Lysinibacillus sphaericus C3-41

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Classification

Higher order taxa

Domain: Bacteria Kingdom: Bacteria Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Planococcaceae Genus: Lysinibacillus Species: Lysinibacillus sphaericus

Also known as Bacillus sphaericus

Species

Lysinibacillus sphaericus C3-41 or Bacillus sphaericus C3-41

Description and significance

Lysinibacillus sphaericus C3-41 is a naturally occurring soil bacterium. It is a Gram-positive, mesophilic, rod-shaped bacterium. Under harsh conditions, Lysinibacillus sphaericus can form dormant endospores that are resistant to heat, chemicals, and ultraviolet light. These spores may remain viable for a long time. Although it is typically a facultative anaerobe, L. sphaericus may be anaerobic under certain conditions (Todar, K). The organism was isolated from a mosquito breeding site in China in 1987 (Pei, G.). It is a common environmental organism which produces an insecticidal toxin similar to that produced by Bacillus thuringiensis (see the NCBI Entrez Genome Project webpage at http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj).

Lysinibacillus sphaericus C3-41 is an important organism to study because it can be used as an insecticidal toxin that controls mosquito growth. This organism, along with similar organisms, is utilized in insect control programs to reduce the population of disease vector species that transmit diseases such as malaria, yellow fever, and West Nile virus (for more info, see the Washington State Department of Health webpage at http://www.doh.wa.gov/). Genome sequencing of this organism is useful because it increases our knowledge of the bacilli and also offers insight for future improvement of important biological control agents (Hu, et al., 2008).

Another name for this organism is Bacillus sphaericus (NCBI Entrez Genome Project). The genus change was proposed by Ahmed et al. in 2007 based on distinctive peptidoglycan composition of the cell wall, as well as phylogenetic and physiological analyses (Ahmed, et al., 2007)


Genome structure

The complete genome of Lysinibacillus sphaericus C3-41 is composed of a circular chromosome of 4,639,821 bp containing 4,786 protein-coding sequences. The genome also includes a large plasmid (177,642 bp containing 186 protein-coding sequences). The circular chromosome has an average GC content of 37.29% and the plasmid has an average GC content of 33.10% (Hu, et al., 2008).

Within the chromosome, 85 tRNA genes representing 20 amino acids were identified as were 10 rRNA operons. A likely origin of replication was identified by comparing the genome to corresponding regions in Bacillus subtilis. Four genes (rpmH, dnaA, dnaN, and recF) near the origin, the presence of multiple dnaA boxes, and AT-rich sequences upstream of the dnaA gene helped to identify the origin of replication (Hu, et al., 2008).

Although the replication termination site is usually found opposite to the origin, Lysinibacillus sphaericus C3-41 differs from B. subtilis in this case, and is found slightly offset from the point opposite to the origin. Although this is not unique to Lysinibacillus sphaericus C3-41, it is unusual (Hu, et al., 2008).

CcpA (catabolite control protein A), HPr, and Crh were identified in the genome of Lysinibacillus sphaericus C3-41. CcpA is a transcriptional regulator. CcpA is a global transcriptional regulator found in Gram positive bacteria, and acts to regulate carbon and nitrogen metabolism by interacting with regulatory sites in the control regions of the regulated operon to repress or activate transcription (Hu, et al., 2008).

Formation of Lysinibacillus sphaericus C3-41 spores is triggered by a deficiency in nutrients and is linked to the expression of several genes that function as sporulation initiation proteins. Based on the comparison of Lysinibacillus sphaericus C3-41 genome to other Bacilli, four CDSs found in the chromosome and plasmid of Lysinibacillus sphaericus C3-41 may regulate the developmental pathway for sporulation. These include Bsph_0057, Bsph_113, Bsph_0076, and Bsph_2825 (Hu, et al., 2008).

The toxin genes of Lysinibacillus sphaericus C3-41 are distributed throughout the chromosome. This is unlike many other bacterial pathogens whose virulent genes are grouped within the genome. The binary toxin genes (binA and binB) of Lysinibacillus sphaericus C3-41 are the main source of toxicity toward mosquito larvae. Interestingly, these genes are found in a 35 kb duplicate in both the chromosome and the plasmid. In addition to the binary toxin genes, a Bsph_3195/Bsph_157 encodes a protein homologous to Mtx2/3 toxins, which are another family of mosquitocidal toxins synthesized during vegetative growth (Wirth, et al., 2007; Hu, et al., 2008).

The binary toxin genes and the 35 kb duplicate fragment are unique to Lysinibacillus sphaericus. This may be a result of a phage infection that only occurred in Lysinibacillus sphaericus and not in any other Bacillus species (Hu, et al., 2008).

