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Lysinibacillus sphaericus:
Classification:
Kingdom: Bacteria Phylum: Bacillota Class: Bacilli Order: Bacillales Family: Bacillaceae Genus: Lysinibacillus Species: sphaericus
Fig: Evolutionary relationships between Lysinibacillus type strains and a selection of additional strains based on the alignment of partial 16S rRNA sequences. (Failor et al. ,2019)
Using 16s rRNA, investigation of the evolutionary relationships was done within the Bacillaceae family and specifically focused on the genus Lysinibacillus. The analysis revealed that Lysinibacillus shares a close evolutionary relationship with several genera, such as Bhargavaea, Domibacillus, Falsibacillus, Kurthia, Paenisporosarcina, Planomicrobium, Planococcus, Psychrobacillus, Solibacillus, Sporosarcina, and Viridibacillus (Failor et al.,2019). Aditionally, a specific 16S rRNA tree focused on Lysinibacillus species and confirmed that Lysinibacillus forms a monophyletic group with strong support. The monophyletic nature of the Lysinibacillus genus was further confirmed using 260 protein sequences from genomes of 52 strains. Genome sequences 20 type strains, additional strains were confirmed from the NCBI database, and five Bacillus strains. Most of the genome-sequenced Lysinibacillus strains belonged to the same clade as Lysinibacillus fusiformis or Lysinibacillus boronitolerans, or to a clonal lineage of mosquitocidal isolates. Importantly, the analysis revealed that the mosquitocidal Lysinibacillus lineage is distinct from the type strain L. sphaericus Gibson 1013T, suggesting the possibility of additional species within the genus (Failor et al.,2019).
Cell structure
Colony morphology: Lysinibacillus sphaericus is a Gram-positive bacteria, indicating that it has a thick peptidoglycan layer in its cell wall. It is aerobic and motile. The shape of these bacteria is rod-like, with a length range of 0.6–1.0 µm and a width range of 1.5–5.0 µm (Failor et al., 2019). It can produce spores, which are round-shaped and formed in terminally located swollen sporangia. Their colonies have a circular shape with wavy edges and a smooth and glossy surface. Their cell wall is composed of L-Lys-D-Asp (A4α) type. Its primary menaquinone is MK-7. The primary polar lipids include DPG, PG, PE, PNL, and one unidentified lipid. The three major fatty acids present in them are iso-C15:0, C16:1 ω7c alcohol, and iso-C17:1 ω10c (Failor et al., 2019). L. sphaericus strains can produce bacteriocins, which are protein antibiotics that are specifically effective against other strains of the same species (Jamal and Ahmad, 2022). These bacteriocins can be considered antibacterial toxins. Bacteriocins produced by Lysinibacillus sphaericus might help eliminate competing strains, secure resources, and defend against harmful microorganisms, increasing their chances of survival. They are resistant to several antibiotics such as ampicillin, cephalothin, gentamycin, lincomycin, polymixin B, and streptomycin. They also display weak resistance to other antibiotics such as chloramphenicol, erythromycin, and rifampicin.
Ecological habitat
They are found in a variety of ecological habitats, including soil, water, and insect gut environments, particularly mosquitoes. This bacterium is known to be a versatile microorganism that can adapt to a variety of environmental conditions, including temperature, pH, and nutrient availability. They are capable of growing in environments with temperatures of around 30°C. They can survive and reproduce in the alkaline environment of mosquito larval midgut which has a pH of around 11 (Glare et al., 2017). They are known for their ability to survive harsh environments due to their formation of endospores. This allows the bacteria to remain dormant until more favorable conditions are present. Therefore, this ability to survive under unfavorable conditions enables them to colonize a wide range of environments.
Lysinibacillus sphaericus: It is capable of growing and forming spores inside the gut, which then release toxin crystals that kill the larvae. (Arrow shows the toxin crystal) https://doi.org/10.1016/j.jip.2011.11.008
Ecological Lifestyle and Interactions
L. sphaericus is pathogenic to mosquito larvae. They can produce proteins that are toxic to mosquito larvae, with the most potent being the binary (Bin) toxin. This toxin is effective against larvae when ingested, binding to specific receptors in the midgut and causing cell damage, ultimately leading to larval death. This Bin toxin produced by them has a specific mode of action against certain mosquito species, particularly Culex and Anopheles, which are of medical importance as vectors of diseases such as malaria and filariasis. They have also been found to be effective in managing populations of black flies and non-biting midges. Importantly, the toxin is safe to non-target organisms. Therefore, their effectiveness in killing mosquitoes varies depending on the type of toxins produced and the species of mosquito. In some cases, they can have antagonistic effects on other organisms. For example, L. sphaericus strains have been shown to produce antimicrobial compounds that inhibit the growth of fungi. One study investigated the potential of L. sphaericus ZA9 to promote plant growth and combat phytopathogenic fungi (Naureen et al., 2017). The bacterium enhanced seedling vigor and promoted shoot length in cucumber and tomato seeds. The bacterium also showed varied antagonistic behavior against several fungi such as Alternaria. alternata, Sclerotinia sp, Curvularia lunata, and Trichophyton spp. Two compounds, 2-pentyl-4-quinoline carboxylic acid, and 1-methylcyclohexane isolated from its fermentation broth culture were responsible for exhibiting antifungal activity (Naureen et al., 2017).
