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11 Bacillus FERGUS G. PRIEST Edinburgh, Scotland, U.K. 1 Introduction 368 2 Taxonomy 368 2.1 Traditional Approaches 368 2.2 Numerical Phenetics and Chemotaxonomy 369 2.3 Phylogenetic Analyses and Evolution 372 2.4 Identification of Bacillus Species 372 3 Ecology 373 3.1 Distribution and Habitats 374 3.2 Associations with Plants 375 3.3 Associations with Animals 376 3.3.1 Pathogens of Man and Mammals 376 3.3.2 Pathogens of Insects 376 4 Extremophiles 378 4.1 Thermophiles 378 4.2 Psychrophiles 379 4.3 Alkaliphiles 379 4.4 Acidophiles 381 5 Physiology 381 5.1 Relations to Oxygen 381 5.2 Carbon Metabolism 383 5.2.1 Catabolite Repression 386 5.3 Nitrogen Metabolism 387 6 The Endospore 388 6.1 Endospore Structure and Properties 388 6.2 Sporulation 391 6.3 Germination 393 7 Genetics and Molecular Biology 394 7.1 Transformation 395 7.2 Plasmids 395 7.3 Recombinant Gene Products from Bacillus 396 8 Concluding Remarks 396 9 References 397 368 11 Bacillus 1 Introduction Bacteria belonging to the genus Bacillus have a long and distinguished history in the realms of biotechnology. They were probably first used by the Japanese in the preparation of a traditional fermented food from rice straw and soybean, itohiki-natto. This derives from the action of “Bacillus natto” (a derivative of B. subtilis) on steamed soybean and results in a viscous, sticky polymer (primarily polyglutamic acid) that forms long, thin threads when touched. Natto has been prepared in Japan for at least four hundred years, and currently consumption is about 108 kg per annum (UEDA, 1989). Exploitation of Bacillus in the west is more recent. Manufacture of extracellular amylases and proteases for industrial applications began early this century, but significant production and usage was delayed until after the 1950s when the advantages of including the alkaline protease (subtilisin Carlsberg) of Bacillus licheniformis in washing detergents was realized. This was followed by developments in the starch processing industry based on the ␣-amylase from B. licheniformis, particularly the conversion of starch to high-fructose corn syrups as sucrose replacements in foods and beverages (PRIEST, 1989 b). The bacilli include many versatile bacteria and the most effective bacterial control agents for various insect pests. Bacillus thuringiensis was isolated early this century in Japan from diseased silkworms and in Germany from the Mediterranean mealmoth. Its value as a control agent for lepidoptera was realized soon thereafter, but only developed commercially in the last 40 years. It now has an annual market value of US $ 107 million which is forecast to reach $ 300 million by the year 1999 (see FEITELSON et al., 1992). Bacilli are also sources of numerous antibiotics, flavor enhancers such as purine nucleosides, surfactants, and various other products (PRIEST, 1989 b). These biotechnological attributes, together with sporulation and germination, the molecular biology of which provides a model system for differentiation, have led to great interest in these bacteria and resulted in B. subtilis becoming the most advanced genetic system available in any Gram-positive bacterium (SONENSHEIN et al., 1993). But as with Escherichia coli, this concentration on one species has resulted in many others being ignored and the great diversity within the genus Bacillus is seldom appreciated. In this chapter, I shall redress the balance and review current knowledge of the biology of these fascinating bacteria. 2 Taxonomy 2.1 Traditional Approaches Gram-positive, rod-shaped bacteria that differentiate into heat-resistant endospores under aerobic conditions are placed in the genus Bacillus. Of the other endospore-forming bacteria, strict anaerobes are allocated to Clostridium, cocci to Sporosarcina and branching filamentous forms to Thermoactinomyces (PRIEST and GRIGOROVA, 1990). It is therefore quite simple to identify bacilli microscopically, and early this century this led to many new “species” being described. It was much simpler to give a new isolate a new species name than to attempt to identify it! By the 1940s some 150 species had been proposed, many synonymous, and most with poor descriptions. The basis of Bacillus classification and identification was established at that time by TOM GIBSON working in Edinburgh and RUTH GORDON, FRANK CLARK and NATHAN SMITH at the Northern Regional Research Laboratory, Peoria, Illinois. SMITH and his