Proc. Natl. Acad. Sci. USA Vol. 84, pp. 955-958, February 1987 Biochemistry Cloning and purification of a unique lysozyme produced by Bacillus phage 4P29 (phage 429 gene 15/phage-type lysozyme/lysozyme evolution) MOHAMMAD S. SAEDI, KEVIN J. GARVEY, AND JUNETSU ITO Department of Microbiology and Immunology, University of Arizona Health Sciences Center, The University of Arizona, Tucson, AZ 85724 Communicated by C. S. Marvel, October 27, 1986 (receivedfor review August 27, 1986)

ABSTRACT A DNA fragment of the bacteriophage 429 end (7). The genes involved in the lytic function ofthis phage chromosome, encoding the entire sequence of 4)29 gene 15, has have not been well characterized. Genetic studies have been cloned into the Escherichia coli expression vector pPLc245 shown that mutations in genes 14 and 15 result in a delayed under the control of the phage X major leftward promoter, PL. lysis phenotype, with normal phage development in infected Upon heat induction, a protein with an apparent molecular cells (8-10). We recently sequenced genes 14 and 15 and mass of 26 kDa was overproduced. The molecular mass of this found that the deduced amino acid sequence of gene 15 has protein corresponds to the 28 kDa predicted for the product of strong homology with the lysozyme of phage P22 and weak gene 15 from its nucleotide sequence. The overproduced but significant homology with the lysozyme of phage T4 (11). protein has been purified to near homogeneity and confirmed However, the DNA sequence data revealed that phage 429 to be the product of gene 15 by amino acid sequence analysis gene 15 encodes a 28-kDa protein, which is substantially of its N terminus. The purified product of gene 15 has a larger than both ofthe T4 and P22 lysozymes; the T4 and P22 lysozyme activity similar to other phage-type lysozymes: prod- lysozymes are 18.7 and 16.1 kDa, respectively (5, 12). ucts of phage T4 gene e and of phage P22 gene 19. However, In this communication, we report the cloning and purifi- to our knowledge 429 lysozyme is structurally unique among cation of phage 429 gene 15 protein. Our results clearly the phage-type lysozymes. establish that the product of gene 15 is a unique phage lysozyme. Lysozymes are widespread in nature and have been isolated from a variety of organisms (1). These enzymes have pro- MATERIALS AND METHODS vided a useful model system for studying protein structure, mechanism of enzyme action, immunochemistry, and evo- All enzymes were purchased from Bethesda Research Lab- lution (1). Lysozymes are generally classified into four oratories, and chemicals were from Sigma. Escherichia coli distinct families: chicken-type lysozyme, goose-type lyso- strain K12AHlAtrp and plasmid pPLc245 were obtained from zyme, phage-type lysozyme, and the bacterial lysozyme Remaut et al. (13). The freeze-dried culture of Micrococcus produced by Streptomyces erythraeus (1). The amino acid lysodeikticus was purchased from United States Biochemical sequences of lysozymes within a given family are clearly (Cleveland, OH). The methionine assay medium was ob- related, but there is no obvious sequence homology between tained from Difco. one family and another (2). However, when the three-dimen- Cloning of Phage 429 Gene 15. Bacteriophage 429 was sional structure oflysozymes from goose-type, chicken-type, prepared by CsCl gradient purification, and DNA was ex- and phage-type are compared, considerable similarities are tracted by NaDodSO4 and proteinase K treatment as de- noted (3). Thus, it has been suggested that these three types scribed (14). Phage 029 HindIII fragment F, containing the of lysozymes have evolved from a common ancestor (3). entire coding region of gene 15, was isolated from a total )29 The goose-type and the chicken-type lysozymes have been HindIII digest by gel electrophoresis (15). The isolated the most intensively investigated; the complete amino acid fragment was ligated into the HindIII site of plasmid sequence of 18 chicken-type lysozymes are already known pPLc245, which is located downstream from a phage