New insights on the reorganization of gene transcription in Pseudomonas putida KT2440 at elevated pressure.
ABSTRACT: BACKGROUND: Elevated pressure, elevated oxygen tension (DOT) and elevated carbon dioxide tension (DCT) are readily encountered at the bottom of large industrial bioreactors and during bioprocesses where pressure is applied for enhancing the oxygen transfer. Yet information about their effect on bacteria and on the gene expression thereof is scarce. To shed light on the cellular functions affected by these specific environmental conditions, the transcriptome of Pseudomonas putida KT2440, a bacterium of great relevance for the production of medium-chain-length polyhydroxyalkanoates, was thoroughly investigated using DNA microarrays. RESULTS: Very well defined chemostat cultivations were carried out with P. putida to produce high quality RNA samples and ensure that differential gene expression was caused exclusively by changes of pressure, DOT and/or DCT. Cellular stress was detected at 7 bar and elevated DCT in the form of heat shock and oxidative stress-like responses, and indicators of cell envelope perturbations were identified as well.Globally, gene transcription was not considerably altered when DOT was increased from 40?±?5 to 235?±?20% at 7 bar and elevated DCT. Nevertheless, differential transcription was observed for a few genes linked to iron-sulfur cluster assembly, terminal oxidases, glutamate metabolism and arginine deiminase pathway, which shows their particular sensitivity to variations of DOT. CONCLUSIONS: This study provides a comprehensive overview on the changes occurring in the transcriptome of P. putida upon mild variations of pressure, DOT and DCT. Interestingly, whereas the changes of gene transcription were widespread, the cell physiology was hardly affected, which illustrates how efficient reorganization of the gene transcription is for dealing with environmental changes that may otherwise be harmful. Several particularly sensitive cellular functions were identified, which will certainly contribute to the understanding of the mechanisms involved in stress sensing/response and to finding ways of enhancing the stress tolerance of microorganisms.
Project description:We investigated the changes of gene expression in PHA-producing Pseudomonas putida KT2440 cultivated under elevated pressure (7 bar) and under combined elevated pressure (7 bar) and elevated dissolved oxygen tension by means of DNA microarrays. RNA samples were isolated from cells cultivated in chemostat under very well defined growth conditions (growth rate, medium, temperature, pH,...)
Project description:The tmoABCDEF genes encode the toluene-4-monooxygenase from Pseudomonas mendocina KR1. Upstream from the tmoA gene an open reading frame, tmoX, encoding a protein 83% identical to TodX (todX being the initial gene in the todXFC1C2BADEGIH operon from Pseudomonas putida DOT-T1E) was found. The tmoX gene is also the initial gene in the tmoXABCDEF gene cluster. The transcription initiation point from the tmoX promoter was mapped, and the sequence upstream revealed striking identity with the promoter of the tod operon of P. putida. The tod operon is regulated by a two-component signal transduction system encoded by the todST genes. Two novel genes from P. mendocina KR1, tmoST, were rescued by complementation of a P. putida DOT-T1E todST knockout mutant, whose gene products shared about 85% identity with TodS-TodT. We show that transcription from P(tmoX) and P(todX) can be mediated by TmoS-TmoT or TodS-TodT, in the presence of toluene, revealing cross-regulation between these two catabolic pathways.
Project description:Bacterial membranes constitute the first physical barrier against different environmental stresses. Pseudomonas putida DOT-T1E accumulates cyclopropane fatty acids (CFAs) in the stationary phase of growth. In this strain the cfaB gene encodes the main cyclopropane synthase responsible of the synthesis of CFAs, and its expression is mediated by RNA polymerase with sigma factor ?(38). We generated a cfaB mutant of P. putida DOT-T1E and studied its response to solvents, acid pH and other stress conditions such as temperature changes, high osmolarity and the presence of antibiotics or heavy metals in the culture medium. A CfaB knockout mutant was more sensitive to solvent stress than the wild-type strain, but in contrast to Escherichia coli and Salmonella enterica, the P. putida cfaB mutant was as tolerant to acid shock as the wild-type strain. The cfaB mutant was also as tolerant as the parental strain to a number of drugs, antibiotics and other damaging agents.
