Vol. 172, No. 2

The cisA Cistron of Bacillus subtilis Sporulation Gene spoIVCEncodes a Protein Homologous to a Site-Specific Recombinase

TSUTOMU SATO, YOSHIHIRO SAMORI, AND YASUO KOBAYASHI*

Department ofApplied Biochemistry, Faculty ofApplied Biological Science, Hiroshima University,Higashi Hiroshima 724, Japan

Received 19 June 1989/Accepted 28 September 1989

The nucleotide sequence of the sporulation gene spolVC cisA in BacUlus subtilis was determined and foundto encode a protein of 500 amino acid residues with a calculated molecular weight of 57,481, which is in goodagreement with the size of the gene product estimated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. The amino acid sequence of the N-terminal region of this protein is homologous to thesite-specific DNA recombinases. Hybridization of a 3.6-kilobase EcoRI fragment carrying the spoIVC cisA genewith the EcoRI-restricted chromosomal DNA prepared from cells of various stages showed that DNArearrangement occurs only in the mother cell in the region adjacent to spoIVC cisA 3 h after the initiation ofsporulation. This result coincides with that of Stragier et al. (P. Stragier, B. Kunkel, L. Kroos, and R. Losick,Science 243:507-512, 1989). The timing of the DNA rearrangement coincides very well with the timing ofspolVC cisA gene expression. The DNA rearrangement was not observed in spoIVC cisA mutants. These resultsstrongly suggest that the spolVC cisA gene encodes a site-specific DNA recombinase having a very importantrole in sporulation.

It is well known that DNA rearrangement occurs in somecases during cellular differentiation. The best known devel-opmentally regulated rearrangements are those of immuno-globulin genes in vertebrates (24). Since endospore forma-tion of Bacillus subtilis is a simple form of cellulardifferentiation, it has been speculated for a long time thatDNA rearrangement occurs during sporulation (14). Sporu-lation of B. subtilis initiates in response to nutrient depriva-tion. In the late stage of sporulation (stages 3 to 7), the cellsare divided into two compartments, mother cell and fore-spore. Each compartment contains a single intact chromo-some. The forespore becomes the mature spore, but themother cell lyses after maturation.The sporulation gene spoIVC of B. subtilis is located in a

3.6-kilobase (kb) EcoRI fragment cloned in temperate bac-teriophage 4105 (6). Farquhar and Yudkin (5) described aseries of new spoIVC sporulation mutations and suggestedthat all the mutations fall into two cistrons, cisA and cisB.We found that spoIVC133 is a mutation in the PvuII Bfragment using a transformation experiment (6) and that thismutation is located in the cisA gene (see Fig. 1). Kunkel etal. (16) suggested that spoIVC23 is located in cisB and thatcisB is expressed from 3.5 h after the initiation of sporulation(T3.5).

Recently, Stragier et al. (23) showed that spoIVC cisB andspoIIIC (4) are fused at T3 as a result ofDNA rearrangementand that the fused gene encodes the late-sporulation-specificsigma factor K (CrK) (15).We show here that spoIVC cisA encodes a protein whose

N-terminal region is highly homologous to DNA recombi-nases and that the DNA rearrangement does not occur inspoIVC cisA mutants. These results suggest that the spoIVCcisA protein is a site-specific DNA recombinase whichrearranges mother cell DNA during sporulation.

* Corresponding author.

MATERIALS AND METHODSBacterial strains. The strains used in the following exper-

iments are listed in Table 1. B. subtilis JH642, 1S47, 23.1,and spoIVC800 mutants provided chromosomal DNA forSouthern hybridization experiments. Escherichia coliMV1184 was used as the host for M13mplO or pUC118 (25).E. coli JM105 (27) was used as the host for expression vectorpKK223-3 (1) and its derivatives.

Nucleotide sequencing. The 3.6-kb EcoRI fragment wasprepared from 4i105spoIVC2 (6). DNA fragments from the3.6-kb EcoRI fragment were subcloned into M13mplO, orthe intact 3.6-kb EcoRI fragment was inserted into pUC118in both directions (pUC118IVC1 or -pUC118IVC2) andtreated with exonuclease III, mung bean nuclease, andKlenow fragment to generate overlapping deletion mutants(12). Sequencing was done by the dideoxy-chain terminationmethod (21).

