This articleis missing information about a description and listing of thespecies group it is in, in reference to the history of many useful bacteria being lumped in or split out of this species. See NCBI tree fortxid653685. Please expand the article to include this information. Further details may exist on thetalk page.(February 2022)
Until 2008,Bacillus globigii was thought to beB. subtilis but is since formally recognized asBacillus atrophaeus.[1][2]
Bacillus subtilis (/bəˈsɪl.əssubˈtiː.lis/),[3][4] known also as thehay bacillus orgrass bacillus, is agram-positive,catalase-positivebacterium, found in soil and thegastrointestinal tract ofruminants, humans and marine sponges.[5][6][7][8] As a member of thegenusBacillus,B. subtilis is rod-shaped, and can form a tough, protectiveendospore, allowing it to tolerate extreme environmental conditions.B. subtilis has historically been classified as anobligate aerobe, though evidence exists that it is afacultative anaerobe.B. subtilis is considered the best studied Gram-positive bacterium and amodel organism to study bacterial chromosome replication and cell differentiation. It is one of the bacterial champions in secretedenzyme production and used on an industrial scale by biotechnology companies.[5][6][7]
Bacillus subtilis is aGram-positive bacterium,rod-shaped andcatalase-positive. It was originally namedVibrio subtilis byChristian Gottfried Ehrenberg,[9] and renamedBacillus subtilis byFerdinand Cohn in 1872[10] (subtilis being the Latin for "fine, thin, slender").B. subtilis cells are typically rod-shaped, and are about 4–10 micrometers (μm) long and 0.25–1.0 μm in diameter, with a cell volume of about 4.6 fL at stationary phase.[6][11]
As with other members of thegenusBacillus, it can form anendospore, to survive extreme environmental conditions of temperature and desiccation.[12]B. subtilis is afacultative anaerobe[6][13] and had been considered as anobligate aerobe until 1998.B. subtilis is heavilyflagellated, which gives it the ability to move quickly in liquids.
This species is commonly found in the upper layers of the soil andB. subtilis is thought to be anormal 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 was too high to be attributed solely to consumption through food contamination.[15] In some bee habitats,B. subtilis appears in the gut flora ofhoney bees.[16]B. subtilis can also be found in marine environments.[6][7]
There is evidence thatB. subtilis issaprophytic in nature. Studies have shown that the bacterium exhibits vegetative growth in soil rich in organic matter, and that spores were formed when nutrients were depleted.[17] Additionally,B. subtilis has been shown to formbiofilms on plant roots, which might explain why it is commonly found in gut microbiomes.[17] Perhaps animals eating plants withB. subtilis biofilms can foster growth of the bacterium in their gastrointestinal tract. It has been shown that the entire lifecycle ofB. subtilis can be completed in the gastrointestinal tract, which provides credence to the idea that the bacterium enters the gut via plant consumption and stays present as a result of its ability to grow in the gut.[17]
Bacillus subtilis can divide symmetrically to make twodaughter cells (binary fission), or asymmetrically, producing a singleendospore that can remain viable for decades and is resistant to unfavourable environmental conditions such asdrought,salinity, extremepH,radiation, andsolvents. 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 becomemotile by producingflagella, take up DNA from the environment, or produceantibiotics.[6][7] 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.[citation needed]
Under stressful conditions, such as nutrient deprivation,B. subtilis undergoes the process ofsporulation. This process has been very well studied and has served as a model organism for studying sporulation.[18]
Although sporulation in B. subtilis is induced by starvation, the sporulation developmental program is not initiated immediately when growth slows due to nutrient limitation. A variety of alternative responses can occur, including the activation offlagellar motility to seek new food sources bychemotaxis, the production ofantibiotics to destroy competing soil microbes, the secretion of hydrolyticenzymes to scavenge extracellular proteins andpolysaccharides, or the induction of 'competence' for uptake of exogenousDNA for consumption, with the occasional side-effect that new genetic information is stably integrated. Sporulation is the last-ditch response to starvation and is suppressed until alternative responses prove inadequate. Even then, certain conditions must be met such aschromosome integrity, the state of chromosomal replication, and the functioning of theKrebs cycle.[19]