Genome Structure, content, and Gene Expression
The genome of L. sphaericus 2362, an entomopathogenic strain, was sequenced in the study by Hernández-Santana et al (2016). This strain was first isolated from adult Simulium damnosum (black fly) in Nigeria in 1984 and has been widely used as a reference in various studies. Once the mosquitocidal activity of L. sphaericus 2362 was confirmed, it was introduced to the WHO Collaborating Centre in Columbus, OH, USA (Hernández-Santana et al., 2016). Since then, this strain has been extensively utilized not just for a reference in research about larvicidal activity but also as a comparative model for exploring its tolerance to metals, the structure of surface proteins such as the S-layer, and the production of biosurfactants (Hernández-Santana et al., 2016). The complete circular chromosome of the genome is 4.67 megabases (Mb) long, with a GC content of 37.3%, and no plasmids were found (Hernández-Santana et al., 2016). The sequencing process generated 102,634 DNA sequences with an average length of 9,902 base pairs (bp) and a coverage of 197 times. The genome annotation identified 4,538 genes, including protein-coding sequences, pseudogenes, and RNA-coding genes (107 tRNAs, 37 rRNAs, and 5 ncRNAs) (Hernández-Santana et al., 2016). In another study by Viersanova et al (2021), L. sphaericus isolate 6.2, pathogenic to Culex quinquefasciatus mosquitoes, was analyzed. Its genomic characteristics, including a G+C content of 37.1% and a genome size of 4.6 Mbp, were similar to the reference strain by WHO L. sphaericus 2362. The sequencing was performed using Pacific Biosciences technology at McGill University and the Génome Québec Innovation Centre in Quebec, Canada. The data were assembled using the Hierarchical Genome Assembly Process (HGAP) and the Celera Assembler methods. HGAP was used for the initial assembly, while the Celera Assembler was employed to correct any sequencing errors specifically on the long reads (Hernández-Santana et al., 2016). The L. sphaericus 2362 genome contains genes encoding larvicidal toxins, such as binary toxins (BinA and BinB) and Mtx toxins. Interestingly, 12 copies of the S-layer gene, which plays a role in pathogenicity, were identified. The expression of the S-layer protein can be regulated through genomic rearrangements among these copies. The S-layer protein in L. sphaericus works in conjunction with other toxin proteins during spore formation and exhibits hemolytic activity, causing damage to the target cells and contributing to the bacterium's ability to control mosquito populations. However, L. sphaericus isolate 6.2 lacks certain toxins (Bin, Cry, and sphaericolysin) but possesses Mtx toxins (Mtx1, Mtx2, Mtx3, and Mtx4) (Viersanova et al.,2021). The isolate also contains genes for the S-layer protein, hemolysin which contributes to its pathogenic activity against Culex quinquefasciatus populations resistant to binary toxins and Aedes aegypti mosquitoes (Viersanova et al.,2021).
