Acid-Soluble Spore Proteins of Bacillus cereus, Bacillus ...

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Apr 10, 1986 - degraded during spore gerniination and which reacted with antibodies raised against B. megaterium SASP. Genes codin for a B.
Vol. 167, No. 1

JOURNAL OF BACTERIOLOGY, July 1986, p. 168-173 0021-9193/86/070168-06$02.00/0 Copyright C 1986, American Society for Microbiology

Cloning and Nucleotide Sequencing of Genes for Small, Acid-Soluble Spore Proteins of Bacillus cereus, Bacillus stearothermophilus, and "Thermoactinomyces thalpophilus" CHARLES A. LOSHON,1 EDWARD R. FLISS,1 BARBARA SETLOW,' HAROLD F. FOERSTER 2 AND PETER SETLOWL* Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032,1 and Division of Life Sciences, Sam Houston State University, Huntsville, Texas 7734J2 Received 7 February 1986/Accepted 10 April 1986

As found previously with other Bacillus species, spores of B. stearothermophilus and "Thermoactinomyces thalpophilus" contained significant levels'of small, acid-soluble spore proteins (SASP) which were rapidly degraded during spore gerniination and which reacted with antibodies raised against B. megaterium SASP. Genes codin for a B. stearothermophilus ad a "T. thalpophilus" SASP as well as for two B. cereus SASP wire cloned, their nucleotide sequences were determined, and the amino acid sequences of the SASP coded for were compared. Strikingly, all of the amino acid residues previously found to be conserved in this group of SASP both within and between two other Bacillus species (B. megaterium and B. subtilis) were also conserved in the SASP coded for by the B. cereus genes as well as those coded for by the genes from the more distantly related organisms B. stearothermophilus and "T. thalpophilus."' This finding strongly suggests that there is significant selective pressure to conserve SASP primary sequence and thus that these proteins serve some function other than shuply amino acid storage.

ously (5, 10). DNA from B. stearothermophilus ATCC 7953 was a gift of H. Cudny. The sources of the Escherichia coli strains, XgtlO, and plasmids were described previously (3, 9). Plasmid-carrying strains were grown at 370C in 2 x YT medium (tryptone, 16 g/liter; yeast extract, 10 gAiter; NaCl, 5 g/liter) with appropriate antibiotics (ampicillin, 50 ,ug/ml; chloramphenicol, 10 ,ug/ml; tetracycline, 10 gxg/ml), and plasmids were isolated by the method of Birnboim and Doly (1). If necessary, plasmid DNA was purified by two cycles of CsCl gradient centrifugation. Spore germination and SASP isolation and analysis. Spores of B. stearothermophilus (1 mg/ml [dry weight]) were activated at 30°C for 24 h in 0.2 M sodium nitrite (pH 8.0). The activated spores (1 mg/ml [dry weight]) were germinated at 65°C in 10 mM sodium phosphate (pH 8.0)-i mM L-valine. Spores of "T. thalpophilus" (1 mg/ml [dry weight]) were activated at 250C for 48 h in distilled water. The activated spores (1 mg/ml [dry weight]) were germinated at 55°C in 10 mM sodium phosphate (pH 8.0)-i mM L-leucine-1 mM L-phenylalanine-l mM L-alanine. After 30 min, >95% of the spores had germinated as determined by phase-contrast microscopy, the culture was centrifuged (10 min, 10,000 x g), and the pellet was lyophilized. Dormant and germinated spores (20 to 30 mg [dry weight]) were broken by dry rupture in a dental amalgamator (Wig-L-Bug) with glass beads (100 mg) as the abrasive (21). The dry powder was extracted twice with 2 ml of 3% acetic acid for 30 min at 40C, and both supernatant fluids were pooled and dialyzed overnight against two changes of 1 liter of 1% acetic acid in Spectrapor no. 3 dialysis tubing (molecular weight cutoff, 3,500). The dialyzed material was centrifuged (10 min, 10,000 x g), and the supernatant fluid was lyophilized. The resulting powder was dissolved in 150 ,ul of 8 M urea plus 75 ,il of acid gel diluent (14, 20). This procedure has been shown to extract >90% of all SASP from spores of several Bacillus species (14, 21). SASP were separated by electrophoresis on acrylamide gels at low pH (20), and proteins were either

