Bacillus subtilis


Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants and humans. A member of the genus Bacillus, B. subtilis is rod-shaped, and can form a tough, protective endospore, allowing it to tolerate extreme environmental conditions. B. subtilis has historically been classified as an obligate aerobe, though evidence exists that it is a facultative anaerobe. B. subtilis is considered the best studied Gram-positive bacterium and a model organism to study bacterial chromosome replication and cell differentiation. It is one of the bacterial champions in secreted enzyme production and used on an industrial scale by biotechnology companies.

Description

Bacillus subtilis is a Gram-positive bacterium, rod-shaped and catalase-positive. It was originally named Vibrio subtilis by Christian Gottfried Ehrenberg, and renamed Bacillus subtilis by Ferdinand Cohn in 1872. B. subtilis cells are typically rod-shaped, and are about 4–10 micrometers long and 0.25–1.0 μm in diameter, with a cell volume of about 4.6 fL at stationary phase. As with other members of the genus Bacillus, it can form an endospore, to survive extreme environmental conditions of temperature and desiccation. B. subtilis is a facultative anaerobe and had been considered as an obligate aerobe until 1998. B. subtilis is heavily flagellated, which gives it the ability to move quickly in liquids.
B. subtilis has proven highly amenable to genetic manipulation, and has become widely adopted as a model organism for laboratory studies, especially of sporulation, which is a simplified example of cellular differentiation. In terms of popularity as a laboratory model organism, B. subtilis is often considered as the Gram-positive equivalent of Escherichia coli, an extensively studied Gram-negative bacterium.

Habitat

This species is commonly found in the upper layers of the soil and B. subtilis is thought to be a normal gut commensal in humans. A 2009 study compared the density of spores found in soil to that found in human feces. The number of spores found in the human gut was too high to be attributed solely to consumption through food contamination. B. subtilis has been linked to grow in higher elevations and act as an identifier for both eco-adaptability and honey bee health.

Reproduction

B. subtilis can divide symmetrically to make two daughter cells, or asymmetrically, producing a single endospore that can remain viable for decades and is resistant to unfavourable environmental conditions such as drought, salinity, extreme pH, radiation, and solvents. The endospore is formed at times of nutritional stress and through the use of hydrolysis, allowing the organism to persist in the environment until conditions become favourable. Prior to the process of sporulation the cells might become motile by producing flagella, take up DNA from the environment, or produce antibiotics. These responses are viewed as attempts to seek out nutrients by seeking a more favourable environment, enabling the cell to make use of new beneficial genetic material or simply by killing off competition.
Under stressful conditions, such as nutrient deprivation, B. subtilis undergoes the process of sporulation. This process has been very well studied and has served as a model organism for studying sporulation.

Chromosomal replication

B. subtilis is a model organism used to study bacterial chromosome replication. Replication of the single circular chromosome initiates at a single locus, the origin. Replication proceeds bidirectionally and two replication forks progress in clockwise and counterclockwise directions along the chromosome. Chromosome replication is completed when the forks reach the terminus region, which is positioned opposite to the origin on the chromosome map. The terminus region contains several short DNA sequences that promote replication arrest. Specific proteins mediate all the steps in DNA replication. Comparison between the proteins involved in chromosomal DNA replication in B. subtilis and in Escherichia coli reveals similarities and differences. Although the basic components promoting initiation, elongation, and termination of replication are well-conserved, some important differences can be found. These differences underline the diversity in the mechanisms and strategies that various bacterial species have adopted to carry out the duplication of their genomes.

Genome

B. subtilis has about 4,100 genes. Of these, only 192 were shown to be indispensable; another 79 were predicted to be essential, as well. A vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics.
The complete genome sequence of B. subtilis sub-strain QB928 has 4,146,839 DNA base pairs and 4,292 genes. The QB928 strain is widely used in genetic studies due to the presence of various markers .
Several noncoding RNAs have been characterized in the B. subtilis genome in 2009, including Bsr RNAs.
Microarray-based comparative genomic analyses have revealed that B. subtilis members show considerable genomic diversity.
FsrA is a small RNA found in Bacillus subtilis. It is an effector of the iron sparing response, and acts to down-regulate iron-containing proteins in times of poor iron bioavailability.

Transformation

involves the transfer of DNA from one bacterium to another through the surrounding medium. In B. subtilis the length of transferred DNA is greater than 1271kb. The transferred DNA is likely double-stranded DNA and is often more than a third of the total chromosome length of 4215 kb. It appears that about 7–9% of the recipient cells take up an entire chromosome.
In order for a recipient bacterium to bind, take up exogenous DNA from another bacterium of the same species and recombine it into its chromosome, it must enter a special physiological state called competence.
Competence in B. subtilis is induced toward the end of logarithmic growth, especially under conditions of amino-acid limitation. Under these stressful conditions of semistarvation, cells typically have just one copy of their chromosome and likely have increased DNA damage. To test whether transformation is an adaptive function for B. subtilis to repair its DNA damage, experiments were conducted using UV light as the damaging agent. These experiments led to the conclusion that competence, with uptake of DNA, is specifically induced by DNA-damaging conditions, and that transformation functions as a process for recombinational repair of DNA damage.
While the natural competent state is common within laboratory B. subtilis and field isolates, some industrially relevant strains, e.g. B. subtilis, are reluctant to DNA uptake due to the presence of restriction modification systems that degrade exogenous DNA. B. subtilis mutants, which are defective in a type I restriction modification system endonuclease, are able to act as recipients of conjugative plasmids in mating experiments, paving the way for further genetic engineering of this particular B. subtilis strain.

