Appl Microbiol Biotechnol DOI 10.1007/s00253-015-7259-9 APPLIED MICROBIAL AND CELL PHYSIOLOGY An in-depth characterization of the entomopathogenic strain Bacillus pumilus 15.1 reveals that it produces inclusion bodies similar to the parasporal crystals of Bacillus thuringiensis Diana C. Garcia-Ramon1 & C. Alfonso Molina1,3 & Antonio Osuna1 & Susana Vílchez1,2 Received: 9 July 2015 /Revised: 13 December 2015 /Accepted: 19 December 2015 # Springer-Verlag Berlin Heidelberg 2016 Abstract In the present work, the local isolate Bacillus microscopy for morphological characterization, and we ob- pumilus 15.1 has been morphologically and biochemically served geometric structures resembling the parasporal crystal characterized in order to gain a better understanding of this inclusions synthesized by Bacillus thuringiensis. Some of the novel entomopathogenic strain active against Ceratitis results obtained here such as the parasporal inclusion bodies capitata. This strain could represent an interesting produced by B. pumilus 15.1 could potentially represent viru- biothechnological tool for the control of this pest. Here, we lence factors of this novel and potentially interesting strain. report on its nutrient preferences, extracellular enzyme pro- duction, motility mechanism, biofilm production, antibiotic Keywords Entomopathogenic bacteria . Crystal inclusions . suceptibility, natural resistance to chemical and physical in- Bacillus pumilus . UV resistance sults, and morphology of the vegetative cells and spores. The pathogen was found to be β-hemolytic and susceptible to penicillin, ampicillin, chloramphenicol, gentamicin, kanamy- Introduction cin, rifampicin, tetracycline, and streptomycin. We also report a series of biocide, thermal, and UV treatments that reduce the Bacillus pumilus was described by Meyer and Gottheil in viability of B. pumilus 15.1 by several orders of magnitude. 1901 (Skerman et al. 1989). B. pumilus belongs to the Heat and chemical treatments kill at least 99.9 % of vegetative Bacillus genus, which comprises of 304 recognized species cells, but spores were much more resistant. Bleach was the to date (9th of December 2015) (Euzeby 1997), several of only chemical that was able to completely eliminate them of high ecological, biotechnological, and medical rele- B. pumilus 15.1 spores. Compared to the B. subtilis 168 vance. B. pumilus is ubiquitous in the environment and has spores, B. pumilus 15.1 spores were between 2.67 and 350 been isolated from soil (Garbeva et al. 2003; Padaria et al. times more resistant to UV radiation while the vegetative cells 2014; Shagimardanova et al. 2014), sea, and marine sediments of B. pumilus 15.1 were almost up to 3 orders of magnitude (Liu et al. 2013; Nithya and Pandian 2010)aswellas more resistant than the model strain. We performed electron fermented foods (Afifah et al. 2014; Yamanaka et al. 2007). The bacterium is associated with animals (Parvathi et al. 2009; Zhang et al. 2009) and plants either as an endophyte (Asraful * Susana Vílchez Islam et al. 2010; Ren et al. 2013)orasanepiphyte(Caoetal. [email protected] 2001). Furthermore, B. pumilus hasbeenisolatedfromunusu- al places such as interior parts of Sonoran desert basalt 1 Institute of Biotechnology, Campus Fuentenueva s/n, University of (Benardini et al. 2003), from a spacecraft assembly facility, Granada, 18071 Granada, Spain and the surface of the International Space Station (La Duc 2 Department of Biochemistry and Molecular Biology I, Campus et al. 2003; Newcombe et al. 2005). Fuentenueva s/n, University of Granada, 18071 Granada, Spain B. pumilus has a high economic relevance owing to the 3 Present Address: International Center for Zoonosis (CIZ), Faculty of wide range of applications that this microorganism and its Veterinary Medicine and Zootechnic, Central University of Ecuador, products have in biotechnology, industry, biopharma, and en- Quito, Ecuador vironmental sectors. Many B. pumilus strains have been used Appl Microbiol Biotechnol for degrading xenobiotic compounds (Hayase et al. 2004; thuringiensis var. israeliensis IPS 78/11 (Ward and Ellar Meyers et al. 1991), as plant growth promoters (de-Bashan 1983), B. thuringiensis var. kurstaki HD1 (Dulmage 1970), et al. 2010;Thomas2004), as antimicrobial agents (Aunpad two B. pumilus strains (B. pumilus M1 and M2) isolated from and Na-Bangchang 2007; Ouoba et al. 2007), or as animal and the sea and kindly provided by Dr. C. Calvo (Uad et al. 2007), human probiotics (Duc le et al. 2004; Prieto et al. 2014). B. subtilis 168 (Burkholder and Giles 1947), Bacillus cereus Several B. pumilus strains are known to exhibit biological 569 (Benedict et al. 1945), Pseudomonas putida MAX10 activity useful for biocontrol of many fungal species of (Manzanera et al. 