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Prokaryotes (2006) 4:530–562 DOI: 10.1007/0-387-30744-3_16 CHAPTER 1.2.16 ehT suneG The Genus Bacillus—Nonmedical RALPH A. SLEPECKY AND H. ERNEST HEMPHILL History One of the earliest bacteria to be described was “Vibrio subtilis” by Ehrenberg in 1835. In 1872, Cohn renamed the organism Bacillus subtilis (Gordon, 1981). That organism was a charter member of a large and diverse genus, initiated by Cohn, that is part of the family Bacillaceae. This family’s distinguishing feature is production of endospores, which are round, oval, or cylindrical highly refractile structures formed within bacterial cells. Spores were first described by Cohn in subtilis and later by Koch in the pathogen, B. anthracis (the only major pathogen of vertebrates in the genus). Cohn demonstrated the heat resistance of spores of B. subtilis and Koch first described in B. anthracis the developmental cycle of sporeformers, vegetative cell to spore and spore to vegetative cell (Keynan and Sandler, 1983). For the reasons of unusual spore resistance to chemical and physical agents; the developmental cycle; ubiquity of its members; and B. anthracis pathogenicity, the genus Bacillus attracted early interest which has continued since. The endospore, either as the free spore or as the structure within the vegetative cell, in which case the whole entity is referred to as a sporangium, is readily detected using the phase contrast microscope (see Fig. 1). This is because the spore at a point in the life cycle (to be detailed later) becomes highly refractile. Early workers used stains and special conditions (such as prolonged heating) to colorize the chemically impermeable spore (Doetsch, 1981). However, a Gram-stain is sufficient to determine the presence of spores because the spore remains unstainable while the vegetative cells or the vegetative part of the sporangia will stain. Because of this ease of microscopic detection of the spore and its heat resistance, many different endosporeformers can be easily found. Using any habitat—soil, water, food, etc.—as the source, sporeformers can be readily isolated by This chapter was taken unchanged from the second edition. suspending a sample in water and heating at 80°C for 10 to 30 min. Vegetative cells and other resting forms such as cysts and exospores are usually killed at that temperature. The heatresistant endospore can then be plated on appropiate media and isolates recovered in 24 to 48 h. An idea of the kinds of habitats from which Bacillus species have been isolated can be obtained from Table 1. Heating the inoculum, when used in conjunction with cultivation at different temperatures, hydrogen ion concentrations, degrees of aeration, and substrates, has resulted in isolating many different species of endosporeformers. The media used for the isolation and cultivation of Bacillus species are listed in Table 2. More often than not since the discovery of bacteria (and in every case since 1913), the possession of an endospore has been used as a premier characteristic in keys for the classification of bacteria. The family Bacillaceae was first formulated by Fisher in 1895 (Gordon, 1981). The features of the members of the genus Bacillus that distinguish it from other Bacillaceae (all endosporeformers) are their aerobic nature, which may be strict or facultative, rod shape, and catalase production. The other genera of sporeformers include Sporolactobacillus, which is microaerophilic and catalase-negative; Clostridium, anaerobic but does not reduce sulfate; Desulfotomaculum, anaerobic but does reduce sulfate; Sporosarcina, a coccus; and Thermoactinomycetes, which while forming endospores displays typical actinomycete characteristics. General Taxonomic Considerations Like the sirens of Greek mythology enticing the unsuspecting sailors, Bacillus species have captured the curiosity of many microbiologists. The first 107 years of the efforts to classify and identify members of the genus Bacillus is chronicled by R.E. Gordon (1981) who with her colleagues (Gordon et al., 1973; Smith et al., 1946, 1952) made many significant contributions on which the current classification (Claus and Berkeley, 1986) was built. The early attempts were “on l ac i demnoN- - su l l i caB CHAPTER 1.2.16 The Genus Bacillus—Nonmedical 531 Fig. 1. B. megaterium sporeforming cells as seen in the phase contrast (A) or in the interference contrast (B) microscope. The refractile bodies in the center are the spores. Bar = 5 µm. A Table 1. Origins of isolates of Bacillus species. Name of Bacillus species Habitats from which isolated B. subtilis B. acidocaldarius B. alcalophilus B. alvei B. amylolyticus B. anthracis B. azotoformans B. badius B. brevis B. cereus B. circulans B. coagulans B. fastidiosus B. firmus B. globisporus B. insolitus B. larvae B. laterosporus B. lautus B. lentimorbus B. lentus B. licheniformis B. macerans B. macquariensis B. marinus B. megaterium B. mycoides B. pabuli B. pantothenicus B. pasteurii B. popilliae B. psychrophilus B. pumilus B. schlegelii B. sphaericus B. stearothermophilus B. thermoglucosidasius B. thuringiensis B. validus Soil, water Thermal acid water and soil pH 10 enrichment from soil Soil, diseased bee larvae Soil Anthrax-diseased animals Soil Feces, foods, marine sources Soil, foods Soil, foods Soil Acid foods Soil, poultry litter Soil, salt marshes Soil, water Soil Diseased bee larvae Soil, water Soil, feces Diseased honeybee larvae Soil, foods Soil Plant materials, food Subantarctic soil Marine sediment Soil Soil Soil, fodder Soil Soil, water, sewage Diseased scarabid beetles Soil, water Soil Lake sediment Soil, water sediments, foods Soil, hot spring, foods Soil Soil, foods Soil Based on Claus and Berkeley (1986). B rocky shoals” because a classification based on only the two characteristics of aerobic growth and endospore formation resulted in grouping together many bacteria possessing different kinds of physiology and occupying a variety of habitats. This heterogeneity in physiology, ecology, and genetics makes it difficult to categorize the genus or to make generalizations about it. The range of physiological life styles is impressive: degraders of most all substrates derived from plant and animal sources including cellulose, starch, proteins, agar, hydrocarbons, and others; antibiotic producers; heterotrophic nitrifiers; denitrifiers; nitrogen fixers; iron precipitators; selenium oxidizers; oxidizers and reducers of manganese; facultative chemolithotrophs; acidophiles; alkalophiles; psychrophiles, thermophiles and others (Slepecky, 1972; Norris et al., 1981; Claus and Berkeley, 1986) (see Table 2). Because of this vast diversity of physiological types, our knowledge of sporeformer ecology is slight (Slepecky, 1972; Norris et al., 1981; Slepecky and Leadbetter, 1977, 1984). In the main, sporeformers as part of the zymogenous flora of the soil are viewed as opportunists. Upon access to the proper germinants and substrates for subsequent outgrowth, they will actively contribute to and participate in the various microhabitats which make up the soil’s heterogeneous environment. Aerial distribution of the dormant spores may explain the occurrence of Bacillus species in most habitats examined. This diversity was apparent even with classical phenotypic characterizations based primarily on morphology (particularly size and position of the endospore within the vegetative cell), nutrition; growth characteristics; and various substrate utilization and physiological assessments. At one time, 145 species made up the genus (Gordon, 1981). The understanding of the genus has been improved by augmenting the phenotypic characterizations with measurements of the DNA base composition and DNA-DNA hybridigation. Currently, there are listed in Bergey’s 532 R.A. Slepecky and H.E. Hemphill CHAPTER 1.2.16 Table 2. Media used for the isolation and cultivation of Bacillus species. B. acidocaldarius B. alcalophilus B. azotoformans B. brevis B. fastidiosus B. lentus B. licheniformis B. marinus B. pantothenicus B. pasteurii B. schlegelii B. stearothermophilus Part A: (NH4)2SO4, 0.4; MgSO4, 1.0; CaCl2 ·2H2O, 0.5; KH2PO4, 6.0; distilled H2O, 1 liter; pH adj. to 4.0 Part B: glucose, 2.0; yeast ext., 2.0 distilled H2O, 1 liter Combine A and B after sterilization Part A: glucose, 1.0; peptone, 5.0; yeast ext., 5.0; KH2PO4, 10.0 MgSO4 ·7H2O, 0.2, distilled H2O, 900ml. Part B: Na2CO3 ·10H2O, 20; distilled H2O, 100ml. Combine A and B after sterilization (final pH = 10.5) Peptone, 10.0; Na2HPO4 ·12H2O, 3.6; MgSO4 ·7H2O, 0.03; MnSO4 ·H2O, 0.05; KH2PO4, 1.0; NH4Cl, 0.5; CaCl2 ·2H2O, 0.1; distilled H2O, 1 liter. K2HPO4, 0.2; MgSO4 ·7H2O, 0.02; NaCl, 0.02; FeSO4 ·7H2O, 0.01; MnSO4 ·H2O, 0.01; betaine, betaine·HCl or valine, 0.05M; agar, 16.0; distilled H2O, 1 liter. K2HPO4, 0.8; KH2PO4, 0.2; MgSO4 ·7H2O, 0.05; CaCl2 ·2H2O, 0.05; FeSO4 ·7H2O, 0.015; MnSO4 ·H2O, 0.01; uric acid, 10.0; distilled H2O, 1 liter. Peptone, 10.0; meat ext., 10.0; agar, 15.0; distilled H2O, 1 liter. adj. pH to 7.0–7.5; after sterilization, add 100g urea, steam for 10min. Peptone, 5.0; meat ext., 3.0; KNO3, 80.0; distilled H2O, 1 liter; adj. pH to 7.0; fill glass-stoppered bottle to top for anacrobic conditions. Peptone, 5.0; yeast ext., 1.0; FePO4 ·4H2O, 0.01; agar, 15.0; aged sea water, 750ml; distilled H2O, 250ml; adj. pH to 7.6. Nutrient broth + 4% (w/v) NaCl Nutrient broth + 2% (w/v) urea Na2HPO4 ·2H2O, 4.5; KH2PO4, 1.5; NH4Cl, 1.0; MgSO4 ·7H2O, 0.2; CaCl2 ·2H2O, 0.01; ferric ammonium citrate, 0.005; NaHCO3, 0.5; trace element soln, 5ml (ZnSO4 ·7H2O, 0.1; MnCl2 ·4H2O, 0.03; H3BO3, 0.3; CoCl2 ·6H2O, 0.02; CuCl2 ·2H2O, 0.001; NiCl2 ·6H2O, 0.02; Na2MoO4 ·2H2O, 0.03; distilled H2O, 1 liter) Other: 65C, atmosphere of 0.05atm. O2 + 0.01atm. CO2 + 0.45atm. H2 Nutrient agar; incubate cultures at 55°C a Numerical amounts are grams unless specified. Most other Bacillus cultures will grow on nutrient broth and nutrient agar. Based on Claus and Berkeley (1986). b Table 3. Major distinguishing characteristics of some Bacillus species. Thermophile Thermophilic acidophiles Psychrophiles B. stearothermophilus B. thermodenitrificans B. caldotenax B. coagulans B. acidocaldarius B. psychrophilus B. macquariensis B. globisporus B. insolitus Alkalophile Facultative chemolithotroph Nitrogen fixers Alginate degraders Aromatic acid and phenol degrader a See Chapter 77. B. psychrosaccharolyticus B. alcalophilus B. schlegelii B. polymyxa B. macerans B. azotofixans B. alginolyticus B. chrondrotinus B. benzoevorans Hydroxy aromatic compounds degrader Nitrate reduction to N2 Growth restricted to uric acid, allantoin or allantoic acid Growth at high pH and NH4Cl Requires pantothenic acid Requires biotin Produces heat stable glucosidase Insect pathogensa Human and animal pathogen B. gordonae B. azotoformans B. licheniformis B. fastidiosus B. pasteurii B. pantothenicus B. pumilus B. thermoglucosidasius B. thuringiensis B. larvae B. popilliae B. sphaericus B. lentimorbus B. anthracis B. cereus CHAPTER 1.2.16 The Genus Bacillus—Nonmedical 533 Table 4. DNA-base composition and sources of the type strains of Bacillus species. GC content (mol%) Bacillus species acidocaldarius alcalophilus alvei amylolyticus anthracis azotoformans badius brevis cereus circulans coagulans fastidiosus firmus globisporus insolitus larvae laterosporus lautus lentimorbus lentus licheniformis macerans macquariensis marinus megaterium mycoides pabuli pantothenicus pasteurii polymyxa popilliae psychrophilus pumilus schlegelii sphaericus stearothermophilus subtilis thermoglucosidasius thuringiensis validus Culture collection number Tma BDb ATCCd DSM NCIB NCTC NRRL 60.3 37.0 44.6 NDc 33.2 ND 43.8 47.3 35.7 35.5 47.1 35.1 41.4 39.8 35.9 ND 40.2 ND 37.7 36.3 46.4 52.2 39.3 37.6 37.3 34.2 ND 36.9 38.5 44.3 41.3 39.7 41.9 64.6 37.3 51.9 42.9 45–46 33.8 ND 62.3 36.7 46.2 53.0 ND 39.0 43.5 47.4 36.2 35.4 44.5 35.1 40.7 39.7 36.1 50.0 40.5 50–52 ND 36.4 44.7 53.2 41.6 38.0 37.6 34.1 48–50 36.8 38.4 45.6 ND 40.5 40.7 66.3 37.1 51.5 43.1 ND 34.3 53–54 27009 27647 6344 446 485 29 3034 11725 10436 9371 4553 6352 NRS1607 B14309 B383 NRS290 9388 10340 9364 9372 9373 9374 9365 11326 9366 11434 11433 10333 2611 2599 2610 10334 9367 6357 11202 8773 9375 9368 9934 4824 10341 6355 10419 9376 10342 8775 8841 8158 8162 4822 10383 9369 10337 9370 8923 3610 10338 10339 3610 14578 29788 14574 8246 14579 4513 7050 29604 14575 23301 23299 9545 64 14707 10840 14580 8244 23464 29841 14581 6462 14576 11859 842 14706 23304 7061 43741 14577 12980 6051 43742 10792 1046 123 30 31 11 1 91 12 4 5 25 3035 2049 9 13 24 2 1297 32 2048 3036 26 33 36 2047 3 27 2000 28 22 10 2542 2046 3037 9134 10335 B14310 NRS663 NRS604 B3711 B380 NRS609 NRS613 NRS1533 B2605 NRS314 NRS666 B2522 B396 NRS1264 B172 B14306 B14321 B14308 NRS273 NRS924 NRS1321 NRS673 NRS1105 B2309 NRS1530 NRS272 B1172 NRS744 B14516 NRS996 NRS1000 a Tm, GC content by thermal melting. BD, GC content by buoyant density. c ND, not determined. d ATCC, American Type Culture Collection; DSM, Deutsche Sammlung von Mikroorganismen; NCIB, National Collection of Industrial Bacteria; NCTC, National Collection of Type Cultures; NRRL, Northern Regional Research Laboratory. b Manual of Systematic Bacteriology (BMSB) 40 recognized species (Claus and Berkeley, 1986), Table 4 lists these with their GC content. There are several validly published new species shown to be genetically and phenotypically distinct from other Bacillus species that have not been described in Bergey’s Manual. These include B. pulvifaciens (Nakamura, 1984); B. alginolyticus and B. chrondrotinus, two alginate-degrading species, (Nakamura, 1987); B. smithii (Nakamura et al., 1988); B. thermoleovorans. an obligately thermophilic hydrocarbon-utilizing organism (Zarilla and Perry, 1987); B. benzoevorans, an aromatic acid and phenol degrader (Pichinoty et al., 1984); and B. gordonae, degrader of hydroxy aromatic compounds (Pichinoty et al., 1986). There are more than 200 species of Bacillus in the category “Species Incertae Sedis” (Claus and Berkeley, 1986). These have been inadequately described or the orginal isolates have been lost. Presumably, these can be revived for listing in Bergey’s Manual after reisolation and more detailed studies. For example, after extensive reconsideration of phenetic and molecular data it has been proposed that B. flexus, B. fusiformis, B. kaustophilus, B. psychrosaccharolyticus, B. 534 R.A. Slepecky and H.E. Hemphill simplex (Priest et al., 1988), and B. thiaminolyticus (Nakumura, 1989) be recognized. The literature contains many important experiments done with Bacillus isolates that have not yet been properly identified to species such as: Bacillus sp. strain SGI, a manganese-oxidizing and reducing organism (Johannes et al., 1986; Rosson and Nealson, 1982); Bacillus sp. strain C59 and Bacillus sp. strain N-6, both alkalophilic organisms with unusual bioenergetic properties (Kitada and Horikoshi, 1987; Kitada et al., 1989); Bacillus sp. strain Gx6638, a novel alkaline and heat stable serine protease-secreting strain (Durham et al., 1987); and Bacillus sp. strain MGA3, a thermophilic methanol-utilizing species, mutants of which are capable of producing large amounts of lysine (Guettler and Hanson, 1988; Schendel et al., 1989). CHAPTER 1.2.16 An extensive list of phenetic characters of most members of the genus have been complied and procedures for the isolation and identification of individual species have been presented (Gordon et al., 1973; Berkeley and Goodfellow, 1981; Norris et al., 1981; Claus and Berkeley, 1986). Summaries of one such rendition are shown in Tables 5 and 6 (Norris et al., 1981). The GC content (32–69 mol%) of the known Bacillus species as well as DNA hybridization experiments have revealed the heterogeneity of the genus (Priest, 1981; Fahmy et al., 1985) (see Table 4). Not only is there variation from species to species but there are differences in GC content within strains of a species identified on other bases. For example, the GC content of the B. megaterium group ranges from 36 to 45% (Hunger and Claus, 1981). It is thus understandable Table 5. Simplified key for the tentative identification of typical strains of Bacillus species. 1. Catalase: positive ..................... 2 negative.................... 17 2. Voges-Proskauer: positive ................ 3 negative ............... 10 3. Growth in anaerobic agar: positive............................ 4 negative ........................... 9 4. Growth at 50°C: positive ............. 5 negative ............ 6 5. Growth in 7% NaCl: positive ............................................. B. licheniformis negative ............................................ B. coagulans 6. Acid and gas from glucose (inorganic N): positive.......... B. polymyxa negative......... 7 7. Reduction of NO3 to NO2: positive............................ 8 negative .......................... B. alvei 8. Parasporal body in sporangium: positive .................. B. thuringiensis negative ................. B. cereus 9. Hydrolysis of starch: positive ...................................... B. subtilis negative..................................... B. pumilus 10. Growth at 65°C: positive ............................................. B. stearothermophilus negative ............................................ 11 11. Hydrolysis of starch: positive ...................................... 12 negative..................................... 15 12. Acid and gas from glucose (inorganic N): positive............... B. macerans negative ............. 13 13. Width of rod 1.0µm or greater: positive ................... B. megaterium negative................... 14 14. pH in V-P broth <6.0: positive .................................... B. circulans negative ................................... B. firmus 15. Growth in anaerobic agar: positive............................ B. laterosporus negative ........................... 16 16. Acid from glucose (inorganic N): positive ................ B. brevis negative ............... B. sphaericus 17. Growth at 65°C: positive ............................................. B. stearothermophilus negative ............................................ 18 18. Decomposition of casein: positive.............................. B. larvae negative............................. 