Proc. Natl Acad. Sci. USAVol. 80, pp. 4074-4078, July 1983Genetics

Isolation and physical mapping of the gene encoding the major arfactor of Bacillus subtilis RNA polymerase

(rpoD/o55/immunological screening/transcription)

CHESTER W. PRICE, MICHAEL A. GITT, AND RoY H. DoiDepartment of Biochemistry and Biophysics, University of California, Davis, California 95616

Communicated by Jesse C. Rabinowitz, April 11, 1983

ABSTRACT At least four a factors separately bind the Ba-ciinw subtilis RNA polymerase core (f8fi'a2), each conferring adifferent promoter specificity on the holoenzyme in vitro. Usingthe Broome-Gilbert immunological screening, we isolated recom-binant A phages that carry rpoD, the gene for the most abundantorfactor, orm. These phages encode a 55,000-dalton protein whosesize, immunological properties, and peptide map identify it as or.All the phages have in common two adjacent 3.5-kilobase EcoRIfragments from the B. subtilis chromosome; most carry additionalgenomic DNA. Deletion analysis localized rpoD to a 1.6-kilobaseregion, suggested the direction of its transcription, and found twoadditional genes near rpoD, which code for proteins of 62,000 and17,000 daltons.

In contrast to the single DNA-dependent RNA polymerase foundin enteric bacteria, RNA polymerase of Bacillus subtilis has atleast four cellular forms, which share the core subunits f3Pf'a2but differ by their associated ofactors. The most abundant formhas a o- factor of 55,000 daltons (oc5) and is analogous to theenteric enzyme (1). The minor polymerase species-compris-ing <10% of total enzyme-have arfactors of 37,000 (2), 29,000(3), and 28,000 (4) daltons. All of the orfactors are found in cellsgrowing logarithmically save .29, which is apparently uniqueto sporulating cells (for a review, see ref. 5).The four holoenzyme forms have been characterized bio-

chemically and by activity on cloned B. subtilis DNA templates,but not genetically. In vitro, the various holoenzyme forms ini-tiate transcription at different sites, and the promoter recog-nition specificity of each holoenzyme depends on the particularo- factor bound (6). Based on these data, Losick and Pero havesuggested that transcription during the sporulation process ispartly regulated by substituting one crfactor for another, therebyaltering the promoter specificity of the polymerase in vivo (6).This model cannot be tested by using in vitro systems alone butrequires a genetic analysis to define the in vivo function of thepolymerase subunits (7).

Little is known about the genetics of the B. subtilis RNApolymerase cistrons. The genes for the A3 and /' subunits arelinked to cysA, mapping in a region containing >20 other genesactive in protein synthesis (8). Nothing is known about the pos-sible operonic structure of this region. The loci of the four ocfactors and the a subunit remain unmapped, no authentic mu-tations in these cistrons have been isolated, and nothing is knownabout their genetic organization.We report here the isolation and characterization of AgtWES

phages that carry rpoD, the gene encoding the major a factorof B. subtilis RNA polymerase, o55. Using deletion analysis andassaying the proteins expressed in E. coli cells bearing appro-priate plasmids, we have physically mapped rpoD to a 1.6-kilo-

base (kb) Ecofd-Sph I fragment, suggested the direction of rpoDtranscription, and found two additional genes in the rpoD re-gion.

MATERIALS AND METHODSBacterial and Phage Strains. Phage were grown on Esche-

richia coli DP50 supF (9) by using the procedures and mediaof Davis et aL (10), unless K-modified phage were required, inwhich case we used BNN45 (10) as host. For UV experiments,we used E. coli S159 (11) to express phage-encoded proteinsand CSR603 (12) for plasmid-encoded peptides, as describedbelow.

Immunological Screening of the B. subtilis Genomic Li-brary. Anti-a antibody was raised in rabbits as described (13).Anti-o5 IgG was purified, labeled with 125I, and used essen-tially as described by Broome and Gilbert (14) to screen theAgtWES library of Ferrari et aL (15). We made autoradiographson Kodak AR5 film, exposed at -70°C using a DuPont CronexLightning Plus intensifying screen.

