scholarly journals Mapping the complex transcriptional landscape of the phytopathogenic bacterium Dickeya dadantii

2020 ◽  
Author(s):  
Raphaël Forquet ◽  
Xuejiao Jiang ◽  
William Nasser ◽  
Florence Hommais ◽  
Sylvie Reverchon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Environmental Changes ◽  
Computational Method ◽  
Fine Tuning ◽  
Transcription Start ◽  
Dickeya Dadantii ◽  
Phytopathogenic Bacterium ◽  
Plant Infection ◽  
Wide Range ◽  
Variable Gene

AbstractDickeya dadantii is a phytopathogenic bacterium that causes soft rot in a wide range of plant hosts worldwide and a model organism for studying gene regulation during the pathogenic process. The present study provides a comprehensive and annotated transcriptomic map of D. dadantii obtained by a computational method combining three independent transcriptomic datasets covering a wide range of conditions which closely reproduce the variations of transcription occurring in the course of plant infection: (1) paired-end RNA-seq data for a precise reconstruction of the RNA landscape, (2) DNA microarray data reflecting gene response to sudden environmental shocks mimicking conditions encountered by bacteria in the plant, (3) dRNA-seq data for a specific high-resolution mapping of transcription start sites. We define transcription units throughout the genome, and map the associated transcription start and termination sites with a quantitative magnitude analysis. Our results show that transcription units sometimes coincide with predicted operons but are generally longer, most of them exhibiting internal promoters and terminators that generate alternative transcripts of variable gene composition. We characterize the occurrence of transcriptional read-through at terminators, which might play a basal regulation role and explain the extent of transcription beyond the scale of operons. We finally highlight the presence of noncontiguous operons and excludons in D. dadantii genome, novel genomic arrangements that might contribute to the basal coordination of transcription. The highlighted transcriptional organization may allow D. dadantii finely adjusting its gene expression program for a rapid adaptation to fast changing environment relevant to plant infection.ImportanceThis is the first transcriptomic map of a phytopathogen, characterized under physiological conditions encountered by the bacteria during the infection process. It might therefore significantly contribute to further progress in the field of phytopathogenicity. Our findings also provide insights into basal rules of coordination of transcription that might be valid for other bacteria, and may raise interest in the field of microbiology in general. In particular, we demonstrate that gene expression is coordinated at the scale of transcription units rather than operons, which are larger functional genomic units capable of generating transcripts with variable gene composition for a fine-tuning of gene expression in response to environmental changes. In line with recent studies, our findings indicate that the canonical operon model is insufficient to explain the complexity of bacterial transcriptomes.

scholarly journals Cell Individuality: The Bistable Gene Expression of the Type III Secretion System in Dickeya dadantii 3937

2012 ◽  
Vol 25 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Quan Zeng ◽  
Michael D. Laiosa ◽  
Douglas A. Steeber ◽  
Eulandria M. Biddle ◽  
Quan Peng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Type Iii Secretion ◽  
Expression Patterns ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Expression Level ◽  
Dickeya Dadantii ◽  
Phytopathogenic Bacterium ◽  
Gram Negative ◽  
Type Iii

Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level. We demonstrated that bistable expression of the HrpL-regulon genes, such as hrpA and hrpN, is controlled by the same regulatory mechanism. We also showed that the expression level of the T3SS master regulatory gene hrpL plays an important role in the development of the bistable expression of hrpA. A high expression level of hrpL is required but unable to guarantee the high-state expression of hrpA in a cell. In addition, bistable expression patterns of T3SS genes in other gram-negative pathogens of the Enterobacteriaceae and Pseudomonadaceae families were also described in this study. This suggests that the T3SS bistability might be a conserved population behavior in several gram-negative bacterial pathogens.

scholarly journals Dual UTR-A novel 5′ untranslated region design for synthetic biology applications

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Simone Balzer Le ◽  
Ingerid Onsager ◽  
Jon Andreas Lorentzen ◽  
Rahmi Lale
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
De Novo ◽  
Expression Profiles ◽  
Regulation Of Gene Expression ◽  
Design Concept ◽  
Fine Tuning ◽  
The Novel ◽  
Wide Range ◽  
Application Potential

