In archaeal microorganisms, the compaction and organization of the chromosome into a dynamic but condensed structure is mediated by diverse chromatin-organizing proteins in a lineage-specific manner. While many archaea employ eukaryotic-type histones for nucleoid organization, this is not the case for the crenarchaeal model species Sulfolobus acidocaldarius and related species in Sulfolobales, in which the organization appears to be mostly reliant on the action of small basic DNA-binding proteins. There is still a lack of a full understanding of the involved proteins and their functioning. Here, a combination of in vitro and in vivo methodologies is used to study the DNA-binding properties of Sul12a, an uncharacterized small basic protein conserved in several Sulfolobales species displaying a winged helix–turn–helix structural motif and annotated as a transcription factor. Genome-wide chromatin immunoprecipitation and target-specific electrophoretic mobility shift assays demonstrate that Sul12a of S. acidocaldarius interacts with DNA in a non-sequence specific manner, while atomic force microscopy imaging of Sul12a–DNA complexes indicate that the protein induces structural effects on the DNA template. Based on these results, and a contrario to its initial annotation, it can be concluded that Sul12a is a novel chromatin-organizing protein.

Neisseria meningitidis is a gram-negative bacterium that often asymptomatically colonizes the human nasopharyngeal tract. These bacteria cross the epithelial barrier can cause life-threatening sepsis and/or meningitis. Antimicrobial peptides are one of the first lines of defense against invading bacterial pathogens. Human beta-defensin 2 (hBD2) is an antimicrobial peptide with broad antibacterial activity, although its mechanism of action is poorly understood. Here, we investigated the effect of hBD2 on N. meningitidis. We showed that hBD2 binds to and kills actively growing meningococcal cells. The lethal effect was evident after 2 h incubation with the peptide, which suggests a slow killing mechanism. Further, the membrane integrity was not changed during hBD2 treatment. Incubation with lethal doses of hBD2 decreased the presence of diplococci; the number and size of bacterial microcolonies/aggregates remained constant, indicating that planktonic bacteria may be more susceptible to the peptide. Meningococcal DNA bound hBD2 in mobility shift assays and inhibited the lethal effect of hBD2 in a dose-dependent manner both in suspension and biofilms, supporting the interaction between hBD2 and DNA. Taken together, the ability of meningococcal DNA to bind hBD2 opens the possibility that extracellular DNA due to bacterial lysis may be a means of N. meningitidis to evade immune defenses.

It is now widely accepted that the first eukaryotic cell emerged from a merger of an archaeal host cell and an alphaproteobacterium. However, the exact sequence of events and the nature of the cellular biology of both partner cells is still contentious. Recently the structures of profilins from some members of the newly discovered Asgard superphylum were determined. In addition, it was found that these profilins inhibit eukaryotic rabbit actin polymerization and that this reaction is regulated by phospholipids. However, the interaction with polyproline repeats which are known to be crucial for the regulation of profilin:actin polymerization was found to be absent for these profilins and was thus suggested to have evolved later in the eukaryotic lineage. Here, we show that Heimdallarchaeota LC3, a candidate phylum within the Asgard superphylum, encodes a putative profilin (heimProfilin) that interacts with PIP₂ and its binding is regulated by polyproline motifs, suggesting an origin predating the rise of the eukaryotes. More precisely, we determined the 3D-structure of Heimdallarchaeota LC3 profilin and show that this profilin is able to: i) inhibit eukaryotic actin polymerization in vitro; ii) bind to phospholipids; iii) bind to polyproline repeats from enabled/vasodilator‐stimulated phosphoprotein; iv) inhibit actin from Heimdallarchaeota from polymerizing into filaments. Our results therefore provide hints of the existence of a complex cytoskeleton already in last eukaryotic common ancestor.

The origin of the eukaryotic cell is an unsettled scientific question. The Asgard superphylum has emerged as a compelling target for studying eukaryogenesis due to the previously unseen diversity of eukaryotic signature proteins. However, our knowledge about these proteins is still relegated to metagenomic data and very little is known about their structural properties. Additionally, it is still unclear if these proteins are functionally homologous to their eukaryotic counterparts. Here, we expressed, purified and structurally characterized profilin from Heimdallarchaeota in the Asgard superphylum. The structural analysis shows that while this profilin possesses similar secondary structural elements as eukaryotic profilin, it contains additional secondary structural elements that could be critical for its function and an indication of divergent evolution.

Sulfolobus acidocaldarius is the closest experimentally tractable archaeal relative of eukaryotes and, despite lacking obvious cyclin-dependent kinase and cyclin homologs, has an ordered eukaryote-like cell cycle with distinct phases of DNA replication and division. Here, in exploring the mechanism of cell division in S. acidocaldarius, we identify a role for the archaeal proteasome in regulating the transition from the end of one cell cycle to the beginning of the next. Further, we identify the archaeal ESCRT-III homolog, CdvB, as a key target of the proteasome and show that its degradation triggers division by allowing constriction of the CdvB1:CdvB2 ESCRT-III division ring. These findings offer a minimal mechanism for ESCRT-III-mediated membrane remodeling and point to a conserved role for the proteasome in eukaryotic and archaeal cell cycle control.

