JOURNAL OF BACTERIOLOGY, Mar. 1976, p. 1120-1126Copyright C) 1976 American Society for Microbiology

Vol. 125, No. 3Printed in U.SA.

Bacteriophage Resistance in Bacillus subtilis 168, W23, andInterstrain Transformants

RONALD E. YASBIN,1 * VERNON C. MAINO,2 AND FRANK E. YOUNGLaboratory ofBiochemical Genetics, National Heart and Lung Institute, Bethesda, Maryland 20014,

and Department of Microbiology, University of Rochester School of Medicine and Dentistry,Rochester, New York 14642

Received for publication 14 October 1975

Strains of Bacillus subtilis 168 deficient in glucosylated teichoic acid vary intheir resistance to bacteriophage infection. Although glucosylated teichoic acidis important for bacteriophage attachment, the results demonstrate that alter-nate receptor sites exist. Non-glucosylated cell wall mutants could be assignedto specific classes (gtaA, gtaB, gtaC) by their pattern of resistance to threeclosely related bacteriophages (G25, Oe, SP82). In addition to glucosylation, thetype of teichoic acid was also important for bacteriophage attachment. B.subtilis strains 168 and W23 have different teichoic acids in their cell walls andhave varied susceptibilities to bacteriophage infection. Transfer of bacterio-phage resistance from strain W23 into a derivative of strain 168 was accom-plished. The resistant bacteria obtained were impaired in their ability to adsorbbacteriophage and in their capacity to be transfected by bacteriophage deoxyri-bonucleic acid.

The importance of teichoic acids as a bacte-riophage receptor site in the wall of the bacte-rium Bacillus subtilis has been demonstrated(2, 8, 24). Specifically, three classes of bacterio-phage-resistant mutants of B. subtilis strain168 have been shown to lack glucosylated tei-choic acid in their cell walls (24, 25). The gluco-sylation of cell wall teichoic acids is regulatedby the products of the gtaA +, gtaB +, and gtaC +

genes (24). These three genes have beenmapped (25), and the proteins coded by two ofthe genes have been characterized (4, 12). Phos-phoglucomutase (EC 2.7.5.1) is the product ofthe gtaC+ gene, whereas uridine 5'-diphos-phate (UDP)-glucose:polyglycerol teichoic acidglucosyltransferase (EC 1.4.1) is a product ofthe gtaA + gene. Although no enzyme has beenidentified for the gtaB + gene, it has been sug-gested that this gene codes for an inactive phos-phoglucomutase monomer (13). In the initialstudies, when only a limited number of bacteri-ophages were examined, it appeared that thebacterial mutants unable to glucosylate tei-choic acid were resistant to certain bacterio-phages regardless of whether the mutation wasat the gtaA, gtaB, or gtaC locus (24). In thisreport, we have expanded these studies and cannow demonstrate that the degree of bacterio-

' Present address: Department of Biology, BrookhavenNational Laboratory, Upton, N.Y. 11973.

2 Present address: Division ofAllergy and Clinical Immu-nology, National Jewish Hospital and Research Center,Denver, Colo. 80206.

phage resistance is strongly affected by the na-ture of the genetic defect.

Glucosylated teichoic acid has also beenshown to play an essential role in the attach-ment of some of the Bacillus bacteriophages toB. subtilis strain W23 (8, 24). B. subtilis strainsW23 and 168 differ in the content of their tei-choic acids (5, 7) and their susceptibility tocertain bacteriophages (3, 16; B. E. Reilly,Ph.D. thesis, Western Reserve Univ., Cleve-land, Ohio, 1965). Furthermore, B. subtilisstrain W23 is lysogenic for defective bacterio-phage PBSZ, whereas strain 168 is lysogenic fordefective bacteriophage PBSX (14, 18). To ex-plore the effect of variability of the cell surfaceon viral infection, we transferred bacteriophageresistance from strain W23 to strain 168 viadeoxyribonucleic acid (DNA)-mediated trans-formation. The results of this transfer as wellas the patterns of bacteriophage resistance instrain W23 indicate the variety and complexityofmechanisms by which bacteria interfere withbacteriophage development.

MATERIALS AND METHODSStrains and methods of propagation. The bacte-

rial strains used in this study are listed in Table 1.The propagation of bacteriophage and the mainte-nance of the bacterial cultures were described previ-ously (21, 22). Bacteriophage SP82 was obtainedfrom M. Green.

