Cloning and purification of a unique lysozyme produced by ... - PNAS

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 (received for review August 27, 1986)

A DNA fragment of the bacteriophage 429 ABSTRACT chromosome, encoding the entire sequence of 4)29 gene 15, has been cloned into the Escherichia coli expression vector pPLc245 under the control of the phage X major leftward promoter, PL. Upon heat induction, a protein with an apparent molecular mass of 26 kDa was overproduced. The molecular mass of this protein corresponds to the 28 kDa predicted for the product of gene 15 from its nucleotide sequence. The overproduced protein has been purified to near homogeneity and confirmed to be the product of gene 15 by amino acid sequence analysis of its N terminus. The purified product of gene 15 has a lysozyme activity similar to other phage-type lysozymes: products of phage T4 gene e and of phage P22 gene 19. However, to our knowledge 429 lysozyme is structurally unique among the phage-type lysozymes.

end (7). The genes involved in the lytic function of this phage have not been well characterized. Genetic studies have shown that mutations in genes 14 and 15 result in a delayed lysis phenotype, with normal phage development in infected cells (8-10). We recently sequenced genes 14 and 15 and found that the deduced amino acid sequence of gene 15 has strong homology with the lysozyme of phage P22 and weak but significant homology with the lysozyme of phage T4 (11). However, the DNA sequence data revealed that phage 429 gene 15 encodes a 28-kDa protein, which is substantially larger than both of the T4 and P22 lysozymes; the T4 and P22 lysozymes are 18.7 and 16.1 kDa, respectively (5, 12). In this communication, we report the cloning and purification of phage 429 gene 15 protein. Our results clearly 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 provided a useful model system for studying protein structure, mechanism of enzyme action, immunochemistry, and evolution (1). Lysozymes are generally classified into four distinct families: chicken-type lysozyme, goose-type lysozyme, phage-type lysozyme, and the bacterial lysozyme produced by Streptomyces erythraeus (1). The amino acid sequences of lysozymes within a given family are clearly related, but there is no obvious sequence homology between one family and another (2). However, when the three-dimensional structure of lysozymes from goose-type, chicken-type, and phage-type are compared, considerable similarities are noted (3). Thus, it has been suggested that these three types of lysozymes have evolved from a common ancestor (3). The goose-type and the chicken-type lysozymes have been the most intensively investigated; the complete amino acid sequence of 18 chicken-type lysozymes are already known (1). Surprisingly, the phage-type lysozymes, with the exception of T4 and T2 lysozymes, are not well explored (1). The phage T2 and T4 lysozymes differ by only three amino acids (4). Recently, phage P22 gene 19 (lysozyme gene) has been sequenced, and the amino acid sequence of the gene has been deduced (5). It was found that there is significant homology between the P22 lysozyme and the T4 lysozyme, indicating that these phage lysozymes are evolutionarily related (2). Perhaps this finding is not surprising because both T4 (a coliphage) and P22 (a Salmonella phage) infect Gram-negative bacteria from the same family, Enterobacteriaceae. Thus, it seems of considerable interest to investigate lysozymes from different phage systems. We have been studying bacteriophage 429, which infects the Gram-positive bacterium Bacillus subtilis (6). 429 is a small lytic phage whose genome is a linear double-stranded DNA with terminal proteins attached covalently at each 5'