X major (1). Surprisingly, the phage-type lysozymes, with the excep- leftward promoter PL, and transformants were obtained in tion of T4 and T2 lysozymes, are not well explored (1). The strain K12AHlAtrp by selecting for resistance to ampicillin phage T2 and T4 lysozymes differ by only three amino acids (100 gg/ml). Among the ampicillin-resistant colonies, those (4). Recently, phage P22 gene 19 (lysozyme gene) has been harboring recombinant plasmids were selected by miniplas- sequenced, and the amino acid sequence ofthe gene has been mid preparation (15). The plasmid with the insert in the same deduced (5). It was found that there is significant homology orientation as the PL promoter, pMS2, and the plasmid with between the P22 lysozyme and the T4 lysozyme, indicating the insert in the reverse orientation, pMS6, were identified by that these phage lysozymes are evolutionarily related (2). restriction enzyme digest analyses. The directions of the Perhaps this finding is not surprising because both T4 (a inserts were also verified by DNA sequence analysis using coliphage) and P22 (a Salmonella phage) infect Gram-nega- the Maxam and Gilbert technique (16). tive bacteria from the same family, Enterobacteriaceae. Protein Analysis of the Induced Clones. To examine the Thus, it seems of considerable interest to investigate lyso- expression ofthe cloned gene 15 in response to a temperature zymes from different phage systems. shift, the cells harboring the above plasmids were grown in We have been studying bacteriophage 429, which infects LB medium (15) with ampicillin (100 ,ug/ml) at 30°C. Bacteria the Gram-positive bacterium Bacillus subtilis (6). 429 is a were then harvested at midlogarithmic phase by centrifuga- small lytic phage whose genome is a linear double-stranded tion and were resuspended in the same volume ofprewarmed DNA with terminal proteins attached covalently at each 5' M9 minimal medium (15) supplemented with 50% methionine assay medium and 50 jig of tryptophan per ml. After 1 hr of to The publication costs of this article were defrayed in part by page charge incubation at 30°C, a portion of the culture was shifted payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: bp, base pair(s).

Downloaded by guest on September 29, 2021 955 956 Biochemistry: Saedi et al. Proc. Natl. Acad. Sci. USA 84 (1987) 420C. After 2 hr, 50 ,ul of the induced and uninduced samples B G K HMI E J D A N F C I Ia aI a I..a A. ..0 were labeled for 10 min with 10 gCi (1 Ci = 37 GBq) of = I" I I I I [35S]methionine (1100 Ci/mmol; New England Nuclear). The labeled cells were then collected and resuspended in 20 .l of sample buffer as described (17). Aliquots of these samples I were then boiled for 5 min prior to loading and were analyzed ,Hindill by NaDodSO4/10% polyacrylamide gel electrophoresis (17). The gel was dried, and the labeled proteins were visualized Hiridlll F by autoradiography. Protein Purification and Amino Acid Sequencing. E. coli K12AHlAtrp carrying pMS2 was grown in 2 liters of LB medium containing ampicillin (100 pug/ml) at 30'C. At mid- logarithmic phase, an equal volume of LB medium pre- warmed to 650C was added, and the culture was incubated at 420C for 2 hr. The cells were harvested, resuspended in 50 ml of TR buffer (50 mM Tris, pH 7.5/5% glycerol/i mM EDTA/1.4 mM 2-mercaptoethanol) containing 0.1% Brij 58, and stored at -70'C. When cell extracts were prepared for lysozyme activity assays, Brij 58 was omitted from the above buffer. The frozen sample was then thawed at 370C (which caused most of the cells to lyse) and was kept on ice. All FIG. 1. Cloning of 429 gene 15. The HindIII restriction map of subsequent steps were then carried out at 4TC. Crude cell bacteriophage 4)29 is schematically diagramed. The HindIII fragment extract was then prepared by sonicating the above cell F containing gene 15 was isolated and ligated into the HindIII site of plasmid pPLc245 (13). The hybrid plasmid pMS2 was then isolated suspension with five bursts of20-sec duration (using a Brason as described. The arrows