Media and procedures for genetic exchange.Modified M agar and broth were prepared as previ-ously described (22). The procedures for transforma-

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BACTERIOPHAGE RESISTANCE 1121

TABLE 1. Bacterial strains used

Strain Genotypea Origin and remarks

BR151 lys-3, trpC2, metBlO Competent derivative of B. subtilis 168RUB807 lys-3, trpC2, metB10, gtaC20 Phage-resistant marker transformed into

BR151 using donor DNA from a spontaneous)29-resistant strain obtained by V. C. Maino

(168/qb29)RUB808 lys-3, trpC2, metB10, gtaA20 Phage-resistant marker transformed into

BR151 using donor DNA from a spontaneous+105-resistant strain obtained by S. Zahler(CU548)

RUB809 lys-3, trpC2, metB10, gtaA21 Isolated similarly to RUB807RUB810 Iys-3, metBlO, gtaB20 Isolated similarly to RUB807RUB811 lys-3, trpC2, metB10, gtaC21 Isolated similarly to RUB807RUB812 lys-3, trpC2, metB10, gtaB21 Isolated similarly to RUB807RUB815 ery-1 A spontaneous erythromycin-resistant deriva-

tive of B. subtilis W23RUB818b A prototrophic derivative of BR141RUB818(SP02) Lysogenic for bacteriophage SP02RUB818(105) Lysogenic for bacteriophage O105RUB821 lys-3, trpC2, metB10, 4Rc Isolated by resistance to' bacteriophages SPP1

and SP02cl2 after transformation of strainBR151 with DNA obtained from strainRUB815

RUB822 lys-3, trpC2, metB10, OR Isolated as was RUB821RUB823 lys-3, trpC2, metB10, OR Isolated as was RUB821RUB824 lys-3, trpC2, metB10, O)R Isolated as was RUB821

a Symbols as defined in reference 26 and references 12, 24, 25, and V. C. Maino (Ph.D. thesis), for gtastrains.

b Obtained after three separate transformations with nonsaturating concentrations of 168 T+ DNA.c Phenotypically phage resistant.

tion, transfection, and isolation ofDNA were identi-cal to those previously used in our laboratory (22,23).

Determination of plating efficiencies. Bacterialcultures were grown at 37 C in modified M broththat had not been supplemented with Ca2 , Mg2+, orMn2+. After the cells had ceased exponential growth(optical density of 120 Klett units on a Klett-Sum-merson colorimeter, filter no. 66), 0.2 ml of the bac-terial culture was added to 2.0 ml of modified Msemisolid agar containing 0.1 ml of the appropriatedilution of the bacteriophage stock, and the mixturewas poured onto modified M agar. This procedurewas used for all bacteriophage studied except 429.The plating efficiency of bacteriophage 429 was de-termined by using a modified M overlay on tryptoseblood agar base (Difco). Assays for bacteriophage429 were done at 30 C, whereas all other quantita-tion of infectious centers were performed at 37 C.

Determination of adsorption efficiencies. Bacte-rial cultures were grown in modified M broth with-out Mg2+, Ca2+, and Mn2+ at 37 C to an opticaldensity of 120 Klett units, centrifuged (12,000 x g for20 min at 4 C), and suspended in Spizizen's minimalsalts (17) containing 3.7% formaldehyde (Fisher).The suspension was kept at 4 C for 30 min before thecells were washed three times with modified Mbroth and finally resuspended in half the originalvolume in modified M broth. The bacteriophage andthe formaldehyde-treated bacteria were mixed toyield a multiplicity of infection of 10-4 to 10-s, incu-

bated at 37 C for 20 min, and centrifuged (8,000 x gfor 5 min at room temperature), and the number ofplaque-forming units in the broth was determined.Enzyme assays. UDP-glucose:pyrophosphorylase

(EC 2.7.7.9), UDP-glucose:polyglycerol teichoic acidglucosyltransferase, and phosphoglucomutase weremeasured as previously specified (4, 12).