OnceB. subtilis commits to sporulation, thesigma factor sigma F is secreted.[20] This factor promotes sporulation. A sporulation septum is formed and a chromosome is slowly moved into the forespore. When a third of one chromosome copy is in the forespore and the remaining two thirds is in the mother cell, the chromosome fragment in the forespore contains the locus for sigma F, which begins to be expressed in the forespore.[21] In order to prevent sigma F expression in the mother cell, an anti-sigma factor, which is encoded by spoIIAB,[22] is expressed. Any residual anti-sigma factor in the forespore (which would otherwise interfere with sporulation) is inhibited by an anti-anti-sigma factor, which is encoded by spoIIAA.[22] SpoIIAA is located near the locus for the sigma factor, so it is consistently expressed in the forespore. Since the spoIIAB locus is not located near the sigma F and spoIIAA loci, it is expressed only in the mother cell and therefore repress sporulation in that cell, allowing sporulation to continue in the forespore. Residual spoIIAA in the mother cell represses spoIIAB, but spoIIAB is constantly replaced so it continues to inhibit sporulation. When the full chromosome localizes to the forespore, spoIIAB can repress sigma F. Therefore, the genetic asymmetry of theB. subtilis chromosome and expression of sigma F, spoIIAB and spoIIAA dictate spore formation inB. subtilis.
Regulation of sporulation inB. subtilis
Sporulation requires a great deal of time and also a lot of energy and is essentially irreversible,[23] making it crucial for a cell to monitor its surroundings efficiently and ensure that sporulation is embarked upon at only the most appropriate times. The wrong decision can be catastrophic: a vegetative cell will die if the conditions are too harsh, while bacteria forming spores in an environment which is conducive to vegetative growth will be out competed.[24] In short, initiation of sporulation is a very tightlyregulatednetwork with numerous checkpoints for efficient control.[citation needed]
Bacillus subtilis is amodel organism used to study bacterial chromosome replication. Replication of the singlecircular chromosome initiates at a single locus, the origin (oriC). Replication proceeds bidirectionally and tworeplication 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 thechromosome 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 inB. subtilis and inEscherichia 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.[25]
Bacillus 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.[26]
The complete genome sequence ofB. 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 [aroI(aroK)906 purE1 dal(alrA)1 trpC2].[27]
Several noncoding RNAs have been characterized in theB. subtilis genome in 2009, includingBsr RNAs.[28]Microarray-based comparative genomic analyses have revealed thatB. subtilis members show considerable genomic diversity.[29]
FsrA is asmall RNA found inBacillus subtilis. It is aneffector of the iron sparing response, and acts to down-regulate iron-containing proteins in times of poor iron bioavailability.[30][31]
A promising fish probiotic,B. subtilis strain WS1A, that possesses antimicrobial activity againstAeromonas veronii and suppressed motileAeromonas septicemia inLabeo rohita. The de novo assembly resulted in an estimated chromosome size of 4,148,460 bp, with 4,288 open reading frames.[6][7]B. subtilis strain WS1A genome contains many potential genes, such as those encoding proteins involved in the biosynthesis ofriboflavin,vitamin B6, andamino acids (ilvD) and in carbon utilization (pta).[6][7]
Natural bacterial transformation involves the transfer of DNA from one bacterium to another through the surrounding medium. InB. subtilis the length of transferred DNA is greater than 1,271kb (more than 1million bases).[32] The transferred DNA is likely double-stranded DNA and is often more than a third of the total chromosome length of 4,215kb.[33] It appears that about 7–9% of the recipient cells take up an entire chromosome.[34]
