Res. MierobioL
c~ |NSTITUT P.~srI~nI~/ELSEVlER Paris 1991
1991, 142, 847-850
Spore germination genes of Bacillus subtilis 168 A . Muir, M . A . Yazdi t'> and E.H+ Kemp Departmen" f .Vlolecutar B~oio&v and Biotecht~ofogy, Uaivers,+) ~2f Sheffield, Sheffield SIO 2UH (UK)
latrod,elion
Genes whose products are specifically concerned with germination in alanine
Baeillu~ ~ubti#s spores will germinate in response to low concentrations of L-alanine, or in an aherna~ive combination of a non-germinative amino acid such as asparagiue plus glucose and fructose. In both of these types of germioanl, potassium ions stimulate germination rates (Wax and Freese, 1968 ; Muir
ct aL, 1979). Germinants (or at least amino acid germinants) are thought In act al!osrericaHy, activating a series o f changes in ~poru structure that are e':semially hydrolytic in nature. As the spore cortex is degraded, the core swells and rehydrates, and the spore in+ net membrane changes its structure and permeability, Ca 2 + and diplcolinate ions being lost from the core at early stages of germinatioa (as reviewed in Foster and Johnstone. 1989). It is impossible as yet to un[avel the causal relationships amongst these events, but a crucial first step would be to determine the nature of the inleractiou oi" the gcrminant '~,tt' the spore :~ad its direct consequences. An analysis of mutants dcfc+:tivc i:3 sl:,rrc wrmination fger mutantz,~ ~,:ientiiies at least some of the genes important in the proc,:~z. O r : tl~a past 15 years, a variety of get mutants have ,een classified on the basis of their genetic and pi~ysiologicai properties; more recently, most of the loci have been cloned (Trow~dale and Smith, 1975 ; Muir and Sroit h, 1990). The presence of two independently-stimulated germination responses in B. subtilis has helped in the allocation of functions to mutaat genes; this short review summarizes the current status of our understanding of the get genes and their products.
Mutations that specifically affect spore germination in L-alaniae and its analo~,ues, but that have no effect on Ihe alternative asparagine + glucose + fructose+ potassium ions (AGFK) pathway, have been described. A{most all of the mutations examined have been mapped to a Iocu~ (gerA) at 289° on the genetic map (Muir es aL, 1979). Some mutants (carrying the gerA38 or gerA44 mutations) require highcr concemrations of germinaat for germination to occur; the ohenoiype of these and other gerA mutants are consistent with this locus encoding the receptor for ak.nine in the spore (Sammoms etal.. 1981). The gerA region encodes an operon of three genes, gerAA, A B and A C (Feavers etal., 1985 ; Zuberi et at., 1987) all of which would encode membraneassociated proteins. The Ger,~A protein (53+5 kDa) contains two hydrophilic domains flanking a hydrophobic, membrane-embedded donmin, of over 200 residues, which contains a number of membranespanning helices, The GerAB protein (41.3 kDa) is entirely membraoe-~ssociated; the gerA38 and gcrA44 mutations map in this gene, suggestmg that GerAB may represent the primary alanine-hinding rote+ The Ge~AC protein (42.4 kDa) is hydrophilic, but contains the signal sequence of a pre-lipoprotein, suggesting that it is located on the outer surface of a membrane, associated via a eysteine-glyeeride link. The gerA genes arc expressed at a very low level in the forespore, tinder the control of the n°-associated RNA polymerase (Feavers e~ aL, 1990), so the likely fate of these proteins is to be rclzined in or at the silrface of the spore inner membrane.
(*) Currenl addresf.: Department of Bia,:hemislr.~'.Clayton Foutldatio;l Rc~eatchtl,klilutc. University of Te'cas, Austin, Te~a~IUSA).
