The Lrs14 family of DNA-binding proteins as nucleoid-associated proteins in the Crenarchaeal order Sulfolobales.

Organization of archaeal chromatin combines bacterial, eukaryotic, and unique characteristics. Many archaeal lineages harbor a wide diversity of small and highly expressed nucleoid-associated proteins, which are involved in DNA structuring. In Sulfolobales, representing model organisms within the Crenarchaeota, Sul7d, Cren7, Sul10a, and Sul12a are well-characterized nucleoid-associated proteins. Here, we combine evidence that the Lrs14 family of DNA binders is part of the repertoire of nucleoid-associated proteins in Sulfolobales. Lrs14-encoding genes are widespread within genomes of different members of the Sulfolobales, typically encoded as four to nine homologs per genome. The Lrs14 proteins harbor a winged helix-turn-helix DNA-binding domain and are typified by a coiled-coil dimerization. They are characterized by distinct sequence- and structure-based features, including redox-sensitive motifs and residues targeted for posttranslational modification, allowing a further classification of the family into five conserved clusters. Lrs14-like proteins have unique DNA-organizing properties. By binding to the DNA nonsequence specifically and in a highly cooperative manner, with a slight preference for AT-rich promoter regions, they introduce DNA kinks and are able to affect transcription of adjacent transcription units either positively or negatively. Genes encoding Lrs14-type proteins display considerable differential expression themselves in response to various stress conditions, with certain homologs being specific to a particular stressor. Taken together, we postulate that members of the Lrs14 family can be considered nucleoid-associated proteins in Sulfolobales, combining a DNA-structuring role with a global gene expression role in response to stress conditions.

YbiB: a novel interactor of the GTPase ObgE.

Obg is a widely conserved and essential GTPase in bacteria, which plays a central role in a large range of important cellular processes, such as ribosome biogenesis, DNA replication, cell division and bacterial persistence. Nevertheless, the exact function of Obg in these processes and the interactions it makes within the associated pathways remain largely unknown. Here, we identify the DNA-binding TrpD2 protein YbiB as an interactor of the Escherichia coli Obg (ObgE). We show that both proteins interact with high affinity in a peculiar biphasic fashion, and pinpoint the intrinsically disordered and highly negatively charged C-terminal domain of ObgE as a main driver for this interaction. Molecular docking and X-ray crystallography, together with site-directed mutagenesis, are used to map the binding site of this ObgE C-terminal domain within a highly positively charged groove on the surface of the YbiB homodimer. Correspondingly, ObgE efficiently inhibits the binding of DNA to YbiB, indicating that ObgE competes with DNA for binding in the positive clefts of YbiB. This study thus forms an important step for the further elucidation of the interactome and cellular role of the essential bacterial protein Obg.

Defining heat shock response for the thermoacidophilic model crenarchaeon Sulfolobus acidocaldarius.

The crenarchaeon Sulfolobus acidocaldarius, growing optimally at temperatures between 75 and 80 °C, thrives in volcanic hot spring habitats that are typified by large temperature gradients, which impose frequent temperature stresses on the cells. Heat shock response is characterized by an upregulation of heat shock proteins, but similar to most (hyper-)thermophilic archaea, S. acidocaldarius seems to be able to bear supra-optimal temperatures with a restricted repertoire of chaperones. Here, we study the physiological consequences of continuous high-temperature stress and rapid heat shock for S. acidocaldarius. Growth experiments and cell viability assays demonstrate that temperatures of 85 °C and higher result in a decreased growth rate and, when the cells are rapidly subjected to a heat shock, a dynamic increase in mRNA levels of all relevant heat shock proteins and a subset of transcription regulators is observed. When exponentially growing cultures are exposed to a heat shock, the survival tipping point is situated around 90 °C, and the rate of heating determines whether cells are able to cope with this stress or whether the defense mechanism immediately fails, leading to extensive cell death. In conclusion, S. acidocaldarius does not seem to be better equipped to handle sudden supra-optimal temperature stress than mesophilic organisms.

