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Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii.

ABSTRACT: BACKGROUND: Sphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was perturbed with either the cell-permeating solute sodium chloride or the non-permeating solute polyethylene glycol with a molecular weight of 8000 (PEG8000). These solutes are assumed to simulate the solute and matric components of the total water potential, respectively. The responses to these perturbations were then assessed and compared using a combination of growth assays, transcriptome profiling, and membrane fatty acid analyses. RESULTS: Under conditions producing a similar decrease in water potential but without effect on growth rate, there was only a limited shared response to perturbation with sodium chloride or PEG8000. This shared response included the increased expression of genes involved with trehalose and exopolysaccharide biosynthesis and the reduced expression of genes involved with flagella biosynthesis. Mostly, the responses to perturbation with sodium chloride or PEG8000 were very different. Only sodium chloride triggered the increased expression of two ECF-type RNA polymerase sigma factors and the differential expression of many genes involved with outer membrane and amino acid metabolism. In contrast, only PEG8000 triggered the increased expression of a heat shock-type RNA polymerase sigma factor along with many genes involved with protein turnover and repair. Membrane fatty acid analyses further corroborated these differences. The degree of saturation of membrane fatty acids increased after perturbation with sodium chloride but had the opposite effect and decreased after perturbation with PEG8000. CONCLUSIONS: A combination of growth assays, transcriptome profiling, and membrane fatty acid analyses revealed that permeating and non-permeating solutes trigger different adaptive responses in strain RW1, suggesting these solutes affect cells in fundamentally different ways. Future work is now needed that connects these responses with the responses observed in more realistic scenarios of soil desiccation.

SUBMITTER: Johnson DR

PROVIDER: S-EPMC3238334 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii.

Johnson David R DR Coronado Edith E Moreno-Forero Silvia K SK Heipieper Hermann J HJ van der Meer Jan Roelof JR

BMC microbiology 20111114

<h4>Background</h4>Sphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was pertur ...[more]

PMID: 22082453

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Project description:Chlorinated congeners of dibenzo-p-dioxin and dibenzofuran are widely dispersed pollutants that can be treated using microorganisms, such as the Sphingomonas wittichii RW1 bacterium, able to transform some of them into non-toxic substances. The enzymes of the upper pathway for dibenzo-p-dioxin degradation in S. wittichii RW1 have been biochemically and genetically characterized, but its genome sequence has indicated the existence of a tremendous potential for aromatic compound transformation, with 56 ring-hydroxylating dioxygenase subunits, 34 extradiol dioxygenases, and 40 hydrolases. To further characterize this enzymatic arsenal, new methodological approaches should be employed. Here, a large shotgun proteomic survey has been performed on cells grown on dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-p-dioxin, and compared to growth on acetate. Changes in the proteome were monitored over time. Peak lists were generated with the Mascot Daemon software (version 2.3.2; Matrix Science, London, UK) using the extract_msn.exe data import filter (Thermo Fisher Scientific) from the Xcalibur FT package (version 2.0.7; Thermo Fisher Scientific). Data import filter options were set to 400 (minimum mass), 5000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans) and 1000 (threshold). The mgf files from technical triplicates were merged, and MS/MS spectra were assigned using the Mascot Daemon 2.3.2 (Matrix Science) and a database containing the nonredundant RefSeq protein entries for S. wittichii RW1 (NCBI Taxonomy ID: 392499), comprising 5345 protein sequences totalling 1 800 684 amino acids. The search was performed using the following criteria: tryptic peptides with a maximum of two miscleavages, mass tolerances of 5 ppm on the parent ion and 0.5 Da on the MS/MS, fixed modification for carbamidomethylated cysteine, and variable modification for methionine oxidation. Mascot results were parsed using the IRMa 1.28.0 software. Peptides were identified with a P-value threshold below 0.05. Proteins were considered validated when at least two distinct peptides were detected. Using a selection of 11 result files and the appropriate decoy database, the false discovery rate for protein identification was estimated to be 0.33% with these parameters.