MATERIALS AND METHODS All enzymes were purchased from Bethesda Research Laboratories, and chemicals were from Sigma. Escherichia coli strain K12AHlAtrp and plasmid pPLc245 were obtained from Remaut et al. (13). The freeze-dried culture of Micrococcus lysodeikticus was purchased from United States Biochemical (Cleveland, OH). The methionine assay medium was obtained from Difco. Cloning of Phage 429 Gene 15. Bacteriophage 429 was prepared by CsCl gradient purification, and DNA was extracted by NaDodSO4 and proteinase K treatment as described (14). Phage 029 HindIII fragment F, containing the entire coding region of gene 15, was isolated from a total )29 HindIII digest by gel electrophoresis (15). The isolated fragment was ligated into the HindIII site of plasmid pPLc245, which is located downstream from a phage X major leftward promoter PL, and transformants were obtained in strain K12AHlAtrp by selecting for resistance to ampicillin (100 gg/ml). Among the ampicillin-resistant colonies, those harboring recombinant plasmids were selected by miniplasmid preparation (15). The plasmid with the insert in the same orientation as the PL promoter, pMS2, and the plasmid with the insert in the reverse orientation, pMS6, were identified by restriction enzyme digest analyses. The directions of the inserts were also verified by DNA sequence analysis using the Maxam and Gilbert technique (16). Protein Analysis of the Induced Clones. To examine the expression of the cloned gene 15 in response to a temperature shift, the cells harboring the above plasmids were grown in LB medium (15) with ampicillin (100 ,ug/ml) at 30°C. Bacteria were then harvested at midlogarithmic phase by centrifugation and were resuspended in the same volume of prewarmed M9 minimal medium (15) supplemented with 50% methionine assay medium and 50 jig of tryptophan per ml. After 1 hr of incubation at 30°C, a portion of the culture was shifted to

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Abbreviation: bp, base pair(s). 955

956

Biochemistry: Saedi et al.

420C. After 2 hr, 50 ,ul of the induced and uninduced samples were labeled for 10 min with 10 gCi (1 Ci = 37 GBq) of [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 were then boiled for 5 min prior to loading and were analyzed by NaDodSO4/10% polyacrylamide gel electrophoresis (17). The gel was dried, and the labeled proteins were visualized 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 midlogarithmic phase, an equal volume of LB medium prewarmed 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 subsequent steps were then carried out at 4TC. Crude cell extract was then prepared by sonicating the above cell suspension with five bursts of 20-sec duration (using a Brason sonic-power sonifier model SilO) and removing the cell debris by centrifugation at 20,000 x g for 30 min. The 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 washed with three-bed-volumes of TR buffer, and the proteins were eluted by a linear gradient of 0-0.4 M NaCl in 200 ml of TR buffer at a rate of 10 ml/hr. Fractions (2 ml) were collected and assayed for lysozyme activity by their ability to lyse M. lysodeikticus (18). Aliquots of each fraction were also subjected to NaDodSO4/polyacrylamide gel electrophoresis. The fractions with peak lysozyme activity, which corresponded to the peak fractions of a 26-kDa protein, were then

pooled. To prepare the sample for amino acid sequence analysis, the pooled fractions were dialyzed against 0.005% NaDodSO4, and the protein concentration was measured (19). This sample was then concentrated in a Savant Speed Vac concentrator to 4 mg/ml. Of this concentrated solution, 0.1 ml was subjected to amino acid sequence analysis using a Beckman 890M sequencer.

RESULTS Cloning and Overproduction of Phage 429 Lysozyme. Our nucleotide sequence analysis revealed that the phage 429 HindIII fragment F contains the complete open reading frame for gene 15 (11). Therefore, we isolated the 1330-base-pair (bp) HindIII fragment F and inserted it into the plasmid pPLc245 (13) (Fig. 1). This plasmid has a single HindIII site located downstream of a phage X PL promoter. Therefore, transcription of the genes inserted at this site will be under the control of the cI repressor. The recombinant plasmid was transferred into E. coli K12AHlAtrp by transformation (15). The clones harboring a plasmid with the insert in the correct orientation relative to the PL promoter were identified by restriction enzyme digestion analysis. One of these plasmids was chosen for further study and designated pMS2 (Fig. 1). Another plasmid, pMS6, carrying the insert in the reverse direction was also selected. The orientation of phage 429 gene 15 was further verified by DNA sequence analysis (data not shown). Strain K12AHlAtrp is a phage X lysogen carrying a thermosensitive mutation in its cI repressor gene, cIts857 (13). When growing cells containing the above plasmids are shifted from 30°C to 42°C, the cI repressor of the prophage is inactivated, and the phage XPL promoter on the plasmid is

