The nucleotide sequence and gene organization of the gerA spore germination operon of Bacillus subtilis 168

Gene. 51 (1987) l-11 Elsevier GEN 01871

The nucleotide

sequence and gene organization

of the gerA spore germination

operon of Bacillus

subtilis 168 (Recombinant DNA; overlapping genes; membrane protein; L&mine; sporulation)

A.R. Zuberi*, Department

fumarase; lac fusion; gene expression;

A. Moir and I.M. Feavers*

of Microbiology,

University of Shefield,

Shefield

SlO 2TN

(U.K.)

Received 27 June 1986 Revised and accepted 6 October 1986

SUMMARY

The nucleotide sequence of the second and third genes in the Bacillus subtilis spore germination locus, gerA, has been determined and the amino acid (aa) sequence was derived. Two open reading fr,ames (ORFs), corresponding to genes II and III, encode 364-aa residue and 313-aa residue polypeptides, respectively. The gene II product, M, 41257, would contain long stretches of hydrophobic aa residues and may be a membrane protein; the gene III product, M, 42 363, is relatively hydrophilic but possesses an apparent signal peptide for transfer across, and perhaps localisation on, a membrane. The ORFs for genes I and II overlap by eleven codons and the termination codon of gene II overlaps the initiation codon of gene III. Insertional inactivation experiments using integrational plasmids have indicated that the gerA locus is a single transcriptional unit. The expression of the gerA genes has been studied using a lacZ transcriptional fusion; they constitute a developmentally regulated operon.

mutants producing spores that fail to germinate normally has revealed 13 different genetic loci whose products are involved in spore germination (Moir et al., 1979; Piggot et al., 1981; Moir and Smith, 1985). The spores of gerA mutants fail to germinate in L-alanine and related aa but germinate normally in aa and sugar combinations; they are blocked at the initiation of germination and it has B. subtilis

INTRODUCTION

Bacterial endospores can be induced to germinate by a variety of small molecules such as amino acids, nucleosides and sugars (Gould, 1969). The study of Correspondence to: Dr. A. Moir, Department of Microbiology, University of Sheffield, Sheffield SlO 2TN (U.K.) Tel. 0742768555.

* Present addresses: (A.R.Z.) Department of Biochemistry and Biophysics, University of California, Davis, CA (U.S.A.) Tel. (916)7523676; (I.M.F.) Friedrich Miescher Institut, CH4002 Base1 (Switzerland) Tel. 061-371111. Abbreviations: aa, amino acid(s); kb, kilobase pair(s); LMP, low-melting point; MLS, macrolide, lincosamide and strepto0378-I 119/87/%03.50

D 1957 Else&r

Science

Publishers

B.V. (Biomedical

gramin B; MU, 4-methylumbelliferone; MUG, 4-methylumbelliferyl-j-D-gdactoside; NA, nutrient agar; nt, nucleotide(s); ORF, open reading frame; PA, polyacrylamide; PolIk, Klenow (large) fragment of E. co/i DNA polymerase I; RBS, ribosome-binding site; rRNA, ribosomal RNA; Tzm, 2,3,5-triphenyl tetrazolium chloride; XGal, 5-bromo 4-chloro 3-indolyl-j%D-galactoside Z buffer, see Miller (1972). Division)

2

been postulated that the gerA locus may encode an L-alanine-specific receptor (Sammons et al., 1981). As part of an investigation of the molecular events leading to spore germination, the gerA region has been cloned into phage ,I by taking advantage of the ability of the closely linked citG gene to complement a fumarase defect of Escherichia coli (Moir, 1983). Various gerA mutants transformed with subcloned segments of DNA from the lcitG phages, have been used in complementation studies that showed the gerA locus contains at least three genes (Zuberi et al., 1985). The nt sequence of the citG-proximal ORF of gerA reveals that the first gene would specify a polypeptide of M, 53 506, containing hydrophobic and hydrophilic domains (Feavers et al., 1985). The presence of a hydrophobic domain in a gerA gene product is consistent with the hypothesis that the gerA region encodes a membrane-associated receptor for L-alanine. This paper presents the nt sequence of the remaining gerA genes and evidence that the gerA genes are transcribed as a polycistronic operon. As the germination apparatus is present in the dormant spore, germination proteins are probably synthesised during sporulation and it is likely that the expression ofger genes is developmentally controlled (Piggot et al., 1981). The use of transcriptional fusions ofgerA to a promoter-less IacZgene, together with the sensitive MUG assay described by Youngman et al. (1985), has permitted a preliminary analysis of the control of gerA gene expression. MATERIALSANDMETHODS (a) Bacteria, plasmids, media and transformation

