Molecular characterisation of the colicin E2 operon and identification of its products

Cole, Stewart T.; Saint‐Joanis, Brigitte; Pugsley, Anthony P.

doi:10.1007/bf00332940

Cited by 126 publications

(86 citation statements)

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“…Several lipoproteins in addition to MLP (1) have been found in bacteria: murein-associated lipoprotein (20), seven new lipoproteins (10), ColE2 lysis protein (4), TraT protein (28), pullulanase (29), and the membrane-bound penicillinases of gram-positive bacteria (15,24,25,30). The precursor forms of these lipoproteins share a common modificationprocessing pathway by virtue of the presence of a consensus pentapeptide sequence, Leu-Leu-Ala-Gly-Cys, in their respective signal sequences (39).…”

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Lipid modification of Escherichia coli penicillin-binding protein 3

et al. 1988

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. 191:1-9, 1983). An amino acid sequence of Leu-26-Leu-Cys-Gly-Cys-30 was found near the amino terminus of the deduced sequence, showing a rather striking homology to the Leu-Leu-Ala-Gly-Cys consensus sequence for the modification and processing of precursors of the E. coli murein lipoprotein and other bacterial lipoproteins. As (29), and the membrane-bound penicillinases of gram-positive bacteria (15,24,25,30). The precursor forms of these lipoproteins share a common modificationprocessing pathway by virtue of the presence of a consensus pentapeptide sequence, Leu-Leu-Ala-Gly-Cys, in their respective signal sequences (39). In Fig. 1 Fig. 2). This polypeptide was not separated with a serum that was not immunized against PBP 3. Rather intense radioactive bands seen on the lower part of the gel in Fig. 2 were presumably degradation products of PBP 3, as has been observed previously (23). These results strongly suggest that PBP 3 contains the covalently linked glycerol and fatty acid. If PBP 3 is not a lipoprotein, it may be processed by SPase I between residues 36 and 37 in the sequence Ala-LeuAla-36-Phe-37 (Fig. 1A), as predicted for the processing site in a precursor protein cleaved by SPase I (27,37,39). PBP 3 contains only two cysteine residues present in the Leu-LeuCys-Gly-Cys consensus sequence preceding the putative site for processing by SPase I. Both cysteine residues would be absent in mature PBP 3 if this second site were used as the processing site. This possibility was examined by labeling of

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Lipid modification of Escherichia coli penicillin-binding protein 3

et al. 1988

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“…Nucleotide and deduced amino acid sequences of the ClaI-PvuI fragment of ColE8-J aligned with those of ColE2-P9 and ColE3-CA38 (3,16,23). Identical nucleotides are connected with vertical lines.…”

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confidence: 87%

“…These E2 and E3 operons have nearly identical nucleotide sequences except in the regions responsible for nuclease activities and immunities. Extensive sequence homology to the E3 operon was also shown for the cloacin operon of plasmid CloDF13 (3,16,25).…”

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Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3

1988

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Colicin E8-J and its immunity protein were characterized with regard to their activities and gene structures. Colicin E8 is a complex of proteins A and B; protein A (the naked E8) exhibits an apparently nonspecific DNase activity that is inhibited by protein B (the immunity protein), as in the case of colicin E2. The nucleotide sequence of the downstream half of the colicin operon of ColE8-J was determined to be highly homologous to that of ColE2-P9, with the exception of the hot spot region of the 3'-terminal segment of the colicin gene and the adjacent immunity gene. The immE2-like gene of ColE3-CA38 was, as assumed previously, extensively homologous to the immE8 gene of ColE8-J, and thus, CoIE8-J was shown to be situated between CoIE2-P9 and ColE3-CA38 in the evolution of the E-group Col plasmids. E-group colicins bind to a common surface receptor, BtuB, when attacking sensitive Escherichia coli cells. A colicin-producing strain is not killed by its own colicin or a closely related colicin, a phenomenon referred to as immunity. Based on their immunity specificities, E-group colicins are divided into 10 subclasses, i.e., colicins A and El to E9 (4,14,18,26).The modes of action for some E-group colicins have been elucidated in vitro; colicins A and El are ionophores, E2 is a DNase, and E3 is a special kind of RNase which effectively acts on the 16S RNA of the 70S ribosome. All these in vitro activities are located on the C-terminal portions of the respective proteins. In both E2 and E3, the C-terminal active fragments obtained by tryptic digestion are referred to as T2A (5,15,20,21). Serological and physiological comparisons have suggested that colicins E4 to E7 resemble either E2 or E3 (14, 18, 26). Both E2 and E3, but not A or El, are purified as a complex of a colicin molecule (Mr, ca. 60,000) and an immunity protein (Mr, ca. 10,000). The immunity proteins are specific inhibitors of the respective colicin nucleases and protect the colicinogenic host cells from the killing action of both extra-and intracellular colicin molecules (16,17).The colicin (cob), immunity (imm), and lysis genes are integrated into an "SOS operon" in both ColE2-P9 and ColE3-CA38 plasmids. These E2 and E3 operons have nearly identical nucleotide sequences except in the regions responsible for nuclease activities and immunities. Extensive sequence homology to the E3 operon was also shown for the cloacin operon of plasmid CloDF13 (3,16,25).We identified an open reading frame between the immE3 and lysis genes of ColE3-CA38 which is highly homologous to the ColE2-P9 immunity gene (12,17) To confirm these assumptions, we determined the nucleotide sequence of the downstream half of the colicin.E8 operon and showed that E8 is a DNase-type colicin that is highly homologous to E2. MATERIALS AND METHODSStrains and plasmids. All the strains used in this work were E. coli K-12 derivatives. The original E8 colicinogenic strain, W3110(CoIE8-J), was obtained from R. James (13). RR1 was used as both a colicin-sensitive indicator strain and a recipien...