Project description:Sphingomonas wittichii RW1 is a bacterium isolated for its ability to degrade the xenobiotic compounds dibenzodioxin and dibenzofuran (DBF). A number of genes involved in DBF degradation have been previously characterized, such as the dxn cluster, dbfB, and the electron transfer components fdx1, fdx3, and redA2. Here we use a combination of whole genome transcriptome analysis and transposon library screening to characterize RW1 catabolic and other genes implicated in the reaction to or degradation of DBF. To detect differentially expressed genes upon exposure to DBF, we applied three different growth exposure experiments, using either short DBF exposures to actively growing cells or growing them with DBF as sole carbon and energy source. Genome-wide gene expression was examined using a custom-made microarray. In addition, proportional abundance determination of transposon insertions in RW1 libraries grown on salicylate or DBF by ultra-high throughput sequencing was used to infer genes whose interruption caused a fitness loss for growth on DBF. Expression patterns showed that batch and chemostat growth conditions, and short or long exposure of cells to DBF produced very different responses. Numerous other uncharacterized catabolic gene clusters putatively involved in aromatic compound metabolism increased expression in response to DBF. In addition, only very few transposon insertions completely abolished growth on DBF. Some of those (e.g., in dxnA1) were expected, whereas others (in a gene cluster for phenylacetate degradation) were not. Both transcriptomic data and transposon screening suggest operation of multiple redundant and parallel aromatic pathways, depending on DBF exposure. In addition, increased expression of other non-catabolic genes suggests that during initial exposure, S. wittichii RW1 perceives DBF as a stressor, whereas after longer exposure, the compound is recognized as a carbon source and metabolized using several pathways in parallel.

Project description:Phenazine-1-carboxylic acid, the main component of shenqinmycin, is widely used in southern China for the prevention of rice sheath blight. However, the fate of phenazine-1-carboxylic acid in soil remains uncertain. Sphingomonas wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources for growth. In this study, dioxygenase-encoding genes, pcaA1A2, were found using transcriptome analysis to be highly upregulated upon phenazine-1-carboxylic acid biodegradation. PcaA1 shares 68% amino acid sequence identity with the large oxygenase subunit of anthranilate 1,2-dioxygenase from Rhodococcus maanshanensis DSM 44675. The dioxygenase was coexpressed in Escherichia coli with its adjacent reductase-encoding gene, pcaA3, and ferredoxin-encoding gene, pcaA4, and showed phenazine-1-carboxylic acid consumption. The dioxygenase-, ferredoxin-, and reductase-encoding genes were expressed in Pseudomonas putida KT2440 or E. coli BL21, and the three recombinant proteins were purified. A phenazine-1-carboxylic acid conversion capability occurred in vitro only when all three components were present. However, P. putida KT2440 transformed with pcaA1A2 obtained phenazine-1-carboxylic acid degradation ability, suggesting that phenazine-1-carboxylic acid 1,2-dioxygenase has low specificities for its ferredoxin and reductase. This was verified by replacing PcaA3 with RedA2 in the in vitro enzyme assay. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) analysis showed that phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation, indicating that PcaA1A2A3A4 constitutes the initial phenazine-1-carboxylic acid 1,2-dioxygenase. This study fills a gap in our understanding of the biodegradation of phenazine-1-carboxylic acid and illustrates a new dioxygenase for decarboxylation.IMPORTANCE Phenazine-1-carboxylic acid is widely used in southern China as a key fungicide to prevent rice sheath blight. However, the degradation characteristics of phenazine-1-carboxylic acid and the environmental consequences of the long-term application are not clear. S. wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources. In this study, a three-component dioxygenase, PcaA1A2A3A4, was determined to be the initial dioxygenase for phenazine-1-carboxylic acid degradation in S. wittichii DP58. Phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation. This finding may help us discover the pathway for phenazine-1-carboxylic acid degradation.

Project description:Sphingomonas wittichii strain RW1 (RW1) is one of the few strains that can grow on dibenzo-p-dioxin (DD). We conducted a transcriptomic study of RW1 using RNA-Seq to outline transcriptional responses to DD, dibenzofuran (DF), and the smectite clay mineral saponite with succinate as carbon source. The ability to grow on DD is rare compared to growth on the chemically similar DF even though the same initial dioxygenase may be involved in oxidation of both substrates. Therefore, we hypothesized the reason for this lies beyond catabolic pathways and may concern genes involved in processes for cell-substrate interactions such as substrate recognition, transport, and detoxification. Compared to succinate (SUC) as control carbon source, DF caused over 240 protein-coding genes to be differentially expressed, whereas more than 300 were differentially expressed with DD. Stress response genes were up-regulated in response to both DD and DF. This effect was stronger with DD than DF, suggesting a higher toxicity of DD compared to DF. Both DD and DF caused changes in expression of genes involved in active cross-membrane transport such as TonB-dependent receptor proteins, but the patterns of change differed between the two substrates. Multiple transcription factor genes also displayed expression patterns distinct to DD and DF growth. DD and DF induced the catechol ortho- and the salicylate/gentisate pathways, respectively. Both DD and DF induced the shared down-stream aliphatic intermediate compound pathway. Clay caused category-wide down-regulation of genes for cell motility and chemotaxis, particularly those involved in the synthesis, assembly and functioning of flagella. This is an environmentally important finding because clay is a major component of soil microbes' microenvironment influencing local chemistry and may serve as a geosorbent for toxic pollutants. Similar to clay, DD and DF also affected expression of genes involved in motility and chemotaxis.

Dataset Information

Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii.

Publications

Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii.

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