Proc. Natl. Acad. Sci. USA 84 (1987) B I

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FIG. 1. Cloning of 429 gene 15. The HindIII restriction map of bacteriophage 4)29 is schematically diagramed. The HindIII fragment F containing gene 15 was isolated and ligated into the HindIII site of plasmid pPLc245 (13). The hybrid plasmid pMS2 was then isolated as described. The arrows represent the direction of transcription. ApR refers to the gene for ampicillin resistance.

activated (13). To determine whether the product of gene 15 would be expressed from pMS2, cells containing this plasmid were grown at 30°C to midlogarithmic phase and then were shifted rapidly to 42°C. After a 2-hr incubation, aliquots were removed and labeled with [35S]methionine, and the total protein was analyzed on NaDodSO4/polyacrylamide gels as described. Cells transformed with the recombinant plasmid pMS2 overproduced at 42°C a labeled polypeptide with an apparent molecular mass of =26 kDa (Fig. 2). The other cells, containing either plasmid pPLc245 or pMS6, did not synthesize this protein. The molecular mass of the overproduced protein at 42°C was consistent with the reported value for the product of phage 429 gene 15 (10). Moreover, it also agrees with the 28-kDa value deduced from the nucleotide sequence analysis of 429 gene 15 (11). 1

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FIG. 2. Detection of the protein overproduced oy temperature induction of E. coli K12AHlAtrp harboring various plasmids. The cells were grown to midlogarithmic phase at 30°C, and a portion of each culture was shifted to 42°C for 2 hr. Aliquots of the induced and uninduced culture of each sample were then labeled with [35S]methionine and subjected to NaDodSO4/polyacrylamide gel electrophoresis as described. Lanes: 1, 3, and 5, uninduced samples of cells harboring pPLc245, pMS2, and pMS6, respectively; 2, 4, and 6, the corresponding induced samples. The positions of the molecular mass markers (in kDa) were determined from prestained markers purchased from Bethesda Research Laboratories. The position of the 26-kDa gene 15 product (gp 15) is also shown.

Proc. Natl. Acad. Sci. USA 84 (1987)

Biochemistry: Saedi et al.

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To determine the enzymatic activity of the gene 15 product, crude extracts of the induced and uninduced cells harboring pMS2, pMS6, or pPLc245 were prepared. Lysozyme activity of each extract was then measured by using M. lysodeikticus as substrate (Table 1). Crude extracts prepared from the induced cells containing pPLc245 or pMS6 and the uninduced cells containing pMS2 showed little lysozyme activity. On the other hand, the induced cells containing pMS2 exhibited increased lysozyme activity under these conditions. These results indicate that the product of phage 429 gene 15 is a lysozyme. Purification of Phage 429 Gene 15 Product. To purify the gene 15 product, cells containing pMS2 were grown at 30'C and shifted to 420C at midlogarithmic phase. After 120 min of incubation at 420C, cells were pelleted and resuspended in TR buffer containing Brij 58, incubated at -70'C, and broken open by sonication to prepare crude extract. The crude extract was then diluted and applied to a CM Bio-Gel A column. Proteins were eluted with a linear salt gradient as described. Aliquots of each fraction were assayed for lysozyme activity and also analyzed on NaDodSO4/polyacrylamide gel electrophoresis. The lysozyme activity was eluted at 0.25-0.3 M NaCl. The peak of lysozyme activity corresponded exactly with the peak fraction of a 26-kDa protein on NaDodSO4/polyacrylamide gel electrophoresis (data not shown). These fractions were then collected and concentrated as described. To determine the purity of the phage 429 lysozyme, about 20 ug of this solution was applied to a NaDodSO4/polyacrylamide gel and stained with Coomassie blue (Fig. 3). The sample was estimated to be =95% pure. N-Terminal Amino Acid Sequence of the Product of Phage qb29 Gene 15. To determine the N-terminal amino acid sequence of the gene 15 product, the purified protein sample was subjected to amino acid sequencing. The sequence of the first 15 amino acids was determined (Fig. 4). This N-terminal sequence agreed perfectly with that deduced from the DNA sequence of gene 15. These results indicate that the 429 gene 15 product was accurately expressed in E. coli cells and that this protein, when overproduced from the E. coli clone, was not modified at its N terminus.

