Proc. Natl. Acad. Sci. USAVol. 82, pp. 7260-7264, November 1985Biochemistry

Deduced product of the stage 0 sporulation gene spoOF shareshomology with the SpoOA, OmpR, and SfrA proteins

(transcriptiosr/regulation/memlbrane proteins)

KATHLEEN A. TRACH*, JOHN W. CHAPMANt, PATRICK J. PIGGOTt, AND JAMES A. HOCH*t*Division of Cellular Biology, BCR2, Department of Basic and Clinical Research, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, LaJolla, CA 92037; and tMicrobiology Divi~ion, National Institute for Medical Research, The Ridgeway, Mill Hill, London, England

Communicated by I. C. Gunsalus, July 8, 1985

ABSTRACT The location of the stage 0 sporulation locusspoOF has been determined on a cloned fragment of Bacilussubtilis DNA. The spoOF gene and surrounding region wassequenced and was shown to code for a protein of Mr 14,286.The amino acid sequence of this deduced protein was 56%homologous to the amino-terminal domain of the spoOA geneproduct. The molecular weight of the SpoOF protein wasapproximately half that of the SpoOA protein, and its sequencewas homologous to the amino-terminal half of the SpoOAprotein. This same portion of the SpoOA protein showedancestral relationship to the OmpR and SfrA regulatoryproteins of Escherichia coli. Mutations in any of the genesencoding these proteins in either organism are highlypleiotropic and result in alterations in the regulation ofmembrane components, suggesting that they may have relatedroles in both organisms and that the stage 0 sporulation defectof spoOA and spoOF mutants is an indirect consequence of thisregulatory system.

The early events in sporulation are thought to be under thecontrol of stage 0 sporulation genes, spoO. Eight spoO locihave been definitively mapped on the Bacillus subtilis chro-mosome although others might exist (1). Five of these loci,spoOA, spoOB, spoOE, spoOF, and spoOH, appear to code forgene products that influence transcription from a-" (2) or a"(3) promoters. Mutations in these genes are highly pleiotropicand result in the loss of a variety of phenotypes associatedwith the sporulation process. Transcription from &37 promot-ers seems to require all five gene products (2), whereas thespoOH gene product is dispensable for transcription from a28promoters (3). The mechanism by which these genes controltranscription from either ofthese promoter types is unknown.

Cloning studies have allowed identification of the geneproducts from the spoOA (4), spoOB (5, 6), spoOF (7), andspoOH (8) genes. Although the primary sequence of theseproteins is known, it has revealed little of the function of thegenes. The spoOA gene codes for a protein of about Mr 29,000with homology to an Escherichia coli protein encoded by theompR gene (4). The product of the ompR gene is involved inthe control of the outer-membrane protein genes ompF andompC by some as-yet-undefined regulatory mechanism (9).In this report we show that the product of the spoOF geneshares homology with the product of the spoOA gene in thatregion ofthe SpoOA protein that is homologous to the productof the ompR locus.

MATERIALS AND METHODSBacterial Strains. E. coli HB101 (F-, hsdS20[Rm-, MB-],

recA13, lacYl, galK2, rpsL20[Smr], xyl-5, mtl-i, supE44, X-)was made competent and transformed in a procedure de-

scribed by Dagert and Ehrlich (10). B. subtilis strains CU474(ctrAl), JH777 (spoOF187), JH1010 (spoOF241), JH775(spoOF124), and JH770 (spoOF221) were transformed accord-ing to the method of Anagnostopoulos and Spizizen (11).Enzymes and Reagents. Restriction enzymes were pur-

chased from New England Bjolabs and were used accordingto the suppliers' recommended assay procedures. Phage T4polynucleotide kinase, E. coli DNA polymerase I largefragment, and calf intestinal alkaline phosphatase were fromBoehringer Mannheim. Phage X DNA, E. coli DNA poly-merase I, and phage T4 DNA ligase were from New EnglandBiolabs.

Nucleic Acid Isolation. The isolation of plasmid DNA andpurified DNA fragments has been described (5, 12). B.subtilis chromosomal DNA was prepared by the procedure ofMarmur (13) with some modifications.

Isolation of the spoOF Locus. B. subtilis 168T DNA wasdigested to completion with Bgl II and fractionated byagarose gel electrophoresis. Fragments of -6 kb were foundto have transforming activity for the spoOF221 allele. DNAfragments of this size were purified from the agarose (14) andligated into the Bcl I site of the positive selection vectorpJHB1 (15). Recombinants were selected as tetracycline-resistant transformants in E. coli. One of these plasmids,pPP41, had transforming activity for spoOF221.

