The membrane channel-forming bacteriocidal protein, colicin El

Cramer, William A.; Dankert, J.; Uratani, Yoshihiko

doi:10.1016/0304-4157(83)90016-3

Cited by 111 publications

(70 citation statements)

References 74 publications

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“…This high degree of sequence conservation may be attributed to the specific interactions required between the colicins and their common chromosomally encoded receptor, the colicin I receptor (cir gene product) (21). Although there is no direct evidence for the location of the colicin I-receptor-binding domain, colicin El does have this organization, the domains for receptor binding and membrane translocation being in the N-terminal two-thirds of the colicin molecules (7).…”

Section: Discussionmentioning

confidence: 99%

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

Mankovich¹,

Hsu²,

Konisky³

1986

J Bacteriol

117

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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.

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Section: Discussionmentioning

confidence: 99%

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

Mankovich¹,

Hsu²,

Konisky³

1986

J Bacteriol

117

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“…In addition to the early electrochemical studies of Fe(ent) (5,11,17), it has been found that reductive removal of Fe from enterobactin is achieved by glutathione at pH values lower than 6 (10). The observed protonation constants of ferric enterobactin (11,12) (6,38). It constitutes up to 40% of the cell volume.…”

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confidence: 98%

Recognition and transport of ferric enterobactin in Escherichia coli

Ecker¹,

Matzanke²,

Raymond³

1986

J Bacteriol

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The specificity of the outer membrane protein receptor for ferric enterobactin transport in Escherichia coli and the mechanism of enterobactin-mediated transport of ferric ions across the outer membrane have been studied. Transport kinetic and inhibition studies with ferric enterobactin and synthetic structural analogs have mapped the parts of the molecule important for receptor binding. The ferric complex of the synthetic structural analog of enterobactin, 1,3,5-N,N',N"-tris-(2,3-dihydroxybenzoyl)triaminomethylbenzene (MECAM), was transported with the same maximum velocity as was ferric enterobactin. A double-label transport assay with [59Fe, 3H]MECAM showed that the ligand and the metal are transported across the outer membrane at an identical rate. Under the growth conditions used, large fractions of the transported complexes were available for exchange across the outer membrane when a large excess of extracellular complex was added to the cell suspension; at least 60% of the internalized [59Felenterobactin exchanged with extracellular [55Fe]enterobactin.Internalized [59Fe, 3H1MECAM was released from the cell as the intact complex when either unlabeled Fe-MECAM or Fe-enterobactin was added extracellularly. The results suggest a mechanism of active transport of unmodified coordination complex across the outer membrane with possible accumulation in the periplasm.

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“…ColE1 belongs to the group A colicins and kills susceptible E. coli cells by forming ion channels in the inner membrane (10). Previous genetic and biophysical studies showed the requirement of both BtuB and TolC for ColE1 entry.…”

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confidence: 99%

Initial Steps of Colicin E1 Import across the Outer Membrane of Escherichia coli

et al. 2007

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Data suggest a two-receptor model for colicin E1 (ColE1) translocation across the outer membrane of Escherichia coli. ColE1 initially binds to the vitamin B 12 receptor BtuB and then translocates through the TolC channel-tunnel, presumably in a mostly unfolded state. Here, we studied the early events in the import of ColE1. Using in vivo approaches, we show that ColE1 is cleaved when added to whole cells. This cleavage requires the presence of the receptor BtuB and the protease OmpT, but not that of TolC. Strains expressing OmpT cleaved ColE1 at K84 and K95 in the N-terminal translocation domain, leading to the removal of the TolQA box, which is essential for ColE1's cytotoxicity. Supported by additional in vivo data, this suggests that a function of OmpT is to degrade colicin at the cell surface and thus protect sensitive E. coli cells from infection by E colicins. A genetic strategy for isolating tolC mutations that confer resistance to ColE1, without affecting other TolC functions, is also described. We provide further in vivo evidence of the multistep interaction between TolC and ColE1 by using cross-linking followed by copurification via histidine-tagged TolC. First, secondary binding of ColE1 to TolC is dependent on primary binding to BtuB. Second, alterations to a residue in the TolC channel interfere with the translocation of ColE1 across the TolC pore rather than with the binding of ColE1 to TolC. In contrast, a substitution at a residue exposed on the cell surface abolishes both binding and translocation of ColE1.

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The membrane channel-forming bacteriocidal protein, colicin El

Cited by 111 publications

References 74 publications

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

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

Recognition and transport of ferric enterobactin in Escherichia coli

Initial Steps of Colicin E1 Import across the Outer Membrane of Escherichia coli

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