19 19. Parasporal body in sporangium: positive .................. B. popilliae negative ................. B. lentimorbus a Numbers on the right indicate the number (on the left) of the next test to be applied until the right-hand number is replaced by a species name. From Norris et al. (1981). CHAPTER 1.2.16 The Genus Bacillus—Nonmedical 535 − + −++−− + − + − − − + V − + + − − − − − V + − + V V + + + − −− + − + V V − − − − − − − − − − − − − − − − − − − − V V − − − − − V V + + + V V + − + V − V + + − − − − − − + + + + + + + + + + + + + + + + − + + + + + V + − V − + + + + − − V Parasporal bodies Rods 1.0µm wide or wider −+ Hydrolysis of casein − V + Acid from glucose − V pH in V-P medium <6.0 a Growth at 65°C a Starch hydrolyzed +−+ +−+ + NO3 reduced to NO2 + −+−++−++++ + − + + + + −++−++−− − + + − − + + + −− + −−+++−− + + − V + V − +−− + −−−−+− + + − Acid and gas in glucose Growth in 7% NaCl −−−+− + + + + + − + + − − − + − −−+−− − − − −−− Growth at 50°C + + + + + + + + + + + V + + + − − − + Growth in anaerobic agar V-P reaction B. megaterium B. cereus B. thuringiensis B. licheniformis B. subtilis B. pumilus B. firmus B. coagulans B. polymyxa B. macerans B. circulans B. stearothermophilus B. alvei B. laterosporus B. brevis B. larvae B. popilliae B. lentimorbus B. sphaericus Catalase Table 6. Summary of the characters used in the simplified key for Bacillus species. − V + − − − − − − − − − + − − + − + +, Greater than 85% of strains examined by Gordon, Haynes, and Pang (1973) positive; −, greater than 85% of strains negative; V, variable character. a Growth in 2% NaCl agar. that Priest et al. (1981, 1988), who have conducted extensive numerical analysis of many unit characters in addition to the DNA studies, have proposed that the genus Bacillus be split into multiple genera, since the intrageneric heterogeneity is as great as exists in most bacterial families. Priest et al. (1988) assigned 80 organisms of species rank to five or more cluster groups. Their studies reemphasized the heterogeneity of the B. brevis, B circulans, B. coagulans, B. sphaericus, and B. stearothermophilus groups. A variety of techniques have been employed to find either a simple approach to Bacillus taxonomy or a quick and painless identification methodology. Assessment of lipid analyses (reviewed by Minnikin and Goodfellow, 1981) indicated that Bacillus could not be separated into discrete groups. On the other hand, some species could be delineated from others. For example, B. acidocaldarius could be characterized by its menaquinone (nine isoprenoid units, MK-9), cyclohexyl fatty acids, triterpenes, and complex lipids. Using the API System (Analytab Products Incorporated) (a rapid identification system wherein many standardized biochemical assessments can be made on test strips) and some supplementary classical determinants, Logan and Berkeley (1981, 1984) have examined 1,075 Bacillus strains. They were able to show that the API System tests were more reproducible than the classical tests. Pyrolysis gas-liquid chromatography has been applied to the problems of Bacillus taxonomy (O’Donnell and Norris, 1981; O’Donnell et al., 1988). Although there are still some problems with the technique, some promise for its use in classification and identification has been shown. For example, as with DNA-DNA hybridization studies, a separation has been made between B. subtilis and B. amyloliquefaciens. When gasliquid chromatography was applied to examine the subgroups of B. megaterium, data were obtained that confirmed the heterogeneity of the group even though there was some difficulty in resolving relationships within the group. Shute et al. 1984 have used Curie-point pyrolysis mass spectrometry as a taxonomic tool. B. subtilis, B. pumilus, B. licheniformis, and B. amyloliquefaciens could be separated using data obtained from nonsporulating cultures (those grown on nutrient agar); however, such was not the case with cultures sporulating on nutrient agar plus manganese. 536 R.A. Slepecky and H.E. Hemphill Ribosomal RNA Sequencing The most effective approach to Bacillus taxonomy may be analysis of 16S rRNA molecules by oligonucleotide sequencing (Fox et al., 1977; Stackebrandt and Woese, 1979). That technique holds much promise for leading microbial taxonomy into natural phylogenetic relationships. However, traditional taxonomists may be dismayed to find that Bacillus species show kinship with nonsporeforming species. Early studies with this powerful tool showed a close relationship among Bacillus, Planococcus, Sporosarcina, Staphylococcus, and Thermoactinomycetes (Stackebrandt et al., 1987; Stackebrandt and Woese, 1981). In a recent study 16S rRNA cataloging showed that B. subtilis and other ellipsoidalsporeforming species, B. cereus, B. megaterium, and B. pumilus, formed a coherent cluster, while the round-sporeforming species, B. sphaericus, B. globisporus, and “B. aminovorans” did not cluster. Furthermore, the latter group were closer phylogenetically to nonsporeforming organisms as follows: B. sphaericus to Caryophanon latum; B. globisporus to Filibacter limicola; B. pasteuri to Sporosarcina urea and “B. aminovorans” to Planococcus citreus. Cell wall composition agreed except with the last case. B. stearothermophilus fell outside the main Bacillus cluster and showed some relationship to Thermoactinomycete vulgaris (Stackebrandt et al., 1987). In a more recent 16S rRNA sequencing survey, three major Bacillus taxonomic cluster groups were defined (Jurtshuk et al., 1989). This was accomplished by determining complete or partial sequences of 16S RNA on 35 recognized neotype reference strains or type species by the technique of Lane et al. 1985. The partial sequences analyzed typically exceeded 1,100 nucleotides. Phylogenetic analyses were performed using three CHAPTER 1.2.16 different approaches (Sneath and Sokal, 1973; Fitch and Margoliash, 1967; Saitou and Nei, 1987) which showed three major groupings of Bacillus spp., hereinafter referred to as clusters I, II, and III (see Table 7). The 16S rRNA Bacillus cluster groups were quite different from those previously noted by Stackebrandt et al. (1987). This is revealed by direct comparison to the commonly used morphological groupings (see Table 7). Except for morphological group II and the Unassigned Subgroup 2E, all strains sequenced fell into the B. subtilis cluster I grouping. Bacillus strains of morphological group II fell into all three 16S rRNA cluster groups and B. macquariensis, unlike other psychrophiles, fell into the B. alvei cluster II group. Comparative 16S rRNA analyses on thermophilic and psychrophilic Bacillus strains (Wisotzkey et al., 1989) showed that the thermophiles, B. stearothermophilus, B. thermodenitrificans and B. caldotenax formed a subgroup within the B. subtilis cluster but separate from both the “thermotolerant” mesophilic, B. subtilis and B. licheniformis strains, and the moderate thermophile, B. coagulans. The psychrophilic strains, B. psychrophilus and B. insolitus, fell into cluster I while B. macquariensis fell into cluster II. Because several species were included in the current study that had previously been examined by 16S rRNA oligonucleotide cataloging, it is possible to compare the two data sets directly. As a result, it is possible to augment the membership of cluster I to include B. fastidiosus, B. firmus, B. badius and B. pasteurii (C. B. Woese, personal communication). In addition, it is extremely likely that at least two nonsporeforming strains, Planococcus citreus (Stackebrandt and Woese, 1979) and Filibacter limicola (Clausen et al., 1985), as well as Sporosarcina ureae (Pechman et al., 1976), are properly regarded as members of cluster I. Table 7. Bacillus 16S rRNA cluster groups. Morphological group I II III Subgroup A Subgroup B Subgroup C Subgroup D Subgroup E1 Subgroup E2 B. subtilis cluster I B. subtilis, B. cereus, B. licheniformis, B. pumilus, B. megaterium strain Mohb, B. coagulans, B. smithil B. circulans, B. larvae, B. stearothermophilus B. alvci cluster II B. alvei, B. polymyxa, B. macerans, B. azotofixans, B. pulvifaciens B. sphaericus “B. thiaminolyticus,” B. alcalophilus B. lentus B. freundenreichii, “B. aneurinolyticus” B. pantothenticus “B. psychrophilus,” B. insolitus Table provided by Peter Jurtshuk. B. macquariensis B. brevis cluster III B. brevis, B. laterosporus CHAPTER 1.2.16 The Genus Bacillus—Nonmedical Life Cycle; Sporulation and Germination as Models for Differentiation 537 Germination and Outgrowth Introduction The processes of resting cell formation and the change back to the vegetative cell in a variety of prokaryotes (Losick and Shapiro, 1984) present excellent models for studying differentiation, with the added attendant advantages of microbial systems: ease of handling, use of large numbers of cells, fast growth, synchrony, and availability of mutants. The endospore models were recognized early and, therefore, more knowledge has been accumulated using them than with other prokaryotic systems. As the attempt to categorize the many species of Bacillus has had a long history, so have the efforts to unravel the many aspects of the life cycle of Bacillus (for a historical treatment see Keynan and Sandler, 1983). Because of the enormous literature in this area, the present treatment of the life cycle relies mainly on reviews of the subject and covers mainly highlights of germination and sporulation. The cycle of germination, outgrowth, growth, and sporulation (shown schematically in Fig. 2) has been studied from many different aspects with many different species of spore-formers but because of the genetic versatility of B. subtilis, most work has focused on this species. Free spores usually must be activated for germination. Activation is a reversible process which conditions the spore for germination and increases the number of spores undergoing germination as well as the rate of germination. Spores can be activated by a variety of treatments, notably exposure to heat. During activation there is a loss of some coat protein, dipicolinic acid (DPA), and Zn2+ along with an increase in membrane fluidity. Germination, the breaking of the spore’s highly dormant state, follows (recent reviews on germination include Setlow, 1983, and Foster and Johnstone, 1989). A series of degradative reactions is triggered in an unknown manner by simple compounds such as certain amino acids and ribosides or mixtures (no universal germinant has been described) or certain nonnutrient conditions, and can be monitored by the loss of spore refractility as seen in the phase-contrast microscope and by decrease in optical density. No metabolic activity can be detected during the first 2 min of germination of spores that require alaine or glucose for germination. Generation of ATP or production of known metabolic products of these initiators has not been found. Mutants deficient in key glycolytic pathway enzymes can germinate, thus ruling out glycolysis in the case of spores requiring glucose for germination. The same spores can be germinated by nonmetabolizable glucose analogs as well. However, metabolism may play a role in the germination of B. fastidiosus, whose GERMINATION OUTGROWTH ACTIVATION GERMINANTS SWELLING FREE SPORE (REFRACTILE) LYSIS OF LARGE CELL MATURATION (STAGE VI) Fig. 2. Cycle of germination, outgrowth, and sporulation of a typical sporeforming bacterium. Also shown are some biochemical and physical events associated with various stages. (modification of a figure in Slepecky, R.A. (1978)). EMERGENCE GERMINATED SPORE (NON-REFRACTILE) SPORULATION (Stage II - Free Spore) COAT FORMATION (STAGE V) CORTEX FORMATION (STAGE IV) ELONGATION MICROCYCLE SPORULATION ASYMMETRICAL DIVISION (STAGE II) VEGETATIVE CELL SYMMETRICAL DIVISION ENGULFMENT (STAGE III) CONTINUED DIVISION UNDER SOME CONDITIONS Alanine Racemase Heat Resistance Cysteine Incorporation Chemical & UV Resistance Refractility Alanine Dehydrogenase Ribisidase Adenosine Deaminase Dipicolinic acid Alkaline Phosphatase Exoenzymes Glucose Dehydrogenase Antibiotics Aconitase Heat-restant Catalase 538 R.A. Slepecky and H.E. Hemphill spores can only be germinated with uric acid, which is the main carbon source for these unique organisms (Aoki and Slepecky, 1973). One hypothesis suggests that germinants act on receptor proteins, possibly in the inner membrane, which then undergo conformational changes that alter permeability (Foster and Johnstone, 1989). This leads to an autocatalytic loss of heat resistance and to changes that initiate metabolism, leading to vegetative growth. Another view, based on the observation that inhibition of the electron transport system affects germination, postulates that respiration and ATP create a proton motive force, lending to the establishment of a proton gradient. The proton motive force is used for transport of ions from core to cortex to neutralize other ions (Gould, 1983). Upon germination, the spores not only lose their resistance to heat but also resistance to radiation and injurious chemicals; their stainability also increases. Concomitantly with “phase” darkening, the spores swell, break out of their coats, and exude up to 30% of their dry weight; about one-half of the exudate consists of a calcium chelate of the spore-specific substance, DPA, and the remainder consists of peptidoglycan fragments (from the action of cortex lytic enzymes) and amino acids. The earliest measurable events are the loss of calcium, DPA, and heat resistance. This is followed by metabolic events using high-energy compounds produced early in germination from energy reserves stored in the dormant spore. RNA synthesis begins rapidly within 2 min of germination. The dormant spore lacks the ability to produce amino acids and amino acid biosynthesis is absent early in germination. During the first minutes of germination, 20% of the spore’s protein is degraded, providing the source of amino acids for biosynthesis of new protein and small molecules (such as nucleotides) during outgrowth (reviewed by Setlow, 1988). The spore’s enzymes are not degraded. Rather, a group of small acid-soluble proteins (SASP) are the source of the amino acids. These unique proteins, located in the core and sensitive to proteolysis, conprise 8 to 20% of the protein in the spore. Their molecular weight is low (5–11 kDa) and although they are not histones, they bind to the spore DNA. The proteins are degraded by a unique protease that has an absolute specificity for these proteins. They are synthesized late in sporulation. Several of the five known SASP genes (referred to as ssp) have been cloned and in addition to coding proteins supplying amino acids in germination their products may have other roles. One has been shown to be involved in ultraviolet (UV) resistance. CHAPTER 1.2.16 Many germination mutants (abnormal germination phenotypes) have been described (reviewed by Moir et al., 1986; Foster and Johnstone, 1989). The ger (germination) genes are considered a subclass of spo (sporulation) loci, and are made up of several classes: I, structural genes for germination mechanism components; II, regulatory genes for class I; III, post-translation processing and assembly genes; and IV, genes for synthesizing spore structure (e.g., cortex) required for germination. Outgrowth is the period during which the spore gradually becomes a vegetative cell and initiates new macromolecular synthesis. Genes associated with this period are out genes. DNA is replicated relatively late in outgrowth, just before division. The vegetative cell is then capable of undergoing various morphological and biochemical changes which lead either to a series of symmetric cell divisions if sufficient nutrients are present, whereas in stressful times (particularly nutrient limitation), subsequent spore formation or the production of a spore without intermittent cell division can occur. The latter is known as microcycle sporulation (Vinter and Slepecky, 1965). Sporulation Electron microscopical analyses of cells during sporulation has revealed seven stages (reviewed by Fitz-James and Young, 1969). The various stages are shown in Fig. 2. Vegetative cells are considered to be Stage 0. Upon induction input prior to actual sporulation, the nuclear material is in an axially disposed filament. This is stage I. However, since such a pattern does not appear to be unique to sporulating cells, current practice refers to cells in stages prior to stage II as preseptation cells. Segregation of the chromatin material to the poles of the cell occurs concomitantly with the invagination of the plasma membrane in an asymmetrical position on the cell which fuses to complete the spore septum. This is stage II. The mode of formation of this septum is similar to the formation of the transverse septum of symmetric vegetative cell division. Indeed, it has been proposed that sporulation because of this and some other similarities is a modified prokaryotic division (Hitchins and Slepecky, 1969) and models for that view have been presented (Freese and Heinze, 1983). In sporulation, the division of the cell is not equal and subsequent proliferation of the larger cell’s plasma membrane leads to complete engulfment of the “forespore” and liberation of the immature spore, surrounded now by a double unit membrane, into the cytoplasm of the larger cell. This completes stage III. This is a key step since this double membrane now has different trans- CHAPTER 1.2.16 port properties owing to the opposing polarity of the two membranes. During sporulation the vegetative cell is divided into two compartments each having a different fate and each displaying different patterns of gene expression (reviewed by Setlow, 1989). At this time, the cell is “committed” to complete the process of sporulation. The small cell eventually becomes the core of the spore while the large cell, the mother cell, goes on to produce the outer protective layers of the spore and then lyses to release the spore. Cortex material similar to vegetative cell peptidoglycan (but differing in the degree of cross-linking and other aspects) is laid down between the unit membranes, and its deposition corresponds in time to the accumulation in the core of DPA and calcium. Stage IV is now completed. Studies with cortex-less mutants show that the cortex is needed for refractility of the spore (when the spores become refractile, they can be seen in the phase-contrast microscope) and for accumulation of DPA. The cortex plays a fundamental role in the dehydration of the spore (reviewed by Gould, 1983). During Stage V, protein coats are synthesized by the mother cell. There are about 10 major coat proteins in B. subtilis and they are encoded by cot genes, seven of which are known (Losick et al., 1986). The coat proteins are placed around the outside of the forespore. In some species, an additional protein layer called an exosporium is synthesized. Since the coat may play important roles in protection of the spore and its subsequent germination, it has been the subject of many investigations (reviewed by Aronson and Fitz-James, 1976; Losick et al., 1986). Electron microscopy reveals that B. cereus contains an outer coat showing a cross-patched pattern, an inner pitted layer, and a thin layer, the undercoat, while other species show distinct differences. B. subtilis possesses a very thick multilayered coat with an outer striped layer and B. thuringiensis has a coat deficient in the outer cross-patched layer. Differences show up as well within the major structural polypeptides and coat-associated proteases. These differences may be responsible for the variation found in germination and certain resistant properties (other than heat and UV resistance) of various species. During vegetative growth and subsequent sporulation, a variety of proteases are produced (reviewed by Priest, 1977). There are six extracellular proteases and at least three major intracellular proteases—ISP, esterase A, and esterase B. They may be involved with turnover of intracellular proteins, the processing of protein precursors for spore coats, or inactivation of later sporulation enzymes, as well as other functions. As with other aspects of B. subtilis physiology, The Genus Bacillus—Nonmedical CW CP SC SCor SM 539 SP Fig. 3. Cross-section of Bacillus megaterium containing a spore and showing the sporangium (cell), cell protoplast (CP) and wall (CW), spore coat (SC), spore cortex (SCor), spore membrane (SM), and spore protoplast (SP). ×120,000. (Norris et al., 1981.) there are other considerations. Even though the genes apr, npr, epr, and isp, which code for the proteases alkaline (subtilisin), neutral (metallo-) “new” serine, and major intracellular serine, respectively, can be deleted, there still is some protease activity (Sloma et al., 1988). This finding suggests that there are other unindentified proteases. As the spore matures (stage VI), it becomes resistant to heat and to a variety of organic solvents. Final lytic enzymes lyse the sporangial or mother cell liberating the free spore (stage VII). Figure 3 shows a cross section of a mature and heat-resistant spore. If the free spore is placed among the proper nutrients it will germinate, completing the cycle. The sporulation process takes 6 to 8 h at 37°C in B. subtilis. Many other biochemical and physiological