Vol. 175, No. 8

Bacillus subtilis Mutants Defective in Bacteriophage 429Head Assembly

BASAVAPATNA S. RAJAGOPAL, BERNARD E. REILLY, AND DWIGHT L. ANDERSON*Departments ofMicrobiology and Oral Science, University ofMinnesota, Minneapolis, Minnesota 55455

Received 27 October 1992/Accepted 14 February 1993

Virus assembly mutants of asporogenous Bacillus subtilis defective in bacteriophage +29 head assembly weredetected by the use of antibodies that reacted strongly with the free dodecameric +29 portal vertex composedof gene product 10 (gplO) but weakly with the portal vertex assembled into proheads or phage. Phageadsorption and the synthesis of phage proteins, DNA-gene product 3, and prohead RNA were normal in thesemutants, but prohead and phage production was greatly reduced. The assembly defect was transferred tocompetent B. subtilis by transformation and transduction. PBS1 transduction showed that the vam locus waslinked to Tn917 located at 3170 on the B. subtilis chromosome.

Bacteriophage 4)29 is an excellent model for studies ofviral morphopoiesis (form determination) and morphogene-sis because it is one of the simplest viruses with a prolatecapsid. Only the portal vertex, scaffold, and major capsidproteins (gplO, gp7, and gp8, respectively) are required forassembly of the precursor capsid or prohead, the earliestassembly intermediate identified (16, 30). The prohead canalso be assembled efficiently in Escherichia coli from theproducts of cloned genes, showing that any host factor(s)needed for head assembly is not unique to Bacillus subtilis(13, 15). The viral genome with covalently bound terminalprotein (DNA-gp3) is packaged efficiently into the prohead ina completely defined in vitro system (14), and the neck andtail are assembled to the DNA-filled head in a single pathway(24, 30).The products of the E. coli genes groEL and groES that

belong to the heat shock proteins are required for assemblyof the heads of the phages X and T4 (reviewed in references11, 12, and 38). Assembly of the X portal protein (connector),a distinctive dodecamer positioned at a special vertex of theprohead that is involved in DNA packaging, may requireGroE function, and cells carrying a missense mutation in oneof the groE genes assemble head-related monsters. GroE-cells infected with phage T4 accumulate membrane-associ-ated lumps of the major capsid protein rather than head-related structures.

Bacteriophage adsorption mutants of B. subtilis can easilybe obtained by direct selection (34). The phages 429 and 4)25were used to select B. subtilis mutants defective in glucosy-lation of polyglycerol teichoic acid (42). By using thesephages and insertional mutagenesis, at least six transcrip-tional units involved in the production of B. subtilis envelopecomponents have been identified and mapped near position3100 of the genetic map (26, 33). Repeated isolation ofadsorption mutants frustrated the search for virus assemblymutants (VAMs). The present report describes the selectionof B. subtilis mutants blocked in assembly of the 4)29prohead by the use of an enzyme immunoassay that distin-guishes free connectors from connectors assembled into theprohead capsid.

* Corresponding author.

MATERIALS AND METHODSBacteria, phages, and media. The B. subtilis 168 strains

used are depicted in Table 1. RAJ strains were constructedby transformation and transduction to aid in map localizationof five vam alleles. For example, VAM5 was selected in B.subtilis 12A, and DNA of this strain was used to transformBR67 (trpC2 ade-J ura-2). vam-S was introduced by congres-sion (31) to give RAJ5(trpC2 vam-5). The VAM phenotypewas identified by cross-streaking 4)29 on transformed clonesand then verified by burst size determination (1). DNA ofSCR115 (spoOA12 thyAl thyBI) was used to transform RAJstrains to prototrophy, and asporogenous clones were testedfor the VAM phenotype to select RAJ1105 (spoOA12 vam-5),and so forth. RAJ2105 and RAJ3105 were selected afterPBS1 transduction of RAJ1105 for Mlsr and are vam-Szii-83: :Tn9l7 and vam-S zjf-85: :Tn9l7, with transposition atmap locations 3170 and 342°, respectively (Table 1).Phages included wild-type 4)29 (35) and the mutant

susl4(1241) (27), which exhibits the delayed lysis phenotype.The suppressor-sensitive mutants sus8(769)-sus14(1241),suslO(302)-sus14(1241), susl6(300)-susl4(1241), and sus7(614)-sus8(769)-sus14(1241) (6-8) are defective for the majorcapsid protein gp8, the connector gplO, the DNA packagingprotein gpl6, and the prohead scaffold gp7 and capsidprotein gp8, respectively.Growth media were tryptose blood agar base (Difco

