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Tag words: bacteria, aerobic bacteria, endospore, spores, Bacillus, B. cereus, B. anthracis, B. thuringiensis, B. subtilis

Gram-positive, aerobic endospore-forming bacteria

TAXONOMY: THE GENUS BACILLUS

Kingdom: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Bacillales
Family: Bacillaceae
Genus: Bacillus










Kenneth Todar currently teaches Microbiology 100 at the University of Wisconsin-Madison.  His main teaching interest include general microbiology, bacterial diversity, microbial ecology and pathogenic bacteriology.

Bacillus cereus bacteria.Print this Page

The Genus Bacillus (page 2)

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Nutrition and Growth

Collectively, the aerobic sporeformers are versatile chemoheterotrophs capable of respiration using a variety of simple organic compounds (sugars, amino acids, organic acids). In some cases, they also ferment carbohydrates in 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 majority 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 growth, Bacillus 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 aerobic 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 for 24 hours up to several days. The plates are examined after 24 hours for typical colonies identified as catalase-positive, Gram-positive, endospore-forming rods. Although many species contain sporangia and free spores within 24 hours, some cultures must be incubated 5-7 days before mature sporangia, and the size and shape of the endospore contained therein, can be observed. The insect pathogens, Paenibacillus larvae, P. popilliae and P. lentimorbus, are more fastidious and must be isolated on J-agar (below). Furthermore, they are typically catalase-negative, and they require special media or inoculation into insect hosts for sporulation.


Mucoid-type colonies of an encapsulated Bacillus species. CDC.

Most Bacillus species can be grown in defined or relatively-simple complex media. For a few bacilli (e.g. B. subtilis, B. megaterium), minimal media have been established. Primary isolations can be performed on either nutrient agar (peptone 5g/l, beef extract 3g/l, agar15g/l, pH6.8) or plates of J-agar (tryptone 5g/l, yeast extract 15g/l, K2HPO4 3g/l, glucose 2g/l, agar20g/l, pH7.4). Stock cultures can be maintained in the laboratory on soil extract agar or on special sporulation media.

Table 2. Minimal medium for the growth of Bacillus megaterium.
Component         Amount
sucrose             10.0 g
K2HPO4              2.5 g
KH2PO4              2.5 g
(NH4)2HPO4             1.0 g
MgSO4 7H2O             0.20 g
FeSO4 7H2O             0.01 g
MnSO4 7H2O             0.007 g
water             985 ml
pH 7.0

Surface Structure of Bacillus

Like most Gram-positive bacteria the surface of the Bacillus is complex and is associated with their properties of adherence, resistance and tactical responses. The vegetative cell surface is a laminated structure that consists of a capsule, a proteinaceous surface layer (S-layer), several layers of peptidoglycan sheeting, and the proteins on the outer surface of the plasma membrane.


Surface of a Bacillus. Transmission E.M. C=Capsule; S=S-layer; P=Peptidoglycan. Pasteur Institute.

S-layers
Crystalline surface layers of protein or glycoprotein subunits, called S-layers, are found in members of the genus Bacillus. As with S-layers 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 negatively charged peptidoglycan sheet in some Gram-positive bacteria and prevent autoagglutination. It has also been proposed that the layer may play some role in bacteria-metal interactions.

Capsules
The capsules of many bacilli, including B. anthracis, B. subtilis, B. megaterium, and B. licheniformis, contain poly-D- or L-glutamic acid. Other Bacillus species, e.g., B. circulans, B. megaterium, B. mycoides and B. pumilus, produce carbohydrate capsules. Dextran and levan are common, but more complex polysaccharides are produced, as well.

Some of the Bacillus polysaccharides cross react with antisera 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 Haemophilus influenzae type B (Hib).

When examined by transmission electron microscopy, some polypeptide and complex polysaccharide capsules appear fibrillar in their arrangement on the cell surface. The capsules are easily observed by light microscopy, especially if the bacteria are prepared ahead of time by growth on media that enhance capsule production. Heavily encapsulated strains may form a mucoid or slimy colony on agar.


FA stain of the capsule of Bacillus anthracis. CDC.


Negative stain (India Ink outline) of the capsule of Bacillus anthracis. CDC.

Bacillus megaterium synthesizes a capsule composed of both polypeptide and polysaccharide. The polypeptide is located laterally along the axis of the cell and the polysaccharide is located at the poles and at the equator of the cell.

The capsule of B. anthracis is composed of a poly-D-glutamic acid. The capsule is a major determinant of virulence in anthrax. The capsule is not synthesized by the closest relatives of B. anthracis, i.e., B. cereus and B. thuringiensis, and this criterion can be used to distinguish the species.

Cell Walls
The variability of cell wall structure that is common in many Gram-positive bacteria does not occur in the genus Bacillus. The vegetative cell wall of almost all Bacillus species is made up of a peptidoglycan 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 cell wall polymer that is nearly universal in Gram-negative bacteria, i.e., containing DAP as the diamino acid in position 3 of the tetrapeptide. In some cases, DAP is directly cross-linked to D-alanine, same as in the Enterobacteriaceae; in other cases, two tetrapeptide side chains of peptidoglycan are spanned by an interpeptide bridge between DAP and D-alanine, which is characteristic of most Gram-positive bacteria.

In addition to peptidoglycan in the cell wall, all Bacillus species contain large amounts of teichoic acids which are bonded to muramic acid residues. The types of glycerol teichoic acids vary greatly between Bacillus species and within species. As in many other Gram-positive bacteria, lipoteichoic acids are found associated with the cell membranes of Bacillus species. These compounds are thought to be involved in the synthesis of wall teichoic acids, as regulators of autolytic activity, and as scavengers of bivalent 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 meso-diaminopimelic acid (DAP) is absent. In addition, all Bacillus spores contain this type of muramic acid subunit in the spore cortex.

Flagella
Most aerobic sporeformers are motile by means of peritrichous flagella. Chemotaxis has been studied extensively in B. subtilis. The flagellar filament of B. firmus, an alkaliphile, has a remarkably low content 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. U.S. Dept. of Agriculture.  A.  B. subtilis;  B. P. polymyxa;  C.  B. laterosporus;  D. P. alvei.




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