Multicopy Single-Stranded DNA Directs Intestinal Colonization of Enteric Pathogens

Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.
Author Summary Multicopy single-stranded DNA (msDNA) is a unique molecule consisting of both an RNA and DNA portion. This molecule is produced by a reverse transcriptase and has no known natural function despite more than 30 years of study. We report that msDNA is important for both Salmonella Typhimurium and an enteropathogenic E. coli, two pathogens that cause diarrhea in susceptible hosts, to survive in the intestine. Using mutant strains incapable of producing msDNA, we show that msDNA is needed for Salmonella to grow in the absence of oxygen. Mutants grown in oxygen-deficient conditions have substantial changes in overall protein composition, including numerous proteins known to be important for anaerobic metabolism and growth in the intestine. Our findings link msDNA to the ability of Salmonella to thrive in an oxygen-deficient environment similar to the conditions inside the gut. We report that msDNA regulates the quantity of proteins, the first natural function attributed to this molecule. msDNA may represent a new class of regulatory molecules.