Ten to twenty percent of the protein of spores of a number of Bacillus species is degraded in the first minutes of spore germination, thus supplying amino acids for both new prote,in synthesis and metabolism (5, 22). The proteins degraded in this process are a group of small, acid-soluble spore proteins (SASP) which are synthesized only during sporulation under transcriptional control (22). In B. megaterium three proteins (termed SASP-A, -B, and .-C) make up -85% of the protein degraded, with SASP-A and -C being very similar in primary sequence and SASP-B being more different (22). Studies of this system at the gene level have revealed that in B. megaterium, in addition to the genes encoding SASP-A and -C, there are five other genes which code for SASP which are very similar to SASP-A and -C (5, 8, 9); this multiplicity of SASP genes' has also been found in B. subtilis (2, 3). Strikingly, the amino acid sequences of the proteins coded for by these different SASP genes exhibit a high degree of homology, with -65% of all residues conserved both within and across species (3, 5, 9). Given this high degree of SASP sequence conservation, we felt that it might be valuable to analyze SASP genes from other sporulating bacteria, including several more distantly related than' are B. megaterium and B. subtilis. Consequently, in this communication we report the cloning and nucleotide sequence of SASP genes from B. cereus, B. stearothermophilus, and "Thermoactinomyces thalpophilus" MATERIALS AND METHODS Organisms used and isolation of DNA. The organisms used were B. cereus T, (originally obtained from H. 0. Halyorson), B. stearothermophilus NGB101 (11), and "T. thalpophilus" HA-O1 (10). Spores of B. stearothermophilus and "T. thalpophilus" were pr-epared and purified as previously described (10, 11). DNA was also isolated and purified from B. cereus and "'T. thalpophilus" as described previ*

Corresponding author. 168

VOL. 167, 1986

a

f~ - w~-2

NUCLEOTIDE SEQUENCE OF SASP GENES

I b

plaque

c

a

b

BL

Dye Frnnt FIG. 1. Analysis of SASP in dormant and germinated spores of (I) B. stearothermophilus NGB101 or (II) "T. thalpophilus." Dormant (lanes a and b) or germinated (lanes c) spores were disrupted, SASP were extracted, samples (5 ,ul in lanes a and b; 15 .Ll in lanes c) were run on acrylamide gel electrophoresis at low pH as described in Materials and Methods, and the proteins were detected by staining with Coomassie blue (lanes a and c) or samples were transferred to nitrocellulose and protein was detected by immunoblotting with a 1/400 dilution of antiserum against B. megaterium SASP-A. The arrows labeled B in lanes a denote the positions of the only bands detected on immunoblotting with antiserum against B. megaterium SASP-B.

stained with Coomassie blue or transferred to nitrocellulose and then detected with a 1/400 dilution of antiserum raised against B. megaterium SASP-A or -B as previously described (14). Total spore protein and protein in the acetic acid supernatant after dialysis was measured as described previously (16). Identification, cloning, and sequencing of SASP genes. Restriction enzyme digests of genomic DNA (1 jig) were run on agarose gel electrophoresis, and fragments were transferred to nitrocellulose (9). These blots were hybridized to one of a variety of SASP gene probes: (i) a B. megaterium SASP-C gene probe, a 0.7-kilobase (kb) BgllI-HaeIII fragment containing the coding sequence of the B. megaterium SASP-C gene (6); (ii) a B. megaterium SASP-C-3 gene probe, a 0.6-kb EcoRI-MspI fragment containing the coding sequence of the B. megaterium SASP-C-3 gene (7), (iii) a B. subtilis sspB gene probe, a 0.7-kb HindIII-PstI fragment containing the complete B. subtilis sspB gene coding sequence (3); (iv) a B. subtilis sspA gene probe, a 1-kb HindIII-PstI fragment containing the coding sequence of the B. subtilis sspA gene (3); (v) a complete B. stearothermophilus SASP-1 gene probe, a 0.7-kb HindIII-HaeIII fragment containing the complete coding sequence of the B. stearothermophilus SASP-1 gene (see Fig. 3); (vi) a partial B. stearothermophilus SASP-1 gene probe, the 0.6-kb EcoRIEcoRI fragment of B. stearothermophilus DNA containing the carboxyl-terminal portion of the sequence coding for SASP-1 (see Fig. 3); and (vii) a "T. thalpophilus" SASP-1 gene probe, a 0.65-kb HaeIII-HaeIII fragment containing the coding sequence of the "T. thalpophilus" SASP-1 gene (see Fig. 3). Hybridization of Southern blots was at 55°C under the conditions described previously (2, 3), except for blots of B. stearothermophilus or "T. thalpophilus" DNA probed with heterologous DNA, which were hybridized at 48°C. Genomic DNA was digested with restriction enzymes, and fragments of appropriate size were isolated as previously described (9). Fragments were cloned into phage or plasmid vectors, and with one exception clones were detected by

