Combined
 sacB
 -Based Negative Selection and
 cre-lox
 Antibiotic Marker Recycling for Efficient Gene Deletion in
 Pseudomonas Aeruginosa

Quenee, Lauriane E.; Lamotte, Danièle; Polack, Benoı̂t

doi:10.2144/05381st01

Cited by 77 publications

(53 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A nonflagellated ΔfliM mutant was obtained by allelic exchange with a truncated version of fliM carrying the Gm-resistance gene aacC1 framed by lox sequences. The aacC1 gene was then excised to yield an antibiotic resistance-free mutant (28). Both strains were tagged by inserting a constitutively expressed fluorescent protein-encoding gene at a neutral position of their genome (29).…”

Section: Methodsmentioning

confidence: 99%

Hydration-controlled bacterial motility and dispersal on surfaces

Dechesne

Wang

Gulez

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

181

240

View full text Add to dashboard Cite

Flagellar motility, a mode of active motion shared by many prokaryotic species, is recognized as a key mechanism enabling population dispersal and resource acquisition in microbial communities living in marine, freshwater, and other liquid-replete habitats. By contrast, its role in variably hydrated habitats, where water dynamics result in fragmented aquatic habitats connected by micrometric films, is debated. Here, we quantify the spatial dynamics of Pseudomonas putida KT2440 and its nonflagellated isogenic mutant as affected by the hydration status of a rough porous surface using an experimental system that mimics aquatic habitats found in unsaturated soils. The flagellar motility of the model soil bacterium decreased sharply within a small range of water potential (0 to −2 kPa) and nearly ceased in liquid films of effective thickness smaller than 1.5 μm. However, bacteria could rapidly resume motility in response to periodic increases in hydration. We propose a biophysical model that captures key effects of hydration and liquid-film thickness on individual cell velocity and use a simple roughness network model to simulate colony expansion. Model predictions match experimental results reasonably well, highlighting the role of viscous and capillary pinning forces in hindering flagellar motility. Although flagellar motility seems to be restricted to a narrow range of very wet conditions, fitness gains conferred by fast surface colonization during transient favorable periods might offset the costs associated with flagella synthesis and explain the sustained presence of flagellated prokaryotes in partially saturated habitats such as soil surfaces.flagella | biophysics | liquid film | fitness | motility D ispersal is recognized as a key ecological process enabling populations' access to new sites and pools of resources (1), thereby affecting structure and productivity of ecosystems (2, 3). Active bacterial motion (motility) takes on many forms that require various appendages (4). If surface-associated modes of motility such as twitching, gliding, or swarming seem restricted to some species (5), the ability to swim by rotating one or more flagella is shared by a large diversity of prokaryotes. This swimming motility has attracted considerable attention, primarily aimed at resolving the biophysical functioning of flagella and to a lesser degree, exploring its adaptive value. In marine environments, a large fraction of bacterial populations are flagellated (6), and swimming motility is often coupled with chemotaxis, conferring a clear benefit to these cells by allowing them to outswim diffusion and exploit transient substrate gradients (7,8).In contrast to water-replete environments where flagellar motility is essentially unrestricted, there exists strong physical limitations to flagellar motility in partially saturated media where aquatic microhabitats are often fragmented and connected only by thin liquid films of bacterial size or smaller (9). The limitations to bacterial motility in thin liquid films have, thus, l...

show abstract

Section: Methodsmentioning

confidence: 99%

Hydration-controlled bacterial motility and dispersal on surfaces

Dechesne

Wang

Gulez

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

181

240

View full text Add to dashboard Cite

show abstract

“…CHA-OST, a ⌬exoS ⌬orf1 ⌬exoT Pseudomonas aeruginosa strain, has been previously described (20). Additional deletions of aroA, lasI, and rhlI were performed in CHA-OST using the Cre-lox system that we previously adapted to P. aeruginosa.…”