Wing phosphorylation is a major functional determinant of the Lrs14-type biofilm and motility regulator AbfR1 in Sulfolobus acidocaldarius.

In response to a variety of environmental cues, prokaryotes can switch between a motile and a sessile, biofilm-forming mode of growth. The regulatory mechanisms and signaling pathways underlying this switch are largely unknown in archaea but involve small winged helix-turn-helix DNA-binding proteins of the archaea-specific Lrs14 family. Here, we study the Lrs14 member AbfR1 of Sulfolobus acidocaldarius. Small-angle X-ray scattering data are presented, which are consistent with a model of dimeric AbfR1 in which dimerization occurs via an antiparallel coiled coil as suggested by homology modeling. Furthermore, solution structure data of AbfR1-DNA complexes suggest that upon binding DNA, AbfR1 induces deformations in the DNA. The wing residues tyrosine 84 and serine 87, which are phosphorylated in vivo, are crucial to establish stable protein-DNA contacts and their substitution with a negatively charged glutamate or aspartate residue inhibits formation of a nucleoprotein complex. Furthermore, mutation abrogates the cellular abundance and transcription regulatory function of AbfR1 and thus affects the resulting biofilm and motility phenotype of S. acidocaldarius. This work establishes a novel wHTH DNA-binding mode for Lrs14-like proteins and hints at an important role for protein phosphorylation as a signal transduction mechanism for the control of biofilm formation and motility in archaea.

The genome-scale DNA-binding profile of BarR, a ß-alanine responsive transcription factor in the archaeon Sulfolobus 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 ß-alanine aminotransferase gene, which is involved in ß-alanine degradation. In contrast to classical Lrp-like transcription factors, BarR is not responsive to any of the α-amino acids but interacts specifically with ß-alanine. Besides the juxtaposed ß-alanine aminotransferase gene, other regulatory targets of BarR have not yet been identified although ß-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 ß-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 ß-alanine aminotransferase. Regulation of glutamine synthase is suggestive of a link between ß-alanine and α-amino acid metabolism in S. acidocaldarius. Furthermore, this work reveals that the BarR regulon overlaps with that of other Lrp-like regulators.

BarR, an Lrp-type transcription factor in Sulfolobus acidocaldarius, regulates an aminotransferase gene in a ß-alanine responsive manner.

In archaea, nothing is known about the ß-alanine degradation pathway or its regulation. In this work, we identify and characterize BarR, a novel Lrp-like transcription factor and the first one that has a non-proteinogenic amino acid ligand. BarR is conserved in Sulfolobus acidocaldarius and Sulfolobus tokodaii and is located in a divergent operon with a gene predicted to encode ß-alanine aminotransferase. Deletion of barR resulted in a reduced exponential growth rate in the presence of ß-alanine. Furthermore, qRT-PCR and promoter activity assays demonstrated that BarR activates the expression of the adjacent aminotransferase gene, but only upon ß-alanine supplementation. In contrast, auto-activation proved to be ß-alanine independent. Heterologously produced BarR is an octamer in solution and forms a single complex by interacting with multiple sites in the 170 bp long intergenic region separating the divergently transcribed genes. In vitro, DNA binding is specifically responsive to ß-alanine and site-mutant analyses indicated that ß-alanine directly interacts with the ligand-binding pocket. Altogether, this work contributes to the growing body of evidence that in archaea, Lrp-like transcription factors have physiological roles that go beyond the regulation of α-amino acid metabolism.

Nanobody(R)-based chromatin immunoprecipitation/micro-array analysis for genome-wide identification of transcription factor DNA binding sites.