Construction of pKKIVCA. Plasmid pCCA32 is a deletionderivative of pUC118IVC1 (see Fig. 4) harboring the intactspoIVC cisA gene in the 2.6-kb insert whose 5' end region is28 base pairs upstream from the initiation codon GTG of thecisA gene. The 2.1-kb HindIII-PstI fragment carrying theentire coding region of cisA was isolated from pCCA32 andinserted downstream of the tac promoter of pKK223-3. Thisplasmid was designated pKKIVCA (6.7 kb) (see Fig. 4).

Production of spoIVC cisA gene product in E. coli. Forgrowth and induction experiments, L broth supplementedwith 50 jig of ampicillin per ml was used. Overnight cultures(0.1 ml) of JM105 harboring the appropriate plasmid wereinoculated into L tubes containing 5 ml of L broth. The cellswere incubated at 37°C with shaking. When the culturereached 50 Klett units (no. 66 filter; Klett-Summerson pho-tocolorimeter), isopropyl-,-D-thiogalactopyranoside (IPTG)was added to a final concentration of 1 mM and incubationwas continued for an additional 5 h. The cells (0.5 ml) wereharvested at hourly intervals and centrifuged at 12,000 x gfor 10 min, and the pellets were suspended in 20 to 100 ,ul of100 mM Tris hydrochloride buffer (pH 6.8) containing 2%sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercap-

1092

JOURNAL OF BACTERIOLOGY, Feb. 1990, p. 1092-10980021-9193/90/021092-07$02.00/0Copyright ©D 1990, American Society for Microbiology

DNA REARRANGEMENT IN B. SUBTILIS 1093

TABLE 1. Bacterial strains used in this study

Strain Genotype Source orreference

Bacillus subtilisJH642 trpC2 pheAl Laboratory stock1S47 trpC2 spoIVCJ33 BGSCa23.1 trpC2 spoIVC23 J. Erringtonb801 lys-J purB6 spo-801 5802 lys-J purB6 spo-802 5804 lys-J purB6 spo-804 5807 lys-J purB6 spo-807 5813 lys-I purB6 spo-813 5814 lys-J purB6 spo-814 5815 lys-J purB6 spo-815 5816 lys-J purB6 spo-816 5818 lys-J purB6 spo-818 5819 lys-l purB6 spo-819 5

Escherichia coliMV1184 A(srl-recA)306::TnlO 25

A(lac-pro) ara thirpsL 48odlacZAM15(Ft proA+B+ lacIqlacZAM15 traD36)

JM105 thi rpsL endA sbcB15 27hspR4 A(lac-proAB)(F' traD36 proABlacIq/DM15)

a Bacillus Genetic Stock Center, Ohio State University, Columbus.b Microbiology Unit, Department of Biochemistry, University of Oxford,

Oxford, United Kingdom.

toethanol, and 0.001% bromophenol blue and heated at100°C for 3 min. A portion of the samples (5 to 30 ,ul) wasanalyzed by SDS-polyacrylamide (12.5%) gel electrophore-SiS.DNA isolation. Cells were cultivated at 37°C in Schaeffer

sporulation medium (22) and harvested at T6 (6 h after theinitiation of sporulation), except for forespore isolation; inthis case, cells were harvested at T7. DNA was purified asdescribed by Marmur (17). The forespore was isolated asdescribed by Nakayama et al. (18). Over 85% of the deter-gent-treated T7 cells were forespores. However, the intactforespore fraction was still contaminated with mother cellDNA. To remove the mother cell DNA, the foresporesuspension was treated with DNase I (final concentration, 10,ug/ml) at 37°C for 20 min and forespores were collected by

c£ t B I

centrifugation at 10,000 x g for 10 min. Then they weresuspended in 5 ml of 0.1 M NaCl-50 mM EDTA and treatedwith lysozyme (final concentration, 5 mg/ml). After 15 min at37°C, SDS (final concentration, 1%) was added and themixture was incubated at 3°C for 5 min. Preboiled (100°C, 10min) RNase A was added to a final concentration of 20,ug/ml, and after 5 min at 37°C, the forespore DNA wasextracted with redistilled phenol and chloroform.

