Applying a New REFINE Approach in Zymomonas mobilis Identifies Novel sRNAs That Confer Improved Stress Tolerance Phenotypes

Haning, Katie; Engels, Sean M; Williams, Paige; Arnold, Margaret; Contreras, Lydia M.

doi:10.3389/fmicb.2019.02987

Cited by 5 publications

(5 citation statements)

References 64 publications

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“…In particular, the existence of large numbers of intragenic and antisense TSSs suggests that Z. mobilis expresses noncoding RNAs from TUs with genomic densities similar to those of E. coli and B. subtilis . These results are consistent with findings that Z. mobilis uses sRNA-based regulation to manage ethanol-induced and other stress responses ( 12 – 14 ). More broadly, however, the function of pervasive noncoding transcription in bacteria remains uncertain ( 40 , 41 ); our results suggest that, in addition to regulatory sRNAs, pervasive noncoding transcription may have functions in Z. mobilis that remain to be discovered.…”

Section: Discussionsupporting

confidence: 92%

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters

et al. 2020

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Zymomonas mobilis is an ethanologenic alphaproteobacterium with promise for the industrial conversion of renewable plant biomass into fuels and chemical bioproducts. Limited functional annotation of the Z. mobilis genome is a current barrier to both fundamental studies of Z. mobilis and its development as a synthetic biology chassis. To gain insight, we collected sample-matched multiomics data, including RNA sequencing (RNA-seq), transcription start site (TSS) sequencing (TSS-seq), termination sequencing (term-seq), ribosome profiling, and label-free shotgun proteomic mass spectrometry, across different growth conditions and used these data to improve annotation and assign functional sites in the Z. mobilis genome. Proteomics and ribosome profiling informed revisions of protein-coding genes, which included 44 start codon changes and 42 added proteins. We developed statistical methods for annotating transcript 5′ and 3′ ends, enabling the identification of 3,940 TSSs and their corresponding promoters and 2,091 transcription termination sites, which were distinguished from RNA processing sites by the lack of an adjacent RNA 5′ end. Our results revealed that Z. mobilis σA −35 and −10 promoter elements closely resemble canonical Escherichia coli −35 and −10 elements, with one notable exception: the Z. mobilis −10 element lacks the highly conserved −7 thymine observed in E. coli and other previously characterized σA promoters. The σA promoters of another alphaproteobacterium, Caulobacter crescentus, similarly lack the conservation of −7 thymine in their −10 elements. Our results anchor the development of Z. mobilis as a platform for synthetic biology and establish strategies for empirical genome annotation that can complement purely computational methods. IMPORTANCE Efforts to rationally engineer synthetic pathways in Zymomonas mobilis are impeded by a lack of knowledge and tools for predictable and quantitative programming of gene regulation at the transcriptional, posttranscriptional, and posttranslational levels. With the detailed functional characterization of the Z. mobilis genome presented in this work, we provide crucial knowledge for the development of synthetic genetic parts tailored to Z. mobilis. This information is vital as researchers continue to develop Z. mobilis for synthetic biology applications. Our methods and statistical analyses also provide ways to rapidly advance the understanding of poorly characterized bacteria via empirical data that enable the experimental validation of sequence-based prediction for genome characterization and annotation.

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Section: Discussionsupporting

confidence: 92%

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters

et al. 2020

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“…Total RNA was extracted following standard methods ( Mihailovic et al., 2018 ) with slight modifications: 300μL instead of 200 μL of 24:1 chloroform:isoamyl alcohol for separation, 1mL IPA with 1 μL GlycoBlue Coprecipitant (Ambion) instead of the sodium citrate and sodium chloride solution for overnight precipitation, and 95% instead of 75% ethanol/water was used for the first pellet wash. The total RNA was then subjected to previously described northern blot analysis ( Haning et al., 2020 ). In summary, DNA oligonucleotide probes designed complementary to a 5’ region of RseX ( Supplementary Table 1 ), as well as the ladder [ΦX174 DNA/HinfI (Promega)], were labeled individually using 20 pmol of olignoucleotide or ladder in a 20 μL kinase reaction consisting of 25 μM [γ-32P]-ATP and 20 units T4 polynucleotide kinase (NEB) at 37°C for 1 hour.…”

Section: Methodsmentioning

confidence: 99%

“…The past decade has marked a shift from fortuitous sRNA discovery to rational sRNA prediction. Indeed, omics studies, often coupled with unique computational screenings, have enabled identification of numerous sRNAs in both model and non-model bacteria ( Leonard et al., 2019 ; Haning et al., 2020 ), finding that sRNAs are pervasive in all eubacterial kingdoms ( Barquist and Vogel, 2015 ). In E. coli alone, over 85 sRNAs have had their expression biochemically confirmed ( Hör et al., 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Uncovering Transcriptional Regulators and Targets of sRNAs Using an Integrative Data-Mining Approach: H-NS-Regulated RseX as a Case Study

Mihailovic

Ekdahl

Chen

et al. 2021

Front. Cell. Infect. Microbiol.