events occur during sporulation in addition to those indicated in Fig. 2 and those that can be surmised to be linked with the morphologically identified stages. Some vegetative enzyme activities disappear, some remain, others are modified, and new sporulation specific enzymes are synthesized. The number of sporulationassociated events is uncertain, but the genetic evidence suggests as many as 200 genes in 40 to 50 operons are involved (Losick et al., 1986; Mandelstam and Errington, 1987; Piggot, 1989; Youngman et al., 1989). Currently the genetic map, which includes sporulation and germination genes as well as all-known vegetative cell genes, contains 700 loci, more than 300 of which have been cloned and 180 of those sequenced (reviewed by Piggott, 1989). The ordered appearance of cytological and biochemical changes 540 R.A. Slepecky and H.E. Hemphill implies a sequential reading of the genome. The genetic data are consistent with there being a single linear dependent sequence to stage III with a more complex pattern of gene expression beyond that stage. The genotype of mutants blocked at the various morphological stages in sporulation (as determined by electron microscopy) are designates spoO, spoII, spoIII, etc. For example, a spoII mutant would be arrested in stage II. Within each genotype, the designation A, B, C, etc. identifies different genetic locations; for example, spoIIA, spoIIB identify two separate mutations that cause arrest of developmental stage II. Eight loci have been shown to be concerned with stage 0, the five principal spoO genes being spo0A, B, E, F, and H. These spo0 mutants have pleiotropic effects on the phenotype, suggesting that their gene products regulate the expression of several genes. They do not make polar septa (the asymmetric division) or Stage 0 associated products—proteases, antibiotics and transformation competent cells. There are suppressors of spo0 mutations, i.e., suppressor mutations can restore sporulation in some spo0 mutants to the wild type level. For example, sof-I can suppress defects in spo0F, B, and E (Hoch et al., 1985). Others include arbA and arbB suppressors of spo0A mutations; rvtA, a spo0 repressor; ssa, a reliever of suppression of alcohols; crs, an alleviator of glucose repression of sporulation; and sapA and sapB. suppressors involved with alkaline phosphate induction (Losick et al., 1986). Studies on these extragenic suppressors have given insights into the role of spo0 mutants. Sporulation, normally repressed at high growth rates in the presence of excess nutrients, will ensue at the beginning of stationary phase (arbritrarily defined as t0) in batch culture upon limitation of carbon, nitrogen, or phosphate (reviewed by Smith, 1989, and Sonenshein, 1989). The initiating signal for sporulation is not known. However, sporulation initiation is associated with a decrease in the intracellular GTP pool (reviewed by Freese and Heinze, 1983). Decoynine, a drug that artificially reduces GTP level, induces sporulation even in the presence of excess nutrients. There may be a connection between GTP levels and a proposed sensing mechanism as follows: spo0A, B, and F share significant homology and A and F products show partial homology with proteins such as OmpR, NtrC, and SfrA which are “sensing proteins” involved in the transduction of various environmental signals (Trach et al., 1985; Nixon et al., 1986). Thus, spo0A and spo0F may play a role in sensing starvation and transferring a signal to other genes (see review by Smith, 1989, on the initiation of sporulation). A gene for a protein CHAPTER 1.2.16 kinase that phosphorylates sporulation regulatory proteins Spo0A and Spo0F has been characterized (Pergo et al., 1989). The gene, kinA, previously known as spo11J, has been shown to code for a protein that is homologous to the transmitter class of proteins (Stragier, 1989). Defects in the methylation of a membraneassociated 40-kDa protein in some B. subtilis spo0 mutants suggests that protein methylation also may be a part of the nutrient-sensing system (Golden and Bernlohr, 1989). Multiple forms of RNA polymerase are of considerable importance in sporulation (reviewed by Doi and Wang, 1986; Losick et al., 1986; Losick and Kroos, 1989; Moran, 1989; Setlow, 1989; Stragier, 1989). The holoenzyme is made up of an enzyme core comprised of subunits beta, beta′ and alpha (the products of genes rpoB, rpoC, and rpoA, respectively) and attached sigma factors (the products of genes to be designated), regulatory proteins that determine the promoter specificity. Currently, there are nine known sigma factors, four associated with vegetative cells and five involved in sporulation (see Table 8). Losick and Pero (1981) suggested there is a succession of different sigma factors governing the transcription of the various temporal classes of sporulation genes. The first acting sporulation sigma is ;gs;xH which is required for the initiation of sporulation and it is involved with the transcription of a later gene, spoVG. A key point in sporulation is stage II, which is the result of the asymmetric division event. stage II mutants and the associated gene products or functions are as follows: spoIIAA σE processing); IIAC (σE factor); IIE (pro σE); IIGAσE processing protease); IIGB (p31; σE); IIN (Fts homology) and IIJ (ntrB protein kinase) (Leighton cited in Stragier (1989), and Stragier (1989)). That the spoIIN product has homology with E. coli Fts, a protein involved in symmetrical cell division, may suggest that the level of Fts controls the asymmetric division. In turn, the functions of the other spoII mutations listed above implies a rather complicated mechanism for the synthesis of σE. The sporulation septum (stage II), dependent on spoIIAC (σF) and spoIIE products, is required for spoIIGA processing activity, i.e., conversion of pro-σE by proteolytic cleavage to σE. The important finding by Stragier (1989) of the coupling of gene expression to morphogenesis may be a recurrent theme throughout other aspects of sporulation. After Stage II, compartmentalization of sigma factors occurs. σE is active both in the mother cell and forespore compartment. σG acts only in the forespore compartment (Setlow, 1989) while σK, the product of sig K, a composite gene from IVCB plus IIC (due to a chromosome σ sig D sig E spoIIGB sig F spoIIAC sig G spoIIIG sig H spoOH spoIVCB + spoIIIC σ34 σSpoIIAC — σ50 σ27 σE σF σG σH σK Physiological role Mother cell gene expression Entry into stationary phase Forespore gene expression Asymmetric division (?) Engulfment; provides compartmentalization Flagellar synthesis Unknown Unknown ctc transcription Housekeeping Modified from Helmann and Chamberlain (1988); Moran (1989); and Stragier (1989). sig C σ38 σD σ 32 3 sig B σ37 σB C σ55, σ43 σA Gene sig A (rpoD) Previous designation Sigma type Table 8. Bacillus subtilis sigma factors. t3.5−t5.5 About t0 t3−t5 About t1−t2 t1.5−t3.5 <t0 <t0 <t0 <t0 Time of action cotA, cotD spoVG sspA-E-spoVA Unknown spoIID Flagellar genes Many Many Many Target genes Unknown CAGGA YGHATR Unknown but similar to σG TTNAAA CTAA AAATC AGGNTT TTGACA Cognate consensus −35 Promoter sequence −10 GAATWWT CAHWHTAH CATATT CCGATAT TANTGNTTNTA GGNATTGNT TATAAT CHAPTER 1.2.16 The Genus Bacillus—Nonmedical 541 542 R.A. Slepecky and H.E. Hemphill rearrangement requiring SpoIIID and a recombinase) acts only in the mother cell (Losick and Kroos, 1989). σK directs the transcription of cotA and cotD, spore coat genes. This brief overview of some of the emerging information on regulation of gene expression during sporulation reflects the current view of a network of dependent pathways in which activation of developmental genes depends on the products of other developmental genes (Losick and Kroos, 1989). Surface Structures of Bacillus S-Layers Crystalline surface layers of protein or glycoprotein subunits, called S-layers, are found in members of the genus Bacillus (reviewed by Sleytr and Messner, 1988). S-layers of individual strains of Bacillus have been shown to differ in molecular weight (40–200 kDa), the degree of glycosylation of the subunits, and the geometry of the S-layer lattice. For example, B. stearothermophilus contains one S-layer consisting of two types of glycan chains, one being a unique type of protein-carbohydrate linkage. On the other hand, B. brevis contains two S-layers, termed the outer wall protein (OWP) and the middle wall protein (MWP), external to the peptidoglycan layer. These form a hexagonal array in the cell wall. The nucleotide sequence of the entire MWP-OWP gene operon is known (Tsuboi et al., 1988). The gene encoding an S-layer protein of B. sphaericus has been cloned and sequenced (Bowditch et al., 1989). Not all Bacillus species contain S-layers and some strains within a species may lack such a layer. Furthermore, the type of lattice may vary from species to species and within strains of a species. B. alvei, B. anthracis, and B. brevis show a hexagonal array; B. cereus, B. fastidiosus, “B. macroides,” B. megaterium, B. psychrophilus, and B. schlegelii present a square lattice, while strains of B. stearothermophilus can be obtained that individually have one of the three types (Claus and Berkeley, 1986). As with S-layers of other bacteria, their function in Bacillus is unknown. However, since it has been demonstrated that the S-layer can physically mask the negatively charged peptidoglycan sacculus in B. stearothermophilus and prevent autoagglutination, it has been postulated that the layer may play a key role in bacteria-metal interactions (Sleytr and Messner, 1988). Capsules The capsule (a homopolypeptide of D-glutamic acid) of B. anthracis as a virulence factor has been CHAPTER 1.2.16 studied extensively (see The Genus Bacillus— Medical in this Volume). Other bacilli, such as B. subtilis, B. megaterium, and B. licheniformis, possess capsules containing the homopolypeptide of D- or L-glutamic acid as well (Makino et al., 1989). Some Bacillus species, e.g., B. circulans, B. mycoides, and B. pumilus, produce carbohydrate capsules. For example, B. circulans forms an extracellular polymer consisting of glucose and glucuronic acid (Claus and Berkeley, 1986). In the case of B. megaterium a heteropolysaccharide composed of D-glucose, D-xylose, D-galactose, and L-arabinose has been found in one strain (Cassity and Kolodziej, 1984). Flagella Most Bacillus species possess peritrichous flagella. Although some use has been made of Hantigens in setting up serotyping schemes, they have not been widely adopted (Claus and Berkeley, 1986). Chemotaxis has been studied extensively in B. subtilis (Ordal and Nettleton, 1985). Cell Walls Almost all Bacillus species tested have vegetative cell walls made up of peptidoglycan containing meso-diaminopimelic acid (m-DAP). The exceptions (B. sphaericus and the related species, B. pasteurii and B. globisporus) contain lysine instead (Bartlett and White, 1985). But even those species, as all others, contain m-DAP in the peptidoglycan of their spore cortex. Cell wall turnover in Gram-positive bacteria, particularly Bacillus species which have been useful models, has been reviewed by Doyle et al. (1988). In addition to peptidoglycan in the cell wall, all Bacillus species contain large amounts of an anionic polymer, such as teichoic acid (a glycerol or ribitol-based polymer joined together by phosphodiester linkages to form a flexible linear strand) or teichuronic acid (uronic acid-based polymer) which are bonded to muramic acid residues. The type of this anionic polymer present depends on the levels of phosphate and magnesium in the growth medium. The glycerol teichoic acids vary a great deal between Bacillus species and within species. For example, B. subtilis can contain either glucosyl α or β (1→2) glycerol or glucosyl α (1→6) galactosyl α (1→1 or 3) glycerol), while B. licheniformis contains galactosyl α (1→2) glycerol. However, they are joined to the peptidoglycan through a common linkage disaccharide, acetylmannosaminyl(1→4)Nacetylglucosamine (Kaya et al., 1984). As in other Gram-positive bacteria, lipoteichoic acids are found associated with most of the cell membranes of Bacillus species. These compounds are involved in the synthesis of wall CHAPTER 1.2.16 teichoic acids as regulators of autholytic activity and as scavengers of bivalent ions. Those so tested in Bacillus fall into three groups based on the presence or absence of N-acetylglucosamine branches in the backbone chains of their lipoteichoic acids—hydrophilic poly (glycerolphosphate) chains and hydrophobic gentiobiosyldiacylglycerol anchors (Iwasaki et al., 1989). Group A is made up of strains of B. subtilis, B. licheniformis, and B. pumilus; group B, other B. subtilis strains and B. cereus, and group C, B. polymyxa and B. circulans. Macrofibers Macrofibers are multicellular and multistranded structures, hundreds of micrometers in length, produced by autocatalytic mutants of B. subtilis (Mendelson, 1978). These left- or right-handed helical structures have been used to study cell wall structure and growth. They are thought to reveal cell-surface molecular organization and force interactions in the cell wall not readily elucidated in the wild type, single-celled organism. The establishment and maintenance of macrofiber structure is influenced by both genetic and physiological factors (Briehl and Mendelson, 1987; Surana et al., 1988). Membranes The membranes of Bacillus species have been studied extensively because of their intrinsic interest; as a model of membrane structure in Gram-positive cells; with regard to their role in sporulation and germination; with respect to explanations for thermophily; and other reasons. For example, one explanation for the ability of thermophilic microorganisms such as B. stearothermophilus to grow at high temperatures is that the physical properties of the membrane are changed due to changes in the lipid composition in response to growth temperature (Gould, 1983). There is great diversity in the range and type of lipids in Bacillus membranes (see review by Minnikin and Goodfellow, 1981) and wide variation in the fatty acids are found. The main phospholipids present are phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine; however, others are found as well. The major isoprenoid quinones are menaquinone, and most species contain menaquinones with seven isoprenoid units (MK-7). B. acidocaldarius is the exception and possesses MK-9 (Minniken and Goodfellow, 1981). Two-dimensional polyacrylamide gel electrophoresis (PAGE) has been used to attempt to resolve all B. subtilis membrane polypeptides (see Shohayer and Chapra [1985] for one such The Genus Bacillus—Nonmedical 543 study). Several membrane enzymes have been isolated and characterized, such as the lactate, malate, glycerate-3-phosphate, NADH, and succinate dehydrogenases. Because of interest in synthesis and modification of the peptidoglycan layer, much attention has been paid to penicillin-binding proteins. Six have been found in B. subtilis, but one of the most penicillin-sensitive binding proteins, number 4, has been found to be absent from B. subtilis 168 (Buchanan, 1987). Genetic Studies The discovery of transformation in B. subtilis strain 168 by Spizizen (1958) was largely responsible for focusing attention on the genetics of the genus Bacillus. Strain 168 is thought to be a derivative of the type strain B. subtilis Marburg (see Hemphill and Whiteley, 1975), and is one of a relatively few bacilli in which competence for DNA uptake has been found to occur as a natural part of the life cycle. As a consequence of Spizizen’s discovery and the later isolation of generalized transducing phages, our knowledge of the chromosomal organization of B. subtilis is second only to that of the enteric bacteria. (About one-half as many genetic markers are known in B. subtilis as in Escherichia coli.) Furthermore, the identification of numerous genes affecting sporulation in B. subtilis is providing a means for analyzing this complex developmental program, which is largely unique to the Gram-positives. Transformation The establishment of a competent state in broth culture is most efficiently brought about in a minimal salts medium (Anagnostopoulos and Spizizen, 1961; Bott and Wilson, 1967). As the bacteria enter stationary phase in this medium, a maximum of 20% become competent, with a 1 to 2% transformation frequency for a given marker. The development of competence is associated with a reduction in macromolecular synthesis that is initiated well before cells are transformable. Competent bacilli are relatively metabolically latent, and have a lower buoyant density compared to noncompetent bacteria (Dooley et al., 1971; Hadden and Nester, 1968). Although the period of competence overlaps the time in which sporulation is initiated, the two forms of physiological differentiation are thought not to be connected. Different media are preferred for the two processes and commitment to competence is reversible. Several lines of research suggest that 544 R.A. Slepecky and H.E. Hemphill the establishment of competence is coincidental with, and perhaps induces, a DNA-repair system analogous to the SOS regulon in E. coli (Love et al., 1985). The development of the capacity of B. subtilis to take up DNA is associated with the appearance of several novel cellular proteins. Among these is a 75-kDa protein complex which has been isolated from the membrane of competent cells (Smith et al., 1985). The complex consists of two subunits of 18 kDa (polypeptide A) and 17 kDa (polypeptide B). Mutants lacking subunit A do not bind DNA to the cell surface whereas those deficient in B are defective in DNA entry. The 75-kDa protein complex also has nuclease activity that is probably associated with polypeptide B. A nuclease is expected, because it is known that one strand of transforming DNA is hydrolyzed in the process of entering the cell, resulting in a single-stranded product on the cytoplasmic side of the membrane (DavidoffAbelson and Dubnau, 1973a, 1973b). Competence factors that interact with the membrane have also been reported in B. stearothermophilus (Streips and Welker, 1971). B. subtilis cells show little specificity with regard to DNA uptake and may be transformed with homologous chromosomal DNA, plasmid DNA, or transfected with bacteriophage DNA. There is evidence that strands of transforming DNA enter the cell at sites on the membrane where the chromosomal DNA is attached (teRiele et al., 1984). The penetrating DNA, now reduced to a single-strand form, is then brought in contact with the homologous region of the recipient chromosome. This step is probably mediated by the 45-kDa protein product of the B. subtilis recE gene and results in a complex in which the transforming DNA begins to displace one strand of the chromosomal DNA while hydrogen-bonding to the complementary strand. A continuation of the displacement reaction allows pairing and integration of several thousand bases of transforming DNA into the chromosome, while an equivalent amount of the homologous strand is removed and degraded. Considerable thought has been given to the question of whether transformation is associated with genetic exchange in natural populations of bacilli (see Stewart and Carlson, 1986, for a review). The complexity of the transformation process with its requirement for unique competence factors appearing only at stationary phase suggests that the capacity to take-up exogenous DNA offers some selective advantage in the evolution of these bacteria. The fact that competence occurs only late in the growth cycle probably means the system is not designed to obtain DNA as a nutrient. EphratiElizur 1968 found that B. subtilis cells excrete CHAPTER 1.2.16 high-molecular-weight DNA into liquid culture as they grow. Under