Uses

1900s

Cultures of B. subtilis were popular worldwide before the introduction of antibiotics as an immunostimulatory agent to aid treatment of gastrointestinal and urinary tract diseases. It was used throughout the 1950s as an alternative medicine, which upon digestion has been found to significantly stimulate broad-spectrum immune activity including activation of secretion of specific antibodies IgM, IgG and IgA and release of CpG dinucleotides inducing INF A/Y producing activity of leukocytes and cytokines important in the development of cytotoxicity towards tumor cells. It was marketed throughout America and Europe from 1946 as an immunostimulatory aid in the treatment of gut and urinary tract diseases such as Rotavirus and Shigellosis. It has been reported that in 1966 the U.S Army dumped bacillus subtilis onto the grates of New York City subway stations for four days in order to observe people's reactions when coated by a strange dust, due to its ability to survive it is thought to still be present there.
Since the 1960s B. subtilis has had a history as a test species in spaceflight experimentation. Its endospores can survive up to 6 years in space if coated by dust particles protecting it from solar UV rays. It has been used as an extremophile survival indicator in outer space such as Exobiology Radiation Assembly, EXOSTACK, and EXPOSE orbital missions.
Wild-type natural isolates of B. subtilis are difficult to work with compared to laboratory strains that have undergone domestication processes of mutagenesis and selection. These strains often have improved capabilities of transformation, growth, and loss of abilities needed "in the wild". And, while dozens of different strains fitting this description exist, the strain designated '168' is the most widely used. Strain 168 is a tryptophan auxotroph isolated after X-rays mutagenesis of B. subtilis Marburg strain and it is widely use in research due to its high transformation efficiency
in a molecular biology laboratory
B. globigii, a closely related but phylogenetically distinct species now known as Bacillus atrophaeus was used as a biowarfare during Project SHAD. Subsequent genomic analysis showed that the strains used in those studies were products of deliberate enrichment for strains that exhibited abnormally high rates of sporulation.
A strain of B. subtilis formerly known as Bacillus natto is used in the commercial production of the Japanese food nattō, as well as the similar Korean food cheonggukjang.

2000s

In other animals

B. subtilis was reviewed by the US FDA Center for Veterinary Medicine and found to present no safety concerns when used in direct-fed microbial products, so the Association of American Feed Control Officials has listed it approved for use as an animal feed ingredient under Section 36.14 "Direct-fed Microorganisms".
The Canadian Food Inspection Agency Animal Health and Production Feed Section has classified Bacillus culture dehydrated approved feed ingredients as a silage additive under Schedule IV-Part 2-Class 8.6 and assigned the International Feed Ingredient number IFN 8-19-119.
On the other hand, several feed additives containing viable spores of B. subtilis have been positively evaluated by the European Food Safety Authority, regarding their safe use for weight gaining in animal production.

In humans

B. subtilis spores can survive the extreme heat during cooking. Some B. subtilis strains are responsible for causing ropiness – a sticky, stringy consistency caused by bacterial production of long-chain polysaccharides – in spoiled bread dough. For a long time, bread ropiness was associated uniquely with B. subtilis species by biochemical tests. Molecular assays revealed greater Bacillus species variety in ropy breads, which all seems to have a positive amylase activity and high heat resistance. B. subtilis CU1 was evaluated in a 16-week study to healthy subjects. B. subtilis CU1 was found to be safe and well tolerated in the subjects without any side effects.
B. subtilis and substances derived from it has been evaluated by different authoritative bodies for their safe and beneficial use in food. In the United States, an opinion letter issued in the early 1960s by the Food and Drug Administration recognized some substances derived from microorganisms as generally recognized as safe, including carbohydrase and protease enzymes from B. subtilis. The opinions were predicated on the use of nonpathogenic and nontoxicogenic strains of the respective organisms and on the use of current good manufacturing practices. The FDA stated the enzymes derived from the B. subtilis strain were in common use in food prior to January 1, 1958, and that nontoxigenic and nonpathogenic strains of B. subtilis are widely available and have been safely used in a variety of food applications. This includes consumption of Japanese fermented soy bean, in the form of Natto, which is commonly consumed in Japan, and contains as many as 108 viable cells per gram. The fermented beans are recognized for their contribution to a healthy gut flora and vitamin K2 intake; during this long history of widespread use, natto has not been implicated in adverse events potentially attributable to the presence of B. subtilis. The natto product and the B. subtilis natto as its principal component are FOSHU approved by the Japanese Ministry of Health, Labour, and Welfare as effective for preservation of health.
B. subtilis has been granted "Qualified Presumption of Safety" status by the European Food Safety Authority.

Identification

Below is a table that helps identify B. subtilis through certain identification tests.
TestResult
Acid from Glucose+.
Acid from Lactose-
Acid from Sucrose+
Acid from Mannitol+
Catalase+
Citrate +
Endospore+
Gas from Glucose-
Gelatin Hydrolysis+
Gram Stain+
Hydrogen Sulfide Production-
Indole Production-
Motility+
Nitrate Reduction+
Oxidase+
Phenylalanine Deaminase-
ShapeRod
Urea Hydrolysis-
Voges-Proskaeur-Proskaeur+