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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