The E. coli K- 12 derivative GM242 (dam-3 recA 1) carrying pAAM was used as the source of plasmid DNA for nt sequencing. The construction of pAAM10, a plasmid based on the shuttle vector pHV33 carrying cloned DNA from the gerA region of B. subtilis, has been described previously (Zuberi et al., 1985). The E. coli strains JMlOl and JMlO5 were used for propagating Ml3 phage derivatives (Messing, 1979; Yanisch-Perron et al., 1985). The B. subtilis strain 1604 (ger + tpC2) was transformed with the insertional plasmids used to investigate gerA gene expression. The construction of pAAM and the insertional plasmids pAAM75, pAAM and pAAM are described in RESULTS

AND DISCUSSION, section c. These plasmids were

maintained in E. coli HBlOl which carries an rpsL mutation (Boyer and Rolland-Dussoix, 1969) as the expression of the plasmid-encoded MLS resistance is detrimental to rpsL + strains (Youngman et al., 1984). All bacterial strains were maintained on Oxoid NA plates. E. coli transformants were selected on NA supplemented with 50 fig of ampicillin per ml. B. subtilis transformants resistant to MLS antibiotics were selected and maintained on NA supplemented with both 1 pg of erythromycin per ml and 25 pg of lincomycin per ml. E. coli strains were transformed by the method of Lederberg and Cohen (1974) while the transformation of competent B. subtilis cells was performed by the method of Anagnostopoulos and Spizizen (1961). (b) DNA manipulation

Large-scale purification of plasmid DNA was performed by the method of Lovett and Keggins (1979). Rapid, small-scale preparation of plasmid DNA was carried out as described by Bimboim and Doly (1979). Specific restriction fragments were purified from LMP agarose (BRL) by phenol extraction according to the manufacturer’s recommended procedure. Restriction and ligation of DNA were performed by standard methods (Maniatis et al., 1982). (c) Nucleotide sequence analysis

The dideoxy chain termination method of nt sequencing (Sanger et al., 1977) was performed using the phages M13mplO and M13mpll (Messing, 1983) and the synthetic Ml3 universal 17-mer primer with deoxyadenosine 5’-[ cr-“S(thio)]triphosphate as the radioactive label. The sequence ladders were resolved on buffer gradient PA gels (Biggin et al., 1983). (d) Test for germination phenotype

The spore germination phenotype was scored by a modification (Zuberi et al., 1985) of the tetrazolium overlay method of Trowsdale and Smith (1975). Wild-type strains (Ger + ) are stained red (Tzm-red)

3

in this test. Germination-defective remain unstained (Tzm-white).

strains (Ger-)

(e) /I-Galactosidase assay

The level of fl-galactosidase in B. subtilis derivatives containing gerA-IucZ transcriptional fusions was determined using the sensitive MUG assay (Youngman et al., 1985). Duplicate 0.5~ml samples of a sporulating culture, obtained using the resuspension method of Sterlini and Mandelstam (1969), were harvested by a 1-min spin at 12 000 rev./min in a microcentrifuge. The cell pellets were stored at -70°C until required, and were then resuspended in 600 ~1 Z buffer (Miller, 1972) containing lysozyme (200 pg/ml) and DNase I (100 pg/ml) and incubated 20 min on ice. The reactions were started by the addition of 200 ~1 MUG (40 pg/ml in Z buffer) and incubated at 30’ C for 40 min before being stopped by the addition of 400 ~1 of 1 M Na,CO,. Following a 5-min spin in a microcentrifuge, the fluorescence of the hydrolysed citG

gerA

1

S

H

1

HE

BC

Bc

gene

gene II

I

c

gene

A

Sn

1

H

E

WI

s

1

I

4 I

L

-I

a--

8

III

I

A

, I

LI

Restriction endonucleases and T4 DNA ligase were purchased from Bethesda Research Laboratories and New England Biolabs. PolIk, deoxy and dideoxynucleoside triphosphates were obtained from Boehringer Corporation Ltd. Ml3 derivatives were purchased from Pharmacia P-L Biochemicals Ltd., primer from Celltech and deoxyadenosine 5’-[~.+~~S(thio)]triphosphate from Amersham International. The 2,3,5-triphenyl tetrazolium chloride, lysozyme, DNase I, XGal, MU and MUG were all from Sigma.

