DNA and amino acid sequence analysis of structural and immunity genes of colicins Ia and Ib

Abstract

The nucleotide sequences for codicin Ia and colicin Tb structural and immunity genes were determined. The two colicins each consist of 626 amino acid residues. Comparison of the two sequences along their lengths revealed that the two colicins are nearly identical in the N-terminal 426 amino acid residues. The C-terminal 220 amino acid residues of the colicins are only 60% The Col group of plasmids endows members of the family Enterobacteriaceae with the capacity to produce plasmidencoded protein toxins, termed colicins (20). Each Col plasmid also confers specific immunity via a second plasmid gene product, the immunity protein to the particular colicin produced. Over the years this laboratory has focused its interest on two closely related colicins, Ia and Ib, and their respective immunity proteins.Colicins Ia and lb are structurally related and adsorb to a common bacterial outer membrane receptor (19,21). Both have identical modes of action, depolarizing the energytransducing cytoplasmic membrane via formation of aqueous channels (45, 49). However, they differ in immunity specificity in that cells harboring the ColIb plasmid are immune to colicin Ib yet sensitive to Ia and vice versa (23). It has been proposed that the colicin I immunity system involves direct interaction of each colicin with its cognate immunity protein, which has been shown to reside in the cytoplasmic membrane (50). We previously reported that the C-terminal halves of the colicin Ta and lb molecules are responsible for immunity recognition (26). Hybrid colicins that contained the Nterminal half of one colicin and the C-terminal half of the other were found to have the immunity specificity of the colicin donating the C terminus. We report here the complete nucleotide sequences for both the colicin Ia and lb structural and immunity genes. Comparison of the two colicin sequences allows a more precise assignment of the immunity domain, localizing it within the C-terminal 200 amino acid residues. MATERIALS AND METHODSBacterial strains, plasmids, and media. Escherichia coli K-12 W3110 containing either pCol Ia-CA53 or pCol Ib-P9 was used as an indicator strain, as a source for colicin purification, and as a source for Col plasmid purification. JK481 (E. coli K-12 294), harboring plasmid pAR29 (50), was the source of the colicin Ia immunity gene. Plasmid pKC30 and E. coli K-12 Hi (4), which was used as a host for pKC30 and its derivatives, were obtained from K. Alton, Amgen, Inc., Thousand Oaks, Calif.LB, M9, and 2 x YT media were prepared as described by Miller (30). Antibiotic plates used for the selection of transformants contained LB medium and 50 ,ug of ampicillin per ml. M9 medium was supplemented with 1% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.2% glucose, 0.1% yeast extract, and 100 ,ug of ampicillin per ml. Methioninedeficient medium for protein labeling was M9 medium supplemented with 0.2% glucose, 0.01% yeast extract, and 0.5 p,g of each amino acid (no methionine) per ml, except for cysteine, which was 0.1 ,ug/ml.

Colicin Biology

“…In all cases, the immunity protein recognized the C-terminal domain of its cognate colicin (21,433). A similar conclusion was reached in experiments with the purified pore-forming domain of colicin E1.…”

“…Except for cloacin DF13, colicin operons con-tain two LexA boxes in tandem but overlapping by one or two bases (106,123,171,411,433,445,466,537,578,599,638,649). The two LexA boxes have been found in every colicin operon studied so far and in the same organization and location.…”

“…The immunity proteins are constitutively expressed at a low level despite the high-level expression of the corresponding colicin (411) (see Colicin Synthesis above), and in order to identify the products of the immunity genes, it has been necessary to clone the genes in appropriate overexpression vectors (215,433). Such techniques have made the demonstration that immunity proteins reside in the inner membrane possible (215,229).…”

“…In all the colicin operons, the first gene is the gene encoding colicin, called cxa, for colicin X activity. It might be the unique gene in the case of operons of pore-forming colicins of group B (347,433,578,649).…”

In vivo inhibition of TonB-dependent processes by a TonB box consensus pentapeptide

“…Colicins B and Ia bind to the iron-regulated outer membrane proteins FepA and Cir, respectively, and require the TonB protein for transport into the cell (1,15). Because these colicins contain a TonB box (11,19), we tested the ability of the TonB box pentapeptide to protect E. coli from colicin killing. Colicinogenic strains of E. coli were obtained from K. Hantke.…”