Characterization of an ORF in a novel orientation in Escherichia coli
Lackey, Melinda K.
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Evidence has been presented in the literature that demonstrates the expendable nature of the C-terminal region for the proper functioning of TolC in the bacterial cell. Recent sequencing of various microbial genomes demonstrated the presence of 3' overlapping, converging open reading frames involving tolC and its homologues found in other species and genera of bacteria. In this study, we examined the Escherichia coli tolC gene and the 3' overlapping, converging ORF found at the same genetic locus, denoted cloT for tolC in reverse. The expression of c/or was examined using reverse transcriptase PCR to identify the presence of mRNA molecules within the bacterial cell. Amplification of a 120 base pair fragment from total RNA isolated from wild-type E. coli suggested that cloT was transcribed, at least at low levels, in the organism. To corroborate these data, the upstream region of the structural gene was analyzed for the ability to drive transcription of a reporter gene, beta-galactosidase. Activity analysis of this reporter fusion demonstrated the ability of the upstream region of the cloT gene to drive transcription in E. coli. Similarly, we have demonstrated an interaction between the upstream region of cloT and soluble proteins isolated from the cytoplasm of wild-type E. coli. Further experiments were attempted to raise antibodies against a portion of the putative CloT protein using both GST fusion technology and a synthetic peptide. Neither has been fully characterized at time of publication. In addition, attempts to isolate mutations in the unique region of the cloTORF in the chromosome of E. coli were also unsuccessful. Examination of the information known about TolC and its homologues in different genera of bacteria revealed interesting information concerning the C-termini of these proteins. Alignment of the amino acid sequences demonstrated lower identity shared in the C-terminal region of these proteins. In addition, examination of the nucleotide sequences of these homologues indicated the presence of a similar open reading frame sharing a convergent, overlapping relationship with its respective tolC homologue. When combined with published data concerning the expendable nature of the last fifty amino acids of TolC, these data suggest that the presence of a conserved open reading frame in this region may play an important role in the regulation or function of the tolC gene or gene product(s).