Plasmid Constructions. Plasmid pPP41 was mapped bysimultaneous digestion with combinations of various restric-tion enzymes. The gel-purified 3.3-kilobase (kb) and 2.2-kbPst I fragments ofpPP41 were ligated to the integrative vectorpJH101 (16), which had been digested with Pst I to giveplasmids pJH4133 and pJH4122, respectively. PlasmidpJH4134 was constructed by ligation of the gel-purified1.0-kb Sac I fragment of X phage DNA into the unique Sac Isite of the Pst I insert of pJH4133. Plasmid DNA from 240 E.coli transformants was analyzed by the method of Grunsteinand Hogness (17) for hybridization to the phage X DNA SacI restriction fragment; 35 were found to be positive, and 1 wasdesignated pJH4134. All plasmids were maintained in E. coliHB1Q1 cells and verified by restriction analysis.

Insertional Inactivation of the Putative spoOF Open ReadingFrame. Plasmid pJH4134, a derivative of the integrativevector pJH101 containing the phage A DNA insertion withinthe putative spoOF open reading frame, was transformed intoB. subtilis CU479 (cytidine-transport-negative; CtrA-) cells.CtrA' recombinants were screened for hybridization to thephage ADNA 1-kb Sac I fragment by the method ofGrunsteinand Hogness (17). Chromosomal DNA from the parentalstrain and two positive recombinants was prepared, and "14pug of the DNA was digested with EcoRI. The digests weresplit in half and electrophoresed along with known sizemarkers in the same 1% nigarose gel (electrode buffer: 50 mMTris HCI/67 mM borate/2 mM EDTA, pH 8.1) containing 50

Abbreviations: kb, kilobase; kbp, kilobase pairs.fTo whom reprint requests should be addressed.

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

7260

Proc. Natl. Acad. Sci. USA 82 (1985) 7261

jig of ethidium bromide per ml. The DNA fragments weretransferred to GeneScreen (New England Nuclear) by themethod of Southern (18); after baking, the filter was cut inhalf and hybridized with either a phage X DNA probe or the1.2-kb HindIII-Pst I probe from plasmid pJH4133. Bothprobes were 32P-labeled by nick-translation (19). Hybridiza-tion at 370C overnight in buffer containing 50% formamide,0.75 M NaCl, 0.075 M sodium citrate, 0.02% polyvinylpyr-rolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 0.02%NaDodSO4, and 100 pg of sonicated, denatured calf thymusDNA per ml. The filters were washed and exposed to KodakXRP film for 2 days at -70'C with double-intensifyingscreens.DNA Sequence Analysis of the spoOF Locus. DNA sequence

data were obtained by the chemical cleavage technique ofMaxam and Gilbert (20) with a reagent kit purchased fromNew England Nuclear. 32P-labeling of 3' and 5' termini andsequencing gels have been described (5, 12).

Genetic Analysis. Genetic analysis of the spoOF mutantsusing cloned plasmid DNA was normally carried out with afragment of the spoOF region cloned into the integrativevector pJH101. Such cloned fragments were used to trans-form strains carrying various alleles of the spoOF gene forchloramphenicol resistance. Among the chloramphenicol-resistant transformants, the Spo+/Spo- phenotype wasscored. In some experiments, the ctrAl marker from strainCU479 was used. This wild-type allele of this mutation wasfound to be present on plasmid pJH4133.

RESULTSInsertion of Phage X DNA into the Putative spoOF Open

Reading Frame. A large Bgl II fragment of the B. subtilischromosome capable of transformation and complementa-tion of spoOF mutations was isolated on a plasmid vector(unpublished data). A portion of this fragment is shown inFig. 1. Shimotsu et al. (7) isolated an EcoRI-EcoRI restric-tion fragment that possessed both transforming and comple-menting activity for the spoOF allele, spoOF221. This EcoRIfragment is identical to that region of the presently clonedfragment shown in Fig. 1 from -1.8 to 4.0 kilobase pairs(kbp). These investigators were able to further subdividetheir EcoRI-EcoRl fragment into smaller fragments andidentify transforming activity for spoOF221 on a 0.3-kb BclI-Bcl I fragment located at -2.5-2.8 kb in Fig. 1. Only asingle large open reading frame was identified from thesequence of the EcoRI-EcoRI fragment; thus, the spoOFlocus was assigned to this open reading frame, which codesfor a protein of approximately Mr 19,065.

