LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Wilson, Charlotte M.; Klingeman, Dawn M.; Schlachter, Caleb R.; Syed, Mustafa; Wu, Chia-Wei; Guss, Adam M.; Brown, Steven D.

doi:10.1128/aem.02751-16

Cited by 16 publications

(17 citation statements)

References 47 publications

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“…Moreover, the genome of C. thermocellum may harbor additional as-yet-unidentified σ I -dependent promoters that may be activated by more than one σ I factor. Therefore, during future studies of a particular σ I factor directly in C. thermocellum , we could expect a cascade of interactions among the different σ I s. Additionally, cellulosomal component genes can also have σ A -dependent promoters 11 , 43 and other regulatory proteins 44 , making the analysis of σ I s directly in C. thermocellum more complex. If these observations are not taken into account during the direct analysis of σ I s in C. thermocellum , the results obtained can be misinterpreted.…”

Section: Discussionmentioning

confidence: 99%

Regulation of biomass degradation by alternative σ factors in cellulolytic clostridia

Ora

Lamed

Liu

et al. 2018

Sci Rep

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Bacteria can adjust their genetic programs via alternative σ factors to face new environmental pressures. Here, we analyzed a unique set of paralogous alternative σ factors, termed σIs, which fine-tune the regulation of one of the most intricate cellulolytic systems in nature, the bacterial cellulosome, that is involved in degradation of environmental polysaccharides. We combined bioinformatics with experiments to decipher the regulatory networks of five σIs in Clostridium thermocellum, the epitome of cellulolytic microorganisms, and one σI in Pseudobacteroides cellulosolvens which produces the cellulosomal system with the greatest known complexity. Despite high homology between different σIs, our data suggest limited cross-talk among them. Remarkably, the major cross-talk occurs within the main cellulosomal genes which harbor the same σI-dependent promoter elements, suggesting a promoter-based mechanism to guarantee the expression of relevant genes. Our findings provide insights into the mechanisms used by σIs to differentiate among their corresponding regulons, representing a comprehensive overview of the regulation of the cellulosome to date. Finally, we show the advantage of using a heterologous host system for analysis of multiple σIs, since information generated by their analysis in their natural host can be misinterpreted owing to a cascade of interactions among the different σIs.

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Section: Discussionmentioning

confidence: 99%

Regulation of biomass degradation by alternative σ factors in cellulolytic clostridia

Ora

Lamed

Liu

et al. 2018

Sci Rep

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“…Zverlov et al observed IS 1447 transposition in the genome of C. thermocellum ATCC27405 under chemical mutagenetic stress induced by ethyl-methanesulfonate [ 24 ]. Wilson et al also detected the insertion of an IS 3 element, which may be IS 1447 , in the genome of C. thermocellum DSM1313 during targeted gene deletions using the hypoxanthine phosphoribosyl transferase gene as the counterselection marker [ 57 ]. There could be transposition precedence for the active IS 1447 with the presence of exotic stress.…”

Section: Discussionmentioning

confidence: 99%

Firmicutes-enriched IS1447 represents a group of IS3-family insertion sequences exhibiting unique + 1 transcriptional slippage

Liu

Zhang

et al. 2018

Biotechnol Biofuels

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BackgroundBacterial insertion sequences (ISs) are ubiquitous mobile genetic elements that play important roles in genome plasticity, cell adaptability, and function evolution. ISs of various families and subgroups contain significantly diverse molecular features and functional mechanisms that are not fully understood.ResultsIS1447 is a member of the widespread IS3 family and was previously detected to have transposing activity in a typical thermophilic and cellulolytic microorganism Clostridium thermocellum. Phylogenetic analysis showed that IS1447-like elements are widely distributed in Firmicutes and possess unique features in the IS3 family. Therefore, IS1447 may represent a novel subgroup of the IS3 family. Unlike other well-known IS3 subgroups performing programmed − 1 translational frameshifting for the expression of the transposase, IS1447 exhibits transcriptional slippage in both the + 1 and − 1 directions, each with a frequency of ~ 16%, and only + 1 slippage results in full-length and functional transposase. The slippage-prone region of IS1447 contains a run of nine A nucleotides following a stem-loop structure in mRNA, but mutagenesis analysis indicated that seven of them are sufficient for the observed slippage. Western blot analysis indicated that IS1447 produces three types of transposases with alternative initiations. Furthermore, the IS1447-subgroup elements are abundant in the genomes of several cellulolytic bacteria.ConclusionOur result indicated that IS1447 represents a new Firmicutes-enriched subgroup of the IS3 family. The characterization of the novel IS3-family member will enrich our understanding of the transposition behavior of IS elements and may provide insight into developing IS-based mutagenesis tools for thermophiles.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1304-8) contains supplementary material, which is available to authorized users.

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“…Total RNA was isolated from 50 ml of chemostat sample using the procedures described in [ 54 ], and quantity and quality were determined using a Nanodrop instrument (ThermoFisher Scientific Waltham, MA). Depletion, library preparation and sequencing were completed at the Joint Genome Institute (JGI, Walnut Creek, CA).…”

Section: Methodsmentioning

confidence: 99%

Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics

Whitham

Moon

Rodríguez

et al. 2018

Biotechnol Biofuels

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BackgroundClostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. C. thermocellum is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand C. thermocellum pH homeostasis we grew strain LL1210 (C. thermocellum DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack), currently the highest ethanol producing strain of C. thermocellum, at different pH values in chemostat culture and applied systems biology tools.ResultsClostridium thermocellum LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h−1. F1F0-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentially expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the F1F0-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6.98, and intercellular concentration reached 1.8 μmol/g pellet at pH 5.80 (stopped flow).ConclusionsClostridium thermocellum LL1210 can grow under slightly acidic conditions, similar to limits reported for other strains. This foundational study provides a detailed characterization of a relatively acid-intolerant bacterium and provides genetic targets for strain improvement. Future studies should examine adding gene functions used by more acid-tolerant bacteria for improved pH homeostasis at acidic pH values.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1095-y) contains supplementary material, which is available to authorized users.

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LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

Cited by 16 publications

References 47 publications

Regulation of biomass degradation by alternative σ factors in cellulolytic clostridia

Regulation of biomass degradation by alternative σ factors in cellulolytic clostridia

Firmicutes-enriched IS1447 represents a group of IS3-family insertion sequences exhibiting unique + 1 transcriptional slippage

Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics

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