CURRENT MICROBIOLOGY Vol. 13 (1986), pp. 299-301 Current Microbiology �9 Springer-Verlag New York Inc, 1986

Molecular Cloning and Sequencing of Lysozyme Gene of Bacteriophage SF6 of Bacillus subtilis

Mukesh Verma

Molecular Genetics Laboratory, Cancer Research Center, Washington, DC, USA

Abstract. Digestion of bacteriophage SF6 (of Bacillus subtilis) DNA with restriction endonu- clease Pst I generates seven fragments (A-G). These fragments were cloned in pBR322 DNA, and the recombinant clones carrying the lysozyme gene were identified by measuring lysozyme activity. The complete nucleotide sequence of 996 bp fragment D, containing lysozymze gene, was determined. One open reading frame was present at 13 bp, and the termination codons were present at 961 bp. The deduced amino acid sequence of the lysozyme gene was also determined.

The isolation and biochemical characterization of bacteriophage SF6 have been well documented [1, 5, 6]. The phage contains single, double-stranded, linear DNA of molecular weight of approximately 42 kbp, and it synthesizes its own lysozyme toward the later stages after infection. In the present inves- tigation lysozyme gene was cloned, and its nucleo- tide and amino-acid sequences were determined. To the best of my knowledge, this is the first report of the complete nucleotide sequence of lysozyme of any of the Bacillus subtilis phage.

Materials and Methods

Growth medium and measurement of growth rate. The cells were grown in minimal medium supplemented with Casamino acids [6], and growth was measured by following the optical density at 610 nm in a Hilger colorimeter. An absorbance of 0.2 of a cell suspension growing in the above medium represents 2.6 x 108 cells/ml.

Preparation of phage lysate, purification of phage, and isolation of phage DNA. The lysate of phage was prepared by infecting exponentially growing B. subtilis cells with phage SF6 (multiplic- ity of infection = 10) and shaking till lysis occurred [6]. A few drops of chloroform were added to ensure complete lysis. Cell debris was removed by centrifugation at low speed, and the puri- fication of the phage was done essentially as described [5]. For the isolation of phage DNA, about l0 TM particles/ml were mixed with an equal volume of 0.1 x SSV (1 • SSV is 0.015 M NaC1, 0.03 M EDTA, pH 8), and the suspension was extracted with SSV-saturated phenol two times. The aqueous layer was col- lected, and the last traces of phenol were removed by washing twice with chloroform. DNA was precipitated by addition of a double volume of ice-cold 95% ethanol and maintenance at -20~ for 2 h. DNA was finally collected by centrifugation and suspended in 0.01 • SSV.

Isolation of pulse-labeled mRNA and D N A - R N A hybridization. The procedure of isolation of pulse-labeled mRNA was de- scribed earlier [8]. Messenger RNA isolated at different times following infection was used as a probe to identify the cloned DNA fragments (to find out which fragment contains early and/ or late genes) by hybridization. The conditions of DNA-RNA hybridization were essentially the same as described by Maniatis et al. [2].

Cloning of lysozyme gene. All the enzymes were the products of the Bethesda Research Laboratory, (Bethesda, Maryland), and the specifications of the manufacturers were strictly followed. Plasmid pBR322 DNA was digested with Pst I and treated with bacterial alkaline phosphatase. SF6 DNA was also digested with Pst I (which generates seven fragments, A-G), and the reaction product was analyzed on 0.7% agarose gel. Each fragment was electroeluted [2] and ligated with pBR322. Transformation of Escherichia coli strain HB 101 by use of the recombination plus- mid was essentially as described [2]. Transformants were en- riched by cycloserine treatment [4].

Two sets (ten plates in each case) of agar plates were inocu- lated in batches of about 30 colonies from a total of about 150 colonies obtained after cycloserine treatment and grown at 37~ for 24 h.

From one plate (the other was kept as a master plate), colo- nies were transferred to 100 ml Luria broth [2] and grown for 150 min at 37~ The cells were sedimented, washed twice with 10 ml of 20 mM Tris-HCl, pH 7.5, containing 1 mM MgC12 and 1 mM 2-mercaptoethanol, and then suspended in 2 ml of the same buffer. The cells were disrupted by sonication for 3 min (15-s pulse each time) and sedimentation at 10,000 g for 10 min at 4~ The lysozyme activity in the supernatant fraction was measured as described [8]. Three colonies, namely, pMV36, pMV51, and pMV77, giving highest lysozyme activity were chosen and ,char- acterized in detail.

