scispace - formally typeset
Search or ask a question
Journal ArticleDOI

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

01 Oct 2011-Environmental Microbiology (John Wiley & Sons, Ltd)-Vol. 13, Iss: 10, pp 2702-2716
TL;DR: 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.
Abstract: Summary The 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, eg 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
Citations
More filters
Journal ArticleDOI
TL;DR: It is argued that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.
Abstract: The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

548 citations


Cites background from "Engineering multiple genomic deleti..."

  • ...This characteristic makes R6K very useful for genome-editing techniques, where chromosomal integration of a specific plasmid is required (31,36)....

    [...]

  • ...Such a formatting (and the resulting six additional vectors, see SEVA-DB) expands the utility of the original procedure (36) to bacteria that are naturally resistant to Km, which was the only selection marker that was available up to that point for the genome-editing method....

    [...]

Journal ArticleDOI
TL;DR: This Review advocate the use of environmental Pseudomonas strains as model organisms that are pre-endowed with the metabolic, physiological and stress-endurance traits that are demanded by current and future synthetic biology and biotechnological needs.
Abstract: Much of synthetic biology research makes use of model organisms, such as Escherichia coli. Here, Victor de Lorenzo and colleagues emphasize the need for a wider choice of model organisms and advocate the use of environmental Pseudomonas strains as model organisms that possess the necessary metabolic traits required to meet current and future synthetic biology and biotechnological needs.

322 citations

Journal ArticleDOI
TL;DR: Pseudomonas putida and related subspecies, traditionally known as well-performing xenobiotic degraders, are becoming efficient cell factories for various products of industrial relevance including a full range of unnatural chemicals.
Abstract: Since their discovery many decades ago, Pseudomonas putida and related subspecies have been intensively studied with regard to their potential application in industrial biotechnology. Today, these Gram-negative soil bacteria, traditionally known as well-performing xenobiotic degraders, are becoming efficient cell factories for various products of industrial relevance including a full range of unnatural chemicals. This development is strongly driven by systems biotechnology, integrating systems metabolic engineering approaches with novel concepts from bioprocess engineering, including novel reactor designs and renewable feedstocks.

300 citations

Journal ArticleDOI
TL;DR: High levels of antibiotics, ARGs, HPB, and HPB carrying ARGs in the manured greenhouse soils compared with those in the field soils were confirmed, and their relative abundance increased with the extension of greenhouse planting years.
Abstract: Antibiotic resistance genes (ARGs), human pathogenic bacteria (HPB), and HPB carrying ARGs pose a high risk to soil ecology and public health. Here, we used a metagenomic approach to investigate their diversity and abundance in chicken manures and greenhouse soils collected from Guli, Pulangke, and Hushu vegetable bases with different greenhouse planting years in Nanjing, Eastern China. There was a positive correlation between the levels of antibiotics, ARGs, HPB, and HPB carrying ARGs in manures and greenhouse soils. In total, 156.2–5001.4 μg/kg of antibiotic residues, 22 classes of ARGs, 32 HPB species, and 46 species of HPB carrying ARGs were found. The highest relative abundance was tetracycline resistance genes (manures) and multidrug resistance genes (greenhouse soils). The dominant HPB and HPB carrying ARGs in the manures were Bacillus anthracis, Bordetella pertussis, and B. anthracis (sulfonamide resistance gene, sul1), respectively. The corresponding findings in greenhouse soils were Mycobacterium tuberculosis and M. ulcerans, M. tuberculosis (macrolide-lincosamide-streptogramin resistance protein, MLSRP), and B. anthracis (sul1), respectively. Our findings confirmed high levels of antibiotics, ARGs, HPB, and HPB carrying ARGs in the manured greenhouse soils compared with those in the field soils, and their relative abundance increased with the extension of greenhouse planting years.