2004), Burkholderia cepacia DSM 9241 Aspergillus, Penicillium, Fusarium, Phytophthora, (Palleroni and Holmes 1981; Yabuuchi et al. 1992), and Rhizoctonia,andPythium (Asraful Islam et al. 2010; Escherichia coli XL1 Blue (Bullock et al. 1987). Luria- Munimbazi and Bullerman 1998). Traditionally, B. pumilus Bertani (LB) medium was routinely used for growing bacteria. has not been considered an entomopathogenic species. When sporulation was required, liquid T3 medium (Travers et However, a few cases of B. pumilus strains with activity al. 1987) was used and cultures were kept at 30 °C and against insects have been reported (Ertürk et al. 2008;Heins 240 rpm for 72 h. E. coli was grown at 37 °C while the rest et al. 1999;Molinaetal.2010; Yaman et al. 2010). The pat- of the strains were grown at 30 °C, unless otherwise stated. ented B. pumilus AQ717 strain, which is active against corn rootworm (Heins et al. 1999), was able to produce metabolites Characterization of metabolic profile in the supernatant of the whole culture that were useful as biocontrol agents. Similarly, strains of B. pumilus were report- Commercially available plates with 95 biochemical tests, fre- ed to be active against the Colorado potato beetle, quently used to type bacterial strains, were used to determine Leptinotarsa decemlineata, larvae (Ertürk et al. 2008) and the biochemical profile of several strains. Biolog GP2 the great spruce bark beetle, Dendroctonus micans, larvae MicroPlates™ (Biolog Catalog #1014) were used following (Yaman et al. 2010). Although these strains are described to manufacturer’s instructions. The Bacillus strains were cul- be active against insects, little is known about them and the tured overnight in LB medium at 30 °C and 240 rpm. The toxicity-causing agent. bacterial cultures were diluted in 18 ml of sterile gelling inoc- We previously reported the isolation of a novel strain, ulating fluid (Biolog Catalog #72101) to a final suspension B. pumilus 15.1, active against Ceratitis capitata (Diptera: with 28 % of transmittance, and 150 μl of the bacterial sus- Tephritidae) larvae, the Mediterranean fruit fly (Molina et al. pensions was transferred into each well. After 4 and 24 h of 2010), one of the worst worldwide distributed insect pests incubation at 30 °C, wells were visually compared with the affecting more than 300 fruits and vegetables of agriculture negative control well. All wells visually similar to the negative importance (Szyniszewska and Tatem 2014). Furthermore, we control were scored as negative and all wells with a noticeable also described a method for increasing the toxicity of this purple color were scored as positive. Each strain was tested strain (Molina et al. 2009). B. pumilus could play an important three times. Those compounds weakly used by the strain role as a biological control agent in pest management in the could show variation in color production in each repetition. future as many other new emerging entomopathogenic bacte- rial species (Ruiu 2015), so an in-depth characterization of it Protease, lipase, catalase, and hemolytic activity was needed in order to get practical knowledge of the strain and to further optimize its virulence. In this work, we centered Extracellular protease activity was assayed by methods previ- our efforts on microbiological, morphological, and biochem- ously described with minor modifications. Very briefly, 10 μl ical characterization of B. pumilus 15.1 in order to acquire a of an overnight culture in LB of the strain was placed on LB better knowledge of this biotechnological interesting strain. plates supplemented with casein 2 % (Montville 1983), or Our results provide some interesting insights into potential skimmed milk powder 3 % (Rahman et al. 2007), and grown virulence factors of B. pumilus 15.1 strain that could be used at optimal temperature. The ability to produce proteases was for controling C. capitata populations. determined by the formation of clearing zones around the colonies on the solid turbid media after 24 and 48 h. Extracellular lipase activity was assayed using LB plates Materials and methods containing olive oil 3 % (v/v) and rhodamine B 0.1 % (w/v) (Solaiman et al. 2001). Ten microliters of an overnight culture Bacterial strains and culture conditions in LB was placed on testing plates and incubated at optimal temperature for 48 h. Extracellular lipase activity was revealed Ten bacterial strains were used in this study: the B. pumilus by the fluorescent halo appearing around the bacterial biomass strain 15.1 (Spanish Type Culture Collection ref. CECT 7462) when visualized under UV light (320 nm). Strains with no with entomopathogenic activity against C. capitata (Molina fluorescence halo were considered negative to extracellular et al. 2010),