Cloning and expression of the ltf gene of bacteriophage T5

Heller, Knut J.; Krauel, Verena

doi:10.1128/jb.167.3.1071-1073.1986

Cited by 10 publications

(8 citation statements)

References 18 publications

(11 reference statements)

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“…This agrees with 150 kDa reported by Heller and Krauel [5] for a precursor of the LTF protein. The direction of ltfgene transcription is from the right to the left (Fig.…”

Section: S G a S A P A I R A M W C D G S L A D T T R Y L G A T Q P 11supporting

confidence: 82%

“…However, it has been proven nonessential for infection since phages lacking the LTF are viable [4], and E. coli strains lacking an appropriate O antigen are infected by bacteriophage T5 [1,3]. The ltf gene was mapped within the BamHI-D fragment ofT5 DNA [5]. It is situated at the left end of the late T5 gene region near the boundary of the early and late genes, and their transcription is accomplished from different DNA strands in the opposite directions [6].…”

Section: Introductionmentioning

confidence: 99%

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The nucleotide sequence of the bacteriophage T5 ltf gene

et al. 1995

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“…This agrees with 150 kDa reported by Heller and Krauel [5] for a precursor of the LTF protein. The direction of ltfgene transcription is from the right to the left (Fig.…”

Section: S G a S A P A I R A M W C D G S L A D T T R Y L G A T Q P 11supporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

The nucleotide sequence of the bacteriophage T5 ltf gene

et al. 1995

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“…The ltf gene codes for a 148-kDa protein (27), and in agreement with the cleavage site proposed here, processing of a 150-kDa precursor protein into a 125-kDa protein was reported (30). The presence of a common cleavage site is also supported by results obtained for the recombinantly expressed lyase of E. coli K5 which depolymerizes the capsular polysaccharide composed of repeating units of 4-linked ␣-N-acetylglucosamine and ␤-glucuronic acid (29,31).…”

Section: Discussionsupporting

confidence: 65%

Proteolytic Processing and Oligomerization of Bacteriophage-derived Endosialidases

Mühlenhoff

Stummeyer

Grove

et al. 2003

Journal of Biological Chemistry

105

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Bacteriophages infecting the neuroinvasive pathogenEscherichia coli K1 require an endosialidase to penetrate the polysialic acid capsule of the host. Sequence information is available for the endosialidases endoNE, endoNF, and endoN63D of the K1-specific phages K1E, K1F, and 63D, respectively. The cloned sequences share a highly conserved catalytic domain but differ in the length of the N-and C-terminal parts. Although the expression of active recombinant enzyme succeeded in the case of endoNE, it failed for endoNF. Protein alignments of all three endosialidase sequences gave rise to the assumption that inactivity of the cloned endoNF is caused by a C-terminal truncation. By reinvestigation of the respective gene locus in the K1F genome, we identified an extended open reading frame of 3195 bp, encoding a 119-kDa protein.Full-length endoNF contains the C-terminal domain conserved in all endosialidases, which may act as an intramolecular chaperone. Comparative studies carried out with endoNE and endoNF demonstrate that endosialidases are proteolytically processed, releasing the C-terminal domain. Using a mutational approach in combination with protein analytical techniques we demonstrate that (i) the C-terminal domain is a common feature of endosialidases and other tail fiber proteins; (ii) the integrity of the Cterminal domain and its presence in the nascent protein are crucial for the formation of active enzymes; (iii) proteolytic processing is not essential for enzymatic activity; and (iv) functional folding is a prerequisite for trimerization of endoNF.

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“…Additional labeled proteins, which exceeded the coding capacity of pUOA1, were seen in the gels. This phenomenon has been reported previously with a similar in vitro system (13). In contrast to pUOA1, pUOA4, its Tcs derivative, did not specify the 68,000-dalton protein.…”

Section: Resultsmentioning

confidence: 47%

Characterization and expression of a cloned tetracycline resistance determinant from Campylobacter jejuni plasmid pUA466

et al. 1987

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A tetracycline resistance (Tcr) determinant previously cloned from the Campylobacterjejwu plasmid pUA466 (D. E. Taylor, J. Bacteriol. 165:1037Bacteriol. 165: -1039Bacteriol. 165: , 1986) was localized by restriction endonucleae mapping, subcloning, and Tnl000 insertion mutagenesis to a 2-kilobase region consisting of 1.8-and 0.2-lobse Hindc fragments. Tcr encoded by the cloned fraent (pUOA1) was expressed constitutively in Escherichia coli. A protein with an apparent molecular weight of 68,000 encoded by pUOA1 was produced in an in vitro transcription-translation system and in minicells. TnlO00 insertions which resulted in inactivation of Tcr expression also resulted in an alteration in the 68,000-molecular-weight protein. Some mutants specified a truncated protein, whereas others completely lost the ability to specify the protein.The protein which appears to be involved in the exprssion of Tcr s lecffied by C. jejuxi pasmids is of approximately the same molkcular weight as the protein specified by the streptococcal dass M determinant. This finling is consistent with our previous results which indicate that homology exists between the Tcr determinant from C. jejuni and a 5-kilobase HincH probe derived from the streptococcal class M determinant. (17) for members of the family Enterobacteriaceae (28). In contrast, some homology was detected between the pUA466 plasmid from C. jejuni and a 5-kb probe for the class M determinant derived from a gram-positive coccus (25). The class M determinant, although originally cloned from Streptococcus agalactiae B109 (5), has now been shown to have a broad host range and has been identified in the chromosome of Streptococcus spp., Staphylococcus spp. (15), Mycoplasma hominis (24), Ureaplasma urealyticum (23), and Gardnerella vaginalis (22) and on plasmids in Neisseria gonorrhoeae (18).Recently the nucleotide sequence of a class M determinant from the streptococcal conjugative shuttle transposon TnJS45 has been determined (16). A TET protein of 68,000 daltons, which corresponded approximately to the tetM DNA coding sequence of 1,917 base pairs, was identified in minicells (16). In this paper we report the characterization of a cloned Tcr determinant from the C. jejuni plasmid pUA466 and the expression of a 68,000-dalton TET protein specified by this determinant. MATERIALS AND METHODSBacterial strains, plasmids, and media. C. jejuni UA466 was grown on Mueller-Hinton agar (Oxoid Ltd., London, England) and incubated at 37°C under 7% CO2 and 85% humidity. Escherichia coli strains used in transformation experiments were JM105 (30) and minicell-producing strain P678-54 (2). E. coli strains were grown in LB broth (GIBCO Diagnostics, Madison, Wis.), brain heart infusion broth (Difco Laboratories, Detroit, Mich.), or nutrient broth (NB) * Corresponding author.(Difco). Tetracycline was added to autoclaved media at 8 pLg/ml, ampicillin was added at 25 ,ug/ml, and carbenicillin was added at 500 ,ug/ml. The C. jejuni plasmid pUA466, pUC8, and plasmid derivatives obtained by cloning fragments...

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Cloning and expression of the ltf gene of bacteriophage T5

Cited by 10 publications

References 18 publications

The nucleotide sequence of the bacteriophage T5 ltf gene

The nucleotide sequence of the bacteriophage T5 ltf gene

Proteolytic Processing and Oligomerization of Bacteriophage-derived Endosialidases

Characterization and expression of a cloned tetracycline resistance determinant from Campylobacter jejuni plasmid pUA466

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