Genetics of Bacillus

The 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 transduction were 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 of Bacillus. 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 called conduction. 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, a conjugative transposon (Tn925) has been identified, which transfers from Enterococcus 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, and Paenibacillus polymyxa.

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

Halophiles: Virgibacillus pantothenticus, Sporosarcina pasteurii. Some strains grow in 10 % NaCl.

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

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

Denitrifiers: include Bacillus azotoformans, Bacillus cereus, Brevibacillus laterosporus, Bacillus licheniformis, Sporosarcina pasteurii, Geobacillus stearothermophilus (over half the type species reduce NO₃ to NO₂). Although Bacillus species are common in agricultural soils, and they are attributed to participate in wasteful denitrification (conversion of the farmer's expensive NO₃ fertilizers 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. Paenibacillus macerans 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 lentimorbus and 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 than P. 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.