Section: Methodsmentioning

confidence: 99%

“…Additional deletions of aroA, lasI, and rhlI were performed in CHA-OST using the Cre-lox system that we previously adapted to P. aeruginosa. This system allows performing multiple successive allelic exchanges in the same strain (20). The genes lasI and rhlI, encoding synthases of the quorum-sensing (QS) homoserine lactones (HSLs; 3-oxo-C12-HSL and C4-HSL, respectively), and the gene aroA were deleted by allelic exchange (see the primer sequences in Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…lasI and rhlI encode the two enzymes producing QS homoserine lactones 3-oxo-C12-HSL and C4-HSL, respectively; P. aeruginosa QS inactivation has been associated with virulence attenuation in various animal models for injury or illness, including those for pneumonia (14,18), burns (23), and pyelonephritis (15). We used the Cre-lox system that we previously adapted to Pseudomonas aeruginosa (20). By successive mutations, we generated three attenuated mutants from CHA-OST: CHA-OA (⌬exoS ⌬exoT ⌬aroA), CHA-OAL (⌬exoS ⌬exoT ⌬aroA ⌬lasI), and CHA-ORL (⌬exoS ⌬exoT ⌬rhlI ⌬lasI).…”

Section: Vol 15 2008 Ttss-based P Aeruginosa Vaccine Vector Optimimentioning

confidence: 99%

See 1 more Smart Citation

Optimization of a Type III Secretion System-Based Pseudomonas aeruginosa Live Vector for Antigen Delivery

Épaulard

Derouazi

Margerit

et al. 2008

Clin Vaccine Immunol

View full text Add to dashboard Cite

During the last few years, the use of type III secretion system-based bacterial vectors for immunotherapy purposes has been assessed in various applications. We showed that a type III secretion-based Pseudomonas aeruginosa vector delivering the ovalbumin (OVA) antigen induced an efficient specific CD8 ؉ T-lymphocyte immune response against OVA-expressing cells. Because of the intrinsic toxicity of the vector, further virulence attenuation was needed. Therefore, we explored the effects of the deletion of quorum-sensing genes and the aroA gene toward toxicity and efficiency of the vector strain. The aroA mutation in our strain (making the strain auxotrophic for aromatic amino acids) conferred a strikingly reduced toxicity, with the bacterial lethal dose being more than 100 times higher than that of the parental strain. The quorum-sensing gene mutation alone was associated with a slightly reduced toxicity. In a prophylactic OVA-expressing melanoma mouse model, an OVA-delivering aroA-deficient mutant was the most efficient at a low dose (10 5 ), but dose enhancement was not associated with a greater immune response. The quorum-sensing-deficient strain was the most efficient at a mild dose (10 6 ), but this dose was close to the toxic dose. Combination of both mutations conferred the highest efficiency at an elevated dose (10 7 ), in agreement with the known negative effects of quorum-sensing molecules upon T-cell activation. In conclusion, we have obtained a promising immunotherapy vector regarding toxicity and efficiency for further developments in both antitumor and anti-infectious strategies.The use of live bacteria and bacterial virulence factors as therapeutic tools in human medicine has been considered for more than a century. The observation that the onset of a bacterial infection could modify the course of a malignant disease (6) was a hallmark in this history, but in the end very few procedures (such as the intravesical administration of an attenuated Mycobacterium bovis strain for the cure of noninvasive urothelial carcinoma) have been routinely used. In the last 10 years, better characterization of bacterial mechanisms (mainly toxins and secretion systems) and extensive progress in genomic studies have allowed engineering of bacteria (mainly Escherichia coli and Salmonella spp.). These domesticated agents can be delivered to mammals for different purposes. Notably, the design of antigen-delivering bacteria that trigger antigen-specific cytotoxic CD8 ϩ T-lymphocyte responses is an emerging field of investigation in vaccine development (5). Antigen delivery can be performed by using intrinsic properties of bacterial toxins (as with a Listeria monocytogenes-derived vector [21]) or secretion pathways normally used by bacteria to release toxins, such as the type III secretion system (TTSS). This system has been considered promising because it allows gram-negative rods to inject toxins into eukaryotic cell cytoplasm; therefore, epitopes delivered by this system are likely to be presented by antigen-presenting c...

show abstract

“…λ Red recombination has been successfully used for convenient generation of gene deletions in E. coli (Datsenko et al, 2000), Salmonella enterica (Hansen-Wester et al, 2002), Pseudomonas aeruginosa (Liang et al, 2010, Quénée et al, 2005, Streptomyces coelicolor (Gust et al, 2004), Shigella spp. (Ranallo et al, 2006), Yersinia pseudotuberculosis (Derbise et al, 2003) and Y. pestis (Sun et al, 2008).…”

Section: Gene Deletionmentioning

confidence: 99%