Group Selection as a Basis for Screening Mutagenized Libraries of Public Goods (Bacillus thuringiensis Cry Toxins).

The pesticidal toxins of Bacillus thuringiensis (Bt) supply the active proteins for genetically modified insect-resistant crops. There is therefore keen interest in finding new toxins, or improving known toxins, in order to increase the mortality of various targets. The production and screening of large libraries of mutagenized toxins are among the means of identifying improved toxins. Since Cry toxins are public goods, and do not confer advantages to producers in competition, conventional directed evolution approaches cannot be used here. Instead, thousands of individual mutants have to be sequenced and assayed individually, a costly and time-consuming process. In this study, we tested a group selection-based approach that could be used to screen an uncharacterized pool of Cry toxin mutants. This involved selecting for infectivity between subpopulations of Bt clones within metapopulations of infected insects in three rounds of passage. We also tested whether additional mutagenesis from exposure to ethyl methanesulfonate could increase infectivity or supply additional Cry toxin diversity during passage. Sequencing of pools of mutants at the end of selection showed that we could effectively screen out Cry toxin variants that had reduced toxicity with our group selection approach. The addition of extra mutagenesis during passage decreased the efficiency of selection for infectivity and did not produce any additional novel toxin diversity. Toxins with loss-of-function mutations tend to dominate mutagenized libraries, and so a process for screening out these mutants without time-consuming sequencing and characterization steps could be beneficial when applied to larger libraries. IMPORTANCE Insecticidal toxins from the bacterium Bacillus thuringiensis are widely exploited in genetically modified plants. This application creates a demand for novel insecticidal toxins that can be used to better manage resistant pests or control new or recalcitrant target species. An important means of producing novel toxins is via high-throughput mutagenesis and screening of existing toxins, a lengthy and resource-intensive process. This study describes the development and testing of an efficient means of screening a test library of mutagenized insecticidal toxins. Here, we showed that it is possible to screen out loss-of-function mutations with low infectivity within a pool without the need to characterize and sequence each mutant individually. This has the potential to improve the efficiency of processes used to identify novel proteins.

Selecting for infectivity across metapopulations can increase virulence in the social microbe Bacillus thuringiensis.

Passage experiments that sequentially infect hosts with parasites have long been used to manipulate virulence. However, for many invertebrate pathogens, passage has been applied naively without a full theoretical understanding of how best to select for increased virulence and this has led to very mixed results. Understanding the evolution of virulence is complex because selection on parasites occurs across multiple spatial scales with potentially different conflicts operating on parasites with different life histories. For example, in social microbes, strong selection on replication rate within hosts can lead to cheating and loss of virulence, because investment in public goods virulence reduces replication rate. In this study, we tested how varying mutation supply and selection for infectivity or pathogen yield (population size in hosts) affected the evolution of virulence against resistant hosts in the specialist insect pathogen Bacillus thuringiensis, aiming to optimize methods for strain improvement against a difficult to kill insect target. We show that selection for infectivity using competition between subpopulations in a metapopulation prevents social cheating, acts to retain key virulence plasmids, and facilitates increased virulence. Increased virulence was associated with reduced efficiency of sporulation, and possible loss of function in putative regulatory genes but not with altered expression of the primary virulence factors. Selection in a metapopulation provides a broadly applicable tool for improving the efficacy of biocontrol agents. Moreover, a structured host population can facilitate artificial selection on infectivity, while selection on life-history traits such as faster replication or larger population sizes can reduce virulence in social microbes.