Nanobodies® are single-domain antibody fragments derived from camelid heavy-chain antibodies. Because of their small size, straightforward production in Escherichia coli, easy tailoring, high affinity, specificity, stability and solubility, nanobodies® have been exploited in various biotechnological applications. A major challenge in the post-genomics and post-proteomics era is the identification of regulatory networks involving nucleic acid-protein and protein-protein interactions. Here, we apply a nanobody® in chromatin immunoprecipitation followed by DNA microarray hybridization (ChIP-chip) for genome-wide identification of DNA-protein interactions. The Lrp-like regulator Ss-LrpB, arguably one of the best-studied specific transcription factors of the hyperthermophilic archaeon Sulfolobus solfataricus, was chosen for this proof-of-principle nanobody®-assisted ChIP. Three distinct Ss-LrpB-specific nanobodies®, each interacting with a different epitope, were generated for ChIP. Genome-wide ChIP-chip with one of these nanobodies® identified the well-established Ss-LrpB binding sites and revealed several unknown target sequences. Furthermore, these ChIP-chip profiles revealed auxiliary operator sites in the open reading frame of Ss-lrpB. Our work introduces nanobodies® as a novel class of affinity reagents for ChIP. Taking into account the unique characteristics of nanobodies®, in particular, their short generation time, nanobody®-based ChIP is expected to further streamline ChIP-chip and ChIP-Seq experiments, especially in organisms with no (or limited) possibility of genetic manipulation.

Expanded target and cofactor repertoire for the transcriptional activator LysM from Sulfolobus.

Previously, Lrp-like transcriptional regulator LysM from the hyperthermoacidophilic crenarchaeon Sulfolobus solfataricus was proposed to have a single target, the lysWXJK operon of lysine biosynthesis, and a single effector molecule, l-lysine. Here we identify ∼70 novel binding sites for LysM in the S. solfataricus genome with a LysM-specific nanobody-based chromatin immunoprecipitation assay coupled to microarray hybridization (ChIP-chip) and in silico target site prediction using an energy-based position weight matrix, and validate these findings with in vitro binding. LysM binds to intergenic and coding regions, including promoters of various amino acid biosynthesis and transport genes. We confirm that l-lysine is the most potent effector molecule that reduces, but does not completely abolish, LysM binding, and show that several other amino acids and derivatives, including d-lysine, l-arginine, l-homoarginine, l-glutamine and l-methionine and branched-chain amino acids l-leucine, l-isoleucine and l-valine, significantly affect DNA-binding properties of LysM. Therefore, it appears from this study that LysM is a much more versatile regulator than previously thought, and that it uses a variety of amino acids to sense nutritional quality of the environment and to modulate expression of the metabolic machinery of Sulfolobus accordingly.

The one-component system ArnR: a membrane-bound activator of the crenarchaeal archaellum.

Linking the motility apparatus to signal transduction systems enables microbes to precisely control their swimming behaviour according to environmental conditions. Bacteria have therefore evolved a complex chemotaxis machinery, which has presumably spread through lateral gene transfer into the euryarchaeal subkingdom. By contrast Crenarchaeota encode no chemotaxis-like proteins but are nevertheless able to connect external stimuli to archaellar derived motility. This raises fundamental questions about the underlying regulatory mechanisms. Recently, we reported that the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius becomes motile upon nutrient starvation by promoting transcription of flaB encoding the filament forming subunits. Here we describe two transcriptional activators as paralogous one-component-systems Saci_1180 and Saci_1171 (ArnR and ArnR1). Deletions of arnR and arnR1 resulted in diminished flaB expression and accordingly the deletion mutants revealed impaired swimming motility. We further identified two inverted repeat sequences located upstream of the flaB core promoter of S. acidocaldarius. These cis-regulatory elements were shown to be critical for ArnR and ArnR1 mediated flaB gene expression in vivo. Finally, bioinformatic analysis revealed ArnR to be conserved not only in Sulfolobales but also in the crenarchaeal order of Desulfurococcales and thus might represent a more general control mechanism of archaeal motility.

Effects of Orange Peel Extract on Laccase Activity and Gene Expression in Trametes versicolor.