Laboratories, Detroit, Mich.), minimal medium (2), and 416medium containing 2% (wt/vol) Difco Bacto-tryptone, 1%Difco yeast extract, and 0.17 M NaCl.

Genetic methods. Competent B. subtilis cells were pre-pared by the method of Anagnostopoulos and Spizizen (2).DNA used for vam transformation was in crude extractsprepared from washed cells in the mid-logarithmic phase ofgrowth. The cells were lysed with lysozyme (1 mg/ml) andsodium dodecyl sulfate (SDS) (1%), the lysates were di-gested with proteinase K (50 ,ug/ml), and the nucleic acidswere precipitated and washed with ethanol. The nucleicacids in SSC (1.5 M NaCl, 0.15 M sodium citrate) werefiltered (0.45 ,um; Gelman Sciences Inc., Ann Arbor, Mich.)and used at saturating concentrations of DNA (>1 ,g/ml) intransformation to increase the frequency of congression (31).The PBS1 lysate preparation and transduction protocols

were modified from the methods of Hoch et al. (22). PBS1(the iP1 strain of the Bacillus Genetic Stock Center) lysatesat 109 phage per ml contained about 103 transducing particles

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2358 RAJAGOPAL ET AL.

TABLE 1. Bacillus subtilis 168 strains

Strain GeoyeSource ordesignation Genotype reference

12A trpC2 spoOA12 2311ONA trp spoA P. Schaeffer (28)SCR115 spoOA12 thyAl thyBi J. Ito (36)BR67 trpC2 ade-l ura-2 B. Reilly

QB944(1A3)a trpC2 cysAl4purA26 BGSCCU973(1A123) trpC2 fur El BGSCPB3292(1A300) trpC2 glyC thyAl thyB1 BGSC

CU4158(1A638) trpC2 zfg-83::Tn9l7(SPPC2) BGSCCU4162(1A642) trpC2 zhi-83::Tn9l7(SPI3C2) BGSCCU4164(1A644) trpC2 zii-83::Tn9l7(SPPC2) BGSCCU4165(1A645) trpC2 zjf-85::Tn9l7(SPI3C2) BGSCCU4665(1A732) trpC2 zjd-89::Tn9l7(SPPC2) BGSC

VAM5b trpC2 spoOA12 vam-S This workVAM6b trpC2 spoOA12 vam-6 This workVAM8b trpC2 spoOA12 vam-8 This workVAM10C trp spoA vam-10 This workVAM11C trp spoA vam-11 This work

RAJ5b trpC2 vam-S This workRAJ6b trpC2 vam-6 This workRAJ8b trpC2 vam-8 This workRAJ10C trpC2 vam-10 This workRAJ11C trpC2 vam-li ura-2 This work

RAJ1105 spoOA12 vam-S This workRAJ1106 spoOA12 vam-6 This workRAJ1108 spoOA12 vam-8 This workRAJ1110 spoOA12 vam-10 This workRAJ1111 spoOA12 vam-li This work

RAJ2105 spoOA12 zii-83::Tn9i7 vam-S This workRAJ3105 spoOA12 zjf-85::Tn9l7 vam-S This work

a Bacillus Genetic Stock Center (BGSC) accession number.b Isolated in B. subtilis 12A.c Isolated in B. subtilis 11ONA.

per ml. The additives for selection of Mlsr included erythro-mycin (1 ,ug/ml) and lincomycin (25 ,ug/ml); those for Furrselection, on minimal medium, included 5-fluorouracil (40,ug/ml) and uracil (40 ,ug/ml). The strain 1A300 was grown ontryptose blood agar base containing thymidine (2 ,ug/ml).

