Identification of Aspergillus niger Aquaporins Involved in Hydrogen Peroxide Signaling.

Aspergillus niger is a robust microbial cell factory for organic acid production. However, the regulation of many industrially important pathways is still poorly understood. The regulation of the glucose oxidase (Gox) expression system, involved in the biosynthesis of gluconic acid, has recently been uncovered. The results of that study show hydrogen peroxide, a by-product of the extracellular conversion of glucose to gluconate, has a pivotal role as a signaling molecule in the induction of this system. In this study, the facilitated diffusion of hydrogen peroxide via aquaporin water channels (AQPs) was studied. AQPs are transmembrane proteins of the major intrinsic proteins (MIPs) superfamily. In addition to water and glycerol, they may also transport small solutes such as hydrogen peroxide. The genome sequence of A. niger N402 was screened for putative AQPs. Seven AQPs were found and could be classified into three main groups. One protein (AQPA) belonged to orthodox AQP, three (AQPB, AQPD, and AQPE) were grouped in aquaglyceroporins (AQGP), two (AQPC and AQPF) were in X-intrinsic proteins (XIPs), and the other (AQPG) could not be classified. Their ability to facilitate diffusion of hydrogen peroxide was identified using yeast phenotypic growth assays and by studying AQP gene knock-outs in A. niger. The X-intrinsic protein AQPF appears to play roles in facilitating hydrogen peroxide transport across the cellular membrane in both Saccharomyces cerevisiae and A. niger experiments.

The genetics of gene expression in a Caenorhabditis elegans multiparental recombinant inbred line population.

Studying genetic variation of gene expression provides a powerful way to unravel the molecular components underlying complex traits. Expression quantitative trait locus (eQTL) studies have been performed in several different model species, yet most of these linkage studies have been based on the genetic segregation of two parental alleles. Recently, we developed a multiparental segregating population of 200 recombinant inbred lines (mpRILs) derived from four wild isolates (JU1511, JU1926, JU1931, and JU1941) in the nematode Caenorhabditis elegans. We used RNA-seq to investigate how multiple alleles affect gene expression in these mpRILs. We found 1789 genes differentially expressed between the parental lines. Transgression, expression beyond any of the parental lines in the mpRILs, was found for 7896 genes. For expression QTL mapping almost 9000 SNPs were available. By combining these SNPs and the RNA-seq profiles of the mpRILs, we detected almost 6800 eQTLs. Most trans-eQTLs (63%) co-locate in six newly identified trans-bands. The trans-eQTLs found in previous two-parental allele eQTL experiments and this study showed some overlap (17.5-46.8%), highlighting on the one hand that a large group of genes is affected by polymorphic regulators across populations and conditions, on the other hand, it shows that the mpRIL population allows identification of novel gene expression regulatory loci. Taken together, the analysis of our mpRIL population provides a more refined insight into C. elegans complex trait genetics and eQTLs in general, as well as a starting point to further test and develop advanced statistical models for detection of multiallelic eQTLs and systems genetics studying the genotype-phenotype relationship.

Punctuated Loci on Chromosome IV Determine Natural Variation in Orsay Virus Susceptibility of Caenorhabditis elegans Strains Bristol N2 and Hawaiian CB4856.

Host-pathogen interactions play a major role in evolutionary selection and shape natural genetic variation. The genetically distinct Caenorhabditis elegans strains, Bristol N2 and Hawaiian CB4856, are differentially susceptible to the Orsay virus (OrV). Here, we report the dissection of the genetic architecture of susceptibility to OrV infection. We compare OrV infection in the relatively resistant wild-type CB4856 strain to the more susceptible canonical N2 strain. To gain insight into the genetic architecture of viral susceptibility, 52 fully sequenced recombinant inbred lines (CB4856 × N2 RILs) were exposed to OrV. This led to the identification of two loci on chromosome IV associated with OrV resistance. To verify the two loci and gain additional insight into the genetic architecture controlling virus infection, introgression lines (ILs) that together cover chromosome IV, were exposed to OrV. Of the 27 ILs used, 17 had an CB4856 introgression in an N2 background, and 10 had an N2 introgression in a CB4856 background. Infection of the ILs confirmed and fine-mapped the locus underlying variation in OrV susceptibility, and we found that a single nucleotide polymorphism in cul-6 may contribute to the difference in OrV susceptibility between N2 and CB4856. An allele swap experiment showed the strain CB4856 became as susceptible as the N2 strain by having an N2 cul-6 allele, although having the CB4856 cul-6 allele did not increase resistance in N2. In addition, we found that multiple strains with nonoverlapping introgressions showed a distinct infection phenotype from the parental strain, indicating that there are punctuated locations on chromosome IV determining OrV susceptibility. Thus, our findings reveal the genetic complexity of OrV susceptibility in C. elegans and suggest that viral susceptibility is governed by multiple genes.IMPORTANCE Genetic variation determines the viral susceptibility of hosts. Yet, pinpointing which genetic variants determine viral susceptibility remains challenging. Here, we have exploited the genetic tractability of the model organism Caenorhabditis elegans to dissect the genetic architecture of Orsay virus infection. Our results provide novel insight into natural determinants of Orsay virus infection.