RESULTSIsolation and identification of bacterio-

phage-resistant mutants of B. subtilis strain168. DNA was isolated from spontaneouslyarising bacteriophage-resistant mutants of B.subtilis, and the resistance trait was trans-formed into strain BR151. These bacteriophage-resistant mutants were found to lack glucosy-lated teichoic acid in their cell walls (V. C.Maino, Ph.D. thesis, Univ. of Rochester, Roch-ester, N.Y., 1972). Previously, we establishedthree classes of bacteriophage-resistant mu-tants ofB. subtilis that lacked glucosylated cellwall teichoic acid due to their inability to syn-thesize UDP-glucose:polyglycerol teichoic acidglucosyltransferase or phosphoglucomutase(24, 25). Bacteria lacking phosphoglucomutaseare defective in the gtaC gene, whereas thosebacteria that lack UDP-glucose:polyglycerolteichoic acid glucosyltransferase have lost a

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1122 YASBIN, MAINO, AND YOUNG

functional gtaA gene. Bacteria that have bothof these enzymes but still lack glucosylated tei-choic acid are deficient in the gtaB gene prod-uct. The class for each of the isolated mutantswas determined, and representatives of eachclass are shown in Table 2.

Previously our laboratory demonstrated thatsome bacteriophages differed in their ability toinfect non-glucosylated teichoic acid cell wallmutants in semisolid agar and in liquid broth(24). To further investigate this phenomenon,representatives of each bacteriophage-resistantclass were infected with nine B. subtilis bacte-riophages. There were major differences in theability of bacteriophages to infect these mu-tants (Table 3). Bacteriophages SPOl and 429were completely dependent on glucosylated cellwall teichoic acid for attachment. Additionally,bacteriophage 425 was unable to successfullyinfect gtaB and gtaC mutants, although itcould infect gtaA mutants with reduced effi-ciency. Bacteriophages 4e and SP82 were alsounable to infect gtaC mutants. BacteriophageSP82 was severely inhibited in its ability toinfect cells carrying gtaB, and bacteriophage

TABLE 2. Enzyme activitiesEnzyme activity (nmol/min/mg

of protein)

Strain Genotype TagPGMa UDPG- transfer-GM PPaseb ase

(x 10-3)

BR151 gta+ 68.5 3.6 3.08RUB807 gtaC20 <0.02 3.8 0.95RUB811 gtaC21 <0.02 3.3 2.60RUB808 gtaA20 51.5 3.6 <0.01RUB809 gtaA21 56.0 2.1 <0.01RUB810 gtaB20 61.0 3.6 3.40RUB812 gtaB21 52.5 4.4 1.80

a PGM, Phosphoglucomutase.b UDPG-PPase, UDP-glucose:pyrophosphorylase.c Tag transferase, UDP-glucose:polyglycerol tei-

choic acid glucosyltransferase.

J. BACTERIOL.

4)105 was totally unable to infectgtaA mutants.In all remaining cases, the bacteriophagescoul,d infect the non-glucosylated cell wall tei-choic acid mutants with reduced efficiency.Bacteriophages SPOl, SPP1, and 41 appearedto be least dependent on cell wall-glucosylatedteichoic acid for infection. It is important tonote that all of these infections occurred insemisolid agar.

Similarly, the ability of these nine bacterio-phages to attach to these bacteria in liquidmedia was determined (Table 4). Although bac-teriophage SPOl was unable to infect gtaA mu-tants, it appeared to attach to these bacteria inliquid media. On the other hand, bacteriophageSPP1 infected gtaA, gtaB, and gtaC mutants,although in liquid media it did not attach tothese same bacteria at wild-type levels. Addi-tionally, bacteriophage 4e infected gtaB mu-tants in agar, but in liquid it attached poorly tothese cells. These conflicting results will bediscussed later. However, it is important tonote that, generally, the lack of a glucosylatedteichoic acid in the cell wall results in a de-crease in the ability of bacteriophages to at-tach. These data confirm the importance of glu-cosylated teichoic acid as a major bacteriophagereceptor.

TABLE 4. Efficiency of adsorption

Bacterio- Efficiency of adsorption (%Pphages BR151 RUB808 RUB810 RUB807studied (gta+) (gtaA) (gtaB) (gtaC)

SPOl 99 92 12 12SPO2 98 65 37 36SP82 94 21 18 18SPP1 91 8 4 74)1 100 50 42 214oe 100 100 8 9)105 84 13 26 24

4)29 48 3 8 54)25 100 74 20 1

a Efficiency of adsorptioneach strain.