169

or colony hybridization with the B. megaterium SASP-C gene probe (B. cereus genes) or the B. subtilis sspB gene probe (B. stearothermophilus gene). The complete B. stearothermophilus SASP-1 gene was cloned in plasmid pBR325 using the partial B. stearothermophilus SASP-1 gene probe for its detection. The "T. thalpophilus" SASP-1 gene was cloned in plasmid pBR325 and identified by Southern blot analysis of plasmids from -300 clones containing HindIII fragment inserts of 2 to 2.5 kb. Clones were grown individually in 1-ml cultures, pooled in groups of five, plasmid isolated, cleaved with HindIII, and run on agarose gel electrophoresis. Fragments were then transferred to nitrocellulose, and the blots were hybridized with the B. subtilis sspB gene probe. The hybridizing regions in DNA fragments were localized by restriction enzyme digestion and Southern blotting as previously described (2, 3), and subfragments were cloned in pUC plasmid vectors. DNA sequence analysis was carried out by the method of Maxam and Gilbert (18); all sequences were determined completely in both directions, and all restriction sites used in sequencing were overlapped.

RESULTS Presence of SASP in B. stearothermophilus and "T. thalpophilus" spores. As found previously with spores of B. cereus, B. megaterium, and B. subtilis (15, 22, 26), dormant spores of B. stearothermophilus and "T. thalpophilus" contained a significant amount of acid-soluble protein-12 and 14% of total spore protein, respectively (data not shown). There were a number of different protein species in

I

_ |

3

/~~~~ I_

,

---.. _

-,",

4-

\

_lo

-

3

-4

a

b

c

d

e

FIG. 2. Southern blots of genomic DNA. Genomic DNA was cut with various restriction enzymes, samples (1 ,ug) were run on a 1% agarose gel, the DNA was transferred to nitrocellulose, and the blots were hybridized as described in Materials and Methods. The lanes contained (a) B. cereus DNA cut with EcoRI and hybridized to the B. megaterium SASP-C gene probe, (b) B. stearothermophilus ATCC 7953 DNA cut with EcoRI and hybridized to the B. subtilis sspB gene probe, (c) "T. thalpophilus" DNA cut with Hindlll and hybridized to the B. subtilis sspB gene probe, (d) B. stearothermophilus ATCC 7953 DNA cut with HindlIl and hybridized to the B. stearothermophilus complete SASP-1 gene probe, and (e) "T. thalpophilus" DNA cut with HindIII and hybridized to the "T. thalpophilus" SASP-1 gene probe. The arrows in lane a denote bands which also hybridized with the B. megaterium SASP-C-3 gene probe; those in lanes b and c denote bands which also hybridized to the B. subtilis sspA gene probe. The lines numbered 1 to 4 show the positions of marker DNAs of sizes 9.6, 4.5, 2.3, and 0.6 kb,

respectively.

170

LOSHON ET AL.

J. BACTERIOL.

TABLE 1. SASP genes cloned from B. cereus, B. stearothermophilus, and "T. thalpophilus'a Size (kb) of fragment cloned (restriction

SASP gene

5 -mAAATVVTATPATTTAACOTTCrCTA

Cloned in:

enzyme used)

5 (EcoRI)'