Analysis of Phage- and Plasmid-Encoded Protein Synthesisin UV-Irradiated Cells. E. coli S159 cells were UV-irradiated,infected with recombinant A phage at a multiplicity of 10, andlabeled with [3S]methionine as described by Yamamoto andNomura (16). After 30 min of labeling, the cells were centri-fuged, resuspended in 100 ,ul of sample application buffer (17),and lysed by heating for 3 min at 90°C. The labeled proteinswere separated on a 12% NaDodSO4/polyacrylamide gel (17)and detected by autoradiography. We included as standards onthe Laemmli gels the P, ,', a, and a55 subunits of B. subtilisRNA polymerase, purified as described by Halling et aL (18).Maxicell assays of plasmid-directed protein synthesis in UV-ir-radiated E. coli CSR603 cells was done by using the modifi-cation of the original Sancar procedure (19) described by Closeand Rodriguez (20).

Detection of 55 and Peptide Fragments by ImmunologicalBlotting. We used a modification of the electroelution methodof Towbin et aL (21) to transfer proteins from NaDodSO4/poly-acrylamide gels to diazophenylthioether paper, which was pre-pared and activated by Seed's procedure (22). The transfer ofproteins from the gels to the activated paper and the subse-quent treatment of the blot was as described by Christmannand Dahmus (23). We incubated the paper gel facsimile withanti-a5 antiserum and then detected the bound anti-o-55 an-tibody by treatment with 125I-labeled goat anti-rabbit IgG andautoradiography. Peptide mapping of plasmid-encoded and au-thentic cr proteins was done by digesting them in situ withStaphylococcus aureus V8 protease (24). After NaDodSO4/polyacrylamide gel electrophoresis, the peptide fragments weretransferred to diazophenylthioether paper and then detectedwith anti-am antibody.

Abbreviation: kb, kilobase(s).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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DNA Methods. Large-scale isolation of plasmid and phageDNA, restriction endonuclease digestions, agarose gel electro-phoresis, ligation, and transformation of E. coli HB101/A (10),BNN45, or CSR603 cells were done as described by Davis etaL (10). Rapid isolation of plasmid DNA was done as describedby Close and Rodriguez (20).

RESULTS

Isolation of AgtWES Phages Encoding or5. To isolate phagescarrying the B. subtilis a' gene, we screened a AgtWES libraryfor phages encoding a protein recognized by anti-a'5 antibody.The library was a gift of E. Ferrari, D. Henner, and J. Hoch,who constructed it using B. subtilis DNA partially digested withEcoRI (15). About 5,000 phages from this library were platedwith E. coli DP50 supF as host. Using the Broome-Gilbertmethod for immunological screening (14), we isolated and pu-rified 13 recombinant phages encoding a product that boundanti-x55 antibody.

Size, Orientation, and EcoRI Restriction Map of ClonedGenomic DNA. We divided the 13 isolates into five classes basedon the composition of their genomic insert (Fig. 1). Xho Idigestions were used to orient the insert with respect to the Aarms. Four of the five cloned EcoRI fragments are found indifferent phages with inserts that either overlap or are in op-posite orientation. Therefore, at least the 1.7-, 1.8-, and two3.5-kb EcoRI fragments shown in Fig. 1 are contiguous on thegenome and are not unlinked regions joined in library con-struction or amplification. All of the recombinant phages carrytwo adjacent 3.5-kb EcoRI fragments of B. subtilis DNA. Thus,all or part of the gene product recognized by anti-a" antibodyis encoded within this 7 kb of common DNA.Four Proteins Are Encoded by the Common 7 kb of DNA.

We chose AgtWES-oa82 to study in greater detail. This is a rep-resentative phage carrying only the 7 kb of B. subtilis DNAcommon to all of the isolates. Heavily UV-irradiated E. coli S159cells virtually cease cellular protein synthesis but can expressgenes encoded by infecting A phage (11). Under these condi-tions, AgtWES-of82 directed the synthesis of four unique pro-teins of molecular masses 62,000 (P62), 55,000 (P55), 31,000(P31), and 17,000 (P17) daltons, as shown in Fig. 2. These pro-teins were not found in cells infected with the AgtWES vectorlacking B. subtilis DNA. Minor proteins of molecular masses

RI RI RI

b

LLi

I--I--<l<

-'13-'/3,

-a

P317

P _-.__

FIG. 2. Four proteins are uniquely encoded by AgtWES-or82. E.coli S159 cells were UV-irradiatedj infected with phage at a multiplici-ty of 10, and labeled with [35S]methionine.as described by Yamamotoand Nomura (16). The cells were broken, and the labeled proteins wereseparated on a 12% NaDodSO4/polyacrylamide gel and then were de-tected by autoradiography. The calculated molecular masses of theunique proteins encoded by AgtWES-o-82 are 62,000 (P62), 55,000 (P55),31,000 (P31), and 17,000 (P17) daltons, as shown on the left. The po-sitions of the B. subtilis RNA polymerase subunits included as stan-dards are shown on the right.

