Conjugative DNA transport in Gram-positive bacteria

Conjugative plasmid transfer in Gram-negative bacteria has been studied in great detail in the last decades, whereas the mechanisms of conjugative plasmid spread in Gram-positive bacteria have remained obscure. In the last few years the complete nucleotide sequences of several conjugative antibiotic resistance plasmids from Gram-positive bacteria have been determined, which revealed a modular organization of their transfer regions and high conservation of the putative transfer genes. Several genes for potential homologues of type IV secretion components involved in secretion of toxins in Gram-negative pathogens and in conjugative transfer of Gram- negative bacteria have been detected in their transfer regions. The transfer region of the multiple antibiotic resistance plasmid pIP501, with the broadest known host range for plasmid transfer in Gram-positive bacteria is organized in an operon encoding fifteen putative transfer proteins. In this project all transfer proteins shall be purified in small scale and checked for solubility and correct fold. All proteins fulfilling the criteria for crystallization will be expressed and purified in large scale. The putative pIP501- encoded type IV homologues, an ATPase, a coupling protein and a lytic transglycosylase will be functionally characterized. Crystallization trials will be performed for the three putative type IV homologous pIP501 proteins, for their analogues from the related plasmids pSK41 and pMRC01 and other members of the transfer operon, which are emerging from the yeast-twohybrid screen and pull-down assays as key players in the conjugative transfer. Interactions found by these assays will be verified and characterized by biophysical methods. This project aims to elucidate the role and importance of the protein components for the assembly of a conjugative type IV-like secretion-machinery in Gram-positive bacteria.

Type IV secretion systems (T4SS) function in many species of Gram-negative (G-) and Gram-positive (G+) bacteria to deliver DNA and protein substrates to target cells. The conjugation machines, a large T4SS subfamily found in most bacterial species to promote widespread transfer of antibiotic resistance and other virulence traits; these systems are especially problematic among the G+ pathogens as a principle mechanism underlying multiple antibiotic resistance in clinically relevant pathogens, e.g., Staphylococci, Streptococci, Enterococci. The aim of this project was the identification and characterization of key-components of the T4SS of pIP501, a multiple antibiotic resistance plasmid of Enterococcus faecalis. We succeeded in characterizing three major components of the T4SS, whose functions were predicted due to a distant homology with components of G- T4SS. Thus we could prove that Orf5, a putative ATPase, and Orf10, the putative coupling protein, possess ATP-binging and ATPase activity. For the Orf7, the putative lytic transglycosilase, which exhibited solubility problems, two domains (SLT and CHAP) were cloned, expressed and purified separately. Both were characterized for their transglycosilase activity and the CHAP-domain showed amidase activity. In addition to these three "known" key components we started to express all other components of the pIP501 T4SS and test the expression products for solubility. So far 5 additional proteins could be expressed and purified in large scale and excitingly three of these yielded diffraction-quality crystals in a variety of crystal screens. High resolution data has been collected for two of these (Orf14 and Orf11) and the structure solution is in progress. Interactions between the components, which have been observed in a yeast-two-hybrid study in our collaborator`s laboratory, were investigate with pull-down assays and thermofluor techniques, yielding a preliminary model of the T4SS. Though this model is far from complete, together with the emerging crystal structures and the enzymatic data it yields a first glimpse of the conjugation machinery of Gram-positive bacteria. These data will contribute to the understanding of the mechanism involved in DNA transfer and exchange of genetic information in this important group of bacteria, which contains several important human pathogens. In the long run, the emerging structure of the T4SS might enable us to identify targets for inhibition of the spread of antimicrobial resistance determinants in Gram-positive pathogens.