B. cereus 1 B. cereus 2

9

0.8 (EcoRI)d 2.3 (HindIII)b 2.2 (HindIII)b

B. stearothermophilus 1 "T. thalpophilus" 1

150

LA ML OCT GM

GIN ALA LRU ASP GLN IQT LYS Tra mx ALA GI OLU PRI OLT VAL GIN T ALA CM OCT CTT GAT CAA ATG AMA TAT GM ATr OCA CM GAO Tr GmT OTA aU1 CMA CrT GO OCA

xgtlo xgto

0.6 (EcoRI)c

B. stearothermophilus 1 B. stearothermophilus 1

10o

SAAACAAT NIT GLY 1.1 AN m S GY SI M A G A U A AM L ATB OGA AM MT MT AgT OGA ArT CUT MT GM QTA TTA GTT COA GOC 200

xgtlo

(EcoRI)b

TOTTAACCACTAAGAAAI

XgtlO pBR325 pBR325

Fragnents were identified and cloned as described in Materials and Methods. b This fragment contained a complete SASP gene coding sequence. C This fragment contained only the amino-terminal coding region of a SASP gene. d This fragment contained only the carboxyl-terminal coding region of a SASP gene. a

ASP GAT

TM TMI ACA ACA

250 ALA AM SIR Am 01. SIR VAL 0LY 0LY (L ILlTM LYS ARSLIU VAL ALA OCT COT TCA MC OGGA TCT oTT GTGOT GM ATr ACA AM CBT TTA OTA OCA 300

GIN GIN LIY OLT0!. AM ALA AmN AMO ON OTAAAT ATO GCA GM * CA CM CTT GOTrGOTAA OC MAC CC TM * MIT

AAAATATAGCATA 400

ALA OUI

350

M M

M _T_A.M.CTMA _

TAAAATATM

450 SOO

*

C _ATTAMA _ _

C

550

TAAMAAI

CMTATGMAACTAOTTrATG

600

the acid-soluble protein fraction (Fig. 1, lanes a), but these proteins disappeared during spore germination (Fig. 1, lanes c). Strikingly, a number of the B. stearothermophilus and "T. thalpophilus" acid-soluble dormant spore proteins reacted strongly with antisera raised against B. megaterium SASP-A (Fig. 1, lanes b) or SASP-B (Fig. 1, arrows labeled B, lanes a). Cloning of SASP genes from B. cereus, B. stearothermophilus, and "T. thalpophUis." With the knowledge that B. stearothermophilus and "T. thalpophilus" spores contained SASP which were quite similar to the B. megaterium SASP-A and -C, we proceeded to analyze these organisms, as well as B. cereus, for SASP genes. Hybridization of Southern blots of restriction enzyme digests of

SASP gene 5 B.

corpus- I

I

i 8

7

10

4

B. cereus-2

32 i 2

5

1O

4?

ATTITCAA_OAGM.TAAMGGTAATAATGATCCAATMGrACATTACOOTGMO 650 700 800

750

MCACCn~MgMOTTTITAgM.GAGC1STA AAfl~AATOTAITO-3 FIG. 4. Nucleotide sequence of the B. cereus SASP-1 gene coding and flanking sequences and the predicted amino acid sequence of SASP-1. The singly underlined bases from positions 155 to 162 show good complementarity to the 3' end of the 16S rRNA of B. cereus. The two regions that are doubly underlined can base pair with each other and may be a transcription stop signal. Dots below nucleotides are positioned every 10 bp.

genomic DNA with B. megaterium SASP-C, -C-3, or B. subtilis sspB gene probes revealed a number of hybridizing bands, some of which hybridized to two different SASP gene probes (Fig. 2, arrows, lanes a, b, and c). These hybridizing bands, in particular those hybridizing with two probes, seemed likely to carry SASP genes. Consequently, two such fragments were cloned from B. cereus, two from B. stearothermophilus ATCC 7953 and one from "T. thalpophilus," and the regions hybridizing to SASP gene probes were localized (Table 1; Fig. 3). Subsequent nucleotide sequence analysis showed that the two B. stearothermophilus EcoRI fragments contained only parts of SASP genes. Consequently, one of the fragments was used s'-

B. stearotiermophilus-

50

E14

Aa

I kb

762 96

TFrTAAcAAAT ACAA TCTLAAACTAOATATTTA ITACACATTATACTCACAA AL

7

4 T tholpophilus-I 4' +4? FIG. 3. Restriction maps of cloned DNA. Restriction maps were determined, and the regions hybridizing to SASP gene probes were localized as described in Materials and Methods and from DNA sequence analysis. The boxed region is the SASP gene coding sequence, and the arrow below it shows the direction of translation. The numbered arrows denote restriction enzyme cleavage sites; the enzymes noted are: 1, AccI; 2, BcIl; 3, CfoI; 4, ClaI; 5, EcoRI; 6, HaeIII; 7, HindIII; 8, HpaI; 9, MluI; and 10, PvuII. The B. cereus SASP-2 gene was initially cloned on a 9-kb EcoRI-EcoRl fragment. The coding region of this SASP gene was further localized on i 3.5-kb PvuII-PvuII fragment, although the location of this fragment in the initial 9-kb fragment was not determined. The exact location of the SASP gene coding sequence in the PvuI fragment was not established, but it is contained on a 430-bp TaqI-TaqI fragment which lies between the HindIII and HpaI sites.