Abstract Bacterial 5′ untranslated regions of mRNA (UTR) involve in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. In this study, we report the design of a novel 5′ UTR, dual UTR, utilizing the transcriptional and translational characteristics of 5′ UTRs in a single expression cassette. The dual UTR consists of two 5′ UTRs, each separately leading to either increase in transcription or translation of the reporter, that are separated by a spacer region, enabling de novo translation initiation. We rationally create dual UTRs with a wide range of expression profiles and demonstrate the functionality of the novel design concept in Escherichia coli and Pseudomonas putida using different promoter systems and coding sequences. Overall, we demonstrate the application potential of dual UTR design concept in various synthetic biology applications ranging from fine-tuning of gene expression to maximization of protein production.

scholarly journals A secreted metal-binding protein protects necrotrophic phytopathogens from reactive oxygen species

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lulu Liu ◽  
Virginie Gueguen-Chaignon ◽  
Isabelle R Gonçalves ◽  
Christine Rascle ◽  
Martine Rigault ◽  
...  
Keyword(s):  
Metal Binding ◽  
Plant Cell Wall ◽  
Metal Accumulation ◽  
Cell Wall Degrading Enzymes ◽  
Dickeya Dadantii ◽  
Phytopathogenic Bacterium ◽  
Gram Positive Bacteria ◽  
Plant Infection ◽  
Ros Formation ◽  
Iron Binding Proteins

Abstract Few secreted proteins involved in plant infection common to necrotrophic bacteria, fungi and oomycetes have been identified except for plant cell wall-degrading enzymes. Here we study a family of iron-binding proteins that is present in Gram-negative and Gram-positive bacteria, fungi, oomycetes and some animals. Homolog proteins in the phytopathogenic bacterium Dickeya dadantii (IbpS) and the fungal necrotroph Botrytis cinerea (BcIbp) are involved in plant infection. IbpS is secreted, can bind iron and copper, and protects the bacteria against H2O2-induced death. Its 1.7 Å crystal structure reveals a classical Venus Fly trap fold that forms dimers in solution and in the crystal. We propose that secreted Ibp proteins binds exogenous metals and thus limit intracellular metal accumulation and ROS formation in the microorganisms.

scholarly journals Inactivation of pecS restores the virulence of mutants devoid of osmoregulated periplasmic glucans in the phytopathogenic bacterium Dickeya dadantii

Microbiology ◽  
2014 ◽  
Vol 160 (4) ◽  
pp. 766-777 ◽  
Author(s):  
Sébastien Bontemps-Gallo ◽  
Edwige Madec ◽  
Jean-Marie Lacroix
Keyword(s):  
Plant Cell Wall ◽  
Transcriptional Regulator ◽  
Soft Rot ◽  
Cellular Functions ◽  
Cell Wall Degrading Enzymes ◽  
Dickeya Dadantii ◽  
Phytopathogenic Bacterium ◽  
Wide Range ◽  
Severe Reduction ◽  
Rot Disease

Dickeya dadantii is a phytopathogenic enterobacterium that causes soft rot disease in a wide range of plant species. Maceration, an apparent symptom of the disease, is the result of the synthesis and secretion of a set of plant cell wall-degrading enzymes (PCWDEs), but many additional factors are required for full virulence. Among these, osmoregulated periplasmic glucans (OPGs) and the PecS transcriptional regulator are essential virulence factors. Several cellular functions are controlled by both OPGs and PecS. Strains devoid of OPGs display a pleiotropic phenotype including total loss of virulence, loss of motility and severe reduction in the synthesis of PCWDEs. PecS is one of the major regulators of virulence in D. dadantii, acting mainly as a repressor of various cellular functions including virulence, motility and synthesis of PCWDEs. The present study shows that inactivation of the pecS gene restored virulence in a D. dadantii strain devoid of OPGs, indicating that PecS cannot be de-repressed in strains devoid of OPGs.

scholarly journals Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

2021 ◽  
Author(s):  
Yu Zhao ◽  
Shiqi Liu ◽  
Zhihui Lu ◽  
Baixiang Zhao ◽  
Shuhui Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Yarrowia Lipolytica ◽  
Isoamyl Alcohol ◽  
Fine Tuning ◽  
Promoter Strength ◽  
Promoter Engineering ◽  
Test Study ◽  
Promoter Library ◽  
Wide Range ◽  
Hybrid Promoter

