Bacillus and related endospore- forming bacteria

The Genus Bacillus


Transmission E.M. of Bacillus megaterium.

Gram-positive, Aerobic or Facultative Endospore-forming Bacteria

In 1872, Ferdinand Cohn, a contemporary of Robert Koch, recognized and named the bacterium Bacillus subtilis. The organism is Gram-positive, capable ofgrowth in the presence of oxygen, and forms a unique type of resting cell called an endospore. The organism represented what was to become a large and diverse genus of bacteria named Bacillus, in the Family Bacillaceae.

Koch relied on Cohn’s observations in his classic work (1876), The etiology of anthrax based on the life history of Bacillus anthracis, which provided the first proof that a specific microorganism could cause a specific disease.


Robert Koch’s original photomicrographs of Bacillus anthracis. In 1876, Koch established by careful microscopy that the bacterium was always present in the blood of animals that died of anthrax. He took a small amount of blood from such an animal and injected it into a healthy mouse, which subsequently became diseased and died. He was able to recover the original anthrax organism from the dead mouse, demonstrating for the first time that a specific bacterium is the cause of a specific disease. 

The genus Bacillus remained intact until 2004, when it was split into several families and genera of endospore-forming bacteria, justifiable on the basis of ssRNA analysis. In order to accommodate former members of the genus Bacilluscovered in this chapter, its title has been changed to “Gram-positive aerobic or facultative endospore-forming bacteria”.

The unifying characteristic of these bacteria is that they are Gram-positive, formendospores, and grow in the presence of O2. The trivial name assigned to them is aerobic sporeformers.

The ubiquity and diversity of these bacteria in nature, the unusual resistance of their endospores to chemical and physical agents, the developmental cycle of endospore formation, the production of antibiotics, the toxicity of their spores and protein crystals for many insects, and the pathogen Bacillus anthracis, have attracted ongoing interest in these bacteria since and Cohn and Koch’s discoveries in the 1870s.

There is great diversity of physiology among the aerobic sporeformers, not surprising considering their recently-discovered  phylogenetic diversity. Their collective features include degradation of most all substrates derived from plant and animal sources, including cellulose, starch, pectin, proteins, agar, hydrocarbons, and others; antibiotic production; nitrification; denitrification; nitrogen fixation; facultative lithotrophy; autotrophy; acidophily; alkaliphily; psychrophily; thermophily; and parasitism. Endospore formation, universally found in the group, is thought to be a strategy for survival in the soil environment, wherein these bacteria predominate. Aerial distribution of the dormant spores probably explains the occurrence of aerobic sporeformers in most habitats examined.


Bacillus coagulans. Gram stain. CDC. Gram-positive or Gram-negative? The cell wall structure of endospore-forming bacteria is consistent with that of Gram-positive bacteria, and young cultures stain as expected. However, many sporeformers rapidly become Gram-negative when entering the stationary phase of growth.

Classification and Phylogeny

Early attempts at classification of Bacillus species were based on two characteristics: aerobic growth and endospore formation.  This resulted in tethering together many bacteria possessing different kinds of physiology and occupying a variety of habitats. Hence, the heterogeneity in physiology, ecology, and genetics, made it difficult to categorize the genus Bacillus or to make generalizations about it.

In Bergey’s Manual of Systematic Bacteriology (1st ed. 1986), the G+C content of known species of Bacillus ranges from 32 to 69%.  This observation, as well as DNA hybridization tests, revealed the genetic heterogeneity of the genus. Not only was there variation from species to species, but there were sometimes profound differences in G+C content within strains of a species. For example, the G+C content of the Bacillus megaterium group ranged from 36 to 45%.

In Bergey’s Manual of Systematic Bacteriology (2nd ed. 2004), phylogenetic classification schemes landed the two most prominent types of endospore-forming bacteria, clostridia and bacilli, in two different Classes of Firmicutes, Clostridiaand Bacilli.  Clostridia includes the Order Clostridiales and FamilyClostridiaceae with 11 genera including, Clostridium. Bacilli includes the OrderBacillales and the Family Bacillaceae. In this family there 37 new genera on the level with Bacillus. This explains the heterogeneity in G+C content observed in the 1986 genus Bacillus.

