Virus-host interactions and microbial ecology This proposal encompasses two very different aspects of microbiology, both at cellular and group levels. (1) Probing E. coli genome organization and chromosome dynamics using phage Mu transposition as our tool. Mu transposition is unique not only in its high efficiency and lack of target specificity, but also in its transposition mechanism, which occurs by a nick-join rather than a cut-and-paste pathway. In the last grant period, we exploited these properties to measure in vivo rates of interactions between genomic loci in E. coli, and studied their proximity using new statistical tools. In a complete reversal of the current view of the E. coli genome, our analysis has revealed an uncompartmentalized, well-mixed genome, where transpositions occur freely between all measured loci. The analysis also revealed that several gene families (for example, six widely distributed ribosomal RNA operons) show `clustering' i.e. strong 3D co-localization regardless of linear genomic distance. The activities of the SMC/condensin complex MukBEF and the nucleoid-compacting protein HU-α are responsible for these properties. We propose to explore these phenomena to obtain a high- resolution view of genome organization, and to understand how it influences gene expression in bacteria. (2) Dissecting the mechanism of antibiotic tolerance under two specific growth conditions: swarming (moving as a collective), and c-di-GMP synthesis catalyzed by the diguanulate cyclase YfiN. Swarming bacteria can withstand exposure to antibiotics at concentrations that are lethal to their planktonic counterparts. We call this swarming-specific (non-genetic) resistance, SR. In the last grant period, we discovered that death of a sub- population as a result of antibiotic-induced killing, is beneficial to the swarm in promoting SR. Introduction of pre-killed cells into a swarm indeed enhanced SR, allowing us to purify the SR factor from killed cells of both E. coli and Salmonella. We identified the SR factor to be AcrA, a periplasmic component of a tripartite RND efflux pump; the outer membrane component of this pump, TolC, is also a constituent of multiple drug efflux pumps. We showed that AcrA stimulates drug efflux in live cells by interacting with TolC from the outside, activating efflux in the short term, and inducing the expression of other classes of efflux pumps in the long term, thus amplifying the response and establishing SR. We have called this phenomenon `necrosignaling', and discovered species-specific necrosignaling in both Gram-positive and Gram-negative bacteria. We also discovered that production of c-di-GMP by the specific cyclase YfiN, arrests cell growth to promote an antibiotic-tolerant persister-like state. We propose to explore both these responses further. Given that non- genetic resistance is a known incubator for evolving genetic resistance, our findings are relevant to the current widespread emergence of genetic resistance to antibiotics, and may be relevant to chemotherapy-resistant cancers, which efflux the drugs prior to acquisition of genetic resistance.

Virus-host interactions and microbial ecology Using E. coli as our model organism, we propose to explore how the DNA-loop extruding condensin motor complex, conserved from bacteria to humans, organizes the genome such that there are widespread contacts between all genomic loci, a finding that has implications for DNA rearrangements, evolution, and cancers. We also propose to understand why, when the bacteria associate in a group and swarm over a surface, they are protected against lethal levels of antibiotics by a subpopulation of dead bacteria. This research has implications for the enhanced drug efflux seen in chemotherapy-resistant cancers.