obtained from the appropriate matings to the number of recombinants obtained from mating with stains containing known auxotrophic markers. Strains c o n t a i n i n g mutations leu-1911 and pro-1401 were mated with H f r K 4 (SA534) which has its point of origin at 137 min and transfers its c h r o m o s o m e in a clockwise direction. The n u m b e r of recombinants obtained were compared to the n u m b e r of recombinants obtained from similar matings with pyrA579 which is known to be at 2 min. Strains containing mutations cysA2462 and ilvA1831 were mated with H F r K 5 (SA535) whose point of origin is at 66 min and transfers in a clockwise direction. The aroD5 mutation, at 73 min, was the known marker in these matings.

207

S.A. Levine: Kat Mutants of Salmonella typhtmurium

Table 4. Mapping of katA1 by interrupted conjugation with TC37 (HfrK5 trp-998)

Results Isolation o f Catalase-deficient M u t a n t s . A b o u t 5000 clones of bacteria f r o m a m u t a t e d culture of S a l m o n e l la LT-2 were tested for catalase activity a n d 5 stable catalase-deficient m u t a n t s were obtained. These m u tants were designated kat-1, kat-2, kat-3, kat-4, a n d kat-5. A d d i t i o n a l catalase m u t a t i o n s were i n d u c e d in strain TC37 ( H f r K 5 trp-998). A b o u t 2000 m u t a t e d clones were e x a m i n e d for catalase activity a n d two stable catalase-deficient m u t a n t s were isolated from i n d e p e n d e n t l y m u t a t e d cultures. These m u t a n t s were n a m e d TC67 ( H f r K 5 trp-998 kat-6) a n d TC70 (HfrK5 trp-998 kat-9). Genetic A n a l y s i s o f kat-1 a n d kat-2. I n u n i n t e r r u p t e d c o n j u g a t i o n experiments with TC37 a n d a u x o t r o p h s of kat-1 t h e kat-1 allele a p p e a r e d to m a p close to the pro region (Table 3). L i n k a g e of kat-1 with the pro locus was higher t h a n linkage with a n y of the other loci tested. This c o n c l u s i o n was s u p p o r t e d by i n t e r r u p t e d c o n j u g a t i o n experiments (Table 4), which show linkage between kat-1 a n d pro-1401 at m i n u t e 10, b u t n o t between k a t - 1 a n d c y s A (rain. 76) or c y s E (min. 116). A u x o t r o p h s of k a t - 2 were m a t e d with 2 different H f r K 5 strains. The results are s u m m a r i z e d in Table 5. There is a clear linkage between k a t - 2 a n d m e t C 1 6 2 1 at m i n u t e 100 o n the c h r o m o s o m e . The k a t - 2 allele appears to be between m e t C (min. 100) a n d s e r A (min. 95). W h e n the d o n o r strain T R l 1 5 0 ( H f r K 5 s e r A 1 3 trp-998 was used, the linkage of k a t + to m e t C + was reduced b u t n o t abolished, T a b l e 5. Since these experiments were p e r f o r m e d o n m i n i m a l m e d i u m the s e r A locus of the d o n o r was selected against a n d few r e c o m b i n a n t colonies had genetic material f r o m the s e r A region of the donor. If the k a t - 2 allele were located before or very close

katSelecteddonor recipient markera

Time of interruption (min)

Frequency of kat + recombinantsb

TC58

cysA + (76')

20 26 34 71

0% 0% 0% 0%

(12) (12) (12) (12)

TC56

eysE + (116')

40 52 68 83

0% 0% 0% 0%

(12) (18) (18) (18)

TC57

pro + (10')

81 91 i00 115

0% 14% 8% 33%

(3) (6) (12) (12)

The numbers in parentheses are the locations of the selected markers on the Salmonella chromosome b The numbers in parentheses are the number of recombinant clones which were isolated and assayed

Table 5. Mapping of katB2 by uninterrupted conjugation Donor

kat recipient

Selected donor marker"

Frequency of kat + recombinantsb

TC37 (HfrK5 trp-998)

TC66 TC62 TC64 TC63 TC65

cysA + (76') guaB+ (77') cysC + (90') metC + (100') cysG + (108")

5% 2% 24% 95% 54%

(96) (143) (72) (20) (139)

TRl150 (HfrK5 serA13 trp-998)