The phylogenetic approach to Bacillus taxonomy has been accomplished largely by analysis of 16S rRNA molecules by oligonucleotide sequencing. This technique, of course, also reveals phylogenetic relationships. Surprisingly, Bacillus species showed a kinship with certain nonsporeforming species, including Enterococcus, Lactobacillus, and Streptococcus at the Order level, and Listeria andStaphylococcus at the Family level. Otherwise, some former members of the genusBacillus were gathered into new Families, including Acyclobacillaceae,Paenibacillaceae and Planococcaceae, now on the level with Bacillaceae. Most of the bacteria discussed in this article come from one of these four Families. Theirtaxonomic hierarchy (Bergey’s 2004) is Kingdom: Bacteria; Phylum: Firmicutes;Class: Bacilli; Order: Bacillales; Family: Acyclobacillaceae (genus:Acyclobacillus); Family: Bacillaceae (genus: Bacillus, Geobacillus); Family:Paenibacillaceae (genus: Paenibacillus, Brevibacillus); Family:Planococcaceae (genus: Sporosarcina).

Notable former members of the genus Bacillus that have been moved to new families and/or genera are given in the table below.

Table 1. Important taxonomic reassignments in the Genus Bacillus (1986-2004).

Bergey’s Manual of Systematic Bacteriology (1st ed. 1986) Bergey’s Manual of Systematic Bacteriology (2nd ed. 2004),
Bacillus acidocalderius Acyclobacillus acidocalderius
Bacillus agri Brevibacillus agri
Bacillus alginolyticus Paenibacillus alginolyticus
Bacillus amylolyticus Paenibacillus amylolyticus
Bacillus alvei Paenibacillus alvei
Bacillus azotofixans Paenibacillus azotofixans
Bacillus brevis Brevibacillus brevis
Bacillus globisporus Sporosarcina globisporus
Bacillus larvae Paenibacillus larvae
Bacillus laterosporus Brevibacillus laterosporus
Bacillus lentimorbus Paenibacillus lentimorbus
Bacillus macerans Paenibacillus macerans
Bacillus pasteurii Sporosarcina pasteurii
Bacillus polymyxa Paenibacillus polymyxa
Bacillus popilliae Paenibacillus popilliae
Bacillus psychrophilus Sporosarcina psychrophilia
Bacillus stearothermophilus Geobacillus stearothermophilus
Bacillus thermodenitrificans Geobacillus thermodenitrificans

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. popilliaeand 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. subtilisB. 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, andB. 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 Streptococcuspneumoniae type III; B. pumilus with Neisseria meningitidis group A. Likewise, the capsular polysaccharide of Paenibacillus alvei is antigenically similar to that ofHaemophilus 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 Bacillusspecies 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.

Endospores

Endospores were first described by Cohn in Bacillus subtilis and later by Koch in the pathogen, Bacillus anthracis. Cohn demonstrated the heat resistance of endospores in B. subtilis, and Koch described the developmental cycle of spore formation in B. anthracis. Endospores are so named because they are formed intacellularly, although they are eventually released from this mother cell or sporangium as free spores. Endospores have proven to be the most durable type of cell found in Nature, and in their cryptobiotic state of dormancy they can remain viable for extremely long periods of time, perhaps millions of years.

When viewed unstained, endospores of living bacilli appear edged in black and are very bright and refractile. Endospores strongly resist application of simple stains or dyes and hence appear as nonstaining entities in Gram-stain preparations. However, once stained, endospores are quite resistant to decolorization. This is the basis of several spore stains such as the Schaeffer-Fulton staining method which also differentiates the spores from sporangia and vegetative cells.


Left. Bacillus thuringiensis phase micrograph. Endospores can be readily recognized microscopically by their intracellular site of formation and their extreme refractility. Right. Bacillus anthracis Crystal violet stain viewed by light microscopy. Endospores are highly resistant to application of basic aniline dyes that readily stain vegetative cells.

Below. Spore stain of a Bacillus species. CDC. The staining technique employed is the Schaeffer-Fulton method. A fixed smear is flooded with a solution of malachite green and placed over boiling water for 5 minutes. After rinsing, the smear is counterstained with safranine. Mature spores stain green, whether free or still in the vegetative sporangium; vegetative cells and sporangia stain red.

