JOURNAL

OF

BACTERIOLOGY, Jan. 1992,

p.

Vol. 174, No. 2

586-594

0021-9193/92/020586-09$02.00/0 Copyright C) 1992, American Society for Microbiology

Characterization of a Sporulation Gene, spoIVA, Involved in Spore Coat Morphogenesis in Bacillus subtilis CHRISTINE M. STEVENS,t RICHARD DANIEL, NICOLA ILLING,t AND JEFFERY ERRINGTON* Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX] 3RE, United Kingdom Received 6 September 1991/Accepted 5 November 1991

Mutations in the spolVA locus of Bacillus subtilis abolish cortex synthesis and interfere with the synthesis and assembly of the spore coat. We have characterized the cloned spoIVA locus in terms of its physical structure and regulation during sporulation. The locus contains a single gene capable of encoding an acidic protein of 492 amino acids (molecular weight, 55,174). The gene is transcribed from a oE-dependent promoter soon after the formation of the spore septum. A genetic test indicated that expression of spolVA is only necessary in the mother cell compartment for the formation of a mature spore. This, together with the phenotypic properties of spolVA mutations, would be in accord with the hypothesis that aE is only active after septation and in the mother cell compartment.

Spore coat formation in Bacillus subtilis is a simple example of cellular morphogenesis involving macromolecular assembly. The coat is composed of at least a dozen relatively abundant proteins deposited in concentric layers on the outer surface of the developing spore (1, 34). The synthesis and order of assembly of the major coat proteins have been studied by using biochemical methods (24, 34, 46). Several mutations affecting the synthesis of specific coat proteins have been identified by using classical methods (31, 32). More recently, several of the genes encoding major proteins of the coat (cot genes) have been cloned and characterized (la, 8, 10, 53, 68, 69). In addition, at least one gene, gerE, involved in the regulation of spore coat synthesis has been extensively characterized (6, 7, 33, 43). The spoIVA locus was originally identified in an abnormal, oligosporogenous mutant isolated by Coote (3). In a strain carrying the mutation spolVA178 the spore began development normally, forming a primordial germ cell wall, but little cortex was made. A unique feature of the mutant was the accumulation of spore coat-like material in partially refractile bodies in the cytoplasm of the mother cell. Apparently spore coat proteins are synthesized but not assembled in their normal place on the prespore outer membrane. The product(s) of spoIVA is thus needed for both cortex synthesis and for an early step in spore coat assembly. The mutation mapped between lys-J and trpC2 (4). A completely asporogenous mutant of similar phenotype, with a mutation (spoIVA67) mapping to the same locus, was isolated subsequently (47). The recombination index between the two mutations was 0.07, indicating that they could be located in the same gene (47). More recently a strain with a Tn917 insertion in spolVA was isolated (54). The effects of spoIVA mutations on expression of other sporulation genes have been studied by several groups. Prespore-specific gene expression does not seem to be affected (5, 18, 20, 40). Certain mother cell-specific genes, spoVJ (19), cotA (53), and gerE (7), are also independent of

spoIVA, but the expression of spoIVC (38) and spolIIC (63) is partly impaired in a spoIVA mutant. The minor effects on expression of some later mother cell-specific genes are presumably due to the impaired processing of pro-uK (39). Surprisingly, cotC expression, which occurs only at a very late stage of sporulation, appeared to be dependent on the culture medium used to induce sporulation; expression was substantially higher when sporulation was induced by nutrient exhaustion rather than by resuspension (69). The spolVA::Tn917 insertion mutation had little effect on the low level of expression in resuspension medium, but it severely reduced expression in the exhaustion medium. The phenotypic properties of spolVA mutants thus suggest that its product(s) is involved in spore cortex and/or coat synthesis, presumably operating in the mother cell compartment. The spoIVA locus is therefore interesting from the point of view of both its regulation and the function of its product(s) in spore coat morphogenesis. In this paper we describe the characterization of the cloned spoIVA gene and studies of its regulation. Similar results obtained independently by Roels et al. (52) are reported in the accompanying paper. MATERIALS AND METHODS

General methods. The bacterial strains and plasmids used listed in Table 1, except for the plasmids containing subcloned fragments of 1405J108 DNA, some of which are illustrated schematically in Fig. 1. These were isolated by standard cloning methods except for those described in detail below. 105J108 was isolated previously (11). All of the spo mutant strains are isogenic with SG38 except for strain 67. The media for growth, transformation, DNA manipulations, and induction of sporulation were as described previously (57). The assay used for measurement of P-galactosidase was described by Errington and Mandelstam (17). One unit of 3-galactosidase catalyzes the production of 1 nmol of 4-methylumbelliferone per min under the standard assay conditions. Induction of sporulation and measurement of the time of spoIVA-lacZ expression. Strains containing a spoIVA-lacZ fusion were induced to sporulate by the method of Sterlini and Mandelstam (56a). Time zero (to) was defined as the time are

* Corresponding author. t Present address: Unipath Ltd., Bedford MK41 OQG, United

Kingdom t Present address: Department of Chemical Pathology, Medical School, Observatory, Cape Town, South Africa.