848
A. MOIR ET AL.
The gerA operon is follo~ved by two open readins frames, encoded by the other strand and reading towards gerA (Zuberi and Muir, unpuUlished). These sequences represent a sensor-regulator gene pair of auk,town function. Disruption of these genes has no effect on sporulation or on germination in alanlne or AGFK (Kemp, unpublished), so they appear 10 have no functional connection with germination. The second alanine-associated locus, gerC(201 °), is in fact concerned with vegetative growth as well as germination. Two mutants were isolated from the same mutagenesis experiment (rrowsdale and Smith, 1975); only one has been examined in detail. The spore germination defect described in the mutant, temperature-sensitive germination in alanine, is a property of a suppressed mutant ; the unsuppressed ,~erC strain barely grows on minimal medium, achieving a colony diameter of approximately 1 ram, and never sporulates. On rich medium, it does not form colonies, but under these conditions mutant derivatives are selected that carry suppressing mutations which restore near-normal growth and successful sporulation, hut leave a residual ts germination defect (Yazdi and Muir, 1990). The gerC locus has been cloned and sequenced (Yazdi and Muir, 1990; Henner e~ el., 1991 ; Ya2di, Cueson, Leatherbarrow and Muir, in preparation), and again encodes an operon of three genes. The gerC58 mutation maps in the third gcnc (gerCC), whose deduced protein product is a hnmologue of the cr~E gene product of Rhodobacter capsutetus. The amino acid sequences align, and share 23 % identical residues. The crtE gene encodes one subunit of the enzyme that condenses two geranyl pyrophosphate molecules to form a C ~ terpenoid (Armstrong et eL, 1989) ; no other enzyme homologues of the carotenoid biosynthetic pathway are present in the gerC operon. The severe growth defect in the gerC58 unsuppressed strain argues that this enzyme is essential for normal growth of B. subtilis, invoking an essential role for some terpenoid ; this is presumably in membrane function, as such molecules are reported to act as membrane stabilizing agents (Taylor, 1984). Ai~ho~gh the nature of the suppression has not yet been established, one could then rationalize the effect of suppression as restoring an almost normal membrane function during growth, but that the membrane environment of the alanine germination apparatus is not entirely normal, so that the conformation and function of the alaoine germination receptor is normal at 30 ° but disturbed at 42 °. As AGFK germination is unaffected, this might suggest that the receptors for the two types of germinant are in slightly different environments,
AGFK = ~5paraginv-rgluco~e÷fruetose+polassiura6ansi.
TLe gerC58 mutation has a pleiotropic effect, and is unlikely therefore to represent a gene with a specific and direct effect in alanine-triggered germination; this leaves the GerA proteins as the only detected candidates for the role of alanine receptor.
Genes concerned specifically with AGFK germination Two distinct loci, gerBat 314 ° (Muir eta/., 1979) and gerK at 32 ° (Irie et eL, 1982) are represented in the collection of mutants specifically defective in AGFK but normal in alanine germination. Both gerB (Corfe and Smith, personal communication) and gerK (lrie, personal communication) have been cloned. Preliminary sequence information from gerB (Corfe, Sammous and Smith, unpublished) suggests that the gerB locus contains at least one homologue of a gerA gen¢. The gerK locus maps between are1 and do1. as does the glucose dehydrogcnase gene, but it is well separated from the glucose dehydrogenase operon Ode, personal commun.); no sequence information is yet available. Mutations in this latter enzyme do not affect germination in AGFK (gamale~, personal communication). Mutations in frog, which cneodes fructose l-P-kinasc, are reported to block the gcrmlnativc contribution of fructose, suggestive of a metabolic role for fructose in the triggering of germination (Prasad at el., 1972).
Genes whose products function in buth pathways of germination Two groups of mutants have a phenotype that is related to germination in both types of germinant. The genes, gerD (at 16 °) and gerF (at 301~), are represented by mutations tkat result in slow germination in alanine and in no germination in AGFK. The long delay in alanine-stimulated germination in these mutants can be reduced in the presence of high concentrations of monovalent cations or by spore formation in resuspenston medium (Warburg et el., 1985 ; Yon er el., 1989) but the defect in AGFK germination is not reversed by these means. The gerD gene has been cloned and sequenced (Yon et aL, 1989). It is a monoclstronlc locus, encoding a protein of 2i.1 kDa; the encoded protein is hydrophilie, but contains a signal sequence similar to that of the Trail protein of the F factor. The promoter of the gerD gene is he'-dependent, and the gene is part of the forespore-expressed repertoire; this protein is likely to be exported from the forcspore into the cortex, so that it is either free in the cortex
SPORE GERMINA 710N GENES OF BACILLUS SUBTILIS 168 or possibly still associated with the outer surface of the membrane (Kemp, Molt. Sun and So[low, submitted). The s e r f gone is located near the hisA locus (t hey are 57 °70cotransdueed by SPPIJ; aRhough gerFwas not carried on hisA clones (Walton et eL, 1984), it should not be too difficult to obtain by chromosome walking. The products of these two genes are essential for germination and, like those that are germinantspecific described above, are essential for the earfiest stages o f spore germination, before toss of heat resistance. Given the observation that they affect germination - - at least to some extent - - in both systems, they may represent a downstream part of the signal transduction mechanism.