Selection of bacterial mutants defective in +29 assembly.4)29 was plated for confluent lysis on asporogenic B. subtilis.Colonies growing in the presence of phage were picked, andmultiple single colony isolations were used to obtain purecultures. Cells infected with 4)29 as described (6) wereincubated for 70 min at 37°C, collected by centrifugation,resuspended at 2 x 109/ml in TMS buffer (0.05 M Tris-HCl,pH 7.8, 0.01 M MgCl2, and 0.1 M NaCl), and lysed withlysozyme (50 ,ug/ml) in the presence of DNase I (10 ,ug/ml).The extracts (2 ,ul) were spotted onto a nitrocellulose mem-brane (Schleicher and Schuell, Keene, N.H.) and incubatedwith rabbit anti-gplO serum (1:10,000 dilution) as the primaryantibody and then with alkaline phosphatase-conjugatedgoat anti-rabbit immunoglobulin G (IgG; Boehringer Mann-heim Biochemicals, Indianapolis, Ind.) as the secondaryantibody. The enzyme immunoassay used methods previ-ously described (17) and color development with the phos-phatase substrate 5-bromo-4-chloro-3-indolyl phosphate incombination with nitroblue tetrazolium. Purified phage, pro-

heads, and portal protein dodecamers (connectors) were alsoincluded in the dot blot assays.

Electrophoresis of proteins. Phage-infected cells were ly-sed and the extracts were separated into pellet and superna-tant fractions prior to SDS-polyacrylamide gel electrophore-sis (PAGE) as described (18). Proteins were transferred toImmobilon-PVDF membranes (Millipore Corporation, Bed-ford, Mass.) by electroblotting, and the membranes were airdried and stored at -20°C. Blots were probed with rabbitanti-4029 serum (diluted 1:100,000) and then with alkalinephosphatase-conjugated goat anti-rabbit immunoglobulin G.Blots of purified connectors and proteins isolated fromsus8(769)-susl4(1241)-infected cell extracts were probedwith rabbit anti-gplO serum (diluted 1:100,000) as the pri-mary antibody.

Isolation of viral particles and connectors. Proheads orisometric particles were isolated from sup mutant bacteriainfected with the mutants susl6(300)-susl4(1241) orsuslO(302)-susl4(1241), which are defective for gpl6 neededfor DNA packaging and gplO needed for prolate particleassembly, respectively (16), as described (41). Head-tailconnectors were isolated from cells infected with the mutantsus8(769)-susl4(1241), defective for the major capsid proteingp8, as follows. Cells were lysed in TMS buffer containinglysozyme (200 ,ug/ml), DNase I (10 ,ug/ml), and phenylmeth-ylsulfonyl fluoride (300 ,ug/ml) at 37°C for 30 min, the lysatewas clarified by centrifugation, and the supernatant wascentrifuged in a 5 to 20% linear sucrose density gradient inTMS buffer in the SW55 rotor at 35,000 rpm for 4 h at 4°C.Fractions containing connectors were identified by SDS-PAGE.

In vitro assembly in extracts. Proheads were purified fromE. coli BL21(DE3)(pARgp7-8-8.5-10) as described (13, 15)and complemented with extracts of VAM strains infectedwith the mutant sus7(614)-sus8(769)-susl4(1241), defectivefor proheads, as described (6).

