Contribution of plasmid proteins to surface associated growth and the induction of conjugative transfer

Gram negative bacteria possess multi-component, membrane spanning secretion systems that enable them to export protein and DNA molecules to the cell exterior and, depending on the system, can deliver their "cargo" directly to another bacterial, plant, or animal cell. The effects on recipient cells impact human health and the economy dramatically. One kind of secretion systems, Type IV (T4SS), spreads antibiotic resistance among bacteria making antibiotic therapies ineffective. They also promote virulence and infection, secrete toxins, or enable a pathogen to evade the immunity of a host. T4SS even transmit bacterial DNA to plant cells inducing disease and substantial losses in agriculture. The research program we propose extends a study of the mechanisms controlling T4SS through regulated expression of the transfer components and steps which prepare substrate molecules for secretion. We use well characterized E. coli K12 strains and plasmid conjugation systems as models. Based on our findings we plan now to investigate how T4SS enable bacteria to adhere to other bacteria and subsequently differentiate into a complex surface-attached community called a biofilm. Biofilms are tenacious coatings of bacteria found on most surfaces in nature, from water pipes to medical implants in patients. In this form bacteria are highly resistant to antibiotics and to host defenses making them difficult to control. Using fluorescent reporter molecules and microscopy we will observe the activity and regulation of the same T4SS that induced formation of the biofilm as it acts to disseminate antibiotic resistance genes to neighboring cells. To gain mechanistic insights we have designed novel molecular tools to visualize intermediate stages of the transport process. Analysis of molecular mechanisms is extended to genetic and biochemical characterization of the enzymes responsible for preparing and delivering export molecules to the transport machinery. These findings in turn may provide insights to inactivate or control these machineries as part of preventive or therapeutic measures.

This research program studied molecular processes which enable bacteria to exchange genes including those required for antibiotic resistance, virulence and intercellular adhesion. In order for genes to be transferred from cell to cell, many bacteria produce complex protein structures that connect the cell interior to the external environment by spanning their own cellular membrane. These protein complexes are not mere "tunnels" but controlled transport systems that enable bacteria to export protein and DNA molecules across membranes and deliver this "cargo" directly into another bacterial, plant, or animal cell. The effects on recipient cells impact human health, in part due to the spread of resistance genes that render antibiotic therapies ineffective. The transport systems we study (T4SS) also enable bacteria to adhere to other bacteria and establish surface-attached communities called biofilm. Biofilms are tenacious coatings of bacteria found on most surfaces in nature, from water pipes to medical implants in patients. In this form bacteria are highly resistant to antibiotics and to host defenses making them difficult to control. Our work aims to understand how T4SS contribute to development of bacterial biofilm and to elucidate the molecular mechanisms controlling gene spread via T4 secretion. The project contributed new knowledge in several areas using well studied laboratory models. Unexpected linkage between gene regulation and gene dosis was discovered resulting in major revision of existing models of transfer control pathways. Genetic and biochemical characterization of the enzymes responsible for preparing and delivering export molecules to the transport machinery led us to publish novel aspects in DNA processing reactions. Previously, what was known about E. coli biofilm formation and the stimulating effect of T4SS on their development has been discovered with laboratory strains. Since pathogenic bacteria are genetically different we published a very broad survey of biofilm formation of hundreds of uncharacterized natural E. coli isolates from humans. Moreover we established a laboratory model where biofilm-stimulating effects due to interactions between genetically diverse E. coli were observed. We found the strongest stimulation of biofilm was T4 mediated transfer of genes among strains. Thus a surprising interplay between gene transfer and bacterial biofilms appears to exist. Not only are regulatory pathways in biofilms supporting T4 mediated gene transfer but we also find that gene transfer events per se are promoting biofilm expansion and persistence. Ultimately these findings may provide insights to inactivate or control T4 conjugative machineries as part of preventive or therapeutic measures.