_~.-OD - i:°=l IIL iCI- aC\(JI C:I lL

I IIJ \ i I i I

IpJH4/22

pIH//4/34

We had some reason to believe that this open reading framedid not, in fact, code for the SpoOF protein. In order to testthe possibility that the open reading frame was indeed spoOF,we set about to obtain mutations in it. The Sac I restrictionsite located at -2.1 kbp in Fig. 1 is close to the middle of theputative spoOF gene. Using the plasmid pJH4133, whichcontains a restriction fragment covering the entire openreading frame as well as flanking sequence cloned in theintegrative vector pJH1O1, we constructed plasmid pJH4134,which carried a 1.0-kb insert of phage X DNA in the Sac Irestriction site. The insert DNA was derived from bacterio-phage X after Sac I digestion and consisted of that fragmentof phage X from bp 24,776 to 25,881. The phage X insert wasintroduced into the chromosome ofB. subtilis by virtue ofthefact that both pJH4133 and pJH4134 contain the wild-typeallele of the ctrA gene, which flanks the spoOF locus.Transformation of a CtrA- strain to CtrA+ was accomplishedby using plasmid pJH4134, and transformants carrying the afragment were identified by hybridization to nick-translatedphage X DNA. That the phage X DNA had indeed insertedinto the correct position was verified by Southern hybridiza-tion analysis of the DNA from two such transformants andthe parental strain. In this analysis, the chromosomal DNAswere digested to completion with EcoRI restriction endonu-clease, the fragments were separated on an agarose gel andtransferred to nitrocellulose by the method of Southern, andthe filter was probed with a labeled HindIII-Pst I fragment ofDNA derived from this region (Fig. 1). The autoradiographfrom this experiment (Fig. 2) shows that the expected 2.2-kbEcoRI-EcoRI fragment is now 3.2 kb in both recombinantscontaining phage X DNA. The parental lane (Fig. 2, lane C)also contains a 3.2-kb piece that hybridized to the probe. Thisfragment is most likely the flanking EcoRI-EcoRI fragmentthat is labeled because the probe overlaps this region, and thefragment is fortuitously the same length as the new phageX-containing EcoRI fragment. Identical lanes from the samegel were similarly transferred and hybridized with nick-translated phage X DNA to identify the location of the Xfragment in these pieces. These results (Fig. 2) show that onlythe 3.2-kb fragment ofboth recombinants hybridized to phageX DNA to which the 3.2-kb fragment in the parental strain hasno homology. Furthermore, in these recombinants there is noindication of any parental size fragment remaining, whichindicates that they are not merodiploid for this region. Theseresults indicate that the construction has yielded an insert ofphage X DNA in the Sac I restriction site of the putativespoOF open reading frame, and it has inserted in the chro-mosome. Neither these extensively tested recombinants norany of the other several hundred transformants containingphage A DNA that were isolated in this experiment had anyof the characteristics of an spoOF mutant, and all were notonly perfectly capable of sporulation but also wereprototrophic when tested on minimal media. In addition, theydid not show any of the phenotypes of ctrA mutations. Thus,

A B C a b

pwmmp ofl* Whll;.c

-3.3 kb

-2.2 kb

2 3kilobase pairs

4 54 5

FIG. 1. Restriction map of the spoOF-containing fragment. Theextent of DNA from this region carried within the three plasmids isindicated by the bars. The shaded box represents that fragment ofthis region nick-translated and used for hybridization in the exper-

iments described in the other figures.

FIG. 2. Southern hybridization of phage X-containing transform-ants. Southern analysis was carried out as described in the text.Lanes: A and a, phage X-containing transformant no. 1; B and b,phage X-containing transformant no. 2; C and c, parental strainCU479. Lanes A, B, and C were probed with the fragment shown inFig. 1. Lanes a, b, and c were probed with nick-translated phage XDNA.

Biochemistry: Trach et al.

Proc. Natl. Acad. Sci. USA 82 (1985)

we concluded that the previously identified open readingframe was not the spoOF gene.