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Bacterial small RNAs (sRNAs) play a vital role in pathogenesis by enabling rapid, efficient networks of gene attenuation during infection. In recent decades, there has been a surge in the number of proposed and biochemically-confirmed sRNAs in both Gram-positive and Gram-negative pathogens. However, limited homology, network complexity, and condition specificity of sRNA has stunted complete characterization of the activity and regulation of these RNA regulators. To streamline the discovery of the expression of sRNAs, and their post-transcriptional activities, we propose an integrative in vivo data-mining approach that couples DNA protein occupancy, RNA-seq, and RNA accessibility data with motif identification and target prediction algorithms. We benchmark the approach against a subset of well-characterized E. coli sRNAs for which a degree of in vivo transcriptional regulation and post-transcriptional activity has been previously reported, finding support for known regulation in a large proportion of this sRNA set. We showcase the abilities of our method to expand understanding of sRNA RseX, a known envelope stress-linked sRNA for which a cellular role has been elusive due to a lack of native expression detection. Using the presented approach, we identify a small set of putative RseX regulators and targets for experimental investigation. These findings have allowed us to confirm native RseX expression under conditions that eliminate H-NS repression as well as uncover a post-transcriptional role of RseX in fimbrial regulation. Beyond RseX, we uncover 163 putative regulatory DNA-binding protein sites, corresponding to regulation of 62 sRNAs, that could lead to new understanding of sRNA transcription regulation. For 32 sRNAs, we also propose a subset of top targets filtered by engagement of regions that exhibit binding site accessibility behavior in vivo. We broadly anticipate that the proposed approach will be useful for sRNA-reliant network characterization in bacteria. Such investigations under pathogenesis-relevant environmental conditions will enable us to deduce complex rapid-regulation schemes that support infection.

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“…These findings, together with the transcriptome analysis, have confirmed the critical role of regulatory and sigma factors in Z. mobilis cellular responses to various stresses. In this context, many efforts are focused on engineering the transcriptional regulators and transcription machinery, small RNAs and RNA chaperones [14][15][16][17][18].…”

Section: Introductionmentioning

confidence: 99%

“…mobilis cellular responses to various stresses. In this context, many efforts are focused on engineering the transcriptional regulators and transcription machinery, small RNAs and RNA chaperones [ 14 – 18 ].…”

Section: Introductionmentioning

confidence: 99%

Impact of hfq and sigE on the tolerance of Zymomonas mobilis ZM4 to furfural and acetic acid stresses

et al. 2020

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Zymomonas mobilis, as an ethanologenic microorganism with many desirable industrial features, faces crucial obstacles in the lignocellulosic ethanol production process. A significant hindrance occurs during the pretreatment procedure that not only produces fermentable sugars but also releases severe toxic compounds. As diverse parts of regulation networks are involved in different aspects of complicated tolerance to inhibitors, we developed ZM4hfq and ZM4-sigE strains, in which hfq and sigE genes were overexpressed, respectively. ZM4-hfq is a transcription regulator and ZM4-sigE is a transcription factor that are involved in multiple stress responses. In the present work, by overexpressing these two genes, we evaluated their impact on the Z. mobilis tolerance to furfural, acetic acid, and sugarcane bagasse hydrolysates. Both recombinant strains showed increased growth rates and ethanol production levels compared to the parental strain. Under a high concentration of furfural, the growth rate of ZM4-hfq was more inhibited compared to ZM4-sigE. More precisely, fermentation performance of ZM4-hfq revealed that the yield of ethanol production was less than that of ZM4-sigE, because more unused sugar had remained in the medium. In the case of acetic acid, ZM4-sigE was the superior strain and produced four and twofold more ethanol compared to the parental strain and ZM4-hfq, respectively. Comparison of inhibitor tolerance between single and multiple toxic inhibitors in the fermentation of sugarcane bagasse hydrolysate by ZM4-sigE strain showed similar results. In addition, ethanol production performance was considerably higher in ZM4-sigE as well. Finally, the results of the qPCR analysis suggested that under both furfural and acetic acid treatment experiments, overproduction of both hfq and sigE improves the Z. mobilis tolerance and its ethanol production capability. Overall, our study showed the vital role of the regulatory elements to overcome the obstacles in lignocellulosic biomass-derived ethanol and provide a platform for further improvement by directed evolution or systems metabolic engineering tools.

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Applying a New REFINE Approach in Zymomonas mobilis Identifies Novel sRNAs That Confer Improved Stress Tolerance Phenotypes

Cited by 5 publications

References 64 publications

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters

Uncovering Transcriptional Regulators and Targets of sRNAs Using an Integrative Data-Mining Approach: H-NS-Regulated RseX as a Case Study

Impact of hfq and sigE on the tolerance of Zymomonas mobilis ZM4 to furfural and acetic acid stresses

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