DNA sequencing. Sequencing was done by chemical cleavage method [3]; the strategy of sequencing is shown in Fig. 1.

Address reprint requests to: Dr. Mukesh Verma, PB 22, Molecular Genetics Laboratory, Cancer Center, 2041 Georgia Avenue NW, Washington, DC 20060, USA.

300 CURRENT MICROBIOLOGY VOI. 13 (1986)

Table 1. Hybridization of 3H-mRNA isolated from Bacillus subtilis cells infected with phage SF6 with cloned DNA fragments

Counts per minute hybridized with cloned phage fragment

3H-mRNA isolated B. sub- from phage- tilis pBR322 infected cells DNA DNA

between A B C D E F G (controls)

0- 2 mln 5- 7 mln 9-11 mm

13-15 mm 17-19 mln 21-23 mln 25-27 mln 30-32 min 35-37 min 40-42 min 45-47 min

55 92 35 82 67 62 32 3521 23 82 2530 57 56 76 66 54 167 41 43 4215 60 32 56 35 55 76 48

265 1546 245 65 123 45 67 56 76 1234 134 987 54 564 67 76 65 43 564 65 765 176 123 67 34 95 34 54 32 65 6578 432 65 98 76 12 23 12 23 2345 543 34 102 54 23 12 23 45 6547 987 65 87 63 93 53 23 65 1265 567 43 65 76 64 32 74 85 85 92 19 48 48 38

The values were corrected for the blank. The input counts per min was 10,000 cpm in each case. The values given are the average of two sets run simultaneously.

C A D E G B I L I

x lO kbp \\

\ \

x 'x \

\

B TM A T A A,S

100 bp

Fig. 1. Strategy of sequencing of SF6 DNA fragment (Pst I D) containing the sequence for lysozyme gene. The upper part of the figure represents the restriction map of phage SF6 DNA for enzyme Pst I (A-G are the fragments generated after Pst 1 diges- tion). The lower portion is the detailed restriction map of frag- ment D. Restriction sites are indicated as: A, Alu I; B, BamH I; M, Msp I; S, Sst I; and T, Taq I. The arrows indicate the direc- tion and extent of sequence analysis performed on each of the restriction fragments.

Results and Discussion

D N A - R N A hybridization. To d e t e r m i n e wh ich f r a g m e n t s o f S F 6 D N A con t a in genes for ea r ly p h a g e f u n c t i o n s and w h i c h ones for la te p h a g e func- t ions , the fo l lowing e x p e r i m e n t was p e r f o r m e d . Ex- p o n e n t i a l l y g rowing B. subtilis cel ls (2.6 • 108/ml) we re i n f e c t e d wi th p h a g e S F 6 (mul t ip l i c i ty o f infec-

t ion = 10), and p u l s e - l a b e l e d 3 H - m R N A s were iso- l a ted at d i f fe ren t t imes fo l lowing infec t ion as de- s c r i be d u n d e r Mater ia ls and Methods . S F 6 D N A was d i g e s t e d wi th Ps t I , and f r agmen t s so g e n e r a t e d we re s e p a r a t e d on a g a r o s e gel . E a c h f r agmen t was e l e c t r o e l u t e d f rom the gel and l o a d e d on n i t roce l lu- lose fi l ters (a f te r hea t dena tu ra t i on ) . F i l t e r s con ta in - ing d i f fe ren t f r a g m e n t s o f the p h a g e D N A were h y b r i d i z e d wi th 3H- labe led m R N A in d i f ferent b a t c h e s ; the r e su l t s a re p r e s e n t e d in Tab le l . As s h o w n in T a b l e 1, 3 H - m R N A i so la t ed b e t w e e n 5 and 7 min (and b e t w e e n 9 and 11 min) h y b r i d i z e s main ly wi th f r a g m e n t B, whi le 3 H - m R N A i so la t ed b e t w e e n 25 and 27 min (and b e t w e e n 30 and 32 min) hybr id - izes wi th f r a g m e n t D~ This sugges t s tha t f r agmen t B con ta in s genes for ea r ly p h a g e func t ions , whi le frag- men t D c o n t a i n s genes for la te p h a g e func t ions . O t h e r r e su l t s can N s o be e x p l a i n e d in the s ame way . S ince lysozyrr ie is a la te p h a g e p ro t e in and p r e v i o u s s tud ies i nd ica t e tha t cod ing s e q u e n c e s for l y s o z y m e a re p r e s e n t in the S F 6 g e n o m e , which is e x p r e s s e d la te a f t e r in fec t ion , f r a g m e n t D was o f i n t e re s t to me.