150

100

OATATAMC ATIT 81X AMSO S8 T A8m ITS ICU ALA VAL PM 01. ALA 0. SR ALA LII ASP TCA COT ASC ACA AMT AAA TTA GCO OTf CCT GOT OCT GM TCA OCA TTA AC 200

GAIN NET 1.1 TR 01. I ALA GIN 01. PS 01 VAL G U 10. ALA ASP ALA WSCALA CAA ATB AAA TAC GM ATC OCT CM GAG OTT OfT OTT CAA CIT GA OCT AT OCA ACA OCT 250 10 0cLu 11 AMO ALA ASm GO 0LY SI VAL 0LY 7 LYS AM LBU VAL SU LIU ALA 01 GUX (Cl OCT AAC Tcr oTT GO GCOTAG AC ACT AM CAT CTA oUT CA cTA OC GA CM 300

0.LY R GIN LYS GAN 10 0T GOT TCC CAM TTA GOC TAC CM AM TM

C

*

A

CI

IIT

350

FIG. 5. Nucleotide sequence of the B. cereus SASP-2 gene coding and flanking sequences and the predicted amino acid sequence of SASP-2. The singly underlined bases from positions 153 to 162 show good complementarity to the 3' end of the 16S rRNA of B. cereus. The two regions that are doubly underlined can base pair with each other and may be a transcription stop signal. Dots below nucleotides are positioned every 10 bp.

NUCLEOTIDE SEQUENCE OF SASP GENES

VOL. 167, 1986 5-AATo1ciATCAAcArACCAAATrrAAcATAATAATABTACAGTCA OCAOccATwOGCAcAGCcGAeAAooCAgro

171

SASP

50

ACTCCCCOGATTCACGOCTICAACTCCAOCMGCCCAACCATACrATATATAAMOOAOATAACA MIT PRO 100 150 ATG CCA

SKR SKR

ASN OLN

SR GLY SER ASN ASN GLN LEU MAC CM TCT GOA AGT AAC TCT TCA MC CM CTA

LSU VAL PRo OLY ALA ALA GUI VAL CTr OTA CCT GGC OCA GCT CAG GTA

ILE ATC

200 ASP GLN IET LYS PSE GLU ILE ALA SEE GLU PSI GLY VAL ASN LWG OLY ALA GLU T TER GAT CM ATG AM TTC GM ATC OCT TCA GAA TTC GOT TO AAC CTT GOT OCT GAA ACA ACT

250 SEE ARO ALA ASN GLY SER VAL OLY GLY GLU ILK TEE LYS ARG LEU VAL SU PSI ALA GLN TCT COC OCT AAT OGA TCT GTC OGA OGA GM ATC ACT MA COT TrA oTr TCT TTC OCT CAA 300 GUI GLN SET GLY GLY OLY VAL GLN NH CTT AATTA TAATC * * CAA CAA ATO GOC GOC GOC OTA CM TM 400 30 350