Significance to Humans
L. sphaericus has several significant influences on human society, particularly in the field of biological control and public health. It is best known for its ability to control mosquito populations, especially those that transmit diseases such as malaria and dengue fever (Santana-Martinez et al., 2019). Their binary toxins are toxic to mosquito larvae and harmless to other organisms. It has been widely used as a biological larvicide in mosquito control programs, helping to reduce the burden of mosquito-borne diseases in many parts of the world. Mosquito-borne diseases have a significant impact on human health, causing widespread illness and death, especially in tropical and subtropical regions. By targeting mosquito larvae, L. sphaericus plays an important role in protecting public health. (Silva-Filha et al., 2021) The use of L. sphaericus-based larvicides offers several environmental benefits as compared to chemical insecticides. Some strains specifically target mosquito larvae, minimizing harm to other organisms and reducing the overall impact on the ecosystem (Santana-Martinez et al., 2019). Additionally, it is biodegradable and poses a lower risk of resistance development compared to some chemical insecticides. It has also shown potential for agricultural applications. Some studies have explored its use as a biopesticide to combat agricultural pests, such as certain beetle larvae and caterpillars (Jamal and Ahmad, 2022). In one study, Lysinibacillus species treated seeds improved the growth and biomass yield of maize plants in both normal and zinc-stressed conditions. The treated plants showed enhanced root development, shoot length, and increased absorption of zinc. The study suggests that using zinc-tolerant Lysinibacillus species can be beneficial for growing maize in agricultural fields with zinc exposure (Jamal and Ahmad, 2022). Some strains have exhibited capabilities in removing or detoxifying heavy metals like lead and arsenic from contaminated environments. These bacteria can be potentially employed in bioremediation processes for cleaning up polluted sites or industrial wastewater. Biodegradation using microorganisms is currently considered the most effective strategy for removing estrogens from the environment which is a naturally occurring hormone that is found in humans and animals. It can be present in the environment as a result of wastewater discharge. A recent study showed that L. sphaericus DH-BO1 which was isolated from wastewater treatment in Beijing can efficiently degrade estrogens. It is the only strain of L. sphaericus known to degrade it. Therefore, it has the potential to be used for bioremediation and environmental restoration in different environments contaminated with estrogen. It also has potential applications in biomining for gold extraction and removing gold from industrial wastewater (Jamal and Ahmad, 2022). The industrialization has brought economic growth but also introduced harmful environmental contaminants. The challenge is to achieve a balance between positive effects and minimizing adverse impacts. Lysinibacillus-based techniques provide a solution by reducing contaminants in water, air, and soil (Jamal and Ahmad, 2022). These beneficial bacteria can replace artificial chemicals, reducing toxicity and ecological disruption. Therefore, Lysinibacillus shows potential in areas such as biopesticides, stimulating plant growth, and bioremediation (Jamal and Ahmad, 2022).
Interesting feature:
The most interesting feature of L. sphaericus is its ability to produce protein toxins that are specifically toxic to certain species of mosquitoes. There are various types of toxins produced by this bacterium, including sphaericolysin, vegetative mosquitocidal toxins, and bin toxins (Berry, 2012). Sphaericolysin is lethal when injected into certain insects like the German cockroach and the common cutworm. L. sphaericus can produce vegetative mosquitocidal toxins during its growth phase, including Mtx1 and Mtx2 toxins (Berry, 2012). Mtx1 toxin is effective against Chironomus riparius larvae, while the Mtx2 protein can kill mosquitoes such as C. quinquefasciatus and A. aegypti (Berry 2012; Santana-Martinez et al., 2019). The Bin or Binary toxin is deposited as a crystal and is highly toxic to mosquito larvae. When ingested by larvae, the Bin toxin is solubilized and undergoes limited proteolysis to increase its toxicity, which occurs in both susceptible and non-susceptible insects. Overall, L. sphaericus is a potent agent for controlling mosquito populations. The bacterium is not able to persist when ponds or pools dry out and then re-flood. However, its spores ingested by invertebrates other than mosquitoes can remain viable, and midges emerging from treated water might carry spores to new locations (Berry, 2012). The bacterium can grow in dead mosquito larvae and produce spores associated with Bin crystals, which are as toxic as those produced in artificial media. This can help maintain the control of mosquito populations if the dead larvae remain in shallow water where new larvae feed. L. sphaericus is beneficial to humans because it can help control certain mosquito populations that can transmit serious diseases like malaria, dengue fever, and filariasis. This is achieved by producing toxins that are harmful to mosquitoes and can reduce their larvae in water sources. As a result, L. sphaericus can be used as a natural and safe alternative to chemical insecticides that can harm both humans and the environment (Santana-Martinez et al., 2019). L. sphaericus is effective at killing insects when lots of its spores are used in the field. However, it is important to understand how it naturally behaves in the environment such as how it persists and cycles and if lower doses of bacteria can be used to control insects (Berry, 2012).
Introduction:
Lysinibacillus sphaericus is found in different environments such as soil, water, and the gut of insects, especially mosquitoes. It is an aerobic, mesophilic, spore-forming, and Gram-positive bacterium. It was previously known as Bacillus sphaericus, but due to its unique cell wall composition and other characteristics, it was later reclassified under the genus Lysinibacillus (Ahmed et al., 2007). Certain strains of L. sphaericus bacteria can be toxic to mosquito larvae, which makes them useful for controlling the spread of diseases such as malaria, filariasis, dengue fever, etc. Therefore, due to its larvicidal effect against mosquito genera Culex and Anopheles, this bacterium is of interest to the World Health Organization (WHO) (Hernández-Santana et al., 2016). Apart from this, it also has been studied for its potential to tolerate metals. It has a high level of tolerance to heavy metals such as arsenic, copper, lead, mercury, zinc etc. and has been demonstrated to be capable of removing these metals from contaminated water sources through both its living and dead cells (Lozano et al., 2013). Lysinibacillus bacteria have the potential to be useful in bioremediation, as they have a diverse range of metabolic capabilities that enable them to break down and remove various pollutants in the environment. However, it cannot metabolize carbohydrates due to the absence of an important enzyme called glucose-6-phosphate isomerase (Gomez-Garzon et al., 2017). This characteristic is shared by all groups of L. sphaericus including those that are toxic to mosquitoes. As a substitute for glucose, L. sphaericus uses acetate or glycerol as a carbon source, which is converted to acetyl-CoA and enters the TCA cycle (Gomez-Garzon et al., 2017). Therefore, acetate or glycerol is used in media to culture L. sphaericus instead of glucose.
