The Genus Bacillus (page 2)
(This chapter has 6 pages)
© Kenneth Todar, PhD
Nutrition and Growth
Collectively, the aerobic sporeformers are versatile
of respiration using a variety of simple organic compounds (sugars,
acids, organic acids). In some cases, they also ferment carbohydrates
a mixed reaction that typically produces glycerol and butanediol. A few
species, such as Bacillus megaterium, require no organic growth
factors; others may require amino acids, B-vitamins, or both. The
are mesophiles, with temperature optima between 30 and 45 degrees, but
some are thermophiles with optima as
high as 65 degrees. Others are true psychrophiles, able to grow and
sporulate at 0 degrees. They are found growing over a range of pH from
2 to 11. In the laboratory, under optimal conditions of
species exhibit generation times of about 25 minutes.
Most aerobic spore-forming species are easily isolated and readily
grown in the
bacteriology laboratory. The simplest technique that enriches for
spore formers is to pasteurize a diluted soil sample at 80 degrees for
15 minutes, then plate onto nutrient agar and incubate at 37 degrees
24 hours up to several days. The plates are examined after 24 hours for
typical colonies identified as catalase-positive,
endospore-forming rods. Although many species contain sporangia and
spores within 24 hours, some cultures must be incubated 5-7 days before
mature sporangia, and the size and shape of the endospore contained
can be observed. The insect pathogens, Paenibacillus larvae, P.
are more fastidious and must be isolated on J-agar (below).
Furthermore, they are typically catalase-negative, and they require
media or inoculation into insect hosts for sporulation.
Mucoid-type colonies of an
Most Bacillus species can be grown in defined or
relatively-simple complex media. For a few bacilli (e.g. B. subtilis,
megaterium), minimal media have been established. Primary
can be performed on either nutrient agar (peptone 5g/l, beef extract
agar15g/l, pH6.8) or plates of J-agar (tryptone 5g/l, yeast extract
K2HPO4 3g/l, glucose 2g/l, agar20g/l, pH7.4).
cultures can be maintained in the laboratory on soil extract agar or on
special sporulation media.
Table 2. Minimal medium for
growth of Bacillus megaterium.
Surface Structure of Bacillus
Like most Gram-positive bacteria the surface of the Bacillus
is complex and is associated with their properties of adherence,
and tactical responses. The vegetative cell surface is a laminated
that consists of a capsule, a proteinaceous surface layer (S-layer),
layers of peptidoglycan sheeting, and the proteins on the outer surface
of the plasma membrane.
Surface of a Bacillus. Transmission
Crystalline surface layers of protein or glycoprotein subunits, called
S-layers, are found in members of the genus Bacillus. As with
of other bacteria, their function in Bacillus is unknown, but
they have been presumed to be involved in adherence. It has been
demonstrated that the S-layer can physically mask the
charged peptidoglycan sheet in some Gram-positive bacteria and
autoagglutination. It has also been proposed that the layer may play
role in bacteria-metal interactions.
The capsules of many bacilli, including B. anthracis, B. subtilis,
B. megaterium, and B. licheniformis, contain poly-D- or
acid. Other Bacillus species, e.g., B. circulans, B.
B. mycoides and B. pumilus, produce carbohydrate capsules.
Dextran and levan are common, but more complex polysaccharides are
Some of the Bacillus polysaccharides cross react with
from other genera of bacteria including human pathogens. For example, B.
mycoides with Streptococcus pneumoniae type III; B.
pumilus with Neisseria meningitidis group A. Likewise, the
capsular polysaccharide of Paenibacillus alvei is
antigenically similar to that of
influenzae type B (Hib).
When examined by transmission electron microscopy, some polypeptide
complex polysaccharide capsules appear fibrillar in their arrangement
the cell surface. The capsules are easily observed by light microscopy,
especially if the bacteria are prepared ahead of time by growth on
that enhance capsule production. Heavily encapsulated strains may form
a mucoid or slimy colony on agar.
FA stain of the capsule of Bacillus
Negative stain (India Ink
of the capsule of Bacillus anthracis. CDC.
Bacillus megaterium synthesizes a capsule composed of both
and polysaccharide. The polypeptide is located laterally along the axis
of the cell and the polysaccharide is located at the poles and at the
of the cell.
The capsule of B. anthracis is composed of a
acid. The capsule is a major determinant of virulence in anthrax. The
is not synthesized by the closest relatives of B. anthracis,
cereus and B. thuringiensis, and this criterion can be used
to distinguish the species.
The variability of cell wall structure that is common in many
bacteria does not occur in the genus Bacillus. The vegetative
wall of almost all Bacillus species is made up of a
containing meso-diaminopimelic acid (DAP). (The cell walls of
Sporosarcina pasteurii and S. globisporus,
contain lysine in the place of DAP.) This is the same type of
that is nearly universal in Gram-negative bacteria, i.e., containing
as the diamino acid in position 3 of the tetrapeptide. In some cases,
DAP is directly cross-linked to
same as in the Enterobacteriaceae; in other cases, two
side chains of peptidoglycan are spanned by an interpeptide bridge
DAP and D-alanine, which is characteristic of most Gram-positive
In addition to peptidoglycan in the cell wall, all Bacillus
contain large amounts of teichoic acids which are bonded to muramic
residues. The types of glycerol teichoic acids vary greatly between
and within species. As in many other Gram-positive bacteria,
acids are found associated with the cell membranes of Bacillus
These compounds are thought to be involved in the synthesis of wall
acids, as regulators of autolytic activity, and as scavengers of
ions for the bacterium.
Structure of the muropeptide
subunit of the peptidoglycan of Bacillus megaterium. In most Bacillus
species, an interpeptide bridge that connects D-alanine to
acid (DAP) is absent. In addition, all Bacillus
spores contain this type of muramic acid subunit in the spore cortex.
Most aerobic sporeformers are motile by means of peritrichous
Chemotaxis has been studied extensively in B. subtilis. The
filament of B. firmus, an alkaliphile, has a remarkably low
of basic amino acids, thought to render it more stable in environmental
pH values up to 11.
Flagellar stains (Leifson's
Method) of various species of bacilli from CDC.
Individual cells of motile
bacilli photographed on nutrient agar. About 15,000X magnification.
Dept. of Agriculture. A. B. subtilis; B. P.
polymyxa; C. B. laterosporus; D. P.
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