Comparative Genomics of Bacillus thuringiensis Reveals a Path to Specialized Exploitation of Multiple Invertebrate Hosts

Abstract

Understanding the genetic basis of host shifts is a key genomic question for pathogen and parasite biology. The Bacillus cereus group, which encompasses Bacillus thuringiensis and Bacillus anthracis, contains pathogens that can infect insects, nematodes, and vertebrates. Since the target range of the essential virulence factors (Cry toxins) and many isolates is well known, this group presents a powerful system for investigating how pathogens can diversify and adapt to phylogenetically distant hosts. Specialization to exploit insects occurs at the level of the major clade and is associated with substantial changes in the core genome, and host switching between insect orders has occurred repeatedly within subclades. The transfer of plasmids with linked cry genes may account for much of the adaptation to particular insect orders, and network analysis implies that host specialization has produced strong associations between key toxin genes with similar targets. Analysis of the distribution of plasmid minireplicons shows that plasmids with orf156 and orf157, which carry genes encoding toxins against Lepidoptera or Diptera, were contained only by B. thuringiensis in the specialized insect clade (clade 2), indicating that tight genome/plasmid associations have been important in adaptation to invertebrate hosts. Moreover, the accumulation of multiple virulence factors on transposable elements suggests that cotransfer of diverse virulence factors is advantageous in terms of expanding the insecticidal spectrum, overcoming insect resistance, or through gains in pathogenicity via synergistic interactions between toxins.

Lineage‐specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group

“…The pXO2 plasmid of Ba was phylogenetically restricted to Ba, although plasmids with homology to pXO1 are widely distributed in clades 1 and 2 (Hu, Swiecicka, et al, 2009;Zheng et al, 2013). A large number of plasmids, including the Cry-bearing plasmids which possess orf156/157 minireplicons (Zheng et al, 2013), were phylogenetically restricted to clade 2 (Figures 4 and 5), as has been found previously (Zheng et al, 2017). Infectious cooperation, on the other hand, predicts that conjugative plasmids carrying social genes such as Cry toxins should be widely distributed across clades and show evidence of recent horizontal transfer.…”

“…Our genomic analysis highlights the inconsistency of Bc , Ba or Bt as species designations based upon phenotype comparisons, particularly for Bc and Bt that can share aspects of their ecology and do not represent discrete cohesive lineage clusters. However, all subsequent phylogenies of B. cereus group isolates, including this work and previous MLST studies, have shown that there are several cohesive genetically distinct clades in the B. cereus group (Cardazzo et al., ; Didelot et al., ; Guinebretière et al., ; Priest et al., ; Raymond, Wyres, et al., ; Sorokin et al., ; Vassileva et al., ; Vilas‐Boas, Sanchis, Lereclus, Lemos, & Bourguet, ; Zheng et al., ). The three major clades originally defined by MLST ( Ba and relatives—clade 1, B. kurstaki and Bc —clade 2 and B. weihenstephanensis —clade 3) were recovered in this study, although the distribution of predicted insecticidal genes and of isolates identified as B. weihenstephanensis and B. mycoides , indicates that there can be additional significant heterogeneity within these clades (Figures and ).…”

“…Isolates are ordered by the phylogeny from Figure 1 and plasmids from which >90% of genes were detected in at least 1 clade 2 isolate (n = 116) are clustered based on gene prevalence patterns inferred by WebGimm (Joshi et al, 2011) using the context-specific infinite mixture model (Freudenberg et al, 2010). The plasmid names in red indicate Cry-harbouring plasmids as inferred from a BtToxin_scanner analysis presented in Table S3 [Colour figure can be viewed at wileyonlinelibrary.com] Bt is also paraphyletic (Cardazzo et al, 2008;Didelot et al, 2009;Priest, Barker, Baillie, Holmes, & Maiden, 2004;Raymond & Bonsall, 2013;Raymond, Wyres, et al, 2010;Tourasse et al, 2011 should be treated as one species (Helgason et al, 2000;Tourasse, Helgason, Økstad, Hegna, & Kolstø, 2006 (Cardazzo et al, 2008;Didelot et al, 2009;Guinebreti ere et al, 2008;Priest et al, 2004;Raymond, Wyres, et al, 2010;Sorokin et al, 2006;Vassileva et al, 2006;Vilas-Boas, Sanchis, Lereclus, Lemos, & Bourguet, 2002;Zheng et al, 2017). In addition, there is abundant evidence for substantial ecological differentiation between clades, either in terms of their ability to colonize plants (Raymond, Wyres, et al, 2010;Vidal-Quist, Rogers, Mahenthiralingam, & Berry, 2013); their carriage of virulence factors such as enterotoxins (Cardazzo et al, 2008); the risks they pose to vertebrates (Cardazzo et al, 2008;Guinebreti ere et al, 2010;Raymond & Bonsall, 2013) or their metabolic and growth characteristics (Guinebreti ere et al, 2008).…”

Spores and soil from six sides: interdisciplinarity and the environmental biology of anthrax (Bacillus anthracis)

“…Bacillus anthracis shares a common chromosomal framework with all main species of the Bacillus cereus ( sensu lato ) super‐species group, including the opportunistic soil bacterium B. cereus , and the entomopathogenic B. thuringiensis , which (because of analogous plasmids occurring throughout the group) sometimes leads to blurred species boundaries (Helgason et al ., ; Priest et al ., ; Kolstø, Tourasse & Økstad, ; Zwick et al ., ; Bazinet, ; Zheng et al ., ). The chromosomal elements principally separating classic B. anthracis ( senso stricto ) from the other closely related species are: (1) the presence of four distinctive chromosomal prophage elements; (2) a specific, inactivating nonsense mutation in the transcription factor PlcR, a positive regulator mainly of chromosomally encoded extracellular virulence factors that are important during mammalian and insect infections by B. cereus and B. thuringiensis ; and (3) being part of the genetically monomorphic B. anthracis cluster by phylogenetic analysis (Kolstø et al ., ).…”

Spores and soil from six sides: interdisciplinarity and the environmental biology of anthrax (Bacillus anthracis)

“…Bacillus anthracis shares a common chromosomal framework with all main species of the Bacillus cereus (sensu lato ) super-species group, including the opportunistic soil bacterium B. cereus , and the entomopathogenic B. thuringiensis , which (because of analogous plasmids occurring throughout the group) sometimes leads to blurred species boundaries. 55–60 The chromosomal elements principally separating classic B. anthracis (senso stricto ) from the other closely related species are: 1) the presence of four distinctive chromosomal prophage elements; 2) a specific, inactivating nonsense mutation in the transcription factor PlcR, a positive regulator mainly of chromosomally encoded extracellular virulence factors that are important during mammalian and insect infections by B. cereus and B, thuringiensis ; and 3) being part of the genetically monomorphic B. anthracis cluster by phylogenetic analysis. 55 In addition, B. anthracis requires two large plasmids for full virulence: pXO1, which encodes the anthrax toxins, and pXO2, which encodes the protective poly-γ-D-glutamate capsule.…”