I I Bc Bc C \/s

I

(f) Enzymes and chemicals

I

A

I---

MUG in the clear supematant was measured with a Perkin-Elmer spectrofluorimeter using excitation and fluorescence wavelengths of 365 nm and 450 nm, respectively. The spectrofluorimeter was calibrated using standard solutions of MU. In this assay 1 unit of j?-galactosidase represents the amount of enzyme that catalyses the production of 1 pmol MU/mm

I

I

-I -

.

I 1 4

*

I-*

44

Fig. 1. Restriction map of the gerA region of the B. subtilis chromosome showing the sequencing strategy in the expanded section. Horizontal arrows represent the direction and extent of sequencing from particular restriction targets. The open bars above the restriction map represent the ORFs corresponding to the three gerA genes. Abbreviations for restriction sites are as follows: A, AhoIII; Bc, MI; Bg, EglII; C, CluI; E, EcoRI; Ev, EcoRV; H, HindUI; P, PSI; Sn, SaaBI; S, &I. The scale bar represents 1 kb in the upper part of the diagram, and 0.5 kb in the expanded lower section.

4 gene I end Gene II start ProLysGluValAsnAsnProAsnGluProLysThrAspSerThrGluThr*~* MetSerG1"LysGlnThrProLeuLysLeuAs"ThrPheGl"GlyIl~SerIleValAldAS"ThrMet CCAAAAGAGGTGAATAATCCAAATGAGCCAAAAACAGACTCCACTGAAACTTAATACCTTTCAAGGTATTTCTATTGTTGCAAATACAAT 1990 1960 1970 1980 iFz-1930 1940 1950

23 2000

Le"GlyAladIyLeuLeuThrLeuProArqAlaLeuThrThrLy~Al~A~"ThrA~pGlyTrpIleThrLe"IleLe"Gl"GlyPheI~~ GCTTGGGG~~GGA~TTTTAACATTG~~G~GAG~G~TGACCA~TAAGGCTAATA~TGA~GG~TGGAT~ACGCTGATATTAGAAGGTTTCAT 2010 2020 2030 2040 2050 2060 2070 2080 AhaIII PheIlePhePheIleTyrLeuAsnThrLeuIleGl"LysLysHisGl"TyrProSerLeuPheGluTyrLeuLysGlUGlyLeUGlyLyS TTTTATTTTCTTCATTTATTTAAACACGCTCATTCAGAAAAAACATCAATACCCTTCACTCTTTGAATATTTGAAGGAAGGGCTTGGGAA 2100 2110 2120 2130 2140 2150 2160 2170

2090 83 2180 113

TrpIleGlySerIleIleGlyLeuLeuIleCysGlyTyrPheLeuGlyValAlaSerPheGluThrArqAldMetAl~GlUMetV~lLYS ATGGATCGGCAGCATCATCGGCCTTTTGAT~TGCGGCTATTTCCTCGGCGTAGCCAGCTTCGAGACACGGGCAATGGCTGAAATGGTGAA 2190 2200 2210 2220 2230 2240 2250 2260 PhePheLeuLeuGluArqThrProIleGl"ValIleIleLeuThrpheIlecysCysGlyIleTyrLeuMetValGlyGlyL~US~rASp GTTTTTCCTGCTGGAGAGAACCCCAATTCAAGTCATTATTTTAACGTTTATTTGCTGCGGCATTTATTTAATGGTTGGGGGCTTAAGCGA 2280 2290 2300 2310 2320 2330 2340 2350 AhaIII ValSerArqLeuPheProPheTyrLeuThrValThrIleIleIleLeuLeuIlevalPheGlyIleSerPheLysIlePheAspIleAs" TGTGTCGCGGCTGTTTCCCTTTTATTTAACGGTAACCATTATTATTTTGCTGATTGTGTTCGGGATCAGTTTTAAAATTTTTGATATCAA 2370 2380 2390 2400 2410 2420 2430 2440 ASnLeuArqProValLeuGlyGluGlyLeuClyLeuGlyProIleAlaAsnSerLeuThrValvalserIleSerPheLeuGlyMetGluValMet TAATTTGCGTCCTGTTTTAGGCGAAGGCCTTGGACCCATTGCAAACTCCCTTACCGTCGTTTCCATCTCTTTTTTAGGAATGGAAGTGAT 2460 2470 2480 2490 2500 2510 2520 2530 LeuPheLeuProGluHisMetLysLysLysLysTyrThrPheArqTyrAlaSerLeuGIyPheLeuIleProIleIleLeuLeuPheLeu GCTGTTTCTTCCTGAACATATGAAGAAAAAGAAATACACGTTCAGATATGCGTCTCTAGGATTTCTGATTCCGATTATCTTATTATTCCT 2550 2560 2570 2580 2590 2600 2610 2620 S"aB1 ThrTyrIleIleValValGlyAlaLeuThrAlaProGluValLysThrLeuIleTrpProThrIleSerLeuPheGlnSerPheGluLeu TACGTATATTATCGTTGTCGGAGCTTTGACCGCTCCCGAGGTGAAAACGCTGATTTGGCCGACTATTTCTCTCTTTCAGTCCTTTGAGCT 2640 2650 2660 2670 2680 2690 2700 2710 LysGlyIlePheIleGluArqPheGluSerPheLeuLeuValValTrpIleIleGlnPhePheThrThrPheValIleTyrGlyTyrPhe TAAAGGCATATTTATTGAACGGTTTGAATCCTTTTTACTGGTGG'rCTGGATTATCCAGTTTTTCACCACATTTGTCATTTACGGATACTT 2730 2740 2750 2760 2770 2780 2790

