Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1

Lacks, Sanford A.; López, Paloma; Greenberg, Bill; Espinosa, Manuel

doi:10.1016/0022-2836(86)90026-4

Cited by 240 publications

(215 citation statements)

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“…Single-stranded DNA replicons are classified into families of highly related plasmids isolated from diverse bacteria (11). The plus origin (plus-on) and the Rep protein of pWVO1 (the parent of the Ts plasmid [25]) have homologies with members of the pE194 family, including pADB201 (2), pLB4 (1), pLS1 (19,30), pSH71 (7a), pFX2 (49), and pHPK255 (16). A Ts derivative of pE194 has already been characterized; two mutations were found, one immediately upstream of its start codon, and the other within the Rep ORF (48).…”

Section: Resultsmentioning

confidence: 99%

“…Sequence analysis (25) revealed that pWV01 is a member of a family of plasmids which replicate by a rolling-circle mechanism, via a single-stranded DNA intermediate (11,25,44). Among its close relatives are streptococcal plasmid pLS1 (19,39) and staphylococcal plasmid pE194 (4,15), which does not replicate in lactococci (6). Plasmid pE194 is naturally Ts at 51°C (43), and a derivative was obtained which is Ts at 39 to 420C (10a, 48); this example suggested the feasibility of our project.…”

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confidence: 99%

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New thermosensitive plasmid for gram-positive bacteria

et al. 1992

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We isolated a replication-thermosensitive mutant of the broad-host-range replicon pVWVOl. The mutant pVE6002 is fuly thermosensitive above 35°C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon TnlO derivative in BaciUus subtilis, we observed transposition frequencies of about 1%.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

New thermosensitive plasmid for gram-positive bacteria

et al. 1992

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“…Bacterial Strains, Growth, and Transformation of Bacteria-S. pneumoniae was grown in a casein hydrolysate-based medium with 0.3% sucrose as an energy source (AGCH medium) and transformed with chromosomal or plasmid DNA as described previously (16). Minimal inhibitory concentrations (MICs) 2 were determined in the same medium by the microdilution method according to the National Committee for Clinical Laboratory Standards (17).…”

Section: Methodsmentioning

confidence: 99%

New Alkaloid Antibiotics That Target the DNA Topoisomerase I of Streptococcus pneumoniae

Garcı́a

Blázquez

Ferrándiz

et al. 2011

Journal of Biological Chemistry

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Streptococcus pneumoniae has two type II DNA-topoisomerases (DNA-gyrase and DNA topoisomerase IV) and a single type I enzyme (DNA-topoisomerase I, TopA), as demonstrated here. Although fluoroquinolones target type II enzymes, antibiotics efficiently targeting TopA have not yet been reported. Eighteen alkaloids (seven aporphine and 11 phenanthrenes) were semisynthesized from boldine and used to test inhibition both of TopA activity and of cell growth. Two phenanthrenes (seconeolitsine and N-methyl-seconeolitsine) effectively inhibited both TopA activity and cell growth at equivalent concentrations (ϳ17 M). Evidence for in vivo TopA targeting by seconeolitsine was provided by the protection of growth inhibition in a S. pneumoniae culture in which the enzyme was overproduced. Additionally, hypernegative supercoiling was observed in an internal plasmid after drug treatment. Furthermore, a model of pneumococcal TopA was made based on the crystal structure of Escherichia coli TopA. Docking calculations indicated strong interactions of the alkaloids with the nucleotide-binding site in the closed protein conformation, which correlated with their inhibitory effect. Finally, although seconeolitsine and N-methyl-seconeolitsine inhibited TopA and bacterial growth, they did not affect human cell viability. Therefore, these new alkaloids can be envisaged as new therapeutic candidates for the treatment of S. pneumoniae infections resistant to other antibiotics.Antibiotic resistance in bacterial pathogens is a serious clinical problem. This problem affects Streptococcus pneumoniae (the pneumococcus), which, in addition to being one of the principal human pathogens, is the main ethyological agent of community-acquired pneumonia. Annually, approximately one million children aged Ͻ5 years die of pneumococcal pneumonia, meningitis, and/or sepsis worldwide (1). Resistance to currently used antimicrobial drugs for the treatment of pneumococcal infections, including ␤-lactams and macrolides, has spread worldwide in the last two decades (2). The new fluoroquinolones, such as levofloxacin and moxifloxacin, which act on type II DNA topoisomerases, are therapeutic alternatives for treatment of adult patients with community-acquired pneumonia (3). However, although resistance to fluoroquinolones in S. pneumoniae is still lower than 3% (4), an increase in resistance is likely to occur.DNA topoisomerases participate in almost all cellular functions involving DNA transactions (5). They solve the topological problems associated with DNA replication, transcription, and recombination. In addition, these enzymes fine-tune the steady-state level of DNA supercoiling, both facilitating protein interactions with the DNA and preventing excessive supercoiling that is deleterious. In bacteria, the homeostasis of DNA supercoiling is maintained by the opposing activities of topoisomerases that relax DNA and gyrase that introduces negative supercoils. The transcriptional response to DNA relaxation involves genes coding for all the DNA topoisomerases from S...

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“…From this vector (pORI) the gene encoding the replication initiation protein RepA was removed. Therefore, pORI is unable to replicate in any Km , Tc , pKVB2 (Kiel et al 1987) carrying the repA gene of pWV01 Law et al (1995) pUC19E Ap , Em , pUC19 (Yanisch-Perron et al 1985) carrying the Em of pE194 (Horinouchi and Weisblum 1982) in the SmaI restriction site Laboratory collection pTC2 Ap , Tc , pMTL25 (Chambers et al 1988) carrying the Tc of pLS1 (Lacks et al 1986) Buist et al (1995) bacterial strain unless RepA is provided in trans. The construction of integration vectors from pORI plasmids is possible by the availability of Escherichia coli, Bacillus subtilis and¸actococcus lactis helper strains that produce the replication protein from a chromosomally incorporated copy of repA (Leenhouts et al 1991a;Law et al 1995;Leenhouts, unpublished data).…”

Section: Introductionmentioning

confidence: 99%

A general system for generating unlabelled gene replacements in bacterial chromosomes

et al. 1996

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A general system for generating unlabelled gene replacements in bacterial chromosomes Leenhouts, K.; Buist, Girbe; Bolhuis, A.; Berge, A. ten; Kiel, Jan; Mierau, I.; Dabrowska, M.; Venema, G.; Kok, Jan IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document VersionPublisher's PDF, also known as Version of record Publication date: 1996Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Leenhouts, K., Buist, G., Bolhuis, A., Berge, A. T., Kiel, J., Mierau, I., ... Kok, J. (1996). A general system for generating unlabelled gene replacements in bacterial chromosomes. Molecular and General Genetics MGG, 253(1-2), 217-224. DOI: 10.1007/s004380050315 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Abstract A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using¸actococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

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Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1

Cited by 240 publications

References 51 publications

New thermosensitive plasmid for gram-positive bacteria

New thermosensitive plasmid for gram-positive bacteria

New Alkaloid Antibiotics That Target the DNA Topoisomerase I of Streptococcus pneumoniae

A general system for generating unlabelled gene replacements in bacterial chromosomes

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