An improved method of transformation in Pseudomonads

Liu, Shie-Chau; Webster, D. R.; Stark, Benjamin C.

doi:10.1007/bf00168480

Cited by 4 publications

(3 citation statements)

References 9 publications

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“…pUCP20 is a high-copy plasmid replicating in E. coli and P. aeruginosa [ 19 ]. PAO1 was transformed by the plasmid pUCP20-[pPA4249-eGFP] according to a protocol previously described [ 20 ] and transformants were selected on LB agar with carbenicillin 200 μg/mL. GFP fluorescence arising from transformants (515 nm) was checked with a Versafluor fluorometer (Biorad).…”

Section: Methodsmentioning

confidence: 99%

Exploring early steps in biofilm formation: set-up of an experimental system for molecular studies

et al. 2014

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BackgroundBacterial biofilms are predominant in natural ecosystems and constitute a public health threat because of their outstanding resistance to antibacterial treatments and especially to antibiotics. To date, several systems have been developed to grow bacterial biofilms in order to study their phenotypes and the physiology of sessile cells. Although relevant, such systems permit analysis of various aspects of the biofilm state but often after several hours of bacterial growth.ResultsHere we describe a simple and easy-to-use system for growing P. aeruginosa biofilm based on the medium adsorption onto glass wool fibers. This approach which promotes bacterial contact onto the support, makes it possible to obtain in a few minutes a large population of sessile bacteria. Using this growth system, we demonstrated the feasibility of exploring the early stages of biofilm formation by separating by electrophoresis proteins extracted directly from immobilized cells. Moreover, the involvement of protein synthesis in P. aeruginosa attachment is demonstrated.ConclusionsOur system provides sufficient sessile biomass to perform biochemical and proteomic analyses from the early incubation period, thus paving the way for the molecular analysis of the early stages of colonization that were inaccessible to date.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0253-z) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Exploring early steps in biofilm formation: set-up of an experimental system for molecular studies

et al. 2014

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“…Plasmids were constructed by using standard recombinant DNA procedures, and E. coli cells were transformed by the routine CaCl 2 procedure (48). Competent cells of P. putida KT2440 and the mutant strain HMR020 were prepared by the MgCl 2 -CaCl 2 method, followed by chemical transformation (30). ).…”

Section: Methodsmentioning

confidence: 99%

MrdH, a Novel Metal Resistance Determinant of Pseudomonas putida KT2440, Is Flanked by Metal-Inducible Mobile Genetic Elements

et al. 2009

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We report here the identification and characterization of mrdH, a novel chromosomal metal resistance determinant, located in the genomic island 55 of Pseudomonas putida KT2440. It encodes for MrdH, a predicted protein of ϳ40 kDa with a chimeric domain organization derived from the RcnA and RND (for resistance-nodulation-cell division) metal efflux proteins. The metal resistance function of mrdH was identified by the ability to confer nickel resistance upon its complementation into rcnA mutant (a nickel-and cobalt-sensitive mutant) of Escherichia coli. However, the disruption of mrdH in P. putida resulted in an increased sensitivity to cadmium and zinc apart from nickel. Expression studies using quantitative reverse transcription-PCR showed the induction of mrdH by cadmium, nickel, zinc, and cobalt. In association with mrdH, we also identified a conserved hypothetical gene mreA whose encoded protein showed significant homology to NreA and NreA-like proteins. Expression of the mreA gene in rcnA mutant of E. coli enhanced its cadmium and nickel resistance. Transcriptional studies showed that both mrdH and mreA underwent parallel changes in gene expression. The mobile genetic elements Tn4652 and IS1246, flanking mrdH and mreA were found to be induced by cadmium, nickel, and zinc, but not by cobalt. This study is the first report of a single-component metal efflux transporter, mrdH, showing chimeric domain organization, a broad substrate spectrum, and a location amid metal-inducible mobile genetic elements.Bacterial efflux systems for inorganic metal cations and anions play an imperative role in the regulatory network governing metal homeostasis. These efflux systems are also important for the environmental adaptability of the bacteria thriving in metal-rich serpentine environments. Bioinformatic and functional genomic analyses have revealed that the efflux systems belonging to the resistance-nodulation-cell division (RND), cation diffusion facilitator (CDF), and P-type ATPases constitute the majority of the multiple layers of heavy metal resistance in an organism (36). Members of the RND protein family include group of bacterial transport proteins involved in heavy metal resistance, nodulation, and cell division. The bestcharacterized RND family members include the efflux systems CzcCBA (Cd 2ϩ , Zn 2ϩ , and Co 2ϩ resistance), CnrCBA (Co 2ϩ

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“…11 The resulting DNA was screened by Agarose gel electrophoresis. This plasmid DNA was transformed into competent cells of P. putida prepared using the protocol developed by Liu et al, 12 with slight modifications. Briefly the procedure adopted was as follows: 1 mL of an overnight LB-broth culture was transferred into 50 mL fresh LB-broth and the incubation was continued at 30…”

Section: Construction Of Recombinant P Putidamentioning

confidence: 99%

Biodesulfurization of dibenzothiophene using recombinant Pseudomonas strain

Meesala¹,

Balomajumder²,

Chatterjee³

et al. 2007

J of Chemical Tech & Biotech

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BACKGROUND: The sulfur content in crude oil available from various sources ranges from 0.03 to values as high as 8.0 wt%. These high quantities of sulfur must be removed before the crude oil is processed because combustion of this oil would result in severe environmental pollution, such as acid rain. Due to high utility and operating costs, the conventional hydrodesulfurization process (HDS) is considered to be uneconomic. The biotechnological option, biodesulfurization (BDS) seems an attractive low cost, environmentally benign technology.

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An improved method of transformation in Pseudomonads

Cited by 4 publications

References 9 publications

Exploring early steps in biofilm formation: set-up of an experimental system for molecular studies

Exploring early steps in biofilm formation: set-up of an experimental system for molecular studies

MrdH, a Novel Metal Resistance Determinant of Pseudomonas putida KT2440, Is Flanked by Metal-Inducible Mobile Genetic Elements

Biodesulfurization of dibenzothiophene using recombinant Pseudomonas strain

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