Sequence, expression and localization of the immunity protein for colicin B

Schramm, Edgar; Ölschläger, Tobias; Tröger, Wilfried; Braun, Volkmar

doi:10.1007/bf00338410

Cited by 34 publications

(26 citation statements)

References 31 publications

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“…The entire colicin B and M gene region was sequenced for 60 E. coli strains selected to represent the diversity present among the strains encoding these genes. Primers for sequencing were designed as needed using published colicin B and M gene sequences (Johnson et al, 2006a;Köck et al, 1987;Ö lschläger & Braun, 1987;Schramm et al, 1987Schramm et al, , 1988 (Supplementary Table S1). DNA fragments were amplified using the PCR conditions described above, and PCR products were purified with the DNA purification reagent ExoSAP-IT (USB).…”

Section: Methodsmentioning

confidence: 99%

“…DNA and amino acid sequence variation in colicins B and M Several previously published colicin B and M sequences were included in the analyses: the colicin region of the plasmid pAPEC-O1-ColBM, and separately published sequences for cmi, cma, cbi and cba (Köck et al, 1987;Ö lschläger & Braun, 1987;Schramm et al, 1987Schramm et al, , 1988. Sequence variation statistics and codon-based Z-tests for selection were performed for each colicin gene (Table 2).…”

Section: Gene Structure Of Colicin Bm Plasmidsmentioning

confidence: 99%

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Evolution of colicin BM plasmids: the loss of the colicin B activity gene

Christenson

Gordon

2009

Microbiology

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Colicins, a class of antimicrobial compounds produced by bacteria, are thought to be important mediators of intra-and interspecific interactions, and are a significant factor in maintaining microbial diversity. Colicins B and M are among the most common colicins produced by Escherichia coli, and are usually encoded adjacently on the same plasmid. In this study, the characterization of a collection of E. coli isolated from Australian vertebrates revealed that a significant fraction of colicin BM strains lack an intact colicin B activity gene. The colicin B and M gene region was sequenced in 60 strains and it was found (with one exception) that all plasmids lacking an intact colicin B activity gene have an identical colicin gene structure, possessing a complete colicin B immunity gene and a 130 bp remnant of the B activity gene. A phylogenetic analysis of the colicin M and B operons and characterization of the plasmids suggested that ColBM plasmids with a truncated B activity gene have evolved on at least three separate occasions. Colicin B immunity was found to be non-functional in strains that have lost colicin B activity, and colicin M was still produced despite the absence of the SOS box believed to regulate its production in colicin BM strains. The presence of a remnant of the microcin V operon next to the truncated colicin B activity gene indicated that these plasmids evolved as a consequence of gene transfer between colicin BM and microcin V plasmids. We suggest that these transfer events most likely involved the transfer of some microcin V genes and associated virulence factors onto ColBM plasmids.

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Section: Methodsmentioning

confidence: 99%

Section: Gene Structure Of Colicin Bm Plasmidsmentioning

confidence: 99%

Evolution of colicin BM plasmids: the loss of the colicin B activity gene

Christenson

Gordon

2009

Microbiology

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“…Six different channel-forming colicins and their Imm proteins have been identified (26,28,(35)(36)(37) lacZ fusion takes advantage of P-galactosidase which, unlike AP, is active in the cytoplasm (29). In-frame lacZ fusions with a periplasmic domain of an integral membrane protein cause the lacZ gene product to be retained in the membrane, resulting in low 3-galactosidase activity (14).…”

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confidence: 99%

“…Six different channel-forming colicins and their Imm proteins have been identified (26,28,(35)(36)(37). The action of an Imm protein is highly specific, meaning that the colicin El Imm protein can protect cells only from exogenous colicin El and not from any of the closely related channel-forming colicins.…”