colleagues examined 1134 strains representing over 150 species and allocated them to just 19 species. In a later, comprehensive monograph (GORDON et al., 1973), strain and species descriptions were provided complete with full methodology, and this became the primary reference for isolation and identification of bacilli. GIBSON and GORDON also promoted the concept of morphological groups of bacilli based on the shape of the endospore (oval or spherical) and its position in the mother cell or sporangium. Thus, group I species, including B. subtilis and many other common bacilli, differentiate into oval spores of the same dia- 2 Taxonomy meter as the mother cell; group II species (B. polymyxa, etc.) possess oval spores that distend the mother cell, and group III species (notably B. sphaericus) produce spherical spores. This division of the genus has been very useful for identification purposes because it reduces the taxon into groups of more manageable size. However, it is sometimes difficult to distinguish the different classes, and this can lead to serious misidentification (GORDON, 1981). Interestingly, the morphological groups of bacilli have recently been shown to have some phylogenetic validity (see Sect. 2.3). 2.2 Numerical Phenetics and Chemotaxonomy With the introduction of modern taxonomic techniques such as numerical phenetics, DNA base composition determinations, and DNA reassociation experiments which allow DNA sequence homology between strains to be estimated, it became apparent that the bacilli were more heterogeneous than hitherto suspected. The range of DNA base composition among strains is a good indicator of genetic diversity; indeed it is generally agreed that species in a genus should vary by no more than 10–12 mol% G + C (JOHNSON, 1989). In the case of Bacillus, the range is about 33 to 65 % although strains of most species cluster between 40 and 50 % (PRIEST, 1981). This indicates considerable genetic diversity among species and suggests that the genus should perhaps be split into several, more homogeneous taxa. Bacilli are also physiologically diverse (see Sect. 5), and this can best be appreciated by numerical classification in which strains are examined for numerous physiological, biochemical and morphological characters and grouped together on the basis of character similarities. Clusters of phenotypically similar strains are revealed by this process and these can usually be equated with species (AUSTIN and PRIEST, 1986). Three comprehensive numerical studies of Bacillus strains (LOGAN and BERKELEY, 1981; PRIEST et al., 1988; KAMPFER, 1991) have produced essentially similar results. The bacteria have been recovered in six large groups or aggregates of clusters which in many ways 369 equate with genera. Within these groups are numerous clusters or species (Tab. 1). The following description is based on the numerical classification of PRIEST et al. (1988). Group I includes B. polymyxa as a reference organism and comprises species such as B. alvei, B. circulans and B. macerans which produce oval spores that distend the mother cell. These bacteria are facultative anaerobes that ferment a variety of sugars and have reasonably fastidious growth requirements in the form of vitamins and amino acids. They secrete numerous extracellular carbohydrases such as amylases, -glucanases including cellulases, pectinases and pullulanases. B. subtilis and its relatives, B. amyloliquefaciens, B. licheniformis and B. pumilus, are included in group II. These bacteria differentiate into oval endospores that do not distend the mother cell. Most of these bacteria are regarded as strict aerobes but many, such as B. subtilis, have a limited ability to ferment sugars and will grow readily anaerobically in the presence of glucose and nitrate as a terminal electron acceptor. Some species, such as B. anthracis, B. cereus, B. licheniformis and B. thuringiensis, are true facultative anaerobes. These bacteria secrete numerous extracellular enzymes including many commercially important amylases, -glucanases and proteases (PRIEST, 1977). Group III species are perhaps taxonomically the least satisfactory and are rather physiologically heterogeneous. The group is based on B. brevis which is a strict aerobe that does not produce appreciable acid from sugars and differentiates into an oval endospore that distends the sporangium. Other species in this group might include B. badius and “B. freudenreichii”. Bacilli which differentiate into spherical