Gene~ whose products ~ffeel spore structure Several types of get mutant affect germination in both pathways, but block germination at a later gage, after loss of heat resistance. In these mutants, spore cortex hydrolysis is only partial. The mutants are pleiotropic, affecting spore resistance properties. The best characterized of these loci isgerE (253~); the irlutaht identified produces coat-defective, lysozyme-sensitive spores (Moir, 198t), The gore gone encodes an 8.5-kDa DNA-binding protein which regulates expression of sporecoat genes (Cutting et el., 1989) at a late stage of sporulatian in the mother cell Two other loci important in spore development, but associated with cortex rather than coat synthesis, are gerJ (206'~; Warburg and M_ojr, 1981)and gerM (251 o ;Sammons et el., 1987). Both genes are switched on earlier in Sporulation, from about 1,5 h. The gerJ mutants are delayed in the expression of spore-specific penicillin-binding proteins (Warburg et oL, 1986}, whereas the gerMmutants are initially defective in septum formation; those leaky germ mutants that successfully make a septum form a defective spore cortex, conferring a lesser degree of heat resistance on the spare (gammons et el., 1987). Ti~e reason for the late block in germination in these mutants must be different in nature; in the gore rantam, some component required for late stages of cortex hydrolysis during germination may be either not synthesized or not retained in the spore, in contrast~ the gerJ and germ germination defects are more likely to be the result of defects in cortex synthesis, affecting the target of lytic enzymes. Possible inechanisms of germination The continued characterization of the ger mutant loci is gradually building a picture of the possible
849
molecular events in/5. subtEis germination. It is possible, for example, to distinguish at least some of the proteins directly concerned with the triggering of germination. The earliest events of atanine germination are mediated by three membrane-associated GerA proteins, which may exist as a complex, in the inner spore membrane, The preliminary but exciting information tb~t gerB may be a homologous system to gerA (Car re, Sammons and Smith, personal communication), along with the observation that a gerAA homologue is located downstream of the fnresporeexpressed spoVA 8enos (Errington, personal communication), raises the intriguing possibility that there may be an extended family of GerA-like proteins in tbe spore, perhaps concerned with germination responses to different stimuli. Nevertheless+ there are qualitative differences between the germination pathways in terms of the number of genes required and some distinctive inhibitor sensitivities (Venkatasubramanian and Johnstone, 198(3tthat will have to be accommodated in any overall model of the process. Key-words: Bacillus stebtilis. Spore, Germinatior~; ger Gone.
Referenc~ Armstrong. G.A.. Albert[. M., Leach, F. &[tearst, J.E. (1989), Nucleotidesequence, organisation, and nature of the protein products of the carotenoid biosynthesis gene cluster of"I~hodobactercepsttlams. MoL sen. Genetics, 2]6, 254-268. Cutting, g., Panzer, S, & Losiek. R. (1989), Regulatory studies on the promoter For a ~ene governing synthesis ~nd assembly of the spore coat in Bacillus rubtiIts. ,I. ntol. BioL, 207, 393-404. reapers, I.M,, Fonlkes, J., So[low, B., Sun, D., NichoIson. W.. So[low, P. & Moir. A. (19901, the regulation of transcription or the gerA spore germination opcron of Bctei/lus subt~lis. MoL Microbial,, 4, 275-282. reapers, l.M.. Miles, ~.S. & Molt, A. {t985), The ~cle~tide sequence of a spore germination gene/geeA) of Bacdlus sub[ills 168. Gene, ]g, 95-102. Foster, S,J. & Johnstone. K. (1989). The trigger mechanism of bacterial spore germination, in "Regulation of pro~aryotic development" (Smith, I., Slcpecky. g., So[low, P.) (pp. 89-108)_ American Society for Microbiology, Washington. Fletcher, DJ-, Molt, A., Yd~di, M.A. & Gollnick, P. ( 19911,Analysiso["the sequence of an 18-kilobazepair region of the Bacillus subtilis chromosome. Proceedings of the 1990 raeeting on Generics of Industrial Microorganisms (in press). ~rie. R., Okamoto, T. & Fuji[a, Y. 11982), A germination mutant of Baeitlus sub[iris deficientin r~ponse to glucose. J. sen. Appl. MicrobiaL, 28, 345-J54. Molt, A. 11981), Germination properIit~sor a spore coat defective rnulant of 8~rcillus subltl~. J. I3act., I46, lt06-t106. Moir, A. & Smith, D,A. (1990). The gent:ticsof bacterial
