Proc. Natl. Acad. Sci. USAVol. 84, pp. 955-958, February 1987Biochemistry

Cloning and purification of a unique lysozyme produced by Bacillusphage 4P29

(phage 429 gene 15/phage-type lysozyme/lysozyme evolution)

MOHAMMAD S. SAEDI, KEVIN J. GARVEY, AND JUNETSU ITODepartment 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)

ABSTRACT A DNA fragment of the bacteriophage 429chromosome, encoding the entire sequence of 4)29 gene 15, hasbeen cloned into the Escherichia coli expression vector pPLc245under the control of the phage X major leftward promoter, PL.Upon heat induction, a protein with an apparent molecularmass of 26 kDa was overproduced. The molecular mass of thisprotein corresponds to the 28 kDa predicted for the product ofgene 15 from its nucleotide sequence. The overproducedprotein has been purified to near homogeneity and confirmedto be the product of gene 15 by amino acid sequence analysisof its N terminus. The purified product of gene 15 has alysozyme activity similar to other phage-type lysozymes: prod-ucts of phage T4 gene e and of phage P22 gene 19. However,to our knowledge 429 lysozyme is structurally unique amongthe phage-type lysozymes.

Lysozymes are widespread in nature and have been isolatedfrom a variety of organisms (1). These enzymes have pro-vided a useful model system for studying protein structure,mechanism of enzyme action, immunochemistry, and evo-lution (1). Lysozymes are generally classified into fourdistinct families: chicken-type lysozyme, goose-type lyso-zyme, phage-type lysozyme, and the bacterial lysozymeproduced by Streptomyces erythraeus (1). The amino acidsequences of lysozymes within a given family are clearlyrelated, but there is no obvious sequence homology betweenone family and another (2). However, when the three-dimen-sional structure oflysozymes from goose-type, chicken-type,and phage-type are compared, considerable similarities arenoted (3). Thus, it has been suggested that these three typesof 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 acidsequence of 18 chicken-type lysozymes are already known(1). Surprisingly, the phage-type lysozymes, with the excep-tion of T4 and T2 lysozymes, are not well explored (1). Thephage T2 and T4 lysozymes differ by only three amino acids(4). Recently, phage P22 gene 19 (lysozyme gene) has beensequenced, and the amino acid sequence of the gene has beendeduced (5). It was found that there is significant homologybetween the P22 lysozyme and the T4 lysozyme, indicatingthat these phage lysozymes are evolutionarily related (2).Perhaps this finding is not surprising because both T4 (acoliphage) and P22 (a Salmonella phage) infect Gram-nega-tive bacteria from the same family, Enterobacteriaceae.Thus, it seems of considerable interest to investigate lyso-zymes from different phage systems.We have been studying bacteriophage 429, which infects

the Gram-positive bacterium Bacillus subtilis (6). 429 is asmall lytic phage whose genome is a linear double-strandedDNA with terminal proteins attached covalently at each 5'

end (7). The genes involved in the lytic function of this phagehave not been well characterized. Genetic studies haveshown that mutations in genes 14 and 15 result in a delayedlysis phenotype, with normal phage development in infectedcells (8-10). We recently sequenced genes 14 and 15 andfound that the deduced amino acid sequence of gene 15 hasstrong homology with the lysozyme of phage P22 and weakbut significant homology with the lysozyme of phage T4 (11).However, the DNA sequence data revealed that phage 429gene 15 encodes a 28-kDa protein, which is substantiallylarger than both of the T4 and P22 lysozymes; the T4 and P22lysozymes are 18.7 and 16.1 kDa, respectively (5, 12).

In this communication, we report the cloning and purifi-cation of phage 429 gene 15 protein. Our results clearlyestablish that the product of gene 15 is a unique phagelysozyme.

MATERIALS AND METHODSAll enzymes were purchased from Bethesda Research Lab-oratories, and chemicals were from Sigma. Escherichia colistrain K12AHlAtrp and plasmid pPLc245 were obtained fromRemaut et al. (13). The freeze-dried culture of Micrococcuslysodeikticus was purchased from United States Biochemical(Cleveland, OH). The methionine assay medium was ob-tained from Difco.