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“…2B, lane h) composition of the fragment and the deduced amino acid sequence, the trypsin fragment is considered to comprise 120 or 121 amino acids, including the carboxyl-terminal part of the large component (13). Figure 3 shows the sequences of the trypsin fragment and T2A, a similar protein of colicin E2, in which processing with trypsin does not increase DNase activity (1,4,8,16). Compared with the regions conserved between the two, the trypsin fragment of pyocin AP41 lacks the 15 or 16 residues from the carboxyl terminus of the large subunit while T2A lacks the 10 residues of the aminoterminal end.…”

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The inherent DNase of pyocin AP41 causes breakdown of chromosomal DNA

Sano

1993

J Bacteriol

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Pyocin AP41 degrades the chromosomal DNA in sensitive strains of Pseudomonas aeruginosa but has little effect on RNA, protein, and lipid syntheses. In vitro experiments showed that the carboxyl-terminal part of the large subunit of pyocin AP41 carries an inherent DNase that is responsible for its killing action.Pyocin AP41 is a typical S-type pyocin, proteinase-susceptible bacteriocin, produced by certain strains of Pseudomonas aeruginosa (3,14). Biochemical studies (11) and molecular analysis of the pyocin determinant (12, 13) have shown that pyocin AP41 is a complex of two protein subunits with molecular weights of 84,000 and 10,000. The large component, as well as pyocin AP41 (LS complex), kills susceptible bacteria, such as the PAO strain. The characteristic action of pyocin AP41 is that it causes production of resident pyocins, like R2 and S2, in the PAO strain and/or bacteriophages (F116 and PS17) in their lysogenic strains in a recA-dependent way (11). In these respects, pyocin AP41 resembles colicin E2, which is known to induce production of phage X in lysogenic Escherichia coli (2) and causes degradation of chromosomal DNA (5, 6, 10). Autoinduction of colicin E2 in cells with broken-down immunity has also been reported (9). Colicin E2 is a complex of two subunits, and its large subunit (the killing or active subunit) has DNA-cleaving activity in vitro which is carried on its C-terminal part (15,16). However, the DNase activity observed in vitro alone is not sufficiently potent to explain the single-hit killing in vivo, since DNA molecules are not considerably degraded in vitro compared with in vivo with equivalent amounts of colicin molecules (16). Our recent findings showed that the large subunits of pyocin AP41 and E2 group colicins share the homologous domain of DNase (13), suggesting that pyocin AP41 possesses DNase activity. Pyocin AP41 also might be multifunctional in its mode of killing, since the large subunit of pyocin AP41 is a rather large molecule having an extra domain compared with E2 group colicins and homologous S-type pyocins Si and S2 (14). Here we report the killing action of pyocin AP41 in vivo, as well as its function in vitro.In this study, we used P. aeruginosa PAF41 and PA03295 (recA) as a producer strain and a strain sensitive to pyocin AP41, respectively (3,11).Effects on macromolecular metabolism in sensitive bacteria. To clarify the molecular mechanisms of the killing action of pyocin AP41, we first examined its effects on protein, nucleic acid, and lipid metabolism in sensitive bacteria. For efficient killing, cells were grown in nutrient broth no. 2 (NB; Oxoid Ltd., London, England) at 25 g/liter in L-shaped tubes with constant seesaw shaking until the cell density reached approximately 2 x 108/ml. Then the radioactive precursor was added to each culture; this was followed by treatment with or without pyocin AP41. Trichloroacetic acid-insoluble counts were measured at a given time. As shown in Fig. 1, uptake of [14C]adenosine into the DNA fraction (c) was remarkably changed...

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Molecular characterisation of the colicin E2 operon and identification of its products

Cited by 126 publications

References 50 publications

Lipid modification of Escherichia coli penicillin-binding protein 3

Lipid modification of Escherichia coli penicillin-binding protein 3

Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3

The inherent DNase of pyocin AP41 causes breakdown of chromosomal DNA

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