ABSTRACT
Nucleotide sequence analysis is increasingly being used to identify bacteria. In this work, a PCR assay based on degenerate primers was used to obtain the partial sequence of infB, the gene encoding translation initiation factor 2 (IF2), in 39 clinical isolates of different Enterobacteriaceae. The partial sequence encodes the GTP-binding domain of IF2. Together with sequences from the literature, a total of 15 species, each represented by one to seven strains, was investigated. Phylogenetic analysis yielded an evolutionary tree which had a topology similar to a tree constructed using available 16S rRNA sequences. It is concluded that the inter-species variation of the infB gene fragment is sufficient for its use in the characterization of strains that have aberrant phenotypic reactions.
Subject(s)
Bacterial Typing Techniques , Enterobacteriaceae Infections/microbiology , Enterobacteriaceae/classification , Genes, Bacterial , Peptide Initiation Factors/genetics , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Enterobacteriaceae/genetics , Humans , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Prokaryotic Initiation Factor-2 , Sequence Analysis, DNAABSTRACT
The Escherichia coli translation initiation factor IF2 is a 97 kDa protein which interacts with the initiator fMet-tRNAfMet, GTP and the ribosomal subunits during initiation of protein biosynthesis. For structural and functional investigations of the factor, we have raised and characterised monoclonal antibodies against E. coli IF2. Twelve epitopes have been localised at the surface of the protein molecule by three different methods: Interactions of the monoclonal antibodies with nested deletion mutants of IF2, comparison of the relative location of the epitopes in a competition immunoassay and cross-reactivity analyses of the monoclonal antibodies towards IF2 from Salmonella typhimurium, Klebsiella oxytoca, Enterobacter cloacae, Proteus vulgaris, and Bacillus stearothermophilus. These data are combined with predicted secondary structure and discussed in relation to a six-domain structural model for IF2. The model describes IF2 as a slightly elongated molecule with a structurally compact C-terminal domain, a well-conserved central GTP-binding domain, and a highly charged, solvent exposed N-terminal with protruding alpha-helical structures.
Subject(s)
Epitopes/chemistry , Escherichia coli/chemistry , Models, Molecular , Peptide Initiation Factors/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Antibodies, Bacterial/immunology , Antigens, Bacterial/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Binding, Competitive , Blotting, Western , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Escherichia coli/immunology , Guanosine Triphosphate/metabolism , Mice , Peptide Initiation Factors/genetics , Peptide Initiation Factors/immunology , Prokaryotic Initiation Factor-2 , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Sequence DeletionABSTRACT
The functionally uncharacterised N-terminal of translation initiation factor IF2 has been found to be extremely variable when comparing different bacterial species. In order to study the intraspecies variability of IF2 the 2670 basepairs nucleotide sequence of the infB gene (encoding IF2) was determined in 10 clinical isolates of E. coli. The N-terminal domains (I, II and III) were completely conserved indicating a specific function of this region of IF2. Only one polymorphic position was found in the deduced 890 amino acid sequence. This Gln/Gly490 is located within the central GTP/GDP-binding domain IV of IF2. The results are further evidence that IF2 from E. coli has reached a highly defined level of structural and functional development.