https://doi.org/10.4056/sigs.4227894
https://doi.org/10.4056/sigs.4227894
https://doi.org/10.4056/sigs.4227894
References:
Ahmed I, Yokota A, Yamazoe A, Fujiwara T. 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. International Journal of Systematic and Evolutionary Microbiology. 2007 May;57(5):1117-25. https://pubmed.ncbi.nlm.nih.gov/17473269/
Berry, C. (2012). The bacterium, Lysinibacillus sphaericus, as an insect pathogen. Journal of Invertebrate Pathology, 109(1), 1-10. https://doi.org/10.1016/j.jip.2011.11.008
Failor, K.C., Tian, L., Monteil, C.L. and Vinatzer, B.A. (2019). Lysinibacillus. In Bergey's Manual of Systematics of Archaea and Bacteria (eds M.E. Trujillo, S. Dedysh, P. DeVos, B. Hedlund, P. Kämpfer, F.A. Rainey and W.B. Whitman). https://doi-org.proxy-tu.researchport.umd.edu/10.1002/9781118960608.gbm01570
Filha MH, Berry C, Regis L. Lysinibacillus sphaericus: Toxins and mode of action, applications for mosquito control and resistance management. InAdvances in insect physiology 2014 Jan 1 (Vol. 47, pp. 89-176). Academic Press. https://www.sciencedirect.com/science/article/abs/pii/B9780128001974000038
Glare TR, Jurat-Fuentes JL, O’callaghan M. Basic and applied research: entomopathogenic bacteria. InMicrobial control of insect and mite pests 2017 Jan 1 (pp. 47-67). Academic press. https://www.sciencedirect.com/science/article/abs/pii/B9780128035276000044?via%3Dihub#kwrds0010
Gomez-Garzon C, Hernandez-Santana A, Dussan J. A genome-scale metabolic reconstruction of Lysinibacillus sphaericus unveils unexploited biotechnological potentials. PLoS One. 2017 Jun 12;12(6):e0179666. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179666
Jamal QM, Ahmad V. Lysinibacilli: A Biological Factories Intended for Bio-Insecticidal, Bio-Control, and Bioremediation Activities. Journal of Fungi. 2022 Dec;8(12):1288. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9783698/
Lozano, L.C., Dussán, J. Metal tolerance and larvicidal activity of Lysinibacillus sphaericus . World J Microbiol Biotechnol 29, 1383–1389 (2013). https://doi.org/10.1007/s11274-013-1301-9
Naureen Z, Rehman NU, Hussain H, Hussain J, Gilani SA, Al Housni SK, Mabood F, Khan AL, Farooq S, Abbas G, Harrasi AA. Exploring the potentials of Lysinibacillus sphaericus ZA9 for plant growth promotion and biocontrol activities against phytopathogenic fungi. Frontiers in microbiology. 2017 Aug 17;8:1477.
https://www.frontiersin.org/articles/10.3389/fmicb.2017.01477/full
Peña-Montenegro, T.D. and Dussán, J., 2013. Genome sequence and description of the heavy metal tolerant bacterium Lysinibacillus sphaericus strain OT4b. 31. Standards in genomic sciences, 9(1), pp.42-56. https://doi.org/10.4056/sigs.4227894
Santana-Martinez JC, Silva JJ, Dussan J. Efficacy of Lysinibacillus sphaericus against mixed-cultures of field-collected and laboratory larvae of Aedes aegypti and Culex quinquefasciatus. Bulletin of entomological research. 2019 Feb;109(1):111-8. https://pubmed.ncbi.nlm.nih.gov/29784071/
Silva-Filha MH, Romão TP, Rezende TM, Carvalho KD, Gouveia de Menezes HS, Alexandre do Nascimento N, Soberón M, Bravo A. Bacterial toxins active against mosquitoes: Mode of action and resistance. Toxins. 2021 Jul 27;13(8):523. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402332/
Wang Y, Zhao X, Tian K, Meng F, Zhou D, Xu X, Zhang H, Huo H. Identification and genome analysis of a novel 17β-estradiol degradation bacterium, Lysinibacillus sphaericus DH-B01. 3 Biotech. 2020 Apr;10:1-1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066359/