Genetic Location of the spoOF Mutations. Using plasmidscontaining fragments of this cloned region, we began a seriesof experiments to locate the spoOF mutant alleles. In theseanalyses we used two plasmids, pJH4122 and pJH4133, thatencompass the entire fragment, yet split it at the Pst I site(Fig. 1). We were able to show (Fig. 3) that the Pst I site waslocated within the spoOF gene; approximately half of thealleles were transformed by one plasmid and halfby the otherplasmid. Yoshikawa et al. (21) had already shown that thespoOF221 mutation was located close to a Bcl I site asdetermined by sequence analysis. The order of markerswithin the spoOF gene had been determined previously byusing the three-factor transformation crosses, with the ctrAlocus as the outside marker (unpublished data). Thus, thespoOF221 allele is representative of four very tightly linkedalleles that appear not to recombine with each other to anyextent. The alleles carried on the plasmid pJH4122 were onlypartially ordered, but the alleles spoOF187, spoOF24J, andspoOF124 were definitively ordered with respect to oneanother. This location of the spoOF mutant alleles waspuzzling, as this region contained no significant open readingframes as determined in either direction. However, wethought it highly likely that the spoOF locus coded for aprotein because we had identified temperature-sensitivemutations in this gene. In order to resolve this question, webegan limited sequencing studies across the region thatcontained the spoOF alleles.

Sequence Analysis of the spoOF Locus. Sequence analysis ofthe region indicated in Fig. 3 was undertaken by using Maxamand Gilbert (20) sequencing techniques. In the region encom-passed by the spoOF mutations, a sequence analysis of bothstrands was carried out to confirm sequences obtained. Theseanalyses showed that the sequence found differed by onebase from that previously reported by Shimotsu et al. (7).This base was an additional G located three bases away fromthe Pst I site (Fig. 4). This additional base connected twopreviously small open reading frames into one large openreading frame (ORF-T, Fig. 3). The sequence of the proteinencoded by this open reading frame is shown in Fig. 5. Sincethere was no independent protein sequence for this gene, weassumed that translation started at either one of the ATGcodons that are just downstream from the ribosome bindingsite shown (Fig. 5). In addition, the location of the spoOF221mutation as determined by Yoshikawa et al. (21) in whichthere is a transversion from a thymidine to an adenosine isshown. This results in the production of a chain-terminationcodon, TAA, which should truncate the product at that point.

/38/9/ /1//97 /29 /70

229 22/ 1 /67/7 24/ /24I . . T

w

C/rA

Cl) m I r.

a

ORF- S I

- 4- Q.o en omo an

-0

I

ORF-T

0 p--

A CG+ +CG TbT

CAGGGGc

GTDGG

IIA C%+ +CG Tb

:"

'46"

.-I

FIG. 4. Location of the missing base in Maxam-Gilbert sequenc-ing ladders. The double-headed arrow indicates the base missed inprevious sequencing of this region. Ladder I was from DNA 5'labeled at the Acc I site (Fig. 3), and ladder II was from DNA 3'labeled at the same restriction site.

The direction of transcription of this open reading frame isopposite from that ofthe original open reading frame (Fig. 3).The location of promoters for this open reading frame has notbeen determined. The calculated molecular weight of thisprotein was 14,286. A protein of this size also was found inminicell analyses of this cloned region (unpublished data).

DISCUSSIONThe results indicate that the mutations that define the spoOFlocus are located within an open reading frame that codes fora protein ofMr 14,286. The previously identified open readingframe (7) with its potential protein product of Mr 19,065 isclearly not the spoOF gene because spoOF mutations do notmap in it and insertion of foreign DNA into the putative genedoes not result in a sporulation-defective phenotype. More-over, strains containing such inserts have no recognizableauxotrophic or other phenotype. This does not rule out a rolefor the Mr 19,065 protein in sporulation. The gene for thisprotein and the spoOF gene are very close together but aretranscribed in opposite directions, and preliminary unpub-

CUCCAC 221

M[GACGAAAATCATAATATTGGGGTGTAAA ATG ATG MT GAA AAA ATT TTA ATC GTT GATMet Met Asn Giu Lys Ile Leu Ile Val Asp

GAT CAA TAC GGC ATT CGT ATT TTG CTA MT GAA GTG TTC MT MA GAA GGC TACAsp Gin Tyr Gly lie Arg Ile Leu Leu Asn Giu Val Phe Asn Lys Giu Giy Tyr

Pst-ICAM ACG m CAM GCT GCG MC GGC CTG CAM GCG CTT GAC ATT GTG ACA AAA GAAGin Thr Phe Gin Ala Ala Asn Giy Leu Gin Ala Leu Asp Ile Val Thr Lys Glu

