Abstract
Clostridium butyricum strains that atypically produce the botulinum neurotoxin type E (BoNT/E) possess a megaplasmid of unknown functions in their genome. In this study, we cured two botulinum neurotoxigenic C. butyricum type E strains of their megaplasmids, and compared the obtained megaplasmid-cured strains to their respective wild-type parental strains. Our results showed that the megaplasmids do not confer beta-lactam resistance on the neurotoxigenic C. butyricum type E strains, although they carry several putative beta-lactamase genes. Instead, we found that the megaplasmids are essential for growth of the neurotoxigenic C. butyricum type E strains at the relatively low temperature of 15°C, and are also relevant for growth of strains under limiting pH and salinity conditions, as well as under favorable environmental conditions. Moreover, the presence of the megaplasmids was associated with increased transcript levels of the gene encoding BoNT/E in the C. butyricum type E strains, indicating that the megaplasmids likely contain transcriptional regulators. However, the levels of BoNT/E in the supernatants of the cured and uncured strains were similar after 24 and 48 h culture, suggesting that expression of BoNT/E in the C. butyricum type E strains is not ultimately controlled by the megaplasmids. Together, our results reveal that the C. butyricum type E megaplasmids exert pleiotropic effects on the growth of their microbial hosts under optimal and limiting environmental conditions, and also highlight the possibility of original regulatory mechanisms controlling the expression of BoNT/E.
Highlights
Microorganisms of the Clostridium butyricum species have been attracting increasing interest as they produce butyric acid which has many chemical, pharmaceutical and biotechnological applications, and are capable of converting glycerol to 1,3-propanediol, a monomer of the production of plastics (Szymanowska-Powałowska et al, 2014)
Except for botulinum neurotoxin (BoNT)/G, which is produced by the rare C. argentinense species, the other BoNT serotypes are formed by heterogeneous microorganisms conventionally grouped within the C. botulinum species; botulinum neurotoxin type E (BoNT/E) and BoNT/F can be produced by sporadic strains of the typically non-neurotoxigenic C. butyricum and C. baratii species, respectively (Hatheway, 1992)
Since the four selected latt, bl, mb, and dp beta-lactamase genes are located within the megaplasmids of the WT C. butyricum type E strains, as previously reported based on the analysis of the available draft genome sequences of two Italian C. butyricum type E strains (GenBank Reference Numbers: NZ_ACOM00000000.1 and NZ_ABDT00000000.1) and on the genetic maps obtained for the megaplasmids (Iacobino et al, 2013), the negative beta-lactamase PCR results suggest the loss of the megaplasmid
Summary
Microorganisms of the Clostridium butyricum species have been attracting increasing interest as they produce butyric acid which has many chemical, pharmaceutical and biotechnological applications, and are capable of converting glycerol to 1,3-propanediol, a monomer of the production of plastics (Szymanowska-Powałowska et al, 2014). Clostridium butyricum Type E Megaplasmids strains have been shown to exert health benefits in humans and animals and are used as probiotics (Shinnoh et al, 2013; Zhao et al, 2013). Certain C. butyricum strains produce the botulinum neurotoxin (BoNT), which is one of the most toxic proteins to humans and some vertebrate animals (Rossetto et al, 2014). Except for BoNT/G, which is produced by the rare C. argentinense species, the other BoNT serotypes are formed by heterogeneous microorganisms conventionally grouped within the C. botulinum species; BoNT/E and BoNT/F can be produced by sporadic strains of the typically non-neurotoxigenic C. butyricum and C. baratii species, respectively (Hatheway, 1992)
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