endospores are allocated to group IV. This is a phylogenetically homogeneous group of species including B. sphaericus, the psychrophiles B. insolitus and B. psychrophilus and some other species. These bacteria are also distinguished from virtually all other bacilli by the replacement of meso-diaminopimelic acid in the peptidoglycan of their cell walls by lysine or ornithine. These bacteria are all strict aerobes and, in the case of B. sphaericus, do not use sugars for growth. Acetate is a pre- 370 11 Bacillus Tab. 1. Allocation of Some Bacillus Speciesa to Groups Based on Phenotypic Similarities Species mol % G+Cb RNA Groupc Group Characteristics Group I. The B. polymyxa group B. alvei B. amylolyticus “B. apiarius” B. azotofixans B. circulans B. glucanolyticus B. larvae B. lautus B. lentimorbus B. macerans B. macquariensis B. pabuli B. polymyxa B. popilliae B. psychrosaccharolyticus B. pulvifaciens B. thiaminolyticus B. validus 46 53 – 52 39 48 38 51 38 52 40 49 44 41 44 44 53 54 3 3 – 3 1 – 3 1 1 3 3 3 3 1 1 3 – – All species are facultative anaerobes and grow strongly in the absence of oxygen. Acid is produced from a variety of sugars. Endospores are ellipsoidal and swell the mother cell. Group lI. The B. subtilis group B. alcalophilus B. amyloliquefaciens B. anthracis B. atrophaeus B. carotarum B. firmus B. flexus B. laterosporus B. lentus B. licheniformis B. megaterium B. mycoides B. niacini B. pantothenticus B. pumilus B. simplex B. subtilis B. thuringiensis 37 43 33 42 – 41 38 40 36 45 37 34 38 37 41 41 43 34 UG 1 1 1 – 1 – 5 1 1 1 1 – 1 1 1 1 1 All species produce acid from a variety of sugars including glucose. Most are able to grow at least weakly in the absence of oxygen, particularly if nitrate is present. Spores are ellipsoidal and do not swell the mother cell. Group IlI. The B. brevis group (B. alginolyticus)d “B. aneurinolyticus” B. azotoformans B. badius B. brevis (B. chondroitinus) “B. freudenreichii” B. gordonae 48 42 39 44 47 47 44 55 – UG 1 1 4 – – 3 Strict aerobes that do not produce acid from sugars; names in parentheses are exceptions. They produce ellipsoidal spores that swell the mother cell. Group IV. The B. sphaericus group (“B. aminovorans”) B. fusiformis 40 36 – 2 All species produce spherical spores which may 2 Taxonomy 371 Tab. 1. Continued Species mol % G+Cb RNA Groupc Group Characteristics B. globisporus B. insolitus B. marinus B. pasteurii (B. psychrophilus) B. sphaericuse 40 36 39 38 42 37 2 2 – 2 2 2 swell the mother cell. They contain L-lysine or ornithine in their cell wall. All species are strictly aerobic but some have limited ability to produce acid from sugars. Group V. The thermophiles B. coagulans “B. flavothermus” B. kaustophilus B. pallidus B. schlegelii B. smithii B. stearothermophilus B. thermocatenulatus B. thermocloacae B. thermodenitrificans B. thermoglucosidasius B. thermooleovorans B. thermoruber B. tusciae 44 61 53 40 64 39 52 69 42 52 45 55 57 58 1 – 5 – – 1 5 – – – 5 – – – All these bacteria grow optimally at 50 °C or above. Physiologically and morphologically they are heterogeneous but most produce oval spores that swell the mother cell. Group Vl. Alicyclobacillus A. acidocaldarius A. acidoterrestris A. cycloheptanicus 60 52 56 6 6 6 Thermophilic, acidophilic species with membraneous -alicyclic fatty acids. Unassigned species B. benzoevorans B. fastidiosus B. naganoensis 41 35 45 1 1 – a Names in quotation marks refer to taxa which do not appear in the Approved Lists of Bacterial Names or its supplements and therefore have not been validly published. b Base composition is given either as the figure for the type strain or as the mean of a range for several strains. c RNA groups are based on the work of ASH et al. (1991) and WISOTZKEY et al. (1992). UG, ungrouped; –, no data available. d Names in parentheses refer to species that are atypical of the general description. e B. sphaericus includes at least five “species” of round-spored strains. Table reproduced from PRIEST (1993) with permission. ferred carbon and energy source, although amino acids such as arginine, glutamate and histidine can also be metabolized. Finally, most numerical classification studies have recovered the thermophilic bacilli as a separate group (group V, Tab. 1). This includes a physiologically and morphologically heterogeneous collection of species with various forms of energy metabolism ranging from strict aerobes to microaerophilic types. Indeed some species such as B. schlegelii are chemolithoautotrophs which can grow with carbon dioxide or carbon monoxide as sole carbon source. The thermophilic bacilli are phylogenetically diverse (ASH et al., 1991), and acidophilic thermophiles have recently been allocated to a new genus Alicyclobacillus (WISOTZKEY et al., 1992) (group VI ). It appears that thermophily has evolved independently in many lineages. 