2800

AlaAlaAsnGlyLeuLysLysThrPheGlyLeuSerThrLysThrSerMetValIleIleGlyIleThrValPheTyrPheSerLeuTrp TGCCGCTAACGGGCTAAAGAAGACATTTGGATTATCGACTAAAACAAGCATGGTGATTATCGGCATAACGGTCTTTTATTTTTCTCTCTG 2820 2830 2840 2850 2860 2870 2880 2890 ProAspAspAlaAsnGlnValMetMetTyrSerAspTyrLeuGlyTyrIlePheValSerLeuPheLeuLeuAlaValHisSerLeuPhe GCCGGATGACGCCAATCAAGTCATGATGTATAGCGATTATCTTGGCTATATTTTTGTCTCCCTGTTTCTGCTTGCCGTTCATTCTCTTTT 2910 2920 2930 2940 2950 2960 2970 2980 Gene III start Gene II end MetLySIleArqIleLeuCySMetPheIleCysThrLeuLeuLeuSerGlyCysTrp HisValAlaLeuLysArqArqIleThrLys"' TCATGTAGCTCTCAAGAGGAGGATTACAACAAAATGAAAATCCGGATTTTATGTATGTTTATATGCACCCTATTGCTGTCCGGATGCTGG 3000 3010 3020 3030 3040 3050 3060 3070 Hind111 ASpSe~G1UAS"IleGluG1uLeuSerLeuV~lIleGlyIl~GlyLeUAspLysProAspAspGluAsnLe~GluLeuThrGlnGlnIle GACAGTGAGAATATCGAGGAATTAAGCTTAGTGATCGGAATAGGGCTAGACAAGCCTGACGATGAAAATTTAGAGCTGACACAGCAAATT 3090 3100 3110 3120 3130 3140 3150 3160 LeuVa1ProLysIleIleSerAlaLysGluGlySerSerSerAspProThrGl"LeuSerIleThrLysGlyLysThrValHisGlnMet CTCGTGCCGAAAATAATCAGCGCCAAGGAGGGCTCTTCATCTGATCCGACACAGCTTTCTATCACAAAGGGTAAAACTGTCCATCAAATG 3180 3190 3200 3210 3220 3230 3240 3250 HetArqThrSerAlaLeuLySHisLysProThrPheSerGlnHisSerArqLeuIleLeuLeuSerLysSerValIleAlaAspGlnIle ATGAGAACCTCTGCATTAAAGCATAAGCCTACGTTTCACAAAGCGTGATTGCGGATCAAATT 3270 3280 3290 3300 3310 3320