TC66 TC62 TC64 TC63 TC65

eysA + (76') guaB + (7T) cysC + (90') metC + (i00 ~) cysG + (108')

4% 0% 0% 42% 13%

(25) (25) (72) (42) (37)

a The numbers in parentheses are the locatlons of the selected markers on the Salmonella chromosome b The numbers in parentheses are the number of recombinant clones which were isolated and assayed

Table 3. Mapping of katAl by uninterrupted conjugation with TC37 (HfrK5" oT-998 ) kat

recipient

TC58 TC60 TC59 TC57

Selecteddonor markera

Frequency of kat + recombinantsb

cysA + (76') ilvA + (i22') /eu+ (3') pro + (i0')

2% 52% 61% 88%

(45) (50) (46) (66)

The numbers in parentheses are the locations of the selected markers on the Salmonella chromosome b The numbers in parentheses are the number of recombinant clones which were isolated and assayed c HfrK5 transfers its chromosome clockwise from an origin at 66 rain

a

to s e r A at m i n u t e 95, very few p r o t o t r o p h i c recombin a n t s w o u l d inherit the d o n o r k a t + allele a n d thus most of the colonies w o u l d have been catalase-deficient. T r a n s d u c t i o n was p e r f o r m e d using phage P 22 int4 grown o n wild type cells a n d different catalasedeficient a u x o t r o p h s as recipients. The resulting prototrophic t r a n s d u c t a n t s were scored for c o i n h e r i t a n c e of the k a t character. Phage were reciprocally grown o n TC55 (kat-1) a n d strains T R 1 7 2 a n d p r o A B 4 7 were used as recipients. The kat-1 was n o t readily cotransducible with either leu or pro (Table 6).

208

S.A. Levine: Kat Mutants of Salmonella typhimurium

1£

2.0 1.0

0.1(

0.10

0.02 0010

]0.02 11)0'01

Table 6. Cotransduction of catalase mutations

2.C

Traus- Reciducpient tional donor a

Selected marker

Unselected marker

Transductants tested

% cotransduction

TC61 TC61 ' TC55 TC55 LT2 LT2 TC67 TC67 TC70 TC70

met + arg+ pro + pro + leu+ pro + pro + pro + pro + pro +

katB2 katB2 katA1 katA1 kat + kat + katC6 katC6 katD9 katD9

837 166 100 65 100 192 128 7 180 100

0 0 0 0 0 0 27 0 0 0

E C

SA950 SB6014 proAB47 TR172 TC59 TC57 proAB47 TR172 proAB47 TR172

" Reciprocal transductions were not possible because the kat + marker is not easily selectable

Since the kat-1 allele is in the Pr01ine r e g i o n o f the c h r o m o s o m e I i n t r o d u c e d the E. coli F ' 1 2 8 (pro + lac +) e p i s o m e into strain TC57 (kat-1 pro-1401) by m a t i n g it w i t h the F ' 1 2 8 (lac ~ pro +) d o n o r strain T C 2 2 on plates o f m i n i m a l lactose m e d i u m . T h e catalase-deficient recipient c a n n o t utilize lactose, since it is a Salmonella a n d it c a n n o t synthesize proline, since it is a p r o - m u t a n t . I n o r d e r to g r o w on m i n i m a l lactose m e d i u m it m u s t receive the F ' 1 2 8 (lac + pro +) episome. L a c t o s e positive clones (25) were deficient in catalase. W h i l e the kat-1 lesion is p r o b a b l y in the pro r e g i o n o f the c h r o m o s o m e , it is n o t c o m p l e m e n t e d b y F'128. W e call this locus k a t A , a n d thus we call the kat-1 allele katA1. T h e kat-2 allele was n o t c o t r a n s d u c i b l e with either the m e t C locus or the argE locus (Table 6). I call this locus k a t B a n d the kat-2 alleie katB2. Genetic Analysis o f kat-6 and kat-9. P r e l i m i n a r y conj u g a t i o n e x p e r i m e n t s using the c a t a l a s e - d e f i c i e n t don o r strains T C 6 7 ( H f r K 5 kat-6) a n d TC70 ( H f r K 5 kat-9) i n d i c a t e d t h a t b o t h kat-6 a n d kat-9 alleles were close to the p r o l i n e r e g i o n o f the c h r o m o s o m e . B o t h alleles are c o m p l i m e n t e d b y the E. coli e p i s o m e F ' 128. The e p i s o m e e x p e r i m e n t s i n v o l v e d the i s o l a t i o n o f F ~ segregants o f TC67 a n d TC70, the i n t r o d u c t i o n o f F ' 1 2 8 , a n d finally the curing o f the newly c o n s t r u c t e d e p i s o m e strains. T h e e x p e r i m e n t a l details are as follows: Cultures o f TC67 a n d T C 7 0 w h i c h h a d g r o w n o v e r n i g h t in c o m p l e t e m e d i u m a n d t h e n s t o r e d at 5 ° C for 4 weeks were s t r e a k e d on petri dishes o f c o m p l e t e m e d i u m . F - segregant clones were identified b y their sensitivity to SP-6, a p h a g e specific for F Salmonella. I n two s e p a r a t e experiments, a t o t a l o f 3 F segregants were