natural conditions this could be the source of donor DNA. Graham and Istock (1978) demonstrated that genetic exchange, thought to be mediated by transformation, occurs at high frequency between genetically labeled strains of B. subtilis in soil. Also, transformation frequencies in cultures in which the bacteria are allowed to attach to sand grains are much higher than in the standard liquid culture procedure (Lorenz et al., 1988). Generalized Transduction Bacteriophage capable of mediating generalized transduction have been reported in many species of Bacillus including B. subtilis, B. cereus, B. megaterium, B. thuringiensis, B. anthracis, and B. stearothermophilus. Thus, transduction offers an immediate advantage for genetic analysis over transformation in that it is applicable to more strains of these bacteria. In addition, some phages transduce fragments of DNA much larger than can be transferred via transformation, and this facilitates linking distant markers. PBS1, a bacteriophage that infects B. subtilis 168, can incorporate 5 to 10% of the bacterial chromosome in a single virion particle, and was instrumental in constructing the complete circular chromosomal map of B. subtilis (LepesantKejzlarova et al., 1975). On the other hand, small DNA molecules such as those of some plasmids are not efficiently packaged in large phages, but can be transduced by a variety of small bacteriophages (Canosi et al., 1982). Some generalized transducing phages have relatively broad host ranges and can transfer plasmids between different species of bacilli (Ruhfel et al., 1984). Little is known about the mechanism by which transducing particles are formed. PBS1 appears to package bacterial DNA randomly. There is little evidence for packaging sites as are indicated in Salmonella phage P22 (Jackson et al., 1978; Schmieger, 1982, 1984). A pac site has been located in the genome of the small generalized transducing phage SPP1. However, its relevance, if any, to packaging transducing DNA is not clear (Deichelbohrer et al., 1982, 1985). The process by which transducing DNA becomes incorporated into the recipient bacterial chromosome differs from that discussed earlier for transformation. In transduction, the donor DNA is thought to enter the infected cell as double-stranded DNA which then synapses with the homologous region of the recipient chromosome. Incorporation of the transducing fragment presumably results from a double CHAPTER 1.2.16 crossover between the two DNA molecules. In support of this model, mutants of B. subtilis deficient in recombination functions have a reduced capacity to be transduced, while they can be transformed (Dodson and Hadden, 1980). Other important Bacillus generalized transducing phages include CP15, a phage originally shown to transduce B. cereus (Thorne, 1968) but which can transfer plasmids between B. cereus, B. thuringiensis, and B. anthracis (Ruhfel et al., 1984). Bacteriophage MP13 has been important in mapping the chromosome of B. megaterium (Vary et al., 1982), and TP-13 and TP18 have been similarly used in genetic studies of B. thuringiensis (Barsomian et al., 1984). Specialized Transduction Specialized transduction has been reported in several B. subtilis phages including π105 (Shapiro et al., 1974), SPβ (Zahler et al., 1977), and π3T (Odebralski and Zahler, 1982) and B. amyloliquefaciens phage H2 (Zahler et al., 1987). The most carefully examined of these is SPβ, a temperate bacteriophage that is normally carried as a prophage in B. subtilis 168 (Warner et al., 1977). SPβ can transduce markers proximal to its normal attachment site attB SPβ between the markers ilvA (threonine dehydratase) and kauA (ketoacid uptake) located near the terminus of chromosomal replication. In addition, SPβ prophage will insert at a variety of aberrant positions when it lysogenizes mutants of B. subtilis lacking attB SPβ. Induction of these lysogens gives rise to specialized transducing phages carrying genetic markers near the novel sites of integration (see Zahler, 1982, for a review). The gene order of SPβ prophage is a circular permutation of that present in the phage DNA (Spancake et al., 1984). Specialized transducing particles are thought to originate from errors in excision when the prophage is induced. The crossingover event that leads to reformation of a circular phage DNA during induction may be displaced, resulting in removal of a portion of the bacterial DNA contiguous to the right or left boundaries of the prophage. Encapsidation of such hybrid DNA produces a specialized transducing particle. Two types of transductants are recognized in the SPβ system (Zahler, 1982). The bacterial portion of the transducing phage DNA may undergo recombination with and replace the homologous region of the genome of the recipient. This so-called “replacement transduction” is similar to generalized transduction, except that only a limited number of genetic markers are involved. On the other hand, the infecting phage-bacterial DNA may incorporate as a prophage to produce a bacterium diploid for the The Genus Bacillus—Nonmedical 545 B. subtilis genes associated with the prophage. Such “addition transductions” are most likely to occur if the recipient bacterium is already lysogenic for SPβ. The resulting merozygote may be used in complementation studies if different alleles are present on the prophage and bacterial chromosomes. Conjugative Plasmids Fertility plasmids capable of bringing about their own transfer from one bacterium to another have been described in several species of Bacillus. The capacity to produce the insecticidal delta toxin crystal protein in B. thuringiensis is encoded in large plasmids. Gonzalez et al. (1982) found that three strains of this bacterium transmitted the crystal-protein phenotype to B. thuringiensis variants which had lost the plasmid. Moreover, these plasmids could also be transferred to B. cereus and yielded transcipients that produce crystal protein. Battisti et al. (1985) reported the transfer of plasmids pXO11 and pXO12 from B. thuringiensis to B. anthracis and B. cereus. The transcipients, in turn, became effective donors, and in the case of those inheriting pXO12, also acquired the ability to produce parasporal crystals. Strains of B. anthracis that acquire plasmid pXO12 can subsequently mobilize and transfer nonconjugative plasmids present in the same cell. Using this system, the tetracycline resistance plasmid pBC16, the B. anthracis toxin plasmid pXO1, and the capsule plasmid pXO2 have been transmitted to B. anthracis and B. cereus recipients lacking these plasmids (Green et al., 1989). The small plasmid pBC16 is transferred at high frequency without direct interaction with pXO12; such transfer of a nonconjunctive plasmid is called donation. The large B. anthracis plasmids are apparently transferred by conduction. The latter involves formation of cointegrative molecules in the donor, and resolution of the cointegrates into pXO12 and the respective B. anthracis plasmid in the recipient. Cell-tocell contact is necessary for plasmid transfer and is resistant to DNase, but little is known about the mechanisms or conjugative structures that may be involved. A strain of B. subtilis (natto) has been found which carries a 55-kb self-transmissible plasmid (pLS20), which can be transferred to closely related strains and to restriction-deficient strains of B. subtilis (Koehler and Thorne, 1987). This plasmid also promotes the transfer of the tetracycline-resistance plasmid pBC16 from B. subtilis (natto) to a wide variety of Bacillus species including B. anthracis, B. megaterium, and B. subtilis. This is a much broader range of conjugative transmission than has been observed with the B. thuringiensis plamid. However, none of 546 R.A. Slepecky and H.E. Hemphill the conjugative plasmids have been found to mobilize and transfer chromosomal markers as is observed with the F plasmid of E. coli. In addition to the naturally occurring transmissible plasmids of Bacillus, Christie et al. (1987) have identified a conjugative transposon (Tn925) which transfers from Streptococcus faecalis to B. subtilis. Bacteriophages Bacteriophages that infect Bacillus are common in soil. In addition, many strains of this genus are naturally lysogenic for one or more prophages. The most extensively studied Bacillus phages are those associated with B. subtilis, and these have been reviewed by Hemphill and Whiteley (1975), Rutberg (1982), and Zahler (1988). With some exceptions, Bacillus phages have relatively narrow host ranges, probably at least in part because of restriction systems that make phage grown in one host incompatible with another strain (Ando et al., 1982). With the exception of the phages of B. subtilis, no scheme of classification has been adopted to organize the phages of this genus. Therefore, the bacteriophages described here will be grouped according to life cycle. Temperate Bacteriophages Most strains of bacilli that have been carefully examined have been found to release phage particles. These are of two types: defective phages that can kill but do not productively infect other strains (see “Defective Bacteriophages,” this chapter), and those that grow on and lysogenize new host bacteria. B. subtilis 168 is lysogenic for phage SPβ and also releases defective phage PBSX. As an extreme, B. thuringiensis subsp. aizawai is polylysogenic for five unique temperate phages (Reynolds et al., 1988). Temperate phages are easily obtained from nature. If samples of soil are placed in broth and the mixture heated 10 min at 80°C, most free phage and nonsporeforming bacteria are destroyed. When the culture is allowed to incubate several hours and CHAPTER 1.2.16 subsequently treated with mitomycin C, temperate phages are induced and released into the medium. (Some investigators inoculate the broth with the Bacillus strain of interest to enrich for phage, and then add mitomycin C.) The phages may then be recovered by filtering the solution and plating on an appropriate indicator. Dean et al. 1978 have divided the temperate phages of B. subtilis and closely related species into four groups based upon serology, immunity, and physical characteristics (Table 9); the defective phages may be considered a fifth class. Several group III phages including B. subtilis phages SPβ and π3T and B. amyloliquefaciens phage H2 can mediate specialized transduction. Group I phage π105 also transduces genes close to its prophage attachment site (Shapiro et al., 1974). In addition, π105, SPβ, ρ11, and others have been used as cloning vehicles, mostly for B. subtilis genes (see Zahler, 1988, for a review). Temperate bacteriophages often alter the biochemistry or phenotype of lysogenic bacteria and several examples of such prophage conversion have been observed in B. subtilis. Strains of this bacterium lysogenic for SPβ release a bacteriocin-like substance called betacin (Hemphill et al., 1980) which kills some Bacillus strains that do not harbor the SPβ prophage. Most group III bacteriophages including π3T, ρ11 and Z (but not SPβ) contain the structural gene for thymidylate synthetase, and express this gene continuously in lysogens. Stains of B. subtilis lysogenic for SP02 cannot be productively infected with virulent phage π1 (Rettenmier and Hemphill, 1974), and bacilli lysogenic for SPβ are protected by a similar interference system active against π1m (Rettenmeir et al., 1979). Defective Bacteriophages Many species of Bacillus including B. subtilis, B. amyloliquefaciens, B. pumilis, and B. laterosporus release defective phages whose presence is revealed by their bactericidal activity against other strains or species of this genus (Hemphill and Whiteley, 1975; Steensma et al., 1978; Zahler, 1988.) For example, B. subtilis strain 168 releases a defective phage called Table 9. Major groups of temperate B. subtilis phages. Virion dimensions (nm) Group Example I II III IV V φ105 SPO2 SPβ SP6 PBSX DNA size (kb) 40 40 126 53 13 Modified from Dean et al. (1978) and Zahler (1988). Head 52 50 72 61 45 × × × × × 52 50 82 61 45 10 10 12 12 20 Tail Other members of group × × × × × ρ14 — φ3T, ρ11, Z, SPR — PBSZ 220 180 358 192 200 CHAPTER 1.2.16 PBSX, which kills the cells of B. subtilis strain W23. Electron microscopic examination of the culture supernatant of strain 168 reveals typical phage particles. However, the PBSX virions cannot replicate and produce plaques; rather, they act much like a bacteriocin. B. subtilis strain W23, in turn, releases defective phage PBSZ, which has a bacteriocin activity against strain 168. Despite being defective, these phages act much like other temperature viruses. Although small numbers are typically present in the culture supernatant, lysogens may be induced with UV light or mitomycin C and lyse and release large numbers of particles. (The situation is often confusing, however, because many bacilli including B. subtilis 168 are simultaneously lysogenic for one or more nondefective temperate phages.) Virions of PBSX are unmistakably phage-like in structure. They have a rather small head (45 nm in diameter) and a cylindrical, contractile tail 18 by 200 nm. The genes for structural proteins of PBSX have been mapped to a chromosomal position between the markers metA and metC (Thurm and Garro, 1975). PBSX and presumably other phages of this group are defective for more than one reason. First, the particles do not contain an identifiable phage DNA. Instead, PBSX packages randomly selected 13-kb fragments of bacterial chromosomal DNA (Anderson and Bott, 1985). Second, although PBSX virions attach efficiently to susceptible strains and the tail appears to contract, the DNA in the phage heads is not injected (Okamoto et al., 1968). Lytic Bacteriophages Lytic bacteriophages that infect Bacillus are also common in soil and water. Again, the most detailed studies are of phages that infect B. subtilis or closely related species (see Hemphill and Whiteley, 1975, for a review.) Although many of these viruses are intrinsically interesting, two groups of subtilis phages have been the focus of studies on the regulation of viral transcription (see Geiduschek and Ito, 1982). The first class, which includes SP01 and SP82, is distinguished by the replacement of thymine by hydroxymethyluracil (hmU) in the viral DNA. The second class includes π29 and relatives, which are very small, linear double-stranded DNA phages. SP01 and SP82 have very complex temporal programs of transcription involving at least three phases of gene expression designated early, middle, and late. Early gene expression starts within 1 min of infection, and is thought to use the host RNA polymerase. Middle genes are activated about 4 min after infection, and the expression of late genes (which are turned on asynchro- The Genus Bacillus—Nonmedical 547 nously) is first detected 10 min into the latent period. Initiation of transcription of the middle and late genes involves structural modifications of the B. subtilis RNA polymerase, such that the enzyme can recognize unique middle and late phage promoters that are not used by the unmodified polymerase (Tarkington and Pero, 1979). The product of at least one SP01/SP82 gene is thought to associate with the enzyme to activate middle genes (Hyde et al., 1986), and at least two other phage-encoded polypeptides appear to modify the polymerase to synthesize late mRNAs (Fox, 1976; Tjian and Pero, 1976). Other viral genes associated with late transcription have been identified, but these may be required to initiate DNA replication, which is also involved in transcriptional control. Even as new mRNAs are made, transcription of at least some early and middle genes is repressed at characteristic times in the latent period of SP82 and SP01. Proteins that bind to phage DNA and block RNA synthesis have been isolated from infected cells, but the actual mechanism of repression is not clear. Although not directly related, the study of gene expression in these subtilis phages has influenced the investigation of somewhat analogous changes in RNA polymerase structure associated with sporulation. Transcription of the π29 genome also involves early and late mRNAs, which in this case are transcribed from different strands of the viral DNA. Early RNA is transcribed from the light (L) and late RNA from the heavy (H) strand. In vitro, B. subtilis RNA polymerase synthesizes early mRNA from as many as eight promoters (Mellado et al., 1988). At least a portion of the host RNA polymerase is also used for synthesis of late RNA, which is apparently initiated from a single promoter called A3. The latter has a −35 region that differs somewhat from the consensus −35 region of B. subtilis promoters (Vlcek and Paces, 1986). Transcription from A3 requires protein P4 encoded by π29 gene 4 (Mellado et al., 1988). Pseudotemperate Phages Pseudotemperate bacteriophages are lytic phages that establish a relatively long-term association with bacteria that mimics true lysogeny (see Hemphill and Whiteley, 1975). Following adsorption and penetration, further advancement of the viral development cycle is often delayed; some infected cells continue to grow, divide, and even sporulate. A portion of the daughter cells may even be cured, and about one-half the spores produced from such cultures do not contain viral DNA. Pseudotemperate bacteriophages characteristically produce turbid plaques, and “pseudolysogens” obtained from 548 R.A. Slepecky and H.E. Hemphill CHAPTER 1.2.16 these plaques may be subcultured. The pseudolysogens apparently consist of a balance between infected cells in a delayed latent period, bacteria containing maturing phage, and uninfected bacilli. Included among the pseudotemperate phages are several of the best-known transducing phages of Bacillus such as B. subtilis phages PBS1, SP10, and SP15 and B. thuringiensis phage TP-13. The genomes of these viruses often contain modified DNA. For instance, the chromosome of PBS1 has a complete replacement of thymine by uracil (Takahashi and Marmur, 1963) and SP15 DNA substitutes 5-(4′5′-dihydroxylpentyl)-uracil for thymine (Brandon et al., 1972). Phage PBS1 (also referred to as PBS2, a clear-plaque mutant) and phage PMB12 carry out their entire development cycle in the presence of rifampin, an antibiotic that inhibits RNA synthesis by interacting with the bacterial RNA polymerase (Rima and Takahashi, 1974; Bramucci et al., 1977). In the case of PBS1 (PBS2), the resistance to rifampin is explained in part by the synthesis of a new phage-induced RNA polymerase that transcribes the viral late functions (Clark et al., 1974). Genetic evidence suggests that early RNA synthesis in PBS1 may be resistant to the drug because the virus, perhaps using a virion protein, induces a modification in the bacterial RNA polymerase that converts the latter to a resistant form (Osburne and Sonenshein, 1980). One of the most intriguing phenomena associated with bacteriophage PMB12 is its capacity to convert pseudolysogens of certain sporulation-negative (spo) mutants of B. subtilis to a Spo+ phenotype (Kinney and Bramucci, 1981). Conversion has been observed in several stage O mutants, as well as in B. subtilis variants deficient in sporulation due to changes in bacterial RNA polymerase or 30S ribosomes. At least three PMB12 genes are involved in spore conversion. The products of these genes apparently interact with host-cell pathways at the earliest stages of sporulation. Conversion of spore-negative mutants has also been observed with B. pumilis phage PMB1 (Bramucci et al., 1977) and numerous other phages which infect this species (Keggins et al., 1978). Bacteriophage TP-13 similarly converts oligosporogenic, acrystalliferous mutants of B. thuringiensis to spore-positive, crystalpositive phenotype (Perlak et al., 1979). This phage also mediates