represent the direction of transcription. sonic-power sonifier model SilO) and removing the cell refers to the for debris by centrifugation at 20,000 x g for 30 min. The ApR gene ampicillin resistance. supernatant was diluted 1:3 in TR buffer and applied to a CM Bio-Gel A (Bio-Grad) column (2.5 x 20 cm). The column was activated (13). To determine whether the product of gene 15 washed with three-bed-volumes of TR buffer, and the pro- would be expressed from pMS2, cells containing this plasmid teins were eluted by a linear gradient of0-0.4 M NaCl in 200 were grown at 30°C to midlogarithmic phase and then were ml of TR buffer at a rate of 10 ml/hr. Fractions (2 ml) were shifted rapidly to 42°C. After a 2-hr incubation, aliquots were collected and assayed for lysozyme activity by their ability to removed and labeled with [35S]methionine, and the total lyse M. lysodeikticus (18). Aliquots ofeach fraction were also protein was analyzed on NaDodSO4/polyacrylamide gels as subjected to NaDodSO4/polyacrylamide gel electrophoresis. described. Cells transformed with the recombinant plasmid The fractions with peak lysozyme activity, which corre- pMS2 overproduced at 42°C a labeled polypeptide with an sponded to the peak fractions of a 26-kDa protein, were then apparent molecular mass of =26 kDa (Fig. 2). The other cells, pooled. containing either plasmid pPLc245 or pMS6, did not synthe- To prepare the sample for amino acid sequence analysis, size this protein. The molecular mass of the overproduced the pooled fractions were dialyzed against 0.005% NaDod- protein at 42°C was consistent with the reported value for the SO4, and the protein concentration was measured (19). This product of phage 429 gene 15 (10). Moreover, it also agrees sample was then concentrated in a Savant Speed Vac with the 28-kDa value deduced from the nucleotide sequence concentrator to 4 mg/ml. Of this concentrated solution, 0.1 analysis of 429 gene 15 (11). ml was subjected to amino acid sequence analysis using a Beckman 890M sequencer. 1 2 3 4 5 6 RESULTS Cloning and Overproduction of Phage 429 Lysozyme. Our 4=- nucleotide sequence analysis revealed that the phage 429 limmoffall HindIII fragment F contains the complete open reading frame 43 -- 4w,-

for gene 15 (11). Therefore, we isolated the 1330-base-pair z.s,.-.. (bp) HindIII fragment F and inserted it into the plasmid .::. pPLc245 (13) (Fig. 1). This plasmid has a single HindIII site +-gp 15 located downstream of a phage X PL promoter. Therefore, ..... transcription ofthe genes inserted at this site will be under the control of the cI repressor. The recombinant plasmid was 18.4-+ dow, transferred into E. coli K12AHlAtrp by transformation (15). vm. _ _ _ ..~~~ IM The clones harboring a plasmid with the insert in the correct orientation relative to the PL promoter were identified by -"Amm- restriction enzyme digestion analysis. One of these plasmids FIG. 2. Detection of the protein overproduced oy temperature was chosen for further study and designated pMS2 (Fig. 1). induction of E. coli K12AHlAtrp harboring various plasmids. The Another plasmid, pMS6, carrying the insert in the reverse cells were grown to midlogarithmic phase at 30°C, and a portion of direction was also selected. The orientation of phage 429 each culture was shifted to 42°C for 2 hr. Aliquots ofthe induced and gene 15 was further verified by DNA sequence analysis (data uninduced culture of each sample were then labeled with [35S]me- thionine and subjected to NaDodSO4/polyacrylamide gel electro- not shown). phoresis as described. Lanes: 1, 3, and 5, uninduced samples ofcells Strain K12AHlAtrp is a phage X lysogen carrying a ther- harboring pPLc245, pMS2, and pMS6, respectively; 2, 4, and 6, the mosensitive mutation in its cI repressor gene, cIts857 (13). corresponding induced samples. The positions ofthe molecular mass When growing cells containing the above plasmids are shifted markers (in kDa) were determined from prestained markers pur- from 30°C to 42°C, the cI repressor of the prophage is chased from Bethesda Research