Southern hybridization. A Photobiotin labeling system anda nonradioactive nucleic acid detection system (BluGENE)were obtained from Bethesda Research Laboratories, Inc.(Gaithersburg, Md.) and used as indicated in the providedinstruction manual. The 3.6-kb EcoRI fragment was labeledwith biotin-11-dUTP and used as a probe DNA. Chromo-somal DNA was digested with EcoRI, electrophoresed onagarose gels, and transferred onto nylon membrane. Hybrid-ization with the biotin-labeled probe DNA (100 ng/ml) wasperformed for 12 h at 42°C in hybridization solution contain-ing 45% formamide, 6x SSC (lx SSC is 0.15 M NaCl plus0.015 M sodium citrate, pH 7.0), Sx Denhardt solution (0.1%Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albu-min), 0.5% SDS, and denatured salmon sperm DNA (20,ug/ml). After hybridization, the unlabeled blots were washedtwice for 3 min at 20°C in 2x SSC containing 0.1% SDS,twice for 3 min at 20°C in 0.2x SSC containing 0.1% SDS,and then twice for 15 min at 65°C in 0.16x SSC containing0.1% SDS. Blocking was performed for 1 h at 65°C in 0.1 MTris hydrochloride buffer (pH 7.5) containing 3% bovineserum albumin and 0.15 M NaCl. After blocking, filters wereincubated in 0.1 M Tris hydrochloride buffer (pH 7.5)containing 0.15 M NaCl and 1.0 ,ug of streptavidin-alkalinephosphatase conjugate per ml. Color development was per-formed at room temperature for 10 to 60 min in 0.1 M Trishydrochloride buffer (pH 9.5) containing 0.1 M NaCl, 50 mMMgCl2, Nitro Blue Tetrazolium (330 ,ug/ml), and bromo-4-chloro-3-indolyl-phosphate (166 ,ug/ml).

Si nuclease mapping. Plasmid pUCIVC is a derivative ofpUC18 (27) carrying the 3.6-kb EcoRI fragment. pUCIVCwas digested with HaeIII, and the HaeIII A fragment wasisolated and purified (see Fig. 8). After dephosphorylationwith calf intestinal alkaline phosphatase, the 5' ends were32p labeled with [_y-32P]ATP and T4 polynucleotide kinase.RNA was isolated from the vegetative and sporulating cellsof B. subtilis as described by Gilman and Chamberlin (7). S1nuclease mapping was performed as modified by Berk and

A

enlll D frn_t

I c I 9A >p.--.-.

4... p

0. 5 kb

FIG. 1. Restriction cleavage map of the 3.6-kb EcoRI fragment, sequence strategy, and organization of the cisA and cisB genes. PvuII Band HaeIII D fragments, which can complement the spoIVC133 mutation by transformation, are shown by a heavy bar. The locations anddirections of sequence determinations are indicated by arrows. Transcription start points are represented by P, and the directions are shownby heavy arrows (16; this study).

VOL. 172, 1990

1094 SATO ET AL. J. BACTERIOL.

I CAGCTGTTACAT AGCCATTACCCAAGGGGTGATGCATTTT ATGAA^AGTGATAATCATCGAGGGACCGCAAGCTG ACAA^ATGCATT AACGATTGCTATCATTATTTAATAAAACTTTATAGG 120

cIsA start121 AAGGAGATTCAG___GAT4AGCAATATATGTAAGGGTATCGACCGAGGAACAAGCGATCAAGGGATCGAGCATCGACAGCCAAATCGAGGCCTGTATAAAGAAAGCAGGGACT0AAAATGT 240

M IA Y V R V S T EE 0Q A KG S S I D S Q E A C I K K A G T X D V

241 CTGAAGTATGCAGATGAAGGATTTTCAGGAGAGCTTTTAGAACGTCCGGCTTTGAATCGCTTGAGGGAGGATGCAAGCAAGGGACTTATAAGTCAAGTCATTTGTTACGATCCTGACCG 340L K Y A D E G F S G E L L E R P A L N R L R E D A S K G L I S Q V I C Y D P D R

361 TCTTTCTC4GA0ATTAATGAATCAGCTAATCATTGATGACGAATTGCGAAAGC4AAACATACCTTTGA8TTTGTAAATGGTGAATACGCCAATTCTCCAGAAGGTCAATTGTTTTTCGC 480L S R K L M N Q L I I D D E L R R9 N I P L I F V N G E Y A N S P E 0Q L F F A

481 AATGCGCGGGGCAATCTCAGAATTTGAAAGCCAAAATCAAAGAACGGACATCAAGCGGCCGACTTCAAAAAATGAAAAGGCATGATCATTAAAGATTCTAAACTATATGGCTATAA 600N R G A I S E F E K A E l K E R T 5 5 G R L Q E M X K G I EK D S K L Y G Y E