586

587

spolVA GENE OF B. SUBTILIS

VOL. 174, 1992

TABLE 1. Bacterial strains and plasmids Genotype or properties

Strain or plasmid

Bacillus subtilis 168 CU267 SG38 1.5 17.4 23.4 36.3 43.9 48.7 55.3 65.4 67

69.17 87.4 131.5 133.1 178.4 298.4 496.1 497.1 561.10 562.5 646 671 713 715 CS4A NI2 N13 N142 N142.7

Escherichia coli DH5a GM48 Plasmids pAT21 pBluescript II KSpBD64

pSG2d pSG28d

pSG119 pSG1301 pTZ19R pUC18 a b

trpC2 ilvB2 leuBJ6 trpC2 trpC2 spolIACI trpC2 spoOH17 trpC2 spolVCA23 trpC2 spoIIIE36 trpC2 spoOA43 trpC2 spoIIE48 trpC2 spoIIGS5 trpC2 spoIIIA65 trpC2 spoIVA67 trpC2 spoIIAA69 trpC2 spoIJIJ87b trpC2 spoIIB131 trpC2 spoIVCB133 trpC2 spoIVA178 trpC2 spoIID298 trpC2 spoIIIC94 trpC2 spoIIID83 trpC2 spoIIAC561 trpC2 spoIIAAS62 trpC2 spoIIIG646 trpC2

Qi(spoIIID::erm)671 trpC2 Qi(spoIVA'-lacZ cat)713 trpC2 Ql(spoIVA: :cat lacZ)715 trpC2 spoIVA::pSG140 trpC2 spoVJ::pSG137 trpC2 spoVA::pSG196 trpC2 trpC2 chr::pSG442 Pp,,c.-spoIIGAB trpC2 fl(spoIVA'-lacZ cat)713 chr::pSG442

P.,P,.-spoIlGAB

Reference or source

Laboratory stock S. Zahler" 17 E1 (47) E22 (64) E31 (64) NG1.67 (47) NG6.21 (47) NG9.3 (47) NG12.12 (47) NG17.17 (47) NG17.23 (47) NG18.6 (47) 27 Z3 (3) Z7 (3) P20 (3) P9(3) 94U (30) 83U (30) 16 16 22 57 This work This work This work 29 29 28 This work

hsdRl7(rK- mK+) supE44 recAl gyrA96 X- thi-l relAI A(lacZYAargF)U169 48odlacZAM15 thr leu thi lacY galK galT ara tonA tsx dam dcm supE44

GIBCO BRL

bla aphA-3 bla, cloning vector

62C Stratagene

cat

26 21 12 This work 55 41 66

(F-) endAl

bla cat, vector for integrational analysis of transcriptional units in B. subtilis bla cat lacZ, vector for constructing transcriptional fusions in B. subtilis pTZ19R + aphA-3 gene from pAT21 pBluescript 1I KS- + cat gene from pBD64 bla, cloning vector bla, cloning vector

Yanisch-Perron et al. (66)

S. A. Zahler, Genetics and Development Department, Cornell University, Ithaca, N.Y. Previously defined as spoOJ87 (27) but now recognized to be a separate locus from spoOJ93 and to block sporulation at stage III rather than at

stage

0

(13,

58). d

Kindly supplied by A. Klier, Institut Pasteur, Paris. Previously given designations beginning pSGMU.

at which the cells were resuspended in the starvation medium. Samples taken usually at 10-min intervals were assayed for P-galactosidase activity, and the results were plotted. The onset of P-galactosidase production was determined as follows. A line was drawn through the first three or more points, at which the enzyme activity was accumulating rapidly and at a relatively uniform rate. The onset of expression was defined as the point at which this line intercepted a line representing the basal activity. The onset of expression determined in this way was quite reproducible as indicated by the relatively low standard deviation of results from repeated experiments (see Results and Discussion).

