Bacillus subtilis

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Bacillus subtilis
Bacillus subtilis.jpg
TEM micrograph of a B. subtilis cell in cross-section (scale bar = 200 nm)
Scientific classification
Domain: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Bacillales
Family: Bacillaceae
Genus: Bacillus
Species: B. subtilis
Binomial name
Bacillus subtilis
(Ehrenberg 1835)
Cohn 1872
Synonyms
  • Vibrio subtilis
  • Bacillus globigii[1][2]

Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium.[3] A member of the genus Bacillus, B. subtilis is rod-shaped, and has the ability to form a tough, protective endospore, allowing the organism to tolerate extreme environmental conditions. B. subtilis has historically been classified as an obligate aerobe, though recent research has demonstrated this is not strictly correct.[4]

Although this species is commonly found in soil, more evidence suggests B. subtilis is a normal gut commensal in humans. A 2009 study compared the density of spores found in soil (about 106 spores per gram) to that found in human feces (about 104 spores per gram). The number of spores found in the human gut is too high to be attributed solely to consumption through food contamination. Soil simply serves as a reservoir, suggesting B. subtilis inhabits the gut and should be considered as a normal gut commensal.[5]

History[edit]

1800s[edit]

In 1835, the bacterium was originally named Vibrio subtilis by Christian Gottfried Ehrenberg,[6] and renamed Bacillus subtilis by Ferdinand Cohn in 1872.[7]

1900s[edit]

Gram-stained B. subtilis
Sporulating B. subtilis

Cultures of B. subtilis were used throughout the 1950s as an alternative medicine due to the immunostimulatory effects of its cell matter, which upon digestion has been found to significantly stimulate broad-spectrum immune activity including activation of secretion of specific antibodies IgM, IgG and IgA[8] and release of CpG dinucleotides inducing INF A/Y producing activity of leukocytes and cytokines important in the development of cytotoxicity towards tumor cells.[9] 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 Shigella,[10] but declined in popularity after the introduction of cheap consumer antibiotics, despite causing fewer allergic reactions and significantly lower toxicity to normal gut flora. 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.[11]

Safety[edit]

B. subtilis is only known to cause disease in severely immunocompromised patients, and can conversely be used as a probiotic in healthy individuals.[12] It rarely causes food poisoning.[13] Some B. subtilis strains produce the proteolytic enzyme subtilisin. B. subtilis spores can survive the extreme heat to which it is exposed 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. Today, molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads which all seems to have a positive amylase activity and high heat resistance.[14]

The B. subtilis microbial strain and substances derived from this microorganism were subjects of evaluation by different authoritative bodies for their safe and beneficial use in food and has been regarded as not presenting safety concerns. In the United States, an opinion letter issued in the early 1960s by the Food and Drug Administration (FDA) recognized some substances derived from microorganisms as GRAS, including carbohydrase and protease enzymes from B. subtilis. The opinions are predicated on the use of nonpathogenic and nontoxicogenic strains of the respective organisms and on the use of current good manufacturing practices (FDA partial list of microorganisms, 2002).

The FDA concluded the enzymes derived from the B. subtilis strain were in common use in food prior to January 1, 1958. The FDA stated the nontoxigenic and nonpathogenic strains of B. subtilis are widely available and have been safely used in a variety of food applications, including the documented consumption of B. subtilis in the Japanese fermented soy bean, natto. Natto, which is commonly consumed in Japan, 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 any adverse events potentially attributable to the presence of B. subtilis.

The natto product and the B. subtilis natto as its principal component are FOSHU (Foods for Specified Health Use) approved in Japan. FOSHU foods are approved by the Ministry of Health, Labour and Welfare as effective for preservation of health by adding certain active ingredients or removing undesirable ones. They are designed to be safe and effective for the maintenance and improvement of health by incorporating them into one’s diet. The Japanese FOSHU products are products which safety and efficacy have been verified scientifically.[15]

The Association of American Feed Control Officials has listed B. subtilis approved for use as a feed ingredient under Section 36.14 Direct-fed Microorganisms. This microorganism was reviewed by the FDA Center for Veterinary Medicine and found to present no safety concerns when used in direct-fed microbial products. 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.

B. subtilis has a long history of safe use. It has been granted Qualified Presumption of Safety status by the European Food Safety Authority[16] and is part of the authoritative list of microorganisms with a documented history of safe use in food established by the International Dairy Federation in collaboration with the European Food and Feed Cultures Association in 2002 and updated in 2012.

Reproduction[edit]

B. subtilis can divide symmetrically to make two daughter cells (binary fission), 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, 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 of competition.[citation needed]

Under stressful conditions, such as nutrient deprivation, B. subtilis undergoes the process of sporulation to ensure the survival of the species. This process has been very well studied and has served as a model organism for studying sporulation.

