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Transcriptional analysis of a gene cluster involved in glucose tolerance in Zymomonas mobilis: evidence for an osmoregulated promoter.

ABSTRACT: Exponentially growing cells of Zymomonas mobilis normally exhibit a lag period of up to 3 h when they are transferred from a liquid medium containing 2% glucose to a liquid medium containing 10% glucose. A mutant of Z. mobilis (CU1) exhibited a lag period of more than 20 h when it was grown under the same conditions, whereas it failed to grow on a solid medium containing 10% glucose. The glucose-defective phenotype of mutant CU1 was due to a spontaneous insertion in a putative gene (ORF4) identified as part of an operon (glc) which includes three additional putative genes (ORF1, ORF2, and ORF3) with no obvious involvement in the glucose tolerance mechanism. The common promoter controlling glc operon transcription, designated P(glc), was found to be osmoregulated and stimulated by the putative product of ORF4 in an autoregulated fashion, as indicated by expression of the gfp reporter gene. Additionally, reverse transcriptase PCR analysis showed that the gene cluster produces a single mRNA, which verified the operon organization of this transcription unit. Further transcriptional analysis demonstrated that glc operon expression is regulated by the concentration of glucose, which supported the hypothesis that this operon is directly involved in the uncharacterized glucose tolerance mechanism of Z. mobilis.

SUBMITTER: Christogianni A

PROVIDER: S-EPMC1196045 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Transcriptional analysis of a gene cluster involved in glucose tolerance in Zymomonas mobilis: evidence for an osmoregulated promoter.

Christogianni Anastasia A Douka Eugenia E Koukkou Anna I AI Hatziloukas Efstathios E Drainas Constantin C

Journal of bacteriology 20050801 15

Exponentially growing cells of Zymomonas mobilis normally exhibit a lag period of up to 3 h when they are transferred from a liquid medium containing 2% glucose to a liquid medium containing 10% glucose. A mutant of Z. mobilis (CU1) exhibited a lag period of more than 20 h when it was grown under the same conditions, whereas it failed to grow on a solid medium containing 10% glucose. The glucose-defective phenotype of mutant CU1 was due to a spontaneous insertion in a putative gene (ORF4) identi ...[more]

PMID: 16030211

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Project description:BackgroundZymomonas mobilis produces near theoretical yields of ethanol and recombinant strains are candidate industrial microorganisms. To date, few studies have examined its responses to various stresses at the gene level. Hfq is a conserved bacterial member of the Sm-like family of RNA-binding proteins, coordinating a broad array of responses including multiple stress responses. In a previous study, we observed Z. mobilis ZM4 gene ZMO0347 showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions.ResultsWe generated a Z. mobilis hfq insertion mutant AcRIM0347 in an acetate tolerant strain (AcR) background and investigated its role in model lignocellulosic pretreatment inhibitors including acetate, vanillin, furfural and hydroxymethylfurfural (HMF). Saccharomyces cerevisiae Lsm protein (Hfq homologue) mutants and Lsm protein overexpression strains were also assayed for their inhibitor phenotypes. Our results indicated that all the pretreatment inhibitors tested in this study had a detrimental effect on both Z. mobilis and S. cerevisiae, and vanillin had the most inhibitory effect followed by furfural and then HMF for both Z. mobilis and S. cerevisiae. AcRIM0347 was more sensitive than the parental strain to the inhibitors and had an increased lag phase duration and/or slower growth depending upon the conditions. The hfq mutation in AcRIM0347 was complemented partially by trans-acting hfq gene expression. We also assayed growth phenotypes for S. cerevisiae Lsm protein mutant and overexpression phenotypes. Lsm1, 6, and 7 mutants showed reduced tolerance to acetate and other pretreatment inhibitors. S. cerevisiae Lsm protein overexpression strains showed increased acetate and HMF resistance as compared to the wild-type, while the overexpression strains showed greater inhibition under vanillin stress conditions.ConclusionsWe have shown the utility of the pKNOCK suicide plasmid for mutant construction in Z. mobilis, and constructed a Gateway compatible expression plasmid for use in Z. mobilis for the first time. We have also used genetics to show Z. mobilis Hfq and S. cerevisiae Lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. The conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development.

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Transcriptional analysis of a gene cluster involved in glucose tolerance in Zymomonas mobilis: evidence for an osmoregulated promoter.

Publications

Transcriptional analysis of a gene cluster involved in glucose tolerance in Zymomonas mobilis: evidence for an osmoregulated promoter.

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