42,000, 24,000, and 23,000 daltons are also encoded by AgtWES-o82.

Identification of the 55,000-Dalton Protein as 55. AAgtWES-o-82 lysate contained a protein that strongly bound anti-o55 antibody in an immunological blot experiment, a protein'with the gel mobility of authentic B. subtilis a' (Fig. 3). A ly-sate of the XgtWES vector had no such protein. Wong and Doi

C\jcob0U

b -<WL <

RI RI RI

1.7 1 3.5 1 3.5 11.8 1 1.29 9

Xhol Xhol

XgtWES -o71,82,92(-),93\gtWES-cr83

oLUb

:0'+-<LU.

XgtWES- a91

I XgtWES-or61,72,81(-),101(-)

a gtWES-<a62,94,102

FIG. 1. B. subtilis genomic DNA carried by recombinant phage. Theupper section of the figure shows an EcoRI (RI) and a Xho I restrictionmap of the B. subtilis DNA carried by the AgtWES phages. The sizes(in kb) of the EcoRI fragments are given within the open boxes. Thelower portion shows the positions of the overlapping DNA inserts car-ried by the 13 phages. We used the Xho I sites to find that most of theinserts are oriented as drawn, with the A right arm to the right of theinsert. Phage with inserts in the reverse orientation are indicated by(-).

FIG. 3. A 55,000dalton protein encoded by AgtWES-o82 binds anti-a155 antibody. E. coli DP50 supF was lysed by AgtWES-oa82 or AgtWESvector, and the lysates were cleared of cells and phage. The lysate pro-teins were run on a 10% NaDodSO4/polyacrylamide gel, together withRNA polymerase holoenzyme standards from B. subtilis (Ecr55) and E.coli (Ea7 ). The proteins were transferred to diazophenylthioether pa-per by electroelution and the paper gel facsimile was incubated withrabbit anti-cr55 antibody. Bound rabbit antibody was detected by au-toradiography after treatment with 125I-labeled goat anti-rabbit an-tibody.

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P62,. _-

rpoD P17?------

O 1 2 3 4 5 6 7kbi I I I I I I II

EcoRI Xhol Sst ) AtEcoRMPBamHI | )t SstO'tIC EcoRIHind Ill Hind III AvoBCla BamHI

PvuIll Pvu 11 Sph I Pvu 11

) pCF

-I pCF(

Expressed inMaxicells

P62 P55 P17PSI + - -

PS2 -- + +

( ) pCPS7 - + +

)-I pCPS9 - - -

FIG. 4. Physical map ofB. subtilis DNA in the rpoD region. Restriction enzyme cleavage sites on the 7-kb AgtWES-a82 insert were determinedas described in the text. Distance is measured (in kb) from the left end of the insert. Various insert fragments were subcloned into plasmid pCP112:the solid lines below the restriction map indicate the fragments carried by the different plasmid constructions and the parentheses enclose regionsdeleted in the subcloning. Maxicell analysis with these plasmids-summarized on the right-allowed us to position the coding sequences for P62,P55 (rpoD), and perhaps P17, as shown by the solid rectangles above the restriction map. The arrows suggest the direction of transcription of rpoDand the P17 gene. No restriction sites were found for Bgl II, Kpn I, Pst I, Sal I, Sma I, Sst II, or Xba I.

(25) found anti-o,55 antibody crossreacts weakly with the E. colio- factor (cr70), and in Fig. 3 a faint o70 band is visible in bothlysates as well as in the E. coli RNA polymerase control.

P55 has the peptide map of o-. We show below that whenthe P55 coding region from AgtWES-o82 was subcloned intopCPS7, a plasmid-encoded protein of 55,000 daltons was cleavedby S. aureus V8 protease into the four fragments characteristicof c5 (13). Thus, the size, immunological properties, and pep-tide map of the 55,000-dalton protein encoded by AgtWES-a82identify it as cr55. Consistent with the nomenclature adoptedfor the E. coli RNA polymerase genes, we refer to the B. subtilis

gene as rpoD.Physical Mapping of rpoD. We constructed a restriction en-

donuclease cleavage map of the AgtWES-o82 insert by com-paring the products of single- and double-digestions of phageDNA. The results are summarized in Fig. 4. Closely spacedrestriction sites were ordered by subcloning various fragmentsinto plasmid vector pCP112. pCP112 is a versatile integrative