Endospores do not form normally during active growth and cell division. Rather, their differentiation begins when a population of vegetative cells passes out of the exponential phase of growth, usually as a result of nutrient depletion. Typically one endospore is formed per vegetative cell. The mature spore is liberated by lysis of the mother cell (sporangium) in which it was formed.


The formation of endospores is a complex and highly-regulated form of development in a relatively simple (procaryotic) cell. In all Bacillus species studied, the process of spore formation is similar, and can be divided into seven defined stages (0-VI). The vegetative cell (a) begins spore development when the DNA coils along the central axis of the cell as an “axial filament” (b).  The DNA then separates and one chromosome becomes enclosed in plasma membrane to form a protoplast (c). The protoplast is then engulfed by the mother cell membrane  to form a intermediate structure called a forespore (d) . Between the two membranes, The core (cell) wall, cortex and spore coats are synthesized (e). As water is removed from the spore and as it matures, it becomes increasingly heat resistant and more refractile (f). The mature spore is eventually liberated by lysis of the mother cell. The entire process takes place over a period of 6-7 hours and requires the temporal regulation of more than 50 unique genes. Pasteur Institute.

Mature spores have no detectable metabolism, a state that is described ascryptobiotic. They are highly resistant to environmental stresses such as high temperature (some endospores can be boiled for several hours and retain their viability), irradiation, strong acids, disinfectants, etc. Although cryptobiotic, they retain viability indefinitely such that under appropriate environmental conditions, they germinate into vegetative cells. Endospores are formed by vegetative cells in response to environmental signals that indicate a limiting factor for vegetative growth, such as exhaustion of an essential nutrient. They germinate and become vegetative cells when the environmental stress is relieved. Hence, endospore-formation is a mechanism of survival rather than a mechanism of reproduction.

Below. Drawing of a cross-section of a Bacillus endospore by Viake Haas, University of Wisconsin. In cross section, Bacillus spores show a more complex ultrastructure than that seen in vegetative cells. The spore protoplast (core) is surrounded by the core (cell) wall, the cortex, and then the spore coat. Depending on the species, an exosporium may be present. The core wall is composed of the same type of peptidoglycan as the vegetative cell wall. The cortex is composed of a unique peptidoglycan that bears three repeat subunits, always contains DAP, and has very little cross-linking between tetrapeptide chains. The outer spore coat represents 30-60 percent of the dry weight of the spore. The spore coat proteins have an unusually high content of cysteine and of hydrophobic amino acids, and are highly resistant to treatments that solubilize most proteins.

Table 3. Differences between endospores and vegetative cells that form them.

Property Vegetative cells Endospores
Surface coats Typical Gram-positive murein cell wall polymer; crystalline S-layer Thick spore coat, cortex, and unique peptidoglycan core wall; no S-layer
Microscopic appearance Nonrefractile Refractile
Calcium dipicolinic acid Absent Present in core
Cytoplasmic water activity High Very low
Enzymatic activity Present Absent
Macromolecular synthesis Present Absent
Heat resistance Low High
Resistance to chemicals and acids Low High
Radiation resistance Low High
Sensitivity to lysozyme Some sensitive; some resistant Resistant
Sensitivity to dyes and staining Sensitive ResistantGenetics of BacillusThe discovery of transformation in a strain of Bacillus subtilis in 1958, focused attention on the genetics of the bacterium. This is one of relatively few bacteria in which competence for DNA uptake has been found to occur as a natural part of the bacterium’s life cycle. Subsequently, generalized and specialized transductionwere observed in B. subtilis, and knowledge of the genetics and chromosomal organization of the bacterium quickly mounted to become second only to that of the enteric bacteria. Furthermore, the identification of numerous genes affecting sporulation in B. subtilis has provided a means for analyzing the complex developmental program of sporulation.

Bacteriophages capable of mediating generalized transduction have also been reported in other species of Bacillus, including B. cereus, B. megaterium, B. thuringiensis, B. anthracis, and in Geobacillus stearothermophilus.

