Engineering multiple genomic deletions in Gram‐negative bacteria: analysis of the multi‐resistant antibiotic profile of Pseudomonas putida KT2440

Wiley - Tập 13 Số 10 - Trang 2702-2716 - 2011
Esteban Martínez‐García1, Vı́ctor de Lorenzo1
1Systems Biology Program. Centro Nacional de Biotecnología‐CSIC, Campus de Cantoblanco, 28049 Madrid, Spain

Tóm tắt

SummaryThe genome of the soil bacterium Pseudomonas putida strain KT2440 has been erased of various determinants of resistance to antibiotics encoded in its extant chromosome. To this end, we employed a coherent genetic platform that allowed the precise deletion of multiple genomic segments in a large variety of Gram‐negative bacteria including (but not limited to) P. putida. The method is based on the obligatory recombination between free‐ended homologous DNA sequences that are released as linear fragments generated upon the cleavage of the chromosome with unique I‐SceI sites, added to the segment of interest by the vector system. Despite the potential for a SOS response brought about by the appearance of double stranded DNA breaks during the process, fluctuation experiments revealed that the procedure did not increase mutation rates – perhaps due to the protection exerted by I‐SceI bound to the otherwise naked DNA termini. With this tool in hand we made sequential deletions of genes mexC, mexE, ttgA and ampC in the genome of the target bacterium, orthologues of which are known to determine various degrees of antibiotic resistance in diverse microorganisms. Inspection of the corresponding phenotypes demonstrated that the efflux pump encoded by ttgA sufficed to endow P. putida with a high‐level of tolerance to β‐lactams, chloramphenicol and quinolones, but had little effect on, e.g. aminoglycosides. Analysis of the mutants revealed also a considerable diversity in the manifestation of the resistance phenotype within the population and suggested a degree of synergism between different pumps. The directed edition of the P. putida chromosome shown here not only enhances the amenability of this bacterium to deep genomic engineering, but also validates the corresponding approach for similar handlings of a large variety of Gram‐negative microorganisms.

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Tài liệu tham khảo

10.1016/S0092-8674(00)81721-3

10.1016/0378-1119(81)90080-9

10.1006/plas.1997.1294

Blatny J.M., 1997, Construction and use of a versatile set of broad‐host‐range cloning and expression vectors based on the RK2 replicon, Appl Env Microbiol, 63, 370, 10.1128/aem.63.2.370-379.1997

10.1073/pnas.0602119103

10.1111/j.1365-2958.2004.04155.x

10.1016/0092-8674(86)90262-X

10.1073/pnas.85.16.6022

10.1002/bies.200900024

10.1073/pnas.120163297

10.2174/138945008785747824

10.1128/MMBR.00020-08

Dominy C.N., 2003, Site‐directed mutagenesis by inverse PCR, Methods Mol Biol, 235, 209

10.1016/0147-619X(87)90044-8

10.1128/jb.179.20.6472-6479.1997

10.1111/j.1462-2920.2008.01576.x

10.1128/AEM.71.2.883-892.2005

10.1111/j.1751-7915.2010.00189.x

10.1093/nar/gnf122

10.1016/0076-6879(83)00066-X

10.1111/j.1462-2920.2009.02124.x

10.1016/S0378-1119(98)00130-9

10.1016/S1369-5274(02)00357-0

10.1016/0378-1119(89)90359-4

10.1128/JB.181.20.6300-6305.1999

10.1101/gr.217202

10.1016/j.jmb.2007.04.025

10.1093/nar/gkp1193

Lewis K., 2001, In search of natural substrates and inhibitors of MDR pumps, J Mol Microbiol Biotechnol, 3, 247

10.1128/CMR.8.4.557

10.1128/AEM.01669-09

10.1016/0076-6879(94)35157-0

10.1111/j.1365-2958.2006.05539.x

10.1111/j.1365-2958.1995.18050851.x

10.1111/j.1574-6976.2008.00157.x

10.1128/jb.167.2.447-454.1986

10.1128/jb.170.6.2575-2583.1988

10.1128/JB.182.4.937-943.2000

10.1371/journal.ppat.1000353

10.1046/j.1462-2920.2002.00366.x

10.1016/0378-1119(92)90263-O

10.1038/ng2051

10.1016/j.copbio.2004.03.008

10.1016/j.molcel.2005.07.025

10.1093/jac/dki171

10.1128/JB.175.22.7363-7372.1993

10.1093/nar/27.22.4409

10.1126/science.1126439

10.1016/0022-2836(90)90358-S

10.1128/JB.180.13.3323-3329.1998

10.1146/annurev.micro.56.012302.161038

10.1046/j.1462-2920.2002.00368.x

10.1128/jb.174.24.8119-8132.1992

10.1111/j.1751-7915.2007.00014.x

Sambrook J., 1989, Molecular Cloning: A Laboratory Manual

10.1111/j.1365-2958.1992.tb01558.x

10.1016/0022-2836(85)90414-0

10.1074/jbc.M511095200

10.1073/pnas.97.18.10191

10.1093/nar/gkn359

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Wiley

Tập 13 Số 10

2702-2716

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