Original Research ArticlepXO16, the large conjugative plasmid from Bacillus thuringiensis serovar israelensis displays an extended host spectrum
Introduction
pXO16 is a large conjugative plasmid associated with the entomopathogen Bacillus thuringiensis serovar israelensis. It is used as biopesticide to control insect larvae of mosquitoes and blackflies, the vectors of animal and human diseases. This entomopathogenic activity results from the production, during sporulation, of delta-endotoxins (Cry) and cytolysins (Cyt), which form crystals in the bacterial sporanges (Lacey, 2007; Palma et al., 2014). B. thuringiensis belongs to Bacillus cereus sensu lato (s.l.), a group of closely related bacteria whose members display differential virulence (Jensen et al., 2003; Okinaka and Keim, 2016). Bacillus anthracis is a mammal and human pathogen, while some strains of B. cereus sensu stricto are known as foodborne pathogens, provoking emesis or diarrhoea with a potentially lethal outcome. Interestingly, most of these bacteria harbour a plethora of plasmids, including conjugative and mobilizable ones. Moreover, some of the extrachromosomal molecules contain the genetic determinants for the virulent genes, as is the case for the virulent plasmids pXO1 and pXO2 of B. anthracis, the causative agent of anthrax (Hu et al., 2009).
One intriguing element of this extrachromosomal pool is pXO16. This 350-kb conjugative plasmid transfers itself in a fast and efficient way (100% within <5 min) (Andrup et al., 1998). Another peculiar feature of pXO16 is its macroscopic aggregation phenotype displayed during conjugation (Andrup et al., 1993; Jensen et al., 1995). Typically, within minutes of contact, donor and recipient cells aggregate and form visible clumps that last for several minutes until pXO16 transfer is achieved. Recently, a 25-kb “aggregation” locus was identified in pXO16 sequence. Deletion of this region hampered the appearance of aggregation. However, although the transfer efficiency was reduced in the aggregation-minus mutant, pXO16 could be transferred at frequencies of ca. 10−4 transconjugants per donor cell (T/D). Similar transfer reductions were observed for mobilization of small plasmids (Makart et al., 2018).
In this paper, we report the transfer of pXO16 aggregation-minus mutant in colony and filter matings. We have also further investigated pXO16 conjugation and mobilization properties among other members of the B. cereus group using filter mating. In these conditions, and contrary to what was previously thought, the host range of this B. thuringiensis serovar israelensis plasmid appears to cover various members of the group, including its most distantly related kin, the thermotolerant Bacillus cytotoxicus.
Section snippets
Bacterial strains and plasmids
All bacterial strains and plasmids used in this study are reported in Table 1. Bacteria were grown in Lysogeny Broth (LB) medium containing NaCl (5 g L−1), yeast extract (5 g L−1) and Tryptone (10 g L−1). LB was solidified with 1.4% (w/v) agar for agar plates. When appropriate, antibiotics (Sigma) were added at the following concentrations (μg mL−1): Sm, streptomycin (100); Nd, nalidixic acid (15); Tc, tetracycline (10); Cm, chloramphenicol (10); Rf, rifampicin (50); Km, kanamycin (50).
Spontaneous antibiotic-resistant mutants
pXO16 aggregation is dispensable in filter and colony matings
One of the predictions of the study on pXO16 aggregation-minus mutant (Makart et al., 2018) was that the transfer deficit, in liquid medium, might be compensated by providing alternative physical contact between donor and recipient cells. In order to test this hypothesis, two complementary experiments were performed: the mating partners were either mixed together as drops on the surface of LB plates (drop-on-drop or colony mating) or were first deposited on a single filter and then incubated on
Discussion
Discovered in 1979, in a mosquito pond in Israel, B. thuringiensis sv. israelensis rapidly became an important biopesticide widely used to control mosquitoes and blackflies, in particular in countries where these insects contribute to the spread of human and animal diseases (Ben-Dov, 2014). Also named serotype H14, these bacteria contain a large pool of extrachromosomal molecules, including small, large and very large circular plasmids, as well as a linear prophage (Bolotin et al., 2017; Gillis
Acknowledgements
This work was supported by the National Foundation for Scientific Research (FNRS, grants to PH and JM), the Université catholique de Louvain (UCLouvain, grants to NF and KMK), the Research Department of the Communauté française de Belgique (Concerted Research Action, ARC N° 17/22-084, grant to AL), and the National Council for Scientific Research in Lebanon (CNRS-L, grant to NF).