Conjugative plasmids are plasmids capable of bringing about their own transfer from one bacterium to another. They have been described in several species ofBacillus. The capacity to produce the insecticidal delta toxin crystal protein in B. thuringiensis is encoded in large plasmids. These plasmids can be transferred to plasmid-deficient strains of B. thuringiensis, as well as to B. cereus, to yield recipients that produce crystal protein.  B. thuringiensis transfers the pXO11 and pXO12 plasmids to B. anthracis and to B. cereus. The recipients, in turn, become effective donors, and in the case of those inheriting pXO12, also acquire 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. The B. anthracis toxin plasmid, pXO1, and the capsule plasmid, pXO2, can be transferred to B. anthracis and B. cereus recipients lacking these plasmids.

The large B. anthracis plasmids are apparently transferred by a process calledconduction. This 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-to-cell 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. None of 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, aconjugative transposon (Tn925) has been identified, which transfers fromEnterococcus faecalis to B. subtilis.

Our understanding of the Bacillus genome, and their means of DNA transfer, has led to its manipulation. So far, this has resulted in numerous medical, agricultural and industrial achievements, involving the use of the organism or its products.


This e.m. image of a spore-forming Bacillus (also at the top of page 1) is that of B. megaterium which has been cloned with the Bt gene and is expressing Bt in the form of the bipyramidal “parasporal” crystal adjacent to the spore.Bt is an insecticidal protein produced by Bacillus thuringiensis.

Ecology

Due to the resistance of their endospores to environmental stress, as well as their long-term survival under adverse conditions, most aerobic sporeformers are ubiquitous and can be isolated from a wide variety of sources. Hence, the occurrence of sporeforming bacteria in a certain environment is not necessarily an indication of habitat. However, it is generally accepted that the primary habitat of the aerobic endospore-forming bacilli is the soil. The great Russian microbiologist, Winogradsky, considered them as “normal flora” of the soil.

In the soil environment the bacteria become metabolically-active when suitable substrates for their growth are available, and presumably they form spores when their nutrients become exhausted. This is a strategy used by other microbes in the soil habitat, including the filamentous fungi and the actinomycetes, which also predominate in the aerobic soil habitat. It is probably not a coincidence, rather an example of convergent evolution, that these three dissimilar groups of microbes live in the soil, form resting structures (spores), and produce antibiotics in association with their sporulation processes.

Since many endospore forming species can effectively degrade a series of biopolymers (proteins, starch, pectin, etc.), they are assumed to play a significant role in the biological cycles of carbon and nitrogen.

From soil, by direct contact or air-borne dust, endospores can contaminate just about anything that is not maintained in a sterile environment. They may play a biodegradative role in whatever they contaminate, and thereby they may be agents of unwanted decomposition and decay. Several Bacillus species are especially important as food spoilage organisms.

Ecophysiological groups

Generally, standard bacteriological criteria do not adequately distinguish the aerobic sporeforming bacteria for discussion or positive identification. An artificial, but convenient, way to organize aerobic spore-formers for this purpose is to place them into ecophysiological groups, such as nitrogen-fixers, denitrifiers, insect pathogens, animal pathogens, thermophiles, antibiotic producers, and so on. Such an approach also allows some speculation concerning the natural history, diversity, and ecology of this important group of bacteria.

Acidophiles: include Acyclobacillus acidocalderius, Bacillus coagulans, andPaenibacillus polymyxa.

AlkaliphilesB. alcalophilus and Sporosarcina pasteurii. The optimum pH is 8, and some strains grow at pH 11.

HalophilesVirgibacillus pantothenticus, Sporosarcina pasteurii. Some strains grow in 10 % NaCl.

Psychrophiles or psychrotrophsSporosarcina globisporus, Bacillus insolitus, Marinibacillus marinus, Paenibacillus macquariensis, Bacillus megaterium,Paenibacillus polymyxa. Two species will grow and form spores at 0oC.

Thermophiles: include Acyclobacillus acidocalderius, Bacillus schlegelii, andGeobacillus stearothermophilus. Acidophiles and Lithoautotrophs are found in this group, too. The upper temperature limit is 65oC.