850
A. MOIR ET AL.
spore germination, Ann. Rev. MicrobiaL, 44, 531-553. Molt, A., Lafferty, E. & Smith, D.A. (1979), Genetic analysis of spore germination mutants of Bacillus subtiIts 168: the correlation of phenotype with map location. Z sen. Microbic/., I 1 I, 165-180. Prasad. C., Dicslerhaft. M. & Preese. E. (19727. Initiation of spore germination in glycolytic mutants of Bacillus subtilis. £ Butt., 110. ]21-328. Sammons, R.L., Muir, A. & Smith, D.A. 09517, Isolation and properties of spore germination mutants of Bacillus subtilis 168 deficient in the initiation of germinallon, d. gcn. ?dicrobloL, 124, 229-241. Sammons, R.L., Slynn, O.M. & Smith, D,A. 0957), Geaetical and molecular studies on gerM, a new developmemal locus of ~gacillus subtills. J. sen. MicrobigL, 133, 3299-3312. Taylor, R.F. (1954), Bacterial triterpenoids. Microbial. Ray., 48, Igl-198. Trowsdale, J. & Smith, D.A. 0975), Isolation, characterization and mapping Of Bacillus $ubtilis 168 germination mutants. J. Beet., 123.83~95. Venkatasubramanian, P. & Johnstone, K. (1989), Biochemical analysis of the Bacillus subtilis 1604 spore germination response, J. gem Microbial., 135, 2723-2733. Walton, D.A., Moir, A., Morse, R., Roberts, I. & Smith,
D,A, {1984), The isolation of phage carrying D N A from the histidine and isolcucine-valine regions of the Badllus subtili$ chromosome. 3". sen. Microbial., 130, 1577-1586. Warbarg, R.J., Buchanan, C.E., Parent, K. & Halvorson, H.O. (19857, A detailed study of gerJ mutants of Bacillus Subtili& J. ~en. Microbiol., 132, 2309-2319. Warburg, R.J. & M o i l A. (1981), Properties of a mutant ofBacillussabtilis 16g in which spore 8ermlnation is blocked at a late stage, d. Sen. Microbiol., 124, 243-25]. Warhurg, R.J,, Molt, A. & Smith, D.A. (19857, Influence of alkali metal cations on the germination of spores of wild-type and gerD mutants of Bacillus subtllis. J. sen. Microbiol., 131,221-230. Wax, R. & Freese, E. 0968), Initiation of the germination of Bacillus sub/ills spores by a combination of compounds in place of L-alanine..L Bact, 95, 433-43g. Yazdi, M.A. & Mvir, A. (1990), Characterisation and cloning of t he gerC locus of Bacillus subtilis 168. Jr. sen. MierobioL, 136, 1335-1342. Yon, J.R., Sammons, R.L. & Smith, D.A, (1989), Cloning and sequencing of the gerD gene of Baeirlus sub. tills. J. sea. MierobioL, 135, 3431.3445. Zuberi, A.R., Molt, A, & Feavers, I.M. (19877, The nucleotlde sequence and gone organization of the gerA spore germination operon of Bacillus subrilis 16g. Gene, 51, 1-11.
