Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa

West, Susan E. H.; Schweizer, Herbert P.; Dall, C.; Sample, Allen K.; Runyen-Janecky, Laura J.

doi:10.1016/0378-1119(94)90237-2

Cited by 538 publications

(261 citation statements)

References 26 publications

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“…The generation of recombinant NosR derivatives took advantage of the replication function of the broad-host-range plasmid pUCP22 in Pseudomonas sp. (46). Since NosZ biosynthesis depends on auxiliary gene products that affect its expression level and protein translocation efficiency (8,13,18,47), we cloned the entire nos cluster, including the tatE gene for protein transport, into pUCP22, which resulted in the nosR expression vector pPRE-3 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

Wunsch

Zumft

2005

J Bacteriol

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Bacterial nitrous oxide (N 2 O) respiration depends on the polytopic membrane protein NosR for the expression of N 2 O reductase from the nosZ gene. We constructed His-tagged NosR and purified it from detergentsolubilized membranes of Pseudomonas stutzeri ATCC 14405. NosR is an iron-sulfur flavoprotein with redox centers positioned at opposite sides of the cytoplasmic membrane. The flavin cofactor is presumably bound covalently to an invariant threonine residue of the periplasmic domain. NosR also features conserved CX 3 CP motifs, located C-terminally of the transmembrane helices TM4 and TM6. We genetically manipulated nosR with respect to these different domains and putative functional centers and expressed recombinant derivatives in a nosR null mutant, MK418nosR::Tn5. NosR's function was studied by its effects on N 2 O respiration, NosZ synthesis, and the properties of purified NosZ proteins. Although all recombinant NosR proteins allowed the synthesis of NosZ, a loss of N 2 O respiration was observed upon deletion of most of the periplasmic domain or of the C-terminal parts beyond TM2 or upon modification of the cysteine residues in a highly conserved motif, CGWLCP, following TM4. Nonetheless, NosZ purified from the recombinant NosR background exhibited in vitro catalytic activity. Certain NosR derivatives caused an increase in NosZ of the spectral contribution from a modified catalytic Cu site. In addition to its role in nosZ expression, NosR supports in vivo N 2 O respiration. We also discuss its putative functions in electron donation and redox activation.

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Section: Resultsmentioning

confidence: 99%

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

Wunsch

Zumft

2005

J Bacteriol

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“…PCR amplicons were cloned into the pUCP24 plasmid, a generous gift of Dr Schweizer (West et al, 1994). This is a shuttle vector which replicates in E. coli and in P. aeruginosa, and contains a multiple cloning site downstream from lacZa.…”

Section: Methodsmentioning

confidence: 99%

Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa

Latino

2016

Microbiology

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Coevolution between bacteriophages (phages) and their prey is the result of mutualistic interactions. Here, we show that pseudolysogeny is a frequent outcome of infection by virulent phages of Pseudomonas aeruginosa and that selection of resistant bacterial mutants is favoured by continuous production of phages. We investigated the frequency and characteristics of P. aeruginosa strain PAO1 variants resisting infection by different combinations of virulent phages belonging to four genera. The frequency of resistant bacteria was 10 25 for single phage infection and 10 26 for infections with combinations of two or four phages. The genome of 27 variants was sequenced and the comparison with the genome of the parental PAO1 strain allowed the identification of point mutations or small indels. Four additional variants were characterized by a candidate gene approach. In total, 27 independent mutations were observed affecting 14 genes and a regulatory region. The mutations affected genes involved in biosynthesis of type IV pilus, alginate, LPS and O-antigen. Half of the variants possessed changes in homopolymer tracts responsible for frameshift mutations and these phase variation mutants were shown to be unstable. Eleven double mutants were detected. The presence of free phage DNA was observed in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Upon further growth of these pseudolysogens, some variants with new chromosomal mutations were recovered, presumably due to continuous evolutionary pressure.

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“…PAI plates (2 % Bacto peptone, 1 % K 2 SO 4 , 0.03 % cetyltrimethylammonium bromide, 14.7 mM MgCl 2 , 1 % glycerol, 1.5 % agar) were used to select P. aeruginosa cells. E. coli cells carrying pUCP20T (Schweizer, 1991;West et al, 1994), pEX100T (Hoang et al, 1998), or their derivatives were cultured in L medium with 100 mg ampicillin ml 21 . P. aeruginosa cells carrying pUCP20T (Schweizer, 1991;West et al, 1994) or its derivatives were cultured in L medium with 100 mg carbenicillin ml 21 .…”

Section: Methodsmentioning

confidence: 99%

“…Primers Mux forward 1 and Mux reverse 1 were used to amplify the first half of muxABC-opmB in PMX7 or PMX725. Each PCR product was digested with EcoRI and KpnI and ligated into pUCP21T (Schweizer, 1991;West et al, 1994). Then the plasmid was digested with Eco52I to remove the Eco52I fragment (0.69 kbp).…”

Section: Methodsmentioning

confidence: 99%

Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa

Mima

Kohira

et al. 2009

Microbiology

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muxA-muxB-muxC-opmB (formerly PA2528-PA2527-PA2526-opmB), encoding a putative resistance nodulation cell division (RND)-type multidrug efflux pump system, was cloned from Pseudomonas aeruginosa PAO1. Introduction of muxABC-opmB into P. aeruginosa YM64, a drug-hypersusceptible strain, led to elevated MICs of aztreonam, macrolides, novobiocin and tetracycline. Since muxB and muxC, both of which encode RND components, were essential for function, MuxABC-OpmB is thought to be a drug efflux pump with four components. One novobiocin-resistant mutant, PMX725, isolated from P. aeruginosa PMX7 showed elevated resistance not only to novobiocin but also to aztreonam, macrolides and tetracycline. Increased mRNA expression of muxABC-opmB was observed in the mutant PMX725 compared with the parental strain. Sequencing analysis revealed that a single-nucleotide insertion had occurred in the deduced promoter region for muxABC-opmB in PMX725. In this study, we have characterized the last RND-type multidrug efflux pump predicted from the genome sequence in P. aeruginosa.

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Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa

Cited by 538 publications

References 26 publications

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa

Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa

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