Denitrifiers: include Bacillus azotoformans, Bacillus cereus, Brevibacillus laterosporus, Bacillus licheniformis, Sporosarcina pasteurii, Geobacillus stearothermophilus (over half the type species reduce NO3 to NO2). AlthoughBacillus species are common in agricultural soils, and they are attributed to participate in wasteful denitrification (conversion of the farmer’s expensive NO3fertilizers to volatile N2O or N2) their exact role in the economy of this processes has not been clarified. A related process conducted by some Bacillus species, called dissimilatory nitrate reduction, reduces NO3 to ammonia (NH3), but this is not considered denitrification.

Nitrogen-fixers: Paenibacillus macerans and Paenibacillus polymyxa. Paenibacillusmacerans is a fairly prominent bacterium in soil and in decaying vegetable material. The bacteria only fix nitrogen under anaerobic conditions because they do not have a mechanism for protection of their nitrogenase enzyme from the damaging effects of O2.  In the same way as the role of the bacilli in denitrification and nitrification, their overall contribution to non symbiotic global nitrogen fixation is not known.

Antibiotic Producers: antibiotics produced by the aerobic sporeformers are often, but not always, polypeptides. Known antibiotic producers are Brevibacillus brevis(e.g. gramicidin, tyrothricin), Bacillus cereus (e.g. cerexin, zwittermicin), Bacillus circulans (e.g. circulin), Brevibacillus laterosporus (e.g. laterosporin), Bacillus licheniformis (e.g. bacitracin), Paenibacillus polymyxa (e.g. polymyxin, colistin),Bacillus pumilus (e.g. pumulin) and Bacillus subtilis (e.g. polymyxin, difficidin, subtilin, mycobacillin).

Bacillus antibiotics share a full range of antimicrobial activity: bacitracin, pumulin, laterosporin, gramicidin and tyrocidin are effective against Gram-positive bacteria; colistin and polymyxin are anti-Gram-negative; difficidin is broad spectrum; and mycobacillin and zwittermicin are anti-fungal.

As in the case of the actinomycetes, antibiotic production in the bacilli is accompanied by cessation of vegetative growth and spore formation. This has led to the idea that the ecological role of antibiotics may not rest with competition between species, but with the regulation of sporulation and/or the maintenance of dormancy.

Pathogens of Insects: Paenibacillus larvae, Paenibacillus lentimorbusand Paenibacillus popilliae are invasive pathogens. Bacillus thuringiensis forms a parasporal crystal that is toxic to Lepidoptera.

P. larvae, P. lentimorbus and P. popilliae are a related cluster of species, being insect pathogens with swollen sporangia and typically catalase-negative. They also are unable to grow in nutrient broth, probably because it is insufficient in thiamin, which they need as a growth factor. Yeast extract (15g/l) must be added to their media for growth. Also, P. lentimorbus and P. popilliae are quite similar in their biochemical properties, virulence and host range. They sometimes occur in coinfections.

P. larvae is the causative agent of American foulbrood of honeybees, which is the most widespread and persistent of the honeybee brood diseases. The organism can be isolated repeatedly from infected brood and honeycomb, usually in a pure culture. It has been noted on many occasions that the natural habitat of the bacterium is remarkably free of contaminants. Presumably, the bacterium can be isolated from soil around the hives of infected bees, but it has not been isolated from other sources. This is indicative of a very close and specific type of host-parasite interaction between the bacterium and the honeybee.

P. popilliae is the cause of the most widespread of two milky diseases of the Japanese beetle, Popillia japonica. Their spores, in a swollen sporangium, are frequently accompanied by a parasporal crystal. Interestingly, the bacterium sporulates with ease in the hemolymph of the infected insect, but it will not form mature spores in most artificial media. Special media have been designed that induce P. popilliae and P. lentimorbus to form mature spores. The prospect that P. popilliae, together with P. lentimorbus, might be used to control or eliminate the Japanese beetle and the European chafer (Amphimallon majalis) has drawn attention to these bacteria. P. popilliae is encountered in naturally-infected grubs far more frequently than P. lentimorbus, which also causes milky disease.