mentioning

confidence: 99%

Membrane topography of ColE1 gene products: the immunity protein

Song

1991

J Bacteriol

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The topography of the colicin El immunity (Imm) protein was determined from the positions of TnphoA and complementary lacZ fusions relative to the three long hydrophobic segments of the protein and site-directed substitution of charged for nonpolar residues in the proposed membrane-spanning segments. Inactivation of the Imm protein function required substitution and insertion of two such charges. It was concluded that the 113-residue colicin El Imm protein folds in the membrane as three trans-membrane a-helices, with the NH2 and COOH termini on the cytoplasmic and periplasmic sides of the membrane, respectively. The approximate spans of the three helices are Asn-9 to . An extrinsic highly charged segment, Lys-66 to Lys-74, containing seven charges in nine residues, extends into the cytoplasmic domain. The specificity of the colicin El Imm protein for interaction with the translocation apparatus and the colicin El ion channel is proposed to reside in its peripheral segments exposed on the surface of the inner membrane. These regions include the highly charged segment Lys-66 to Lys-83 (loop 2) and the short (approximately eight-residue) NH2 terminus on the cytoplasmic side, and Glu-29 to Val-44 (loop 1) and the COOH-terminal segment Gly-105 to Asn-113 on the periplasmic side.Colicin El exerts its bactericidal effect by forming a highly conductive ion channel in the cytoplasmic membrane that depolarizes and deenergizes the cell (9). The channel domain, localized in the COOH-terminal third of the 522-residue colicin molecule, is susceptible to the protective effect of the 113-residue colicin El immunity (Imm) protein, a cytoplasmic membrane protein (3).Six different channel-forming colicins and their Imm proteins have been identified (26,28,(35)(36)(37) lacZ fusion takes advantage of P-galactosidase which, unlike AP, is active in the cytoplasm (29). In-frame lacZ fusions with a periplasmic domain of an integral membrane protein cause the lacZ gene product to be retained in the membrane, resulting in low 3-galactosidase activity (14). The dependence of lacZ fusion activity on the trans-membrane orientation of the fusion site is thus complementary to that of phoA fusions (29), so that this method can often, but not always (19), provide an additional determinant of the topography of integral membrane proteins. MATERIALS AND METHODSConstruction of plasmid pSL630. Plasmid pDMS630 (40), a ColEl derivative with an ampicillin resistance gene (constructed by insertion of Tn3 into ColEl) was digested with EcoRI and SmaI, each of which cuts at only one site. Two linear DNA fragments were generated, a 1.3-kb fragment that spans most of cea and a 10.5-kb fragment containing imm. These were blunt-ended by digestion with mung bean nuclease, self-ligated for 3 h at 16°C, and transformed into JM83. Colonies that grew on ampicillin were chosen randomly, plasmid DNA was isolated by a modified alkaline lysis method (34a), and a plasmid produced from self-ligation of the 10.5-kb fragment without the 1.3-kb fragment was isolated by d...

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“…Colicin-producing cells produce a specific immunity protein that protects them from the action of their own toxin (2,16). Proteins with immunity to pore-forming colicins are integral inner membrane proteins.…”

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confidence: 99%

Colicin A Immunity Protein Interacts with the Hydrophobic Helical Hairpin of the Colicin A Channel Domain in the Escherichia coli Inner Membrane

et al. 2001

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The colicin A pore-forming domain (pfColA) was fused to a bacterial signal peptide (sp-pfColA). This was inserted into the Escherichia coli inner membrane in functional form and could be coimmunoprecipitated with epitope-tagged immunity protein (EpCai). We constructed a series of fusion proteins in which various numbers of sp-pfColA ␣-helices were fused to alkaline phosphatase (AP). We showed that a fusion protein made up of the hydrophobic ␣-helices 8 and 9 of sp-pfColA fused to AP was specifically coimmunoprecipitated with EpCai produced in the same cells. This is the first biochemical evidence that Cai recognizes and interacts with the colicin A hydrophobic helical hairpin.

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Sequence, expression and localization of the immunity protein for colicin B

Cited by 34 publications

References 31 publications

Evolution of colicin BM plasmids: the loss of the colicin B activity gene

Evolution of colicin BM plasmids: the loss of the colicin B activity gene

Membrane topography of ColE1 gene products: the immunity protein

Colicin A Immunity Protein Interacts with the Hydrophobic Helical Hairpin of the Colicin A Channel Domain in the Escherichia coli Inner Membrane

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