3330

3340

GlyHetASpAlaIleIl~AS"Gl"PheV~lArqASpAs"GlyThrArqArqSerSerTyrVdlPh~IleThrAs"GlyArgThrLysAsp GGAATGGACGCCATTATTAACCAGTTTGTCAGAGACAACGGAACAAGGAGGAGTTCCTACGTCTTTATCACCAACGGCCGAACTAAGGAT 3360 3370 3380 3390 3400 3410 3420 3430 IlePheAsnHetAsnAspGluGlyGluProAlaSerAsnValIleTyrAspLeuThrGluAsnAsnLysValThrIleArqThrMetGl~ ATATTTAACATGAATGATGAAGGCGAACCCGCATCCAACGTGATATATGATCTGACAGAGAATAATAAGGTGACGATCAGAACGATGGAG 3450 3460 3470 3480 3490 3500 3510 3520 89111 ProValThrLeuGlyGluIleSerGluHisLeuThrSerAspAspSerPheLeuIleProHisValGlyLysGluAsnGlyLysL~~Al~ CCAGTTACATTGGGGGAGATCTCAGAACACTTAACCTCTGACGATTCATTCCTCATCCCTCATGTCGGTAAGGAAAACGGCAAGCTCGCG 3540 3550 3560 3570 3580 3590 3600 3610 IleAsnGlyAlaSerIleIleLysAsnLysLeuTrpHisArqAspLeuThrProIleGluValGlnAsnIleSerLeuPheSerGlyThr ATAAACGGAGCCTCTATCATAAAAAATAAATTATGGCACCGGGACCTCACCCCGATTGAGGTTCAAAATATCAGCTTATTTTCAGGCACT 3630 3640 3650 3660 3670 3680 3690 3.700

53

2270 133

EcoRV

2360 163 2450 193 2540 223 2630 253 2720 283 2810 313 2900 343 2990 19

3080 49 3170 79 3260 109 3350 139 3440 169 3530 199 3620 229 3710

5 259 ValCluGlyGlyValIleAspLeuLysAeqAspGlyHisLeuPheSerTy~GluValTyrSerSerAsnArqLysIleLysThrAlaTyr GTCGAGGGAGGCGTCATTGACTTGAAGCGCGATGGCCATCTCTTTTCTTATGAAGTATATTCAAGCAATCGGAAGATCAAGACGGCATAC 3720 3730 3740 3750 3760 3770 3780 3790 LysASpGlyLysPheLysPheThrValThrArqASnIleGluGlyArqLeuSerGluAspTrpAsnProAsnGluAsp~erPheLysAsp AAGGACGGAAAGTTCAAATTCACCGTTACCCGCAATATTGAAGGTCGTCTATCTGAGGACTGGAATCCCAATGAAGACTCGTTTAAGGAT 3810 3820 3830 3840 3850 3860 3870 3880 est1 SerTyrIleLysSerIleGluLysThrValGluLysArgValHisGluThrValThrSerPheIleThrGluLysLeuGlnLysGluIle TCATATATTAAAAGTATTGAAAAGACAGTCGAAAAACGAGTACACGAAACAGTAACGTCATTTATTACAGAGAAGCTGCAGAAAGAGATC 3900 3910 3920 3930 3940 3950 3960 3970 EcoRI LysAlaAspValThrGlyLeuGlyAsnGluValArgIleHisTyrProGlnLysTrpLysLysIleSerArgLysTrpAspAspAspTyr AAAGCGGACGTAACCGGGCTGGGAAACGAGGTCAGAATTCATTATCCTCAAAAGTGGAAAAAGATTTCAAGAAAATGGGATGATGATTAT 3990 4000 4010 4020 4030 4040 4050 4060 PheSerAsnAlaGluIleAspTyrArqValAsnValIleValArqAspPheGlyThrLysGlyAlaAsnLys*** TTCAGCAATGCGGAAATAGACTACCGGGTCAATGTGATTGTCAGAGACTTCGGAACGAAAGGCGCAAACAAATAGCAGCCGCCTAATTCA 4080 4090 4100 4110 4120 4130 4140 4150 CGAGATGATTTCGGTGTFCGTACACCGCCGCCTGCGTCCGGTCACTGACATCCAGCTTTGATAAAATATTCGTAATATGTGTTTTGACTG 4170 4180 4190 4200 4210 4220 4230 4240 sst1 TTTTAATCGTAATAAACAGTTCCTCGCCTATTTCTTTGTTTGTCTTTCCTTCTGCGATCAGGCAGAGTATTTCGAGCTC 4260 4270 4280 4290 4300 4310 4320

3800 289 3890 319 3980 349 4070

4160 4250

Fig. 2. The nucleotide sequence of the part of the B. subfilis chromosome that encodes the second and third gerA genes. The predicted amino acid sequence of genes II and III, and their potential RBSs (underlined) are shown. The stop codons are marked with asterisks. The nucleotides are numbered to follow on from the previously published sequence of gene I (Feavers et al., 1985).