Chromosomal replication[edit]

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 (oriC). 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 (Ter sites) 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 (such as one bacterium missing proteins essential in the other). These differences underline the diversity in the mechanisms and strategies that various bacterial species have adopted to carry out the duplication of their genomes.[17]

Uses[edit]

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. It is also heavily flagellated, which gives it the ability to move quickly in liquids. In terms of popularity as a laboratory model organism, B. subtilis is often used as the Gram-positive equivalent of Escherichia coli, an extensively studied Gram-negative bacterium.

Monsanto has isolated a gene from B. subtilis that expresses cold shock protein B and spliced it into their drought-tolerant corn hybrid MON 87460, which was approved for sale in the United States in November 2011.[18][19]

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 (uptake and integration of environmental DNA), 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.

Colonies of B. subtilis grown on a culture dish in a molecular biology laboratory

As a model organism, B. subtilis is commonly used in laboratory studies directed at discovering the fundamental properties and characteristics of Gram-positive spore-forming bacteria.[20] In particular, the basic principles and mechanisms underlying formation of the durable endospore have been deduced from studies of spore formation in B. subtilis.

B. subtilis is also used as a soil inoculant in horticulture and agriculture.

The high stability of B. subtilis in harsh environmental conditions makes this microorganism a perfect candidate for probiotics applications either in baked and pasteurized foods/beverages or in other galenic forms like tablets, capsules, and powder.

B. globigii, a closely related but phylogenetically distinct species now known as B. atrophaeus [21][22] was used as a biowarfare simulant during Project SHAD (aka Project 112).[23] 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.[24]

Enzymes produced by B. subtilis and B. licheniformis are widely used as additives in laundry detergents.

Its other uses include:

  • A strain of B. subtilis formerly known as Bacillus natto is used in the commercial production of the Japanese food natto, as well as the similar Korean food cheonggukjang.
  • It was popular worldwide before the introduction of consumer antibiotics as an immunostimulatory agent to aid treatment of gastrointestinal and urinary tract diseases. It is still widely used in Western Europe and the Middle East as an alternative medicine.
  • It can convert some explosives into harmless compounds of nitrogen, carbon dioxide, and water.
  • Its surface-binding properties play a role in safe radionuclide waste [e.g. thorium (IV) and plutonium (IV)] disposal.
  • Recombinant strains pBE2C1 and pBE2C1AB were used in production of polyhydroxyalkanoates (PHA), and malt waste can be used as their carbon source for lower-cost PHA production.
  • It is used to produce amylase.
  • It is used to produce hyaluronic acid,[25] which is useful in the joint-care sector in healthcare.
  • It may provide some benefit to saffron growers by speeding corm growth and increasing stigma biomass yield.[26]
  • It is often used as an "indicator organism" during gas sterilization procedures, to ensure a sterilization cycle has completed successfully.[27][28] This is due to the difficulty in sterilizing endospores.
  • It has been used as an extremophile survival indicator in outer space such as Exobiology Radiation Assembly,[29][30] EXOSTACK,[31][32] and EXPOSE orbital missions.[33][34][35]

Genome[edit]

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.[36]

Several noncoding RNAs have been characterized in the B. subtilis genome, including Bsr RNAs.[37]

Transformation[edit]

Natural bacterial transformation involves the transfer of DNA from one bacterium to another through the surrounding medium. Transformation depends on the expression of numerous bacterial genes whose products appear to be specifically designed to carry out this process.[38] In order for a recipient bacterium to bind, take up and recombine exogenous DNA into its chromosome, it must enter a special physiological state called competence.[39] Ordinarily (with rare exceptions), the DNA integrated into the recipient bacterial chromosome is from another bacterium of the same species, thus the donated DNA is usually homologous to the resident chromosome.

In B. subtilis, the length of the transferred DNA is greater than 1271 kb (more than 1 million bases).[40] The length transferred is likely double-stranded DNA and is often more than a third of the total chromosome length of 4215 kb.[41] It appears that about 7-9% of the recipient cells take up an entire chromosome.[42]

Competence in B. subtilis is induced toward the end of logarithmic growth, especially under conditions of amino-acid limitation.[43] Under these stressful conditions of semistarvation, the cells typically have just one copy of their chromosome and likely have increased DNA damage. To test whether the adaptive function of transformation is repair of DNA damages, a sequence of experiments was conducted with B. subtilis using UV light as the damaging agent[44][45][46] and also reviewed by Michod et al.[47] 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.[47]

See also[edit]

References[edit]

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