CCT-JICETTOTOTO CACCTI0LT1 j-rCCTA7TCCACATAECCTAOOGAAAG 0 0

~~~~450

0

GOAATAGTGTCTATOAEU

GcgTITOTCAETrGGOCEGOACAgTFrAAOrATATBoAT-3' 500

FIG. 6. Nucleotide sequence of the B. stearothermophilus ATCC 7953 SASP-1 gene coding and flanking sequences and the predicted amino acid sequence of SASP-1. The singly underlined bases from positions 137 to 144 show good complementarity to the 3' end of the 16S rRNA of B. stearothermophilus. The two regions that are doubly underlined can base pair with each other and may be a transcription stop signal. Dots below nucleotides are positioned every 10 bp.

as a probe to isolate the complete SASP gene on an HindIII fragment; this fragment includes the hybridizing regions of both EcoRI fragments (Table 1; Fig. 3). Nucleotide sequence of B. cereus, B. stearothermophilus, and "T. thalpophilus" SASP genes. DNA sequence analysis of the regions of the cloned fragments which hybridized to SASP gene probes confirmed that they did encode SASP genes (Fig. 4, 5, 6, and17). All of these genes had a strong gram-positive-type ribo tsome binding site 6 to 7 base pairs (bp) upstream from the site of initiation of translation and a region of dyad symmetrry shortly after the translational stop codon (Fig. 4, 5, 6, and 7). Strikingly, the proteiins coded for by these four SASP genes were extremely ssimilar, and the amino acid residues previously found to be conserved in SASP coded for by B. megaterium and B. subttilis genes were also conserved in the TMOAC LACMGCCFFgECAOTrrCATCTMTCrCTATOCTrCOG 5'-GOCCOCMACCurrylFCi 50

GUI GOLY AM Se MN CATAAMQqWUCAC ATGETWA CM CM esCGC MC CT

OCAWACACMTATAAGACCMCAOAC 100

A

15C *

LYE GLU SEE SIR ASN GULN LU LEU VAL ALLA GLY ALA MA ALA ILE "AT sGA AGC TCC MC CM CTO CTr0 gET 01Cr GOT GCA 0CC CM GCG ATr OAT CM AMM * ILE ALA GUI OLU PSI OLE VAL TE ATC OCA CM GtM mT tOTrOTA AC

CC

CTA

*

LGCTALACA25SP

200 C MCea CT TCC CSAT

*

*

TCe

B. cereus-1

7ll SE PE #NI TAATMTAATCTr NOTCCWTAOGAMCCCGO@AOTACrTATrOTGCArAAAAG ACO AGC TTC TAA 350 400 **

=OAAAGAATAT'I'C

*

Q

DT A S

70 AM E QL RANR-COOH

A S L MY

Q

Q

DA A

65 SL E QL YQK-COOH

Conserved in B. negaterium A/C family

NELE GA QAIDQKYEEIASEFGVNLG AEQQLGG CDTTARANGSVGGEITKRLVQ A s qan s vAfL Y F

Conserved in B. subtilis a/B family

N LLVPGVEQAIDQMKLEIASEFGVNLGADTTSRANGSVGGEI TKRLV q se va m vi pais le f v q

h

S

B. stearothermophilus-I

NH2-MPNQSGSNSS Q V

T. thalpophilus-l

NH2-MAQQGRNRSS Q

AAQVI M F A AAO I M F

Q

AQQQMGG s Nln

N

ET S

70 SF Q QM GVQ-COOH

T

DT S

71 SL Q QL GTSF-COOH

FIG. 8. Comparison of SASP amino acid sequences. The residues in the center two lines are those which are conserved in all B. megaterium and B. subtilis SASP whose sequences have been determined to date. Residues in capitals are those which are either the only residue found at a particular position in all SASP from the organism noted or one of the only two residues found; residues in lowercase letters are those found only once at this position in the SASP from the organism listed. Residues in B. cereus, B. stearothermophilus, or "T. thalpophilus" SASP which are blank are identical to those residues conserved in all, or all but one, SASP of B. megaterium and B. subtilis or in a few cases in only one of these organisms. Sequences were aligned for maximum homology. The number over the carboxyl-terminal residue shows the length of the protein in amino acids, including the amino-terminal methionine residue. The residues underlined in B. cereus SASP-2 are identical to those found when a major B. cereus SASP was sequenced (26). The arrow denotes the site of cleavage of these SASP by the amino acid sequence-specific spore protease as determined experimentally or inferred (5). The data are taken from references 2, 3, 5, 9, and 26 and Fig. 4 to 7.

SASP from B. cereus, B. stearothermophilus, and "T. thalpophilus" (Fig. 8). Whereas we have not shown definitively that these four new SASP genes are all expressed, previous work has shown that all B. megaterium and B. subtilis genes are expressed in parallel during sporulation (3, 5). In addition, the sequence of the first 15 amino acids of the protein coded for by the B. cereus SASP-2 gene is identical to that determined for one of the major SASP of B. cereus spores (26) (Fig. 8), suggesting that this gene is expressed at a high level during sporulation. Presence of multiple SASP genes in B. cereus, B.