601 ATTTGTTA7AGAGAAAAGAACTCTTGAGATATTAGAAGAGGAAGCAAAAATCATTCGGATGATTTTTAACTATTTCACCGATC0ATAAAGCCCTTTTTTCGGCAGAGTAAATGGTATTGC 720F V K E K R T L E l L E E E A X I 1 P M I F N Y F T D H K S P F F G R V N G IA

721 TCTACATTTAACTCAGATGGGGGTTAAAACAAAAAAAGCGCC4AAAGTATGGCACAGGCAGGTTGTTCGGCAAATATTAATGAACTCTTCCTATAAGGGTGA0CATAGACAGTATAAAT 40L H L T Q M G V K T X K G A X V W H R Q V V R Q I L N N S S Y K G E H R Q Y K Y

841 T9ATACAGAGGGTTCCTATGTTTCAAAGCAGGCAGGGAACAAATCTATAATT0AAATAAGGCCTGAAGAAGAACAAATCACTGTGACAATTCCAGCAATTGTTCCAGCTGAACAATGGGA 960D T E G S Y V S K Q A G N X S 1 1 K R P E E E Q I T V T P A VV P A E6 W D

961 TT1ATGCTCAGAACTCTTAGGTCAAAGTAAAAGAAAACACTTGAGTATCAGCCCTCACAATTACTTGTTATCGGGTTTGGTTAGATGCGGAAAATGCGG6AATACCATGACAGGGAAGAA 1060Y A Q E L L G Q S X R K H L S I S P H N Y L L S G L V R C G X C G N T M T G E K

1081 A1GA2A0TCACATGGT0AAGACTACTATGTATATACTTGCCGGAAAAATTATTCTGGCGCAAAGGACCGCGGCTGCGGAAAAGAAATGTCTGAGAAT0AAATGAACCGGCATGTATGGGG 1200R K S H GO D YY V Y T C R E N Y S G A K D R G C G K E N S E N K L N R M v W G

1201 TG1AATTTTT3AATTCATCACAAATCCTCAAAAGTATGTTTCTTTTAAAGAGGCTGAACAATCAAATCACCTGTCTGATGAATTAGAACTTATTGAAAAAGAGATAGAGAAAACAAAAAA 1320E I FP F T N P Q K Y vS F K E A E Q S N H L S D E L E L 1 E X E 1 E K T E K

1321 AGGCCGCA1GCGTCTTTTAACGCT1AATCAGCCTAGCGATGACGATGATTTAGACATAGATGAAATCAAAGCACAAATTATTGAACTGCAAAAAAAGCAAAATCAGCTTACTGAAAAGTG 1440G R E R L L T L I S L S D D D D L D 1 D E XK A Q0 1 E L Q K Q N Q L T E K C

B end

1441 15T6CAGATCCAGTCAAAAAT0AAAGTCCTAGATGAT0ACGAGCTCAAGTGAAATGCTCTAAAAAGAGCCATCGACTATTTTCAATCAATCGGTGCAGATAACTTAACTCTTGAAGATAA 1560N R I Q S E N K V L D D T 5S S E N A L E R A I D Y F Q S I G A D N L T L E D K

cisA end1561 ACT1ACA6TTGTT6ACTTTATCGTGAAAGAAGTTACCATTGTGGATTCTGACACCATATATATTGAAACG60AAAGAGGGGTGTATGCACCCCCCTTTTGTAATTACAATC TT 1680

K T I V ' F V XRE V6 TT V D S D T I Y E T Y

1681 CA1TACACCTCGCTGCATACGTCGCCACCTTTGTCCCTTTTCCAGCGGAATAGCTTTCAATTCCTTTAATAAGCCCGATCGTTCCGATGGAGATTAAGTCCTCTGCATCCTCACCTGTAT 1800

1801 TTTCGAACTTTTTCACA1TATGGGCGACCAAGCGAAGATTATGC CAATCAGCATGTTTCTGGCATGTTCATCCCCTTTAGCCATGAGCTCT AAGTATTTTTTTCTTCGCTGCTTGAGA 1920ciSB startM GGAGG

1921 GCGGTTGTGGAAAGGCATTGTTTTTCACGTAAGATACTAAAAAGACAAGCTCTTTAACAACAAAGCCGAGCGCTGCGAAAACACCTGTCAC A CGTCACCTCCACAAAAGTATGTAGGC 2040