P. lentimorbus is similar in most ways to P. popilliae. The most obvious difference is that P. lentimorbus does not form a parasporal body. The bacteria also differ morphologically and culturally. P. lentimorbus likewise causes one of two milky diseases in the Japanese beetle. The bacterium can only be isolated from the hemolymph of scarabaeid beetles, although it most certainly exists in soil inhabited with infected larvae.

The principal interest in P. lentimorbus arises from its ability to cause disease of Japanese beetle and European chafer larvae, which together cause millions of dollars in damage each year to a variety of plants. P. lentimorbus is more widespread thanP. popilliae, which also causes milky disease in the same hosts. The reason the infections are called “milky disease” is that as the disease develops, the larvae become milky in appearance. This is caused by the prolific production of spores in the insect hemolymph.


Spores of the the insect pathogens seen by phase microscopy. U.S. Dept. of Agriculture.  A. Paenibacillus larvae spores from a comb infected with American foulbrood; B. Paenibacillus lentimorbus spores from hemolymph of infected Japanese beetle larvae; C. Spores of Paenibacillus popilliae from hemolymph of infected Japanese beetle larvae.

Bacillus thuringiensis is a variety of B. cereus and is therefore considered in the B. cereus-B. anthracis-B. thuringiensis group. B thuringiensis is distinguished from B. cereus or B. anthracis by its pathogenicity for lepidopteran insects and by production of an intracellular parasporal crystal in association with spore formation. The bacteria and protein crystals are marketed as “Bt” insecticide, which is used for the biological control of certain garden and crop pests.

Pathogens of AnimalsBacillus anthracis and B. cereus are the predominant pathogens of medical importance. Paenibacillus alvei, B. megaterium, B. coagulans, Brevibacillus laterosporus, B. subtilis, B. sphaericus, B. circulans, Brevibacillus brevis, B. licheniformis, P. macerans, B. pumilus and B. thuringiensis have been occasionally isolated from human infections.

B. anthracis is the causative agent of anthrax, and B. cereus causes food poisoning. Nonanthrax Bacillus species can also cause a wide variety of other infections, and they are being recognized with increasing frequency as pathogens in humans.

Anthrax
Anthrax is primarily a disease of domesticated and wild animals, particularly herbivorous animals, such as cattle, sheep, horses, mules and goats. Humans become infected incidentally when brought into contact with diseased animals, which includes their flesh, bones, hides, hair and excrement. In the United States, the incidence of naturally-acquired anthrax is extremely rare (1-2 cases of cutaneous disease per year). Worldwide, the incidence is unknown, although B. anthracis is present in most of the world’s soils.

The most common form of the disease in humans is cutaneous anthrax, which is usually acquired via injured skin or mucous membranes. A minor scratch or abrasion, usually on an exposed area of the face or neck or arms, is inoculated by spores from the soil or a contaminated animal or carcass. The spores germinate, vegetative cells multiply, and a characteristic gelatinous edema develops at the site. This develops into papule within 12-36 hours after infection. The papule changes rapidly to a vesicle, then to a pustule (malignant pustule), and finally into a necrotic ulcer, from which infection may disseminate, giving rise to septicemia. Lymphatic swelling also occurs within seven days. In severe cases, where the blood stream is eventually invaded, the disease is frequently fatal.

Another form of the disease, inhalation anthrax (woolsorters’ disease), results most commonly from inhalation of spore-containing dust where animal hair or hides are being handled. The disease begins abruptly with high fever and chest pain. It progresses rapidly to a systemic hemorrhagic pathology and is often fatal if treatment cannot stop the invasive aspect of the infection.

Gastrointestinal anthrax is analogous to cutaneous anthrax but occurs on the intestinal mucosa. As in cutaneous anthrax, the organisms probably invade the mucosa through a preexisting lesion. The bacteria spread from the mucosal lesion to the lymphatic system. Intestinal anthrax results from the ingestion of poorly cooked meat from infected animals. Gastrointestinal anthrax is rare but may occur as explosive outbreaks associated with ingestion of infected animals.

The pathology of anthrax is mediated by two primary determinants of bacterial virulence: presence of an antiphagoytic capsule, which promotes bacterial invasion, and production of a powerful lethal toxin, the anthrax toxin.