tD

(5

+ Time, hours

Fig. la-d. Growth curves in nutrient broth media, a ( o - - o ) control strain LT2; (o o) katA1 mutant; strain TC55; (A---A) katC6 mutant; strain TC67. b Growth medium is 3 ml of nutrient broth in 15 mm test tube, innoculum size 0.1 ml of stat-culture. Growth cultures shaken at 37°. e Optical density measurements were made spectrophotometrically at 650 nm. d The growth curve of the kat mutant, katB2, is similar to that of katA1 and the growth curve of katD9 is similar to that of katC6

o b t a i n e d f r o m 52 clones o f TC67 a n d 2 F - segregants f r o m 76 clones o f TC70. A s expected, these F segregants were trp- a n d k a t - . I then i n t r o d u c e d the F ' 1 2 8 (pro + lac +) e p i s o m e by crossing d o n o r strain T C 2 2 with the F - segregants o f TC67 a n d TC70, a n d e x a m i n e d a b o u t 25 p r o g e n y clones. A l l were trp a n d catalase-positive. T w o clones o f each o f these nex~ F ' strains were then c u r e d o f their e p i s o m e s b y being g r o w n o v e r n i g h t in c o m p l e t e m e d i u m s u p p l e m e n t e d with 1 % s o d i u m dodecyl sulfate a n d t h e n s t r e a k e d o n t o solid M a c C o n k e y ' s lactose m e d i u m . A few p e r c e n t o f the clones f r o m each c u l t u r e h a d lost the e p i s o m e a n d were lac-. E a c h o f the lac- clones was also catalase-deficient. I c o n c l u d e t h a t b o t h the kat-6 a n d the kat-9 alleles can be c o m p l e m e n t e d by a n E. coli F'lac pro e p i s o m e a n d t h a t they are p r o b a b l y in the pro region o f the c h r o m o s o m e . T r a n s d u c t i o n s were p e r f o r m e d with p h a g e g r o w n on TC67 a n d T C 7 0 a n d recipients c a r r y i n g v a r i o u s p r o l i n e m u t a t i o n s . T h e kat-6 allele was 27% c o n t r a n s ducible with p r o A B 4 7 ; however, the kat-9 allele was n o t c o n t r a n s d u c i b l e with p r o A B 4 7 (Table 6) suggesting t h a t kat-6 a n d kat-9 were in different genes. W e thus call these alleles k a t C 6 a n d katD9. Growth Curves and Specific Catalase Activities during Growth. T o further c h a r a c t e r i z e the c a t a l a s e - d e f i c i e n t m u t a n t s n u t r i e n t b r o t h cultures were m o n i t o r e d for g r o w t h a n d c a t a l a s e activity. G r o w t h curves (Fig. 1) suggest t h a t a l t h o u g h the m u t a n t s h a d a slightly longer lag p e r i o d t h a n the LT-2 strain, they are n o t

S.A. Levine: Kat Mutants of Salmonella typhimurium

2o

~, ~o 6_ if?

l/"