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 calledcompetence.Competence inB. subtilis is induced toward the end of logarithmic growth, especially under conditions of amino-acid limitation.[35] 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 forB. subtilis to repair its DNA damage, experiments were conducted using UV light as the damaging agent.[36][37][38] 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.[39]
While the natural competent state is common within laboratoryB. subtilis and field isolates, some industrially relevant strains, e.g.B. subtilis (natto), are reluctant to DNA uptake due to the presence of restriction modification systems that degrade exogenous DNA.B. subtilis (natto) 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 particularB. subtilis strain.[40]
By adopting Green Chemistry in the use of less hazardous materials, while saving cost, researchers have been mimicking nature's methods of synthesizing chemicals that can be useful for the food and drug industry, by "piggybacking molecules on shorts strands of DNA" before they are zipped together during their complementary base pairing between the two strands. Each strand will carry a particular molecule of interest that will undergo a specific chemical reaction simultaneously when the two corresponding strands of DNA pairs hold together like a zipper, allowing another molecule of interest, to react with one another in controlled and isolated reaction between those molecules being carried into these DNA complementary attachments. By using this method with certain bacteria that naturally follow a process replication in a multi-step fashion, the researchers can simultaneously carry on the interactions of these added molecules to interact with enzymes and other molecules used for a secondary reaction by treating it like a capsule, which is similar to how the bacteria performs its own DNA replication processes.[41]
Cultures ofB. subtilis were popular worldwide, before the introduction ofantibiotics, as an immunostimulatory agent to aid treatment ofgastrointestinal andurinary tract diseases. It was used throughout the 1950s as analternative medicine, which upon digestion has been found to significantly stimulatebroad-spectrum immune activity including activation of secretion of specificantibodiesIgM,IgG andIgA[42] and release ofCpG dinucleotides inducinginterferonIFN-α/IFNγ producing activity ofleukocytes andcytokines important in the development ofcytotoxicity towardstumor cells.[43] It was marketed throughout America and Europe from 1946 as an immunostimulatory aid in the treatment of gut and urinary tract diseases such asRotavirus andShigellosis. In 1966, the U.S. Army dumpedbacillus subtilis onto the grates of New York City subway stations for five days in order to observe how a biological agent dispensed around the subway trains would disperse and potentially affect unsuspecting passengers.[44] Due to its ability to survive, it is thought to still be present there.[45]
The antibioticbacitracin was first isolated from a variety ofBacillus licheniformis named "Tracy I"[46] in 1945, then considered part of theB. subtilis species. It is still commercially manufactured by growing the variety in a container of liquidgrowth medium. Over time, the bacteria synthesizes bacitracin and secretes the antibiotic into the medium. The bacitracin is then extracted from the medium using chemical processes.[47]
Wild-type natural isolates ofB. subtilis are difficult to work with compared to laboratory strains that have undergone domestication processes ofmutagenesis 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. Strain 168 is atryptophanauxotroph isolated after X-ray mutagenesis ofB. subtilis Marburg strain and is widely used in research due to its high transformation efficiency.[56]
Bacillus globigii, a closely related butphylogenetically distinct species now known asBacillus atrophaeus[57][58] was used as a biowarfaresimulant duringProject SHAD (akaProject 112).[59] Subsequent genomic analysis showed that the strains used in those studies were products of deliberate enrichment for strains that exhibited abnormally high rates ofsporulation.[60]
A strain ofB. subtilis formerly known asBacillus natto is used in the commercial production of the Japanese foodnattō, as well as the similar Korean foodcheonggukjang.
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.[29] In particular, the basic principles and mechanisms underlying formation of the durable endospore have been deduced from studies of spore formation inB. subtilis.