For more information on anthrax, including use and detection of Bacillus anthracis as an agent of bioterrorism, please see the chapter on Bacillus anthracis and Anthrax.


Bacillus anthracis Gram stain. CDC.

Bacillus cereus food poisoning
Bacillus cereus causes two types of food-borne intoxications. One type is characterized by nausea and vomiting and abdominal cramps and has an incubation period of 1 to 6 hours. It resembles Staphylococcus aureus food poisoning in its symptoms and incubation period. This is the “short-incubation” or emetic form of the disease. The second type is manifested primarily by abdominal cramps and diarrhea with an incubation period of 8 to 16 hours. Diarrhea may be a small volume or profuse and watery. This type is referred to as the “long-incubation” or diarrheal form of the disease, and it resembles more food poisoning caused by Clostridium perfringens. In either type, the illness usually lasts less than 24 hours after onset.

The short-incubation form of disease is caused by a preformed heat-stable enterotoxin. The mechanism and site of action of this toxin are unknown. The long-incubation form of illness is mediated by a heat-labile enterotoxin, which apparently activates intestinal adenylate cyclase and causes intestinal fluid secretion.

This bacterium is dealt with separately in the medical section of the text at Bacillus cereus and Food Poisoning.


Colonies of Bacillus anthracis (right) and Bacillus cereus (left) on a plate of blood agar. CDC.


Table 4. Characteristics of Bacillus and related aerobic endospore-forming bacteria


Acyclobacillus acidocaldarius Thermoacidophile. Limits of temperature for growth are  45o and  65oC. The limits of pH for growth around 2. Found in hot acidic environments. Spores have surprisingly weak thermal resistance.


Bacillus alcalophilus Alkaliphile. Tolerant to alkaline conditions and does not grow at pH 7. Capable of growth at pH >10.


Paenibacillus alvei Isolated from soil and from honeybee larvae suffering from European foulbrood disease. Not classified as an insect pathogen.


Bacillus anthracis The causative agent of anthrax in humans and in animals. Spores persist for long periods on contaminated materials.


Bacillus azotoformans. Has a negative Gram reaction. Can respire anaerobically using NO3, NO2, SO4 or fumarate as a final electron acceptor. A vigorous denitrifying bacterium in soils, it converts NO3, NO2 and N2O to large amounts of N2.


Bacillus badius Forms a distinct colony with rhizoid outgrowths. Has been isolated from feces, dust, marine sources, foods and antacids.



Brevibacillus brevis Has been isolated chiefly from soils and foods. Requires a mixture of amino acids without vitamins for growth.


Bacillus cereus A close relative of B. anthracis, B. mycoides and B. thuringiensis. Spores are widespread in soil and air. Usually observed multiplying in foods such as cooked rice and may lead to food poisoning. Produces antibiotics.


Bacillus circulans Some strains are cellulolytic. Has a distinct rhizoid colony.



Bacillus coagulans Includes acidophilic strains. Spores are relatively sparse in soils. May multiply in acid foods such as canned tomato juice and silage. Found in medicated creams and antacids


Bacillus fastidiosis Uses only uric acid, allantoic acid or allantoin as an energy source. Isolated from soil and from poultry litter.


Bacillus firmus Isolated chiefly from soil. Pigmented strains occur in salt marshes.


Sporosarcina globisporus Forms spherical spores. Found in soil and river water.


Bacillus insolitus Growth and sporulation occur at 0 degrees. Vegetative cells are short and stout. Found in Arctic soils.


Paenibacillus larvae Causes American foulbrood in honeybees.



Brevibacillus laterosporus Produces a canoe-like body attached to the side of spore forcing the spore into a lateral position in the sporangium. Rarely isolated, but has been found in dead honeybee larvae, soil, water and antacids.


Paenibacillus lentimorbus More fastidious nutritionally and more widespread thanP. popilliae, it also infects the Japanese beetle and the European chafer. Isolated from diseased larvae or infected honeycombs.


Bacillus lentus Similar to B. firmus, but more nutritionally-versatile. Isolated from soil, food and spices.



Bacillus licheniformis Produces same type of poly-D-glutamate capsule as B. anthracis. Red pigment produced by many strains. Spores occur in soil. Growth in foods, especially if held between 30 and 50 degrees. Industrial source of bacitracin, a medically useful antibiotic.