c~ |NSTITUT P.~srI~nI~/ELSEVlER Paris 1991
1991, 142, 847-850
Spore germination genes of Bacillus subtilis 168 A . Muir, M . A . Yazdi t'> and E.H+ Kemp Departmen" f .Vlolecutar B~oio&v and Biotecht~ofogy, Uaivers,+) ~2f Sheffield, Sheffield SIO 2UH (UK)
latrod,elion
Genes whose products are specifically concerned with germination in alanine
Baeillu~ ~ubti#s spores will germinate in response to low concentrations of L-alanine, or in an aherna~ive combination of a non-germinative amino acid such as asparagiue plus glucose and fructose. In both of these types of germioanl, potassium ions stimulate germination rates (Wax and Freese, 1968 ; Muir
ct aL, 1979). Germinants (or at least amino acid germinants) are thought In act al!osrericaHy, activating a series o f changes in ~poru structure that are e':semially hydrolytic in nature. As the spore cortex is degraded, the core swells and rehydrates, and the spore in+ net membrane changes its structure and permeability, Ca 2 + and diplcolinate ions being lost from the core at early stages of germinatioa (as reviewed in Foster and Johnstone. 1989). It is impossible as yet to un[avel the causal relationships amongst these events, but a crucial first step would be to determine the nature of the inleractiou oi" the gcrminant '~,tt' the spore :~ad its direct consequences. An analysis of mutants dcfc+:tivc i:3 sl:,rrc wrmination fger mutantz,~ ~,:ientiiies at least some of the genes important in the proc,:~z. O r : tl~a past 15 years, a variety of get mutants have ,een classified on the basis of their genetic and pi~ysiologicai properties; more recently, most of the loci have been cloned (Trow~dale and Smith, 1975 ; Muir and Sroit h, 1990). The presence of two independently-stimulated germination responses in B. subtilis has helped in the allocation of functions to mutaat genes; this short review summarizes the current status of our understanding of the get genes and their products.
Mutations that specifically affect spore germination in L-alaniae and its analo~,ues, but that have no effect on Ihe alternative asparagine + glucose + fructose+ potassium ions (AGFK) pathway, have been described. A{most all of the mutations examined have been mapped to a Iocu~ (gerA) at 289° on the genetic map (Muir es aL, 1979). Some mutants (carrying the gerA38 or gerA44 mutations) require highcr concemrations of germinaat for germination to occur; the ohenoiype of these and other gerA mutants are consistent with this locus encoding the receptor for ak.nine in the spore (Sammoms etal.. 1981). The gerA region encodes an operon of three genes, gerAA, A B and A C (Feavers etal., 1985 ; Zuberi et at., 1987) all of which would encode membraneassociated proteins. The Ger,~A protein (53+5 kDa) contains two hydrophilic domains flanking a hydrophobic, membrane-embedded donmin, of over 200 residues, which contains a number of membranespanning helices, The GerAB protein (41.3 kDa) is entirely membraoe-~ssociated; the gerA38 and gcrA44 mutations map in this gene, suggestmg that GerAB may represent the primary alanine-hinding rote+ The Ge~AC protein (42.4 kDa) is hydrophilic, but contains the signal sequence of a pre-lipoprotein, suggesting that it is located on the outer surface of a membrane, associated via a eysteine-glyeeride link. The gerA genes arc expressed at a very low level in the forespore, tinder the control of the n°-associated RNA polymerase (Feavers e~ aL, 1990), so the likely fate of these proteins is to be rclzined in or at the silrface of the spore inner membrane.
(*) Currenl addresf.: Department of Bia,:hemislr.~'.Clayton Foutldatio;l Rc~eatchtl,klilutc. University of Te'cas, Austin, Te~a~IUSA).
