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.

Different elongation factors distinctly modulate RNA polymerase II transcription in Arabidopsis.

Various transcript elongation factors (TEFs) including modulators of RNA polymerase II (RNAPII) activity and histone chaperones tune the efficiency of transcription in the chromatin context. TEFs are involved in establishing gene expression patterns during growth and development in Arabidopsis, while little is known about the genomic distribution of the TEFs and the way they facilitate transcription. We have mapped the genome-wide occupancy of the elongation factors SPT4-SPT5, PAF1C and FACT, relative to that of elongating RNAPII phosphorylated at residues S2/S5 within the carboxyterminal domain. The distribution of SPT4-SPT5 along transcribed regions closely resembles that of RNAPII-S2P, while the occupancy of FACT and PAF1C is rather related to that of RNAPII-S5P. Under transcriptionally challenging heat stress conditions, mutant plants lacking the corresponding TEFs are differentially impaired in transcript synthesis. Strikingly, in plants deficient in PAF1C, defects in transcription across intron/exon borders are observed that are cumulative along transcribed regions. Upstream of transcriptional start sites, the presence of FACT correlates with nucleosomal occupancy. Under stress conditions FACT is particularly required for transcriptional upregulation and to promote RNAPII transcription through +1 nucleosomes. Thus, Arabidopsis TEFs are differently distributed along transcribed regions, and are distinctly required during transcript elongation especially upon transcriptional reprogramming.

TFIIS Is Crucial During Early Transcript Elongation for Transcriptional Reprogramming in Response to Heat Stress.

In addition to the stage of transcriptional initiation, the production of mRNAs is regulated during elongation. Accordingly, the synthesis of mRNAs by RNA polymerase II (RNAPII) in the chromatin context is modulated by various transcript elongation factors. TFIIS is an elongation factor that stimulates the transcript cleavage activity of RNAPII to reactivate stalled elongation complexes at barriers to transcription including nucleosomes. Since Arabidopsis tfIIs mutants grow normally under standard conditions, we have exposed them to heat stress (HS), revealing that tfIIs plants are highly sensitive to elevated temperatures. Transcriptomic analyses demonstrate that particularly HS-induced genes are expressed at lower levels in tfIIs than in wildtype. Mapping the distribution of elongating RNAPII uncovered that in tfIIs plants RNAPII accumulates at the +1 nucleosome of genes that are upregulated upon HS. The promoter-proximal RNAPII accumulation in tfIIs under HS conditions conforms to that observed upon inhibition of the RNAPII transcript cleavage activity. Further analysis of the RNAPII accumulation downstream of transcriptional start sites illustrated that RNAPII stalling occurs at +1 nucleosomes that are depleted in the histone variant H2A.Z upon HS. Therefore, assistance of early transcript elongation by TFIIS is required for reprogramming gene expression to establish plant thermotolerance.

Distinct role of subunits of the Arabidopsis RNA polymerase II elongation factor PAF1C in transcriptional reprogramming.

Transcript elongation by RNA polymerase II (RNAPII) is dynamic and highly regulated, thereby contributing to the implementation of gene expression programs during plant development or in response to environmental cues. The heterohexameric polymerase-associated factor 1 complex (PAF1C) stabilizes the RNAPII elongation complex promoting efficient transcript synthesis. In addition, PAF1C links transcriptional elongation with various post-translational histone modifications at transcribed loci. We have exposed Arabidopsis mutants deficient in the PAF1C subunits ELF7 or CDC73 to elevated NaCl concentrations to provoke a transcriptional response. The growth of elf7 plants was reduced relative to that of wildtype under these challenging conditions, whereas cdc73 plants exhibited rather enhanced tolerance. Profiling of the transcriptional changes upon NaCl exposure revealed that cdc73 responded similar to wildtype. Relative to wildtype and cdc73, the transcriptional response of elf7 plants was severely reduced in accord with their greater susceptibility to NaCl. The data also imply that CDC73 is more relevant for the transcription of longer genes. Despite the fact that both ELF7 and CDC73 are part of PAF1C the strikingly different transcriptional response of the mutants upon NaCl exposure suggests that the subunits have (partially) specific functions.

Combining a robust thermophilic methanogen and packing material with high liquid hold-up to optimize biological methanation in trickle-bed reactors.

The hydrogen gas-to-liquid mass transfer is the limiting factor in biological methanation. In trickle-bed reactors, mass transfer can be increased by high flow velocities in the liquid phase, by adding a packing material with high liquid hold-up or by using methanogenic archaea with a high methane productivity. This study developed a polyphasic approach to address all methods at once. Various methanogenic strains and packings were investigated from a microbial and hydrodynamic perspective. Analyzing the ability to produce high-quality methane and to form biofilms, pure cultures of Methanothermobacter performed better than those of the genus Methanothermococcus. Liquid and static hold-up of a packing material and its capability to facilitate attachment was not attributable to a single property. Consequently, it is recommended to carefully match organism and packing for optimized performance of trickle-bed reactors. The ideal combination for the ORBIT-system was identified as Methanothermobacter thermoautotrophicus IM5 and DuraTop®.