Plasmids containing cos ends inhibit the replication of phage φCTX in Pseudomonas aeruginosa

Guan, Xiong; Oepen, P.; Geiben, Ralf; El-Idrissi, Ahmed H.; Lutz, F.

doi:10.1016/0168-1702(95)01279-6

Cited by 3 publications

(5 citation statements)

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“…CAT, chloramphenicol acetyltransferase During the infection of P. aeruginosa E40 cells by /CTX at 37°C, cos site ligation, /CTX DNA integration, and CTX production exhibited dierent kinetics. Primary ligation of the cos site of /CTX DNA was observed within 1 h (Table 2), indicating that the replication process had occurred (Xiong et al 1996). The time required for cos ligation is in accordance with experiments E. coli phages where a latent period of <1 h was observed (Ellis and DelbruÈ ck 1939;Schwienhorst et al 1996).…”

Section: Discussionsupporting

confidence: 60%

“…ctx expression in P. aeruginosa correlates with chromosomal integration of /CTX DNA P. aeruginosa 158 has steadily produced CTX in large quantities for 20 years under laboratory conditions (Scharmann 1976;Xiong et al 1996). The EcoRI-digested whole DNA extracts of all colonies that hybridized with a ctx probe, 70 single colonies tested, contained a 12 000-bp fragment derived from the chromosomally integrated /CTX genome (Elsabbagh et al 1993).…”

Section: Resultsmentioning

confidence: 99%

“…A non-lysogenic strain, E40 (horse abscess; Xiong et al 1996), was used for transformation/expression experiments. P. aeruginosa and Escherichia coli HB101, both of which are sensitive to b-lactam antibiotics, were grown in a 180-rpm shaker at 37°C in 2 ml of tryptic soy broth medium unless otherwise indicated.…”

Section: Methodsmentioning

confidence: 99%

“…Transformed cells were selected in the presence of 300 lg/ml carbenicillin (E40) or 80 lg/ml ampicillin (A r ; HB101). /CTX preparation, storage, and infection of host cells were performed as described earlier (Xiong et al 1996). Plasmids used were pHA10 (A r , P tac , 80±120 copies/cell; Arai et al 1991) and pCM7 (Pharmacia).…”

Section: Methodsmentioning

confidence: 99%

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Control of effective expression of the phage φCTX-encoded ctx gene in Pseudomonas aeruginosa by a promoter upstream of the cos site

et al. 1998

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In the DNA of bacteriophage phiCTX, the attachment site (attP), the cohesive end site (cos), and the gene (ctx) encoding the pore-forming cytotoxin, are clustered. Deletion variants and PCR-generated fragments of the DNA were cloned into the Pseudomonas aeruginosa and Escherichia coli vector pHA10. Recombinant plasmids carrying the chloramphenicol acetyltransferase gene, cat, were used for promoter studies. Two promoters responsible for ctx expression are located between attP and cos and between cos and ctx, the promoter upstream of cos being stronger than the one downstream. The promoters and the ribosomal binding site are recognized by both the P. aeruginosa and E. coli transcriptional systems. The results indicate that chromosomal integration of phiCTX DNA, which requires cos site ligation, is necessary for high ctx expression in phiCTX-infected P. aeruginosa strains.

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Section: Discussionsupporting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Control of effective expression of the phage φCTX-encoded ctx gene in Pseudomonas aeruginosa by a promoter upstream of the cos site

et al. 1998

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“…P. aeruginosa strain GuA18 (Xiong et al 1996) was used for isolation of bacterial membranes, flagellin filaments, phage propagation, phage inhibition assay, and the phage-overlay protein blot assay. Bacteria were cultivated at 37°C in tryptic soy broth and tryptic soy agar (Hedinger, Stuttgart).…”

Section: Methodsmentioning

confidence: 99%

Flagellin inhibits Myoviridae phage φCTX infection of Pseudomonas aeruginosa strain GuA18: purification and mapping of binding site

Geiben-Lynn

Sauber

Lutz

2001

Archives of Microbiology

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PhiCTX is a double-stranded DNA phage of the Myoviridae family that converts Pseudomonas aeruginosa into a cytotoxin producer. A 42-kDa phiCTX-inhibiting protein was purified from the outer membrane fraction of P. aeruginosa strain GuA18 by octyl-beta-glucoside extraction, DEAE-chromatography, and mono-Q HPLC. This protein had an isoelectric point of 5.4 and bound specifically [125I]-labeled phiCTX. The N-terminal amino acid sequence of six out of seven Lys-C fragments was highly similar (87%) to that of the entire of type-a flagellin of P. aeruginosa strain PAK. At a concentration of 14 nM, purified flagellin protein caused a 50% decrease in the phage titer after a 20-min incubation at 37 degrees C (PhI50). The presence of ethanol was necessary to reconstitute the inhibitory activity. In contrast, no ethanol treatment was necessary for the inhibitory activity of the sheared flagellin filaments from P. aeruginosa strain GuA18, which consists of the 42-kDa flagellin subunits and the synthesized 17-mer phage-binding-peptide NGSNSDSERTALNGEAK, representing flagellin residues 100-116 of P. aeruginosa strain PAK. The PhI50 was 10 nM and 200 nM, respectively. Antisera against the flagellin filament protein as well as against the 17-mer peptide neutralized phage infection. These results indicated that the amino acid region 100-116 of the flagellin subunit of strain GuA18 is involved in phiCTX binding. This region might play a role in phage attachment.

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Mutational analysis of bacteriophage φCTX cos site

Guan

Lutz

1996

Virus Research

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Plasmids containing cos ends inhibit the replication of phage φCTX in Pseudomonas aeruginosa

Cited by 3 publications

References 24 publications

Control of effective expression of the phage φCTX-encoded ctx gene in Pseudomonas aeruginosa by a promoter upstream of the cos site

Control of effective expression of the phage φCTX-encoded ctx gene in Pseudomonas aeruginosa by a promoter upstream of the cos site

Flagellin inhibits Myoviridae phage φCTX infection of Pseudomonas aeruginosa strain GuA18: purification and mapping of binding site

Mutational analysis of bacteriophage φCTX cos site

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