generalized transduction of large DNA fragments. Plasmids Plasmids are of widespread occurrence in the genus Bacillus and have been found in most species that have been screened. The vast majority are cryptic plasmids, that is, their presence has not been correlated with any unique property of the bacterial host. Although efforts have been made to analyze the relationship between different plasmids in a few species by comparing DNA-restriction fragment profiles, there is no accepted scheme for systematically classifying plasmids within the bacilli. Further confusing the study of plasmids in Bacillus is the fact that some of those used as vectors for gene cloning, such as pUB110, were actually derived from Staphylococcus aureus (Gryczan et al., 1978). As shown in Table 10, some of the Bacillus plasmids do confer a recognizable phenotype to cells which carry them, such as antibiotic resistance, synthesis of toxins, unique metabolic activities, and transconjugation. Because of the medical and economic importance of their hosts, the plasmids conferring virulence in B. anthracis Table 10. Bacillus plasmids. Bacterium Plasmid B. anthracis pXO1 DNA size (kb) 168 pXO2 pBC7 pBC16 pBL10 pIM13 pLS19 pLS20 85.6 69 4.3 6.8 2.2 5.4 55 B. thuringiensis pXO12 112.5 Bacillus species (thermophilic) pTB19 pTB20 26 4.3 B. cereus B. pumilus B. subtilis B. subtilis (natto) Phenotype associated with plasmid Reference Exotoxin (lethal factor, edema factor, protective antigen) Capsule Bacteriocin Tetracycline resistance Bacteriocin Erythromycin resistance Polyglutamate production Self-transmissible plasmid, which also promotes transfer of other plasmids Production of insecticidal crystal protein, and is a self-transmissible plasmid, which can co-transfer unrelated plasmids Kanomycin and tetracycline resistance Tetracycline resistance Mikesell et al., 1983 Tippetts and Robertson, 1988 Green et al., 1985 Bernhand et al., 1978 Bernhand et al., 1978 Lovett et al., 1976 Mahler and Halvorson, 1980 Hara et al., 1982 Koehler and Thorne, 1987 Green et al., 1989 Imanaka et al., 1981 Imanaka et al., 1981 CHAPTER 1.2.16 and B. thuringiensis have been of increasing interest. Virulence in B. anthracis requires the production of a capsule composed of poly-Dglutamic acid and an exotoxin consisting of three components: protective antigen, lethal factor, and edema factor. The capacity to produce the capsule is associated with plasmid pXO2 (Green et al., 1985), and the constituents of the toxin are encoded in plasmid pXO1 (Mikesell et al., 1983). Strains losing either plasmid are rendered avirulent. With the help of fertility plasmid pXO12 derived from B. thuringiensis, both pXO1 and pXO2 have been transferred to bacilli lacking these plasmids (Green et al., 1989). In the case of pXO1, cured strains of B. anthracis have been restored to toxin production (Thorne, 1985). The association of the toxin with plasmids is of historical interest. The original Pasteur vaccine strains of B. anthracis were produced by subculturing this bacterium at high temperatures which are now known to inhibit replication of pXO1. Indeed, two existing Pasteur strains lack this plasmid (Mikesell and Vodkin, 1985). The genes for delta-exotoxin crystal proteins of B. thuringiensis strains are carried in large plasmids (see The Genus Bacillus—Insect Pathogens in this Volume). These plasmids are somewhat divergent in size and the crystal proteins produced by B. thuringiensis strains differ in serology and toxicity to various insects (Höfte and Whiteley, 1989). Moreover, several of these plasmids, such as pXO12 of B. thuringiensis subsp. thuringiensis 4042, (Reddy et al., 1987; Green et al., 1989) have been shown to mediate conjugal transfer of themselves and other plasmids (see “Genetic Studies” this chapter). The production of the parasporal body containing the crystal protein toxin is coordinated with sporulation; crystal protein gene expression begins about stage II (see Whiteley and Schnepf, 1986, for a review). Changes in transcription during sporulation are associated with modifications of RNA polymerase which enable the enzyme to recognize sporulation-specific promoters (see “Sporulation”, this chapter). It has been suggested the activation of the crystal protein genes is also regulated by polymerase changes. DNA sequence analysis of the 133-kDa crystal protein gene from B. thuringiensis subsp. kurstaki HD-1Dipel revealed two overlapping promoters, one used early in sporulation (BtI) and the other (BtII) activated midway through development (Wong et al., 1983). Comparison of these promoters with those of the carefully studied B. subtilis system show little sequence consensus with promoters that bind vegetative RNA polymerase. Brown and Whiteley (1988) have isolated an RNA polymerase from strain HD-1Dipel that directs transcription in vitro from the BtI promoter and also transcribes crystal protein The Genus Bacillus—Nonmedical 549 genes from two other strains of B. thuringiensis. This polymerase contains a unique 35-kDa sigma subunit that is different from the major sigma subunit present in vegetative cells of B. thuringiensis. In addition, the presence of two regions of hyphenated dyad symmetry near the BtI and BtII promoters suggests that binding of other regulatory proteins in this region could be associated with gene expression. Transcriptional Regulation in Biosynthetic and Catabolic Pathways Bacillus species demonstrate great catabolic and biosynthetic versatility. It is only recently, however, that significant progress has been made in understanding how synthesis of enzymes is regulated. Again, most studies have concentrated on B. subtilis and closely related species. Control of Biosynthetic Gene Expression B. subtilis can be grown in minimal salts media with glucose as carbon source. Thus, this bacterium must have the capacity to synthesize de novo all amino acids, nucleotides, etc. As in the enteric bacteria, the structural genes for enzymes of common biosynthetic pathways are clustered on the B. subtilis chromosome (see Zalkin and Ebbole, 1988, for a review.) Such operon-like organization has been observed for the genes of the tryptophan, arginine, and isoleucine-valineleucine synthesis. In addition, the sequences encoding the enzymes for purine (pur genes) and pyrimidine (pyr genes) occur in multicistron clusters in B. subtilis (Ebbole and Zalkin, 1987; Lerner et al., 1987), which is not the case in E. coli. Gene clustering is associated with coordinate control of gene expression at the level of transcription by the products of the pathways. The trp, arg, and ilv operons are repressed, respectively, by tryptophan, arginine, and leucine. The pur genes are repressed by adenine and guanine nucleotides and pyr by pyrimidines. It does not appear that regulation of the biosynthetic operons is mediated primarily through repressors, although the arg pathway may be an exception (Smith et al., 1986). Rather, the favored means of control appears to be termination/antitermination (attenuation) mechanisms which affect the elongation of nascent mRNA molecules. In several operons studied, the 5′-end of the mRNA contains a leader of 100 to 300 nucleotides which does not encode part of the first structural gene. Within this leader is a region that can fold into two mutually exclusive secondary structures, one of which is a procaryote transcription termination signal. The other con- 550 R.A. Slepecky and H.E. Hemphill figuration is an antiterminator stem-and-loop (hairpin) which allows full-length message to be made. Control then resides in changing the ratio of termination vs. antitermination secondary structure. In enteric bacteria, the leader sequence of amino acid biosynthetic gene clusters includes an open reading frame (ORF) for a short peptide, which is generally enriched in codons for the amino acid synthesized by that pathway. The formation of termination vs. antitermination hairpins is modulated by the rate at which the leader polypeptide is synthesized. In the E. coli trp operon, for example, the peptide is made slowly if intracellular levels of tryptophan are low, and the antitermination structure is generated. The converse is true if tryptophan levels are high. In B. subtilis those leader sequences that have been analyzed do not contain an ORF. Moreover, transcription of pur and pyr gene clusters is also thought to be controlled by termination/antitermination, and it is unlikely these operons could be regulated by a mechanism precisely analogous to the E. coli system. In the B. subtilis trp operon, attenuation is regulated in part by a trans-acting factor thought to be the product of the methyltryptophan resistance locus mtr (Shimotsu et al., 1986). Mutations in this gene result in constitutive expression of the trp genes. It is likely that the mtr product, when activated by tryptophan, binds to the leader region of the nascent mRNA in such a manner as to prevent formation of the antiterminator structure, thus favoring termination. In addition, binding of tryptophan-activated mtr gene product to already completed mRNA molecules is thought to inhibit the initiation of translation, thereby further reducing the production of trp enzymes (Kuroda et al., 1988). Transcriptional regulation of the pur operon of B. subtilis shows some similarities to that of the trp system, but may be even more complicated. Synthesis of inosine monophosphate (IMP) is repressed by both adenine and guanine nucleotides, but apparently by different mechanisms. The latter are thought to promote premature cessation of transcription at a rho-independent termination site in the leader sequence, while adenine nucleotides appear to repress transcription initiation (Zalkin and Ebbole, 1988). It is thought likely that when purine levels are high, a guanine nucleotide activates a regulatory molecule, which in turn binds to the leader region of the nascent mRNA and blocks formation of the antiterminator secondary structure. The products of polycistronic mRNAs are sometimes subunits in a common enzyme complex. In these instances, the polypeptides are required in 1 : 1 stoichiometry. Sequence analysis of the trp and pur operons shows that both are CHAPTER 1.2.16 characterized by many gene overlaps; that is, the 3′-end of one coding sequence overlaps with the 5′-end of a contiguous downstream coding sequence (Henner et al., 1984; Ebbole and Zalkin, 1987). The overlaps are thought to result in “translational coupling” in which synthesis of one polypeptide from a polycistronic mRNA is at least partially dependent on the translation of the contiguous upstream gene. In some instances, this coupled translation is thought to result in 1 : 1 stoichiometric synthesis of the products of the overlapping genes. Control of Catabolic Gene Expression Members of the family Bacillaceae catabolize a wide variety of simple and complex organic compounds, including mono-and disaccharides, and polysaccharides such as starch which are partially digested by extracellular enzymes. As in the enteric bacteria, the structural genes for enzymes involved in degradation of these substrates are often clustered on the Bacillus chromosome. All operons so far studied are transcribed from σxA promoters, and control of gene expression involves repressors and termination/antitermination systems (see Klier and Rapport, 1988, for a review). The pathway involved in gluconate catabolism in B. subtilis is apparently regulated by negative control mediated by a repressor. Two inducible enzymes are required: the transport protein gluconate permease and gluconate kinase which phosphorylates gluconate. The genes for these functions, gntP and gntK, respectively, are part of a four cistron operon gntR-gntK-gntP-gntZ. Mutations that inactivate gntR are constitutive. This gene encodes a 243 amino acid protein thought to inhibit transcription by binding near the promoter located 40 bp upstream from gntR (Fujita and Fujita, 1987). Curiously, there is a five base overlap between the coding sequence of gntR and gntK suggesting there may be a translational coupling between these two proteins (Fujita et al., 1986). The genes required for the growth of B. subtilis on xylose polymers are also organized in a five cistron regulon, in which one of the genes, xylR, codes for a repressor that regulates expression of the entire gene cluster (Hastrup, 1988). Synthesis of the enzymes is induced by the presence of xylose in the medium. A repressor system has also been found to regulate the induction of penicillinase in B. licheniformis (Wittman and Wong, 1988). Perhaps the most extensively studied catabolic system in the bacilli is that involved in sucrose catabolism. B. subtilis produces two β,Dfructofuranosidases, sucrase and levansucrase, after induction by sucrose. The former is an intracellular enzyme while the latter (which also cat- CHAPTER 1.2.16 alyzes the formation of the fructose polymer levan) is secreted (see Klier and Rapoport, 1988). The structural genes for sucrase, sacA, and that for levansucrase, sacB, are widely separated on the B. subtilis chromosome. The sacA gene is tightly linked to sacP, which is thought to be a membrane-associated protein that transports and phosphorylates sucrose. The sacB gene and the sacA-sacP cistrons are linked to regulatory loci, sacR and sacT, respectively. Mutations in either of the regulatory loci results in constitutive synthesis of the corresponding enzyme. The most extensive studies of the mechanisms of control have been made in sacR. The sacR locus contains the sacB promoter. Between the transcription start site and the sacB coding sequence is a 199-bp region that is the target of several regulatory effectors of expression of the levansucrase gene (Aymerich et al., 1986). Part of this control is thought to involve a region of dyad symmetry that could form a transcription termination signal in the nascent mRNA. Deletion of this perspective terminator results in constitutive synthesis of levansucrase, and previously isolated constitutive sacR mutants map to this putative stem-and-loop structure (Shimotsu and Henner, 1986; Steinmetz and Aymerich, 1986). Messenger RNA synthesis is thought to stop at this terminator unless an antiterminator protein, presumably regulated by sucrose, interacts with the nascent RNA to prevent formation or to allow bypass of the loop. One candidate for the antiterminator maps to sacS, a genetic locus that affects both the synthesis of levansucrase and sucrase. The sacS locus consists of two cistrons, sacY and sacX (Aymerich and Steinmetz, 1987; Steinmetz et al., 1988; Zukowski et al., 1988). The sacY gene is thought to encode a sucrose-dependent antiterminator protein whose target probably is the region of secondary structure in sacR, where it presumably acts to prevent formation of the stem-and-loop. Deletion of sacY abolishes levansucrase synthesis. Current data suggest the sacX gene directs the production of a negative regulator of sacY; mutants in this cistron produce levansucrase constitutively. The products of two other regulatory genes, sacU and sacQ, have been implicated in regulation of levansucrase synthesis. The target of the products of these genes also appears to be the sacR region, and they may serve to modulate the levels of gene expression (Shimotsu and Henner, 1986; Zukowski and Miller, 1986). sacQ encodes a 46 amino acid polypeptide which appears to stimulate the transcription of several secreted enzymes. Some mutations in sacQ lead to higher levels of expression of target loci, and genes thought to be similar to sacQ have been cloned from B. licheniformis and B. amylolique- The Genus Bacillus—Nonmedical 551 faciens. It now appears that the sacQ polypeptide is one of many small polypeptides which somehow regulate the transcription of degradative enzymes in the family Bacillaceae. Others may include the products of the sacV, sin, and ptrR genes (see Klier and Rapoport, 1988). The enzymes for the histidase pathway responsible for the degradation of histidine are encoded in a cluster of four genes hutH-hutU-hutI-hutG in B. subtilis. Expression of this hut operon is induced by histidine and is also subject to catabolite repression. Between the σA promoter and the first gene, hutH, is a regulatory locus, hutR. The latter has been cloned and sequenced and found to contain two regions of possible regulatory significance (Oda et al., 1988). One is an ORF (ORF1) for a 151 amino acid protein that acts trans in heterozygotes to regulate the hut operon. A mutant having an amino-acid substitution in this protein has been isolated and found to be uninducible for the histidase pathway enzymes. This suggests that the ORF1 protein is a positive regulator in expression of the hut operon, although the possibility that the mutant is a superrepressor cannot be entirely excluded. The second regulatory region located between ORF1 and the first structural gene hutH (ORF2) is a region of dyad symmetry which could form a rho-independent terminator. It is possible that the product of ORF1, presumably activated by histidine, acts as an antiterminator to allow complete transcription of the hut operon. The picture emerging from these and other studies suggests that regulation of catabolic pathways in B. subtilis involves a combination of repressors and positive controls through antitermination. It should be noted that in the attenuation systems observed in the biosynthetic pathways the leader sequences appear to be able to form two mutually exclusive secondary structures, one of which is the terminator. In the several leader sequences studied in catabolic pathways, only the terminator stem-and-loop is indicated. Thus, the putative antiterminator proteins in catabolic systems probably act by blocking formation of or allowing bypass of the terminator, rather than acting to favor one secondary structure over the other. Catabolite Repression Catabolic pathways in Bacillus are subject to catabolite repression by high levels of glucose and a variety of other rapidly metabolized substrates (Nihashi and Fujita, 1984). This phenomenon is of special interest in bacteria of this genus because sporulation is also repressed by glucose. In the enteric bacteria, catabolite 552 R.A. Slepecky and H.E. Hemphill repression is mediated in part by the levels of cAMP and the interaction of this nucleotide with catabolite activator protein (CAP). However, cAMP apparently is not present in Bacillus, and thus could not be involved in catabolite repression in these organisms. The promoters and associated control regions of many catabolic operons from Bacillus have been cloned and sequenced (Laoide et al., 1989; Melin et al., 1987; Oda et al., 1988). In some instances, these cloned regulatory systems, which for experimental purposes are sometimes fused to an indicator gene such as E. coli lacZ, continue to be subject to glucose suppression of transcription. Among the most thoroughly studied is the complex amyR1-amyE, the regulatory and structural genes for alpha-amylase of B. subtilis, and amyR1-amyL, the corresponding regulatory region and structural gene for alphaamylase of B. licheniformis. Amylase synthesis is actually under two forms of regulation. One is a temporal control manifested by the fact that amylase genes are not fully induced until the onset of stationary phase. Temporal control may not occur in all Bacillus strains (Rothstein et al., 1986). In addition, in both B. subtilis and B. licheniformis FDO2 production of alphaamylase is repressed about 10-fold in glucosecontaining cultures in early stationary phase, even when multiple copies of the complex are present on a plasmid (Laoide et al., 1989). Both forms of regulation are associated with the amyR regions. A plasmid-borne construct of amyL missing the promoter but including all transcribed sequences