Pseudomonas putida mutants in the exbBexbDtonB gene cluster are hypersensitive to environmental and chemical stressors

113

In this work we investigated the correlations between global gene expression patterns and environmental parameters in natural ecosystems. We studied the preferential gene expression of the iron oxidizer bacterium Leptospirillum ferrooxidans to adapt its physiology to changes in the physicochemical parameters in its natural medium. Transcriptome analysis by DNA microarrays can proportionate an instant picture about the preferential gene expression between two different environmental samples. However, this type of analysis is very difficult and complex in natural ecosystems, mainly because of the broad biodiversity and multiple environmental parameters that may affect gene expression. The necessity of high-quality RNA preparations as well as complicated data analysis are also technological limitations. The low prokaryotic diversity of the extremely acidic and iron-rich waters of the Tinto River (Spain) ecosystem, where L. ferrooxidans is abundant, allows the opportunity to achieve global gene expression studies and to associate gene function with environmental parameters. We applied a total RNA amplification protocol validated previously for the amplification of the environmental transcriptome (meta-transcriptome). The meta-transcriptome of two sites from the Tinto River mainly differing in the salt and oxygen contents were amplified and analysed by a L. ferrooxidans DNA microarray. The results showed a clear preferential induction of genes involved in certain physicochemical parameters like: high salinity (ectAB, otsAB), low oxygen concentration (cydAB), iron uptake (fecA-exbBD-tonB), oxidative stress (carotenoid synthesis, oxyR, recG), potassium (kdpBAC) or phosphate concentrations (pstSCAB), etc. We conclude that specific gene expression patterns can be useful indicators for the physiological conditions in a defined ecosystem. Also, the upregulation of certain genes and operons reveals information about the environmental conditions (nutrient limitations, stresses, etc.).

Section: Resultsmentioning

confidence: 99%

Analysis of environmental transcriptomes by DNA microarrays

Parro¹,

Moreno‐Paz²,

González-Toril

2006

113

“…This conformational change favours the interaction of TonB with outer membrane receptors, leading to the internalization of bound ligands (reviewed by Moeck and Coulton, 1998; Braun, 2003). Pseudomonas putida and P. aeruginosa mutants in exbB , exbD or tonB show reduced iron uptake and their growth is severely limited in iron‐deficient medium (Poole et al ., 1996; Godoy et al ., 2001, 2004). Given the role that seed‐borne iron plays in the initiation of bacterial attachment to seeds, we hypothesized that a deficiency in the TonB system might result in a more limited adhesion capacity, as a consequence of reduced iron transport and detection by the bacterial cells.…”

Section: Resultsmentioning

confidence: 99%

Role of iron and the TonB system in colonization of corn seeds and roots by Pseudomonas putida KT2440

Molina

Godoy

Ramos-González

et al. 2005

Self Cite

Iron, which is abundant in corn (Zea mays L.) seeds, plays an important role in the initial establishment of Pseudomonas putida KT2440 populations on seeds. Sequestration of seed-borne iron by chelators decreases the capacity of KT2440 to initiate attachment to corn seeds. The importance of iron for this plant-bacteria interaction is further supported by the fact that mutations in the TonB system, which is key for iron uptake, result in reduced seed colonization. TonB is also a primary determinant of the fitness of P. putida in the rhizosphere, as a deletion mutant shows a clear competitive disadvantage during colonization of corn roots.

“…We have shown here that pGRT1 is stable under non‐selective conditions, and that the rate of plasmid loss is below 10 −8 per cell and per cell generation, even when cells are cultured under suboptimal laboratory conditions. This is of interest because the property of solvent tolerance is a phenotype that can be exploited in the bioremediation of heavily polluted sites (Ramos et al ., 1995) or in industrial processes in which solvent‐tolerant bacteria are used as whole‐cell biocatalysts in double‐phase systems for the production of toxic compounds that partition in the organic phase of a solvent/water mixture (Godoy et al ., 2004; Rojas et al ., 2004; Wery and de Bont, 2004; Meyer et al ., 2005; Neumann et al ., 2005; Nijkamp et al ., 2005). Therefore, the presence of the ttgGHI genes in a self‐transmissible plasmid opens a window for applications of this plasmid in biotechnological processes for which biochemically relevant properties are present in other strains.…”

Section: Discussionmentioning

confidence: 99%

The ttgGHI solvent efflux pump operon of Pseudomonas putida DOT‐T1E is located on a large self‐transmissible plasmid

Rodríguez‐Herva

García

Hurtado

et al. 2007

Self Cite

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of > 1% (v/v) toluene in the culture medium. A set of multidrug efflux pumps have been found to play a major role in the tolerance of this bacterium to organic solvents (Rojas et al., J Bacteriol 183: 3967-3973). In the course of studies of the mechanisms underlying solvent tolerance in DOT-T1E, we isolated a spontaneous solvent-sensitive mutant derivative which had lost the genes encoding the TtgGHI efflux pump, the most important extrusion element in quantitative terms. Genomic comparisons between the mutant and its parental strain by microarray analysis revealed that in addition to the ttgVW-ttgGHI gene cluster, another group of genes, highly similar to those found in the Tn4653A and ISPpu12 transposable elements of the TOL plasmid pWW0 from P. putida mt-2, were also absent from this strain. Further analysis demonstrated that strain DOT-T1E harboured a large plasmid (named pGRT1) that was lost from the solvent-sensitive mutant. Mapping analysis revealed that the ttgVW-ttgGHI genes and the Tn4653A-like transposon are borne by the pGRT1 plasmid. Plasmid pGRT1 is highly stable and its frequency of loss is below 10(-8) per cell per generation under a variety of growth conditions, including nutritional and physical stresses. The pGRT1 plasmid is self-transmissible, and its acquisition by the toluene-sensitive P. putida KT2440 and Pseudomonas aeruginosa PAO1 increased the recipient's tolerance to toluene up to levels similar to those exhibited by P. putida DOT-T1E. We discuss the importance and potential benefits of this plasmid for the development of bacteria with enhanced solvent tolerance, and its potential impact for bioremediation and whole-cell biotransformations.