2041 2A1AGCCTAT0ATGTATGTATGTGACCGGGAGGCTGTCTGTGTCTGTACCGGG0AATTTCTCGCGGGGGAGTCCGGCTTTGGTAAGCAGCAT6ACT0AC TCATGTTAAAAATATTCT 2160

2161 TCATCAAGCGCCCATACATTGAAATGAACAAAGGTATGGGGGGATGGGGATGAAAAAATGGATGGCAGGCCTCTTTCTTGCTGCAG 2246

FIG. 2. Sequence analysis of the cisA and cisB genes (GenBank accession number M29040). Only the coding DNA strand of cisA is shown.Numbering is from the S' end at the PvuII site of the far left and ends downstream at the PstI site (nt 2246) shown in Fig. 1. The putativeinitiation codon of cisA is located at nt 134 and that of cisB is located at nt 2014 on the antisense strand of cisA DNA. The presumptiveribosome-binding sites are underlined. The stop codons are indicated by asterisks. The DNA breakpoint (nt 1674 to 1678) is boxed (23). Apair of arrows with broken lines indicate an inverted repeat sequence with several mismatches.

IEVOOl ...., A*AUS Am Ao

!A3VIISW~ siO}U1DE 4 .;.3' bt

.WQH ,RtwDI. N*. 4HS,Q3GDI' 0E-i

\F^*~~~~MJ^ svZ kri ............. i E*G

go ORGSSN

*AS|A| AAIR&R L :' 1r . N ?..'G*AFA RSL R GRECIAiLA0IR A. " 3 ~-AIA AQ RQR L 'INEWQ-AMA W,'*hAN RE V LA A.A _ARA R- OhARMtF UIUQRAI 9; .452.R A k1: - .. * ;* i}^ A|llM R . I wMA A. :A ^ R RC^ li t:" * ..r..

FIG. 3. Alignment of the peptide sequence of the cisA product, resolvases of Tn3, Tn21, and 'y8, and invertases Hin, Gin, and Pin.Residues identical to CisA are shaded. *-, C-terminal region of the cisA product. aa, Amino acids.

DNA REARRANGEMENT IN B. SUBTILIS 1095

3.6 kb EcoRI fragment

* 'i { pUC1180s g :; i

cis '

L igase I FuRI= I

. .. E I6CCSGTTGCAT0TMCXTGCTA i3GTAMMTOTTC e- ^z i s

HAMi Spnh Slst WI EcoRI Pvullr PvuII

Multiple cloning site pUC118ii16.8kb

PeasesI IsBo

Exnc IeseI 1, Mvbi nMcIaOMKIen frapet. ligse

P tacHindl Pst

AmprpKK223-34. 6kb

PvulPst / \ Hindl

/V I\VU2.6kb fragment 2.0kb fragment

AACTTGATAMCAT1 6 ATTCA TG---rHindlIl SD cisA start *

HiadillPVUII

Asr

Hindlll Pstl2.1kb fragment

IliigaseHindl PSt

PvuIIP tac

pK ICA6. 7kb

L~~~~~~~~~DVUNulPSt~~~~~~~~~v I kPstH iII

FIG. 4. Structure of plasmid pKKIVCA. The construction of pKKIVCA is described in the text. The DNA fragment containing the intactcisA gene is shown by heavy bars. Ampr, Ampicillin resistance.

Sharp (2). RNA samples (50 jig) were mixed with 50,000 cpmof the probe DNA. Hybridization was performed for 3 h at49°C. Samples were treated with 25 U of S1 nuclease andelectrophoresed on a 5% polyacrylamide-8 M urea gel inTBE (90 mM Tris base, 90 mM boric acid, 2 mM EDTA, pH7.6) buffer.

