Conjugation across Bacillus cereus and kin: A review.

Horizontal gene transfer (HGT) is a major driving force in shaping bacterial communities. Key elements responsible for HGT are conjugation-like events and transmissible plasmids. Conjugative plasmids can promote their own transfer as well as that of co-resident plasmids. Bacillus cereus and relatives harbor a plethora of plasmids, including conjugative plasmids, which are at the heart of the group species differentiation and specification. Since the first report of a conjugation-like event between strains of B. cereus sensu lato (s.l.) 40 years ago, many have studied the potential of plasmid transfer across the group, especially for plasmids encoding major toxins. Over the years, more than 20 plasmids from B. cereus isolates have been reported as conjugative. However, with the increasing number of genomic data available, in silico analyses indicate that more plasmids from B. cereus s.l. genomes present self-transfer potential. B. cereus s.l. bacteria occupy diverse environmental niches, which were mimicked in laboratory conditions to study conjugation-related mechanisms. Laboratory mating conditions remain nonetheless simplistic compared to the complex interactions occurring in natural environments. Given the health, economic and ecological importance of strains of B. cereus s.l., it is of prime importance to consider the impact of conjugation within this bacterial group.

Conjugation-mediated transfer of pXO16, a large plasmid from Bacillus thuringiensis sv. israelensis, across the Bacillus cereus group and its impact on host phenotype.

pXO16, the 350 kb-conjugative plasmid from Bacillus thuringiensis sv. israelensis promotes its own transfer at high efficiency, triggers the transfer of mobilizable and non-mobilizable plasmids, as well as the transfer of host chromosomal loci. Naturally found in B. thuringiensis sv. israelensis, pXO16 transfers to various strains of Bacillus cereus sensu lato (s.l.) at a wide range of frequencies. Despite this host diversity, a paradox remains between the relatively large host spectrum and the natural occurrence of pXO16, so far restricted to B. thuringiensis sv. israelensis. Proposing first insights exploring this paradox, we investigated the behaviour of pXO16 amongst different members of the B. cereus group. We first looked at the transfer of pXO16 to two new host clusters of B. cereus s.l., Bacillus mycoides and Bacillus anthracis clusters. This examination brought to light the impairment of the characteristic rhizoidal phenotype of B. mycoides in presence of pXO16. We also explored the stability of pXO16 at different temperatures as some B. cereus group members are well-known for their psychro- or thermo-tolerance. This shed light on the thermo-sensitivity of the plasmid. The influence of pXO16 on its host cell growth and on swimming capacity also revealed no or limited impact on its natural host B. thuringiensis sv. israelensis. On the contrary, pXO16 affected more strongly both the growth and swimming capacity of other B. cereus s.l. hosts. This reinforced the running hypothesis of a co-evolution between pXO16 and B. thuringiensis sv. israelensis, enabling the plasmid maintenance without impairing the host strain development.

New Insights into the Potential Cytotoxic Role of Bacillus cytotoxicus Cytotoxin K-1.

The thermotolerant representative of the Bacillus cereus group, Bacillus cytotoxicus, reliably harbors the coding gene of cytotoxin K-1 (CytK-1). This protein is a highly cytotoxic variant of CytK toxin, initially recovered from a diarrheal foodborne outbreak that caused the death of three people. In recent years, the cytotoxicity of B. cytotoxicus has become controversial, with some strains displaying a high cytotoxicity while others show no cytotoxicity towards cell lines. In order to better circumscribe the potential pathogenic role of CytK-1, knockout (KO) mutants were constructed in two B. cytotoxicus strains, E8.1 and E28.3. The complementation of the cytK-1 KO mutation was implemented in a mutant strain lacking in the cytK-1 gene. Using the tetrazolium salt (MTT) method, cytotoxicity tests of the cytK-1 KO and complemented mutants, as well as those of their wild-type strains, were carried out on Caco-2 cells. The results showed that cytK-1 KO mutants were significantly less cytotoxic than the parental wild-type strains. However, the complemented mutant was as cytotoxic as the wild-type, suggesting that CytK-1 is the major cytotoxicity factor in B. cytotoxicus.

TipB, a novel cell wall hydrolase, is required for efficient conjugative transfer of pXO16 from Bacillus thuringiensis sv. israelensis.

pXO16, a large plasmid from Bacillus thuringiensis serovar israelensis, exhibits unique features. Not only is pXO16 able to transfer at high frequencies within few minutes, but it is also able to transfer among virtually all members of the Bacillus cereus group. Among the proteins encoded in the tip transfer locus of pXO16, TipB displays an atypical organization compared to known conjugative cell wall hydrolases with the large central soluble lytic transglycosylase (SLT) domain missing from the protein. We constructed a tipB deletion mutant which led to significant reduction in transfer efficiencies in both liquid and filter mating. The initial transfer frequencies could be restored when complementing tipB in trans thus showing the TipB implication in pXO16 conjugative transfer. Additionally, truncated versions of TipB were expressed in Escherichia coli to assess the protein lytic activity. When applied exogenously, TipB-2TM, in which the two N-terminal TM domains were removed, yielded a decrease of ca. 40% in optical density of B. thuringiensis sv. israelensis, a lytic activity that could partially be explained by the C-terminal CHAP-like domain. These results confirm TipB conjugative hydrolase function and provide new insights into pXO16 unique conjugative apparatus.

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

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.

A novel T4SS-mediated DNA transfer used by pXO16, a conjugative plasmid from Bacillus thuringiensis serovar israelensis.

The entomopathogenic Bacillus thuringiensis serovar israelensis displays peculiar conjugative transfer capabilities, accounted for by the large conjugative plasmid pXO16 (350 kb). The efficient and fast conjugative transfers are accompanied by a macroscopic aggregation of bacterial partners. Moreover, pXO16 has proven capable of effective mobilization and the retro-transfer of both mobilizable and 'non-mobilizable' plasmids. In this work, the aggregation phenomenon is shown to promote pXO16 transfer while not being mandatory for transfer. Transfer of pXO16 to B. thuringiensis recipient strains that do not display aggregation is observed as well, hence enlarging the previously defined host range. The use of variant calling analysis of transconjugants allowed for observation of up to 791 kb chromosomal regions mobilization. Previous analysis of pXO16 did not reveal any Type IV Secretion System (T4SS) homologs, which suggested the presence of an unusual conjugative system. A FtsK/SpOIIIE ATPase gene proved here to be necessary for conjugative transfer. Additionally, the analysis of natural restriction-modification systems in both conjugative partners gave credit to a ssDNA transfer mechanism. A 'transfer israelensis plasmid' (tip) region containing this ATPase gene was shown to code for other potential T4SS proteins, illustrating a conjugative system distantly related to the other known Gram-positive T4SSs.