A Microbial Biorealm page on the genus Bacillus clausii
Higher order taxa
Eubacteria (kingdom); Bacteria (domain); Firmicutes (phylum); Bacilli (class); Bacillales (order); Bacillaceae (family); Bacillus (genus)
Description and significance
Bacillus clausii is Gram-positive, motile, spore-forming and like most of the Bacillus bacteria, it is rod-shaped. Colonies of B. clausii form filamentous margins that appear cream-white in color. B. clausii is alkaliphilic and produces a class of subtilisins known as high-alkaline proteases. The protease from Bacillus clausii strain 221, the H-221 protease, was the first enzyme to be identified in an alkaliphilic Bacillus.  The alkaliphilic nature of the organism has also proved it to be useful in preventing and treating various gastrointestinal disorders as an oral bacteriotherapy.  This organism can be found in many alkaline environments, including soil and marine habitat.
The B. clausii strain KSM-K16 was obtained from soil samples, and its phylogenic position as a member of B. clausii was identified using Bacillus clausii DSM 8716 as a reference strain- also isolated from a soil sample. DSM 8716 was identified as a novel Bacillus species by Nielsen et. all, with unique characteristics detailed in Cell Structure and Metabolism. The techniques used to determine the classification of KSM-K16 included 16S rRNA sequencing, which directly compares two or more strains of rRNA sequences to determine sequence homology- in this case the sequence of KSM-K16 with that of DSM 8716. Other classification techniques including fatty acid analysis, which identifies fatty acids in the membrane, and carbohydrate utilization tests, which establish the metabolic characteristics of the organism. Growth of KSM-K16 was observed in the temperature range of 15-50°C and the pH range of 7-10.5, with optimal growth at 40°C and pH 9.0. The KSM-K16 strain produces the high-alkaline protease, M-protease, which is hyperproduced by a mutant used in industrial scale compact heavy-duty laundry detergent. This protease, among other enzymes used by B. clausii organisms, are being extensively studied to understand their ability to function in such alkaline conditions for possible biotechnology use, making the genome of B. clausii a necessary tool.
The Bacillus clausii KSM-K16 complete genome is one circular chromosome. The chromosome is composed of 4,303,871 nucleotides. This genome contains 4204 genes, of which 4096 are protein coding and 96 code for RNAs. The GC content of B. clausii KSM-K16 is 44%, one of the highest GC contents amongst the Bacillus microbes. The most studied genes on the chromosome include M-protease and other alkaline-adapted proteases which have been X-ray crystallized based on genome sequence and erm-related genes (see Current Research). 
Cell structure and metabolism
Bacillus clausii is a rod shaped, gram-positive microbe, meaning it is surrounded by a thick cell wall. The cell wall is made up of the peptidoglycan murien. B. clausii cells tend to line up into chain-like formation, observable as a long rod cell. B. clausii is an endospore producing microbe that creates ellipsoidal spored located subterminally or paracentrally in the sporangium. Spores of B. clausii are resistant to many antibiotics including erythromycin, lincomycin, cephalosporins, and cycloserine. 
Bacillus clausii strain DSM 8716 was originally noticed as a novel species upon the characteristics of ability to hydrolyze casein, ability to reduce nitrate, and ability to grow at 50°C. Further tests showed Bacillus clausii were able to use multiple sources of carbon including: L-aribose, xylitol, galactose, dulcitol, sorbitol, methyl a-D-mannoside, mannose, N-acetylglucosamine, D-tagose, 2-ketogluconate. Part of current classification tests probe for which carbon sources are used by a Bacillus strands to identify its species. 
Nitrate reduction uses nitrate as the terminal electron acceptor during anaerobic respiration. The use of nitrate as the electron acceptor into reduced nitrite is not as efficient as the use of oxygen- and microbes such as B. clausii will prefer the use of oxygen over nitrate in terms of energy production. But in environments of low oxygen such as soil, where B. clausii is usually found, nitrate reduction can be used to keep electron transport in operation to maintain an electron gradient for ATP synthesis. 