RESULTS AND DISCUSSION

Nucleotide sequencing. To obtain the exact location of thespoIVC genes, we determined the nucleotide sequence of the3.6-kb EcoRI fragment. The sequencing strategy is shown inFig. 1, and the nucleotide sequence of the PvuII-PstI region,2,246 nucleotides (nt), of the fragment is shown in Fig. 2.There are two large open reading frames, cisA and cisB, inthe opposite direction facing each other (Fig. 1). A transfor-

mation experiment indicated that the spoIVC133 locus wasin the HaeIII D fragment in the cisA gene (data not shown;Fig. 1). The potential start codon of cisA, extending from nt134 to nt 1633, is the GTG at nt 134. Preceding this GTG by6 nt is the sequence 5'-GGAAGGAGA-3', which could beused as a ribosome-binding site. The cisA gene would thusencode a polypeptide of 500 amino acid residues with acalculated molecular weight of 57,481. The nucleotide se-quence of the cisB gene (155 codons) is identical to thatreported by Stragier et al. (23). This gene encodes theamino-terminal region of the sigK gene.Amino acid sequence homology. Computer analysis of the

deduced amino acid sequence of the cisA gene product withthe National Biochemical Research Foundation data baseusing the rapid search program IDEAS (integrated data base

--i

VOL. 172, 1990

Iww.6

1096 SATO ET AL.

rIN

I_. tk.

FIG. 5. Overproduction of the CisA protein in E. coli. JM105 carrying pKK223-3 or pKKIVCA was induced with IPTG (finalconcentration, 1 mM), and at the indicated times, the total cellular proteins were analyzed by SDS-polyacrylamide gel electrophoresis.Noninduced JM105 cells carrying pKK223-3, pKKIVCA, or no plasmids were harvested at the early stationary phase when the cells reacheda density of 250 Klett units. The position of the overproduced protein is shown (55 K). The positions of the molecular weight markers areindicated at the left margin (K, x 103).

and extended analysis system developed by the NationalInstitute of Genetics, Mishima, Japan) revealed that theN-terminal half ofCisA has significant homology with almostthe whole sequence of the site-specific recombination pro-teins (DNA invertases) Hin (28), Gin (19), and Pin (19) andthe resolvases of transposons Tn3 (11), Tn2J (13), and -yb(20). The amino acid sequences of these recombinases werealigned with that of CisA. These aligned sequences exhibiteda remarkable similarity to the N-terminal region (amino acids1 to 144) of CisA; 43 (29.9%) amino acid residues of the Tn3resolvase and 44 (30.6%) amino acid residues of Hin areidentical (Fig. 3). This result suggests that the cisA geneencodes a recombinase. However, compared with thesesite-specific recombinases, CisA has a high molecular weightand its C-terminal half has no homology with the recombi-nases. Data base analysis of the C-terminal half of CisAshowed that amino acid residues 372 to 447 of CisA share 20of 76 identities with residues 141 to 216 of the colicin Elprotein (26) and that residues 149 to 206 of CisA share 22 of58 identities with residues 231 to 292 of the 32,000-daltonthyracoid membrane protein (10), respectively. The functionof the C-terminal region of CisA is unknown at present, butit may have an important function in DNA rearrangement,since DNA rearrangement did not occur in the cisA mutantshaving defects in the C-terminal region (see Fig. 6b).

Overexpression of CisA in E. coli. We wished to determinedirectly the size of the cisA gene product to confirm the sizepredicted from the open reading frame. We constructed ahigh expression vector, pKKIVCA, carrying the cisA gene(Fig. 4). This plasmid contains the strong tac promoterwhich can be induced at an appropriate time by the addi-tion of IPTG. E. coli JM105 cells harboring the plasmidpKKIVCA were grown at 370C to the log phase, and then theexpression of the spoIVC cisA gene was induced by theaddition of 1 mM IPITG. After an additional 5 h of incuba-tiQn, the cells were harvested and the total cellular proteinswere analyzed by SDS-12.5% polyacrylamide gel electro-phoresis. A protein band of approximately 55,000 molecularweight, which was in good agreement with the predicted

molecular weight of the CisA protein, was observed 1, 3, and5 h after induction (Fig. 5). This result suggests that thespoIVC cisA gene encodes the 55,000-molecular-weight pro-tein. Deletion analysis of the cisA gene also supported thisconclusion (data not shown).DNA rearrangement. If the spoIVC cisA gene product is a

site-specific recombinase, DNA recombination should occuraround the cisA gene, since the invertible DNA segments arelocated in the adjacent regions of all the DNA invertasegenes. With this idea in mind, we did Southern hybridizationexperiments and found, independently of the results ofStragier et al. (23), that DNA rearrangement occurs aroundthe cisA region (T. Sato, Y. Samori, and Y. Kobayashi, 11thAnnu. Meet. Mol. Biol. Soc. Jpn., Tokyo, 20 to 23 Decem-ber 1988). Furthermore, we found that the rearrangementdoes not occur in the spoIVC cisA mutants.Chromosomal DNA was prepared from wild-type and