immediately adjacent and downstream from the promoter has been produced. When this fragment is attached at various distances from heterologous promoters, transcription continues to be subject to catabolite repression. This suggests the target of repression is not in the promoter itself or in regions upstream from it. One possibility is that a regulatory protein involved in mediating catabolite repression binds to a cis-acting site very close to the transcription start site, which in the case of amyL is only 29 to 31 nucleotides from the translation initiation codon (121 nucleotides for amyE). A search for consensus sequences downstream from several promoters of B. subtilis genes subject to catabolite repression has yielded a candidate sequence: 5′-ATTGTNA-3′ (Laoide et al., 1989). In the regulatory loci for amyL and amyE, this sequence is contained within an inverted repeat sequence that overlaps the transcription start site, and in the case of amyL, the translation initiation codon. A point mutation (gra-10) in this region of dyad symmetry relieves catabolite repression of amyE (Nicholson et al., 1987). In addition, Weickert and Chambliss (1989) have reported that deletion of DNA 3′ to CHAPTER 1.2.16 the amyR1 promoter does not impair temporal activation of chloramphenicol acetyltransferase in amyR1-cat86 transcriptional fusions, but abolishes catabolite repression. Mutants of B. subtilis that are resistant to at least some manifestations of catabolite repression have been isolated. Among mutants originally selected for resistance to glucose-mediated repression of sporulation (Takahashi, 1979) is crysA43, which maps to rpoD, the structural gene for σA (Price and Doi, 1985). The alteration in σA in this variant also relieves catabolite repression in an amyRi=lacZ fusion cloned in B. subtilis (Laoide and McConnell, 1989). This may suggest a connection between the mechanism of glucose repression affecting sporulation and catabolite repression of at least those genes activated in stationary phase. In addition, Sun and Takahashi (1984) localized another catabolite-resistant mutation, crysE1, to the rpo operon. This locus encodes the β and β′ subunits of RNA polymerase. Taken together the two types of mutations occurring in subunits of RNA polymerase may suggest this enzyme is directly involved in catabolite repression or interacts with a protein, perhaps bound to the regulatory region, which mediates this phenomenon. Resistance of Spores The resting forms of bacteria are usually more resistant to various environmental stresses than their counterpart vegetative forms. The structure and composition of the resting (and dormant) form of endosporeforming bacteria, however, are quite different from other bacterial-resting forms (see Fig. 3). The core or protoplast containing the heat labile DNA, RNA, ribosomes, enzymes, and other proteins is surrounded by a primitive “germ cell wall.” Moving toward the surface, there is a layer called the cortex which consists of peptidoglycan of a similar nature to that of the vegetative cell wall but with less cross linking in the peptides among other differences (Warth, 1978). A second cell membrane surrounds the cortex. The protoplast and cortex and their membranes are enclosed by layers of protein coat. A loose-fitting exosporium, appendages, and internal protein crystals may be found in some species. Compared with vegetative cells, spores are more resistant to heat by a factor of 105 or more, to UV and ionizing radiation by 100-fold or more, and to desiccation, antibiotics, disinfectants, and other chemicals (reviewed by Roberts and Hitchins, 1969; Russell, 1982; Gould and Dring, 1974; Gould, 1983). Since spore resistance has been recognized for a long time and is important in food processing and sterilization CHAPTER 1.2.16 considerations, it has been extensively studied particularly with the genus Bacillus. Spore resistance to physical and chemical agents other than heat and irradiation indicated above is also noteworthy. Vegetative cells are killed at 88,000 p.s.i. for 14 h hydrostatic pressure while their spores have been shown to require 176,000 p.s.i. for the same time period (Gould and Dring, 1974). Spores are about 10,000 times more resistant to hyperchlorites than are vegetative cells. Two hundred roentgens (r) × 103 of x-rays were required to kill 50% of treated B. megaterium spores, while 50% of treated E. coli cells were killed at 5.6 × 103 r. In every process designed for killing microorganisms, the spores are more resistant than vegetative cells and with few exceptions are the most resistant biological entities known. One exception is the nonsporeformer Deinococcus radiodurans, the most radiationresistant organism yet discovered. Its resistance is presumably due to the possession of efficient repair mechanisms for radiation-induced changes in DNA. The degree of heat resistance has been shown to depend not only on the species but also on the physiological environment in which the spores were formed. B. stearothermophilus spores are more heat resistant than B. subtilis spores which are more resistant than spores of B. megaterium (Roberts and Hitchins, 1969; Khoury et al., 1987). In addition, the spore resistance of each species depends on the temperature at which it was grown. For example, B. subtilis grown and sporulated at 20, 30, and 45°C produced spores having D90 values (the time required to kill 90% of the spores at 90°C) of 37, 78, and 99 min, respectively (Khoury et al., 1987). The spores were more temperature sensitive when formed in ethanol-supplemented media. Since temperature and ethanol are known to perturb the degree of order within membranes, this suggests that alteration of membrane function is an additional factor in the multifactorial nature of heat resistance. Other factors to consider in explaining heat resistance include protection of essential spore macromolecules (Murrell, 1981; Lindsay et al., 1985; Gerhardt and Marquis, 1989); specific effects of calcium dipicolinate (Lindsay and Murrell, 1986); mineralization (Marquis, 1989), and possibly foremost, dehydration (Gerhardt and Marquis, 1989). Current hypotheses on the heat resistance of endospores center on the dehydration of the protoplast (core) and the expandable cortex with its counterions (Gould, 1983). In the heat-resistant form, the spore coat is relatively impermeable to multivalent cations. The cortex (of high water content) contains an expanded electronegative peptidoglycan and mobile counterions exerting high osmotic pressure. The protoplast, of low water The Genus Bacillus—Nonmedical 553 content, is osmotically dehydrated by the surrounding cortex and is, therefore, heat resistant. In the heat-sensitive form, there is a modified coat leaky to multivalent cations. The neutralized cortex, collapsed and free of counterions, exerts low osmotic pressure. The protoplast becomes partly hydrated and, therefore, heat sensitive. The theory of heat resistance, called the osmoregulatory expanded cortex theory, fits all the known facts but has yet to be proven or disproven. Germinated spores lose their heat resistance and yet, under special conditions, can be dehydrated to become both heat resistant and dormant once again (Gould, 1983). It is noteworthy that the heat resistance of some nonsporeformers can be increased in dehydrated cells. The expansion or contraction of the cortex is thought to account for the dehydrated state of the spore protoplast. This reduced water content may also play a role in radiation resistance. It is thought that conformational differences between DNA in spore and vegetative cells may be associated with differences in hydration levels. The greater resistance of spores to UV radiation is also related to repair processes. The photoproduct formed during sporulation, 5-thyminyl-5,6-dehydrothymine (TDHT), is different from the thymine dimers formed in vegetative cells, both types of photoproducts being deemed as the cause of death. Dark repair mechanisms in germinating spores convert TDHT to thymine. The greater spore resistance to UV radiation is attributed to the more efficient removal of the unusual photoproduct (reviewed by Russell, 1982). However, as with the mechanism of spore heat resistance, the picture is complex and many factors are involved. For example, dipicolinic acid has been suggested as a protectant of spore DNA (Lindsay and Murrell, 1986) and it has been shown that the SASP may be involved (Setlow, 1988). Likewise, explanations for ionizing radiation resistance of spores are not yet complete (Russell, 1982). Different spore DNA conformation (possibly due to dehydration), possession of coat layer radioprotectant substances, dipicolinic acid protection, and more efficient repair systems have all been implicated. Spore coat layers, both coat protein and cortex, act as permeability barriers to toxic agents. At the present, this is the accepted explanation for the greater spore chemical resistance and resistance to lytic agents (Russell, 1982). Spores as Biological Indicators Endospores are often used as biological indicators. Known numbers of spores of various Bacil- 554 R.A. Slepecky and H.E. Hemphill lus species of predictable death rate can be placed on various solid substrates (usually strips of filter paper) and placed with the items to be sterilized. The strips are then checked for retention of viable organisms by immersion into culture medium (various strategies are employed) after the sterilization process is completed if growth occurs then survival of the spores has occurred and hence the procedure for processing was inadequate. A D-value (decimal reduction time or time to kill 90% of the population at a particular temperature or treatment) of 3.0 min for B. subtilis spores is required for ethylene oxide sterilization (600 mg ethylene oxide per liter at 50% relative humidity and 54°C). For moist heat (121°C) B. stearothermophilus spores with a D-value of 1.5 min and for dry heat (170°C) spores of B. subtilis with a D-value of 0.8 min are preferred. B. pumilus spores (Dvalue of 0.17 Mrad) are used when ionizing radiation is used for sterilization (Korczynski, 1981). Production of Antibiotics Members of the genus Bacillus are capable of producing antibiotics as secondary metabolites in the late logarithmic or early stationary phase CHAPTER 1.2.16 of growth of batch cultures. As many as 169 of these secondary metabolites have been recorded; for example, various strains of B. subtilis have been shown to produce 68 antibiotics while B. brevis can produce 23 (Katz and Demain, 1977). A partial updated listing modified from that of Katz and Demain (1977) is presented (Table 11) to illustrate that there are other Bacillus antibiotic producers. Most of the antibiotics are active against Gram-positive organisms, although there are exceptions. The majority are peptide antibiotics but some belong to other chemical classes (e.g., butirosin is an aminoglycoside and protocin is a phosphorus-containing triene). Also indicated in Table 11 are those antibiotics whose structural genes have been mapped and the few whose genes have been cloned. The latter types of studies are important in their own right and serve as models for the study of expression of the genes of other antibiotics. A controversy has existed for some time concerning the function of these antibiotics. Since they usually appear upon the onset of sporulation, it has been proposed that they may be important factors in the transition of vegetative cells to spores (reviewed by Katz and Demain, 1977). Support for this hypothesis that Table 11. Some Bacillus antibiotics. Species B. subtilis B. brevis B. licheniformis B. pumilus B. mesentericus B. polymyxa B. thiaminolyticus B. circulans B. laterosporus B. cereus a Anti-Gram-positive bacteria. Inhibitor of fibrin clotting. c Broad spectrum antibiotic. d Anti-fungal antibiotic. e Anti-Gram-negative bacteria. b Antibiotic a Subtilin Surfactinb Bacilysina Difficidinc Oxydifficidinc Bacillomycin Fd Mycobacillind Gramicidin Sa Lincar Gramicidina Tyrocidina Bacitracina Proticin Pumilina Tetain Esperina Polymyxine Colistine Octopytina Baciphelacina Circuline Butirosina Laterospuraminea Laterosporina Biocerina Cerexina Genes mapped Genes cloned + + + − − − − + − + + − Remaining antibiotics have neither been mapped nor cloned. + − − − − − − + − + + − CHAPTER 1.2.16 antibiotic production and sporulation may be regulated by the same or similar control mechanisms is now available. Transcription of the B. subtilis tycA gene (coding for tyrocidin synthesis) is dependent on the products of certain Stage 0 sporulation regulatory genes (Marahiel et al., 1987). Thus it does appear that the two physiological events are partially coupled to regulatory events occurring at the onset of sporulation. In addition, it has recently been demonstrated that gramicidin S functions as an inhibitor of outgrowth after germination (Daher et al., 1985). Protein Secretion Members of the genus Bacillus are able to secrete a wide variety of enzymes into the culture medium (reviewed by Priest, 1977; Mezes and Lampen, 1985). Every Bacillus species which has been checked produces at least one extracellular enzyme. These include many different carbohydrates, several kinds of proteases, penicillinases, nucleases, phosphatases, lipase, phospholipase C, thiaminase, and bacteriolytic enzymes. A vast literature exists on the use of Bacillus enzyme models for studying secretion mechanisms, cellular location, and regulation. For some time, there has been considerable interest in producing large quantities of enzymes for industrial purposes— proteases for detergent supplementation, the brewing industry, various uses in the food industry and in leather manufacturing; and different amylases for brewery use, in bread making, and in the paper industry (see review by Debabov, 1982). Since many prokaryotic and eukaryotic genes can be fused to B. subtilis-derived regulatory regions and signal peptide sequences, such genetically manipulated organisms can be used for expressing and secreting many different heterologous proteins (for molecular cloning in B. subtilis, see Gryczan, 1982; Ganeson et al., 1982; Mezes and Lampen, 1985; Ganeson and Hoch, 1988). Interferon (Palva et al., 1983; Schien et al., 1986); human growth hormone (Honjo et al., 1986); and human interleukin-1 (Motley and Graham, 1988) are three such examples. However, yields of such proteins can be low, because extracellular neutral protease, subtilisin, esterases, and other proteases may degrade the secreted proteins. One strategy to overcome the problem and stabilize proteins has been to use strong glucose-insensitive promoters, protease deficient mutants, and catabolite repression of sporulation (Wong et al., 1986). The widespread interest in protein secretion by B. subtilis has stimulated many studies on the genetics of secretion. There appears to be a general mechanism for regulating the synthesis The Genus Bacillus—Nonmedical 555 of extracellular proteins. The activity of degradative enzymes can be increased by mutations at a number of loci that are unlinked to the structural genes for the affected enzymes. One family of genes, senN, sacU, prtR and hpr, codes for small regulatory proteins. For example, mutations at the sacU and sacQ loci can increase the expression of levan sucrase, alkaline protease, neutral protease, xylanase, betagluconase, alpha-amylase, and intracellular serine protease (Henner et al., 1988a). This type of stimulation of degradative enzymes is thought to be a global regulatory system turned on by a requirement for other carbon or nitrogen sources. The SacU product shares homology with other two-component sensorregulator systems (Ronson et al., 1987; Henner et al., 1988; Kunst et al., 1988). Other Considerations Just as Bacillus species have been important as models for studying differentiation or secretion, they have been employed extensively to study other important biological problems. These include DNA replication (Winston and Sueoka, 1982) and repair (Yasbin, 1985); chemotaxis (Ordal and Nettleton, 1985); genetic transformation (Dubnau, 1982); and translation apparatus (Smith, 1982; Hager and Rabinowitz, 1985). Literature Cited Anagnostopoulos, C. J. Spizizen, 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741– 746. Anderson, L. M. K. F. Bott, 1985. DNA packaging by the Bacillus subtilis defective bacteriophage PBSX. J. Virol. 54:773–780. Ando, T. E. Hayase, S. Ikawa, T. Shibata, 1982. Site-specific restriction endodeoxyribonucleases in Bacilli. 66–70. D. Schlessinger (ed.) Microbiology-1982. American Society of Microbiology. Washington, D.C. Aoki, H. R. A. Slepecky, 1973. Inducement of a heat-shock requirement for germination and production of increased heat resistance in Bacilla fastidiosus spores by manganous ins. J. Bacterial 114:137–143. Aronson, A. I. P. C. Fitz-James, 1976. Structure and morphogenesis of the bacterial spore coat. Bacteriol. Rev. 40:360–402. Aymerich, S. G. Gonzy-Treboul, M. Steinmetz, 1986. 5′Noncoding region sacR is the target of all identified regulation affecting the levansucrase gene in Bacillus subtilis. J. Bacteriol. 166:993–998. Aymerich, S. M. Steinmetz, 1987. Cloning and preliminary characterization of the sacS locus from Bacillus subtilis, which controls the regulation of the exoenzyme levansucrase. Mol. Gen. Genet. 208:114–120. Barsomian, G. D. N. J. Robillard, C. B. Thorne, 1984. Chromosomal mapping of Bacillus thuringiensis by transduction. J. Bacteriol. 157:746–750. 556 R.A. Slepecky and H.E. Hemphill Bartlett, A. T. M. P. J. White, 1985. Species of Bacillus that make a vegetative peptidoglycan containing lysine lack diaminopimelate epimerase but have diaminopimelate dehydrogenase. J. Gen. Microbiol. 131:2145–2152. Battisti, L. B. D. Green, C. B. Thorne, 1985. Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. J. Bacteriol. 162:543–550. Berkeley, R. C. W. M. Goodfellow (ed.), 1981. The Aerobic endosporeforming bacteria: classification and identification. Academic Press. London, Bernhard, K. H. Schrempf, W. Goebel, 1978. Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. J. Bacteriol. 133:897–903. Bott, K. F. G. A. Wilson, 1967. Development of competence in the Bacillus subtilis transformation system. J. Bacteriol. 94:562–570. Bowditch, R. D. P. Baumann, A. A. Yousten, 1989. Cloning and sequencing of the gene encoding a 125-Kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene. J. Bacteriol. 171:4178–4188. Bramucci, M. G. K. Keggins, P. S. Lovett, 1977. Bacteriophage conversion of spore-negative mutants to sporepositive in Bacillus pumilis. J. Virol. 22:194–202. Brandon, C. P. M. Gallop, J. Marmur, H. Hayashi, N. Nakanishi, 1972. Structure of a new pyrimidine from Bacillus subtilis phage SP-15 nucleic acid. Nature New Biol 239:70–71. Briehl, M. N. H. Mendelson, 1987. Helix hand fidelity in Bacillus subtilis macrofibers after spheroplast regeneration. J. Bacteriol. 169:5838–5840. Brown, K. L. H. R. Whiteley, 1988. Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes. Proc. Natl. Acad. Sci. USA. 85:4166–4170. Buchanan, C. E. 1987. Absence of penicillin-binding protein 4 from an apparently normal strain of Bacillus subtilis. J. Bacteriol. 169:5301–5303. Canosi, V. G. Luber, T. A. Trautner, 1982. SPP1-mediated plasmid tranduction. J. Virol. 44:431–436. Cassity, R. R. B. J. Kolodziej, 1984. Isolation partial characterization and utilization of a polysaccharide from Bacillus megaterum ATCC 19213. J. Gen. Microbiol. 130:535–539. Christie, P. J. R. Z. Korman, S. A. Zahler, J. C. Adsit, G. M. Dunny, 1987. Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and B. subtilis. J. Bacteriol. 169:2529–2536. Clark, S. R. Losick, J. Pero, 1974. New RNA polymerase from Bacillus subtilis infected with phage PBS2. Nature 252:21–24. Claus, D. R. C. W. Berkeley, 1986. The genus Bacillus. 