of bacteriophages for

TABLE 3. Efficiency ofplatinga

Bacteriophages Bacterial strainsstudied BR151 (gta+) RUB808 (gtaA) RUB810 (taB) RUB807 (gtaC)SP01 1.0 0 (<10-8) 0 (<10-8) 0 (<10-8)SPO2 1.0 4.3 x 10-1 3.5 x 10-' 2.3 x 10-1SP82 1.0 4.0 x 10-2 <1.0 X 10-4 0 (<10-i)SPP1 1.0 6.5 x 10-1 7.6 x 10-1 3.0 x 10-1)1 1.0 4.7 x 10-l 5.9 x 10-l 7.9 X 10'

4)e 1.0 6.7 x 10-' 3.1 x 10-1 0 (<10-8)4)105 1.0 0 (<10-8) 4.3 x 10-l <1 x 10-4029 1.0 0 (<10-8) 0 (<10-8) 0 (<10-8)425 1.0 5.0 x 10-1 0 (<10-8) 0 (<10-8)

a Efficiency ofthe bacteriophages plating on strains RUB808, RUB810, and RUB807 as compared to strainBR151.

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Bacteriophage resistance in B. subtilisstrain W23. B. subtilis strain W23 differs sig-nificantly from B. subtilis strain 168 both in thechemical composition of the cell wall and thesusceptibility to bacteriophage infection. StrainW23 has both polyribitol phosphate and gluco-syl polyribitol phosphate in its cell wall (5),whereas the teichoic acid in the cell wall ofstrain 168 contains glucosyl polyglycerol-phos-phate (1, 7). Several of the Bacillus bacterio-phages can successfully infect only strain W23or only strain 168 (Reilly, Ph.D. thesis, 1965).Additionally, several studies have revealedthat certain of the bacteriophages that have 5-hydroxymethyluracil in their DNA, instead ofthymine, plate with a reduced efficiency onstrain W23 as compared to strain 168 (15, 16;Reilly, Ph.D. thesis, 1965). This reduced plat-ing efficiency occurs even if the bacteriophageshave been previously grown on strain W23.Therefore, bacteriophages are either unable toattach to one of the strains or the two strainshave different capabilities for supporting bacte-riophage replication. To investigate these pos-sibilities, a derivative of strain W23 (RUB815)and our standard laboratory strain BR151 (aderivitive of 168) were examined for their ca-pacity to adsorb and to replicate several of theBacillus bacteriophages (Table 5). The eightbacteriophages used can be divided into twogroups. Group I was unable to infect or adsorbto strain RUB815. However, group II could ad-sorb equally well to both strains, although itinfects strain RUB815 with a reduced efficiency(Table 5). The results imply that polyglycerol-phosphate cannot be substituted by polyribitol-

TABLE 5. Bacteriophage resistance in strainRUB815

B. subtilis 168, B. subtilis W23, strainBacterio- strain BRi51 RUB815

phages stud-ied PE AEb AE

(%) P

Group ISP02cl-2 1.0 100 0 (<10-9) 11SPP1 1.0 98 0 (<10-8) 14105c1-z 1.0 90 0 (<10-8) 0429 1.0 65 0 (<10-7) 20

Group IISPOl 1.0 100 5.5 x 10-4 100SP82 1.0 95 4.5 x 10-4 99425 1.0 100 8.2 x 10- 100q6e 1.0 100 3.6 x 10-4 100

a Plating efficiency (PE) of bacteriophages foreach strain as compared to strain BR151.

b Adsorption efficiency (AE) of bacteriophages ex-pressed as percentage of adsorption for each strain.

phosphate teichoic acid as a receptor site forgroup I bacteriophages. On the other hand,group II bacteriophages can attach equally wellto both types of teichoic acid. In addition, thedata also suggest that strain RUB815 is unableto complete some step(s) in the growth cycle ofthe group II bacteriophages as efficiently asstrain BR151.Transfer of bacteriophage resistance from

