Mode of action of the cloacin DF13-immunity protein

Oudega, Bauke; Klaasen-Boor, P; Graaf, Frits K. de

doi:10.1016/0304-4165(75)90179-8

Cited by 30 publications

(18 citation statements)

References 17 publications

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“…The conformation of the cloacin moiety of the cloacin DF13 complex changes upon dissociation of the complex into cloacin and immunity protein (13,24,25). Conformational changes or dissociation of the complex or both might result in a loss of specific epitopes or an unmasking of epitopes at the interface of cloacin and immunity protein.…”

Section: Resultsmentioning

confidence: 99%

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Uptake of cloacin DF13 by susceptible cells: removal of immunity protein and fragmentation of cloacin molecules

Krone¹,

Vries²,

Koningstein³

et al. 1986

J Bacteriol

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The bacteriocin cloacin DF13 is capable of killing susceptible cells of Enterobacter cloacae, Klebsiella edwardsii, and Escherichia coli (9). Cloacin DF13 conlsists of a complex of two polypeptides, designated cloacin and immunity protein, with relative molecular masses of 59,293 and 9,974 daltons, respectively (29,30).Three consecutive stages can be distinguished in the lethal action of cloacin DF13 on susceptible cells: (i) binding of cloacin DF13 onto outer membrane receptor proteins which are involved in the uptake of the iron chelator aerobactin (32), (ii) transport of the cloacin, or a cloacin fragment, across the cell envelope into the cytoplasm, and (iii) endoribonucleolytic cleavage of 16S rRNA, which results in defective protein synthesis and ultimately in cell death (8,23). The immunity protein inhibits the endoribonucleolytic activity of cloacin (24) and may be involved in the uptake and translocation process (25). To account for the lethal activity of cloacin DF13, the immunity protein is supposed to be removed from the cloacin complex or to be inactivated during the uptake of the cloacin.Although the mechanism of uptake of cloacin DF13 by susceptible cells has been studied extensively during the last decade, it is still unknown at which stage the immunity protein is removed from the cloacin complex and whether cloacin molecules are proteolytically fragmented during their uptake. The major problem in these studies is the identification of cloacin molecules or fragments and of immunity protein within the susceptible cells.To facilitate studies of cloacin DF13 uptake, we isolated and characterized various monoclonal antibodies which recognized epitopes located in different regions of the cloacin * Corresponding author. molecule or on the immunity protein. Cloacin uptake was studied with E. coli cells harboring a recombinant plasmid encoding the cloacin DF13 receptor protein (18). Evidence is presented that the immunity protein is removed from the cloacin DF13 complex and released into the culture medium upon the binding of the complex to the receptor protein and that the removal of the immunity protein is hampered by conditions which induce decreased cloacin DF13 susceptibility. Furthermore, we show that cloacin is fragmentated during its uptake.MATERIALS AND METHODS Bacterial strains, media and culture conditions. The cloacin DF13-resistant strain E. coli K-12 C600 thr leu thi lacYfhuA supE (strain F176) was used as host for the different CloDF13 mutant plasmids. Strain F176, harboring the recombinant plasmid pFS8 encoding the cloacin DF13-aerobactin outer membrane receptor protein (strain F344 [18]), was used for cloacin DF13 uptake studies. E. cloacae S458(CloDF13) was used for the isolation of cloacin DF13. Klebsiella edwardsii var. edwardsii S15 was used as indicator organism in in vivo killing activity assays of cloacin DF13 (9). The strains were grown in broth containing Lab-Lemco powder (8 g/liter; Oxoid Ltd., London, England) and sodium chloride (5 g/liter) or in brain heart infusion (BHI, 3...

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Section: Resultsmentioning

confidence: 99%

“…The immunity protein inhibits the endoribonucleolytic activity of cloacin (24) and may be involved in the uptake and translocation process (25). To account for the lethal activity of cloacin DF13, the immunity protein is supposed to be removed from the cloacin complex or to be inactivated during the uptake of the cloacin.…”

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Uptake of cloacin DF13 by susceptible cells: removal of immunity protein and fragmentation of cloacin molecules

Krone¹,

Vries²,

Koningstein³

et al. 1986

J Bacteriol

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“…The nucleases, for example, colicins E2, E3, and cloacin DF13, are released by the cells as equimolar complexes with the immunity proteins (11,13,26,35). In contrast, the channelforming colicins A (41), B (28), El (7,37), 1 (16), and K (27) are released in free form.…”

