Organismal development is the most dynamic period of the life cycle, yet we have only a rough understanding of the dynamics of gene expression during adolescent transition. Here we show that adolescence in Caenorhabditis elegans is characterized by a spectacular expression shift of conserved and highly polymorphic genes. Using a high resolution time series we found that in adolescent worms over 10,000 genes changed their expression. These genes were clustered according to their expression patterns. One cluster involved in chromatin remodelling showed a brief up-regulation around 50 h post-hatch. At the same time a spectacular shift in expression was observed. Sequence comparisons for this cluster across many genotypes revealed diversifying selection. Strongly up-regulated genes showed signs of purifying selection in non-coding regions, indicating that adolescence-active genes are constrained on their regulatory properties. Our findings improve our understanding of adolescent transition and help to eliminate experimental artefacts due to incorrect developmental timing.
A transcriptomics and proteomics approach was employed to study the expression changes associated with p-hydroxybenzoate production by the engineered Pseudomonas putida strain S12palB1. To establish p-hydroxybenzoate production, phenylalanine-tyrosine ammonia lyase (pal/tal) was introduced to connect the tyrosine biosynthetic and p-coumarate degradation pathways. In agreement with the efficient p-hydroxybenzoate production, the tyrosine biosynthetic and p-coumarate catabolic pathways were upregulated. Also many transporters were differentially expressed, one of which--a previously uncharacterized multidrug efflux transporter with locus tags PP1271-PP1273--was found to be associated with p-hydroxybenzoate export. In addition to tyrosine biosynthesis, also tyrosine degradative pathways were upregulated. Eliminating the most prominent of these resulted in a 22% p-hydroxybenzoate yield improvement. Remarkably, the upregulation of genes contributing to p-hydroxybenzoate formation was much higher in glucose than in glycerol-cultured cells.
C. elegans is extensively used to study the Wnt-pathway and most of the core-signalling components are known. Four β-catenins are important gene expression regulators in Wnt-signalling. One of these, bar-1, is part of the canonical Wnt-pathway. Together with Wnt effector pop-1, bar-1 forms a transcription activation complex which regulates the transcription of downstream genes. The effects of bar-1 loss-of-function mutations on many phenotypes have been studied well. However, the effects on global gene expression are unknown. Here we report the effects of a loss-of-function mutation bar-1(ga80). By analysing the transcriptome and developmental phenotyping we show that bar-1(ga80) impairs developmental timing. This developmental difference confounds the comparison of the gene expression profile between the mutant and the reference strain. When corrected for this difference it was possible to identify genes that were directly affected by the bar-1 mutation. We show that the Wnt-pathway itself is activated, as well as transcription factors elt-3, pqm-1, mdl-1 and pha-4 and their associated genes. The outcomes imply that this response compensates for the loss of functional bar-1. Altogether we show that bar-1 loss-of function leads to delayed development possibly caused by an induction of a stress response, reflected by daf-16 activated genes.
Summary Pseudomonas putida S12.49, a mutant stain of P. putida S12 that tolerates up to 20 mM benzene, was obtained by evolutionary selection. The genetic basis for the strongly enhanced benzene tolerance was investigated by proteome and transcriptome analysis. Indications were found that the highly benzene‐tolerant phenotype is the resultant of multi‐level systemic changes. The solvent extrusion pump SrpABC was constitutively expressed in P. putida S12.49, which could be attributed to the disruption of the srpS regulator gene by the indigenous mutator element IS S12 . The occurrence of this and two additional transposition events was in good agreement with the increased transcriptional activity of transposase‐encoding genes in strain S12.49. These observations suggested that transposition events are an important force driving the generation of the genetic diversity apparently required to obtain highly solvent‐tolerant phenotypes. In addition, various expression responses relating to energy generation indicated system changes that accommodated the energy demand associated with the high‐level expression of the proton‐driven solvent extrusion pump. The relatively modest effect of a respiratory chain uncoupler on benzene tolerance in P. putida S12.49 indicated the involvement of an alternative, non‐respiratory mechanism to maintain the proton gradient.
The aim of this study was to assess the cellular response of the solvent-tolerant Pseudomonas putida S12 to toluene as the single effector. Proteomic analysis (two-dimensional difference-in-gel-electrophoresis) was used to assess the response of P. putida S12 cultured in chemostats. This approach ensures constant growth conditions, both in the presence and absence of toluene. A considerable negative effect of toluene on the cell yield was found. The need for energy in the defence against toluene was reflected by differentially expressed proteins for cell energy management. In toluene-stressed cells the balance between proton motive force (PMF) enforcing and dissipating systems was shifted. NAD(P)H generating systems were upregulated whereas the major proton-driven system, ATP synthase, was downregulated. Other differentially expressed proteins were identified: outer membrane proteins, transport proteins, stress-related proteins and translation-related proteins. In addition, a protein with no assigned function was found. This study yielded a more detailed view of the effect of toluene on the intracellular energy management of P. putida S12 and several novel leads have been obtained for further targeted investigations.
Pseudomonas putida S12 is exceptionally tolerant to various organic solvents. To obtain further insight into this bacterium's primary defence mechanisms towards these potentially harmful substances, we studied its genome wide transcriptional response to sudden addition of toluene. Global gene expression profiles were monitored for 30 minutes after toluene addition. During toluene exposure, high oxygen-affinity cytochrome c oxidase is specifically expressed to provide for an adequate proton gradient supporting solvent efflux mechanisms. Concomitantly, the glyoxylate bypass route was up-regulated, to repair an apparent toluene stress-induced redox imbalance. A knock-out mutant of trgI, a recently identified toluene-repressed gene, was investigated in order to identify TrgI function. Remarkably, upon addition of toluene the number of differentially expressed genes initially was much lower in the trgI-mutant than in the wild-type strain. This suggested that after deletion of trgI cells were better prepared for sudden organic solvent stress. Before, as well as after, addition of toluene many genes of highly diverse functions were differentially expressed in trgI-mutant cells as compared to wild-type cells. This led to the hypothesis that TrgI may not only be involved in the modulation of solvent-elicited responses but in addition may affect basal expression levels of large groups of genes.
Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type strain (DSM291(T)), plasmid pWW0-containing KT2440-derivative strain mt-2 (DSM3931), the solvent-tolerant P. putida S12, and several other Pseudomonas strains. Depending on the strain tested, 22 to 99% of all genetic elements were identified in the genomic DNAs. The efficacy of these microarrays to study cellular function was determined for all strains included in the study. The vast majority of DSM6125 genes encoding proteins of primary metabolism and genes involved in the catabolism of aromatic compounds were identified in the genomic DNA of strain S12: a prerequisite for reliable transcriptomics analyses. The genomotypic comparisons between Pseudomonas strains were used to construct highly discriminative phylogenetic relationships. DSM6125 and DSM3931 were indistinguishable and clustered together with strain S12 in a separate group, distinct from DSM291(T). Pseudomonas monteilii (DSM14164) clustered well with P. putida strains.