CGG CCC GAC CTT GTG CTG TTG GAC ATG AAA ATT CCC GGC ATG GAC GGA ATC GAAArg Pro Asp Leu Val Leu Leu Asp Met Lys Ile Pro Gly Met Asp Gly Ile Glu

ATC TTA AMA CGG ATG AAM GTC ATT GAC GAM MC ATC CGG GTC ATT ATC ATG ACGIle Leu Lys Arg Met Lys Val Ile Asp Glu Asn Ile Arg Val Ile Ile Met Thr

GCA TAC GGA GAM CTC GAC ATG ATC CAG GM TCG MAG GAM TTG GGC GCT CTG ACGAla Tyr Giy Giu Leu Asp Met Ile Gin Glu Ser Lys Glu Leu Giy Ala Leu Thr

CAC TTT GCC AAG CCG iTT GAC ATC GAC GM ATC AGA GAC GCC GTC AAA AAA TATHis Phe Ala Lys Pro Phe Asp Ile Asp Glu Ile Arg Asp Ala Val Lys Lys Tyr

FIG. 3. Genetic fine structure analysis and sequencing strategyfor the spoOFlocus. The top line shows the genetic fine structure mapof the spoOF allele as determined from three factor crosses using ctrAas an outside marker. The arrows at the bottom of the figure indicatethe sequencing strategy for this DNA: o, 5' labeling offragments; x,3' labeling offragments. The arrows at either end of the open readingframes (ORFs) indicate the direction of transcription for each ORF.

CTG CCC CTG AAG TCT MC TGA CAAAAAGAAGAAACMATGMTCATGTCATTATGTTGCCGATTLeu Pro Leu Lys Ser Asn OP

FIG. 5. Sequence of the spoOF gene. The putative ribosomebinding site and its potential pairing to the messengerRNA are shownin the top line. The "221" marks the location of the spoOF221mutation, which is an alteration of the middle T of the codon to an A.

7262 Biochemistry: Trach et al.

Biochemistry: Trach et al.

lished studies suggest that their promoters might overlap. Atpresent, it is clear only that inactivation of the gene does notresult in defective sporulation.The location of the spoOF gene impinges on the observed

phenomenon that multicopy plasmids carrying DNA fragmentsof this region inhibit sporulation in otherwise sporulation-proficient strains (21, 22). Strong inhibition of sporulation waslocated to that region of the DNA "upstream" of the putativespoOF gene (21). Since this region is now known to contain theactual spoOF gene, the inhibition phenomenon most likelyresults from the overproduction of the spoOF gene product.Thus, we have the paradox that a protein required for normalsporulation can be inhibitory if present in excess copy number.The sequence of the spoOF gene was compared, via

computer, to other proteins in the National BiomedicalResearch Foundation Protein Sequence Database with thesurprising result that the spoOF, spoOA, and ompR geneproducts shared substantial homology. A side-by-side com-parison of these proteins is shown in Fig. 6. The spoOF geneproduct shows 56% homology to the spoOA gene product, ifhomologous and related substitutions are used. We previ-ously had shown that the spoOA gene product shared homol-ogy with the ompR gene product of E. coli (4). The spoOA andompR gene products are most homologous in the amino-terminal half of each protein and show little homology in thecarboxyl half of their proteins. The spoOF gene productshares homology with the amino-terminal half or domain ofthe spoOA gene product and completely lacks the carboxyldomain. The consequences of such homology for the functionof these proteins is unknown. Structurally related proteinsmight form a complex in vivo. This possibility might explainthe ability of missense mutations in the SpoOA protein tocompensate for mutations in the spoOF gene (4). However,

Proc. Natl. Acad. Sci. USA 82 (1985) 7263

similar proteins may have similar but independent functionsin sporulation, and mutations in the spoOA protein thatcompensate for lack of spoOF gene product might simply alterthe spoOA gene product to allow it to accomplish the functionof the spoOF gene product.The ompR gene product is a positive regulatory factor that

controls the synthesis of the major porins of E. coli encodedby the ompR and ompC genes. The OmpR protein is thoughtto consist of two domains from studies of mutations in ompR(9). The carboxyl-terminal domain can be mutated or partiallydeleted (23), with loss of the control ofOmpC while retainingcontrol over OmpF. Thus, different domains of the OmpRprotein may have unique functions for any given genecontrolled by ompR. This situation is reminiscent of muta-tions in the spoOA gene, where mutations leading to alter-ations of the carboxyl domain of the SpoOA protein (4) areless pleiotropic than other spoOA mutations and allow at leastone a37 promoter to be transcribed (2). More extensivestudies will be required to prove such a domain hypothesis forSpoOA, however.

