Dead but Not Forgotten: How Extracellular DNA, Moisture, and Space Modulate the Horizontal Transfer of Extracellular Antibiotic Resistance Genes in Soil.

Antibiotic-resistant bacteria and the spread of antibiotic resistance genes (ARGs) pose a serious risk to human and veterinary health. While many studies focus on the movement of live antibiotic-resistant bacteria to the environment, it is unclear whether extracellular ARGs (eARGs) from dead cells can transfer to live bacteria to facilitate the evolution of antibiotic resistance in nature. Here, we use eARGs from dead, antibiotic-resistant Pseudomonas stutzeri cells to track the movement of eARGs to live P. stutzeri cells via natural transformation, a mechanism of horizontal gene transfer involving the genomic integration of eARGs. In sterile, antibiotic-free agricultural soil, we manipulated the eARG concentration, soil moisture, and proximity to eARGs. We found that transformation occurred in soils inoculated with just 0.25 µg of eDNA g-1 soil, indicating that even low concentrations of soil eDNA can facilitate transformation (previous estimates suggested ∼2 to 40 µg eDNA g-1 soil). When eDNA was increased to 5 µg g-1 soil, there was a 5-fold increase in the number of antibiotic-resistant P. stutzeri cells. We found that eARGs were transformed under soil moistures typical of terrestrial systems (5 to 30% gravimetric water content) but inhibited at very high soil moistures (>30%). Overall, this work demonstrates that dead bacteria and their eARGs are an overlooked path to antibiotic resistance. More generally, the spread of eARGs in antibiotic-free soil suggests that transformation allows genetic variants to establish in the absence of antibiotic selection and that the soil environment plays a critical role in regulating transformation. IMPORTANCE Bacterial death can release eARGs into the environment. Agricultural soils can contain upwards of 109 ARGs g-1 soil, which may facilitate the movement of eARGs from dead to live bacteria through a mechanism of horizontal gene transfer called natural transformation. Here, we track the spread of eARGs from dead, antibiotic-resistant Pseudomonas stutzeri cells to live antibiotic-susceptible P. stutzeri cells in sterile agricultural soil. Transformation increased with the abundance of eARGs and occurred in soils ranging from 5 to 40% gravimetric soil moisture but was lowest in wet soils (>30%). Transformants appeared in soil after 24 h and persisted for up to 15 days even when eDNA concentrations were only a fraction of those found in field soils. Overall, our results show that natural transformation allows eARGs to spread and persist in antibiotic-free soils and that the biological activity of eDNA after bacterial death makes environmental eARGs a public health concern.

Transposable Elements Mediate Adaptive Debilitation of Flagella in Experimental Escherichia coli Populations.

Although insertion sequence (IS) elements are generally considered genomic parasites, they can mediate adaptive genetic changes in bacterial genomes. We discovered that among 12 laboratory-evolved Escherichia coli populations, three had experienced at least six different IS1-mediated deletions of flagellar genes. These deletions all involved the master flagellar regulator flhDC, and as such completely incapacitate motility. Two lines of evidence strongly suggest that these deletions were adaptive in our evolution experiment: (1) parallel evolution in three independent populations is highly unlikely just by chance, and (2) one of these deletion mutations swept to fixation within ~1000 generations, which is over two million times faster than expected if this deletion was instead selectively neutral and thus evolving by genetic drift. Because flagella are energetically expensive to synthesize and operate, we suspect that debilitating their construction conferred a fitness advantage in our well-stirred evolution experiment. These findings underscore the important role that IS elements can play in mediating adaptive loss-of-function mutations in bacteria.

Complete Genome Sequence of the Highly Transformable Pseudomonas stutzeri Strain 28a24.

Here, we report the complete genome sequence for an isolate of Pseudomonas stutzeri that is highly competent for natural transformation. This sequence enables insights into the genetic basis of natural transformation rate variations and provides an additional data point for genomic comparisons across a ubiquitous and highly diverse bacterial species.

The molecular basis of host specialization in bean pathovars of Pseudomonas syringae.

Biotrophic phytopathogens are typically limited to their adapted host range. In recent decades, investigations have teased apart the general molecular basis of intraspecific variation for innate immunity of plants, typically involving receptor proteins that enable perception of pathogen-associated molecular patterns or avirulence elicitors from the pathogen as triggers for defense induction. However, general consensus concerning evolutionary and molecular factors that alter host range across closely related phytopathogen isolates has been more elusive. Here, through genome comparisons and genetic manipulations, we investigate the underlying mechanisms that structure host range across closely related strains of Pseudomonas syringae isolated from different legume hosts. Although type III secretion-independent virulence factors are conserved across these three strains, we find that the presence of two genes encoding type III effectors (hopC1 and hopM1) and the absence of another (avrB2) potentially contribute to host range differences between pathovars glycinea and phaseolicola. These findings reinforce the idea that a complex genetic basis underlies host range evolution in plant pathogens. This complexity is present even in host-microbe interactions featuring relatively little divergence among both hosts and their adapted pathogens.

Relaxed natural selection alone does not permit transposable element expansion within 4,000 generations in Escherichia coli.

Insertion sequences (ISs) are transposable genetic elements in bacterial genomes. IS elements are common among bacteria but are generally rare within free-living species, probably because of the negative fitness effects they have on their hosts. Conversely, ISs frequently proliferate in intracellular symbionts and pathogens that recently transitioned from a free-living lifestyle. IS elements can profoundly influence the genomic evolution of their bacterial hosts, although it is unknown why they often expand in intracellular bacteria. We designed a laboratory evolution experiment with Escherichia coli K-12 to test the hypotheses that IS elements often expand in intracellular bacteria because of relaxed natural selection due to (1) their generally small effective population sizes (N (e)) and thus enhanced genetic drift, and (2) their nutrient rich environment, which makes many biosynthetic genes unnecessary and thus selectively neutral territory for IS insertion. We propagated 12 populations under four experimental conditions: large N (e) versus small N (e), and nutrient rich medium versus minimal medium. We found that relaxed selection over 4,000 generations was not sufficient to permit IS element expansion in any experimental population, thus leading us to hypothesize that IS expansion in intracellular symbionts may often be spurred by enhanced transposition rates, possibly due to environmental stress, coupled with relaxed natural selection.

Transposable element loads in a bacterial symbiont of weevils are extremely variable.

Not only are transposable elements profuse in the bacterial endosymbiont of maize weevils, but we found that their quantities also vary approximately 10-fold among individual weevils. Because multicopy elements can facilitate homologous recombination, this insertion sequence (IS) load variability suggests that these essentially asexual bacteria may exhibit substantial intraspecific genomic variation.