Laboratories. The position of the inactivated, and the phage XPL promoter on the plasmid is 26-kDa gene 15 product (gp 15) is also shown. Downloaded by guest on September 29, 2021 Biochemistry: Saedi et al. Proc. Natl. Acad. Sci. USA 84 (1987) 957 Table 1. Comparison of the lysozyme activities of E. coli A B K12AH1A&trp harboring various plasmids Lysozyme specific activity, * 43 Plasmid Condition units per mg of protein pPlc245 Induced 0.19 pMS6 Induced 0.15 pMS2 Uninduced 0.36 pMS2 Induced 20.70 gp 15 25.7 Crude extracts of each culture were prepared, and the lysozyme activity and protein concentration were measured. To measure lysozyme activity, a freeze-dried culture of M. lysodeikticus was suspended in 0.05 M Tris (pH 7.5), and 0.6 ml of this suspension (turbidity of 0.5 absorbance unit at 450 nm) was incubated with 5 1.d 18.4 ofeach extract. The lysozyme unit was defined as the decrease in the _110 absorbance of this solution after 5 min at room temperature. - 14.3 To determine the enzymatic activity of the gene 15 prod- uct, crude extracts of the induced and uninduced cells harboring pMS2, pMS6, or pPLc245 were prepared. Lyso- zyme activity ofeach extract was then measured by using M. lysodeikticus as substrate (Table 1). Crude extracts prepared FIG. 3. NaDodSO4/polyacrylamide gel electrophoresis pattern from the induced cells containing pPLc245 or pMS6 and the ofthe purified product of phage 429 gene 15 (gp 15). Purified gene 15 uninduced cells containing pMS2 showed little lysozyme product (20 ug) was subjected to NaDodSO4/10%0 polyacrylamide activity. On the other hand, the induced cells containing gel electrophoresis and stained with Coomassie blue (lane A). Lane pMS2 exhibited increased lysozyme activity under these B shows the positions of molecular mass markers in kDa. conditions. These results indicate that the product of phage 429 gene 15 is a lysozyme. has an apparent molecular mass of 26 kDa on NaDodSO4/ Purification of Phage 429 Gene 15 Product. To purify the polyacrylamide gel (Fig. 3) and comprises about 15% of the gene 15 product, cells containing pMS2 were grown at 30'C total cellular protein in the induced clones (data not shown). and shifted to 420C at midlogarithmic phase. After 120 min of The above molecular mass estimation is comparable to the incubation at 420C, cells were pelleted and resuspended in TR 28-kDa value predicted from the nucleotide sequence (11) and buffer containing Brij 58, incubated at -70'C, and broken is in agreement with the molecular mass previously reported open by sonication to prepare crude extract. The crude for the product of 429 gene 15 (9, 10). extract was then diluted and applied to a CM Bio-Gel A The gene 15 product was purified to near homogeneity column. Proteins were eluted with a linear salt gradient as (Fig. 3) and was shown to have bacteriolytic properties described. Aliquots of each fraction were assayed for lyso- (Table 1). This fact is consistent with the slow lysis pheno- zyme activity and also analyzed on NaDodSO4/polyacryl- type exhibited by 429 gene 15 mutants (8) and also agrees amide gel electrophoresis. The lysozyme activity was eluted with our finding that the amino acid sequence of 429 gene 15 at 0.25-0.3 M NaCl. The peak of lysozyme activity corre- product is homologous with the amino acid sequences of P22 sponded exactly with the peak fraction ofa 26-kDa protein on and T4 lysozymes (11). We also have sequenced the first 15 NaDodSO4/polyacrylamide gel electrophoresis (data not amino acids ofthe N terminus ofthe purified protein and have shown). These fractions were then collected and concentrat- shown that it is in complete agreement with the sequence ed as described. To determine the purity of the phage 429 deduced from the nucleotide sequence (Fig. 4). This confirms lysozyme, about 20 ug of this solution was applied to a the assigned reading frame of gene 15 product in our nucle- NaDodSO4/polyacrylamide gel and stained with Coomassie otide sequence and also shows that the protein was neither blue (Fig. 3). The sample was estimated to