Project Grant R01GM154193

CONTRIBUTION OF INSERTION SEQUENCE MEDIATED TANDEM CHROMOSOMAL AMPLIFICATION TO BACTERIAL HETEROGENEITY - THE LONG-TERM GOAL OF THE APPLICATION IS TO UNDERSTAND HOW INSERTION SEQUENCES (IS) IN BACTERIAL GENOMES CREATE HETEROGENEOUS SUBPOPULATIONS BY AMPLIFYING CHROMOSOMAL SEGMENTS. IS ARE SHORT, MOBILE DNA ELEMENTS INTEGRATED INTO BACTERIAL CHROMOSOMES AND PLASMIDS THAT ENCODE A TRANSPOSE FLANKED BY INVERTED TERMINAL REPEATS (ITR). IS ACTIVITY PLAYS A MAJOR ROLE IN SHAPING PROKARYOTIC GENOMES, WHERE THEY CAN DESTROY GENE FUNCTION THROUGH INTRAGENIC TRANSPOSITION, INDUCE EXPRESSION BY JUXTAPOSITION OF OUTWARD FACING PROMOTER ELEMENTS, REMOVE/REARRANGE LARGE SEGMENTS OF GENOMES BY RECOMBINATION EVENTS, AND TANDEM AMPLIFY DNA SEGMENTS INTERVENING BETWEEN TWO IS ELEMENTS. THIS PROPOSAL FOCUSES ON THE LATTER PROCESS, AS IT IS THE LEAST WELL UNDERSTOOD DESPITE ARGUABLY BEING THE MOST COMMON AND EFFECTIVE MECHANISM FOR GENERATING HETEROGENEOUS POPULATIONS. CHROMOSOMAL TANDEM AMPLIFICATION CATALYZED BY IS ELEMENTS GENERALLY OCCURS AT MUCH HIGHER FREQUENCIES IN COMPARISON TO POINT MUTATIONS, AMPLIFIES INTERVENING SEGMENTS OF DNA THAT CAN BE HUNDREDS OF KILOBASES LONG, AND RESULTS IN HEAD-TO-TAIL TANDEM COPIES OF DNA SEGMENTS SEPARATED BY A THIRD, HYBRID IS ELEMENT. IN GENOMES WITH HIGH IS CONTENT, AMPLIFICATION THUS GENERATES TREMENDOUS HETEROGENEITY AND TRANSIENT SINGLE CELL INDIVIDUALITY THAT IS REVERTIBLE THROUGH SUBSEQUENT RECOMBINATION BETWEEN THE AMPLIFIED SEGMENTS. HETEROGENEITY IN BACTERIAL POPULATIONS HELPS ENSURE SURVIVAL IN THE EVENT OF ENVIRONMENTAL STRESS (INCLUDING ANTIBIOTIC TREATMENT) BY SAMPLING A LARGER POOL OF CELLS WITH DISTINCT PHENOTYPES. THE CONNECTION BETWEEN IS ELEMENT ACTIVITY, GENOME AMPLIFICATION, AND HETEROGENEITY WILL BE EXPLORED IN THIS PROPOSAL USING A MODEL SYSTEM OF ESCHERICHIA COLI B WITH THE ELEMENT IS1. IN THE FIRST AIM, HOW GENOMIC IS1 ELEMENT DISTRIBUTION IMPACTS FREQUENCY OF AMPLIFICATION AND THE REQUIREMENTS FOR AMPLIFICATION WILL EXPLORED. IN THE SECOND AIM, GENETIC AND ENVIRONMENTAL FACTORS WILL BE IDENTIFIED USING A TN-SEQ ASSAY THAT HAS BEEN DEVELOPED TO BE SPECIFIC FOR DEFINED IS1-FLANKED CHROMOSOMAL AMPLIFICATION EVENTS. IN THE THIRD AIM, THE CONTRIBUTION OF IS1 TO CREATING DISTINCT LIPOPOLYSACCHARIDE COMPOSITIONS AND GENE EXPRESSION PROFILES AT THE SINGLE CELL LEVEL WILL BE EXPLORED USING A PAIRED IS1-LESS STRAIN. BY UNDERSTANDING IS1-INDUCED HETEROGENEITY IN A MODEL BACTERIAL STRAIN, THE PROJECT WILL HELP FORMULATE RATIONALE STRATEGIES TO MITIGATE THE IMPACT OF HETEROGENEITY ON ANTIMICROBIAL BACTERIAL RESISTANCE.