Cloning of SF6 Pst I fragments into pBR322 and identification of clones containing gene for lyso- zyme. T h e p r o c e d u r e has a l r e a d y been d e s c r i b e d ill de ta i l (unde r Mater ia ls and Methods ) . Differen t c lones w e r e a n a l y z e d for l y s o z y m e ac t iv i ty , and it was c o n c l u d e d tha t c lones pMV36 , pMV51, and p M V 7 7 c o n t a i n the gene for l y s o z y m e (in each

M. V e r m a : S e q u e n c i n g o f L y s o z y m e G e n e 3 0 1

5 ' - C T G C A G A T C G T A ATG GAT CCT CGC CTA CGT GAA GAA GTA GTA CGG CTG ATA ATC GCA TTA ACG AGT GAT )~AT GGA GCA TCA CTG TCA AAA CGG CTT CAA TCA AGG GTC TCG GCG CTC GAG AAG ACG TCT CAA ATA CAC TCT GAT ACT ATC CTC CGG ATC ACC CAG GGA CTC GAT GAT GCA AAC AAA CGA ATC ATC GCT CTT GAG CAA AGT CGG GAT GAC TTG GTT GCA TCA GTC AGT GAT GCT CAA CTT GCA ATC TCC AGA TTG GAA AGC TCT ATC GGA GCC CTC CAA ACA GTT GTC AAT GGA CTT GAT TCG AGT GTT ACC CAG TTG GGT GCT CGA GTG GGA CAA CTT GAG ACA GGA CTT GCA GAC GTA CGC GTT GAY CAC GAC AAT CTC GTT GCG AGA GTG GAT ACT GCA GAA CGT AAC ATT GGA TCA TTG ACC ACT GAG CTA TCA ACT C;FG ACG TTA CGA GTA ACA TCC ATA CAA GCG GAT TTC GAA TCT AGG A-FA TCC ACG TTA GAG CGC ACG GCG GTC ACT AGC GCG GGA GCT CCC CTC TCA ATC CGT AAT AAC CGT ATA ACC ATG GGA TTA AAT GAT GGA CTC ACG TTG TCA GGG AAT AAT CTC GCC ATC CGA TTG CCA GGA AAT ACG GGT CTG AAT ATT CAA AAT GGT GGA CTT CAG TTT CGA TTT AAT ACT GAT CAA TTC CAG ATA GTT AAT AAT AAC TTG ACT CTC AAG ACG ACT GTG TTT GAT TCT ATC AAC TCA AGG ATA GGC GCA ACT GAG CAA AGT TAC GTG GCG TCG GCA GTG ACT CCC TTG AGA TTA AAC AGT AGC ACG AAG GTG CTG GAT ATG CTA ATA GAC ATG TCA ACA CTT GAA ATT AAT TCT AGT GGA CAG CTA ACT GTT AGA TCG ACA TCC CCG AAT TTG AGG TAT CCG ATA GCT GAT GTT AGC GGC GGT ATC GGA ATG AGT CCA

AAT TAT AGG TTT AGG ]TGA] GGA TCA GAC CAC CCC GCG GCA CTG GGG CTG CAG 3' I I

met asp pro arg leu arg glu glu val val arg leu ile ile ala leu thr ser asp ash gly ala set leu set lys giy leu glu ser arg val ser ala leu glu lys thr ser gin lie his set asp thr lie leu arg ile thr gin gly leu asp asp ala asn lys arg ile ile ala leu glu gin ser arg asp asp leu val ala ser val set asp ala gin leu ala ile ser arg leu glu ser ser lie gly ala leu gln thr val val ash gly leu asp ser ser val thr gin leu gly ala arg val giy gin leu giu thr gly leu ala asp val arg val asp his asp asn leu val ala arg val asp thr ala glu arg ash ile gly set leu thr thr glu leu ser thr leu thr leu arg val thr set ile gin ala asp phe glu ser arg ile ser thr leu glu arg thr ala val thr ser ala gly ala pro leu ser ile arg asn asn arg met thr met gly leu asn asp gly leu thr leu ser gly ash asn leu ala ile arg leu pro gly a s h

thr gly leu asn ile gin asn gly gly leu gln phe arg phe ash thr asp gin phe gin ile val asn asn asn leu thr leu lys thr thr val phe asp ser ile asn ser arg ile gly ala thr giu gin set tyr val ala set ala val thr pro leu arg leu asn ser ser thr lys val leu asp met leu ile asp ser ser thr leu glu ile ash ser ser gly gin leu thr val arg ser thr set pro asn leu arg tyr pro ile ala asp val ser gly gly ile gly met ser pro a s n tyr arg phe arg