Dissecting the eQTL Micro-Architecture in Caenorhabditis elegans.

The study of expression quantitative trait loci (eQTL) using natural variation in inbred populations has yielded detailed information about the transcriptional regulation of complex traits. Studies on eQTL using recombinant inbred lines (RILs) led to insights on cis and trans regulatory loci of transcript abundance. However, determining the underlying causal polymorphic genes or variants is difficult, but ultimately essential for the understanding of regulatory networks of complex traits. This requires insight into whether associated loci are single eQTL or a combination of closely linked eQTL, and how this QTL micro-architecture depends on the environment. We addressed these questions by testing for independent replication of previously mapped eQTL in Caenorhabditis elegans using new data from introgression lines (ILs). Both populations indicate that the overall heritability of gene expression, number, and position of eQTL differed among environments. Across environments we were able to replicate 70% of the cis- and 40% of the trans-eQTL using the ILs. Testing eight different simulation models, we suggest that additive effects explain up to 60-93% of RIL/IL heritability for all three environments. Closely linked eQTL explained up to 40% of RIL/IL heritability in the control environment whereas only 7% in the heat-stress and recovery environments. In conclusion, we show that reproducibility of eQTL was higher for cis vs. trans eQTL and that the environment affects the eQTL micro-architecture.

In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions.

BACKGROUND: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.

Pseudomonas putida KT2440 is HV1 certified, not GRAS.

Pseudomonas putida is rapidly becoming a workhorse for industrial production due to its metabolic versatility, genetic accessibility and stress-resistance properties. The P. putida strain KT2440 is often described as Generally Regarded as Safe, or GRAS, indicating the strain is safe to use as food additive. This description is incorrect. P. putida KT2440 is classified by the FDA as HV1 certified, indicating it is safe to use in a P1 or ML1 environment.

A multi-parent recombinant inbred line population of C. elegans allows identification of novel QTLs for complex life history traits.

BACKGROUND: The nematode Caenorhabditis elegans has been extensively used to explore the relationships between complex traits, genotypes, and environments. Complex traits can vary across different genotypes of a species, and the genetic regulators of trait variation can be mapped on the genome using quantitative trait locus (QTL) analysis of recombinant inbred lines (RILs) derived from genetically and phenotypically divergent parents. Most RILs have been derived from crossing two parents from globally distant locations. However, the genetic diversity between local C. elegans populations can be as diverse as between global populations and could thus provide means of identifying genetic variation associated with complex traits relevant on a broader scale. RESULTS: To investigate the effect of local genetic variation on heritable traits, we developed a new RIL population derived from 4 parental wild isolates collected from 2 closely located sites in France: Orsay and Santeuil. We crossed these 4 genetically diverse parental isolates to generate a population of 200 multi-parental RILs and used RNA-seq to obtain sequence polymorphisms identifying almost 9000 SNPs variable between the 4 genotypes with an average spacing of 11 kb, doubling the mapping resolution relative to currently available RIL panels for many loci. The SNPs were used to construct a genetic map to facilitate QTL analysis. We measured life history traits such as lifespan, stress resistance, developmental speed, and population growth in different environments, and found substantial variation for most traits. We detected multiple QTLs for most traits, including novel QTLs not found in previous QTL analysis, including those for lifespan and pathogen responses. This shows that recombining genetic variation across C. elegans populations that are in geographical close proximity provides ample variation for QTL mapping. CONCLUSION: Taken together, we show that using more parents than the classical two parental genotypes to construct a RIL population facilitates the detection of QTLs and that the use of wild isolates facilitates the detection of QTLs. The use of multi-parent RIL populations can further enhance our understanding of local adaptation and life history trade-offs.

Contribution of trans regulatory eQTL to cryptic genetic variation in C. elegans.