Construction of plasmid pSG119. An integrational plasmid vector carrying a selectable kanamycin resistance gene,

aphA-3, was constructed by cloning the 1.5-kb ClaI fragment from pAT21 into the unique AhaII site of pTZ19R. This disrupts the bla gene, but aphA-3 is selectable in both Escherichia coli (50 ,ug of kanamycin sulfate per ml) and B. subtilis (5 ,ug/ml; for a single copy of the gene). Construction of plasmid pSG1301. Plasmid pBluescript II KS- was partially digested with PvuII and ligated to the 1.0-kbp HpaII fragment from plasmid pBD64 after the cohesive ends were filled in. A chloramphenicol-resistant transformant conferring a blue colony color on plates containing

5-bromo-4-chloro-3-indolyl-o-D-galactopyranoside

was

iso-

588

STEVENS ET AL.

J. BACTERIOL.

IlacZ H

RHLM VA

S VPV H

I I 5II;f

VVCRS RR}C

3

4

nspoil/A

pSG129

_----- H------q m

pSG127

F-

aPSG F-pSG 136

(P)

))I I I II (I )

1

0

ca

1009

pSG328

5.4

pSG1005 RF ---H

pSG1018

RH Hs

pSGIO16

mG322

p

5

P'SG140

- _-_ pSG320 -

pSG142

pSG1014

FIG. 1. Restriction map of the spoIVA region and localization of the gene by integration plasmid analysis. The upper part shows a partial restriction map of the insert in 4105J108. The ends of the inserts correspond to MboI sites, because the DNA fragments were prepared for cloning by partial digestion with MboI (11). The abbreviations for restriction endonuclease sites are as follows: A, ApaI; C, AccI; H, HindIll; L, Sall; M, Smal; P, PstI; R, EcoRI; S, Sstl; V, EcoRV. The unsequenced region was not mapped for AccI sites. The Pstl site within parentheses lies in the polylinker of 4105J108. Of the other enzymes tested, there were not sites for BamHI, BglII, or XbaI. The fragments subcloned in various plasmids are shown below. Where necessary, the restriction endonuclease used to derive the subcloned fragment is indicated with a letter to the side of the bar. The inserts of some of the integration plasmids used to define the spoIVA transcriptional unit are shown by thick bars. The bar is solid if the plasmid generated a Spo- phenotype (insert internal to the spoIVA transcription unit) and dotted if the plasmid did not give a recognizable Spo- phenotype. All of these plasmids were derived from pSG2 (21) except pSG320, which was derived from pSG119, and pSG1009, which was derived from pSG1301 (see Materials and Methods). The inserts of two other plasmids are shown with thin bars: pSG142, a derivative of pUC18, and pSG10014, which was essentially derived from pSG2 but with part of the cat gene deleted. Below the restriction map the box indicates the spoIVA coding region as deduced from the integration plasmid experiments and DNA sequencing. Shown at the top of the figure are the location and orientation of the lacZ cat insertion in strain 713.

lated and found to have the cat gene fragment inserted into the PvuII site in the lacI gene (55). This provided an integration plasmid with a polylinker that was more extensive than that of pSG2. Construction of plasmid pSG1005. The rightmost extremity of the insert in 4105J108 (to the right of the rightmost EcoRI site shown in Fig. 1) resisted all attempts at subcloning directly in plasmids. It was eventually isolated by the following approach. First, plasmid pSG2 was inserted by

prophage transformation (36) into a derivative of the fl05J108 prophage in place of all of the insert except for the uncloned region. Insertion was arranged such that digestion of the purified phage DNA with PstI released the vector together with the desired fragment from the spoIVA region. Ligation at a low DNA concentration gave only minute colonies that underwent spontaneous DNA rearrangements during subculturing. Ligation at higher concentrations gave some colonies that grew well and contained stable plasmids. Restriction mapping of several of the plasmids from these strongly growing colonies revealed that they all carried an additional PstI fragment of phage origin (PstI fragment L, 1.04 kb [2]). This fragment of phage DNA thus suppressed the apparently lethal effects of the DNA from the promoterdistal part of the spoIVA region. The nature of this stabilizing influence is not yet clear. Construction of plasmid pSG1014. pSG1005 was integrated into the chromosome of strain 168. Chromosomal DNA was digested with Ncol and SmaI, and the cohesive ends were filled in by treatment with the Klenow fragment of DNA polymerase I. The DNA was ligated at low DNA concentration and transformed into E. coli DH5cx. This procedure resulted in the isolation of a plasmid in which part of the cat gene was deleted. DNA sequencing. Double-stranded plasmid DNA was sequenced by the chain terminator method with Sequenase and 35S-labeled nucleotides as described previously (57). Where necessary, synthetic oligonucleotides were used, so that all of the sequenced region was determined from both DNA strands. Construction of a spoIVA'-lacZ transcriptional fusion. The spoIVA'-lacZ fusion was constructed by cutting plasmid pSG142 at its unique BcII site, which lies in the spoIVA coding region (Fig. 1 and 2), and ligating to a lacZ cat cartridge released from pSG28 by digestion with BamHI. B. subtilis 168 was transformed with selection for chloramphenicol resistance. Stable chloramphenicol-resistant Spo- Lac' and Spo- Lac- colonies were isolated and designated strains 713 and 715, respectively. Southern hybridization (56) showed that these strains had insertions of the expected structure with the lacZ cat cartridge inserted at the appropriate BclI site in spoIVA (data not shown). The orientation of the lacZ gene was the same as that of spoIVA in strain 713 and the reverse in strain 715. Integration plasmid excision experiments. Cells containing integrated plasmids were grown in hydrolyzed casein medium containing chloramphenicol (5 ,ugIml) and resuspended in antibiotic-free sporulation medium. After 10 h at 37°C, samples were plated on NA (nutrient agar) or NA containing chloramphenicol, either immediately to measure viable cells or after 10 min at 85°C to select heat-resistant spores (29). Primer extension analysis of mRNA. The oligonucleotides used had the following sequences: 5'-GACAGCACCTACG ACTC-3'; 5'-TCCTTGAAAATATCGACC-3'. RNA was isolated from sporulating cells as described by Foulger and