RESULTS

Isolation of VAMs of B. subtilis. An antiserum raisedagainst the purified 4)29 portal vertex protein gplO (connec-tor) interacted weakly with phage, proheads, and extracts of429-infected and uninfected cells in the dot blot enzymeimmunoassay (Fig. 1, Al, A2, B2, A4, and B4). On thecontrary, the antiserum interacted strongly with purifiedconnectors (Fig. 1, A3 and B1) or extracts of B. subtilisinfected with the mutant sus7(614)-sus8(769)-susl4(1241),defective for the major capsid (gp8) and prohead scaffold(gp7) proteins, in which the connector is free (Fig. 1, B3).This difference may be due to inaccessibility of epitopes onconnectors that have been assembled into particles and arein contact with the scaffold and major capsid proteins. Theantiserum and dot blot enzyme immunoassay were then usedto select VAMs among single colony isolates that grew in thepresence of 4)29. Eleven of a total of 60 4)29-resistant strainstested were VAMs, and 5 of the VAMs were used in thepresent study (Table 1).VAMs produced +29 proteins but no proheads or phage.

Although 4)29 adsorption by VAMs was equivalent to that ofthe parent strain (>95%), the burst size (18 to 22 per cell)was approximately 2% of that of the wild type (900 to 1,200per cell). Proteins in extracts of 429-infected VAM andVAM+ cells were separated by SDS-PAGE and transferredto Immobilon-PVDF membranes, and 4)29 proteins weredetected by the use of 4)29 antiserum and enzyme immunoas-says. The 4)29-infected VAM5, VAM8, and VAM10 strains

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HOST MUTANTS DEFECTIVE IN 4)29 ASSEMBLY 2359

1 2 3 4

A

B

C

D

FIG. 1. Dot blot enzyme immunoassay for the selection of virusassembly mutants. Al, A2, and A4, 2 x 109 429, 2 x 109 proheads,and 4 x 105 cell equivalents of uninfected VAM' (strain 12A),respectively; A3 and Bi, purified connectors (gplO), 1.3 x 107 and1.3 x 1010, respectively; B2, B3, and B4, 4 x 105 cell equivalents(each) of VAM' (12A) infected with 4)29, VAM' infected with7-8-14- phage, and uninfected VAM', respectively; Cl and C2, 4x 105 cell equivalents of VAM5 infected with 429 and uninfectedVAM5, respectively; C3 and C4, VAM6, infected and uninfected,respectively; Dl and D2, VAM8, infected and uninfected, respec-

tively; D3 and D4, VAM10, infected and uninfected, respectively.

produced the major capsid (gp8) and head fiber (gp8.5)proteins in quantity (Fig. 2). After infection of VAM5 orVAM10 with the mutant sus8(769)-susl4(1241), defective forthe major capsid protein, the assembled form of the portalvertex protein gplO was detected in sucrose density gradientfractions of the lysate as the typical ring-shaped connectorstructure (21) by negative staining and electron microscopyand by Western blotting (immunoblotting) and enzyme im-munoassays with gplO antiserum (data not shown).

4)29 protein production was also normal in VAMs infectedwith the mutant susl6(300)-susl4(1241). This mutant is de-fective for the DNA packaging protein gpl6 and thereforeaccumulates proheads in infection of the nonpermissiveVAM' host. However, VAMs infected with this mutant didnot produce enough proheads to be detected by sucrose

density gradient centrifugation of the lysates followed bySDS-PAGE of the fractions (Fig. 3A). Similarly, VAMsinfected with the mutant suslO(302)-susl4(1241), which isdefective for the portal protein gplO, did not produce theisometric particles (16) found in the infected VAM' cells(data not shown).VAM extracts complemented prohead donor extracts ef-

ficiently in in vitro assembly but were unable to provideproheads in a reciprocal experiment (Table 2). Purifiedproheads produced either in phage-infected B. subtilis or inE. coli containing the plasmid pARgp7-8-8.5-10, which en-codes all of the 429 prohead proteins, were complementedeffectively with either VAM' or VAM gpl6 donor extracts,demonstrating that prohead RNA (pRNA), DNA-gp3, and4)29 neck and tail proteins were present in the VAM extracts(Table 2). Taken together, the results showed that reducedphage yield in VAM strains was due to inefficient assemblyof the 4)29 precursor capsid or prohead, even though the

VAM VAM+0 5 8 10 5 (12A)