Project description:We report the application of a high-throughput technique, RNA-seq, to study the transcriptomic response of P. putida DOT-T1E and E. coli growing in co-cultures Overall design: P. putida DOT-T1E and E. coli mRNA profiles comparing their growth in single-cultures and in co-cultures in LB at exponential phase
Project description:A novel strain of Pseudomonas putida LS46 was isolated from wastewater on the basis of its ability to synthesize medium chain-length polyhydroxyalkanoates (mcl-PHAs). P.putida LS46 was differentiated from other P.putida strains on the basis of cpn60 (UT). The complete genome of P.putida LS46 was sequenced and annotated. Its chromosome is 5,86,2556 bp in size with GC ratio of 61.69. It is encoding 5316 genes, including 7 rRNA genes and 76 tRNA genes. Nucleotide sequence data of the complete P. putida LS46 genome was compared with nine other P. putida strains (KT2440, F1, BIRD-1, S16, ND6, DOT-T1E, UW4, W619 and GB-1) identified either as biocontrol agents or as bioremediation agents and isolated from different geographical region and different environment. BLASTn analysis of whole genome sequences of the ten P. putida strains revealed nucleotide sequence identities of 86.54 to 97.52%. P.putida genome arrangement was LS46 highly similar to P.putida BIRD1 and P.putida ND6 but was markedly different than P.putida DOT-T1E, P.putida UW4 and P.putida W619. Fatty acid biosynthesis (fab), fatty acid degradation (fad) and PHA synthesis genes were highly conserved among biocontrol and bioremediation P.putida strains. Six genes in pha operon of P. putida LS46 showed >98% homology at gene and proteins level. It appears that polyhydroxyalkanoate (PHA) synthesis is an intrinsic property of P. putida and was not affected by its geographic origin. However, all strains, including P. putida LS46, were different from one another on the basis of house keeping genes, and presence of plasmid, prophages, insertion sequence elements and genomic islands. While P. putida LS46 was not selected for plant growth promotion or bioremediation capacity, its genome also encoded genes for root colonization, pyoverdine synthesis, oxidative stress (present in other soil isolates), degradation of aromatic compounds, heavy metal resistance and nicotinic acid degradation, manganese (Mn II) oxidation. Genes for toluene or naphthalene degradation found in the genomes of P. putida F1, DOT-T1E, and ND6 were absent in the P. putida LS46 genome. Heavy metal resistant genes encoded by the P. putida W619 genome were also not present in the P. putida LS46 genome. Despite the overall similarity among genome of P.putida strains isolated for different applications and from different geographical location a number of differences were observed in genome arrangement, occurrence of transposon, genomic islands and prophage. It appears that P.putida strains had a common ancestor and by acquiring some specific genes by horizontal gene transfer it differed from other related strains.
Project description:The basic mechanisms underlying solvent tolerance in Pseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant P. putida DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of P. putida DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to P. putida DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn5-'phoA mutagenesis of wild-type P. putida DOT-T1E cells. The mutant, called P. putida DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[14C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[14C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of 14C-labeled substrate in whole cells increased in competition assays in which toluene-and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in P. putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene mexB, which belongs to the efflux pump family of the resistant nodulator division type.
Project description:We report the application of a high-throughput technique, RNA-seq, to study the transcriptomic response of P. putida DOT-T1E in the presence of antibiotics with different mechanisms of action with the aim to study in more detail the defense mechanisms that bacteria use to resist against toxic compounds. We find that P. putida DOT-T1E responde in a different way against each antimicrobial compound, what clearly shows that bacteria defense in different ways depending on the targets that compounds uses to attack. Our work is the first global transcriptomic analysis done in P. putida DOT-T1E in the presence of a considerable range of antibiotics. P. putida DOT-T1E mRNA profiles in the presence of control condition (LB) and 8 different antibiotics (ampicillin, chloramphenicol, kanamycin, ciprofloxacin, tetracycline, spectinomycin, gentamicin and rifampicin)
Project description:Frameshift mutations in a poly(G) track at the flhB gene of Pseudomonas putida DOT-T1E are responsible for the diminished swimming of this strain on semisolid medium, which contrasts with the high swimming ability of P. putida KT2440, which does not exhibit a poly(G) track at the flhB gene. We previously showed that a mutant lacking FlhB was more sensitive to solvents than the wild-type strain (Segura et al., J. Bacteriol., 183:4127-4133, 2001). In this study, we show that swimming ability correlates with solvent tolerance in P. putida DOT-T1E, so that growth conditions favoring a functional flhB gene (growth on semisolid medium) resulted in increased innate tolerance to a sudden toluene shock.
Project description:Proteins containing a Bin/Amphiphysin/Rvs (BAR) domain regulate membrane curvature in the cell. Recent simulations have revealed that BAR proteins assemble into linear aggregates, strongly affecting membrane curvature and its in-plane stress profile. Here, we explore the opposite question: do mechanical properties of the membrane impact protein association? By using coarse-grained molecular dynamics simulations, we show that increased surface tension significantly impacts the dynamics of protein assembly. While tensionless membranes promote a rapid formation of long-living linear aggregates of N-BAR proteins, increase in tension alters the geometry of protein association. At high tension, protein interactions are strongly inhibited. Increasing surface density of proteins leads to a wider range of protein association geometries, promoting the formation of meshes, which can be broken apart with membrane tension. Our work indicates that surface tension may play a key role in recruiting proteins to membrane-remodelling sites in the cell.
Project description:According to the cohesion-tension theory, mangrove trees desalinate salty water using highly negative pressure (or tension) that is generated by evaporative capillary forces in mangrove leaves. Here, we demonstrate a synthetic mangrove that mimics the main features of the natural mangrove: capillary pumping (leaves), stable water conduction in highly metastable states (stem), and membrane desalination (root). When using nanoporous membranes as leaves, the maximum osmotic pressures of saline feeds (10 to 30 bar) allowing pure water uptake precisely correspond to expected capillary pressures based on the Young-Laplace equation. Hydrogel-based leaves allow for stable operation and desalination of hypersaline solutions with osmotic pressures approaching 400 bar, fivefold greater than the pressure limits of conventional reverse osmosis. Our findings support the applicability of the cohesion-tension theory to desalination in mangroves, provide a new platform to study plant hydraulics, and create possibilities for engineered membrane separations using large, passively generated capillary pressures.