Recently the deduced sequence ofthe dye or sfrA gene wasobtained from cloned DNA, and it was shown that thisprotein shares homology with OmpR most highly in theamino-terminal domain (24). dye mutants have increases inthe amounts of certain inner membrane proteins, loss of theexpression of periplasmic alkaline phosphatase, and reducedexpression of traJ gene presumably because of a defect inantitermination (25). Thus, two related genes with effects onthe control of membrane proteins in E. coli are both relatedto the spoOA and spoOF genes ofB. subtilis, whose functionsare unknown but certainly control gene expression of avariety of genes including those for membrane functions.Perhaps the sporulation defect of spoOA and spoOF mutants

* * * * * * * *

spoOF M N E K I I L I V D D Q Y G I R E L L N E V F N K E G Y Q T F Q A A N G L Q A L D I* ** * * * ** * * * * * * * * * * *

*** * * * * * * * *

spoOA N E K I K V C V A D D N R E L V S L L S E Y I E G Q E D N E V I G V A Y N G Q E C L S* * * * * * * ** * * * * * * * * *

* * * * * * * * * * * * * *

ampR MQ E N Y K N L V V D D D N R L R A L L E R Y L T E Q G F Q V R S V A N A E QM D R L* * * * * * * *

* * * * * *

spoOF V T K E R P D L V L L D M K I P G M D G I E I L K RM K V I D E N I R V I I M T A Y G E L* * ** * * *** * ** * * * * ** * * * ** *********

* * * * * * * * * * * * * * * * *

spoOA L F K E K D P D V L V L D I I N P H L D G L A V L E R L R E S D L K K Q P N V I ML T A F G Q E* * * ** ** * **** ** * ** * ** * * * * ** * ** * *

* * * ** * * ** * ** * * * * * * * *

apR L T R E S F H L M V L D L M L P G E D G L S I C R R L R S Q S N PM P I I M V T A K G L E* * * * * *

* *

spoOF D M I Q E S K E L G A L T H F A K P F D I D E I R D A V K K Y L P L K S N* * * * * ** * ** * * *** * * *

* * ** * * ** *

spoOA D V T K K A V D L G A S Y F I L K P F D M E N L V G H I R Q V S G N A S S V T H R A P S S Q S* * * * ** * * * * ** * * ** ** * * * ** *

* * * * * ** * * * ****rpR V D R I V G L E I G A D D Y I P K P F N P R E L L A R I R A V L R R Q A D E L P G A P S Q E E

* *

spoOA S I I R S S Q P E P K K K N L D A S I T S I I H E I G V D A H I K G Y L Y L R E A I S N V Y N D* ** * * * * * *

* *

ampR A V I A F G K F K L N L G T R E N F R E D E P N P L T S G E F A V L K A L V S H P R E P L S R D

spoOA I E L L G S I T K V L Y P D0I A K K F N T T A S R V E R A I R H A I E V AW S R G N I D S I* * * * * * * * ** * * * * *

* * * * * *

OPR K L N N L A R G R E Y S A M E R S I D V Q I S R L R R W W K K I Q R F R V T F R P S G V W A

spoOA S S L F G Y T V S 1 T K A K P T N S E F I A N V A D K L R L E H K A S

* * * * * *

OWR T S L Y R T A L K H E A I A L L A T K F I C P Y V I A H R H L A V R Q PG D D L S GG A E L R D

FIG. 6. Comparison of a sequence of the SpoOF, SpoOA, and OmpR proteins. The three proteins are aligned for best fit utilizing gaps. Twostars indicate identical sequences, whereas one star indicates a conservative amino acid change. Stars on the outside of SpoOF and OmpRsequences indicate homology between these sequences that is not shared by the SpoOA protein.

Proc. Natl. Acad. Sci. USA 82 (1985)

is an indirect consequence of sporulation being regulated bya more general regulatory mechanism.

We thank S. M. H. Howard for excellent genetic analyses. Thisresearch was supported in part by Public Health Service GrantGM19416 from the National Institute of General Medical Sciences.This is publication number 3915-BCR from the Research Institute ofScripps Clinic.

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(1985) in Molecular Biology of Microbial Differentiation, eds.Hoch, J. A. & Setlow, P. (Am. Soc. Microbiol., Washington,DC), pp. 29-34.

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7264 Biochemistry: Trach et al.