Abstract Background: In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research. Results: In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. We also report for the first time that upstream activation sequences (UAS) of Saccharomyces cerevisiae promoters can be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1-30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica.Conclusion: We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

scholarly journals The role of transcriptional repressor activity of LexA in salt-stress responses of the cyanobacterium Synechocystis sp. PCC 6803

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kosuke Takashima ◽  
Syota Nagao ◽  
Ayumi Kizawa ◽  
Takehiro Suzuki ◽  
Naoshi Dohmae ◽  
...  
Keyword(s):  
Gene Expression ◽  
Salt Stress ◽  
Posttranslational Modification ◽  
Environmental Changes ◽  
Heterotrophic Bacteria ◽  
Fine Tuning ◽  
Oligomeric State ◽  
Digital Switching ◽  
Pcc 6803

Abstract Different from typical LexA repressors in heterotrophic bacteria exerting SOS response by auto-cleavage, cyanobacterial LexAs, especially that of Synechocystis sp. PCC 6803 (S.6803), have been suggested be involved in regulation of a number of genes related to various cellular processes, rather than the typical SOS regulon. When and how cyanobacterial LexAs are triggered to regulate its target genes have remained unknown. In this study, we found the profound repressing effect of LexA on salt-stress inducible genes in S.6803. The repressing activity of LexA was likely to persist during salt stress and the salt response of these genes was mainly achieved by other regulators than LexA, suggesting that the physiological role of LexA is fine-tuning of gene expression in response to environmental changes. Although the amount and oligomeric state of LexA were unchanged upon salt stress, two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry analyses detected a change in posttranslational modification in a small fraction of LexA molecules, possibly dephosphorylation of Ser173, after 30 min upon the upshift in salt concentration. Activity of LexA in S.6803 may be under gradual control by posttranslational modification to fine-tune gene expression, which is contrasted with the digital switching-off regulation by auto-cleavage in heterotrophic bacteria.

scholarly journals Robust gene expression control in human cells with a novel universal TetR aptamer splicing module

2019 ◽  
Vol 47 (20) ◽  
pp. e132-e132 ◽  
Author(s):  
Adam A Mol ◽  
Florian Groher ◽  
Britta Schreiber ◽  
Ciaran Rühmkorff ◽  
Beatrix Suess
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Dynamic Range ◽  
Intron Retention ◽  
Human Cells ◽  
Fine Tuning ◽  
Genomic Context ◽  
Mammalian Cell Lines ◽  
Gene Expression Control ◽  
Wide Range

Abstract Fine-tuning of gene expression is desirable for a wide range of applications in synthetic biology. In this context, RNA regulatory devices provide a powerful and highly functional tool. We developed a versatile, robust and reversible device to control gene expression by splicing regulation in human cells using an aptamer that is recognized by the Tet repressor TetR. Upon insertion in proximity to the 5′ splice site, intron retention can be controlled via the binding of TetR to the aptamer. Although we were able to demonstrate regulation for different introns, the genomic context had a major impact on regulation. In consequence, we advanced the aptamer to develop a splice device. Our novel device contains the aptamer integrated into a context of exonic and intronic sequences that create and maintain an environment allowing a reliable and robust splicing event. The exon-born, additional amino acids will then be cleaved off by a self-cleaving peptide. This design allows portability of the splicing device, which we confirmed by demonstrating its functionality in different gene contexts. Intriguingly, our splicing device shows a high dynamic range and low basal activity, i.e. desirable features that often prove a major challenge when implementing synthetic biology in mammalian cell lines.

scholarly journals The versatile interactome of chloroplast ribosomes revealed by affinity purification mass spectrometry

2020 ◽  
Author(s):  
Lisa Désirée Westrich ◽  
Vincent Leon Gotsmann ◽  
Claudia Herkt ◽  
Fabian Ries ◽  
Tanja Kazek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Environmental Changes ◽  
Affinity Purification ◽  
Small Subunit ◽  
Fine Tuning ◽  
Chloroplast Gene ◽  
Purification Method ◽  
Chloroplast Gene Expression ◽  
Associated Proteins