1105– 1139. P. H. A. Sneath (ed.) Bergey’s manual of systematic bacteriology, vol. 2. Williams and Wilkins. Baltimore. Clausen, V. J. G. Jones, E. Stackebrandt, 1985. 16S ribosomal RNA analysis of Filibacter limicola indicates a close relationship to the genus Bacillus. J. Gen. Microbiol. 131:2659–2663. Cowan, S. T. K. J. Steel, 1974. Manual for the identification of medical bacteria, 2nd ed. Cambridge University Press. London. Daher, E. E. Rosenberg, A. L. Demain, 1985. Germinationinitiated spores of Bacillus brevis nagano retain their resistance properties. J. Bacteriol. 161:47–50. CHAPTER 1.2.16 Davidoff-Abelson, R. D. Dubnau, 1973a. Conditions affecting the isolation from transformed cells of Bacillus subtilis of high-molecular-weight single-stranded deoxyribonucleic acid of donor origin. J. Bacteriol. 116:146– 153. Davidoff-Abelson, R. D. Dubnau, 1973b. Kinetic analysis of the products of donor deoxyribonucleate in transformed cells of Bacillus subtilis. J. Bacteriol. 116:154–162. Dean, D. H. C. L. Fort, J. A. Hoch, 1978. Characterization of temperate phages of Bacillus subtilis. Curr. Microbiol. 1:213–217. Debabov, V. G. 1982. The industrial use of Bacilli. 331–370. D. A. Dubnau (ed.) The molecular biology of the bacilli. Academic Press. New York, Diechelbohrer, I. J. C. Alonso, G. Luder, T. Trautner, 1985. Plasmid transduction by Bacillus subtilis bacteriophage SPP1: Effects of DNA homology between plasmid and bacteriophage. J. Bacteriol. 162:1238–1243. Diechelbohrer, I. W. Messer, T. A. Trautner, 1982. Genome of Bacillus subtilis bacteriophage SPP1: structure and nucleotide sequence of pac, the origin of DNA packaging. J. Virol. 42:83–90. Dodson, L. A. C. T. Hadden, 1980. Capacity for postreplication repair correlated with transducibility in Rec mutants of Bacillus subtilis. J. Bacteriol. 144:608–615. Doetsch, R. N. 1981. Determinative methods of light microscopy. 21–33. P. Gerhart (ed.) Manual of methods for general microbiology.. American Society for Microbiology. Washington, D.C. Doi, R. H. L. F. Wang, 1986. Multiple procaryotic ribonucleic acid polymerase sigma factors. Microbiol. Rev. 50:227– 243. Dooley, D. C. C. T. Hadden, E. W. Nester, 1971. Macromolecular synthesis in Bacillus subtilis during development of the competent state. J. Bacteriol. 108:668–679. Doyle, R. J. J. Chaloupka, V. Vinter, 1988. Turnover of cell walls in microorganisms. Microbiol. Rev. 52:554–567. Dubnau, D. A. 1982. Genetic transformation in Bacillus subtilis. 148–178. D. A. Dubnau (ed.) The molecular biology of the bacilli, vol. 1. Bacillus subtilis Academic Press. New York. Dubnau, D. A. (ed.) 1982. The molecular biology of the bacilli, vol. 1. Bacillus subtilis Academic Press. New York. Dubnau, D. A. (ed.) 1985. The molecular biology of the bacilli, vol. 2. Academic Press. New York. Durham, D. R. D. B. Stewart, E. J. Stellwag, 1987. Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. Strain GX 6638. J. Bacteriol. 169:2762– 2768. Ebbole, D. J. H. Zalkin, 1987. Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis. J. Biol. Chem. 262:8274–8287. Ephrati-Elizur, E. 1968. Spontaneous transformation in Bacillus subtilis. Genet. Res. 11:83–96. Fahmy, F. J. Flossdorf, D. Claus, 1985. The DNA base composition of the type strains of the genus Bacillus. Syst. and Appl. Microbiol. 6:60–65. Fitch, W. M. E. Margoliash, 1967. Construction of phylogenetic trees: A method based on mutational distances as estimated from cytochrome c sequences is of general applicability. Science 155:279–284. Fitz-James, P. C. E. Young, 1969. Morphology of sporulation. 39–72. G. W. Gould and A. Hurst (ed.) The bacterial spore. Academic Press. New York. CHAPTER 1.2.16 Foster, S. J. K. Johnstone, 1989. The trigger mechanism of bacterial germination. 89–108. I. Smith, R. A. Slepecky, and P. Setlow (ed.) Regulation of procaryotic development, structural and functional analysis of bacterial sporulation and germination.. American Society for Microbiology. Washington, D.C. Fox, G. E. K. R. Pechan, C. R. Woese, 1977. Comparative cataloging of 16s ribosomal ribonucleic acid: molecular approach to prokaryotic systematics. Int. J. Syst. Bacteriol. 27:44–57. Fox, T. D. 1976. Identification of phage SP01 proteins coded by regulatory genes 33 and 34. Nature 262:748–753. Freese, E. J. Heinze, 1983. Metabolic and genetic control of bacterial sporulation. 101–172. A. Hurst and G. W. Gould (ed.) The bacterial spore, vol. 2. Academic Press. New York. Freese, E. J. Heinze, T. Mitani, E. B. Freese, 1978. Limitation of nucleotides induces sporulations. 277–285. G. Chambliss and J. C. Vary (ed.) Spores VII.. American Society for Microbiology. Washington, D.C. Fujita, Y. T. Fujita, 1987. The gluconate operon gnt of Bacillus subtilis encodes its own transcriptional negative regulator. Proc. Natl. Acad. Sci. USA. 84:4524–4528. Fujita, Y. T. Fujita, Y. Miwa, J. Nihashi, Y. Aratani, 1986. Organization and transcription of the gluconate operon, gnt, of Bacillus subtilis. J. Biol. Chem. 261:13744–13753. Ganesan, A. T. S. Chang, J. A. Hoch (ed.), 1982. Molecular cloning and gene regulation in bacilli. Academic Press. New York. Ganesan, A. T. J. A. Hoch (ed.), 1988. Genetics and biotechnology of bacilli, vol. 2. Academic Press, New York.. Geiduschek, E. P. J. Ito, 1982. Regulatory mechanisms in the development of lytic bacteriophages in Bacillus subtilis. 203–245. D. A. Dubnau (ed.) The molecular biology of the bacilli, vol. 1. Academic Press. New York. Gerhardt, P. R. E. Marquis, 1989. Spore thermo-resistance mechanisms. 43–64. I. Smith, R. A. Slepecky, and P. Setlow (ed.) Regulation of procaryotic development structural and functional analysis of bacterial sporelation and generation. Am. Soc. Microbiol. Washington, D.C. Golden, K. J. R. W. Bernlohr, 1989. Defects in the nutrientdependent methylation of a membrane-associate protein in spo mutants of Bacillus subtilis. Mol. and Gen. Genet 220:1–7. Gonzalez, J. M., Jr. B. J. Brown, B. C. Carlton, 1982. Transfer of Bacillus thuringiensis plasmids coding for deltaendotoxin among strains of B. thuringiensis and B. cereus. Proc. Natl. Acad. Sci. USA. 79:6951–6955. Gordon, R. E. 1981. One hundred and seven years of the genus Bacillus. R. C. Berkeley, and M. Goodfellow (ed.) The aerobic endosporeforming bacteria. Academic Press. London, Gordon, R. E. W. C. Haynes, C. H.-N. Pang, 1973. The genus Bacillus. Handbook No. 427. U.S. Department of Agriculture. Washington, D.C. Gould, G. W. 1983. Mechanisms of resistance and dormancy. 173–209. A. Hurst and G. W. Gould (ed.) The bacterial spore, vol. 2. Academic Press. New York. Gould, G. W. G. J. Dring, 1974. Mechanisms of spore heat resistance. Adv. Microbiol. Physiol. 2:137–161. Graham, J. B. C. A. Istock, 1978. Genetic exchange in Bacillus subtilis in soil. Mol. Gen. Genet. 166:287–290. Green, B. D. L. Battisti, T. M. Koehler, C. B. Thorne, B. E. Ivins, 1985. Demonstration of a capsule plasmid in Bacillus anthracis. Infect. Immun. 49:291–297. The Genus Bacillus—Nonmedical 557 Green, B. D. L. Battisti, C. B. Thorne, 1989. Involvement of Tn 4430 in transfer of Bacillus anthracis plasmids mediated by Bacillus thuringiensis plasmid pXO12. J. Bacteriol. 171:104–113. Gryczan, T. J. 1982. Molecular cloning in Bacillus subtilis. 307–330. D. A. Dubnau (ed.) The molecular biology of the bacilli. vol. 1. Academic Press. New York. Gryczan, T. J. S. Contente, D. Dubnau, 1978. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J. Bacteriol. 134:318–329. Guettler, M. R. S. Hanson, 1988. Characterization of a methanol oxidizing member of the genus Bacillus. Abs. Ann. Mtg. Am. Soc. Microbiol I-95:196. Hadden, C. E. W. Nester, 1968. Purification of competent cells in the Bacillus subtilis transformation system. J. Bacteriol. 95:876–885. Hager, P. W. J. C. Rabinowitz, 1985. Translation specificity in Bacillus subtilis. 1–32. D. A. Dubnau (ed.) The molecular biology of the bacilli, vol. 2. Academic Press. New York. Hara, T. A. Aumayr, Y. Fujio, S. Veda, 1982. Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange. Appl. Environ. Microbiol 44:1456–1458. Hastrup, S. 1988. Analysis of the Bacillus subtilis xylose regulon. 79–83. A. T. Ganesan and J. A. Hoch (ed.) Genetics and biotechnology of bacilli, vol. 2. Academic Press. New York. Helmann, J. D. M. J. Chamberlain, 1988. Structure and function of bacterial sigma factors. Ann. Rev. Biochem. 57:839–872. Hemphill, H. E. I. Gage, S. A. Zahler, R. Korman, 1980. Prophage-mediated production of a bacteriocinlike substance by Spβ lysogens of Bacillus subtilis. Can. J. Microbiol. 23:45–51. Hemphill, H. E. H. R. Whiteley, 1975. Bacteriophages of Bacillus subtilis. Bacteriol. Rev. 39:257–315. Henner, D. J. L. Band, H. Shimotsu, 1984. Nucleotide sequence of the Bacillus subtilis tryptophon operon. Gene 34:169–177. Henner, D. J. E. Ferrari, M. Perego, J. A. Hoch, 1988a. Location of the targets of the hpr-97, sacU32(hy) and sacQ 36(Hy) mutations in upstream regions to the subtilis promoter. J. Bacteriol. 170:296–300. Henner, D. J. M. Yang, E. Ferrari, 1988b. Localization of B. subtilis sacU (Hy) mutations to two linked genes with similarities to the conserved procaryotic family of twocomponent signalling systems. J. Bacteriol. 170:5102– 5109. Hitchins, A. D. R. A. Slepecky, 1969. Bacterial sporulation as a modified procaryotic cell division. Nature London, 223:804–807. Hoch, J. A. K. Trach, I. Kawamura, H. Saito, 1985. Identification of the transcriptional suppressor SOF-1 as an alteration in the SpoOA protein. J. Bacteriol. 161:552– 555. Höfte, H. H. R. Whiteley, 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53:242– 255. Honjo, M. A. Akaoka, A. Nakayama, Y. Furutani, 1986. Secretion of human growth hormone in B. subtilis using prepropeptide coding region of B. amyloliquefaciens neutral protease gene. J. Biotechnol. 4:63–71. Hunger, W. D. Claus, 1981. Taxonomic studies on Bacillus megaterium and on agarolytic Bacillus strains. 217–239. 558 R.A. Slepecky and H.E. Hemphill R. C. Berkeley and M. Goodfellow (ed.) The aerobic endosporeforming bacteria. Academic Press. London. Hyde, E. I. M. D. Hilton, H. R. Whiteley, 1986. Interactions of Bacillus subtilis RNA polymerase with subunits determining the specificity of initiation. J. Biol. Chem. 261:16565–16570. Imanaka, T. M. Fujii, S. Aiba, 1981. Isolation and characterization of antibiotic resistance plamids from thermophilic bacilli and construction of deletion plasmids. J. Bacteriol. 146:1091–1097. Iwasaki, H. A. Shimada, K. Yokoyama, E. Iyo, 1989. Structure and glycosylation of lipoteichoic acids in Bacillus strains. J. Bacteriol. 171:424–429. Jackson, E. N. D. A. Jackson, R. J. Deans, 1978. Eco R1 analysis of bacteriophage P22 DNA packaging. J. Mol. Biol. 118:365–388. Johannes, P. M. de V. F. G. Boogerd, E. W. deV-deJong, 1986. Manganese reduction by a marine Bacillus species. J. Bacteriol. 167:30–34. Jurtshuk, R. J. C. Lin, P. Candela, J. D. Wisotzkey, P. Jurtshuk, Jr, G. E. Fox, 1989. 16S Ribosomal RNA sequencing studies on organisms of the Bacillus species. Abst. Ann. Meet. Am. Soc. Microbiol. R-10:281. Katz, E. A. L. Demain, 1977. The peptide antibiotics of Bacillus: chemistry, biogenesis and possible functions. Bacteriol. Rev. 41:449–474. Kaya, S. K. Yokoyama, Y. Araki, E. Ito, 1984. N-acetylmannos-aminyl (14) N-acetylglucosamine, a linkage unit between glycerol teichoic acid and peptidoglycan in cell walls of several Bacillus strains. J. Bacteriol. 158:990– 996. Keggins, K. M. R. K. Nauman, P. S. Lovett, 1978. Sporulation-converting bacteriophages for Bacillus pumilis. J. Virol. 27:819–822. Keynan, A. N. Sandler, 1983. Spore research in historical perspective. 1–48. A. Hurst and G. W. Gould (ed.) The bacterial spore, vol. 2. Academic Press. New York. Khoury, P. H. S. L. Lombardi, R. A. Slepecky, 1987. The perturbation of the heat resistance of bacterial spores by sporulation temperatures and ethanol. Current Microbiol. 15:15–19. Kinney, D. M. M. G. Bramucci, 1981. Analysis of Bacillus subtilis sporulation with spore-converting bacteriophage PMB12. J. Bacteriol. 145:1281–1285. Kitada, M. K. Horikoshi, 1987. Bioenergetic properties of alkalophilic Bacillus sp. strain C-59 on an alkaline medium containing K2CO3. J. Bacteriol 169:5761–5765. Kitada, M. K. Onda, K. Horikoshi, 1989. The sodium/proton antiport system in a newly isolated alkalophilic Bacillus sp. J. Bacteriol. 171:1879–1884. Klier, A. F. G. Rapoport, 1988. Genetics and regulation of carbohydrate catabolism in Bacillus. Ann. Rev. Microbiol. 42:65–95. Koehler, T. M. C. B. Thorne, 1987. Bacillus subtilis (natto) plasmid pLS20 mediates interspecies plasmid transport. J. Bacteriol. 169:5271–5278. Korczynski, M. 1981. Sterilization. 476–486. P. Gerhardt (ed.) Manual of methods for general microbiology.. American Society of Microbiology. Washington, D.C. Kunst, F. M. Debarbouille, T. Msadek, M. Young, C. Mauel, D. Karomata, A. Klier, G. Rapoport, R. Dedonder, 1988. Deduced polypeptides encoded by the Bacillus subtilis sac U locus share homology with two-component sensor-regulator systems. J. Bacteriol. 170:5093–5101. Kuroda, M. I. D. Henner, C. Yanofsky, 1988. cis-Acting sites in the transcript of the Bacillus subtilis trp operon regu- CHAPTER 1.2.16 late expression of the operon. J. Bacteriol. 170:3080– 3088. Lane, D. J. B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, N. R. Pace, 1985. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analysis. Proc. Natl. Acad. Sci. USA. 82:6955–6959. Laoide, B. G. H. Chambliss, D. J. McConnell, 1989. Bacillus licheniformis α-amylase gene, amyL, is subject to promoter-independent calabotite repression in Bacillus subtilis. J. Bacteriol. 171:2435–2442. Laoide, B. M. D. J. McConnell, 1989. cis Sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation and catabolite repression resistance mutations on expression. J. Bacteriol. 171:2443–2450. Lepesant-Kejzlarova, J. J.-A. Lepeseant, J. Walle, A. Billaut, R. Dedonder, 1975. Revision of the linkage map of Bacillus subtilis 168: indications for circularity of the chromosome. J. Bacteriol. 121:823–834. Lerner, C. G. B. T. Stephenson, R. L. Switzer, 1987. Structure of the Bacillus subtilis pyrimidine biosynthetic (Pyr) gene cluster. J. Bacteriol. 169:2202–2206. Lindsay, J. A. W. G. Murrell, 1986. Solution spectroscopy of dipicolinic acid interaction with nucleic acids: role in spore heat resistance. Curr. Microbiol. 13:255–259. Lindsay, J. A. W. G. Murrell, A. D. Warth, 1985. Spore resistance and the basic mechanism of heat-resistance. 162– 186. L. E. Harris and A. J. Skopek (ed.) Sterilization of medical products, Vol. 3. Johnson & Johnson Pty., Ltd. Botany NSW Australia. Logan, N. A. R. C. W. Berkeley, 1981. Classification and identification of members of the genus Bacillus using API tests. 105–140. R. C. W. Berkeley and M. Goodfellow (ed.) The aerobic endosporeforming bacteria: classification and identification.” Academic Press. London, Logan, N. A. R. C. W. Berkeley, 1984. Identification of Bacillus strains using the API system. J. Gen. Microbiol. 130:1871–1882. Lorenz, M. G. B. W. Aardema, W. Wackernagel, 1988. Highly efficient genetic transformation of Bacillus subtilis attached to sand grains. J. Gen. Micro. 134:107–112. Losick, R. L. Kroos, 1989. Dependence pathways for the expression of genes involved in endospore formation in Bacillus subtilis. 223–242. I. Smith, R. A. Slepecky, and P. Setlow (ed.) Regulation of procaryotic development, structural and functional analysis on bacterial sporulation and germination.. American Society for Microbiology. Washington, D.C. Losick, R. J. Pero, 1981. Cascades of sigma factors. Cell 25:582–584. Losick, R. L. Shapiro (ed.), 1984. Microbial development. Cold Spring Harbor Laboratory. NY. Losick, R. P. Youngman, P. J. Piggot, 1986. Genetics of endospore formation in Bacillus subtilis. Annv. Rev. Genet. 20:625–669. Love, P. E. M. V. Lyle, R. E. Yasbin, 1985. DNA-damage inducible (din) loci are transcriptionally activated in competent Bacilus subtilis. Proc. Natl. Acad. Sciences 82:6201–6205. Lovett, P. S. E. J. Duvall, K. M. Keggins, 1976. Bacillus pumilis plasmed pPL10: Properties and insertion into Bacillus subtilis 168 by transformation. J. Bacteriol 127:817–828. Mahler, I. H. O. Halvorson, 1980. Two erythromycin resistance plasmids of diverse origin and their effect on sporulation in Bacillus subtilis. J. Gen. Microbiol. 120:259–263. CHAPTER 1.2.16 Makino, S-II. Uchida, N. Terakado, C. Sasakawa, M. Yoshikawa, 1989. Molecular characterization and protein analysis of the cap region which is essential for encapsulation in Bacillus anthracis. J. Bacteriol. 171:722– 730. Mandelstam, J. J. Errington, 1987. Dependent sequences of gene expression controlling spore formation in Bacillus subtilis. Microbiol. Sciences 4:238–244. Marahiel, M. A. P. Zuber, G. Czekay, R. Losick, 1987. Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis. J. Bacteriol. 169:2215–2222. Marquis, R. E. 1989. Minerals and bacterial spores. 147–161. T. J. Beveridge and R. J. Doyle (ed.) Bacterial interactions with metal ions. J. Wiley and Sons. New York. Melin, L. K. Magnusson, L. Rutberg, 1987. Identification of the promoter of the Bacillus subtilis sdh operon. J. Bacteriol. 169:3232–3236. Mellado, R. P. I. Barthelemy, M. Salas, 1988. Transcription initiation and termination signals of the Bacillus subtilis phage π29 DNA. 215–219. A. T. Ganesan and J. A. Hoch (ed.) Genetics and biotechnology of bacilli, vol. 2. Academic Press. New York. Mendelson, N. H. 1978. Helical Bacillus subtilis macrofibers: morphogenesis of a bacterial multicellular macroorganism. Proc. Natl. Acad. Sci. USA. 75:2478–2482. Mezes, P. S. J. O. Lampen, 1985. Secretion of proteins by Bacilli. 151–185. D. A. Dubnau (ed.) The molecular biology of the bacilli, vol. 2. Academic Press. New York. Mikesell, P. B. E. Ivins, J. D. Ristroph, T. M. Dreier, 1983. Evidence for plasmid-mediated toxin production in Bacillus anthracis. Infect. Immun. 39:371–376. Mikesell, P. M. Vodkin, 1985. Plasmids of Bacillus anthracis. 52–55. L. Leive (ed.) Microbiology-1985.. American Society for Microbiology. Washington, D.C. Minnikin, D. E. M. Goodfellow, 1981. Lipids in the classification of Bacillus and related taxa. 59–103. Berkeley, R. C. and M. Goodfellow (ed.) The aerobic endosporeforming bacteria.. Academic Press. London, Moir, A. I. M. Feavers, A. R. Zuberi, 1986. A spore germination operon in Bacillus subtilis 168. 183–194. A. T. Ganesan and J. A. Hoch (ed.) Bacillus molecular genetics and biotechnology applications.. Academic Press. London, Moran, C. P. 1989. Sigma factors and the regulation of transcription. 167–184. I. Smith, R. A. Slepecky and P. Setlow (ed.) Regulation of prokaryotic development, structural and functional analysis of bacterial sporulation and germination. American Society for Microbiology. Washington, D.C. Motley, S. T. S. Graham, 1988. Expression and secretion of human interleukin-1 in Bacillus subtilis. 371–376. A. T. Ganeson and J. A. Hoch (ed.) Genetics and biotechnology of bacilli, vol. 2. Academic Press. New York. Murrell, W. G. 1981. Biophysical studies on the molecular mechanisms of spore heat resistance and dormancy. 64– 77. H. S. Levinson, A. L. Sonenshein, and D. J. Tupper (ed.) Sporulation and germination.. American Society for Microbiology. Washington, D.C. Nakamura, L. K. 1984. Bacillus pulvifaciens sp. nov., nom. rev. Int. J. Syst. Bacteriol. 34:410–413. Nakamura, L. K. 1987. Bacillus alginolyticus sp. nov. and Bacillus chondritinus sp. nov. Int. J. Syst. Bacteriol. 37:284–286. Nakamura, L. K. 1989. Bacillus thiaminolyticus sp. nov., nom. rev. Abst. Annu. Mtg. Am. Soc. Microbiol. R-11:282. The Genus Bacillus—Nonmedical 559 Nakamura, L. K. I. Blumenstock, D. Claus, 1988. Taxonomic study of Bacillus coagulans Hammer 1915 with a proposal for Bacillus smithii sp. nov. Int. J. Syst. Bacteriol. 