BACKGROUND: Cryptic genetic variation (CGV) is the hidden genetic variation that can be unlocked by perturbing normal conditions. CGV can drive the emergence of novel complex phenotypes through changes in gene expression. Although our theoretical understanding of CGV has thoroughly increased over the past decade, insight into polymorphic gene expression regulation underlying CGV is scarce. Here we investigated the transcriptional architecture of CGV in response to rapid temperature changes in the nematode Caenorhabditis elegans. We analyzed regulatory variation in gene expression (and mapped eQTL) across the course of a heat stress and recovery response in a recombinant inbred population. RESULTS: We measured gene expression over three temperature treatments: i) control, ii) heat stress, and iii) recovery from heat stress. Compared to control, exposure to heat stress affected the transcription of 3305 genes, whereas 942 were affected in recovering animals. These affected genes were mainly involved in metabolism and reproduction. The gene expression pattern in recovering animals resembled both the control and the heat-stress treatment. We mapped eQTL using the genetic variation of the recombinant inbred population and detected 2626 genes with an eQTL in the heat-stress treatment, 1797 in the control, and 1880 in the recovery. The cis-eQTL were highly shared across treatments. A considerable fraction of the trans-eQTL (40-57%) mapped to 19 treatment specific trans-bands. In contrast to cis-eQTL, trans-eQTL were highly environment specific and thus cryptic. Approximately 67% of the trans-eQTL were only induced in a single treatment, with heat-stress showing the most unique trans-eQTL. CONCLUSIONS: These results illustrate the highly dynamic pattern of CGV across three different environmental conditions that can be evoked by a stress response over a relatively short time-span (2 h) and that CGV is mainly determined by response related trans regulatory eQTL.

Natural Genetic Variation Differentially Affects the Proteome and Transcriptome in Caenorhabditis elegans.

Natural genetic variation is the raw material of evolution and influences disease development and progression. An important question is how this genetic variation translates into variation in protein abundance. To analyze the effects of the genetic background on gene and protein expression in the nematode Caenorhabditis elegans, we quantitatively compared the two genetically highly divergent wild-type strains N2 and CB4856. Gene expression was analyzed by microarray assays, and proteins were quantified using stable isotope labeling by amino acids in cell culture. Among all transcribed genes, we found 1,532 genes to be differentially transcribed between the two wild types. Of the total 3,238 quantified proteins, 129 proteins were significantly differentially expressed between N2 and CB4856. The differentially expressed proteins were enriched for genes that function in insulin-signaling and stress-response pathways, underlining strong divergence of these pathways in nematodes. The protein abundance of the two wild-type strains correlates more strongly than protein abundance versus transcript abundance within each wild type. Our findings indicate that in C. elegans only a fraction of the changes in protein abundance can be explained by the changes in mRNA abundance. These findings corroborate with the observations made across species.

Dynamic Response of Pseudomonas putida S12 to Sudden Addition of Toluene and the Potential Role of the Solvent Tolerance Gene trgI.

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.

Remarkably Divergent Regions Punctuate the Genome Assembly of the Caenorhabditis elegans Hawaiian Strain CB4856.

The Hawaiian strain (CB4856) of Caenorhabditis elegans is one of the most divergent from the canonical laboratory strain N2 and has been widely used in developmental, population, and evolutionary studies. To enhance the utility of the strain, we have generated a draft sequence of the CB4856 genome, exploiting a variety of resources and strategies. When compared against the N2 reference, the CB4856 genome has 327,050 single nucleotide variants (SNVs) and 79,529 insertion-deletion events that result in a total of 3.3 Mb of N2 sequence missing from CB4856 and 1.4 Mb of sequence present in CB4856 but not present in N2. As previously reported, the density of SNVs varies along the chromosomes, with the arms of chromosomes showing greater average variation than the centers. In addition, we find 61 regions totaling 2.8 Mb, distributed across all six chromosomes, which have a greatly elevated SNV density, ranging from 2 to 16% SNVs. A survey of other wild isolates show that the two alternative haplotypes for each region are widely distributed, suggesting they have been maintained by balancing selection over long evolutionary times. These divergent regions contain an abundance of genes from large rapidly evolving families encoding F-box, MATH, BATH, seven-transmembrane G-coupled receptors, and nuclear hormone receptors, suggesting that they provide selective advantages in natural environments. The draft sequence makes available a comprehensive catalog of sequence differences between the CB4856 and N2 strains that will facilitate the molecular dissection of their phenotypic differences. Our work also emphasizes the importance of going beyond simple alignment of reads to a reference genome when assessing differences between genomes.

Loss-of-function of ß-catenin bar-1 slows development and activates the Wnt pathway in Caenorhabditis elegans.

C. elegans is extensively used to study the Wnt-pathway and most of the core-signalling components are known. Four ß-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.