B. subtilis strain W23 into strain 168. Theresults presented in Tables 3 and 4 demonstratethe requirement of glucosylated teichoic acidfor the attachment of bacteriophages 429 andSPOl. However, the data shown in Table 5 indi-cate that the type of teichoic acid is of impor-tance for the attachment of bacteriophagesSP02, '105, SPP1, and 029, but not for bacterio-phage SPOl. Therefore, an attempt was made totransfer the bacteriophage resistance pattern ofstrain RUB815 into the genetic background ofstrain BR151. DNA isolated from strainRUB815 was added to competent bacteria ofstrain BR151. After transformation, the recipi-ent cells were placed on trytose blood agar baseagar for 3 h at 37 C and then challenged withboth bacteriophages SPP1 and SP02cl-2. Thebacteriophage-resistant colonies were found ata frequency of 10-7. This low frequency oftransformation was not the result of mutationsince no colonies occurred in the absence ofDNA or if the DNA was pretreated with deoxy-ribonuclease. Four of the resistant colonieswere isolated, cloned, and tested for their abil-ity to be infected by several of the Bacillusbacteriophages (Table 6). The four isolatedstrains (RUB821-824) were all resistant to in-fection by bacteriophages 029 and 4105. How-ever, the resistance patterns for the other twogroup I bacteriophages (SPP1, SP02) were com-plicated. Bacteriophage SP02 plated with a re-duced efficiency on strains RUB821, RUB822,and RUB823, whereas bacteriophage SPP1plated with a reduced efficiency on strainsRUB821 and RUB822 (Table 6). Strain RUB823was totally resistant to bacteriophage SPP1 andstrain RUB824 was totally resistant to bacterio-phages SPP1 and SP02. Interestingly, thegroup II bacteriophages were also reduced intheir ability to successfully infect these resist-ant strains. Bacteriophage SPOl was unable toinfect any ofthe resistant strains, whereas bac-teriophage SP82 infected strains RUB821,RUB822, and RUB823 at reduced efficiency.Strain RUB824 was totally resistant to infec-tion from bacteriophage SP82.The ability of these resistant strains to ad-

sorb the bacteriophages was also determined(Table 7). Bacteriophages SP02, SPP1, 4105,SPOl, and SP82 did adsorb to strain RUB821,

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TABLE 6. Efficiency ofplatinga

Bacteriophages 168, strain B. subtilis W Intergenote strains'BRi51 ra RUB821 RUB822 RUB823 RUB824

Group ISP02 1.0 0 (<10-8) 3.3 x 10-4 3.7 x 10-4 1.3 x 10-3 0 (<10-')SPP1 1.0 0 (<10-') 2.8 x 10-5 4.3 x 10-5 0 (<10-') 0 (<10-')qb105 1.0 0 (<10-') 0 (<10-') 0 (<10-') 0 (<10-8) 0 (<10-8)qb29 1.0 0 (<10-') 0 (<10-9) 0 (<10-') 0 (<10-9) 0 (<10-9)

Group IISPOl 1.0 2.6 x 10-4 0 (<10-') 0 (<10-8) 0 (<10-') 0 (<10-8)SP82 1.0 1.1 x 10-4 2.0 x 10-5 2.0 x 10-5 1.6 x 10-2 0 (<10-9)

a Efficiency of plating of bacteriophages for each strain as compared to strain BR151.I Intergenote strains resulted from the transformation of strain BR151 with DNA isolated from strain

RUB815, as described in Table 1.

TABLE 7. Efficiency ofadsorptiona

B. subtilis 168, B. subtilis W23, Intergenote strinBacteriophages studied strain BR151 strain RUB815 RUB821 RUB822 RUB823 RUB824

Group ISP02 100 0 84 83 100 0SPP1 91 4 15 0 30 0q6105 99 0 46 53 99 0q629 58 0 0 0 46 18

Group IISPOl 100 100 95 95 100 98SP82 100 100 49 55 100 89

aEfficiency of adsorption of bacteriophages for each strain, expressed as percent.

although bacteriophages 105 and SPOl were un-able to infect this strain. Similarly, bacterio-phages 4105 and SPOl were able to adsorb to,but not infect, strain RUB822. However, bacte-riophage SPP1 did not seem able to adsorb tostrain RUB822 in liquid culture, although itcan infect that strain. Additionally, bacterio-phages SPP1, 4105,4)29, and SPOl could adsorbto, but not infect, strain RUB823, and bacterio-phages SPOI and SP82 adsorbed to, but wereunable to infect, strain RUB824 at wild-typelevels. The results suggest that, in addition tothe alteration of bacteriophage receptor sites,the transfer of bacteriophage resistance fromstrain RUB815 to strain BR151 also impairedthe ability of the bacteria to process the infect-ing bacteriophage.Transformation and transfection of bacte-

riophage-resistant strains. The strains of B.subtilis (Table 1) that lack glucosylated teichoicacid in their cell walls can be transformed andtransfected with essentially the same efficiencyas strain BR151 (23; unpublished data). There-fore, these non-glycosylated teichoic acidstrains are capable of processing bacteriophageDNA, although they cannot be infected by in-tact bacteriophage. However, strains RUB821,RUB822, RUB823, and RUB824 were partially