257 citations

Journal ArticleDOI
TL;DR: This review provides an overview of applications of P. putida as a host organism for the recombinant biosynthesis of such natural products, including rhamnolipids, terpenoids, polyketides and non-ribosomal peptides, and other amino acid-derived compounds.
Abstract: The biosynthesis of natural products by heterologous expression of biosynthetic pathways in amenable production strains enables biotechnological access to a variety of valuable compounds by conversion of renewable resources. Pseudomonas putida has emerged as a microbial laboratory work horse, with elaborated techniques for cultivation and genetic manipulation available. Beyond that, this bacterium offers several particular advantages with regard to natural product biosynthesis, notably a versatile intrinsic metabolism with diverse enzymatic capacities as well as an outstanding tolerance to xenobiotics. Therefore, it has been applied for recombinant biosynthesis of several valuable natural products. This review provides an overview of applications of P. putida as a host organism for the recombinant biosynthesis of such natural products, including rhamnolipids, terpenoids, polyketides and non-ribosomal peptides, and other amino acid-derived compounds. The focus is on de novo natural product synthesis from intrinsic building blocks by means of heterologous gene expression and strain engineering. Finally, the future potential of the bacterium as a chassis organism for synthetic microbiology is pointed out.

205 citations

References
More filters
Journal ArticleDOI
TL;DR: A simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s), which should be widely useful, especially in genome analysis of E. coli and other bacteria.
Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

14,389 citations

Journal ArticleDOI
15 Apr 1989-Gene
TL;DR: Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase.

3,339 citations

Journal ArticleDOI
TL;DR: Certain environmental signals (i.e., osmolarity and the presence of amino acids) are tightly coupled to the expression of toxR-regulated proteins and therefore may be signals that are directly sensed by the ToxR protein.
Abstract: The toxR gene of Vibrio cholerae encodes a transmembrane, DNA-binding protein that activates transcription of the cholera toxin operon and a gene (tcpA) for the major subunit of a pilus colonization factor. We constructed site-directed insertion mutations in the toxR gene by a novel method employing the chromosomal integration of a mobilizable suicide plasmid containing a portion of the toxR coding sequence. Mutants containing these new toxR alleles had an altered outer membrane protein profile, suggesting that two major outer membrane proteins (OmpT and OmpU) might be under the control of toxR. Physiological studies indicated that varying the concentration of the amino acids asparagine, arginine, glutamate, and serine caused coordinate changes in the expression of cholera toxin, TcpA, OmpT, and OmpU. Changes in the osmolarity of a tryptone-based medium also produced coordinate changes in the expression of these proteins. Other environmental signals (temperature and pH) had a more pronounced effect on the expression of cholera toxin and TcpA than they did on the outer membrane proteins. These results suggest that certain environmental signals (i.e., osmolarity and the presence of amino acids) are tightly coupled to the expression of toxR-regulated proteins and therefore may be signals that are directly sensed by the ToxR protein.

2,066 citations

Journal ArticleDOI
TL;DR: The ability of the prevalent beta-Lactamases to cause resistance to widely used beta-lactams, whether resistance is accurately reflected in routine tests, and the extent to which the antibiogram for an organism can be used to predict the type of beta- lactamase that it produces are considered.
Abstract: beta-Lactamases are the commonest single cause of bacterial resistance to beta-lactam antibiotics. Numerous chromosomal and plasmid-mediated types are known and may be classified by their sequences or phenotypic properties. The ability of a beta-lactamase to cause resistance varies with its activity, quantity, and cellular location and, for gram-negative organisms, the permeability of the producer strain. beta-Lactamases sometimes cause obvious resistance to substrate drugs in routine tests; often, however, these enzymes reduce susceptibility without causing resistance at current, pharmacologically chosen breakpoints. This review considers the ability of the prevalent beta-lactamases to cause resistance to widely used beta-lactams, whether resistance is accurately reflected in routine tests, and the extent to which the antibiogram for an organism can be used to predict the type of beta-lactamase that it produces.

1,882 citations

Journal ArticleDOI
28 May 1998-Gene
TL;DR: An improved method for gene replacement in Pseudomonas aeruginosa was developed and a cassette was constructed that contains a GmR selectable marker next to the green fluorescent protein structural gene, with both markers being flanked by Flp recombinase target (FRT) sites.

1,858 citations

Related Papers (5)