Additionally, the chromosome contains homologs of several genes involved in pathogenicity of many other gram-positive bacteria including "B. cereus" and Listeria monocytogenes. These genes might be part of a common attack mechanism found in some gram-positive pathogenic bacteria (Hu, et al., 2008).


Cell structure and metabolism

The vegetative Lysinibacillus sphaericus cells are Gram-positive. This indicates that they contain peptidoglycan layers that are penetrated by teichoic acids, and a proteinaceous S-layer. Cell wall polysaccharides help anchor the S-layer to the cell wall (Hu, et al., 2008).

Lysinibacillus sphaericus are unable to metabolize polysaccharides. Their lack of specific enzymes and sugar transport systems are most likely the reason for this inability. The abundance of proteolytic enzymes and transport systems allow Lysinibacillus sphaericus to use exclusive metabolic pathways to use a wide variety of organic compounds and amino acids. Although the inability to metabolize carbohydrates is a known characteristic of Lysinibacillus sphaericus, the details of its energy metabolism remain to be investigated (Hu, et al., 2008).

When the vegetative cells are deprived of nutrients, they may form endospores. A septum forms within the vegetative cell that produces a mother cell and a genome-containing compartment called the forespore. When the spore is formed, the mother cell lyses and releases the spore into the environment. Spores can survive in the soil and other harsh environments for many years because of their resistance to physical and chemical damage. When conditions are normal again and proper nutrients are available, the spores germinate and grow as vegetative cells (Slonczewski and Foster, 2009).

The Lysinibacillus sphaericus endospores are eaten by mosquito larvae and release toxins into the larvae’s gut. This causes the larvae to stop eating and die. These toxic endospores do not affect mosquito pupae or adults (Washington State Department of Health).


Ecology

Lysinibacillus sphaericus C3-41 is a naturally occurring, mesophilic, soil bacterium. It is able to kill mosquito larvae present in water that is rich in organic matter. The organism is able to survive, reproduce, and release endotoxins in the intestine of mosquito larvae. Because the midgut of the mosquito larvae is an alkaline environment of approximately pH 11, we know that the spores of Lysinibacillus sphaericus C3-41 are alkaliphiles (Boudko et al., 2001).

B. sphaericus is also used for probiotic purposes as a component of yoghurt starter. It is not significantly harmful to humans when ingested and is considered as a probiotic ingredient. This probiotic comprises enzymes, amino acids, anti-inflammatory compounds and colostrum. (Chandan, 2006)

B. sphaericus is one of approximately 400 bacteria that live in the human digestive tract. (Cichoke, 1997)

Pathology

Lysinibacillus sphaericus C3-41 is toxic to mosquito larvae. When bacteria are consumed by mosquito larvae, they penetrate the intestines of the mosquito larvae and enter the hemocoel. In the hemocoel, the bacteria release an endotoxin that causes the larvae to stop eating and die (Pham et al., 1998).

The insecticidal property of this organism is due to two proteins produced during sporulation. These proteins are the binary toxins, which accumulate as crystal inclusions. These proteins bind to the gastric cavity and midgut of the mosquito larvae that ingested the spores, disrupt its feeding, and cause it to die. Mosquitocidal toxins (Mtx proteins) are produced during vegetative growth of the bacteria and also contribute to the organism’s insecticidal property. Some Lysinibacillus sphaericus strains also produce another two-component toxin (Cry48 and Cry49 proteins) during sporulation (Hu, et al., 2008).

Lysinibacillus sphaericus is only effective against feeding mosquito larvae. It does not affect mosquito pupae or adults (see http://www.doh.wa.gov/). For this reason, certain Lysinibacillus sphaericus strains have been developed as commercial larvicides and are used to control mosquito populations in order to reduce mosquito-born diseases. Lysinibacillus sphaericus C3-41 is one such strain and has been used to control mosquito larvae population in China for more than 10 years (Hu, et al., 2008). Commercially, Lysinibacillus sphaericus is known as VectoLex. It is highly toxic to the Culex genus of mosquitoes and somewhat toxic to the Anopheles and Aedes genera of mosquitoes (Pham et al., 1998).

Though Bacillus sphaericus is toxic to mosquitos, and larvae, has not been reported to be a harmful bacteria in either human or animal populations. It may cause a skin rash with heavy or prolonged exposure in some people. The Department of Health recommends washing with soap and water as a method of cleansing in the unlikely event of heavy exposure. (probiotic.org)

Application to Biotechnology

Lysinibacillus sphaericus C3-41 is used as an insecticide because its spores release endotoxins that kill mosquito larvae. These toxins are ideal agricultural tools. Instead of using chemicals that have adverse effects on humans, other animals, and aquatic life, these bacteria can be used to control mosquito population and, therefore, control disease vectors. Because the bacteria is short-lived and are not harmful to humans and other animals, Lysinibacillus sphaericus C3-41 is an ideal insecticide (Hu, et al., 2008). The organism is used commercially by licensed applicators to control mosquito populations. Granules containing this organism can be added to standing water in mosquito breeding areas or can be sprayed in the air in a liquid form.