RESULTS

AND

DISCUSSION

(a) The nucleotide sequence of genes II and III of the gerA locus

The overlapping 2.6-kb ClaI-PsrI and 1.8-kb BgflI fragments of pAAM were gel-purified. The relevant restriction sites in pAAM are underlined in Fig. 4. Both fragments were then further digested with enzymes of 6-bp recognition sequence for cloning into the appropriately cut Ml3 vector. The sequence obtained from these specific clones was overlapped by sequence from MspI, Sau3A or TaqI fragments shotgun cloned into M13mplO. This sequencing strategy is summarized in the expanded section of Fig. 1. The nt sequence of the second and third genes of the gerA locus is shown in Fig. 2. This sequence overlaps and extends the previously published sequence of gene I in this locus (Feavers et al., 1985). A computer search of both strands for ORFs, using the FIUMESCAN program (Staden and McLachlan, 1982) revealed two extensive ORFs on the same strand as gene I. The first, overlapping the end of gene I, extends from nt 1933 to a stop codon at nt 3025. There is a potential RBS 9 nt upstream from its initiation codon that has a calculated free energy of interaction (dG) with the 3’ end of B. subtilis 16s rRNAofdG = -18.6 kcal/mol (Tinoco et al., 1973).

The second ORF extends from an initiation codon at nt 3024 to a stop codon at nt 4143 and overlaps the last two codons of the first ORF. The potential RBS 11 nt upstream from this second ORF has a calculated dG = -16.2 kcal/mol. The position of these two ORFs correlates well with the position and extent of complementation units II and III of gerA (Zuberi et al., 1985). The polypeptides corresponding to the products of genes II and III contain 364 and 373 aa, respectively, and have predicted A4,s 41257 and 42363. Analysis of chromosomal gerA mutations, by complementation with subcloned fragments of gerA DNA, has revealed that this locus consists of at least three complementation units (Zuberi et al., 1985). Since the only extensive ORF downstream from gene III is specified by the complementary strand of DNA (Zuberi, 1985) it seems unlikely that there are more than three gerA genes. No sequence similar to a Rho-independent terminator can be found downstream from gene III. The three ORFs appear to be tightly coupled. Genes I and II overlap by eleven codons, and the initiation codon for gene III overlaps the termination codon of gene II. Such coupling is a feature of polycistronic mRNA and could encourage efficient translational reinitiation within the transcript to facilitate the synthesis of equimolar amounts of the three encoded proteins (Das and Yanofsky, 1984).

6

(b) Analysis of the deduced amino acid sequences

The hydropathy profiles of the gerA gene II and gene III products (Fig. 3) have been determined. The deduced polypeptide product of gene II, containing long stretches of hydrophobic residues, has the hydropathy profile reminiscent of an integral membrane protein. The gene III product is relatively hydrophilic (Fig. 3b) but includes an N-terminal sequence with the characteristics of a signal peptide. The potential cleavage site, between residues 17 and 18, is contained in aa sequence LSGC which

a k 2 8

4-

E 1

2-

! \I

oI’-/

-,-i -4

I

I

! 200

lb0

I 300 AMINO

b

I

ACIDS

1

1

I

IA0

1 260

I 300

AMINO

1

ACIDS

Fig. 3. Hydropathy profiles of (a) the predicted gene II product and (b) the predicted gene III product of gerA. The average hydropathy value for 7-aa windows centred on each residue along the sequence is plotted according to the computer program of Kyte and Doolittle (1982). In this program, hydrophobic residues are assigned high hydropathy values. The horizontal bar represents the average hydropathy of a large number of sequenced proteins.