/'7\

0--

.?i j s

Time,hours

209

20

lo

1'0//--20

Fig. 2a-e. Catalase activities during growth, a ( e - - e ) control strain LT2; ( o - - - o ) katB2 mutant; strain TC61; ( v l v ) katA1 mutant; strain TC55. b Error flags are larger in the earlier growth because dilute samples contained low levels of catalase activity that was near the level of resolution of the assay system, e Catalase specific activity is expressed as gmoles of hydrogen peroxide decomposed per minute per mg of dry cells, d The catalase activity curves of katC6 and katD9 are very similar to that of katA1. e The corresponding growth curves of these strains are presented in Figure 1

grossly abnormal in aerobic growth. The quantitative catalase activities (Fig. 2) of the parent LT-2 strain are high in lag phase and in stationary phase (reached at 6 1/2 h after innoculation) and reach maximum levels in late stationary phase, after 24 h of growth. Catalase-deficient strains TC-55, TC-67 and TC=70 (mutations katA1, katC6 and katD9 respectively) exhibit only low activity (0-4 units sp. act.) throughout the growth cycle. Catalase-deficient strain TC61 (katB2) exhibits normal lag phase activity but no significant activity in stationary phase.

alMic w i t h k a t C 6 or katD9 we have assigned them to different loci. KatC and katD are designated as different genetic loci because katC6 was found to be cotransducible with proAB47 but katD9 was not. Since katC6, katD9 and katA1 all map in the proline region, it is possible that they are in a single operon or their close location may have some other regulatory significance. Mutations katA1, katC6, and katD9 result in low levels of catalase activity throughout the growth cycle. The katB2 mutation results in low stationary phase activity but the early activity is not decreased. The katB2 mutation may be in a regulatory gene which controls the expression of the high catalase activity in stationary phase. Since all of the four kat mutations genetically characterized map in different loci, it is likely that other kat genes exist. All four mutations do not cause a total deficiency in catalase activity; in each case there is some residual activity throughout the growth cycle. This activity may be due to another catalase enzyme or to the residual activity of a single mutated catalase enzyme. Acknowledgments. I thank Mr. R. Altenhof for his cheerful technicaI assistance, Drs. G.W. Chang, B.N. Ames, A.J. Clark and J.R. Roth for advice and bacterial strains. This work was supported m part by a grant from the California Cancer Research Coordinating Committee and NIH training grant H367-14 administered by the Graduate Group in Genetics, University of California, Berkeley.

References Discussion

I have isolated seven catalase-deficient mutants, four of which were genetically characterized in conjugation, complementation, and transduction experiments. The mutants are probably not affected in heme assembly because the strains grow well on nonfermentable medium. The inability to synthesize heme would lead to a loss in respiratory ability since the prosthetic group is an essential component in cytochrome enzymes. The location of the katB locus at about minute 100 on the Salmonella chromosome is clearly different from that of katA, katC, and katD at about minute 10. The conclusion that katA is different from katC and katD rests upon the ability of the E. coli episome F'128 to restore catalase activity to katC6 and katD9 but not to katA1 mutants. KatA1 could be an allele of the katC or katD genes if for some reason the katA1 allele was dominant and katC6 and katD9 alleles were recessive when tested with the E. coli episome. However, in the absence of evidence that katA1 is

Beers, R.F., Sizer, I.W. : A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. biol. Chem. 159, 133-140 (1952) Clayton, R. : Purified catalase from Rhodopseudomonas spheroides. Biochim. biophys. Acta (Amst.) 36, 40 47 (1952) Deisseroth, A., Dounce, A. : Catalase: Physical and chemical properties, mechanism of catalysis, and physiological role. Physiol. Rev. 50, 319 375 (1970) Gottstein, A.: lJber die Zerlegung des Wasserstoff Superoxyds durch die Zellen, mit Bemerkungen fiber eine makrospopische Reaction ffir Bakterien. Virchows Arch. path. A n a l 133, 295307 (1893) Herbert, D., Pinset, J.: Crystalline bacterial catalase. Biochem. J. 43, 193-202 (1948) McCord, J., Keele, B., Jr., Fridovich, I. : An enzyme-based theory of obligate anaerobiosis : The physiological function of superoxide dismutase. Proc. nat. Acad. Sci. (Wash.) 68, 1024-1027 (1971) Sanderson, K.E., Demerec, M.: The linkage map of Salmonella typhimurium. Genetics 51, 897-913 (1965) Vogel, H., Bonner, D. : Acetylornithase of E. coli: Partial purification and some properties. J. biol. Chem. 218, 97-106 (1956)

Communicated by W. Arber Received March 15 / Accepted June 18, 1976