stearothermophilus, and "T. thalpophilus." One of the striking findings about the genes coding for this group of SASP in B. megaterium and B. subtilis is that they are members of a multigene family with a least seven members in B. megaterium (9) and four to seven members in B. subtilis (3). B. cereus also appears to contain multiple SASP genes, since the B. megaterium SASP-C gene probe recognized at least five EcoRI fragments in B. cereus DNA (Fig. 2, lane a). coding sequence probes from the B.

stearothermophilus or "T. thalpophilus" SASP genes de-

30

TA

Q

NH2-MSRST K A

B. cereus-2

ALEU GLYOLY GLY VAL OLY OLY OLU ILK MI LYS AlKOLIU AL MR LUWALAGMA Similarly, C gTT TCT 779 GCT C CAG CMA C CA CTO esr Off esc' AM COAT CTO gET GOT OGA GGM ATC ACC 0 *

ATITAACA

NH2-MGKNNSGSR EV R A Q L M Y

45.

3

FIG. 7. Nucleotide sequence of the "T. thalpophilus" SASP-1 gene coding and flanking sequences and the predicted amino acid sequence of SASP-1. The singly underlined bases from positions 113 to 122 show good complementarity to the 3' end of the 16S rRNA of Bacillus species and, by analogy, with thermoactinomycetes as well. The two regions that are doubly underlined can base pair with each other and may be a transcription stop signal. Dots below nucleotides are positioned every 10 bp.

tected at least four bands in addition to the fragment from

which the probe was derived (Fig. 2, lanes d and e). Thus, these species also appear to contain a number of closely related SASP genes.

DISCUSSION The work presented in this communication allows a number of conclusions to be drawn concerning SASP and SASP genes. (i) SASP are present in B. stearothermophilus and "T. thalpophilus" spores and apparently serve the same function in these organisms, i.e., generating amino acids by

172

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their degradation during germination, as they do in B. cereus, B. megaterium, and B. subtilis. This is not an unexpected conclusion, since SASP have also been demonstrated in spores of the more distantly related organism Clostridium bifermentans (23). However, the presence of SASP which cross-react with antiserum against B. megaterium SASP-A does substantiate the close relationship of the thermoactinomycetes to the bacilli which has been suggested based on physiological data and rRNA oligonucleotide analysis (4, 12, 13). (ii) B. cereus, B. stearothermophilus, and "T. thalpophilus" all have multiple SASP genes. This is indicated by the multiple bands in genomic digests which hybridize to SASP gene probes and suggests that the events which gave rise to multiple SASP genes may have taken place prior to the evolutionary divergence of the bacilli and thermoactinomycetes. (iii) Probably the most significant conclusion to be drawn from this work concerns the conservation of SASP amino acid sequence between species. Previous work has shown that when the sequences of the proteins coded for by the seven B. megaterium SASP-A and -C-like genes are aligned for maximum homology, there are 55 amino acid residues between the first and last conserved residues (9). Of these 57 amino acids, 34 residues are conserved exactly in all seven B. megaterium SASP, with an additional 15 residues either conserved in all but one SASP or being one of two generally similar amino acids (Fig. 8). This is a total of 49 of 57 residues which are conserved in the proteins coded for by these seven genes (Fig. 8). Strikingly, in the four B. subtilis SASP which have been analyzed to date, 43 of the residues conserved in B. megaterium are also conserved in B. subtilis (Fig. 8). Even in positions where conservation is not absolute between B. subtilis and B. megaterium SASP, analogous amino acid residues are found or an infrequent residue in the SASP of one organism becomes the predominant residue in the other (Fig. 8) (3). Comparison of the sequences of B. megaterium and B. subtilis SASP with those of B. cereus shows that essentially all residues conserved within or between B. megaterium and B. subtilis SASP are also conserved in B. cereus SASP (Fig. 8). This is not completely unexpected, since B. cereus, B. subtilis, and B. megaterium are thought to have diverged from each other at about the same time in evolution (13). However, what is even more striking is that the SASP from the much more distantly related organisms B. stearothermophilus and "T. thalpophilus" (12, 13, 24) have also retained essentially all of the amino acid residues conserved in the SASP from B. megaterium and B. subtilis (Fig. 8). It can, of course, be argued that the method we used to isolate the SASP genes from B. stearothermophilus and "T. thalpophilus" resulted in the isolation of the only