Bacillus clausii is found in the soil where it can reduce nitrate to nitrite. The use of nitrite to become other reduced forms of nitrogenous compounds is possible, and some bacteria such as Pseudomonas aeruginosa are capable of the complete reduction of nitrite.  But the extent of nitrate reduction has not been studied thoroughly in Bacillus clausii. A possible relationship with other organisms that use nitrites may therefore exist.
B. clausii DSM 8716 were oberved in linked chain form with each other.  In terms of other organisms, no published journals discuss a direct relationship with B. clausii. B. clausii spores have been used in a European probiotic called Enterogermina, which stimulates GI tract immune system function by increasing production of secretory A immunoglobulin- indirectly acting as an antagonist to other bacterial pathogens that infect the gastrointestinal tract (see Current Research and Application to Biotechnology).
Bacillus clausii resistance to many antibiotics makes it seem capable of harm to humans, but Bacillus clausii sporulated strains are actually used in the treatment of gastrointestinal illnesses to restore intestinal flora because of their antibiotic resistance and ability to stimulate immune activity- a class of bacteria dubbed probiotics (see Application to Biotechnology) .
Application to Biotechnology
B. clausii genome sequence is being studied for its importance in biotechnology:
"[Bacillus clausii and other relatives] are now being investigated in order to better understand the physiology, biochemistry, and especially molecular genetics underlying the behavior of alkaliphilic bacteria . Most of the studies have been performed to examine enzyme biotechnology, as alkaliphilic Bacillus strains produce enzymes, such as xylanases, cellulases, amylases, and proteases, that are very useful in industry and domestic life" 
B. clausii strain KSM-K16, for example, produces especially useful proteases known as of M, H, and N-proteases. A proteolytic enzyme cleaves polypeptides into smaller pieces of amino acids. Like other Bacillus organisms, KSM-K16 secretes its proteases directly into the medium, especially during periods of low nutrition, coupled with the process of sporulation. The control of protease release has been studied in more detail with Bacillus subtilis. B. subtilis studies has shown that regulatory events during periods of cellular stress can lead to a cascade of events that include the increased release of proteloytic enzymes; more specifically, the regulatory phosphorylation of the transcription factor Spo0A inhibits the repression of a gene that encodes the B. subtilis protease.  Microbial control of M-protease is similarly studied to implement for industrial use for mass production by B. claussi strain KSM-K16.
This most extensively studied protease produced by KSM-K16, M-protease, is used in heavy-duty detergents to remove protein containing spots from laundry. M-protease has a maximum enzyme activity at pH 12.3 and 55°C in phosphate-NaOH buffer. The ability for M-protease to function at such high pH was remarkable, and the enzyme characteristics were studied to determine what modifications exist on the structural level of the protein to enable its activity in such alkaline conditions, using X-ray crystallography and genome sequencing. Results indicated that the unique protease contained a lower number of negatively charged amino acids and lysine residues, with an increase in arginine and nuetral amino acids than proteases not adapted to such alkaline environments. This in effect increases the isoelectric point of the enzyme to enable its function in high pH , .
With this important information, bioengineers can design novel proteins in the lab to be used in such extreme conditions. For example, alkaline proteases are currently finding newer uses, including their usage to create useful biomass from fibrous proteins such as horn, feather or hair. A couple other uses include hydrolysis of gelatine layers of X-ray films and the recovery of silver .
The spores of B. clausii and other related Bacilli are used as probiotics to improve the intestinal microbial balance during periods of antibiotic usage, modify the immune system function of the GI tract, and act as anti-microbial agents themselves. Probiotic-containing treatments are available for human nutrition, animal feed supplements, and also for aquaculture. An antibiotic resistant probiotic known as Enterogermina consists of 4 strains of Bacillus microbes (O/C, N/R, SIN, and T), all of which were recently reclassified from B. subtilis to B. clausii. Enterogermina is notably used in the treatment of diarrhea and prevention of infectious gastrointestinal diseases. Though not completely understood, the enzyme secretions of B. clausii during sporulation are believed to lead to these positive effect on the GI tract; during sporulation, strains from Enterogermina were found to release antimicrobial compounds and modulate immune activity by increasing production of secretory immunoglobin A. The spore resistance to antibiotics makes it especially useful for use in conjunction with antibiotic treatment for other pathogens. With further knowledge of the function of B. clausii activity as Enterogermina, the usage of this mibrobe in medicine can be optimized and implemented in more effective ways .
