Genome and transcriptome architecture in Pyrococcus furiosus

Archaea nowadays are acknowledged for representing the second domain of life and for playing significant roles in the Earth ́s biogeochemical cycles. Before their initial discovery by Carl Woese and colleagues in the late 1970s, Archaea have not been recognised and erroneously confused with look-alike Bacteria under the microscope for decades. Since their classification as the third primary “kingdom” in 1990, not only their position in the universal tree of life has changed, defining the archaeal ancestry of Eukaryotes. Also, the knowledge about their ecology, diversity, evolution and molecular principles has been extended tremendously. Notably, it has been revealed that on the molecular level, Archaea share remarkably striking characteristics with both Eukarya and Bacteria, with transcription as one of the prime examples. Here, we have primarily been interested in the genome and transcriptome architecture, the regulatory roles of transcription factors and post-transcriptional mechanisms in the hyperthermophilic model archaeon Pyrococcus furiosus. To obtain the most accurate and informative background for further studies, we re- sequenced the culture collection strain DSM 3638 employing state-of-the-art hybrid Illumina and PacBio DNA sequencing and extensively expanded the annotation on the transcript level by using a differential RNA sequencing approach. Digestion of all non 5 ́- triphosphorylated transcripts by a Terminator-exonuclease allowed us to specifically enrich primary transcripts. The redefinition of the transcriptional landscape of P. furiosus included the genome-wide detection of transcription start sites, promoter architectures, sense- and antisense-RNAs. Interestingly, we discovered bidirectional transcription from symmetric promoters as an extensive source of antisense transcription, which is presumably a widespread feature of archaeal transcription. Additionally, we could prove that despite the relatively high abundance of insertion sequences in the 2 Mbp genome, the handling of a lab culture for two years did not lead to genomic rearrangements. Although we did not specifically challenge the genomic integrity, this still suggests that the genome is more stable than previously anticipated, which is an essential prerequisite for the comparability and feasibility of future genome-wide studies in P. furiosus. For rapid and cost-efficient re- sequencing of archaeal strains, we established 3rd generation long-read Nanopore sequencing technology in the lab, which allowed us to sequence the lab strain with high consensus accuracy. Next, we established a protocol for direct RNA sequencing in prokaryotes using the Nanopore technology, which is currently the only option for single-molecule sequencing of transcripts in their native context. The plethora of transcriptional and post-transcriptional events and features are usually tackled by short-read sequencing approaches that specifically have to be tailored to the respective research question by making adaptions to the library preparation protocol or by chemical treatment. In contrast, we evaluated the potential of native RNA sequencing to address multiple transcriptomic features simultaneously in a bacterial (Escherichia coli) and archaeal (Haloferax volcanii, P. furiosus) model organisms. Performing meta-data and single-molecule analysis we could (re-)annotate large transcriptional units and map transcription boundaries. Besides, we showed that long reads are a valuable tool for heterogeneous 3 ́-end detection and that diverse termination mechanisms occur in Archaea. Next, we used the single-molecule potential of Nanopore reads for the identification of previously known and unknown intermediates in the poorly understood rRNA maturation pathway in Archaea. Moreover, we were able to detect RNA base modifications in the form of systematic basecalling errors and shifts in the ionic current, which allowed us to follow the relative timely order of KsgA- dependent di-methylation and N4-cytidine acetylation in mature and precursor 16S rRNAs in archaeal species. Third, using the new reference genome of P. furiosus, we performed an integrative RNA-seq and ChIP-seq based approach to decipher the function of the transcriptional regulator CopR during copper detoxification in P. furiosus. To get a global view on the transcriptomic response and find components of the CopR-regulon, we performed differential gene expression analysis and ChIP-seq analysis after copper shock. We discovered that CopR, which is essential in copper detoxification, binds to the upstream regions of highly copper-induced genes, that all share a common palindromic motif. Additionally, negative-stain transmission electron microscopy and image analysis by 2D class averaging revealed that CopR binds to DNA in an octameric conformation similar to other factors of the Lrp family. Finally, we proposed a model for allosteric regulation of CopR upon copper-binding and revealed different layers of copper detoxification in P. furiosus. The findings of the studies that make up this thesis contribute to a deeper understanding of basic and regulatory principles of transcription in Archaea and update the genomic and transcriptomic landscape of P. furiosus. Also, the application of Nanopore- based native RNA sequencing not only represents a significant extension of the transcriptomic toolbox in prokaryotes but also provided us with a wealth of information, especially regarding transcriptional and post-transcriptional events during rRNA maturation