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confidence: 99%

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

Ölschläger

Braun

1987

J Bacteriol

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Escherichia coli strains carrying the cmi locus on plasmids are immune against colicin M, which primarily inhibits murein biosynthesis, followed by lysis of cells. The nucleotide sequence of the cmi region was determined. It contains an open reading frame for a polypeptide with a molecular weight of 19,227. However, the major protein band observed on polyacrylamide gels after transcription and translation in an in vitro system or in minicells had an apparent molecular weight between 15,000 and 16,000. The nucleotide sequence contained internal ATG codons, two of which could serve for the synthesis of polypeptides with molecular weights of 15,349 and 15,996, respectively. A subclone with a DNA fragment that encoded these two shorter polypeptides exhibited full immunity. The colicin M immunity protein was found in the cytoplasmic membrane. The colicin M activity and immunity genes were transcribed in opposite directions. Both properties are typical of the channel-forming colicins and are in contrast to the colicins with endonuclease activities. However, colicin M does not form channels and exhibits no structural similarity to channel-forming colicins.Colicins are plasmid-encoded proteins which are synthesized by Escher-ichia coli and which kill susceptible strains. With regard to their mode of action, they can be subdivided into several classes. One class impairs the membrane potential by forming small aqueous channels in the cytoplasmic membrane (8,15). A second class consists of endonucleases which degrade the cellular DNA or the 16S rRNA (15).Strains are protected against the colicin they produce and against the isolated colicin added to the culture by an immunity protein that is highly specific for the colicin. The nucleases, for example, colicins E2, E3, and cloacin DF13, are released by the cells as equimolar complexes with the immunity proteins (11,13,26,35). In contrast, the channelforming colicins A (41), B (28), El (7, 37), 1 (16), and K (27) are released in free form. In the latter cases the immunity proteins could not be detected in the culture supernatant or in the cells. After cloning on multicopy vectors, the immunity proteins for colicins A (18), El (10), and Ia (44) MATERIALS AND METHODSBacterial strains. E. coli HB101 recA hsdM hsdR supE lacY leu thi pro was used for transformation. E. coli JM101 A&(lac pro) thi supE F' traD36 proAB lacIqZAM5 served to screen for transformants with pUC-derived plasmids.Tabor and Richardson constructed vectors in which inserted genes are transcribed by the T7 polymerase under the control of a T7 promoter (39). Plasmid pT7-5 contains a polylinker preceded by the promoter of T7 gene 10. In addition, they contain the bla gene with a transcription polarity opposite to that of the +10 promoter region (S. Tabor, private communication). The TaqI-BamHI fragment of pTO195 was cloned into pT7-5 digested with ClaI and BamHI. The cmi fragment was isolated from an agarose gel and ligated into pT7-5. E. coli WM1576 containing plasmid pGP1-2, which carries the gene for the T7 R...

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“…Although colicins E2 and E3 have different biochemical targets, they share a common receptor protein (9), a btu B gene product utilized also by vitamin B12 and phage BF23 (10,11). Each bacteriocin is released from the producing cells as an equimolar complex with another plasmid-coded product known as the 'immunity' protein whose primary function is to protect the colicinogenic cells from the lethal action of its own toxin (4,12,13). The basis for the immunity system has been attributed to the specific electrostatic interactions of the acidic immunity protein with the basic nuclease domain localized at the C-terminus of each colicin (5,(14)(15)(16)(17)(18).…”

Section: Introductionmentioning

confidence: 99%

“…Because the neutralization of the toxin action requires specific interactions between the colicin and an immunity protein of a 'homologous' but not that of a 'heterologous' system (1,2,13), the various colicin-immunity proteins and their genes (19,20) offer an excellent system to study protein-protein interactions and protein/gene evol ution.…”

Section: Introductionmentioning

confidence: 99%

Comparative nucleotide sequences encoding the immunity proteins and the carboxyl-terminal peptides of colicins E2 and E3

Lau¹,

Rowsome²,

Zuker³

et al. 1984

Nucl Acids Res

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Using INTRODUCTIONThe plasmid-coded bactericidal proteins, colicins E2 and E3 and the E3-related cloacin DF13, constitute an interesting group of toxins in that each protein with an approximate Mr of 60,000 contains information for i) translocation across cell membranes, ii) receptor binding on the surface of sensitive E. coli cells and iii) nucleolytic activities (for recent reviews, see ref. 1-3). Colicin E2 is an endonuclease active on both single and double stranded DNA but with undefined specificity (4,5) whereas colicin E3

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Mode of action of the cloacin DF13-immunity protein

Cited by 30 publications

References 17 publications

Uptake of cloacin DF13 by susceptible cells: removal of immunity protein and fragmentation of cloacin molecules

Uptake of cloacin DF13 by susceptible cells: removal of immunity protein and fragmentation of cloacin molecules

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

Comparative nucleotide sequences encoding the immunity proteins and the carboxyl-terminal peptides of colicins E2 and E3

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