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FIG. 3. NaDodSO4/polyacrylamide gel electrophoresis pattern of the purified product of phage 429 gene 15 (gp 15). Purified gene 15 product (20 ug) was subjected to NaDodSO4/10%0 polyacrylamide gel electrophoresis and stained with Coomassie blue (lane A). Lane B shows the positions of molecular mass markers in kDa.

has an apparent molecular mass of 26 kDa on NaDodSO4/ polyacrylamide gel (Fig. 3) and comprises about 15% of the total cellular protein in the induced clones (data not shown). The above molecular mass estimation is comparable to the 28-kDa value predicted from the nucleotide sequence (11) and is in agreement with the molecular mass previously reported for the product of 429 gene 15 (9, 10). The gene 15 product was purified to near homogeneity (Fig. 3) and was shown to have bacteriolytic properties (Table 1). This fact is consistent with the slow lysis phenotype exhibited by 429 gene 15 mutants (8) and also agrees with our finding that the amino acid sequence of 429 gene 15 product is homologous with the amino acid sequences of P22 and T4 lysozymes (11). We also have sequenced the first 15 amino acids of the N terminus of the purified protein and have shown that it is in complete agreement with the sequence deduced from the nucleotide sequence (Fig. 4). This confirms the assigned reading frame of gene 15 product in our nucleotide sequence and also shows that the protein was neither processed nor modified during or after expression in E. coli cells. Based on the above results, we conclude that phage 429 gene 15 product is a 28-kDa basic protein with lysozyme activity. To our knowledge, this lysozyme is unique in being purified from a bacteriophage that infects Gram-positive bacteria. The lysozyme of bacteriophage T4, a product of gene e, is the prototype of phage-type lysozymes (1). Recently, a lysozyme from phage P22 has been purified (5). This lysozyme is the second member of phage-type lysozymes reported (2). The amino acid sequence of P22 lysozyme, a product of P22 gene 19, has a 26% homology with that of the T4 lysozyme (2). Weaver et al. (2) have concluded that the P22 lysozyme may provide an evolutionary link between the T4 lysozyme and the goose-type lysozyme. Such a link would is 10

DISCUSSION We have cloned gene 15 of bacteriophage 429 into an E. coli expression vector and overproduced its product. This protein 1 ATG Met

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Table 1. Comparison of the lysozyme activities of E. coli K12AH1A&trp harboring various plasmids Lysozyme specific activity, units per mg of protein Condition Plasmid 0.19 Induced pPlc245 0.15 Induced pMS6 0.36 Uninduced pMS2 20.70 Induced pMS2 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 of each extract. The lysozyme unit was defined as the decrease in the absorbance of this solution after 5 min at room temperature.

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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.

958 Phage

Biochemistry: Saedi et al. Host

Property

Proc. Natl. Acad. Sci. USA 84 (1987) Genome Size

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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 phage-type lysozymes have all evolved from a common ancestor (3). As mentioned above, the amino acid sequence of the product of phage 429 gene 15 shows strong homology with the P22 lysozyme (38%) and weak homology with the T4 lysozyme (18%) (11). Moreover, a T4 lysozyme-deficient mutation can be complemented by the 429 gene 15 product (11). This was demonstrated by the plaque-forming ability of a T4 gene e mutant when plated on E. coli cells harboring the pMS2 plasmid (20). These findings clearly indicate that the 429 lysozyme is structurally and functionally related to the T4 and P22 lysozymes. Since these three phages are considered to be totally unrelated, a close relationship between their lysozymes is quite remarkable (Fig. 5). Some of the properties of T4, P22, and 429 bacteriophages and their lysozymes are compared in Fig. 5. The genomic size of 029 is much smaller than those of P22 and T4, but its lysozyme is much larger than the lysozymes of T4 and P22. However, the homology of 429 lysozyme with the lysozymes of T4 and P22 is located entirely at the N terminus of this enzyme. DNA sequence results indicated that there are long tandem repeats at the nonhomologous C terminus of the 429 gene 15 product, suggesting a gene duplication (11); this unusual structure could be due to involvement in other functions in addition to lysozyme activity. One such function has been suggested to be in phage morphogenesis (8, 10). Grutter et al. (21) have suggested that the C-terminal lobe of T4 lysozyme is directly involved in substrate binding of this protein. If this is also true for the 429 lysozyme, then the unusual structure of this protein's C terminus could be a common requirement for the lysozymes of phages that infect Gram-positive bacteria. However, this hypothesis can only be analyzed when more information about the lysozymes of other phages infecting Gram-positive bacteria becomes available. Although bacteriolytic enzymes produced by various phages have been studied, very few of them belong to the family of phage-type lysozymes. For example, phage X has two genes, R and Rz, that are responsible for cell-wall hydrolysis (22). Gene R codes for a transglucosylase, and the Rz product has endopeptidase activity (22). Both of these enzymes are different from the phage-type lysozymes (22). Bacteriophage T7 gene 3.5 encodes a bacteriolytic enzyme as well. However, the product of this gene is an amidase and is functionally and structurally different from phage-type lysozymes (23). Therefore, if one considers T4 and T2 lysozymes as one member, 429 lysozyme is only the third member of the family of phage-type lysozymes whose pri-