spoIVC133 mutant cells of B. subtilis harvested at thevegetative stage and at different times after the initiation ofsporulation. The extracted DNAs were digested with endo-nuclease EcoRI, electrophoresed, blotted, and hybridized tothe biotin-labeled 3.6-kb EcoRI fragment. When the probeDNA was hybridized with the DNAs from vegetative-to-T2cells of wild type and from the vegetative-to-T6 cells of thespoIVC133 mutant which has the mutation in the cisA gene,only one EcoRI band (3.6 kb) was observed (Fig. 6a).However, in addition to the 3.6-kb band, two new bands (2.8and 5.4 kb) appeared when DNA of the wild-type cellsobtained after T3 was used (Fig. 6a). These results indicatethat (i) rearrangement occurs around the cisA gene from 3 hafter the initiation of sporulation, (ii) one end of the DNAbreakpoint should exist within the 3.6-kb EcoRI fragment,and (iii) this rearrangement is promoted by CisA.

Recently, Farquhar and Yudkin (5) have analyzed a seriesof new spoIVC mutations and have shown that the spoIVClocus comprises two cistrons, cisA and cisB. A Southernhybridization experiment with the EcoRI-digested chromo-somal DNAs of these mutants showed that the DNA rear-rangement did not occur in the cisA mutants spoIVC801,

J. BACTERIOL.

..A. -Afts II..I.: %..:

,-, 4

i10Ift..j%.4t

DNA REARRANGEMENT IN B. SUBTILIS 1097

A B C D E F G H J K L M

*.*, 0* - -11 - - 5.4_ _gm as a& fg a& am a& -.- _.6 _.a _- 3.6

- xi*a-- 2. 8

5. 4-3.6-2.8-

b 22 - 0 O 0Oco _ co X 0 co co co:c

1,.- ....... .......III II

CiSA

A B C D E F G H

aN It 0

a:> N 4m mI I #.... ^X

cisB

I J K

EcoRI HindUilMF F MF F

-7.2- 4.4

4. 2

FIG. 7. Southern hybridization experiments with T7, DNAs ob-tained from whole cells (lanes MF) and from purified forespores(lanes F). EcoRI-digested chromosomal DNA (EcoRI) and HindIll-digested chromosomal DNA (Hindlll) were hybridized with the3.6-kb EcoRI fragment. The numbers show sizes in kilobases.

- -5.4-A-- - 3.6 _ _ _ 3. 6

.r -2. 8

A I E IO I C I a

FIG. 6. (a) Time course of DNA rearrangement during sporula-tion. DNAs were extracted from B. subtilis JH642 at the vegetativestage (lane A), To (lane B), T7 (lane C), T2 (lane D), T3 (lane E), T4(lane F), T5 (lane G), T6 (lane H), and T8 (lane I) and from 1S47(spoIVC133) at the vegetative stage (lane J), T2 (lane K), T4 (lane L),and T6 (lane M). DNA samples were digested with EcoRI, electro-phoresed on a 0.8% agarose gel, and blotted onto Hybond-N filterpaper (Amersham Corp., Arlington Heights, Ill.) and hybridizedwith the 3.6-kb EcoRI fragment. (b) DNA rearrangement in spoIVCcisA and cisB mutants. DNA samples were prepared from T6 cells ofthe mutants. EcoRI-digested DNA samples were hybridized withthe 3.6-kb EcoRI fragment. Lanes A to H are DNAs from cisAmutants (spoIVC813, -133, -819, -818, 401, -815, -807, and -816,respectively), and lanes I to K are DNAs from cisB mutants(spoIVC802, -23, and -814, respectively). The numbers to the sidesshow sizes in kilobases.

-807, -813, -815, -816, -818, and -819 but did occur in the cisBmutants spoIVC802, -814, and -23 (Fig. 6b). These resultssuggested that the cisA gene product is a recombinase.