Current Research

Although Lysinibacillus sphaericus C3-41 is an attractive biological insecticide, efforts are being made to increase its toxicity and manufacturing techniques. For example, it has been found that growing Lysinibacillus sphaericus in an egg yolk-based media increases toxicity and early sporulation. This results in a more cost-effective means, shorter fermentation time, higher activity yield, and increased biomass when compared to conventional growth mediums (Prabakaran et al., 2008).

A toxin taken from B. thuringiensis called israelensis dipteran toxin has been inserted into Lysinibacillus sphaericus to enhance its toxic activity against certain mosquito larvae. These modifications are being made by inserting genes on plasmid vectors that also carry antibiotic resistance. This and other manipulations to Lysinibacillus sphaericus can increase the range of mosquito genera that are affected by its toxins (Poncet et al., 1994). Potentially, this approach could be used to control mosquito populations that help spread significant human diseases such as malaria and Dengue.

Recently, B. sphaericus has been used in genetic engineering due to its characteristic of being unable to use carbon-based sugars. (Pérez-Martínez , 2003).

The longevity of the B. sphaericus strain is of interest to many researchers. In 1995, a researcher examining insects in fossilized amber estimated to be 25 to 40 million years old, recovered bacterial spores of this specific bacteria. Laboratory investigation following up with the finding showed some distinctions in the genetic structure of the recovered bacteria and modern bacteria, eliminating the possibility of the original sample as contaminated. (Monastersky, 1995).

References

[1] Ahmed, et al. Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov. Int J Syst Evol Microbiol. 2007 May;57(Pt 5):1117-25.

[2] Bacillus sphaericus. Agave Biosystems. 30 Nov. 2008 <http://www.agavebio.com/Catalog/contentframe.html>

[3] Boudko, Dimitri et al., “In situ Analysis of pH Gradients in Mosquito Larvae Using Noninvasive, Self-Referencing, pH-Sensitive Microelectrodes.” Journal of Experimental Biology. February 2001; 204: 691-699.

[4] Hu, Xiaomin et al., “Complete Genome Sequence of the Mosquitocidal Bacterium Bacillus sphaericus C3-41 and Comparison with Those of Closely Related Bacillus Species.” Journal of Bacteriology. April 2008; 190(8): 2892-2902.

[5] NCBI Entrez Genome Project. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj.

[6] Pham, Chuong et al., “Investigating the Effects of Bacillus sphaericus (Vectolex) on Aedes Larvae and Non-Target Organisms.” Northeastern Mosquito Control Association. December 1998.

[7] Poncet, S. et al., “Transfer and Expression of the cryIVB and cryIVD Genes of Bacillus thuringiensis subspecies israelensis in Bacillus sphaericus 2297. FEMS Microbial Lett. 1994; 117: 91-96.


[8] Prabakaran, G et al., “Egg Yolk Enhances Early Sporulation and Toxicity of Bacillus sphaericus H5a5b for Small-Scale Production of a Mosquito Control Agent. Acta Tropica. October 2008; 108: 50-53.

[9] Todar, K. "Todar's Online Textbook of Bacteriology."

[10] Washington State Department of Health. “Larvicide: Bacillus sphaericus.” February 2008. <http://www.doh.wa.gov/ehp/ts/ZOO/WNV/larvicides/Bsphaericus.html> .

[11] Wirth, M. et al., “Mtx Toxins Synergize Bacillus sphaericus and Cry11Aa against Susceptible and Insecticide-Resistant Culex quinquefasciatus Larvae.” Applied and Environmental Microbiology. October 2007; 73(19): 6066-6071.

Manufacturing yogurt and fermented milks By Ramesh C. Chandan, Feb 2006, pg 312

[12] PROBIOTICS BALANCE: DIGESTION AND IMPROVE OVERALL HEALTH By Anthony Cichoke, D.C. ; Nutrition Science news August 1997-VoI.2, No. 8, pp.380-382

[13] "Bacillus Sphaericus." Bacillus Sphaericus. Probiotic.org,

[14] The genes encoding functional components of the phosphoenolpyruvate phosphotransferase system (HPr,EI) and N-acetyl glucosamine metabolism form a gene cluster in Bacillus sphaericus. Microbiology, 149, 1687-1698 (2003) by Alice AF, Pérez-Martínez G, y Sánchez-Rivas C.

[15] Science News, 20 May 1995, Ancient bacteria brought back to life by Monastersky, R. thefreelibrary.com/.


Edited by, Zehra Samani, Megan Foxworth, Bridget Paliwoda, and Niloufar Aghakasiri, students of Dr. Maia Larios-Sanz at University of St. Thomas , Irina Rojas

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