38:63–73. Nicholson, W. L. Y.-K. Paris, T. M. Henkin, M. Won, M. J. Weickert, J. A. Gaskell, G. H. Chambliss, 1987. Catabolite repression-resistant mutations of the Bacillus subtilis alpha-amylase promoter affect transcription levels and are in an operator-like sequence. J. Mol. Biol. 198:609– 618. Nihashi, J-I. Y. Fujita, 1984. Catabolite repression of inositol dehydrogenase and gluconate kinase synthesis in Bacillus subtilis. Biochimica et Biophysica Acta 798:88–95. Nixon, B. T. C. W. Ronson, F. M. Ausubel, 1986. Two component regulating systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc. Natl. Acad. Sci. USA. 83:7850–7854. Norris, J. R. R. C. W. Berkeley, N. A. Logan, A. G. O’Donnell, 1981. The genera Bacillus and Sporalactobacillus. 1711– 1742. M. P. Starr, A. Stolp, A. G. Truper, A. Balows, and H. G. Schlegel (ed.) The prokaryotes, vol. 2. SpringerVerlag. Berlin, Oda, M. A. Sogishita, K. Furukawa, 1988. Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon. J. Bacteriol 170:3199–3205. Odebralski, J. M. S. A. Zahler, 1982. Specialized transduction of the kauA and citK genes of Bacillus subtilis by bacteriophage π3T. Abstr. Am. Soc. Microbial. 130. O’Donnell, A. G. H. J. H. Macfie, J. R. Norris, 1988. An assessment of taxononic congruence between DNADNA hybridization and pyrolysis gas-liquid chromatographic classifications. J. Gen. Microbiol. 134:743–749. O’Donnell, A. G. J. R. Norris, 1981. Pyrolysis gas-liquid chromatographic studies in the genus Bacillus. 141–179. R. C. W. Berkeley and M. Goodfellow (ed.) The aerobic endosporeforming bacteria, classification and identification.. Academic Press. New York. Okamoto, K. J. A. Mudd, J. Mangan, W. M. Huang, T. V. Subbaiah, J. Marmur, 1968. Properties of the defective phage of Bacillus subtilis. J. Mol. Biol. 34:413–428. Ordal, G. W. D. O. Nettleton, 1985. Chemotaxis in Bacillus subtilis. 53–73. D. A. Dubnau (ed.) The molecular biology of the bacilli, vol. 2. Academic Press. New York. Osburne, M. A. L. Sonenshein, 1980. Inhibition by Lipiarmycin of bacteriophage growth in Bacillus subtilis. J. Virol. 33:945–953. Palva, I. P. Lehtovaara, L. Kaariainen, M. Sibakov, L. Cantell, C. H. Schein, K. Kashiwagi, C. Weismann, 1983. Secretion of interferon by Bacillus subtilis. Gene 22:229– 235. Pechman, K. J. B. J. Lewis, C. R. Woese, 1976. Phylogenetic status of Sporosarcina ureae. Int. J. Syst. Bacteriol. 261:305–310. Pergo, M. S. P. Cole, D. Burbulys, K. Trach, J. A. Hoch, 1989. Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory rooteins SpoOA and SpoOF of Bacillus subtilis. J. Bacteriol. 171:6187–6196. Perlak, F. J. C. L. Mendelsohn, C. B. Thorne, 1979. Converting bacteriophage for sporulation and crystal-formation in Bacillus thuringiensis. J. Bacteriol. 140:699–706. Pichinoty, F. J. Asselineau, M. Mandel, 1984. Characterisation biochimique de Bacillus benzoevorans sp. nov., une nouvelle espèce filamenteuse, engainée et mesophile, 560 R.A. Slepecky and H.E. Hemphill dégradant divers acides aromatiques et phenols. Ann. Microbiol. 135B:209–217. Pichinoty, F. J. B. Waterbury, M. Mandel, J. Asselineau, 1986. Bacillus gordonae sp. nov., Une nouvelle espèce appartenant au second groupe morphologique, dégradant divers composes aromatiques. Ann. Inst. Pasteur 137A:65–78. Piggot, P. 1989. Revised genetic map of B. subtilis 168. 1–42. I. Smith, R. A. Slepecky, and P. Setlow (ed.) Regulation of procaryotic development, structural and functional analysis of bacterial sporulation and germination. American Society for Microbiology. Washington, D.C. Potvin, B. W. R. J. Kelleher, Jr., H. Gooder, 1975. Pyrimidine biosynthelic pathway of Bacillus subtilis. J. Bacteriol. 123:604–615. Price, C. W. R. H. Doi, 1985. Genetic mapping of rpoD implicates the major sigma factor of Bacillus subtilis RNA polymerase in sporulation intiation. Mol. Gen. Genet. 201:88–95. Priest, F. G. 1977. Extracellular enzyme synthesis in the genus Bacillus. Bacterol. Rev. 41:711–753. Priest, F. G. 1981. DNA homology in the genus Bacillus. 33– 57. R. C. Berkeley and M. Goodfellow (ed.) The aerobic endosporeforming bacteria. Academic Press. London, Priest, F. G. M. Goodfellow, C. Todd, 1981. The genus Bacillus: A numerical analysis. 91–103. R. C. Berkeley and M. Goodfellow (ed.) The aerobic endosporeforming bacteria.. Academic Press. London, Priest, F. G. M. Goodfellow, C. Todd, 1988. A numerical classification of the genus Bacillus. J. Gen. Microbiol. 134:1847–1882. Reddy, A. L. Battisti, C. B. Thorne, 1987. Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies. J. Bacteriol. 169:5263–5270. Rettenmier, C. W. H. E. Hemphill, 1974. Abortive infection of lysogenic Bacillus subtilis 168(SPO2) by bacteriophage π1. J. Virol. 13:870–880. Rettenmeir, C. W. B. Gingell, H. E. Hemphill, 1979. The role of temperate bacteriophage SPβ in prophage-mediated interference in Bacillus subtilis. Can. J. Microbiol. 25:1345–1351. Reynolds, R. B. A. Reddy, C. B. Thorne, 1988. Five unique temperate phages from a polylysogenic stain of Bacillus thuringiensis subsp. aizawai. J. Gen. Microbiol. 134:1577–1585. Rima, B. K. I. Takahashi, 1974. The synthesis of nucleic acids in Bacillus subtilis infected with phage PBSI. Can. J. Biochem. 51:1219–1224. Roberts, T. A. A. D. Hichins, 1969. Resistance of spores. 611– 670. G. W. Gould and A. Hurst (ed.) The bacterial spore. Academic Press. London. Ronson, C. W. B. T. Nixon, F. Ausubel, 1987. Conserved domains in bacterial regulatory proteins that respond to environmental stimuli. Cell 49:579–581. Rosson, A. R. K. H. Nealson, 1982. Manganese binding and oxidation by spores of a marine Bacillus. J. Bacteriol. 151:1027–1034. Rothstein, D. M. P. E. Devlin, R. L. Cate, 1986. Expression of α-amylase in Bacillus licheniformis. J. Bacteriol 168:839–842. Ruhfel, R. E. N. J. Robillard, C. B. Thorne, 1984. Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis. J. Bacteriol. 157:708–711. Russell, A. D. 1982. The bacterial spore. 1–24. A. D. Russell (ed.) The destruction of bacterial spores. Academic Press. London. CHAPTER 1.2.16 Rutberg, L. 1982. Temperate bacteriophages of Bacillus subtilis. 247–268. D. A. Dubnau (ed.) The molecular biology of the bacilli, vol. 1. Academic Press. New York. Saitou, N. M. Nei, 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molec. Biol. and Evol. 4:406–425. Schendel, F. J. C. E. Bremmon, M. G. Flickinger, M. Guettler, R. S. Hanson, 1989. L-Lysine production from methanol at high cell densities of MGA3, a thermophilic Bacillus. Abst. Annu. Mtg. Am. Soc. Microbiol. 316. Schien, C. H. K. Kashiwagi, A. Fujisawa, C. Weissmann, 1986. Secretion of mature IFN-α2 and accumulation of uncleared precursor by Bacillus subtilis transformed with a hybrid α-amylase signal sequence-IFN-α2 gene Bio/Technology. 4:719–725. Schmieger, H. 1982. Packaging signals for phage P22 on the chromosome of Salmonella typhimureim. Mol. Gen. Genet. 187:516–518. Schmieger, H. 1984. Pac sites are indispensible for in vivo packaging of DNA by P22. Mol. Gen. Genet. 195:252– 255. Setlow, P. 1983. Germination and outgrowth. 211–254. A. Hurst, and G. W. Gould (ed.) The bacterial spore, vol. 2. Academic Press. London. Setlow, P. 1988. Small, acid-soluble spore proteins of Bacillus species: Structure, synthesis, genetics, function and degradation. Annv. Rev. Microbiol. 42:319–338. Setlow, P. 1989. Forespore specific genes of Bacillus subtilis: function and regulation of expression. 211–222. I. Smith, R. A. Slepecky, and P. Setlow (ed.) Regulation of procaryotic development, structural and functional analysis of bacterial sporulation and germination. American Society for Microbiology. Washington, D.C. Shapiro, J. M. D. H. Dean, H. O. Halvorson, 1974. Lowfrequency specialized transduction with Bacillus subtilis bacteriophage π105. Virology 62:393–403. Shimotsu, H. D. J. Henner, 1986. Modulation of Bacillus subtilis levansucrase gene expression by sucrose and regulation of the steady state mRNA level by sac V and sac Q genes. J. Bacteriol. 168:380–388. Shimotsu, H. M. I. Kuroda, C. Yanofsky, D. J. Henner, 1986. Novel form of transcription attenuation regulates expression of the Bacillus subtilis tryptophan operon. J. Bacteriol. 166:461–471. Shohayer, M. I. Chapra, 1985. Composition of membranes from whole cells and minicells of Bacillus subtilis. J. Gen. Microbiol. 131:345–354. Shute, L. A. C. S. Gutteridge, J. R. Norris, R. C. W. Berkeley, 1984. Curepoint pyrolysis mass spectrometry applied to characterization and identification of selected Bacillus species. J. Gen. Microbiol. 130:343–355. Slepecky, R. A. 1972. Ecology of bacterial sporeformers. 297– 313. H. G. Halvorson, R. Hanson, and L. L. Campbell (ed.) Spores V. American Society for Microbiology. Washington, D.C. Slepecky, R. A. 1978. Resistant forms. 14/1–14/31. J. R. Norris and M. H. Richmond (ed.) Essays in microbiology. John Wiley & Sons. New York. Slepecky, R. A. E. R. Leadbetter, 1977. The diversity of spore-forming bacteria: some ecological implications. 869–877. A. N. Barker, J. Wolf, D. J. Ellar, G. J. Dring, and G. W. Gould (ed.) Spore Research. 1976. Academic Press. London. Slepecky, R. A. E. R. Leadbetter, 1984. On the prevalence and roles of sporeforming bacteria and their spores in CHAPTER 1.2.16 nature. 79–99. A. Hurst and G. W. Gould (ed.) The bacterial spore, vol. 2. Academic Press. London. Sleytr, U. B. P. Messner, 1988. Crystalline surface layers in procaryotes. J. Bacteriol. 170:2891–2897. Sloma, A. A. Ally, D. Ally, J. Pero, 1988. Gene encoding a minor extracellular protease in Bacillus subtilis. J. Bacteriol. 170:5557–5563. Smith, H. K. Wiersma, G. Venema, S. Bron, 1985. Transformation in Bacillus subtilis: Further characterization of a 75,000-dalton protein complex involved in binding and entry of donor DNA. J. Bacteriol. 164:201–206. Smith, I. 1982. The translational apparatus of Bacillus subtilis. 111–147. D. A. Dubnau (ed.) The molecular biology of the bacilli, vol. 1. Academic Press. New York. Smith, I. 1989. The initiation of sporulation. 185–210. I. Smith, R. A. Slepecky and P. Setlow (ed.) Regulation of procaryotic development, structural and functional analysis of bacterial sporulation and germination. American Society for Microbiology. Washington, D.C. Smith, M. C. M. AMountain, S. Baumberg, 1986. Sequence analysis of the Bacillus subtilis argC promoter region. Gene 49:53–60. Smith, N. R. R. E. Gordon, F. E. Clark, 1946. Aerobic mesophilic sporeforming bacteria. US Dept. Agri. Misc. Publication 559. Washington, D.C. Smith, N. R. R. E. Gordon, F. E. Clark, 1952. Aerobic sporeforming bacteria. US Dept. Agr. Washington, D.C. Spancake, G. A. H. E. Hemphill, P. S. Fink, 1984. Genome organization of SPβc2 bacteriophage carrying the thy P3 gene. J. Bacterial. 157:428–434. Sneath, P. H. A. R. R. Sokal, 1973. Numerical Taxonomy. W. H. Freeman. San Francisco. Sonenshein, A. L. 1989. Metabolic regulation of sporulation and other stationary-phase phenomena. 109–130. I. Smith, R. A. Slepecky, and P Setlow (ed.) Regulation of procaryotic development, structural and functional analysis of bacerial sporulation and germination. American Society Microbiology. Washington, D.C. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. USA. 44:1072–1078. Stackebrandt, E. C. R. Woese, 1979. A phylogenetic dissection of the family Micrococcaceae. Curr. Microbiol. 2:317–322. Stackebrandt, E. C. R. Woese, 1981. The evolution of prokaryotes. 1–31. M. J. Carlile, J. F. Collins, and B. E. B. Moseley (ed.) Molecular and cellular aspects of microbial evolution.. Cambridge University Press. Cambridge. Stackebrandt, E. W. Ludwig, M. Weizenegger, S. Dorn, T. J. McGill, G. E. Fox, C. R. Woese, W. Schubert, K-H. Schleifer, 1987. Comparative 16S RNA oligonucleotide analyses and murein types of round-sporeforming bacilli and nonsporeforming relatives. J. Gen. Microbiol. 133:2523–2529. Steensma, H. Y. L. A. Robertson, J. D. Van Elsas, 1978. The occurrence and taxonomic value of PBSX-like defective phages in the genus Bacillus. Antonie van Leeuwenhock 44:353–366. Steinmetz, M. S. Aymerich, 1986. Analysis genetique de sacR, regulateur en cis de la synthese de la levanesaccharose de Bacillus subtilis. Ann. Microbiol. Paris, 137A:3–14. Steinmetz, M. S. Aymerich, G. Goney-Treboul, D. LeCoq, 1988. Levansucrase induction by sucrose in Bacillus subtilis involves an antiterminator. Homology with the The Genus Bacillus—Nonmedical 561 Escherichia coli bgl operon. 11–15. A. T. Ganesan and J. A. Hock (ed.) Genetics and biotechnology of bacilli, vol. 2. Academic Press. New York. Stewart, G. J. C. A. Carlson, 1986. The biology of natural transformation. Ann. Rev. Microbiol. 40:211–35. Stragier, P. 1989. Temporal and spatial control of gene expression during sporulation: from facts to speculations. 243–254. I. Smith, R. A. Slepecky, and P. Setlow (ed.) Regulation of procaryotic development, structural and functional analysis of bacterial sporulation and germination.. American Society for Microbiology. Washington, D.C. Streips, U. N. N. E. Welker, 1971. Competence-inducing factor of Bacillus stearothermophilus. J. Bacteriol. 106:955– 959. Sun, D. I. Takahashi, 1984. A catabolite-resistant mutation is localized in the rpo operon of Bacillus subtilis. Can. J. Microbiol. 30:423–429. Surana, U. A. J. Wolfe, N. H. Mendelson, 1988. Regulation of Bacillus subtilis macrofibe twist development by Dalanine. J. Bacteriol. 170:2328–2335. Takahashi, I. 1979. Catabolite repression-resistant mutants of Bacillus subtilis. Can. J. Microbiol. 25:1283–1287. Takahashi, I. J. Marmur, 1963. Replacement of thymidylic acid by deoxyuridylic acid in the deoxyribonucleic acid of a transducing phage for Bacillus subtilis. Nature 197:794–795. Tarkington, C. J. Pero, 1979. Distinctive nucleotide sequences of promoters recognized by RNA polymerase containing a phage-coded “σ-like” protein. Proc. Nat. Acad. Sci. USA. 76:5465–5469. teRiele, H. P. J. G. Venema, 1984. Heterospecific transformation in Bacillus subtilis: protein composition of a membrane DNA complex containing a heterologous donor-recipient complex. Mol. Gen. Genetics. 197:478– 485. Thorne, C. B. 1968. Transducing bacteriophage for Bacillus cereus. J. Virol. 2:657–682. Thorne, C. B. 1985. Genetics of Bacillus anthracis. 56– 62.L. Leive, P. F. Bonventure, J. A. Morello, S. Schlesinger, S. D. Silver, and H. C. Wu (ed.) Microbiology-1985. American Society for Microbiology, Washington, D.C. Thurm, P. A. J. Garro, 1975. Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX. J. Virol. 16:184–191. Tippetts, M. T. D. L. Robertson, 1988. Molecular cloning and expression of the Bacillus anthrasis edema factor toxin gene: a calmodulin-dependent adenylcyclase. J. Bacteriol. 170:2263–2266. Tjian, R. J. Pero, 1976. Bacteriophage SPO1 regulatory proteins directing late gene transcription in vitro. Nature 262:753–757. Trach, K. A. J. W. Chapman, P. J. Piggot, J. A. Hoch, 1985. Deduced product of the stage O sporulation gene spoOF shares homology with the SpoOA, OmpR and SfrA proteins. Proc. Natl. Acad. Sci. USA. 82:7260–7264. Tsuboi, A. R. Uchihi, T. Adachi, T. Sasaki, S. Hayakawa, H. Yamagata, N. Tsukagoshi, S. Udaka, 1988. Characterization of the genes for the hexagonally arranged surface layer proteins in protein-producing Bacillus brevis 47: Complete nucleotide sequence of the middle wall protein gene. J. Bacteriol. 170:935–945. Vary, P. S. J. C. Garbe, M. Franzen, E. W. Frampton, 1982. MP13, a generalized transducing bacteriophage for Bacillus megaterium. J. Bacteriol. 149:112–119. 562 R.A. Slepecky and H.E. Hemphill Vinter, V. R. A. Slepecky, 1965. Direct transition of outgrowing bacterial spores to new sporangia without intermediate cell division. J. Bacteriol. 90:803–807. Vlcek, C. V. Paces, 1986. Nucleotide sequence of the late region of Bacillus phage π29 complete the 19285-bp sequence of π29 genome. Comparison with the homologous sequence of phage PZA. Gene 46:215–225. Warner, F. D. G. A. Kitos, M. P. Romano, H. E. Hemphill, 1977. Characterization of SPβ: a temperate bacteriophage from Bacillus subtilis 168M. Can. J. Microbiol. 23:45–51. Warth, A. D. 1978. Molecular structure of the bacterial spore. Adv. Microb. Physiol. 17:1–45. Weickert, M. J. G. H. Chambliss, 1989. Genetic analysis of the promoter region of the Bacillus subtilis α-amylase gene. J. Bacteriol. 171:3656–3666. Whiteley, H. R. H. E. Schnepf, 1986. The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. Ann. Rev. Microbiol. 40:549–576. Winston, S. N. Sueoka, 1982. DNA replication in Bacillus subtilis. 36–71. D. A. Dubnau (ed.) The molecular biology of the bacilli, vol. 1. Academic Press. New York. Wisotzkey, J. D. P. Jurtshuk, Jr., G. E. Fox, 1989. Comparative 16S rRNA analyses on thermophilic and psychrophilic bacillus species. Abst. Ann. Meet. Am. Soc. Microbiol. 281. Wittman, V. H. C. Wong, 1988. Regulation of the penicillinase genes of Bacillus licheniformis: Interaction of the pen repressor with its operators. J. Bacteriol. 170:3206– 3212. Wolf, J. A. N. Barker, 1968. The genus Bacillus: aids to the identification of its species. 93–109. M. Gibbs and D. A. Shapton (ed.) Identification methods for microbiologists. Part B. Academic Press. London, Wong, H. C. H. E. Schnepf, H. R. Whiteley, 1983. Transcriptional and translational start sites for the Bacillus thuringiensis crystal protein gene. J. Biol. Chem. 258:1960– 67. Wong, S. F. Kawamura, R. H. Doi, 1986. Use of Bacillus subtilis subtilisin signal peptide for efficient secretion of TEMB-lactamase during growth. J. Bacteriol. 168:1005– 1009. Yasbin, R. E. 1985. DNA repair in Bacillus subtilis. 33–52. Dubnau (ed.) The molecular biology of the bacilli, vol. 2. Academic Press. New York. CHAPTER 1.2.16 Yasbin, R. E. P. I. Fields, B. J. Andersen, 1980. Properties of Bacillus subtilis 168 derivatives freed of their natural prophages. Gene 121:155–159. Yasbin, R. E. G. A. Wilson, F. E. Young, 1975. Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells. J. Bacteriol. 121:296–304. Youngman, P. H. Poth, B. Green, K. York, G. Olmedo, K. Smith, 1989. Methods for genetic manipulation, cloning and functional analysis of sporulation genes in Bacillus subtilis. 65–88. I. Smith. R. A. Slepecky, and P. Setlow (ed.) Regulation of procaryotic development, structural and functional analysis of bacterial sporulation and germination. American Society for Microbiology. Washington, D.C. Zahler, S. A. 1982. Specialized transduction in Bacillus subtilis. 269–305. D. A. Dubnau (ed.) The molecular biology of the bacilli, vol. 1. Academic Press. New York. Zahler, S. A. 1988. Temperate bacteriophages of Bacillus subtilis. 559–592. R. Calendar (ed.) The bacteriophages, vol. 1. Plenum Press. New York. Zahler, S. A. R. Z. Korman, R. Rosenthal, H. E. Hemphill, 1977. Bacillus subtilis bacteriophage SPβ: localization of the prophage attachment site and specialized transduction. J. Bacteriol 129:556–558. Zahler, S. A. R. Z. Korman, C. Thomas, P. S. Fink, M. P. Weiner, J. M. Odebralski, 1987. H2, a temperate bacteriophage isolated from Bacillus amyloliquefaciens strain H. J. Gen. Microbiol. 133:2937–2944. Zalkin, H. D. J. Ebbole, 1988. Organization and regulation of gene encoding biosynthetic enzymes in Bacillus subtilis. J. Biol. Chem. 263:1595–1598. Zarilla, K. A. J. J. Perry, 1987. Bacillus thermoleovorans, sp.nov., a species of obligately thermophilic hydrocarbon utilizing endosporeforming bacteria. System. App. Microbiol. 9:258–264. Zukowski, M. M. L. Miller, 1986. Hyperproduction of an intracellular heterologous protein in a sac Uh mutant of Bacillus subtilis. Gene 46:247–55. Zukowski, M. L. Miller, P. Cogswell, K. Chen, 1988. Inducible expression system based on sucrose metabolism genes of Bacillus subtilis. 17–22. A. T. Ganesan and J. A. Hoch (ed.) Genetics and biotechology of bacilli, vol. 2. Academic Press. New York.
