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Global Transcriptomic and Proteomic Responses of Dehalococcoides ethenogenes Strain 195 to Fixed Nitrogen Limitation

ABSTRACT: Bacteria of the genus Dehalococcoides play an important role in the reductive dechlorination of chlorinated ethenes. A systems level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing D. ethenogenes strain 195 to fixed nitrogen limitation (FNL). As expected, the nitrogen-fixing (nif) genes were differentially up-regulated in the transcriptome and proteome of strain 195 during FNL. Aside from the nif operon, a putative methylglyoxal synthase-encoding gene (DET1576), which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly up-regulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally down regulated in response to FNL and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition while others were associated with general stress responses. A number of genes related to the biological functions of nucleotide synthesis, replication, transcription, translation, and post-translational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate cellular nitrogen status in the environment.

ORGANISM(S): Dehalococcoides mccartyi 195

PROVIDER: GSE28855 | GEO | 2012/04/26

SECONDARY ACCESSION(S): PRJNA140685

REPOSITORIES: GEO

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Project description:Bacteria of the genus Dehalococcoides play an important role in the reductive dechlorination of chlorinated ethenes. A systems level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing D. ethenogenes strain 195 to fixed nitrogen limitation (FNL). As expected, the nitrogen-fixing (nif) genes were differentially up-regulated in the transcriptome and proteome of strain 195 during FNL. Aside from the nif operon, a putative methylglyoxal synthase-encoding gene (DET1576), which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly up-regulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally down regulated in response to FNL and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition while others were associated with general stress responses. A number of genes related to the biological functions of nucleotide synthesis, replication, transcription, translation, and post-translational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate cellular nitrogen status in the environment. Dehalococcoides ethenogenes strain 195 was grown with or without ammonium in batch cultures with trichloroethene as the electron acceptor, acetate as the carbon source, and hydrogen as the electron donor. Microarrays were used to compare the transcriptome during fixed nitrogen limitation and triplicate biological samples were prepared for each condition. Differentially expressed genes between the two growth conditions were identified using a two-tailed Student's t-test, a false discovery rate of less than 1%, an absolute hybridization signal intensity of 300 or greater in one of the two conditions, and an expression ratio of two-fold or greater.

Project description:Tetrachloroethene (PCE) and trichloroethene (TCE) are prevalent groundwater contaminants that can be completely reductively dehalogenated by Dehalococcoides organisms. A Dehalococcoides-containing microbial consortium (ANAS) with the ability to degrade TCE to ethene, an innocuous end-product, was previously enriched from contaminated soil. A whole-genome photolithographic microarray was developed based on the genome of Dehalococcoides ethenogenes 195 (strain 195). This microarray contains probes designed to hybridize to >99% of the predicted protein-coding sequences in the strain 195 genome. DNA from ANAS was hybridized to the microarray to characterize the genomic content of the ANAS enrichment. The microarray revealed that the genes associated with central metabolism including an apparently incomplete carbon fixation pathway, cobalamin salvaging system, nitrogen fixation pathway, and five hydrogenase complexes are present in both strain 195 and ANAS. Although the gene encoding the TCE reductase tceA was detected, 13 of the 19 reductive dehalogenase genes present in strain 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, consistent with these elements being genetically mobile. Sections of the tryptophan operon and an operon encoding an ABC transporter in strain 195 were also not detected in ANAS. These insights into the diversity of Dehalococcoides genomes will improve our understanding of the physiology and evolution of these bacteria which is essential in developing effective strategies for bioremediation of PCE and TCE in the environment. Keywords: comparative genomic hybridization Genomic DNA from each culture was divided into replicate samples which were independently fragmented, labeled, and hybridized to arrays. Two microarrays were processed for the positive control (strain 195), two for the negative control (D. restrictus), and five for the ANAS enrichment culture(two analyses from one biological sample followed one year later by three analyses of a second biological sample).

Project description:Purpose: Klebsiella oxytoca M5a1 (previously Klebsiella pneumonia M5a1) is a principle model organism for free-living biological nitrogen fixation. This strain has been used for decades in the characterisation of nitrogenase function and the genetic regulation of nitrogen fixation physiology. Currently it represents a key model for synthetic biology and biotechnology approaches. This project involves a multi-omics approach to modelling nitrogen physiology in Klebsiella oxytoca M5a1, in order to inform rational genetic engineering for improved nitrogen fixation activity. Here we studied the transition from nitrogen replete (high NH4Cl) to nitrogen starved (NH4Cl run-out) conditions, the latter of which induce nif gene expression and synthesis of the nitrogenase enzyme. We mapped RNA-sequencing (Illumina) reads to our assembled M5aI genome in order to compare the transcriptome between nitrogen replete and nitrogen fixing conditions using differential gene expression (DEG) analysis. Methods: Total RNA was extracted from fixed M5a1 cell samples after culturing in nitrogen replete (10 mM NH4Cl; one replicate) or nitrogen starved (3 hours after complete run-out of 0.5 mM NH4Cl; two replicates) growth medium (NFDM). cDNA libraries were prepared for Illumina (NextSeq 500) sequencing, with a read length of 75 nt and a depth of 100 million reads. Following processing and quality control, sequence reads were mapped to a M5a1 genomic template (GenBank WGS accession JAFHKG010000000; BioSample SAMN17288411) and gene count normalisation performed (RPKM). Differential gene expression analysis was performed using the DEseq2 package utilising settings of regularized logarithm (rlog) normalisation of read counts, Wald hypothesis testing and p-value adjustment using a False Discovery Rate threshold of 0.05. Log2 fold changes between conditions were calculated for differentially expressed genes (DEGs) with an adjusted p value of <0.05. Results: Across the three samples, between 22.0-25.1 million reads (81.4-87.7%) were mapped to the genome, including 56.7-68.5% mapped to annotated genes. More than 50% of genes (2694/5279 genes) were differentially expressed (adjusted p-value <0.05) in the nitrogen starved condition with respect to the nitrogen replate condition. 925 genes were upregulated and 1117 genes downregulated by more than 2-fold. Of these, 1515 differentially expressed genes correspond to annotated proteins in the KEGG functional orthology (KO) database. The 20 nitrogen fixation (nif) genes were ranked among the 200 genes with highest calculated expression values (RPKM) in the nitrogen starved condition. Conclusions: Our study provides a detailed insight into the global gene expression profile associated with diazotrophic (nitrogen fixing) growth, revealing a dominant role for nif and other nitrogen regulatory genes in the adaptation to nitrogen stress, in addition to a large array of secondary genes indirectly associated with shifts in metabolism and growth phenotype (e.g. metabolic enzymes, gene expression machinery, transporters).

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Global Transcriptomic and Proteomic Responses of Dehalococcoides ethenogenes Strain 195 to Fixed Nitrogen Limitation

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