+ + +-_- +- 1 *r

:;St-A~ ~ I;

gp8.5-

FIG. 2. Synthesis of the major capsid (gp8) and head fiber (gp8.5)proteins in VAM was comparable to that in the VAM' host strain12A. 4)29 proteins in the envelope fraction of lysates of VAM andVAM' cells, 4)29 infected (+) or uninfected (-), were resolved bySDS-PAGE and detected on a Western blot by 4)29 antiserum andenzyme immunoassay. Lane 4) contained 4)29 to mark the positionsof gp8 and gp8.5; other lanes contained lysates of VAM5, -8, and -10and lysates of VAM' as shown.

presumed initiator of prohead assembly, the portal protein orconnector, was assembled.Map position of the VAM locus. A set of cryptic chromo-

somal insertions of Tn9l7 were constructed at specificpositions on the B. subtilis genetic map (39). PBS1 transduc-ing lysates were prepared with strains having the transposonpositioned at 2160, 2850, 3170, and 3420 (Table 1). Each of fiveVAM strains was transduced with lysates representing eachmap position by selecting forMlop transductants, subcloningon selective media, and testing for the VAM phenotype bycross-streaking with 4)29. The burst size increase was mea-sured for at least 10 clones for each transduction. Onaverage, the burst size increased from about 20 to 840 phageper cell when the wild-type allele was introduced (data notshown). Proheads produced in VAM' transductants ofRAeu1e8 for transposon Tn917 at 3170 were readily detectedby sucrose density gradient centrifugation (Fig. 3B).

Introduction of Tn917at map positions 2160 and 2850 didnot affect the Bam allele. Of 500 clones transduced for MIS%none showed change to VAM', and the average burst sizeremained at 14 phage per cell (n = 100). In contrast, at mapposition 3170, with VAM alleles taken as a group, 238 of 250Ml5r clones were changed to VAM'. On average, the burstsize had increased to about 840 per cell (n = 50) (data notshown). With Tn917 at342s, the cotransfer of Mlsr andVAMo was 41 of 250, and all VAM clones retained thereduced burst size. Each VAM was also transduced with alysate prepared on a strain having theTn9Ja7 transposon at3370, and 38 of 250 MlSr clones wereVAMi (data notshown). Thus, the frequency of cotransfer of Mls' andVAMI at positions 3420 and 3370 was about 15%, in contrastto about 95% cotransfer at position 3170. These results

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2360 RAJAGOPAL ET AL.

A 1

gp8-

gplO-

gp8.5-

gp7-

B

gpB-

gpiO-

gp8.5-

8 p

8 p

16

16

gp7

FIG. 3. Prohead production in RAJ1108 was not detected bysucrose density gradient centrifugation, but PBS1 VAM' transduc-tants of RAJ1108 for transposon Tn917 at 3170 produced proheads.Lysates of RAJ1108 (A) or RAJ1108 VAM' transductants (B) werecentrifuged in sucrose density gradients to isolate proheads, andfractions of the gradient were analyzed by SDS-PAGE. Fraction 1 isat the bottom of the gradient and 16 is near the top. P depictsproheads which mark the positions of the major capsid (gp8), portal(gplO), head fiber (gp8.5), and scaffold (gp7) proteins, respectively.The peak of the proheads occurs at fractions 5 and 6 (B). The fourmajor proteins of fractions 6 through 8 of panel A and fraction 8 ofpanel B are constituents of the B. subtilis pyruvate dehydrogenasecomplex.

suggest that the vam locus as defined by these five mutationsmaps relatively near the 3170 position by this type oftransduction.PBS1 transducing phage were then made on the newly

constructed strains RAJ2105 and RAJ3105, having the mu-tation vam-S and Tn917 at 3170 and 3420, respectively. Foreach transduction, 100 MlSr clones were tested for introduc-tion of the vam-5 allele and any other genetic event (Table 3).By using Tn917 (317°), 333 of 400 clones gave cotransfer ofMlsr and VAM. At the glyC (3200) locus, 70 of 82 VAMtransductants were prototrophs, and at thefurE (3250) locus65 of 65 VAM clones were fluorouracil sensitive. In contrast,none of 88 VAM clones had been transduced to prototrophyfor purA (3480).With Tn917 at 342°, cotransfer of Mlsr and VAM was

reduced to 5 of 400 clones, and there was no transduction forVAM and glyC (3200) (0 of 100) or VAM andfiirE (3250) (0 of

