Analysis of the tsx gene, which encodes a nucleoside-specific channel-forming protein (Tsx) in the outer membrane of Escherichia coli

The tsx gene ofEscherichia coli encodes an outer membrane protein, Tsx, which constitutes the receptor for colicin K and bacteriophage T6, and functions as a substrate-specifie channel for nucleosides and deoxynucleosides. The mini-Mu element pEG5005 was used to prepare a gene bank in vivo, and this bank was used to identify T6-sensitive strains carrying the cloned tsx gene. Subcloning of the tsx gene into the multicopy plasmid, pBR322, resulted in a strong verproduction of Tsx. The sequence of a 1477-bp DNA segment containing tsx and its flanking regions was determined. An open reading frame (ORF)
was found which was followed by a pair of repetitive extragenic palindromic sequences. This ORF translated into a protein of 294 amino acids (aa), the first 22 aa of which showed the characteristic features of a bacterial signal sequence peptide.
The putative mature form of Tsx is composed of 272 aa with a calculated Mr of 31418. The aa sequence of Tsx shows an even distribution of charged residues (52 aa) and lacks extensive hydrophobic stretches. No significant homologies of Tsx to the channel-forming proteins OmpC, OmpF, PhoE and LamB from the E. coil outer membrane were detected. Using nuclease S 1, we identified two transcription start points for the tsx mRNA which were separated by approx. 150 bp. Genetic data suggest that the synthesis of the larger mRNA species is directed by a weak promoter (t'I) that is controlled by the DeoR repressor, whereas the smaller mRNA species is directed by the main promoter 1'2, which is negatively controlled by the CytR repressor and positively affected by the cyclic AMP/catabolite activator protein complex.