Paenibacillus macerans Most strains fix N2 under anaerobic conditions. Degrades pectin and plant polysaccharides. Some strains moderately thermophilic. Also has been found in canned fruit  at pH 3.8.


Paenibacillus macquariensis Grows and sporulates at 0 degrees. Otherwise similar to B. circulans.


Marinibacillus marinus Grows at 5-30 degrees but not at 37 degrees. Has an obligate requirement for Na+. Isolated routinely from marine sediments.



Bacillus megaterium “Megaterium” means “big beast”.  The largest cell diameter of any aerobic spore former (1.2 -1.5 micrometers). Grows in minimal medium without any added growth factors. Spores are common in soil. Subject of many basic studies of Gram-positive bacteria in the laboratory.


Bacillus mycoides Similar to B. cereus but non motile, and forms distinctive rhizoid colonies. High degree of relatedness with B. anthracis, B. cereus and B. thuringiensis.



Sporosarcina pasteurii
 Converts urea to ammonium carbonate more actively than any known bacterium. Requires alkaline medium (pH 9) for growth. Isolated from soil, water, sewage and encrustations on urinals.



Paenibacillus polymyxa Colonies are mucoid, slimy and tend to spread. Synthesizes profuse levan capsule from sucrose. Spores have longitudinal surface ridges so are star-shaped in cross-section. Degrades pectin and plant polysaccharides. Nitrogen fixed under anaerobic conditions. Spores are widespread. Multiplication occurs chiefly in decaying vegetation. Often isolated from foods. Found in medicated creams and antacids. Source of the antibiotic polymyxin. A very versatile and widespread sporeformer.


Paenibacillus popilliae Pathogen of scarabeid beetles that causes (one variety of) milky disease in the Japanese beetle. Together with B. lentimorbus, it is a biological agent for the Japanese beetle and the European chafer. The larvae become milky white because of the prolific production of spores in the insect hemolymph. Forms a distinctive parasporal crystal that distinguishes it from B. lentimorbus. Isolated from hemolymph of Japanese beetle grubs.


Bacillus pumilus Spores are ubiquitous; occurs in soil more frequently than those of B. subtilis.


Bacillus schlegelii Thermophile similar to  B. sphaericus in its high G+C content, but differentiated because it is a facultative lithoautotroph. The bacterium can derive energy from the oxidation of H2 or CO while obtaining carbon from either CO2 or CO. Isolated from lake sediments and sugar factory sludge.


Bacillus sphaericus Isolated from soil, marine and fresh water sediments, milk and foods.


Geobacillus stearothermophilus Grows at 65o C and has tolerance to acid. Occurs in soil, hot springs, desert sand, Arctic waters, ocean sediments, food and compost.



Bacillus subtilis Grows as a unicellular rod, seldom as chains. Degrades pectin and polysaccharides in plant tissues, and some strains cause rots in live potato tubers. Grows in a minimal defined medium with no added growth factors. Endospores are widespread. Vegetative organisms take part in various stages in the early breakdown of materials of plant and animal origin. Grows in non acid food under aerobic conditions. Causative agent of ropy (slimy) bread. This bacterium is the experimental “E. coli” of Gram-positive bacteria. Much of the information we have on the biology, biochemistry and genetics of the Gram-positive cell, indeed, of bacteria in general, has been derived from the study of B. subtilis.


Bacillus thuringiensis Distinguished from B. cereus by pathogenicity for lepidopteran insects, and production of a parasporal crystal in association with spore formation. In the larval gut, the protein (crystal) is toxic. The spores and crystals are marketed in garden centers as BT, for biological control of lepidopterans that attack garden and crop plants. Bt is encoded on a plasmid which can be spontaneously transferred to B. cereus, endowing it with the ability to produce the toxic crystal. Some taxonomists have argued that this is evidence of such a close genetic relationship between the two bacteria that B. thuringiensis should be considered a variant subspecies of B. cereus. The same argument has been made for the relationship between B. anthracis, the plasmid-encoded anthrax toxin, and B. cereus.

 

source:http://textbookofbacteriology.net/Bacillus.html




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