EcoRI HindillPvuI Bar HI

Pst 1 0 SalApr 4 Tcr

or Ava I

Sal

FIG. 5. Restriction map of integrative plasmid pCP112. The mapofpCP112 is derived from the published DNA sequences ofE. coli plas-mid pBR327 (26) and S. aureus plasmid pC194 (27). The thin line de-notes the DNA from pBR327, which provides the tetracycline (Tcr) andampicillin (Apr) resistance genes together with a ColEl origin of rep-lication (ori). The heavy line indicates the inserted 1,137-base pair MspI-Taq I fragment from pC194, containing the Gram-positive chlor-amphenicol resistance (Cmr) gene. Total length of pCP112 is 4.4 kb, asshown by the circular scale within the map. The restriction sites forenzymes that cut pCP112 infrequently are shown on the map perim-eter.

plasmid that carries a chloramphenicol resistance gene ex-pressed in both Gram-positive and Gram-negative organismsand that replicates autonomously in E. coli but not B. subtilis.We show a restriction map of pCP112 in Fig. 5 and will de-scribe elsewhere its construction and use in mapping the chro-mosomal locus of rpoD.We located the rpoD coding sequence on the cloned DNA

by a process equivalent to deletion mapping. We subdloned intopCP112 restriction fragments from the AgtWES-of82 insert andassayed gene expression in UV-irradiated E. coli CSR603 cells(maxicells), which preferentially synthesize plasmid-encodedproducts (19, 20). Four plasmids allowed us to localize the se-quences necessary for expression of P62, P55, and P17. Fig. 4gives a summary of the data, with the B. subtilis DNA carriedby each of the four plasmids shown beneath the restriction mapand the unique proteins encoded by each subcloned fragmentindicated on the right. Maxicell data are given in Fig. 6.The data from cells containing pCPSl and pCPS2 show that

the left 3.5-kb EcoRI fragment encodes P62, whereas the right3.5-kb EcoRI fragment specifies both P55 and P17 (Figs. 4 and6). The 1.6-kb EcoRI-Sph I fragment carried by pCPS7 is suf-ficient to express P55. We show in Fig. 7 that the 55,000-daltonprotein encoded by pCPS7 has the same peptide map as a".This protein and authentic o-55 were both completely digestedwith S. aureus V8 protease to four peptide fragments whosemolecular masses total 56,000 daltons. Therefore, we concludethat the 1.6-kb fragment contains the entire coding sequenceof rpoD.We believe that the P17 gene lies astride the Sph I site on

the right EcoRI fragment, because pCPS7 does not express P17but instead expresses a 14,000-dalton protein, P14 (Figs. 4 and6). If P14 is a large NH2-terminal fragment of P17, then the P17gene is transcribed in the direction indicated in Fig. 4. NeitherP55 nor a smaller derivative was apparent in cells containingpCPS9 (Fig. 6). Thus, the Pvu II deletion of pCPS9 cuts intorpoD or sequences required for its expression, perhaps thoseof the rpoD promoter. Therefore, we suggest that rpoD is tran-scribed in the direction indicated in Fig. 4. This analysis did notlocate the gene for the P31 protein encoded by AgtWES-of82.Either the 31,000-dalton ,B-lactamase of pCP112 masked thepresence of P31 or the EcoRI sites lie within sequences es-sential for P31 expression.

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r) N - N

QL CL QL0 U) U+U) Q~ QC) + + +

U) U)

V) C)

+ +

P62-p55 .

-a

TCrApr

P 7-P14-_

FIG. 6. Expression in UV-irradiated E. coli cells ofB. subtilis pro-teins encoded by recombinant plasmids. E. coli CSR603 cells weretransformed with the recombinant plasmids shown in Fig. 4 and thenwere treated as described by Close and Rodriguez (20) to preferentiallylabel plasmid-encoded proteins with [35Slmethionine. The cells werebroken, and the proteins were separated on a 12% NaDodSO4/poly-acrylamide gel and then- visualized by autoradiography. The sizes (inkilodaltons) of the major proteins specified by the recombinant plas-mids are given on the left, together with the positions of the proteinsassociated with the plasmid tetracycline resistance (Tcr) and ampicillinresistance (Apr) phenotypes. Positions ofB. subtilis RNA polymerasesubunits used as standards are shown on the right.