Cloning of Phage 429 Gene 15. Bacteriophage 429 wasprepared by CsCl gradient purification, and DNA was ex-tracted by NaDodSO4 and proteinase K treatment as de-scribed (14). Phage 029 HindIII fragment F, containing theentire coding region of gene 15, was isolated from a total )29HindIII digest by gel electrophoresis (15). The isolatedfragment was ligated into the HindIII site of plasmidpPLc245, which is located downstream from a phage X majorleftward promoter PL, and transformants were obtained instrain K12AHlAtrp by selecting for resistance to ampicillin(100 gg/ml). Among the ampicillin-resistant colonies, thoseharboring recombinant plasmids were selected by miniplas-mid preparation (15). The plasmid with the insert in the sameorientation as the PL promoter, pMS2, and the plasmid withthe insert in the reverse orientation, pMS6, were identified byrestriction enzyme digest analyses. The directions of theinserts were also verified by DNA sequence analysis usingthe Maxam and Gilbert technique (16).

Protein Analysis of the Induced Clones. To examine theexpression ofthe cloned gene 15 in response to a temperatureshift, the cells harboring the above plasmids were grown inLB medium (15) with ampicillin (100 ,ug/ml) at 30°C. Bacteriawere then harvested at midlogarithmic phase by centrifuga-tion and were resuspended in the same volume ofprewarmedM9 minimal medium (15) supplemented with 50% methionineassay medium and 50 jig of tryptophan per ml. After 1 hr ofincubation at 30°C, a portion of the culture was shifted to

Abbreviation: bp, base pair(s).

955

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

420C. After 2 hr, 50 ,ul of the induced and uninduced sampleswere labeled for 10 min with 10 gCi (1 Ci = 37 GBq) of[35S]methionine (1100 Ci/mmol; New England Nuclear). Thelabeled cells were then collected and resuspended in 20 .l ofsample buffer as described (17). Aliquots of these sampleswere then boiled for 5 min prior to loading and were analyzedby NaDodSO4/10% polyacrylamide gel electrophoresis (17).The gel was dried, and the labeled proteins were visualizedby autoradiography.

Protein Purification and Amino Acid Sequencing. E. coliK12AHlAtrp carrying pMS2 was grown in 2 liters of LBmedium 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 at420C for 2 hr. The cells were harvested, resuspended in 50 mlof TR buffer (50 mM Tris, pH 7.5/5% glycerol/i mMEDTA/1.4 mM 2-mercaptoethanol) containing 0.1% Brij 58,and stored at -70'C. When cell extracts were prepared forlysozyme activity assays, Brij 58 was omitted from the abovebuffer. The frozen sample was then thawed at 370C (whichcaused most of the cells to lyse) and was kept on ice. Allsubsequent steps were then carried out at 4TC. Crude cellextract was then prepared by sonicating the above cellsuspension with five bursts of20-sec duration (using a Brasonsonic-power sonifier model SilO) and removing the celldebris by centrifugation at 20,000 x g for 30 min. Thesupernatant was diluted 1:3 in TR buffer and applied to a CMBio-Gel A (Bio-Grad) column (2.5 x 20 cm). The column waswashed with three-bed-volumes of TR buffer, and the pro-teins were eluted by a linear gradient of 0-0.4 M NaCl in 200ml of TR buffer at a rate of 10 ml/hr. Fractions (2 ml) werecollected and assayed for lysozyme activity by their ability tolyse M. lysodeikticus (18). Aliquots ofeach fraction were alsosubjected to NaDodSO4/polyacrylamide gel electrophoresis.The fractions with peak lysozyme activity, which corre-sponded to the peak fractions of a 26-kDa protein, were thenpooled.To prepare the sample for amino acid sequence analysis,

the pooled fractions were dialyzed against 0.005% NaDod-SO4, and the protein concentration was measured (19). Thissample was then concentrated in a Savant Speed Vacconcentrator to 4 mg/ml. Of this concentrated solution, 0.1ml was subjected to amino acid sequence analysis using aBeckman 890M sequencer.

