A panel of Tn7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site

Koch, Birgit; Jensen, Linda; Nybroe, Ole

doi:10.1016/s0167-7012(01)00246-9

Cited by 242 publications

(175 citation statements)

References 32 publications

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“…P. putida KT2440::miniTn 7 ‐Gm:GFP and P. putida KT2440 Δ fcs ::miniTn 7 ‐Gm:GFP (Table 1) were constructed by transformation of plasmid pBK‐miniTn7‐gfp2 (Koch, Jensen, & Nybroe, 2001) as previously described (Choi, Kumar, & Schweizer, 2006), using plasmid pTNS2 (Choi et al, 2005) as a helper. The correct integration of the transposon was checked by colony PCR using primers Tn7‐GlmS and Tn7R109 (Table 2).…”

Section: Methodsmentioning

confidence: 99%

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

Calero

Jensen

Bojanovič

et al. 2017

Biotech & Bioengineering

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The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p‐coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome‐wide approach, Tn‐seq. Libraries containing a large number of miniTn5‐Km transposon insertion mutants were grown in the presence and absence of p‐coumaric acid, and the enrichment or depletion of mutants was quantified by high‐throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p‐coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p‐coumaric acid is related to transport and membrane integrity.

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Section: Methodsmentioning

confidence: 99%

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

Calero

Jensen

Bojanovič

et al. 2017

Biotech & Bioengineering

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“…The 1.8 kb XbaI-SacI fragment from pBK-miniTn7-VGm (Koch et al, 2001) carrying a mini-Tn7-VGm cassette was inserted into plasmid pGP704 L (Pavel et al, 1994) cut with XbaI and SacI. Then, the overexpression cassette of P. putida fis was cloned into pJB785TT (Santos et al, 2001).…”

Section: Methodsmentioning

confidence: 99%

“…The suicide vector pBK-miniTn7-VSm1 (Koch et al, 2001) was used to insert the mini-Tn7 transposon into the chromosome of P. putida strain PSm, and this strain was subsequently used as the wild-type strain in our experiments.…”

Section: Vgmmentioning

confidence: 99%

Fis regulates the competitiveness of Pseudomonas putida on barley roots by inducing biofilm formation

et al. 2012

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An important link between the environment and the physiological state of bacteria is the regulation of the transcription of a large number of genes by global transcription factors. One of the global regulators, Fis (factor for inversion stimulation), is well studied in Escherichia coli, but the role of this protein in pseudomonads has only been examined briefly. According to studies in Enterobacteriaceae, Fis regulates positively the flagellar movement of bacteria. In pseudomonads, flagellar movement is an important trait for the colonization of plant roots. Therefore we were interested in the role of the Fis protein in Pseudomonas putida, especially the possible regulation of the colonization of plant roots. We observed that Fis reduced the migration of P. putida onto the apices of barley roots and thereby the competitiveness of bacteria on the roots. Moreover, we observed that overexpression of Fis drastically reduced swimming motility and facilitated P. putida biofilm formation, which could be the reason for the decreased migration of bacteria onto the root apices. It is possible that the elevated expression of Fis is important in the adaptation of P. putida during colonization of plant roots by promoting biofilm formation when the migration of bacteria is no longer favoured.

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“…E. coli donor and helper strains used for random transposon mutagenesis with mini-Tn5[Km1] have been described elsewhere (de Lorenzo et al, 1990). Derivatives of KT2440 and ibi-626 carrying gfp in single copy in the chromosome were obtained using mini-Tn7, as described by Koch et al (2001).Unless otherwise stated, cultures were grown in liquid Luria-Bertani (LB) medium (Lennox, 1955) or 1 : 10 LB, and on LB plates with 1.5 % agar, at 30 uC (P. putida) or 37 uC (E. coli). When appropriate, antibiotics were used at the following concentrations (in mg ml 21 ): ampicillin, 100; chloramphenicol, 30; kanamycin, 50; gentamicin, 10 and 100 for E. coli and P. putida, respectively.…”

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confidence: 99%

Selection of hyperadherent mutants in Pseudomonas putida biofilms

et al. 2011

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A number of genetic determinants required for bacterial colonization of solid surfaces and biofilm formation have been identified in different micro-organisms. There are fewer accounts of mutations that favour the transition to a sessile mode of life. Here we report the isolation of random transposon Pseudomonas putida KT2440 mutants showing increased biofilm formation, and the detailed characterization of one of them. This mutant exhibits a complex phenotype, including altered colony morphology, increased production of extracellular polymeric substances and enhanced swarming motility, along with the formation of denser and more complex biofilms than the parental strain. Sequence analysis revealed that the pleiotropic phenotype exhibited by the mutant resulted from the accumulation of two mutations: a transposon insertion, which disrupted a predicted outer membrane lipoprotein, and a point mutation in lapG, a gene involved in the turnover of the large adhesin LapA. The contribution of each alteration to the phenotype and the possibility that prolonged sessile growth results in the selection of hyperadherent mutants are discussed. INTRODUCTIONThe development of a multicellular community associated with a surface and surrounded by an exopolymeric matrix, referred to as a biofilm, is common to a variety of bacteria under different environmental conditions. Biofilm formation has received increasing attention due to its importance in medicine, since biofilm populations are considered relevant to chronic infection, and are more resistant to the action of antibiotics and biocidals than planktonic populations (Anderson & O'Toole, 2008;Høiby et al., 2010). Biofilm development is also relevant in industrial settings and in the design of bioreactors (Nicolella et al., 2000;Singh et al., 2006). Genetic determinants that play a role in biofilm formation have been unravelled in different bacterial species, and environmental and cellular signals that influence this process have also been described. These include iron availability, quorum sensing, and the intracellular secondary messenger cyclic di-GMP (Banin et al., 2006;Patriquin et al., 2008;Ueda & Wood, 2009;Coenye, 2010). Changes in the levels of cyclic di-GMP correlate with phenotypic changes associated with virulence, motility, colony morphology, production of exopolysaccharides, and the transition between planktonic and sessile lifestyles (Hengge, 2009; Römling & Simm, 2009, and references therein). Changes in the expression of different functions are also associated with one or the other lifestyle; in Pseudomonas aeruginosa and Pseudomonas putida, for example, differential expression of flagellar components has been observed when comparing planktonic and biofilm populations, and even during the various stages of biofilm development (Sauer & Camper, 2001;Sauer et al., 2002;Toutain et al., 2007). In the former organism, swarming motility and surface attachment are inversely regulated through a pathway that involves BifA, a cyclic-di-GMP phosphodiesterase, and the membra...

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A panel of Tn7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site

Cited by 242 publications

References 32 publications

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

Fis regulates the competitiveness of Pseudomonas putida on barley roots by inducing biofilm formation

Selection of hyperadherent mutants in Pseudomonas putida biofilms

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