mary sequences have been analyzed so far. For this reason, 429 lysozyme could potentially be a valuable source for investigating lysozyme evolution and the evolutionary origin of bacteriophages. Now that large quantities ofthe product of phage 429 gene 15 can be isolated, it is feasible to elucidate the tertiary structure of this protein and to approach the evolutionary questions at the molecular level. We thank Drs. Harris and Carol Bernstein for their critical evaluation of this manuscript. This investigation was supported by National Institutes of Health Grant GM28013. 1. Jolles, P. & Jolles, J. (1984) Mol. Cell. Biochem. 63, 165-189. 2. Weaver, L. H., Rennell, D., Poteete, A. R. & Matthews, B. W. (1985) J. Mol. Biol. 184, 739-741. 3. Weaver, L. H., Grutter, M. G., Remington, S. J., Gray, T. M. & Matthews, B. W. (1985) J. Mol. Evol. 21, 97-111. 4. Tsugita, A. & Inouye, M. (1968) J. Mol. Biol. 37, 201-212. 5. Rennell, D. & Poteete, A. (1985) Virology 43, 280-289. 6. Reilly, R. E. (1965) Dissertation (Case Western Reserve University, Cleveland, OH). 7. Geiduscheck, E. P. & Ito, J. (1982) in The Molecular Biology of Bacillus, ed. Dabanau, D. (Academic, New York), p. 203-244. 8. Carrascosa, J., Camacho, A., Morreno, F., Mellado, R., Vinuela, E. & Salas, M. (1976) Eur. J. Biochem. 66, 229-241. 9. Hagen, E. W., Reilly, B. E., Tosi, M. E. & Anderson, D. C. (1976) J. Virol. 19, 501-517. 10. Jimenez, F., Camacho, A., De La Torre, J., Vinuela, E. & Salas, M. (1977) Eur. J. Biochem. 73, 57-72. 11. Garvey, K. J., Saedi, M. S. & Ito, J. (1987) J. Nucleic Acids Res., in press. 12. Inouye, M., Imada, M. & Tsugita, A. (1970) J. Biol. Chem. 245, 3479-3484. 13. Remaut, E., Stanssens, P. & Fiers, W. (1983) Nucleic Acids Res. 11, 4677-4689. 14. Ito, J. (1978) J. Virol. 28, 895-904. 15. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 16. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65,

499-560. 17. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 18. Morita, T., Hara, S. & Matsushima, Y. (1978) J. Biochem. 83, 893-903. 19. Bradford, M. (1976) Anal. Biochem. 72, 248-254. 20. Garvey, K. J. (1986) Dissertation (University of Arizona, Tucson, AZ). 21. Grutter, M. G. & Matthews, B. W. (1982) J. Mol. Biol. 154, 525-535. 22. Bienkowski-Szewcyk, K. & Taylor, A. (1980) Biochim. Biophys. Acta 615, 489-4%. 23. Inouye, M., Arnheim, N. & Sternglanz, R. (1973) J. Biol. Chem. 248, 7247-7252.