matches the consensus found in prelipoprotein signal sequences, and includes the cysteine residue which is the site of lipophilic modification (Pugsley and Schwartz, 1985). It is likely therefore that the gene III protein is transferred across a membrane and is anchored at the outer surface as a lipoprotein. As gene I product also has a very hydrophobic domain (Feavers et al., 1985), it is likely that all three gerA proteins are membrane-associated; they may therefore form a complex in the membrane. Some indirect evidence that membrane-associated proteins are involved in the germination process has been presented previously (Keynan, 1978; Rossignol and vary, 1979). The deduced aa sequences of the three gerA genes have been compared, using the DIAGON computer program (Staden, 1982). This analysis failed to show any extensive homologies between the three sequences. The deduced sequences of the three ORFs have been compared with the Protein Identification Resource (Release No. 7.0 of the NBRF). Using the rapid search program PEPSCAN (M. Bishop, personal communication) no extensive homologies were detected with sequences in the database, although a short homology with the periplasmic maltose-binding protein (malE; Duplay et al., 1984) was observed. Residues 24-37 of gene III protein share 9114 identities with aa residues 28-41 of malE preprotein. The significance of this homology (if any) is unknown, although the similarity in position close after the signal sequence in both cases - is intriguing. No such sequence is present in the periplasmic binding proteins encoded by hisJ (Higgins et al., 1982), rbsB (Groarke et al., 1983) or phoS (Grin et al., 1984). (c) Analysis of the transcriptional gerA using insertional plasmids

organization

of

A plasmid, encoding an antibiotic resistance that is expressed in B. subtilis but that is incapable of autonomous replication, can only be maintained by insertion into the chromosome. Plasmid integration can occur by homologous recombination between a chromosomal DNA fragment cloned in the plasmid, via a Campbell-type crossover event (Haldenwang et al., 1980). When the cloned fragment contains either end of a transcriptional unit, a complete copy

Fig. 4. The construction of insertional plasmids used to analyse the transcriptional organisation of gerA. The plasmids pTV32 (Youngman et al., 1984; Perkins and Youngman, 1986), pAAM and pAAM (Zuberi et al., 1985) that form the basis ofthese plasmid constructions have been described elsewhere. The insertional plasmid pAAM was constructed by cloning the gel-purified 5.6-kb SmoI-Hind111 fragment of pTV32 into SnuBI + HindHI-cut pAAM53, effectively replacing the pC194 replicon of pAAM with the segment of pTV32 that both carries a promoterless 1ueZ gene and specifies MLS resistance. Plasmids pAAM and pAAM 1 were generated by cloning the 1.2-kb BclI fragment of pAAM that contains the diverging promoters of cifG and gerA into the BumHI site of pAAM in each orientation. pAAM itself was the product of cloning into pBR322 the 5.6-kb BornHI-Hind111 fragment of pTV32 that specifies MLS resistance. The insertional plasmid pAAM consists of the 4.6-kb BumHI-XhoI fragment of pTV32 cloned between the BglII and Sal1 sites of pAAM71. pAAM had been constructed by cloning the 4-kb ClaI fragment that contains genes II and III from pAAM into pBR322. Restriction sites in pAAM delimiting fragments used for sequencing are underlined. The etm gene encodes MLS resistance; amp, cat and ret genes encode ampicillin, chloramphenicol and tetracycline resistance, respectively. The Tn917-luc transposon in pTV32 is indicated by the dashed region between arrowheads.

8

of that unit will still exist after plasmid integration; if, on the other hand, the cloned fragment only contains an internal portion of the transcriptional unit, it will be disrupted by the insertion and as a result the bacteria will have the mutant phenotype. This approach has already been used to study operon organisation in B. subtilis (Piggot et al., 1984). The construction of plasmids used to study the transcriptional organization of the gerA locus is summarised in Fig. 4. The germination phenotype of B. subtilis strains transformed by the insertional plasmids was determined using the tetrazolium overlay method on at least twelve independent isolates from each transformation (Fig. 5). Both pAAM and pAAM appear to contain one end of the gerA transcriptional unit, as the insertional transformants of a wild-type B. subtilis strain remained Ger’ . Insertion of pAAM generated a Ger - phenotype. As the orientation of the ORFs in gerA is known, this would suggest that gene III is expressed from a promoter that lies upstream from the C/u1 site in gene I (Fig. 5)

and that genes I, II and III are part of the same transcriptional unit. The simplest interpretation of these results is that the three gerA genes constitute an operon and the close coupling of the ORFs would be consistent with this interpretation. It was previously reported, however, that each gerA gene was individually able to be expressed from a multicopy plasmid at a sulIiciently high level to permit complementation (Zuberi et al., 1985). This was the case whether the downstream genes were cloned separately or in circumstances where they were separated from the upstream genes of the gerA locus by a T&000 insertion. It is likely that a low level of readthrough from spurious promoters in either the plasmid or the transposon provided sufficient gene product for complementation when the region was present in multiple copies. As experiments described below suggest that the normal levels ofgerA expression in wild-type cells are relatively low, this explanation is plausible, and therefore these experiments do not conflict with our conclusion that gerA is an operon.

lkb

Tzm phenotype of transformants.