DISCUSSIONThe multiple af factors of the B. subtilis RNA polymerase arewell characterized biochemically, but a genetic analysis is neededto establish their physiological and developmental role. There-fore, we have adapted to B. subtilis the methods found usefulin analyzing the E. coli transcriptional apparatus (16, 28-30),isolating and characterizing A phages carrying rpoD; the genefor the B. subtilis o,5 factor.The lack of known B. subtilis a mutations prevented us from

isolating or genes by complementation, as was done for the E.

) C\JI) C) C) C

b Q a

7.5

11.5

coli rpoD gene (29, 30). We used instead an immunologicalmethod (14) to screen a B. subtilis genomic bank for A phageswhose product was recognized by anti-oM antibody. The lackof a B. subtilis rpoD mutation also excluded complementationas a means to confirm the function of the cloned gene, so werelied on three independent biochemical criteria to identify thegene product as o, . The 55,000-dalton protein encoded by therecombinant phage has the same size, immunological proper-ties, and peptide map as authentic or.By restriction mapping, deletion analysis, and assay of plas-

mid gene expression in E. coli maxicells, we have establisheda physical map of the rpoD region and have located the entirerpoD coding sequence on a 1.6-kb EcoRI-Sph I fragment, asshown in Fig. 4. The a5 subunit has an apparent molecularmass of 55,000 on NaDodSO4/polyacrylamide gels, which sug-gests rpoD occupies about 1.5 kb of DNA. Thus, the 1.6-kbEcoRI-Sph I fragment is barely larger than the expected sizeof rpoD, and little coding capacity remains for the promoter-proximal part of the P17 gene, which we believe lies on thesame fragment. The or5 subunit might be smaller than indi-cated by its gel mobility, as is the case for the 70,000-dalton E.coli aofactor, which migrates as an 82,000- to 90,000-dalton pro-tein (31).Our deletion analysis also provides an indication of the tran-

scriptional organization of the rpoD region. The rpoD and P17gene promoters may lie within a 0.6-kb Pvu II-Sph I fragment,as shown in Fig. 4, but because the data are from plasmidexpression in the heterologous E. coli system, these results arenot conclusive. By determining the nucleotide sequence of therpoD region and by nuclease Si mapping of B. subtilis in vivotranscripts, the physiological promoters for rpoD and nearbygenes can be located. The DNA sequence, gene organization,and expression of this region can then be compared to the E.coli rpoD operon, which contains genes active in transcription,translation, and replication (32).

Although the existence of the P62 and P17 genes in the rpoDregion is suggested by their expression in E. coli, we do notknow their functions in B. subtilis. Nuclease S1 mapping ex-periments will show whether these regions are expressed in B.subtilis but cannot address the question of function. Once weknow the location of rpoD on the B. subtilis chromosome, wecan identify P17 or P62 gene function by complementing knownmutations that map nearby. In this regard, our preliminarymapping experiments find rpoD linked to aroD and strC byPBS1 transduction, lying at.about 2250 on the B. subtilis geneticmap (8). Thus; rpoD is distant from the cysA region that con-tains most of the known genes for transcription and translation(8).The methods we used to find and identify rpoD can be used

for other genes, particularly those whose functions are eitherdifficult to assay or unknown, such as the minor ogenes. Basedon peptide mapping data, at least the 37,000- and 29,000-daltonor factors are encoded by unique genes (25) and are thus ame-nable to genetic analysis.

FIG. 7. Peptide mapping of a 55,000-dalton protein encoded bypCPS7. E. coli CSR603 cells containing pCPS7, pCPS9, or pCP112(Figs. 4 and 5) were UV-irradiated, treated to maximize expression ofplasmid-encoded genes, and then lysed. Proteins from each of these ly-sates were separated on an 8% NaDodSO4/polyacrylamide gel. The gelregions containing proteins of55,000 daltons were cut out, the proteinswere completely digested in situ with 0.5 ug of S. aureus V8 protease(24), and the peptide fragments were separated on a 15% NaDodSO4/polyacrylamide gel. The peptide fragments recognized by anti-cr55 an-tibody were detected by the immunological blotting procedure de-scribed in the legend to Fig. 3. The calculated size (in kilodaltons) ofeach of the four V8 fragments is shown on the left.

We thank James Hoch for generously providing the-AgtWES bank ofB. subtilis DNA which was constructed in his laboratory. This researchwas supported in part by National Research Service Award GM 06826from the National Institute of General Medical Sciences (C.W.P.), byNational Science Foundation Grant PCM 7924872 (R.H.D.), and byNational Institute of General Medical Sciences Grant GM 19673(R.H.D.).

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