848
A. MOIR ET AL.
The gerA operon is follo~ved by two open readins frames, encoded by the other strand and reading towards gerA (Zuberi and Muir, unpuUlished). These sequences represent a sensor-regulator gene pair of auk,town function. Disruption of these genes has no effect on sporulation or on germination in alanlne or AGFK (Kemp, unpublished), so they appear 10 have no functional connection with germination. The second alanine-associated locus, gerC(201 °), is in fact concerned with vegetative growth as well as germination. Two mutants were isolated from the same mutagenesis experiment (rrowsdale and Smith, 1975); only one has been examined in detail. The spore germination defect described in the mutant, temperature-sensitive germination in alanine, is a property of a suppressed mutant ; the unsuppressed ,~erC strain barely grows on minimal medium, achieving a colony diameter of approximately 1 ram, and never sporulates. On rich medium, it does not form colonies, but under these conditions mutant derivatives are selected that carry suppressing mutations which restore near-normal growth and successful sporulation, hut leave a residual ts germination defect (Yazdi and Muir, 1990). The gerC locus has been cloned and sequenced (Yazdi and Muir, 1990; Henner e~ el., 1991 ; Ya2di, Cueson, Leatherbarrow and Muir, in preparation), and again encodes an operon of three genes. The gerC58 mutation maps in the third gcnc (gerCC), whose deduced protein product is a hnmologue of the cr~E gene product of Rhodobacter capsutetus. The amino acid sequences align, and share 23 % identical residues. The crtE gene encodes one subunit of the enzyme that condenses two geranyl pyrophosphate molecules to form a C ~ terpenoid (Armstrong et eL, 1989) ; no other enzyme homologues of the carotenoid biosynthetic pathway are present in the gerC operon. The severe growth defect in the gerC58 unsuppressed strain argues that this enzyme is essential for normal growth of B. subtilis, invoking an essential role for some terpenoid ; this is presumably in membrane function, as such molecules are reported to act as membrane stabilizing agents (Taylor, 1984). Ai~ho~gh the nature of the suppression has not yet been established, one could then rationalize the effect of suppression as restoring an almost normal membrane function during growth, but that the membrane environment of the alanine germination apparatus is not entirely normal, so that the conformation and function of the alaoine germination receptor is normal at 30 ° but disturbed at 42 °. As AGFK germination is unaffected, this might suggest that the receptors for the two types of germinant are in slightly different environments,
AGFK = ~5paraginv-rgluco~e÷fruetose+polassiura6ansi.
TLe gerC58 mutation has a pleiotropic effect, and is unlikely therefore to represent a gene with a specific and direct effect in alanine-triggered germination; this leaves the GerA proteins as the only detected candidates for the role of alanine receptor.
Genes concerned specifically with AGFK germination Two distinct loci, gerBat 314 ° (Muir eta/., 1979) and gerK at 32 ° (Irie et eL, 1982) are represented in the collection of mutants specifically defective in AGFK but normal in alanine germination. Both gerB (Corfe and Smith, personal communication) and gerK (lrie, personal communication) have been cloned. Preliminary sequence information from gerB (Corfe, Sammous and Smith, unpublished) suggests that the gerB locus contains at least one homologue of a gerA gen¢. The gerK locus maps between are1 and do1. as does the glucose dehydrogcnase gene, but it is well separated from the glucose dehydrogenase operon Ode, personal commun.); no sequence information is yet available. Mutations in this latter enzyme do not affect germination in AGFK (gamale~, personal communication). Mutations in frog, which cneodes fructose l-P-kinasc, are reported to block the gcrmlnativc contribution of fructose, suggestive of a metabolic role for fructose in the triggering of germination (Prasad at el., 1972).
Genes whose products function in buth pathways of germination Two groups of mutants have a phenotype that is related to germination in both types of germinant. The genes, gerD (at 16 °) and gerF (at 301~), are represented by mutations tkat result in slow germination in alanine and in no germination in AGFK. The long delay in alanine-stimulated germination in these mutants can be reduced in the presence of high concentrations of monovalent cations or by spore formation in resuspenston medium (Warburg et el., 1985 ; Yon er el., 1989) but the defect in AGFK germination is not reversed by these means. The gerD gene has been cloned and sequenced (Yon et aL, 1989). It is a monoclstronlc locus, encoding a protein of 2i.1 kDa; the encoded protein is hydrophilie, but contains a signal sequence similar to that of the Trail protein of the F factor. The promoter of the gerD gene is he'-dependent, and the gene is part of the forespore-expressed repertoire; this protein is likely to be exported from the forcspore into the cortex, so that it is either free in the cortex
SPORE GERMINA 710N GENES OF BACILLUS SUBTILIS 168 or possibly still associated with the outer surface of the membrane (Kemp, Molt. Sun and So[low, submitted). The s e r f gone is located near the hisA locus (t hey are 57 °70cotransdueed by SPPIJ; aRhough gerFwas not carried on hisA clones (Walton et eL, 1984), it should not be too difficult to obtain by chromosome walking. The products of these two genes are essential for germination and, like those that are germinantspecific described above, are essential for the earfiest stages o f spore germination, before toss of heat resistance. Given the observation that they affect germination - - at least to some extent - - in both systems, they may represent a downstream part of the signal transduction mechanism.