FIG. 2. DNA sequence and translation of the spoIVA region. The sequence given is of 1,950 bp extending from a SmaI site upstream of the spoIVA transcription unit as defined by integration plasmid analysis (50) to beyond the putative transcription terminator. The last 26 bp overlap the sequence reported by Micka et al. (42). Some of the restriction sites in the sequence, those indicated in Fig. 1 and Bcll sites, are underlined. The Bcll site beginning at bp 656 was used in the construction of the spoIVA'-lacZ fusion. The transcription start site detected by primer extension analysis is indicated by an arrow. A likely ribosome binding site sequence is underlined, and promoter sequences similar to those used by 'E-dependent genes are highlighted in boldface type. Conspicuous inverted and directly repeated sequences are indicated by arrows above the sequences; each sequence has been given a number. The putative SpoIVA amino acid sequence is indicated in the standard one-letter code. The amino acid sequence motifs in proteins capable of binding mononucleotides are highlighted in boldface type.

spoIVA GENE OF B. SUBTILIS

VOL. 174, 1992

OCOGOCACTGAAATCCAGGATATGATTTGGCATGAAACCCCCTTTGTCCCCGCGTTTTCTGTAAAATATACGGTAAATGACAAACAGGAA 50

SmaI

2

2

1

CCTGTTTTTTTAGAAAATGATATGAAAAAGGAATGAACCTTTCTCCCTTGCATACAAATAGGGAGAAAGGTTTTTTTATATTAATAGATT 150

100

-10 -35 TATAGTGTCCTGTGTTTGATTGAAAGAGCTTAATA GAGGATGAGAAATTTTCTAAAGATGPCATATTCAAATAGGACAACGTCTC 250 200

spol VA M

E

K

D

V

I

F

K D

I

A

E

R

T

G

G

D

I

AAATTGAAAAGGATAGGAAGTCCGfiaGiGA=ACTTGGAAAAGGTCGATATTTTCAAGGATATCGCTGAACGAACAGGAGGCGATATA 300

Y

L

G

V

aa V R T a

V

350

EcoRV

X 8

T

F

K

I

K

F

M

E

L

V

V

L

P

N

I

S

N

TACTTAGGAGTCGTAGGTGCTGTCCGTACAGGAAAATCCACGTTCATTAAAAAATTTATGGAGCTTGTGGTGCTCCCGAATATCAGTAAC 450 400 E

A

D

R

R

A

A Q

D

E

L

Q S

P

A

G

A

K

T

I

M

T

T

E

P

K

F

V

P

N

GAAGCAGACCGGGCCCGAGCGCAGGATGAACTGCCGCAGAGCGCAGCCGGCAAAACCATTATGACTACAGAGCCTAAATTTGTTCCGAAT 500

ApaI Q A

M

S

H

V

V

S

D

G

L

D

V

N

X

R

L

V

D

C

V

G

Y

T

V

P

G

A

K

G

CAGGCGATGTCTGTTCATGTGTCAGACGGACTCGATGTGAATATAAGATTAGTAGATTGTGTAGGTTACACAGTGCCCGGCGCTAAAGGA 600 550 Y

E

D

E

G

N

P

R

M

I

N

T

P

W

Y

E

E

P

I

P

F

H

E

A

A

E

I

G

T

R

TATGAAGATGAAAACGGGCCGCGGATaGATCATACGCCTTGGTACGAAGAACCGATCCCATTTCATGAGGCTGCTGAAATCGGCACACGA 700 650 BclI K