Abstract In plant cells, chloroplast gene expression is predominantly controlled through post-transcriptional regulation. Such fine-tuning is vital for precisely orchestrating protein complex assembly as for the photosynthesis machinery and for quickly responding to environmental changes. While regulation of chloroplast protein synthesis is of central importance, little is known about the degree and nature of the regulatory network, mainly due to challenges associated with the specific isolation of transient ribosome interactors. Here, we established a ribosome affinity purification method, which enabled us to broadly uncover putative ribosome-associated proteins in chloroplasts. Endogenously tagging of a protein of the large or small subunit revealed not only interactors of the holo complex, but also preferential interactors of the two subunits. This includes known canonical regulatory proteins as well as several new proteins belonging to the categories of protein and RNA regulation, photosystem biogenesis, redox control and metabolism. The sensitivity of the here applied screen was validated for various transiently interacting proteins. We further provided evidence for the existence of a ribosome-associated Nα-acetyltransferase in chloroplasts and its ability to acetylate substrate proteins at their N-terminus. The broad set of ribosome interactors underscores the potential to regulate chloroplast gene expression on the level of protein synthesis.

scholarly journals Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

2021 ◽  
Author(s):  
Yu Zhao ◽  
Shiqi Liu ◽  
Zhihui Lu ◽  
Baixiang Zhao ◽  
Shuhui Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Yarrowia Lipolytica ◽  
Isoamyl Alcohol ◽  
Fine Tuning ◽  
Promoter Strength ◽  
Promoter Engineering ◽  
Test Study ◽  
Promoter Library ◽  
Wide Range ◽  
Hybrid Promoter

Abstract Background In biological cells, promoters drive gene expression by binding to RNA polymerase specifically. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for metabolic engineering applications to optimize biosynthetic pathways has recently become an active area of research. Results In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. We also report for the first time that upstream activation sequences (UAS) of Saccharomyces cerevisiae promoters can be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. Under the control of PUAS1B8−LEUm, the strongest promoter we constructed, overexpression of a key pathway gene led to 7.7-fold increase in the titer of isoamyl alcohol. Interestingly, a much weaker promoter PUAS1B4−EXPm increase the isoamyl alcohol titer by 30.3-fold. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica. Conclusion We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

scholarly journals Regulatory Mechanisms of Exoribonuclease PNPase and Regulatory Small RNA on T3SS of Dickeya dadantii

2010 ◽  
Vol 23 (10) ◽  
pp. 1345-1355 ◽  
Author(s):  
Quan Zeng ◽  
A. Mark Ibekwe ◽  
Eulandria Biddle ◽  
Ching-Hong Yang
Keyword(s):  
Gene Expression ◽  
Small Rna ◽  
Mrna Stability ◽  
Type Iii Secretion ◽  
Single Gene ◽  
Expression Patterns ◽  
Dickeya Dadantii ◽  
Phytopathogenic Bacterium ◽  
Effector Genes

The type III secretion system (T3SS) is an essential virulence factor for many bacterial pathogens. Polynucleotide phosphorylase (PNPase) is one of the major exoribonucleases in bacteria and plays important roles in mRNA degradation, tRNA processing, and small RNA (sRNA) turnover. In this study, we showed that PNPase downregulates the transcription of T3SS structural and effector genes of the phytopathogenic bacterium Dickeya dadantii. This negative regulation of T3SS by PNPase occurs by repressing the expression of hrpL, encoding a master regulator of T3SS in D. dadantii. By reducing rpoN mRNA stability, PNPase downregulates the transcription of hrpL, which leads to a reduction in T3SS gene expression. Moreover, we have found that PNPase downregulates T3SS by decreasing hrpL mRNA stability. RsmB, a regulatory sRNA, enhances hrpL mRNA stability in D. dadantii. Our results suggest that PNPase decreases the amount of functional RsmB transcripts that could result in reduction of hrpL mRNA stability. In addition, bistable gene expression (differential expression of a single gene that creates two distinct subpopulations) of hrpA, hrpN, and dspE was observed in D. dadantii under in vitro conditions. Although PNPase regulates the proportion of cells in the high state and the low state of T3SS gene expression, it appears that PNPase is not the key switch that triggers the bistable expression patterns of T3SS genes.

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