372 11 Bacillus Numerical classification has also helped clarify relationships between bacilli at the species level, although in most cases this is better done by DNA reassociation studies. It is reassuring that, in general, numerical classification and DNA homology have given concordant results. In many areas, for example B. circulans, B. megaterium, B. sphaericus, B. stearothermophilus and B. subtilis, examination of strains by these techniques has revealed that GORDON et al. (1973) “lumped” strains into species rather too enthusiastically and that each of these species probably represents several taxa. B. subtilis sensu lato, for example, is now known to include B. amyloliquefaciens and B. atropheus as well as B. subtilis itself and B. circulans sensu lato encompasses numerous species including B. alginolyticus, B. amylolyticus, B. chondroitinus, B. glucanolyticus, B. lautus, B.pabuli, and B. validus as well as some unnamed DNA homology groups. These revisions of several taxa, together with the isolation and naming of new strains, has led to the expansion of Bacillus, and the genus now includes at least 67 validly described species (Tab. 1). 2.3 Phylogenetic Analyses and Evolution There are essentially two types of relationships on which classifications of organisms can be based. Phenetic relationships are those which encompass the complete organism and describe the genotypes and/or phenotypes of the organisms under study, and phylogenetic relationships are intended to represent the evolutionary branching patterns of the bacteria under study. Classifications based on phylogenetic relationships are termed cladistic. Such classifications can be derived from comparisons of gene sequences. In particular ribosomal (r)RNA sequences are amenable to this type of analysis (WOESE, 1987). Such studies reveal possible evolutionary patterns among the bacilli and are used to group Bacillus species into phylogenetically-based taxa (ASH et al., 1991; RÖSSLER et al., 1991). It is becoming apparent that many strains cluster around B. subtilis, and indeed most of these are members of the phenetic group II. There are anomalies, however, including B. circulans and B. coagulans which apparently diverged from B. subtilis relatively recently and yet are phenetically dissimilar. One of the more robust groups is that based on B. sphaericus which is distinct both phenetically and phylogenetically indicating that these bacteria diverged from the main Bacillus lineage at an early date and have subsequently evolved at a constant rate. Similarly, the B. polymyxa group diverged from B. subtilis at a very early stage and is the equivalent of a separate genus. The thermophilic bacilli are placed in several areas of the tree including two nuclei based on B. stearothermophilus and Alicyclobacillus acidocaldarius. The latter is a new genus established to accommodate “B. acidocaldarius” and some other acidophilic thermophiles which show an early divergence from the main Bacillus lineage and are considered sufficiently different to warrant separate genus status (WISOTZKEY et al., 1992). 2.4 Identification of Bacillus Species An important goal of the taxonomist is to produce an accurate and simple identification system from the classification. Such schemes should be multi-purpose and enable the identification of strains of, for example, medical, ecological or biotechnological relevance, in an unambiguous manner. The traditional approach to the identification of bacilli was based on the three morphological groups mentioned in Sect. 2.1. Having assigned an isolate to one of these groups, the bacterium was identified to the species level using a panel of physiological and biochemical tests. This system was workable, but familiarity with these bacteria was often necessary in order to distinguish spore morphologies. Largely because of this, later schemes disregarded spore morphology (CLAUS and BERKELEY, 1986), but then the number of tests to effect an identification had to be increased. There was also the problem of the ever-increasing number of new species to be included in the tables. The expansion