TABLE 2. In vitro assembly of 429a

Prohead donors gpl6 donor Mean/ml + SD PFUor proheads extract ± SD (n)

Prohead donor extractSpoOA12 SpoOA12 7.2 ± 3.2 x 1011 (4)SpoOA12 VAM5, -6, -8, 5.9 ± 2.3 x 1011 (16)

-10VAM5, -6, -8, -10 SpoOA12 0.7 ± 0.7 x 108 (16)SpoOA12 None 0.4 ± 0.1 x 108 (4)VAM5, -6, -8, -10 None 0.2 ± 0.2 x 108 (16)None SpoOA12 0.09 ± 0.02 x 108 (4)None VAM5, -6, -8, 0.06 ± 0.04 X 108 (16)

-10

Purified proheadsSpoOA12 SpoOA12 1.7 ± 0.5 x 1011 (7)SpoOA12 VAM5, -6 1.6 ± 0.6 x 1011 (14)E. coli(pARgp7-8-8.5-10) SpoOA12 1.5 ± 0.3 x 1011 (7)E. coli(pARgp7-8-8.5-10) VAM5, -6 1.4 ± 0.5 x 1011 (14)None SpoOA12 0.1 ± 0.1 x 108 (7)None VAM5, -6 0.03 ± 0.01 x 108 (14)

a Cells infected with one of the 4)29 mutants, susl6(300)-susl4(1241) andsus7(614)-sus8(769)-susl4(1241), which are defective for the DNA packagingprotein gpl6 and proheads (gp7 is the prohead scaffold and gp8 is the majorcapsid protein), respectively, were concentrated and lysed to give proheaddonor and gpl6 donor extracts. The extracts were mixed and incubated andthe mixture was assayed for phage as described in Materials and Methods.

100) (Table 3). Of 60 MlsrpurA (3480) clones, only 2 wereVAM. These results suggest that the site of the vam-Smutation is close to the glyC (3200) and furE (325°) loci.

Transformation with DNA of RAJ1105 introduced vam-Sinto 55% of glyC (3200) prototrophs in contrast to theabsence of cotransfer of vam and purA (3480) (data notshown). The data are consistent with a vam locus positionednear min 320 of the B. subtilis genetic map (see reference 32for the B. subtilis genetic map).

DISCUSSION

The majority of B. subtilis strains that form colonies in thepresence of 429 are adsorption mutants (26, 42). However,among these resistant cells were VAMs that produced o29proteins, DNA-gp3, and prohead RNA but were blocked inassembly of the precursor capsid or prohead and thereforedid not produce phage. The mutants were selected by the useof polyclonal antibodies directed against a specific prohead

TABLE 3. Mapping of the vam-5 allele by PBS1 transduction

Source of No. of No. of No. of transductantstransducing Recipient primary VAMstrain selections glyC furE purAphagea for MlsrC clones (320°) (3250) (3480)

RAJ2105 1A300 100 82 781A123 100 65 651A3 100 88 0

RAJ3105 1A300 100 0 01A123 100 0 01A3 100 2 60

RAJ2105 and RAJ3105 have Tn917 at map positions 3170 and 3420,respectively.

b For details, see Table 1. In addition, cotransfer of Mlsr and VAM forstrain 12A was 98 of 100 and 3 of 100 for Tn917 at 3170 and 3420, respectively.

C Transduced cells were first selected for Mlsr, and subclones were testedfor additional genetic exchange (see Materials and Methods and Resultssections).