deficient in their ability to produce bacterio-phage after transfection (Table 8). These bacte-riophage-resistant strains (RUB821-824) wereas competent as strain BR151 (Table 8) sincethey all had the same relative frequency oftransformation. Therefore, the reduced effi-ciency of transfection must be related to a re-duced capacity to promote bacteriophage devel-opment. This decrease was effectively illus-trated by the ratio of Trp+ transformants toplaque-forming units after transfection (Table8). This ratio was between 0.7 to 4.6 for strainBR151, whereas the four resistant strains hadratios that ranged from 28 to 1,215. It is impor-tant to note that the four resistant strains werereduced in their abilities to process mature aswell as prophage DNA.

DISCUSSIONA successful bacteriophage infection involves

attachment ofthe bacteriophage, penetration ofthe bacteriophage nucleic acid, and the replica-tion and release of progeny bacteriophage. Avariety of bacteriophage receptors has beenidentified (11). Bacteriophage can specificallyadsorb to pili, flagella, proteins, polysaccharideside chains, and teichoic acid moieties. In B.subtilis, cell wall teichoic acids have been iden-

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BACTERIOPHAGE RESISTANCE 1125

TABLE 8. Transformation and transfection ofstrain BR151 and bacteriophage-resistant intergenotic strains

Donor DNA

818/SP82d818/SP82818/SP82818/SP82

818(0105)818(4105)818(4105)818(0105)818(0105)818(SP02)818(SP02)818(SP02)818(SP02)818(SP02)

Trp CFU/108 CFUa

2.57 x 1054.89 x 1051.71 x 1054.11 X 105

7.1 x 1053.4 x 1062.6 x 1063.1 x 1054.2 x 105

1.3 x 1062.7 x 1062.6 x 1061.5 x 1054.3 x 105

PFUb/108 CFU

3.72 x 1054.11 x 1023.95 x 1023.38 x 102

2.9 x 1051.3 x 1045.4 x 1032.2 x 1031.5 x 104

2.8 x 1052.2 x 1041.2 x 1041.2 x 1033.4 x 103

Trp+ CFU/PFUC

0.71,189.8432.9

1,215.9

2.4261.5634.1140.928.0

4.6122.7216.6125.0126.4

a CFU, Colony-forming units.b PFU, Plaque-forming units.c Ratio of number of Trp+ transformants/108 CFU divided by the number of PFU/108 CFU.d Either RUB818 DNA or DNA isolated from bacteriophage SP82 was added.

tified as important bacteriophage receptors (1,2, 8, 24, 25). The data presented in Tables 2, 3,and 4 demonstrate the importance of glucosy-lated cell wall teichoic acid in bacteriophageattachment. These experiments also establishthat certain bacteriophages infect these non-

glucosylated teichoic acid strains under specificconditions. For instance, bacteriophages SPP1and 4e infected these resistant strains whengrowth occurred in an agar medium but were

unable to attach to the walls of these strains inliquid media (Tables 3 and 4). Therefore, theseresults support previous conclusions (24) thatcell wall-glucosylated teichoic acid cannot bethe sole determinant for adsorption of bacterio-phages toB. subtilis. This contention was basedon the diminution of bacteriophage adsorptionafter hydrolysis of the amide bonds between N-acyl muramic acid and L-alanine and the obser-vation that most of the bacteriophages studiedwere not inactivated by macromolecular tei-choic acid (24). Since the publication of theseearlier results, evidence has accumulated forthe existence of membrane teichoic acid (6) andof membrane bacteriophage attachment sites(10; A. P. Hirvonen and 0. E. Landman, Abstr.Annu. Meet. Am. Soc. Microbiol. 1973, p. 91, p.

