Unknown,Transcriptomics,Genomics,Proteomics

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Genome changes associated with a Zymomonas mobilis sodium acetate-tolerant mutant (AcR)

ABSTRACT: We report the genome changes associated with a Zymomonas mobilis sodium acetate-tolerant mutant (AcR). We used comparative genomics, transcriptomics, and genetics to show nhaA over-expression conferred sodium acetate (NaAc) tolerance in Z. mobilis. We observed a synergistic effect for sodium and acetate ions that enhanced toxicity against the wild-type strain (ZM4), which was not observed for similar concentrations of potassium and ammonium acetate under controlled laboratory conditions. We extended our studies and demonstrated that Saccharomyces cerevisiae sodium-proton antiporter genes contribute to NaAc tolerance for this important ethanologen. The application of classical and systems biology tools is a paradigm for industrial strain improvement and combines benefits of few a priori assumptions with detailed, rapid, mechanistic studies. Finally, our studies reinforce the idea that one obtains what one selects for in mutant screens and that a genetic system is important for industrial strain development. ZM4_ACr_NaCl_NaAc_study. Whole-genome expression profiles of exponential and stationary phase cells were analyzed for the wild-type Zymomonas mobilis ZM4 and the acetate-tolerant mutant AcR under 12g/L sodium acetate and same molar concentration of sodium chloride (8.55g/L) control conditions.

ORGANISM(S): Zymomonas mobilis

SUBMITTER: Stan Martin

PROVIDER: E-GEOD-18106 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae.

Yang Shihui S Land Miriam L ML Klingeman Dawn M DM Pelletier Dale A DA Lu Tse-Yuan S TY Martin Stanton L SL Guo Hao-Bo HB Smith Jeremy C JC Brown Steven D SD

Proceedings of the National Academy of Sciences of the United States of America 20100519 23

The application of systems biology tools holds promise for rational industrial microbial strain development. Here, we characterize a Zymomonas mobilis mutant (AcR) demonstrating sodium acetate tolerance that has potential importance in biofuel development. The genome changes associated with AcR are determined using microarray comparative genome sequencing (CGS) and 454-pyrosequencing. Sanger sequencing analysis is employed to validate genomic differences and to investigate CGS and 454-pyrosequen ...[more]

PMID: 20484677

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Project description:Zymomonas mobilis is an excellent ethanologenic bacterium. Biomass pretreatment and saccharification provides access to simple sugars, but also produces inhibitors such as acetate and furfural. Our previous work has identified and confirmed the genetic change of a 1.5-kb deletion in the sodium acetate tolerant Z. mobilis mutant (AcR) leading to constitutively elevated expression of a sodium proton antiporter encoding gene nhaA, which contributes to the sodium acetate tolerance of AcR mutant. In this study, we further investigated the responses of AcR and wild-type ZM4 to sodium acetate stress in minimum media using both transcriptomics and a metabolic labeling approach for quantitative proteomics the first time. Proteomic measurements at two time points identified about eight hundreds proteins, or about half of the predicted proteome. Extracellular metabolite analysis indicated AcR overcame the acetate stress quicker than ZM4 with a concomitant earlier ethanol production in AcR mutant, although the final ethanol yields and cell densities were similar between two strains. Transcriptomic samples were analyzed for four time points and revealed that the response of Z. mobilis to sodium acetate stress is dynamic, complex and involved about one-fifth of the total predicted genes from all different functional categories. The modest correlations between proteomic and transcriptomic data may suggest the involvement of posttranscriptional control. In addition, the transcriptomic data of forty-four microarrays from four experiments for ZM4 and AcR under different conditions were combined to identify strain-specific, media-responsive, growth phase-dependent, and treatment-responsive gene expression profiles. Together this study indicates that minimal medium has the most dramatic effect on gene expression compared to rich medium followed by growth phase, inhibitor, and strain background. Genes involved in protein biosynthesis, glycolysis and fermentation as well as ATP synthesis and stress response play key roles in Z. mobilis metabolism with consistently strong expression levels under different conditions. A sixteen array study using total RNA recovered from wild-type cultures of Zymomonas mobilis subsp mobilis ZM4 and acetate-tolerant mutant AcR at different time points of 130, 148, 166, and 190h post-inoculation with 10g/L sodium acetate (NaAc) treatment were carried out to investigate the expression differences between ZM4 and AcR. Two biological replicates for treatment or control condition.

Project description:Background Zymomonas mobilis ZM4 is a capable ethanologenic bacterium with high ethanol productivity and ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of its ethanol stress response have not been elucidated fully. Methodology/Principal Findings In this study, ethanol stress responses were investigated using systems biology approaches. Medium supplementation with an initial 47 g/L (6% v/v) ethanol reduced Z. mobilis ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 55% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated that the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Transcriptomic data were used to update Z. mobilis ZM4 operon models. Furthermore, correlations among the transcriptomic, proteomic and metabolic data were examined. Among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher. Conclusions Our study has provided insights into the responses of Z. mobilis to ethanol stress through an integrated “omics” approach for the first time. This systems biology study elucidated key Z. mobilis ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress. A sixteen array study using total RNA recovered from wild-type cultures of Zymomonas mobilis subsp mobilis ZM4 at different time points of 6, 10, 13.5, and 26h post-inoculation with 6% (v/v) treatment compred to that of control without ethanol supplementation. Two biological replicates for treatment and control condition.

Dataset Information

Genome changes associated with a Zymomonas mobilis sodium acetate-tolerant mutant (AcR)

Publications

Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae.

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