Plasmid citG 1L

gerA

gene 1

gene

II

gene

I

red

pAAM

I

I

III

I

pAAM pAAM I

I

pAAM

red 1 white

Fig. 5. The effect of integration of the insertional plasmid into the B. subtilir chromosome on the spore germination phenotype of the bacteria. The position of the ORFs, determined from the nucleotide sequence, and the extents of the fragments cloned in the insertional plasmids are shown below the restriction map of the citG-gerA region. The Tzm phenotype of transformants of a wild-type Ger’ (Tzm-red) strain into which the insertional plasmid has integrated is indicated (see MATERIALS AND METHODS, section d).

9

(d) Analysis of gerA expression using luc fusions The construction of some of the insertional plasmids generated fusions of the gerA promoter region upstream from a promoter-less IacZ gene that possesses a Bacillus ribosome-binding sequence (Perkins and Youngman, 1986; Fig. 4); one such construction, pAAM 1, has been used to investigate the transcriptional control of gerA. The plasmid pAAM80, in which the citG promoter is fused to the IacZ gene, was used as a positive control in some of these experiments. Preliminary experiments with B. subtilis 1604 derivatives carrying integrated copies of pAAM or pAAM showed that the citG, but not the gerA, promoter was active at a level sufficient to turn the colonies blue on NA containing XGal (Zuberi, 1985). This suggests that the gerA promoter was active at a considerably lower level than that of citG.

50

00

The level of fi-galactosidase in sporulating cells was determined at hourly intervals during the sporulation of Ger + bacteria bearing a copy of pAAM 1 integrated into the chromosome (Fig. 6). The control strain, without a copy of pAAM in its chromosome, showed a slight increase in the level of /3-galactosidase during sporulation similar to the endogenous low-level activity reported previously (Smith et al., 1985). The synthesis of fi-galactosidase by the gerA-IacZ fusion strain increased significantly above this background activity, from t, to t3 onwards (for to-t6, see Fig. 6). This apparent time of expression correlates well with the time during sporulation at which gerA mRNA can be detected in S 1 nuclease protection experiments (I.M.F., unpublished). These results are consistent with the data of Dion and Mandelstam (1980), who used intrasporangial germination to show that the germination apparatus essential for the spore to respond to L&nine as germinant is assembled by t, and is the expression of events in protein synthesis that occur before t,. As little is known about the rate of turnover of mRNA, or proteins such as fi-galactosidase, during the developmental cycle of B. subtilis, it is not possible to quantitate accurately expression of the gerAIacZ fusion. However, the maximal level of fi-galactosidase (at to) detected in the strain bearing the citG-lacz fusion (not shown) was more than 150-fold higher than peak expression from the gerA-1acZ fusion; gerA is clearly expressed at a relatively low level. (e) Conclusions

0

0 t1

12 Time

t3 after

t4 resuspension

t5

t6

(h.1

Fig. 6. The activity of @-galactosidase expressed from a gerAlucZ fusion during sporulation. Transfer into the resuspension medium, which induces the initiation of sporulation, is defined as I,. Samples were collected at intervals from I h to 6 h after resuspension (I, tot,, respectively). Cultures were B. subtiiis 1604 (0) and a derivative carrying an integrated copy of pAAM (m). The percentage of cells that contained phase-bright prespores (0) was monitored by phase-contrast microscopy and was identical for both cultures.

The data presented in this paper indicate that gerA is a weakly expressed polycistronic operon that is transcribed only during sporulation. Its polypeptide products, required for L-alanine-stimulated germination, are expressed coordinately, and probably form a membrane-associated complex. This is the first operon known to be concerned with spore germination that has been amenable to an analysis of gene expression. It is clear that it is indeed subject to developmental control. As evidence suggests that the gerA operon is expressed during sporulation, after the asymmetric cell division that generates sporangium and forespore, the question of whether gerA is transcribed in the mother cell or forespore compartment must now be addressed.

10

ACKNOWLEDGEMENTS

We would like to thank Jennifer Naylor for valuable technical assistance. We are also grateful to the N.E.R.C. Unit in the Department of Botany for the use of its spectrofluorimeter, Dr. Jeff Errington of Oxford University for advice on the MUG assay and Dr. Phil Youngman for supplying the plasmid pTV32. This work was supported by the Science and Engineering Research Council.

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