be =95% pure. processed nor modified during or after expression in E. coli N-Terminal Amino Acid Sequence of the Product of Phage cells. Based on the above results, we conclude that phage 429 qb29 Gene 15. To determine the N-terminal amino acid gene 15 product is a 28-kDa basic protein with lysozyme sequence of the gene 15 product, the purified protein sample activity. To our knowledge, this lysozyme is unique in being was subjected to amino acid sequencing. The sequence ofthe purified from a bacteriophage that infects Gram-positive first 15 amino acids was determined (Fig. 4). This N-terminal bacteria. sequence agreed perfectly with that deduced from the DNA The lysozyme of bacteriophage T4, a product of gene e, is sequence ofgene 15. These results indicate that the 429 gene the prototype of phage-type lysozymes (1). Recently, a 15 product was accurately expressed in E. coli cells and that lysozyme from phage P22 has been purified (5). This lyso- this protein, when overproduced from the E. coli clone, was zyme is the second member ofphage-type lysozymes report- not modified at its N terminus. ed (2). The amino acid sequence of P22 lysozyme, a product of P22 gene 19, has a 26% homology with that of the T4 DISCUSSION lysozyme (2). Weaver et al. (2) have concluded that the P22 We have cloned gene 15 of bacteriophage 429 into an E. coli lysozyme may provide an evolutionary link between the T4 expression vector and overproduced its product. This protein lysozyme and the goose-type lysozyme. Such a link would 1 5 10 is ATG CAA ATU TCA CAA GCG GGT ATC MC TTA AUT MG AGC TTT GAG Met Gln Ile Ser Gln Ala Gly Ile Asn Leu Ile Lys Ser Phe Glu FIG. 4. The sequence of the first 15 amino acids of the N terminus of the 429 gene 15 product. The top line is the nucleotide sequence of the first 45 bp of the 5' end of gene 15, which are described elsewhere (11). The bottom line represents the respective amino acid sequence of gene 15 product obtained by sequencing the purified protein. Downloaded by guest on September 29, 2021 958 Biochemistry: Saedi et al. Proc. Natl. Acad. Sci. USA 84 (1987)

Genome Structural Lysozyme Phage Host Property Size Gene Mr P22 S. typhimurium Lysogenic 43 kb 19 16.1 kDa NH2///////////////// ////> COOH (Gram -) 029 B. subtilis Lyt i c 19 kb 15 28 kDa NH12 *HOOH (Gram +)

18.7 kfa NH . . . OOH T4 E. coli Lytic 166 kb e 2... OOH (Gram -) FIG. 5. Comparison of the properties of lysozymes of phages P22, 429, and T4. The structure of each protein is schematically illustrated. The black area in the 429 lysozyme represents the portion of this protein that is homologous to the P22 and T4 lysozymes. The hatched area in P22 lysozyme represents the strong homology and the stippled area in T4 lysozyme represents the weak homology observed between these proteins and the 429 lysozyme. The details have been described elsewhere (11).

strongly favor the notion that goose-type, chicken-type, and mary sequences have been analyzed so far. For this reason, phage-type lysozymes have all evolved from a common 429 lysozyme could potentially be a valuable source for ancestor (3). As mentioned above, the amino acid sequence investigating lysozyme evolution and the evolutionary origin of the product of phage 429 gene 15 shows strong homology ofbacteriophages. Now that large quantities ofthe product of with the P22 lysozyme (38%) and weak homology with the T4 phage 429 gene 15 can be isolated, it is feasible to elucidate lysozyme (18%) (11). Moreover, a T4 lysozyme-deficient the tertiary structure of this protein and to approach the mutation can be complemented by the 429 gene 15 product evolutionary questions at the molecular level. (11). This was demonstrated by the plaque-forming ability of a T4 gene e mutant when plated on E. coli cells harboring the We thank Drs. Harris and Carol Bernstein for their critical pMS2 plasmid (20). These findings clearly indicate that the evaluation of this manuscript. This investigation was supported by 429 lysozyme is structurally and functionally related to the National Institutes of Health Grant GM28013. T4 and P22 lysozymes. Since these three phages are consid- a 1. Jolles, P. & Jolles, J. (1984) Mol. Cell. Biochem. 