B G K HMI E J Da a a .. . ..0

,Hindill

A N F C

I

Hiridlll F

FIG. 1. Cloning of 429 gene 15. The HindIII restriction map ofbacteriophage 4)29 is schematically diagramed. The HindIII fragmentF containing gene 15 was isolated and ligated into the HindIII site ofplasmid pPLc245 (13). The hybrid plasmid pMS2 was then isolatedas 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 15would be expressed from pMS2, cells containing this plasmidwere grown at 30°C to midlogarithmic phase and then wereshifted rapidly to 42°C. After a 2-hr incubation, aliquots wereremoved and labeled with [35S]methionine, and the totalprotein was analyzed on NaDodSO4/polyacrylamide gels asdescribed. Cells transformed with the recombinant plasmidpMS2 overproduced at 42°C a labeled polypeptide with anapparent molecular mass of =26 kDa (Fig. 2). The other cells,containing either plasmid pPLc245 or pMS6, did not synthe-size this protein. The molecular mass of the overproducedprotein at 42°C was consistent with the reported value for theproduct of phage 429 gene 15 (10). Moreover, it also agreeswith the 28-kDa value deduced from the nucleotide sequenceanalysis of 429 gene 15 (11).

1 2 3 4 5 6

RESULTSCloning and Overproduction of Phage 429 Lysozyme. Our

nucleotide sequence analysis revealed that the phage 429HindIII fragment F contains the complete open reading framefor gene 15 (11). Therefore, we isolated the 1330-base-pair(bp) HindIII fragment F and inserted it into the plasmidpPLc245 (13) (Fig. 1). This plasmid has a single HindIII sitelocated downstream of a phage X PL promoter. Therefore,transcription ofthe genes inserted at this site will be under thecontrol of the cI repressor. The recombinant plasmid wastransferred into E. coli K12AHlAtrp by transformation (15).The clones harboring a plasmid with the insert in the correctorientation relative to the PL promoter were identified byrestriction enzyme digestion analysis. One of these plasmidswas chosen for further study and designated pMS2 (Fig. 1).Another plasmid, pMS6, carrying the insert in the reversedirection was also selected. The orientation of phage 429gene 15 was further verified by DNA sequence analysis (datanot shown).

Strain K12AHlAtrp is a phage X lysogen carrying a ther-mosensitive mutation in its cI repressor gene, cIts857 (13).When growing cells containing the above plasmids are shiftedfrom 30°C to 42°C, the cI repressor of the prophage isinactivated, and the phage XPL promoter on the plasmid is

43 --

18.4-+_ _ _ ..~~~

FIG. 2. Detection of the protein overproduced oy temperatureinduction of E. coli K12AHlAtrp harboring various plasmids. Thecells were grown to midlogarithmic phase at 30°C, and a portion ofeach culture was shifted to 42°C for 2 hr. Aliquots of the induced anduninduced culture of each sample were then labeled with [35S]me-thionine and subjected to NaDodSO4/polyacrylamide gel electro-phoresis as described. Lanes: 1, 3, and 5, uninduced samples of cellsharboring pPLc245, pMS2, and pMS6, respectively; 2, 4, and 6, thecorresponding induced samples. The positions ofthe molecular massmarkers (in kDa) were determined from prestained markers pur-chased from Bethesda Research Laboratories. The position of the26-kDa gene 15 product (gp 15) is also shown.

I I I a=

I a A I" I I I I

-"Amm-

956 Biochemistry: Saedi et al.

4=-

limmoffall

4w,-z.s,.-..

.::.

+-gp 15.....

dow,vm.IM

Proc. Natl. Acad. Sci. USA 84 (1987) 957

Table 1. Comparison of the lysozyme activities of E. coliK12AH1A&trp harboring various plasmids

Lysozymespecific activity,

Plasmid Condition units per mg of protein

pPlc245 Induced 0.19pMS6 Induced 0.15pMS2 Uninduced 0.36pMS2 Induced 20.70

Crude extracts of each culture were prepared, and the lysozymeactivity and protein concentration were measured. To measurelysozyme activity, a freeze-dried culture of M. lysodeikticus wassuspended 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.dofeach extract. The lysozyme unit was defined as the decrease in theabsorbance of this solution after 5 min at room temperature.