Localization of DNA rearrangement in the mother cell. Todetermine whether the DNA rearrangement occurred in themother cell or forespore or both, we isolated whole-cell andforespore DNAs from T7 cells. The DNAs were digestedwith either EcoRI or Hindlll, electrophoresed, blotted, andhybridized with the 3.6-kb probe DNA. RepresentativeSouthern hybridization data are shown in Fig. 7. One strongband (3.6 kb with EcoRI or 7.2 kb with HindIII) was

detected with DNA prepared from both forespore and wholecells. In addition, weak bands (5.4 and 2.8 kb with EcoRI or

4.4 and 4.2 kb with HindIII) were detected with DNAprepared from the whole cell but not with DNA purified fromthe forespore. These results indicate that the DNA rear-

rangement specifically occurred in the mother cell and thatthe effects of the DNA rearrangement should not be inher-ited by the next generation, since the mother cell is dis-carded after maturation of the forespore.Time of expression of cisA gene. Using sporangium proto-

plasts prepared 3 h after induction of sporulation, Dancerand Mandelstam (3) showed that the spoIVC133 (cisA)

P

I c Is A jIP robe

kb

200 bp

l} A 8 C 0 E F G H I I K L

Iw

S0200 bP

4b

FIG. 8. Time course of expression of spoIVC cisA and transcrip-tion initiation site of the cisA gene. (a) HaeIII restriction map of the3.6-kb EcoRl fragment containing spoIVC cisA and cisB and theprobe DNA fragment. Plasmid pUCIVC was constructed by insert-ing the 3.6-kb EcoRI fragment into the EcoRl site of pUC18. Thesolid box represents a 228-base-pair (bp) fragment from pUC18DNA. H, HaeIII; E, EcoRI. mRNA hybridizes to the antisensestrand of the cisA DNA. (b) S1 nuclease mapping was conductedwith RNAs from the cells at the vegetative stage (lane E), To (laneF), T, (lane G), T2 (lane H), T3 (lane I), T4 (lane J), and T. (lane K).Lanes A and L are M13mpll-HpaII size markers. Lane B is the5'-end-labeled probe. Lane C is the 5'-end-labeled probe digestedwith EcoRI. Lane D is a control experiment with RNA from E. colicells. Arrow indicates the location of the transcript of spoIVC cisA.

a

IXC1UVIC 6. 4 kO

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GI FI.......~~ .. ... .....__....._....... . ....

VOL. 172, 1990

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1098 SATO ET AL. J. BACTERIOL.

mutant can be complemented by protoplast fusion withspoIVC+ cells. This result indicates that the spoIVC cisAgene is probably expressed in the mother cell. Using Sinuclease mapping, the time of cisA gene expression andlocation of the transcriptional start point of the cisA genewere determined. The experimental strategy is shown in Fig.8a. RNA samples were prepared from wild-type cells atdifferent times after the initiation of sporulation. Figure 8bshows that the transcription of the cisA gene starts at T2about 130 base pairs upstream from the initiation codon andreaches a maximum at T3. Note that the timing of the cisAgene expression precedes the occurrence ofDNA rearrange-ment.

Recently, Stragier et al. (23) showed that spoIVCB (cisB)joined in frame to spoIIIC (4) to create a composite struc-tural gene for uK (15) by a chromosomal rearrangementoccurring in the mother cell at an intermediate stage ofsporulation. This observation coincides with our findingsthat the cisA gene product is homologous to site-specificrecombinases and that the DNA rearrangement occurs in themother cell in the adjacent region of the spoIVC cisA gene at3 h after the initiation of sporulation in B. subtilis.Developmentally regulated DNA rearrangement in pro-

caryotes has been demonstrated in the DNA adjacent to thenitrogen fixation (nij) genes of Anabaena species duringheterocyst formation. This DNA rearrangement results inthe deletion of 11 kb ofDNA between an 11-base-pair directrepeat sequence flanking the nifK and nifD genes and 55 kbof DNA in the adjacent region (8, 9). The heterocyst differ-entiation is initiated in response to nitrogen deprivation. Thisphenomenon is similar to DNA rearrangement occurringduring sporulation, since the DNA rearrangement occursonly in the heterocyst, which is specifically differentiated toprovide nitrogen to the vegetative cells and has no effect onthe descendants. These findings are very interesting becausethey clearly indicate that differentiation can be mediated byDNA rearrangement in procaryotes as well as in vertebrates.

ACKNOWLEDGMENTS

We thank R. Losick and M. D. Yudkin for communicating theirresults before publication and K. Kutsukake for many useful com-ments and for critical reading of the manuscript. T. lino and F.Kawamura are thanked for helpful advice.

This work was supported in part by a grant-in-aid for scientificresearch and for special project research from the Ministry ofEducation, Science, and Culture of Japan.

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