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Principal Investigator: Rasika M Harshey
Organization: UNIVERSITY OF TEXAS AT AUSTIN
Fiscal Year: 2024
Award: $560,773
Funding agency: National Institute of General Medical Sciences
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.
Terms: <3-D><3-Dimensional><3D><Antibiotic Agents><Antibiotic Drugs><Antibiotic Resistance><Antibiotics><Bacteria><Bacteriophage mu><Bionomics><Cancers><Cell Communication><Cell Interaction><Cell-to-Cell Interaction><Cellular Expansion><Cellular Growth><Cessation of life><Chemoresistance><Chromatin Loop><Chromatin Loop Domains><Chromosomes><Coliphage mu><Complex><DNA Loop><DNA Rearrangement><Death><Drug Efflux><E coli><E. coli><Ecology><Enterobacteria phage Mu><Escherichia coli><Evolution><Exposure to><Gene Expression><Gene Family><Gene Rearrangement><Generalized Growth><Genome><Genomics><Gram-Negative Bacteria><Grant><Growth><HU Protein><Human><Incubators><Malignant Neoplasms><Malignant Tumor><Measures><Membrane><Microbiology><Miscellaneous Antibiotic><Modern Man><Motor><Operon><Paste substance><Pastes><Pathway interactions><Periplasmic Space><Phage mu><Production><Property><Pump><Research><Resistance><Resistance to antibiotics><Resistant to antibiotics><Resolution><Ribosomal RNA><Salmonella><Specificity><Surface><Tissue Growth><antibiotic drug resistance><antibiotic resistant><antibiotic tolerance><cell growth><cell killing><chemoresistant><chemotherapy resistance><chemotherapy resistant><condensin><efflux pump><gene locus><genetic locus><genetic resistance><genomic location><genomic locus><in vivo><malignancy><membrane structure><microbial><model organism><neoplasm/cancer><non-genetic><nongenetic><ontogeny><pathway><periplasm><rRNA><refractory cancer><resistant><resistant cancer><resolutions><response><three dimensional><tolerance to antibiotics><tolerate antibiotics><tool><virus host interaction>