V

I

Q

E

H

S

T

I

G

V

I

V

T

T

D

G

T

I

G

D

I

A

R

S

D

Y

I

E

A

AAAGTCATTCAAGAACACTCGACCATCGGAGTTGTCATTACGACAGACGGCACCATTGGAGATATCGCCAGAAGTGACTATATAGAGGCT EcoRV 800 750 E

E

R

V

I

E

E

L

K

E

V

K

G

P

F

I

M

V

I

N

S

V

R

P

Y

H

P

E

T

E

GAAGAAAGAGTCATTGAAGAGCTGAAAGAGGTTGGCAAACCTTTTATTATGGTCATCAACTCAGTCAGGCCGTATCACCCGGAAACGGAA 900 850 A

M

R

L

D

Q

S

E

K

Y

D

P

I

V

L

A

M

S

V

E

S

M

R

E

S

D

V

L

S

V

GCCATGCGCCAGGATTTAAGCGAAAAATATGh&ATCCCGGTATTGGCAATGAGTGTAGAGAGCATGCGGGAATCAGATGTGCTGAGTGTG 950

EcoRV L

R

E

L

A

Y

E

F

S

V

P

V

L

V

E

N

V

N

L

P

S

W

V

M

V

L

K

E

N

H

W

L

CTCAGAGAGGCCCTCTACGAGTTTCCGGTGCTAGAAGTGAATGTCAATCTCCCAAGCTGGGTAATGGTGCTGAAAGAAAACCATTGGTTG 1050 1000 R

E

S

Y Q E

K

E

T

K

V

D

I

K

R

L

R

D

V

D

R

V

V

G Q

F

S

E

CGTGAAAGCTATCAGGAGTCCGTGAAGGAAACGGTTAAGGATATTAAACGGCTCCGGGACGTAGACAGGGTTGTCGGCCAATTCAGCGAG AccI 1150 1100 F

E

F

I

E

S

A

G

L

A

G

I

E

L

G

Q

G

V

A

E

I

D

L

Y

A

P

D

H

L

Y

TTTGAA&LATTGAAAGTGCCGGATTAGCCGGAATTGAGCTGGGCCAAGGGGTGGCAGAAATTGATTTGTACGCGCCTGATCATCTATAZ 1250

1200

EcoRI

D Q I L K E V V G V E I R G R D H L L E L M Q D F A H A K T GATC AATCCTAAAAGAAGTTGTGGGCGTCGAAATCAGAGGAAGAGACCATCTGCTTGAGCTCATGCAAGACTTCGCCCATGCGAAAACA 1350 SstI 1300 BcdI E

Y

D

Q V

E

P

E

I

A

L

K

R Q G

S

R

S

D

M

V

K

Q

T

R

L

G

Y

G

I

A

A

P

A

L

A

D

M

S

L

D

GAATATGATCAAGTGTCTGATGCCTTAAAAATGGTCAAACAGACGGGATACGGCATTGCAGCGCCTGCTTTAGCTGATATGAGTCTCGAT 1400 I

F

G

V

K

A

V

A

P

S

I

H

M

I

K V

D

V

E

GAGCCGGAAATTATAAGGCAGGGCTCGCGATTCGGTGTGAGGCTGAAAGCTGTCGCTCCGTCGATCCATATGATCAAAGTAGATGTCGAA BclI 1500 1450 S

E

F

A

P

I

I

G

T

E

K

O

S

E

E

L

V

R

Y

L

M Q

D

F

E

D

D

P

L

S

AGCMAATTCGCCCCGATTATCGGAACGGAAAAACAAAGTGAAGAGCTTGTACGCTATTTAATGCAGGACTTTGAGGATGATCCGCTCTCC 1600 1550 EcoRI I

W

N

D

S

ATCT GAATTC

EcoRI R

Y

K

I

F

G

R

S

L

S

S

I

V

R

E

G

I

Q A

K

L

S

L

M

P

E

N

A

TAATTGTGAGAGAAGGGATTCAGGCAAAGCTGTCATTGATGCCTGAAAACGCA fiATATCTTCGGAAGGTCGCTQa = 1650

EcoRV

L K E

T

L

E

R

1700

SstI

I

I

4 N

E

G

S

G

G

L

I

A

I

I

L

*

CGGTATAAATTAAAAGAAACATTAGAAAGAATCATAAACGAAGGCTCTGGCGGCTTAATCGCCATCATCCTGTAATACCGSIAGaCTCT 4

AccI 1800

1750

.