Research performed by Bülent Bozdogan at the Service de Microbiologie in Caen, France has revealed a 846 base pair erm-related gene in Bacillus clausii that confers resistance to certain antibiotics. Erm proteins are ribosomal methylases that monomethylate or dimethylate a certain adenine in 23S rRNA, which when methylated could bind macrolides (antibiotics) such as erythromycin, azithromycin, spiramycin, lincomycin, clindamycin, and pristinamycin I. This erm-related gene in B. clasii strain DSM 8716 was named erm(34) .
The process of by which Enterogermina strains of B. clausii alleviate gastrointestinal disorder is not completely understood. Gabriella Casula and Simon M. Cutting of the Royal Holloway University of London have developed a method to study colonization characteristics of Enterogermina in mouse gut. They have shown that B. clausii in Enterogermina can in fact colonize for brief periods of time on the intestinal wall of the gut, and provoke immune response in mice to rid of pathogenic bacteria. Studies such as these can shed light on the mechanism of gastrointestinal revival and protection induced by Enterogermina supplementation .
M-protease from B. clausii KSM-K16 is still heavily studied to determine the unique structural components that allow its function at such high pH. A recent study by Kobayashi and Kageyama from the Tochigi Research Laboratories in Japan have revealed a salt bridge triad (Arg19–Glu271–Arg275) that exists in M-protease but not in proteases that cannot withstand such alkaline conditions. This salt bridge was found to increase the thermostability of the enzyme at such high pH. Findings such as these are furthering our knowledge of alkaline-adapted enzymes for the possible utilization in novel enzyme engineering .
Kageyama Y, Takaki Y, Shimamura S, Nishi S, Nogi Y, Uchimura K, Kobayashi T, Hitomi J, Ozaki K, Kawai S, Ito S, and Horikoshi K. Intragenomic diversity of the V1 regions of 16S rRNA genes in high-alkaline protease-producing Bacillus clausii spp. Extremophiles. 2007. Online.
Senesi S, Celandroni F, Tavanti A, and Ghelardi E. Molecular characterization and identification of Bacillus clausii strains marketed for use in oral bacteriotherapy. Appl Environ Microbiol. 2001. Volume 67. p.834–839.
Kobayashi T, Hakamada Y, Adachi S, Hitomi J, Yoshimatsu T, Koike K, Kawai S, and Ito S. Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16. Appl Microbiol Biotechnol. 1995. Volume 43. p.473–481.
Shirai, T., A. Suzuki, T. Yamane, T. Ashida, T. Kobayashi, J. Hitomi, and S. Ito. High-resolution crystal structure of M-protease: phylogeny aided analysis of the high-alkaline adaptation mechanism. Protein Eng. 1997. Volume 10. p. 627-634.
Christiansen, Torben and Nielsen J. Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state transient continuous cultures. J Biotechnol. 2002. Volume 97. p. 265–273.
A.A. Denizci, D. Kazan, E.C.A. Abeln, and A. Erarslan Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under higly alkaline conditions. Journal of Applied Microbiology. 2004. Volume 96. p. 320–327.
Bozdogan, B., S. Galopin, and R. Leclercq. Characterization of a new erm-related macrolide resistance gene present in probiotic strains of Bacillus clausii. Appl. Environ. Microbiol. 2004. Volume 70. p. 280-284.
Kobayashi T, Kageyama Y, Sumitomo N, Saeki K, Shirai T, and Ito S. Contribution of a salt bridge triad to the thermostability of a highly alkaline protease from an alkaliphilic Bacillus strain. World J Microbiol Biotechnol. 2005. Volume 21. p. 961–967.
Edited by Ankur Patel of Rachel Larsen and Kit Pogliano