Gene~ whose products ~ffeel spore structure Several types of get mutant affect germination in both pathways, but block germination at a later gage, after loss of heat resistance. In these mutants, spore cortex hydrolysis is only partial. The mutants are pleiotropic, affecting spore resistance properties. The best characterized of these loci isgerE (253~); the irlutaht identified produces coat-defective, lysozyme-sensitive spores (Moir, 198t), The gore gone encodes an 8.5-kDa DNA-binding protein which regulates expression of sporecoat genes (Cutting et el., 1989) at a late stage of sporulatian in the mother cell Two other loci important in spore development, but associated with cortex rather than coat synthesis, are gerJ (206'~; Warburg and M_ojr, 1981)and gerM (251 o ;Sammons et el., 1987). Both genes are switched on earlier in Sporulation, from about 1,5 h. The gerJ mutants are delayed in the expression of spore-specific penicillin-binding proteins (Warburg et oL, 1986}, whereas the gerMmutants are initially defective in septum formation; those leaky germ mutants that successfully make a septum form a defective spore cortex, conferring a lesser degree of heat resistance on the spare (gammons et el., 1987). Ti~e reason for the late block in germination in these mutants must be different in nature; in the gore rantam, some component required for late stages of cortex hydrolysis during germination may be either not synthesized or not retained in the spore, in contrast~ the gerJ and germ germination defects are more likely to be the result of defects in cortex synthesis, affecting the target of lytic enzymes. Possible inechanisms of germination The continued characterization of the ger mutant loci is gradually building a picture of the possible
849
molecular events in/5. subtEis germination. It is possible, for example, to distinguish at least some of the proteins directly concerned with the triggering of germination. The earliest events of atanine germination are mediated by three membrane-associated GerA proteins, which may exist as a complex, in the inner spore membrane, The preliminary but exciting information tb~t gerB may be a homologous system to gerA (Car re, Sammons and Smith, personal communication), along with the observation that a gerAA homologue is located downstream of the fnresporeexpressed spoVA 8enos (Errington, personal communication), raises the intriguing possibility that there may be an extended family of GerA-like proteins in tbe spore, perhaps concerned with germination responses to different stimuli. Nevertheless+ there are qualitative differences between the germination pathways in terms of the number of genes required and some distinctive inhibitor sensitivities (Venkatasubramanian and Johnstone, 198(3tthat will have to be accommodated in any overall model of the process. Key-words: Bacillus stebtilis. Spore, Germinatior~; ger Gone.
Referenc~ Armstrong. G.A.. Albert[. M., Leach, F. &[tearst, J.E. (1989), Nucleotidesequence, organisation, and nature of the protein products of the carotenoid biosynthesis gene cluster of"I~hodobactercepsttlams. MoL sen. Genetics, 2]6, 254-268. Cutting, g., Panzer, S, & Losiek. R. (1989), Regulatory studies on the promoter For a ~ene governing synthesis ~nd assembly of the spore coat in Bacillus rubtiIts. ,I. ntol. BioL, 207, 393-404. reapers, I.M,, Fonlkes, J., So[low, B., Sun, D., NichoIson. W.. So[low, P. & Moir. A. (19901, the regulation of transcription or the gerA spore germination opcron of Bctei/lus subt~lis. MoL Microbial,, 4, 275-282. reapers, l.M.. Miles, ~.S. & Molt, A. {t985), The ~cle~tide sequence of a spore germination gene/geeA) of Bacdlus sub[ills 168. Gene, ]g, 95-102. Foster, S,J. & Johnstone. K. (1989). The trigger mechanism of bacterial spore germination, in "Regulation of pro~aryotic development" (Smith, I., Slcpecky. g., So[low, P.) (pp. 89-108)_ American Society for Microbiology, Washington. Fletcher, DJ-, Molt, A., Yd~di, M.A. & Gollnick, P. ( 19911,Analysiso["the sequence of an 18-kilobazepair region of the Bacillus subtilis chromosome. Proceedings of the 1990 raeeting on Generics of Industrial Microorganisms (in press). ~rie. R., Okamoto, T. & Fuji[a, Y. 11982), A germination mutant of Baeitlus sub[iris deficientin r~ponse to glucose. J. sen. Appl. MicrobiaL, 28, 345-J54. Molt, A. 11981), Germination properIit~sor a spore coat defective rnulant of 8~rcillus subltl~. J. I3act., I46, lt06-t106. Moir, A. & Smith, D,A. (1990). The gent:ticsof bacterial
850
A. MOIR ET AL.
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