Nanopore sequencing of RNA and cDNA molecules in Escherichia coli

High-throughput sequencing dramatically changed our view of transcriptome architectures and allowed for ground-breaking discoveries in RNA biology. Recently, sequencing of full-length transcripts based on the single-molecule sequencing platform from Oxford Nanopore Technologies (ONT) was introduced and is widely used to sequence eukaryotic and viral RNAs. However, experimental approaches implementing this technique for prokaryotic transcriptomes remain scarce. Here, we present an experimental and bioinformatic workflow for ONT RNA-seq in the bacterial model organism Escherichia coli, which can be applied to any microorganism. Our study highlights critical steps of library preparation and computational analysis and compares the results to gold standards in the field. Furthermore, we comprehensively evaluate the applicability and advantages of different ONT-based RNA sequencing protocols, including direct RNA, direct cDNA, and PCR-cDNA. We find that (PCR)-cDNA-seq offers improved yield and accuracy compared to direct RNA sequencing. Notably, (PCR)-cDNA-seq is suitable for quantitative measurements and can be readily used for simultaneous and accurate detection of transcript 5′ and 3′ boundaries, analysis of transcriptional units, and transcriptional heterogeneity. In summary, based on our comprehensive study, we show nanopore RNA-seq to be a ready-to-use tool allowing rapid, cost-effective, and accurate annotation of multiple transcriptomic features. Thereby nanopore RNA-seq holds the potential to become a valuable alternative method for RNA analysis in prokaryotes

Coupling of Transcription and Translation in Archaea: Cues From the Bacterial World

The lack of a nucleus is the defining cellular feature of bacteria and archaea. Consequently, transcription and translation are occurring in the same compartment, proceed simultaneously and likely in a coupled fashion. Recent cryo-electron microscopy (cryo-EM) and tomography data, also combined with crosslinking-mass spectrometry experiments, have uncovered detailed structural features of the coupling between a transcribing bacterial RNA polymerase (RNAP) and the trailing translating ribosome in Escherichia coli and Mycoplasma pneumoniae. Formation of this supercomplex, called expressome, is mediated by physical interactions between the RNAP-bound transcription elongation factors NusG and/or NusA and the ribosomal proteins including uS10. Based on the structural conservation of the RNAP core enzyme, the ribosome, and the universally conserved elongation factors Spt5 (NusG) and NusA, we discuss requirements and functional implications of transcription-translation coupling in archaea. We furthermore consider additional RNA-mediated and co-transcriptional processes that potentially influence expressome formation in archaea

Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea

Ribosomal RNA (rRNA) maturation in archaea is a complex multistep process that requires well-defined endo- and exoribonuclease activities to generate fully mature linear rRNAs. However, technical challenges prevented detailed mapping of rRNA processing steps and a systematic analysis of rRNA maturation pathways across the tree of life. In this study, we used long-read (PCR)-cDNA and direct RNA nanopore-based sequencing to study rRNA maturation in three archaeal model organisms, namely the Euryarchaea Haloferax volcanii and Pyrococcus furiosus and the Crenarchaeon Sulfolobus acidocaldarius. Compared to standard short-read protocols, nanopore sequencing facilitates simultaneous readout of 5′- and 3′-positions, which is required for the classification of rRNA processing intermediates. More specifically, we (i) accurately detect and describe rRNA maturation stages by analysis of terminal read positions of cDNA reads and thereupon (ii) explore the stage-dependent installation of the KsgA-mediated dimethylations in H. volcanii using base-calling and signal characteristics of direct RNA reads. Due to the single-molecule sequencing capacity of nanopore sequencing, we could detect hitherto unknown intermediates with high confidence, revealing details about the maturation of archaea-specific circular rRNA intermediates. Taken together, our study delineates common principles and unique features of rRNA processing in euryarchaeal and crenarchaeal representatives, thereby significantly expanding our understanding of rRNA maturation pathways in archaea