156; E; D. Jacobson and 0. E. Landman, Abstr.Annu. Meet. Am. Soc. Microbiol. 1975, K223, p.184). Taken collectively, these results indicatethat glucosylated teichoic acid is of primaryimportance in bacteriophage attachment to B.subtilis. However, in the absence of glucosy-lated cell wall teichoic acid, secondary sites ofattachment may exist in the cell wall as well asin the bacterial cell membrane. The complexityof bacteriophage adsorption can be seen by the

patterns of bacteriophage resistance demon-strated by each class of non-glucosylated cellwall teichoic acid mutants (Tables 3 and 4).Only bacteriophages SPOl and 429 were unableto infectgtaA, gtaB, andgtaC mutants. In fact,the three classes of mutants could be distin-guished by their abilities to be infected by bac-teriophages 4e, 425, and SP82. Specifically,bacteriophages 4e and SP82 were unable toinfect gtaC mutants, bacteriophage SP82 was

severely reduced in its ability to infect gtaBmutants, and bacteriophage 425 was unable toinfect gtaC and gtaB mutants. Therefore, bycomparing the plating efficiency of these threebacteriophages, one can obtain a good indica-tion of the type of mutation responsible for theloss of glucosylated cell wall teichoic acid. Simi-larly, these bacterial cell wall mutants can beused to distinguish these three closely relatedbacteriophages (9, 19).

Strains RUB821-824 were originally isolatedby their resistance to bacteriophages SPP1 andSP02cl2. However, whereas all four strainswere resistant to some extent, only strainRUB824 was totally resistant to these two bac-teriophages (Tables 6-7). In addition, those bac-teriophages (group II) that were able to adsorbto strain RUB815 but displayed a reduction intheir ability to infect strain RUB815 were alsoimpaired in their ability to infect the four re-sistant strains constructed by interspecifictransformation (Table 6). Furthermore, DNAisolated from bacteriophages representing bothgroups I and II was not as infectious in the fourresistant strains as it was with strain BR151(Table 8). Interestingly, bacteriophage 41plates with similar efficiencies on strains

Recipient

BR151RUB821RUB822RUB823

BR151RUB821RUB822RUB823RUB824

BR151RUB821RUB822RUB823RUB824

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1126 YASBIN, MAINO, AND YOUNG

BR151, RUB815, RUB821, RUB822, RUB823,and RUB824 (unpublished data). These resultsindicated that the bacteriophage resistancetransferred from strain RUB815 to strainBR151 was of a complex nature. This resistanceconsists of the failure of some bacteriophages toadsorb to the resistant strains as well as theinability of these strains to successfully processthe bacteriophage DNA (Tables 6-8). This defi-ciency in the proper processing ofbacteriophagegenetic material can be the result of blocks inany one of the steps involving penetration, rep-lication, translation, and/or transcription oftheDNA. In addition, this deficiency in the properprocessing may be the result of the activationand/or introduction of a restricting nuclease.Our laboratory has recently identified such arestriction endonuclease (Bam-1) in Bacillusamyloliquefaciens (20). These possibilities arepresently being investigated. Irrespective ofthe cause, these new resistant bacterial strainsshould aid in the dissection of the processesinvolved in a successful bacteriophage infec-tion.

ACKNOWLEDGMENTSThis study was aided by grant VC-27-J from the Ameri-

can Cancer Society and Public Health Service grant 5-TOl-GM-00592 from the National Institute of General MedicalSciences. R.E.Y. was supported by a fellowship from theNational Cancer Institute (6 F22 CAO1113).We would like to thank E. Freese for his generous cooper-

ation and help. We also acknowledge the helpful advice ofE. Eisenstadt and R. Lemons in the preparation of themanuscript.

LITERATURE CITED1. Archibald, A. R., and H. E. Coapes. 1972. Blocking of

bacteriophage receptor sites by concanavalin A. J.Gen. Microbiol. 73:581-585.

2. Boylan, R. B., N. H. Mendelson, D. Brooks, and F. E.Young. 1972. Regulation of the bacterial cell wall:analysis of a mutant of Bacillus subtilis defective inbiosynthesis of teichoic acid. J. Bacteriol. 110:281-290.

3. Brodesky, A. M., and W. R. Romig. 1965. Characteriza-tion ofBacillus subtilis bacteriophages. J. Bacteriol.90:1655-1663.

4. Brooks, D., L. L. Mays, Y. Hatefi, and F. E. Young.1971. Glucosylation of teichoic acid: solubilizationand partial characterization of the uridine diphospho-glucose:polyglycerol teichoic acid glucosyl transfer-ase from membranes ofBacillus subtilis. J. Bacteriol.107:223-229.

5. Chin, T., M. M. Burger, and L. Glaser. 1966. Synthesisof teichoic acids. VI. The formation of multiple wallpolymers in Bacillus subtilis W23. Arch. Biochem.Biophys. 116:358-367.