of the genus and growing interest in these bacteria prompted the development of computerized identification sys- 3 Ecology tems. BERKELEY et al. (1984) used API 50 CH trays (miniaturized kits each enabling the examination of 50 phenotypic tests for one bacterium) to characterize a large number of strains and from the results developed a computerized identification matrix. An alternative computer-assisted identification matrix is available from this laboratory. This is based on 30 tests and 44 species. The tests include common physiological and biochemical features such as starch and casein hydrolysis, acid production from sugars, etc., and the 44 taxa represent all of the common species. With this system we can routinely identify about 50 % of environmental isolates of Bacillus. Unidentified strains presumably represent undescribed taxa which we do not have in the matrix or variants of established species. Of the various chemotaxonomic approaches available for identification of Bacillus strains, pyrolysis mass spectrometry seems to hold particular promise (BERKELEY et al.,1984). Pyrolysis involves the combustion of a sample in an inert atmosphere. The fragments produced are then separated on the basis of their mass/ charge ratio using a mass spectrometer. A library of pyrograms or mass spectra is held in a computer and when a new isolate is to be identified its pyrogram is compared with the library using multivariate statistics. This procedure suffers from lack of reproducibility and drift over a period; pyrograms prepared at sixmonth intervals often give different results. The procedure is very quick, however, a few minutes per sample for colonies from a Petri plate, so standards can be readily examined alongside the unknown to overcome the drift of the machine. This approach lends itself to rapid and highly specific typing of bacteria including Bacillus spp. A second chemotaxonomic approach of promise for routine identification of bacilli is based on fatty acid composition. A microbial identification system using gas chromatographic analysis of methyl esters of cellular fatty acids is marketed by Hewlett-Packard. The production of a profile takes about 60–90 minutes (including preparation time which can be reduced when samples are prepared in batches) and the profile is compared with a library of profiles for Bacillus species. Matching of the profile with one from the library effects an 373 identification. This approach has recently been successfully used for the identification of B. sphaericus strains including types pathogenic for mosquitoes (FRACHON et al., 1991). The few reports of DNA probes and related technology for the identification and typing of bacilli concentrate on specific groups of particular environmental or biotechnological importance although a probe for B. subtilis based on the 23S rRNA has been prepared. The insect pathogen B. thuringiensis has received most attention in this respect. Oligonucleotide probes directed at the toxin genes have been successful for the detection and identification of B. thuringiensis (reviewed in PRIEST and GRIGOROVA, 1990), and a typing procedure using polymerase chain reaction (PCR) products of toxin genes has also been reported (CAROZZI et al., 1991). 3 Ecology The aerobic endospore-forming bacteria are widespread and can be recovered from almost every environment in the biosphere. Bacilli have been isolated from dry antarctic valleys and from thermal sites. They are prevalent in marine and other aquatic sites and, of course, they comprise a major proportion of the soil microflora. One of the most interesting aspects of the ecology of these bacteria is their role in these varied locations. Bacilli are almost invariably isolated from environmental samples by heat destruction of vegetative cells (usually by incubating samples at 80 °C for about 10 min) or some other procedures such as ethanol inactivation. The spores are then germinated and colonies grown on suitable media in air. This simple process is absolutely specific for aerobic, endospore-forming bacteria; hence its attraction (see PRIEST and GRIGOROVA, 1990). However, this process only recovers spores from the environment and no indication is provided as to the contribution of these microorganisms to the habitat from which they were isolated. For example, the isolation of strict thermophiles from garden soils, or alkaliphilic bacilli from acid soils, suggests that the spores had simply accumulated but were unable to