To determine the enzymatic activity of the gene 15 prod-uct, crude extracts of the induced and uninduced cellsharboring pMS2, pMS6, or pPLc245 were prepared. Lyso-zyme activity ofeach extract was then measured by using M.lysodeikticus as substrate (Table 1). Crude extracts preparedfrom the induced cells containing pPLc245 or pMS6 and theuninduced cells containing pMS2 showed little lysozymeactivity. On the other hand, the induced cells containingpMS2 exhibited increased lysozyme activity under theseconditions. These results indicate that the product of phage429 gene 15 is a lysozyme.

Purification of Phage 429 Gene 15 Product. To purify thegene 15 product, cells containing pMS2 were grown at 30'Cand shifted to 420C at midlogarithmic phase. After 120 min ofincubation at 420C, cells were pelleted and resuspended in TRbuffer containing Brij 58, incubated at -70'C, and brokenopen by sonication to prepare crude extract. The crudeextract was then diluted and applied to a CM Bio-Gel Acolumn. Proteins were eluted with a linear salt gradient asdescribed. Aliquots of each fraction were assayed for lyso-zyme activity and also analyzed on NaDodSO4/polyacryl-amide gel electrophoresis. The lysozyme activity was elutedat 0.25-0.3 M NaCl. The peak of lysozyme activity corre-sponded exactly with the peak fraction ofa 26-kDa protein onNaDodSO4/polyacrylamide gel electrophoresis (data notshown). These fractions were then collected and concentrat-ed as described. To determine the purity of the phage 429lysozyme, about 20 ug of this solution was applied to aNaDodSO4/polyacrylamide gel and stained with Coomassieblue (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 acidsequence of the gene 15 product, the purified protein samplewas subjected to amino acid sequencing. The sequence of thefirst 15 amino acids was determined (Fig. 4). This N-terminalsequence agreed perfectly with that deduced from the DNAsequence ofgene 15. These results indicate that the 429 gene15 product was accurately expressed in E. coli cells and thatthis protein, when overproduced from the E. coli clone, wasnot modified at its N terminus.

DISCUSSIONWe have cloned gene 15 of bacteriophage 429 into an E. coliexpression vector and overproduced its product. This protein

1 5

A B

* 43

gp 15 25.7

_110 18.4

- 14.3

FIG. 3. NaDodSO4/polyacrylamide gel electrophoresis patternof the purified product of phage 429 gene 15 (gp 15). Purified gene 15product (20 ug) was subjected to NaDodSO4/10%0 polyacrylamidegel electrophoresis and stained with Coomassie blue (lane A). LaneB 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 thetotal cellular protein in the induced clones (data not shown).The above molecular mass estimation is comparable to the28-kDa value predicted from the nucleotide sequence (11) andis in agreement with the molecular mass previously reportedfor 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 pheno-type exhibited by 429 gene 15 mutants (8) and also agreeswith our finding that the amino acid sequence of 429 gene 15product is homologous with the amino acid sequences of P22and T4 lysozymes (11). We also have sequenced the first 15amino acids ofthe N terminus ofthe purified protein and haveshown that it is in complete agreement with the sequencededuced from the nucleotide sequence (Fig. 4). This confirmsthe assigned reading frame of gene 15 product in our nucle-otide sequence and also shows that the protein was neitherprocessed nor modified during or after expression in E. colicells. Based on the above results, we conclude that phage 429gene 15 product is a 28-kDa basic protein with lysozymeactivity. To our knowledge, this lysozyme is unique in beingpurified from a bacteriophage that infects Gram-positivebacteria.The lysozyme of bacteriophage T4, a product of gene e, is

the prototype of phage-type lysozymes (1). Recently, alysozyme from phage P22 has been purified (5). This lyso-zyme is the second member of phage-type lysozymes report-ed (2). The amino acid sequence of P22 lysozyme, a productof P22 gene 19, has a 26% homology with that of the T4lysozyme (2). Weaver et al. (2) have concluded that the P22lysozyme may provide an evolutionary link between the T4lysozyme and the goose-type lysozyme. Such a link would