F ig . 2. N u c l e o t i d e s e q u e n c e ( u p p e r part) and d e d u c e d a m i n o ac id s e q u e n c e s o f P s t I f r a g m e n t D o f S F 6 D N A . T h e t r a n s l a t i o n in i t i a t ion

c o d o n (pos i t ion 13) is u n d e r l i n e d , and the t e r m i n a t i o n c o d o n is b o x e d . A f t e r e v e r y ten n u c l e o t i d e s , one do t is m a r k e d at t he top o f t he

nuc l eo t i de .

clone fragment D was cloned). The detailed restric- tion map of the insert was constructed (Fig. 1), and the nucleotide and amino acid sequences were de- termined.

Sequencing of the insert containing lysozyme gene. The majority of the sequence (90%) was determined (according to the strategy shown in Fig. 1) in both directions, and unambiguous data were obtained for the remaining regions. The complete nucleotide se- quence of the insert (996 bp) is shown in Fig. 1. The quantitative distribution of the four bases was found to be relatively even: 27% A, 21% C, 26% G, and 26% T. There is an open reading frame of 948 bp (316 amino acids) which starts from bp 13. The ter- mination codons are present at 961 position. The deduced amino acid sequence of the polypeptide encoded by the reading frame is shown in Fig. 1. Its length (316 amino acids) is sufficient to account for the size of the enzyme (unpublished results).

The lysozyme gene may be transcribed by ei- ther its own promoter or the promoter present on the plasmid. The transcript from the pBR322 pro- moter, P2 [7], codes for the tetracycline-resistant protein which is initiated at the ATG (86-88 bp) and stops at TGA (1274-1276 bp) on the plasmid DNA

[7]. Experiments to analyze the translation product are under way, which will indicate which promoter was really utilized.

Literature Cited

1. B r o d e t s k y A M , R o m i g W R (1965) C h a r a c t e r i z a t i o n o f Bacil- lus subtilis b a c t e r i o p h a g e s . J B a c t e r i o l 90 :1655-1663

2. M a n i a t i s T , F r i t s c h E F , S a m b r o k J (1982) M o l e c u l a r c lon ing :

a laboratory m a n u a l . Co ld S p r i n g H a r b o r N Y : C o l d S p r i n g

H a r b o r L a b o r a t o r y

3. M a x a m A M , G i l b e r t W (1977) A n e w m e t h o d fo r s e q u e n c i n g

D N A . P r o c N a t l A c a d Sci U S A 7 4 : 5 6 0 - 5 6 4

4. R o d r i g u e z R L , B o l i v a r F , G o o d m a n H M , B o y e r H W , B e t l a c h

M (1976) In : N i e r u l i c h D P , R u t t e r W J , F o x C F (eds) M o l e c u -

lar m e c h a n i s m s in the con t ro l o f g e n e e x p r e s s i o n . N e w Y o r k :

A c a d e m i c P r e s s , pp 4 7 1 - 4 7 7

5. S t e e n s m a H Y , B l o k J (1979) E f f e c t o f c a l c i u m on the i n f e c t i o n

of Bacillus subtilis b y b a c t e r i o p h a g e SF6 . J G e n Vi ro l 4 2 : 3 0 5 -

314

6. S t e e n s m a H Y , H a v e l a r A H , L e y d e n s H M , W i k e n T O (1974)

B a c t e r i o p h a g e s o f Bacillus subtilis: e x p l a n a t i o n o f the influ-

e n c e o f t he n u m b e r o f i n d i c a t o r b a c t e r i a on the e f f i c i e n c y o f

p la t ing . A n n M i c r o b i o l 2 4 : 1 1 - 1 9

7. S t u b e r D , B u j a r d H (1981) O r g a n i z a t i o n o f t r a n s c r i p t i o n a l s ig-

nals in p l a s m i d s p B R 3 2 2 a n d p A C Y C 184. P r o c N a t l A c a d Sc i

U S A 78 :167 -171

8. V e r m a M, S i d d i q u i J Z , C h a k r a v o r t y M (1985) B a c t e r i o p h a g e

P22 he lp s b a c t e r i o p h a g e M B 7 8 to o v e r c o m e the t r a n s c r i p t i o n

i nh ib i t i on in r i f a m p i c i n r e s i s t a n t m u t a n t o f Salmonella typhi- murium. B i o c h e m In t 11 :177-186