A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: Application for DNA fragment transfer between chromosomes and plasmid transformation

Choi, Kyoung-Hee; Kumar, Ayush; Schweizer, Herbert P.

doi:10.1016/j.mimet.2005.06.001

Cited by 904 publications

(785 citation statements)

References 24 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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“…Plasmids were propagated in Escherichia coli DH5a or DH5alpir (for pUIC3 derivatives). Transformation of DNA into E. coli and P. fluorescens was accomplished by chemical transformation, electroporation or conjugation according to standard protocols 29,30 . E. coli was grown on Luria broth (LB) media at 37°C and P. fluorescens on LB or minimal (M9) media at 28°C.…”

Section: Methodsmentioning

confidence: 99%

Adaptive synonymous mutations in an experimentally evolved Pseudomonas fluorescens population

2014

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Conventional wisdom holds that synonymous mutations, nucleotide changes that do not alter the encoded amino acid, have no detectable effect on phenotype or fitness. However, a growing body of evidence from both comparative and experimental studies suggests otherwise. Synonymous mutations have been shown to impact gene expression, protein folding and fitness, however, direct evidence that they can be positively selected, and so contribute to adaptation, is lacking. Here we report the recovery of two beneficial synonymous single base pair changes that arose spontaneously and independently in an experimentally evolved population of Pseudomonas fluorescens. We show experimentally that these mutations increase fitness by an amount comparable to non-synonymous mutations and that the fitness increases stem from increased gene expression. These results provide unequivocal evidence that synonymous mutations can drive adaptive evolution and suggest that this class of mutation may be underappreciated as a cause of adaptation and evolutionary dynamics.

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“…Karsten, while Burkholderia species applied were B. glathei, B. hospita, B. fungorum and B. caledonica (Table 1). To visualize the interaction with fungi under the fluorescent microscope, all Burkholderia strains were tagged with either the green fluorescent protein (GFP) or the dsRED protein using the electroporation protocol described by Choi et al (2006). In short, overnight cultures of Burkholderia strains were washed twice in 0.3 M sucrose and subsequently electroporated with the plasmid pBBR1MCS-2-gfp mut3 Km r or pin62: DsRed Cm r .…”

Section: Co-occurrence Analysismentioning

confidence: 99%

Molecular mechanisms underlying the close association between soil Burkholderia and fungi

Stopnišek¹,

Zühlke²,

Carlier³

et al. 2015

The ISME Journal

109

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Bacterial species belonging to the genus Burkholderia have been repeatedly reported to be associated with fungi but the extent and specificity of these associations in soils remain undetermined. To assess whether associations between Burkholderia and fungi are widespread in soils, we performed a co-occurrence analysis in an intercontinental soil sample collection. This revealed that Burkholderia significantly co-occurred with a wide range of fungi. To analyse the molecular basis of the interaction, we selected two model fungi frequently co-occurring with Burkholderia, Alternaria alternata and Fusarium solani, and analysed the proteome changes caused by cultivation with either fungus in the widespread soil inhabitant B. glathei, whose genome we sequenced. Co-cultivation with both fungi led to very similar changes in the B. glathei proteome. Our results indicate that B. glathei significantly benefits from the interaction, which is exemplified by a lower abundance of several starvation factors that were highly expressed in pure culture. However, co-cultivation also gave rise to stress factors, as indicated by the increased expression of multidrug efflux pumps and proteins involved in oxidative stress response. Our data suggest that the ability of Burkholderia to establish a close association with fungi mainly lies in the capacities to utilize fungalsecreted metabolites and to overcome fungal defense mechanisms. This work indicates that beneficial interactions with fungi might contribute to the survival strategy of Burkholderia species in environments with sub-optimal conditions, including acidic soils.

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A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: Application for DNA fragment transfer between chromosomes and plasmid transformation

Cited by 904 publications

References 24 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

Adaptive synonymous mutations in an experimentally evolved Pseudomonas fluorescens population

Molecular mechanisms underlying the close association between soil Burkholderia and fungi

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