The transcriptional regulator EarA and intergenic terminator sequences modulate archaellation in Pyrococcus furiosus

The regulation of archaellation, the formation of archaeal-specific cell appendages called archaella, is crucial for the motility, adhesion, and survival of archaeal organisms. Although the heavily archaellated and highly motile Pyrococcus furiosus is a key model organism for understanding the production and function of archaella in Euryarchaea, the transcriptional regulation of archaellum assembly is so far unknown. Here we show that the transcription factor EarA is the master regulator of the archaellum (arl) operon transcription, which is further modulated by intergenic transcription termination signals. EarA deletion or overexpression strains demonstrate that EarA is essential for archaellation in P. furiosus and governs the degree of archaellation. Providing a single-molecule update on the transcriptional landscape of the arl operon in P. furiosus, we identify sequence motifs for EarA binding upstream of the arl operon and intergenic terminator sequences as critical elements for fine-tuning the expression of the multicistronic arl cluster. Furthermore, transcriptome re-analysis across different Thermococcales species demonstrated a heterogeneous production of major archaellins, suggesting a more diverse composition of archaella than previously recognized. Overall, our study provides novel insights into the transcriptional regulation of archaellation and highlights the essential role of EarA in Pyrococcus furiosus. These findings advance our understanding of the mechanisms governing archaellation and have implications for the functional diversity of archaella

Methanofollis propanolicus sp. nov., a novel archaeal isolate from a Costa Rican oil well that uses propanol for methane production

A novel methanogenic strain, CaP3V-MF-L2AT, was isolated from an exploratory oil well from Cahuita National Park, Costa Rica. The cells were irregular cocci, 0.8–1.8 μm in diameter, stained Gram-negative and were motile. The strain utilized H2/CO2, formate and the primary and secondary alcohols 1-propanol and 2-propanol for methanogenesis, but not acetate, methanol, ethanol, 1-butanol or 2-butanol. Acetate was required as carbon source. The novel isolate grew at 25–40 °C, pH 6.0–7.5 and 0–2.5% (w/v) NaCl. 16S rRNA gene sequence analysis revealed that the strain is affiliated to the genus Methanofollis. It shows 98.8% sequence similarity to its closest relative Methanofollis ethanolicus. The G + C content is 60.1 mol%. Based on the data presented here type strain CaP3V-MF-L2AT (= DSM 113321T = JCM 39176T) represents a novel species, Methanofollis propanolicus sp. nov

Next Generation DNA-Seq and Differential RNA-Seq Allow Re-annotation of the Pyrococcus furiosus DSM 3638 Genome and Provide Insights Into Archaeal Antisense Transcription

