Surface exclusion specificity of the TraT lipoprotein is determined by single alterations in a five-amino-acid region of the protein.
The TraT protein is a highly cell-surface-exposed lipoprotein specified by F-like plasmids that confers serum resistance and blocks the conjugative transfer of plasmids to cells bearing identical or closely related plasmids, a process known as surface exclusion. The TraT protein specified by the antibiotic-resistance plasmid R6-5 was purified to apparent homogeneity. When added to mating mixtures, TraT blocked the transfer of plasmids belonging to Surface Exclusion Group IV (Sfx IV) but had no significant effect on the transfer of plasmids belonging to other groups. Additionally, the purified protein has a protective effect on bacterial cells incubated in serum, indicating that it does not have to be located on the cell surface to mediate serum resistance. To localize regions of the protein that were responsible for surface exclusion specificity, the amino acid sequence of the TraT protein specified by CoIB2-K98 (Sfx II) was determined by cloning and sequencing of the corresponding gene. Comparison of the derived sequence with those of the F and R100-1 proteins indicated that surface exclusion specificity of TraT is determined by single alterations in a five-amino-acid region (residues 116-120). This was confirmed by segment swapping experiments in which the specificity of the R6-5 TraT protein (Sfx IV) was switched to that of the CoIB2-K98 protein (Sfx II). Our results suggest that the region defined by residues 116-120 is located on the external face of the outer membrane and interacts specifically with the donor cell in surface exclusion.
Department of Biochemistry, University of Southampton, Bassett Crescent East, UK.
J L
JL
Harrison
I M
IM
Taylor
K
K
Platt
C D
CD
O'Connor
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001770:Blood Bactericidal Activity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D029968:Escherichia coli Proteins; D005144:F Factor; D010861:Fimbriae, Bacterial / Q000502:physiology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C036297:TraT protein, E coli