10 is

ATG CAA ATU TCA CAA GCG GGT ATC MC TTA AUT MG AGC TTT GAGMet 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 ofthe first 45 bp of the 5' end of gene 15, which are described elsewhere (11). The bottom line represents the respective amino acid sequence ofgene 15 product obtained by sequencing the purified protein.

Biochemistry: Saedi et al.

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

Phage Host

P22 S. typhimurium(Gram -)

029 B. subtilis(Gram +)

T4 E. coli(Gram -)

Genome Structural LysozymeProperty Size Gene Mr

Lysogenic 43 kb 19 16.1 kDa NH2///////////////// ////> COOH

Lyt i c

Lytic

19 kb 15 28 kDa NH

166 kb e

12 *HOOH

18.7 kfa NH . . . OOH2... OOH

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 areain P22 lysozyme represents the strong homology and the stippled area in T4 lysozyme represents the weak homology observed between theseproteins and the 429 lysozyme. The details have been described elsewhere (11).

strongly favor the notion that goose-type, chicken-type, andphage-type lysozymes have all evolved from a commonancestor (3). As mentioned above, the amino acid sequenceof the product of phage 429 gene 15 shows strong homologywith the P22 lysozyme (38%) and weak homology with the T4lysozyme (18%) (11). Moreover, a T4 lysozyme-deficientmutation can be complemented by the 429 gene 15 product(11). This was demonstrated by the plaque-forming ability ofa T4 gene e mutant when plated on E. coli cells harboring thepMS2 plasmid (20). These findings clearly indicate that the429 lysozyme is structurally and functionally related to theT4 and P22 lysozymes. Since these three phages are consid-ered to be totally unrelated, a close relationship between theirlysozymes 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 sizeof 029 is much smaller than those of P22 and T4, but itslysozyme is much larger than the lysozymes of T4 and P22.However, the homology of 429 lysozyme with the lysozymesof T4 and P22 is located entirely at the N terminus of thisenzyme. DNA sequence results indicated that there are longtandem repeats at the nonhomologous C terminus of the 429gene 15 product, suggesting a gene duplication (11); thisunusual structure could be due to involvement in otherfunctions in addition to lysozyme activity. One such functionhas been suggested to be in phage morphogenesis (8, 10).Grutter et al. (21) have suggested that the C-terminal lobe ofT4 lysozyme is directly involved in substrate binding of thisprotein. If this is also true for the 429 lysozyme, then theunusual structure of this protein's C terminus could be acommon requirement for the lysozymes of phages that infectGram-positive bacteria. However, this hypothesis can onlybe analyzed when more information about the lysozymes ofother phages infecting Gram-positive bacteria becomes avail-able.Although bacteriolytic enzymes produced by various

phages have been studied, very few of them belong to thefamily of phage-type lysozymes. For example, phage X hastwo genes, R and Rz, that are responsible for cell-wallhydrolysis (22). GeneR codes for a transglucosylase, and theRz product has endopeptidase activity (22). Both of theseenzymes are different from the phage-type lysozymes (22).Bacteriophage T7 gene 3.5 encodes a bacteriolytic enzyme aswell. However, the product of this gene is an amidase and isfunctionally and structurally different from phage-typelysozymes (23). Therefore, if one considers T4 and T2lysozymes as one member, 429 lysozyme is only the thirdmember 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 forinvestigating lysozyme evolution and the evolutionary originofbacteriophages. Now that large quantities ofthe product ofphage 429 gene 15 can be isolated, it is feasible to elucidatethe tertiary structure of this protein and to approach theevolutionary questions at the molecular level.

We thank Drs. Harris and Carol Bernstein for their criticalevaluation of this manuscript. This investigation was supported byNational Institutes of Health Grant GM28013.

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