Fatty acid metabolism and its regulation are known to play important roles in bacteria and eukaryotes. By contrast, although certain archaea appear to metabolize fatty acids, the regulation of the underlying pathways in these organisms remains unclear. Here, we show that a TetR-family transcriptional regulator (FadR(sa)) is involved in regulation of fatty acid metabolism in the crenarchaeon Sulfolobus acidocaldarius. Functional and structural analyses show that FadR(sa) binds to DNA at semi-palindromic recognition sites in two distinct stoichiometric binding modes depending on the operator sequence. Genome-wide transcriptomic and chromatin immunoprecipitation analyses demonstrate that the protein binds to only four genomic sites, acting as a repressor of a 30-kb gene cluster comprising 23 open reading frames encoding lipases and beta-oxidation enzymes. Fatty acyl-CoA molecules cause dissociation of FadR(sa) binding by inducing conformational changes in the protein. Our results indicate that, despite its similarity in overall structure to bacterial TetR-family FadR regulators, FadR(sa) displays a different acyl-CoA binding mode and a distinct regulatory mechanism.

Super-resolution microscopy (SRM) has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for SRM designed to combine high performance and ease of use. We named it NanoJ-a reference to the popular ImageJ software it was developed for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image reconstruction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.

In bacteria, the GntR family is a widespread family of transcription factors responsible for the regulation of a myriad of biological processes. In contrast, despite their occurrence in archaea only a little information is available on the function of GntR-like transcription factors in this domain of life. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius harbors a GntR-like regulator belonging to the YtrA subfamily, encoded as the first gene in an operon with a second gene encoding a putative membrane protein. Here, we present a detailed characterization of this regulator, named YtrA(Sa), with a focus on regulon determination and mechanistic analysis with regards to DNA binding. Genome-wide chromatin immunoprecipitation and transcriptome experiments, the latter employing a ytrA(Sa) overexpression strain, demonstrate that the regulator acts as a repressor on a very restricted regulon, consisting of only two targets including the operon encoding its own gene and a distinct genetic locus encoding another putative membrane protein. For both targets, a conserved 14-bp semi-palindromic binding motif was delineated that covers the transcriptional start site and that is surrounded by additional half-site motifs. The crystallographic structure of YtrA(Sa) was determined, revealing a compact dimeric structure in which the DNA-binding motifs are oriented ideally to enable a specific high-affinity interaction with the core binding motif. This study provides new insights into the functioning of a YtrA-like regulator in the archaeal domain of life.

DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N-6-methyladenine (m6A), N-4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we began by investigating the DNA methylome of Sulfolobus acidocaldarius. The strategy behind this initial study entailed the use of combined digestion assays, dot blots, and genome resequencing, which utilizes specific restriction enzymes, antibodies specifically raised against m6A and m5C and single-molecule real-time (SMRT) sequencing, respectively, to identify DNA methylations occurring in exponentially growing cells. The previously identified restriction-modification system, specific of S. acidocaldarius, was confirmed by digestion assay and SMRT sequencing while, the presence of m6A was revealed by dot blot and identified on the characteristic Dam motif by SMRT sequencing. No m5C was detected by dot blot under the conditions tested. Furthermore, by comparing the distribution of both detected methylations along the genome and, by analyzing DNA methylation profiles in synchronized cells, we investigated in which cellular pathways, in particular the cell cycle, this m6A methylation could be a key player. The analysis of sequencing data rejected a role for m6A methylation in another defense system and also raised new questions about a potential involvement of this modification in the regulation of other biological functions in S. acidocaldarius.

Background: The Leucine-responsive Regulatory Protein (Lrp) family is a widespread family of regulatory transcription factors in prokaryotes. BarR is an Lrp-like transcription factor in the model archaeon Sulfolobus acidocaldarius that activates the expression of a beta-alanine aminotransferase gene, which is involved in beta-alanine degradation. In contrast to classical Lrp-like transcription factors, BarR is not responsive to any of the alpha-amino acids but interacts specifically with beta-alanine. Besides the juxtaposed beta-alanine aminotransferase gene, other regulatory targets of BarR have not yet been identified although beta-alanine is the precursor of coenzyme A and thus an important central metabolite. The aim of this study is to extend the knowledge of the DNA-binding characteristics of BarR and of its corresponding regulon from a local to a genome-wide perspective. Results: We characterized the genome-wide binding profile of BarR using chromatin immunoprecipation combined with high-throughput sequencing (ChIP-seq). This revealed 21 genomic binding loci. High-enrichment binding regions were validated to interact with purified BarR protein in vitro using electrophoretic mobility shift assays and almost all targets were also shown to harbour a conserved semi-palindromic binding motif. Only a small subset of enriched genomic sites are located in intergenic regions at a relative short distance to a promoter, and qRT-PCR analysis demonstrated that only one additional operon is under activation of BarR, namely the glutamine synthase operon. The latter is also a target of other Lrp-like transcription factors. Detailed inspection of the BarR ChIP-seq profile at the beta-alanine aminotransferase promoter region in combination with binding motif predictions indicate that the operator structure is more complicated than previously anticipated, consisting of multiple (major and auxiliary) operators. Conclusions: BarR has a limited regulon, and includes also glutamine synthase genes besides the previously characterized beta-alanine aminotransferase. Regulation of glutamine synthase is suggestive of a link between beta-alanine and alpha-amino acid metabolism in S. acidocaldarius. Furthermore, this work reveals that the BarR regulon overlaps with that of other Lrp-like regulators.