Its surface-binding properties play a role in safe radionuclide waste [e.g. thorium (IV) and plutonium (IV)] disposal.[citation needed]
Due to its excellent fermentation properties, with high product yields (20 to 25 gram per litre) it is used to produce various enzymes, such asamylase andproteases.[61]
It may provide some benefit tosaffron growers by speeding corn growth and increasing stigma biomass yield.[65]
It is used as an "indicator organism" during gas sterilization procedures, to ensure a sterilization cycle has completed successfully. SpecificallyB. subtilis endospores are used to verify that a cycle has reached spore-destroying conditions.[66][67]
B. subtilis has been found to act as a useful bioproduct fungicide that prevents the growth ofMonilinia vaccinii-corymbosi, a.k.a. the mummy berry fungus, without interfering with pollination or fruit qualities.[68]
Both metabolically active and non-metabolically activeB. subtilis cells have been shown to reduce gold (III) to gold (I) and gold (0) when oxygen is present. This biotic reduction plays a role in gold cycling in geological systems and could potentially be used to recover solid gold from said systems.
Novel strains ofB. subtilis that could use 4-fluorotryptophan (4FTrp) but not canonical tryptophan (Trp) for propagation were isolated. AsTrp is only coded by a single codon, there is evidence that Trp can be displaced by 4FTrp in the genetic code. The experiments showed that the canonical genetic code can be mutable.[69]
Recombinant strains pBE2C1 and pBE2C1AB were used in production ofpolyhydroxyalkanoates (PHA), and malt waste can be used as their carbon source for lower-cost PHA production.[citation needed]
It is used to producehyaluronic acid, which is used in the joint-care sector in healthcare[70] and cosmetics.
Monsanto has isolated a gene fromB. 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 US in November 2011.[71][72]
A new strain has been modified to convert nectar intohoney by secreting enzymes.[73]
Bacillus subtilis was reviewed by the US FDACenter for Veterinary Medicine and found to present no safety concerns when used in direct-fed microbial products, so theAssociation of American Feed Control Officials has listed it approved for use as ananimal feed ingredient under Section 36.14 "Direct-fed Microorganisms".[citation needed]TheCanadian Food Inspection Agency Animal Health and Production Feed Section has classifiedBacillus culture dehydrated approved feed ingredients as asilage additive under Schedule IV-Part 2-Class 8.6 and assigned the International Feed Ingredient number IFN 8-19-119.[citation needed]On the other hand, several feed additives containing viable spores ofB. subtilis have been positively evaluated by theEuropean Food Safety Authority, regarding their safe use for weight gaining in animal production.
Bacillus subtilis spores can survive the extreme heat generated during cooking. SomeB. subtilis strains are responsible for causing ropiness or rope spoilage – a sticky, stringy consistency caused by bacterial production of long-chainpolysaccharides – in spoiled bread dough and baked goods.[74] For a long time, bread ropiness was associated uniquely withB. subtilis species by biochemical tests. Molecular assays (randomly amplified polymorphic DNA PCR assay,denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of16S ribosomal DNA) revealed greaterBacillus species variety in ropy breads, which all seems to have a positive amylase activity and high heat resistance.[75]
B. subtilis CU1 (2 × 109 spores per day) was evaluated in a 16-week study (10 days administration of probiotic, followed by 18 days wash-out period per each month; repeated same procedure for total 4 months) to healthy subjects.B. subtilis CU1 was found to be safe and well tolerated in the subjects without any side effects.[76]
Bacillus subtilis and substances derived from it have 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 theFood and Drug Administration (FDA) designated some substances derived from microorganisms asgenerally recognized as safe (GRAS), includingcarbohydrase and protease enzymes fromB. 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.[77] The FDA stated that the enzymes derived from theB. subtilis strain were in common use in food prior to January 1, 1958, and that nontoxigenic and nonpathogenic strains ofB. 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 ofNatto, 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 andvitamin K2 intake; during this long history of widespread use,natto has not been implicated in adverse events potentially attributable to the presence ofB. subtilis.[citation needed] The natto product and theB. subtilis natto as its principal component are FOSHU (Foods for Specified Health Use) approved by the JapaneseMinistry of Health, Labour, and Welfare as effective for preservation of health.[78]
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