SASP gene in these organisms which is similar to those in B. megaterium and B. subtilis. However, this seems unlikely for two reasons. (i) Hybridization probes made from the single B. stearothermophilus and "T. thalpophilus" genes cloned readily detect what are probably other SASP genes in the genomes of these organisms. (ii) Results with the SASPA and -C-like genes in B. megaterium and B. subtilis showed that conservation of SASP sequence within a species is actually slightly higher than conservation across species (Fig. 8). Consequently, the conservation of SASP protein sequence is extremely striking. In contrast to the high degree of sequence conservation between B. subtilis and B. stearothermophilus SASP, B. subtilis and B. stearothermophilus amylase and neutral protease exhibit very little amino acid sequence homology (19, 25). The high degree of SASP amino acid sequence conservation throughout the

evolution of the spore formers we analyzed suggests that the SASP genes must be under significant selective pressure to maintain the amino acid sequence of the proteins for which they code. This in turn suggests that the primary sequence of the SASP is extremely important in its function. One sequence-specific function of these SASP has already been identified. This is the ability of SASP to be recognized and cleaved by a specific spore protease which initiates proteolysis during spore germination. This protease requires a specific pentapeptide sequence surrounding its cleavage site (Fig. 8, arrow) (5, 22). However, the extensive sequence conservation in these proteins in regions far removed from the protease cleavage site strongly suggests that SASP may serve functions other than simply amino acid storage. We have previously suggested that SASP might play a role in the resistance of spores to UV light via interaction between SASP and spore DNA (1, 2), and recently we have obtained strong evidence supporting this suggestion (17). Such a role for these proteins might explain their high degree of amino acid sequence conservation throughout evolution. ACKNOWLEDGMENTS This work was supported by a Public Health Service grant from the National Institutes of Health (GM-19698) and an equipment grant from the Army Research Office. We are grateful for the technical assistance of Susan Goldrick and Elisabeth Murphy. Jennifer Setlow assisted in the isolation of the SASP-2 gene from B. cereus. LITERATURE CITED 1. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 2. Connors, M. J., and P. Setlow. 1985. Cloning of a small, acid-soluble spore protein gene from Bacillus subtilis and determination of its complete nucleotide sequence. J. Bacteriol. 161:333-339. 3. Connors, M. J., J. M. Mason, and P. Setlow. 1986. Cloning and nucleotide sequencing of genes for three small, acid-soluble proteins from Bacillus subtilis spores. J. Bacteriol. 166:417-425. 4. Cross, T., P. D. Walker, and G. W. Gould. 1968. Thermophilic actinomycetes producing resistant endospores. Nature (London) 220:352-354. 5. Fliss, E. R., M. J. Connors, C. A. Loshon, E. Curiel-Quesada, B. Setlow, and P. Setlow. 1985. Small, acid-soluble spore proteins of Bacillus: products of a sporulation-specific, multigene family, p. 60-66. In J. A. Hoch and P. Setlow (ed.), Molecular biology of microbial differentiation. American Society for Microbiology, Washington, D.C. 6. Fliss, E. R., and P. Setlow. 1984. Complete nucleotide sequence and start sites for transcription and translation of the Bacillus megaterium protein C gene. J. Bacteriol. 158:809-813. 7. Fliss, E. R., and P. Setlow. 1984. Bacillus megaterium spore protein C-3: nucleotide sequence of its gene and the amino acid sequence at its spore protease cleavage site. Gene 30:167-170. 8. Fliss, E. R., and P. Setlow. 1985. Genes for Bacillus megaterium small, acid-soluble spore proteins: nucleotide sequence of two genes and their expression during sporulation. Gene 35:151-157. 9. Fliss, E. R., C. A. Loshon, and P. Setlow. 1986. Genes for Bacillus megaterium small, acid-soluble spore proteins: cloning and nucleotide sequence of three additional genes from this multigene family. J. Bacteriol. 65:467-473. 10. Foerster, H. F. 1978. Effects of temperature on the spores of thermophilic actinomycetes. Arch. Microbiol. 118:257-264. 11. Foerster, H. F. 1983. Activation and germination characteristics observed in endospores of thermophilic strains of Bacillus. Arch. Microbiol. 134:175-181. 12. Fox, G. E., K. R. Pechman, and C. R. Woese. 1977. Comparative

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