Leah H. Hochanadel

Horizontal gene transfer is an important evolutionary process by which DNA is exchanged between cells that are physically co-located but not direct evolutionary descendants. Horizontal transfer of highly divergent DNA is relatively easy to detect and can produce major phenotypic changes, exemplified by the acquisition of antibiotic resistance determinants. However, transfer of high-identity DNA, for example, between strains of the same species, is likely to be more frequent, harder to detect, and highly impactful in aggregate. In this work, we demonstrate that soil isolates of the alphaproteobacterium Novosphingobium aromaticivorans can exchange chromosomal DNA, leading to multiple unselected recombination events spanning approximately 10% of the chromosome. Chromosomal recombination was directional and more efficient near an integrative and conjugative element (ICE), and required a relaxase found in the ICE. Recombination could not be observed in strains from closely related Novosphingobium species. In combination, these results suggest that ICE-mediated recombination can efficiently recombine DNA within N. aromaticivorans , increasing the adaptive potential of the species while also enforcing species boundaries through preferential intraspecific recombination. IMPORTANCE: Horizontal gene transfer is a key process in bacterial evolution. Mechanisms for transfer of mobile genetic elements are well-characterized, but less is known about how chromosomal DNA is recombined. In this work, we demonstrate that integrative and conjugative elements can efficiently recombine chromosomal DNA between strains of Novosphingobium aromaticivorans but not between different Novosphingobium species. We conclude that integrative and conjugative element-mediated chromosomal recombination can be an important adaptive mechanism within a species, due to its ability to recombine nearby chromosomal alleles, but also serves to delineate species-specific gene pools as a result of its limited phylogenetic range.

Genetic mapping is a powerful tool for eukaryotic genetics that has only been applied to bacteria in limited circumstances. Quantitative trait locus (QTL) mapping generally relies on sexual recombination to break linkages between genes, yet bacteria rarely undergo sufficient homologous recombination to generate suitable mapping populations. In this work, we used iterative biparental genome shuffling by protoplast fusion in Bacillus subtilis to generate a population of bacteria with substantial random recombination throughout their genomes. Individual shuffled progeny were arrayed in well plates, resequenced, and characterized for a range of complex phenotypes, including spore germination and swarming motility. Genetic mapping of the resulting phenotypes identified high-confidence QTLs of moderate size (~15 kb), and these associations were validated through targeted genetic swaps. This B. subtilis QTL population can easily be used to map additional phenotypes and the general approach for QTL mapping is applicable to a wide range of Gram-positive and Gram-negative bacteria using diverse methods for genome-wide recombination.

Horizontal gene transfer is an important evolutionary process by which DNA is exchanged between cells that are physically co-located but not direct evolutionary descendants. Horizontal transfer of highly divergent DNA is relatively easy to detect and can produce major phenotypic changes, exemplified by acquisition of antibiotic resistance determinants. However, transfer of high-identity DNA, for example between strains of the same species, is likely to be more frequent, harder to detect, and highly impactful in aggregate. In this work, we demonstrate that recombination between soil isolates of the alphaproteobacterium Novosphingobium aromaticivorans can exchange chromosomal DNA, leading to multiple unselected recombination events spanning approximately 10% of the chromosome. Chromosomal recombination was directional, more efficient near an integrative and conjugative element (ICE), and required a relaxase found in the ICE. Recombination could not be observed into strains from closely related Novosphingobium species. In combination, these results suggest that ICE-mediated recombination can efficiently recombine DNA within N. aromaticivorans , increasing the adaptive potential of the species while also enforcing species boundaries through preferential intraspecific recombination. Importance: Horizontal gene transfer is a key process in bacterial evolution. Mechanisms for transfer of mobile genetic elements are well-characterized, but less is known about how chromosomal DNA is recombined. In this work, we demonstrate that integrative and conjugative elements can efficiently recombine chromosomal DNA between strains of Novosphingobium aromaticivorans but not between different Novosphingobium species. We conclude that ICE-mediated chromosomal recombination can be an important adaptive mechanism within a species, due to its ability to recombine nearby chromosomal alleles, but also serves to delineate species-specific gene pools as a result of its limited phylogenetic range.