6. Coley, J., M. Duckworth, and J. Baddiley. 1972. Theoccurrence of lipoteichoic acids in the membranes ofgram-positive bacteria. J. Gen. Microbiol. 73:587-591.

7. Glaser, L., and M. M. Burger. 1964. The synthesis of

teichoic acids. III. Glucosylation of polyglycerolphos-phate. J. Biol. Chem. 239-.3187-3191.

8. Glaser, L., H. Ionesco, and P. Schaeffer. 1966. Teichoicacids as components of a specific phage receptor inBacillus subtilis. Biochim. Biophys. Acta 124:415-417.

9. Hemphill, H. E., and H. R. Whiteley. 1975. The bacte-riophages ofBacillus subtilis. Bacteriol. Rev. 39:257-315.

10. Jacobson, E. D., and 0. E. Landman. 1975. Interactionof protoplasts, L forms, and bacilli ofBacillus subtiliswith 12 strains of bacteriophage. J. Bacteriol.124:445-458.

11. Lindberg, A. A. 1973. Bacteriophage receptors. Annu.Rev. Microbiol. 27:205-241.

12. Maino, V. C., and F. E. Young. 1974. Regulation ofglucosylation of teichoic acid. I. Isolation of phos-phoglucomutase in Bacillus subtilis 168. J. Biol.Chem. 249.5169-5175.

13. Maino, V. C., and F. E. Young. 1974. Regulation ofglucosylation ofteichoic acid. II. Partial characteriza-tion of phosphoglucomutase in Bacillus subtilis 168.J. Biol. Chem. 249.5176-5181.

14. Okamoto, K., J. A. Mudd, J. Mangan, W. M. Huang, T.- V. Subbaiah, and J. Marmur. 1968. Properties of thedefective phage of Bacillus subtilis. J. Mol. Biol.34:413-428.

15. Okubo, S., B. Strauss, and M. Stodolsky. 1964. Thepossible role of recombination in the infection ofcom-petent Bacillus subtilis by bacteriophage deoxyribo-nucleic acid. Virology 25:552-562.

16. Rettenmier, C. W., and H. E. Hemphill. 1973. Pro-phage-mediated interference affecting the develop-ment of Bacillus subtilis bacteriophage q6e. J. Virol.11:372-377.

17. Spizizen, J. 1958. Transformation of biochemically defi-cient strains of Bacillus subtilis by deoxyribonu-cleate. Proc. Natl. Acad. Sci. U.S.A. 44:1072-1078.

18. Subbaiah, T. V., C. D. Goldthwaite, and J. Marmur.1965. Nature of bacteriophages induced in Bacillussubtilis, p. 435-446. In W. Bryson and H. J. Vogel(ed.), Evolving genes and proteins. Academic PressInc., New York.

19. Truffaut, N., B. Revet, and M. Soulie. 1970. ltudecomperative des DNA de phages 2C, SP8, SP82, 4e,SPOl et SP50. Eur. J. Biochem. 15:391-400.

20. Wilson, G. A., and F. E. Young. 1975. Isolation of asequence specific endonuclease (Bam-1) from Ba-cillus amyloliquefaciens H. J. Mol. Biol. 97:123-125.

21. Yasbin, R. E., A. T. Ganesan, and F. E. Young. 1974.Bacteriophage interference in Bacillus subtilis 168. J.Virol. 13:916-921.

22. Yasbin, R. E., G. A. Wilson, and F. E. Young. 1973.Transformation and transfection in lysogenic strainsofBacillus subtilis 168. J. Bacteriol. 113:540-548.

23. Yasbin, R. E., G. A. Wilson, and F. E. Young. 1975.Transformation and transfection in lysogenic strainsofBacillus subtilis: evidence for selective induction ofprophage in competent cells. J. Bacteriol. 121:296-304.

24. Young, F. E. 1967. Requirement of glucosylated tei-choic acid for adsorption of phage in Bacillus subtilis168. Proc. Natl. Acad. Sci. U.S.A. 58:2377-2384.

25. Young, F. E., C. Smith, and B. E. Reilly. 1969. Chro-mosomal location of genes regulating resistance tobacteriophage in Bacillus subtilis. J. Bacteriol.98:1087-1097.

26. Young, F. E., and G. A. Wilson. 1974. Practical guideto techniques for genetic analysis in Bacillus subtilis,p. 69-114. In R. C. King (ed.), Handbook of genetics,vol. 1. Plenum Press, New York.

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