A rapid and massive gene expression shift marking adolescent transition in C. elegans.

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.

Gene-environment and protein-degradation signatures characterize genomic and phenotypic diversity in wild Caenorhabditis elegans populations.

BACKGROUND: Analyzing and understanding the relationship between genotypes and phenotypes is at the heart of genetics. Research on the nematode Caenorhabditis elegans has been instrumental for unraveling genotype-phenotype relations, and has important implications for understanding the biology of mammals, but almost all studies, including forward and reverse genetic screens, are limited by investigations in only one canonical genotype. This hampers the detection and functional analysis of allelic variants, which play a key role in controlling many complex traits. It is therefore essential to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map in wild C. elegans populations. RESULTS: We used multiple wild C. elegans populations freshly isolated from local sites to investigate gene sequence polymorphisms and a multitude of phenotypes including the transcriptome, fitness, and behavioral traits. The genotype, transcriptome, and a number of fitness traits showed a direct link with the original site of the strains. The separation between the isolation sites was prevalent on all chromosomes, but chromosome V was the largest contributor to this variation. These results were supported by a differential food preference of the wild isolates for naturally co-existing bacterial species. Comparing polymorphic genes between the populations with a set of genes extracted from 19 different studies on gene expression in C. elegans exposed to biotic and abiotic factors, such as bacteria, osmotic pressure, and temperature, revealed a significant enrichment for genes involved in gene-environment interactions and protein degradation. CONCLUSIONS: We found that wild C. elegans populations are characterized by gene-environment signatures, and we have unlocked a wealth of genotype-phenotype relations for the first time. Studying natural isolates provides a treasure trove of evidence compared with that unearthed by the current research in C. elegans, which covers only a diminutive part of the myriad of genotype-phenotype relations that are present in the wild.

Comparative transcriptomics and proteomics of p-hydroxybenzoate producing Pseudomonas putida S12: novel responses and implications for strain improvement.

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.

Hydrophobic substances induce water stress in microbial cells.

Ubiquitous noxious hydrophobic substances, such as hydrocarbons, pesticides and diverse industrial chemicals, stress biological systems and thereby affect their ability to mediate biosphere functions like element and energy cycling vital to biosphere health. Such chemically diverse compounds may have distinct toxic activities for cellular systems; they may also share a common mechanism of stress induction mediated by their hydrophobicity. We hypothesized that the stressful effects of, and cellular adaptations to, hydrophobic stressors operate at the level of water : macromolecule interactions. Here, we present evidence that: (i) hydrocarbons reduce structural interactions within and between cellular macromolecules, (ii) organic compatible solutes - metabolites that protect against osmotic and chaotrope-induced stresses - ameliorate this effect, (iii) toxic hydrophobic substances induce a potent form of water stress in macromolecular and cellular systems, and (iv) the stress mechanism of, and cellular responses to, hydrophobic substances are remarkably similar to those associated with chaotrope-induced water stress. These findings suggest that it may be possible to devise new interventions for microbial processes in both natural environments and industrial reactors to expand microbial tolerance of hydrophobic substances, and hence the biotic windows for such processes.

Isolation and genetic characterization of an improved benzene-tolerant mutant of Pseudomonas putida S12.

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 ISS12. 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.

TrgI, toluene repressed gene I, a novel gene involved in toluene-tolerance in Pseudomonas putida S12.

Pseudomonas putida S12 is well known for its remarkable solvent tolerance. Transcriptomics analysis of this bacterium grown in toluene-containing chemostats revealed the differential expression of 253 genes. As expected, the genes encoding one of the major solvent tolerance mechanisms, the solvent efflux pump SrpABC and its regulatory genes srpRS were heavily up-regulated. The increased energy demand brought about by toluene stress was also reflected in transcriptional changes: genes involved in sugar storage were down-regulated whereas genes involved in energy generation such as isocitrate dehydrogenase and NADH dehydrogenases, were up-regulated in the presence of toluene. Several flagella-related genes were up-regulated and a large group of transport genes were down-regulated. In addition, a novel Pseudomonas-specific gene was identified to be involved in toluene tolerance of P. putida S12. This toluene-repressed gene, trgI, was heavily down-regulated immediately upon toluene exposure in batch cultures. The relationship of trgI with solvent tolerance was confirmed by the increased resistance to toluene shock and toluene induced lysis of trgI knock-out mutants. We propose that down-regulation of trgI plays a role in the first line of defence against solvents.

Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies.

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.

Chemostat-based proteomic analysis of toluene-affected Pseudomonas putida S12.

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.