References (26)
- et al.
Kinetics of conjugative transfer: a study of the plasmid pXO16 from Bacillus thuringiensis subsp. israelensis
Plasmid
(1998) - et al.
Comparative genomics of extrachromosomal elements in Bacillus thuringiensis subsp. israelensis
Res. Microbiol.
(2017) - et al.
Detection of the cryptic prophage-like molecule pBtic235 in Bacillus thuringiensis subsp. israelensis
Res. Microbiol.
(2017) - et al.
Horizontal transfer of chromosomal markers mediated by the large conjugative plasmid pXO16 from Bacillus thuringiensis serovar israelensis
Plasmid
(2017) - et al.
Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation
J. Bacteriol.
(1993) Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins
Toxins (Basel)
(2014)- et al.
CalY is a major virulence factor and a biofilm matrix protein
Mol. Microbiol.
(2018) - et al.
Bacterial sexuality at the nanoscale
Nano Lett.
(2018) - et al.
Role of plasmid plasticity and mobile genetic elements in the entomopathogen Bacillus thuringiensis serovar israelensis
FEMS Microbiol. Rev.
(2018) - et al.
Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus group occasionally associated with food poisoning
Int. J. Syst. Evol. Microbiol.
(2013)
Genetic diversity of clinical isolates of Bacillus cereus using multilocus sequence typing
BMC Microbiol.
Family portrait of Bacillus cereus and Bacillus weihenstephanensis cereulide-producing strains
Environ. Microbiol. Rep.
Sympatric soil communities of Bacillus cereus sensu lato: population structure and potential plasmid dynamics of pXO1- and pXO2-like elements
FEMS Microbiol. Ecol.
Cited by (13)
Prevalence, antimicrobial susceptibility, and antibiotic resistance gene transfer of Bacillus strains isolated from pasteurized milk
2023, Journal of Dairy ScienceCitation Excerpt :Escherichia coli was grown at 37°C in Luria Bertani broth. Transfer of tetracycline resistance gene tetL was determined by filter mating as described previously (Hinnekens et al., 2019) with slight modification. Both donor and recipient stains were incubated at 30 or 37°C to reach OD600 nm of 0.8, respectively.
Conjugation-mediated transfer of pXO16, a large plasmid from Bacillus thuringiensis sv. israelensis, across the Bacillus cereus group and its impact on host phenotype
2022, PlasmidCitation Excerpt :These data suggest that pXO16 replicon is thermosensitive, providing first hints on pXO16 replication since the delimitation of its putative replication region by Makart et al. (2015). Given that pXO16 was recently demonstrated to mobilise a co-resident plasmid and displace chromosomal loci from one strain of B. cytotoxicus to another (Hinnekens et al., 2019; Koné et al., 2021), a similar scenario could occur amongst other B. cereus species. Besides, as many key features of B. cereus s.l. are located on plasmids, pXO16 might blur the lines by moving about critical plasmids, like the capsule-encoding plasmid pXO2 from B. anthracis (Reddy et al., 1987).
TipB, a novel cell wall hydrolase, is required for efficient conjugative transfer of pXO16 from Bacillus thuringiensis sv. israelensis
2021, Research in MicrobiologyCitation Excerpt :Furthermore, recent work on pXO16 transfer focused on solid mating instead of liquid mating by passing donor and recipient cells through a filter or spotting on plates. When using these artificially compacted cell partners, the plasmid host spectrum was extended to virtually all members of the Bacillus cereus group [13]. Even though phenotypic characterization of pXO16 conjugation has been widely addressed, less information was available on the genetics of its transfer.
Development and regulation of microbial pesticides in the post-genomic era
2021, Biopesticides: Volume 2: Advances in Bio-inoculants