63, 165-189. ered to be totally unrelated, close relationship between their 2. Weaver, L. H., Rennell, D., Poteete, A. R. & Matthews, lysozymes is quite remarkable (Fig. 5). B. W. (1985) J. Mol. Biol. 184, 739-741. Some of the properties ofT4, P22, and 429 bacteriophages 3. Weaver, L. H., Grutter, M. G., Remington, S. J., Gray, T. M. and their lysozymes are compared in Fig. 5. The genomic size & Matthews, B. W. (1985) J. Mol. Evol. 21, 97-111. of 029 is much smaller than those of P22 and T4, but its 4. Tsugita, A. & Inouye, M. (1968) J. Mol. Biol. 37, 201-212. lysozyme is much larger than the lysozymes of T4 and P22. 5. Rennell, D. & Poteete, A. (1985) Virology 43, 280-289. However, the homology of 429 lysozyme with the lysozymes 6. Reilly, R. E. (1965) Dissertation (Case Western Reserve Uni- of T4 and P22 is located entirely at the N terminus of this versity, Cleveland, OH). 7. Geiduscheck, E. P. & Ito, J. (1982) in The Molecular Biology enzyme. DNA sequence results indicated that there are long of Bacillus, ed. Dabanau, D. (Academic, New York), p. tandem repeats at the nonhomologous C terminus of the 429 203-244. gene 15 product, suggesting a gene duplication (11); this 8. Carrascosa, J., Camacho, A., Morreno, F., Mellado, R., unusual structure could be due to involvement in other Vinuela, E. & Salas, M. (1976) Eur. J. Biochem. 66, 229-241. functions in addition to lysozyme activity. One such function 9. Hagen, E. W., Reilly, B. E., Tosi, M. E. & Anderson, D. C. has been suggested to be in phage morphogenesis (8, 10). (1976) J. Virol. 19, 501-517. 10. Jimenez, F., Camacho, A., De La Torre, J., Vinuela, E. & Grutter et al. (21) have suggested that the C-terminal lobe of Salas, M. (1977) Eur. J. Biochem. 73, 57-72. T4 lysozyme is directly involved in substrate binding of this 11. Garvey, K. J., Saedi, M. S. & Ito, J. (1987) J. Nucleic Acids protein. If this is also true for the 429 lysozyme, then the Res., in press. unusual structure of this protein's C terminus could be a 12. Inouye, M., Imada, M. & Tsugita, A. (1970) J. Biol. Chem. common requirement for the lysozymes ofphages that infect 245, 3479-3484. Gram-positive bacteria. However, this hypothesis can only 13. Remaut, E., Stanssens, P. & Fiers, W. (1983) Nucleic Acids Res. 11, 4677-4689. be analyzed when more information about the lysozymes of 14. Ito, J. (1978) J. Virol. 28, 895-904. other phages infecting Gram-positive bacteria becomes avail- 15. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular able. Cloning: A Laboratory Manual (Cold Spring Harbor Labora- Although bacteriolytic enzymes produced by various tory, Cold Spring Harbor, NY). phages have been studied, very few of them belong to the 16. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, family of phage-type lysozymes. For example, phage X has 499-560. 17. Laemmli, U. K. (1970) Nature (London) 227, 680-685. two genes, R and Rz, that are responsible for cell-wall 18. Morita, T., Hara, S. & Matsushima, Y. (1978) J. Biochem. 83, hydrolysis (22). Gene R codes for a transglucosylase, and the 893-903. Rz product has endopeptidase activity (22). Both of these 19. Bradford, M. (1976) Anal. Biochem. 72, 248-254. enzymes are different from the phage-type lysozymes (22). 20. Garvey, K. J. (1986) Dissertation (University of Arizona, Bacteriophage T7 gene 3.5 encodes a bacteriolytic enzyme as Tucson, AZ). well. However, the product of this gene is an amidase and is 21. Grutter, M. G. & Matthews, B. W. (1982) J. Mol. Biol. 154, 525-535. functionally and structurally different from phage-type 22. Bienkowski-Szewcyk, K. & Taylor, A. (1980) Biochim. Bio- lysozymes (23). Therefore, if one considers T4 and T2 phys. Acta 615, 489-4%. lysozymes as one member, 429 lysozyme is only the third 23. Inouye, M., Arnheim, N. & Sternglanz, R. (1973) J. Biol. member of the family of phage-type lysozymes whose pri- Chem. 248, 7247-7252. Downloaded by guest on September 29, 2021