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HOST MUTANTS DEFECTIVE IN 4)29 ASSEMBLY 2361

component, the dodecameric portal vertex structure (head-tail connector) (Fig. 1). Most of the connector is buriedinside the major capsid protein and therefore is essentiallyinaccessible to antibodies in the assembled prohead andvirion. On the contrary, the purified free connector reactedstrongly with its antiserum in the enzyme immunoassay. Thetechnique should have broad applicability for the isolation ofspecific host mutants that are blocked in viral morphogenesisand that accumulate free protein subassemblies that areordinarily surrounded by other proteins in the assembledvirion.The connector of the tailed, double-stranded DNA bacte-

riophages has been considered to be the initiator of headassembly (5, 11, 29). The 4)29 connector is assembled inVAMs into the typical ring-shaped structure with a mass ofabout 0.4 MDa that can be partially purified by sucrosedensity gradient centrifugation. If this structure is assembledcorrectly in the VAMs and the connector is the initiator of4)29 prohead assembly (13), then the required host factor(s)must function in at least one other step of head assembly.Head-related structures of the approximate size of +29 thatpresumably contain only the major capsid protein are pro-duced in the sus7(614)-sus14(1241) mutant infection, whichis defective for the prohead scaffold (16), and in thesus7(614)-susl0(302)-susl4(1241) infection, which is defec-tive for the prohead scaffold and portal proteins (data notshown). Since these particles were not observed in VAMs,the host factor(s) may chaperone the folding of the 429 majorcapsid protein gp8 into oligomers that may subsequently beassembled onto the connector in a T = 3 lattice (3). Recentwork on disassembly and reassembly of the lambdoid phageHK97 suggests that the GroEL and GroES proteins and ATPare needed to assemble the major capsid protein into pen-tamers and hexamers that subsequently assemble to capsidsof the correct size and shape (19).

B. subtilis produces an oligomer that resembles the GroELoligomer from E. coli and that copurifies with the +29connector protein (10). Three-dimensional reconstructionfrom electron micrographs demonstrates a structure withsevenfold symmetry and a central channel (9). Because ofthe role of the groESL operon of E. coli in viral morphogen-esis and because antibodies against the E. coli heat shockproteins DnaK, GroEL, and Lon cross-react with B. subtilisheat shock proteins (4), our genetic studies were directed bythe recent reports of cloning, sequencing, and mapping ofthe B. subtilis dnaK locus (40) and the groESL operon (25,37). PBS1 transduction with dnaK::cat mutant strains wasused to demonstrate cotransduction with the aroD locus andto fix the dnaK locus at about 2230 on the genetic map (40).Again, chloramphenicol resistance was used to mark thetransfer of groEL::cat. The reported gene order is sacSgroESL purA, with groE at about 3420 on the genetic map(37).The transduction results from cryptic insertions of trans-

poson Tn917 show that the vam locus is not positioned nearthe dnaK locus at 2230 (data not shown). Although the vamloci are about 95% linked to Tn917 located at map position3170, there is significant cotransduction (15%) with Tn917 atboth positions 3370 and 3420. However, Tn917 at 2820 and3170 does not cotransduce Mlsr and gtaA at 3090, nor doesTn917 Mlsr at 3170 cotransduce with hisA at 2980 (data notshown). Although PBS1 can transfer about 150 MDa ofDNA, the frequency of cotransduction decreases rapidly asphysical distance between markers increases (20). Theseresults demonstrate that cryptic insertions of Tn917 can beused to place loci on the genetic map, but the results are not

always precise or directly comparable with those fromclassical PBS1 transduction.The results obtained with transformation and transduction

(Table 3) demonstrate that the vam-5 locus is closely linkedtoglyC (3200) andftrE (3250) but is distant frompurA (348°),a marker near the groESL::cat locus at 3420 (37). Our resultsplace the vam locus we have described near map position3200, close to a region concerned with the construction andmaintenance of the cell envelope (3100) (26).The precise localization and fine structure mapping of this

locus will follow a more complete description of the relation-ship of the vam product to the groESL function and thecloning and sequencing of the vam gene(s).

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes ofHealth to D.L.A.We thank Charlene Peterson for her assistance in the preparation

of the manuscript.

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