Rasika M Harshey
Job Title
Research Interests

We have two major research interests: DNA Transposable Elements and Bacterial Signaling & Motility. (1) Transposable Phage Mu and E. coli Genome Organization. DNA transposition is central to the propagation of phage Mu (our model system) as it is to retroviruses. In the last several decades, we have analyzed the proteins and DNA sites that participate in transposition, discovered an enhancer element and worked out its role in building a transpososome, analyzed the topology of the complex, deciphered the mechanism by which Mu integrates into its host upon phage infection, and showed that repair of the integrant is dependent on host replication complex (Pol III). In recent years our attention has been focused on using our knowledge of Mu to understand the organization of the E. coli host chromosome. By placing Mu in different locations around the E. coli genome, which include the four insular MDs, we exploited efficiency and promiscuity of phage Mu transposition to directly measure in vivo interactions between genomic loci in E. coli. Two global organizing principles emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation. Our work advances a new view of the structure and dynamic properties E. coli genome. (2) cyclic-di-GMP- and Necrosignaling. Bacteria use rotation of helical flagella to propel themselves either through bulk liquid (swimming), or through a thin film of liquid on a solid surface (swarming). Chemosensory pathways normally communicate environmental information to the bidirectional rotary motor, modulating its CW/CCW bias to optimize bacterial migration towards favorable locales. These pathways are well-understood. We are currently working on the following three aspects of motility and cell physiology in E. coli and Salmonella: (i) Sensing outer membrane perturbation to arrest growth via ci-di-GMP signaling. While the major role of c-di-GMP signaling is to control the decision to move freely or settle in a biofilm, recent studies show a broader range of output functions for c-di-GMP signaling. We have found an unexpected second role for YfiN, a conserved diguanylate cyclase in Gram-negative bacteria, known to contribute to persistence in the host. We are dissecting the molecular nature of the signal that is generated, the mechanism by which the signal is sensed, and the mechanism by which growth is arrested. (ii) Swarming bacteria exhibit adaptive resistance to multiple antibiotics. Analysis of this phenomenon has revealed the protective power of high cell densities to withstand exposure to otherwise lethal antibiotic concentrations. We observed that antibiotic-induced death of a sub-population that we identify as AcrA, a periplasmic component of RND efflux pumps. The released AcrA interacts on the surface of live cells with an outer membrane component of the efflux pump, TolC, stimulating drug efflux and inducing expression of other efflux pumps. This phenomenon, which we call 'necrosignaling', exists in other Gram-negative and Gram-positive bacteria and displays species-specificity. Given that adaptive resistance is a known incubator for evolving genetic resistance, our findings might be clinically relevant to the rise of multidrug resistance. We are currently examining Necrosignaling in greater depth, including its effect on the flagellar motor.