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Plasmid

Volume 102, March 2019, Pages 46-50
Plasmid

Original Research Article
pXO16, the large conjugative plasmid from Bacillus thuringiensis serovar israelensis displays an extended host spectrum

https://doi.org/10.1016/j.plasmid.2019.02.004Get rights and content

Highlights

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    Filter and colony matings can substitute for the aggregation of the conjugative plasmid pXO16.

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    pXO16 can efficiently conjugate to various members of B. cereus s.l., including its most distantly member B. cytotoxicus.

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    pXO16 mobilization of small, non-conjugative plasmids, is also extended to a broader host range.

Abstract

pXO16, the large conjugative plasmid from Bacillus thuringiensis serovar israelensis is able to efficient self-transfer, to mobilize and retro-mobilize non-conjugative plasmids, including “non-mobilizable” plasmids, and to transfer chromosomal loci. It also displays a remarkable aggregation phenotype associated with conjugation under liquid conditions. However, it was recently shown that aggregation boosts pXO16 transfer but is not mandatory. In this paper, we have further explored pXO16 transfers under various mating conditions and with different members of the Bacillus cereus group. The results indicated that colony or filter mating largely compensate the transfer deficit observed when using a pXO16 aggregation-minus mutant. Using filter mating, pXO16 transfer efficiency and host range were both improved. For instance, pXO16 was shown to transfer itself, and to mobilize the small pUB110 plasmid, from B. thuringiensis serovar israelensis to the thermotolerant Bacillus cytotoxicus at frequencies of 3.3 × 10−3 and 5.2 × 10−4 transconjugants per donor (T/D), respectively. All together, these results indicate that pXO16 can potentially “circulate” among members of the Bacillus cereus group. Yet, this is contrasting with pXO16's known natural distribution, which is apparently limited to the israelensis serovar of B. thuringiensis.

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).

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