Pyrococcus furiosus DSM 3638 is a model organism for hyperthermophilic archaea with an optimal growth temperature near 100 degrees C. The genome was sequenced about 18 years ago. However, some publications suggest that in contrast to other Pyrococcus species, the genome of P. furiosus DSM 3638 is prone to genomic rearrangements. Therefore, we re-sequenced the genome using third generation sequencing techniques. The new de novo assembled genome is 1,889,914 bp in size and exhibits high sequence identity to the published sequence. However, two major deviations were detected: (1) The genome is 18,342 bp smaller than the NCBI reference genome due to a recently described deletion. (2) The region between PF0349 and PF0388 is inverted most likely due an assembly problem for the original sequence. In addition, numerous minor variations, ranging from single nucleotide exchanges, deletions or insertions were identified. The total number of insertion sequence (IS) elements is also reduced from 30 to 24 in the new sequence. Re-sequencing of a 2-year-old "lab culture" using Nanopore sequencing confirmed the overall stability of the P furiosus DSM 3638 genome even under normal lab conditions without taking any special care. To improve genome annotation, the updated DNA sequence was combined with an RNA sequencing approach. Here, RNAs from eight different growth conditions were pooled to increase the number of detected transcripts. Furthermore, a differential RNA-Seq approach was employed for the identification of transcription start sites (TSSs). In total, 2515 TSSs were detected and classified into 834 primary (pTSS), 797 antisense (aTSS), 739 internal and 145 secondary TSSs. Our analysis of the upstream regions revealed a well conserved archaeal promoter structure. Interrogation of the distances between pTSSs and aTSSs revealed a significant number of antisense transcripts, which are a result of bidirectional transcription from the same TATA box. This mechanism of antisense transcript production could be further confirmed by in vitro transcription experiments. We assume that bidirectional transcription gives rise to non-functional antisense RNAs and that this is a widespread phenomenon in archaea due to the architecture of the TATA element and the symmetric structure of the TATA-binding protein

Uncovering the temporal dynamics and regulatory networks of thermal stress response in a hyperthermophile using transcriptomics and proteomics

Facing rapid fluctuations in their natural environment, extremophiles, like the hyperthermophilic archaeon Pyrococcus furiosus, exhibit remarkable adaptability to extreme conditions. However, our understanding of their dynamic cellular responses remains limited. This study integrates RNA-sequencing and mass spectrometry data, thereby elucidating transcriptomic and proteomic responses to heat and cold shock stress in P. furiosus. Our results reveal rapid and dynamic changes in gene and protein expression following these stress responses. Heat shock triggers extensive transcriptome reprogramming, orchestrated by the transcriptional regulator Phr, targeting a broader gene repertoire than previously demonstrated. For heat shock signature genes, RNA levels swiftly return to baseline upon recovery, while protein levels remain persistently upregulated, reflecting a rapid but sustained response. Intriguingly, cold shock at 4°C elicits distinct short- and long-term responses at both RNA and protein levels. Cluster analysis identified gene sets with either congruent or contrasting trends in RNA and protein changes, representing well-separated arCOG groups tailored to their individual cellular responses. Particularly, upregulation of ribosomal proteins and significant enrichment of 5′-leadered sequences in cold-shock responsive genes suggest that translation regulation is important during cold shock adaption. Further investigating transcriptomic features, we reveal that thermal stress genes are equipped with basal sequence elements, such as strong promoter and poly(U)-terminators, facilitating a regulated response of the respective transcription units. Our study provides a comprehensive overview of the cellular response to temperature stress, advancing our understanding of stress response mechanisms in hyperthermophilic archaea and providing valuable insights into the molecular adaptations that facilitate life in extreme environments

CopR, a Global Regulator of Transcription to Maintain Copper Homeostasis in Pyrococcus furiosus

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analyzed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterize the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 μM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the regulatory network that enables Pyrococcus to respond to increased copper concentrations

Coupling of Transcription and Translation in Archaea: Cues From the Bacterial World

The lack of a nucleus is the defining cellular feature of bacteria and archaea. Consequently, transcription and translation are occurring in the same compartment, proceed simultaneously and likely in a coupled fashion. Recent cryo-electron microscopy (cryo-EM) and tomography data, also combined with crosslinking-mass spectrometry experiments, have uncovered detailed structural features of the coupling between a transcribing bacterial RNA polymerase (RNAP) and the trailing translating ribosome in Escherichia coli and Mycoplasma pneumoniae. Formation of this supercomplex, called expressome, is mediated by physical interactions between the RNAP-bound transcription elongation factors NusG and/or NusA and the ribosomal proteins including uS10. Based on the structural conservation of the RNAP core enzyme, the ribosome, and the universally conserved elongation factors Spt5 (NusG) and NusA, we discuss requirements and functional implications of transcription-translation coupling in archaea. We furthermore consider additional RNA-mediated and co-transcriptional processes that potentially influence expressome formation in archaea