The genome of Trametes versicolor encodes multiple laccase isozymes, the expression of which is responsive to various conditions. Here, we set out to investigate the potential of orange peel extract as an inducer of laccase production in this white-rot fungus, in comparison to the previously identified inducing chemical compound, veratryl alcohol. For four geographically distinct T. versicolor strains, a positive correlation has been observed between their oxidative activity and incubation time in liquid cultures. The addition of 20% orange peel extract or 5 mM veratryl alcohol caused a rapid increase in the oxidative potential of T. versicolor M99 after 24 h, with a more pronounced effect observed for the orange peel extract. To elucidate the underlying molecular mechanisms of the induced laccase activity, a transcriptional gene expression analysis was performed for the seven individual laccase genes in T. versicolor, revealing the upregulation of several laccase genes in response to the addition of each inducer. Notably, the gene encoding TvLac5 demonstrated a substantial upregulation in response to the addition of 20% orange peel extract, likely contributing to the observed increase in its oxidative potential. In conclusion, our results demonstrate that orange peels are a promising agro-industrial side stream for implementation as inducing agents in large-scale laccase production with T. versicolor.

A novel endo-1,4-ß-xylanase from Alicyclobacillus mali FL18: Biochemical characterization and its synergistic action with ß-xylosidase in hemicellulose deconstruction.

A novel endo-1,4-ß-xylanase-encoding gene was identified in Alicyclobacillus mali FL18 and the recombinant protein, named AmXyn, was purified and biochemically characterized. The monomeric enzyme worked optimally at pH 6.6 and 80 °C on beechwood xylan with a specific activity of 440.00 ± 0.02 U/mg and a good catalytic efficiency (kcat/KM = 91.89 s-1mLmg-1). In addition, the enzyme did not display any activity on cellulose, suggesting a possible application in paper biobleaching processes. To develop an enzymatic mixture for xylan degradation, the association between AmXyn and the previously characterized ß-xylosidase AmßXyl, deriving from the same microorganism, was assessed. The two enzymes had similar temperature and pH optima and showed the highest degree of synergy when AmXyn and AmßXyl were added sequentially to beechwood xylan, making this mixture cost-competitive and suitable for industrial use. Therefore, this enzymatic cocktail was also employed for the hydrolysis of wheat bran residue. TLC and HPAEC-PAD analyses revealed a high conversion rate to xylose (91.56 %), placing AmXyn and AmßXyl among the most promising biocatalysts for the saccharification of agricultural waste.

Paracoccus kondratievae produces poly(3-hydroxybutyrate) under elevated temperature conditions.

As part of ongoing efforts to discover novel polyhydroxyalkanoate-producing bacterial species, we embarked on characterizing the thermotolerant species, Paracoccus kondratievae, for biopolymer synthesis. Using traditional chemical and thermal characterization techniques, we found that P. kondratievae accumulates poly(3-hydroxybutyrate) (PHB), reaching up to 46.8% of the cell's dry weight after a 24-h incubation at 42°C. Although P. kondratievae is phylogenetically related to the prototypical polyhydroxyalkanoate producer, Paracoccus denitrificans, we observed significant differences in the PHB production dynamics between these two Paracoccus species. Notably, P. kondratievae can grow and produce PHB at elevated temperatures ranging from 42 to 47°C. Furthermore, P. kondratievae reaches its peak PHB content during the early stationary growth phase, specifically after 24 h of growth in a flask culture. This is then followed by a decline in the later stages of the stationary growth phase. The depolymerization observed in this growth phase is facilitated by the abundant presence of the PhaZ depolymerase enzyme associated with PHB granules. We observed the highest PHB levels when the cells were cultivated in a medium with glycerol as the sole carbon source and a carbon-to-nitrogen ratio of 10. Finally, we found that PHB production is induced as an osmotic stress response, similar to other polyhydroxyalkanoate-producing species.

Conditions for CaCO3 Biomineralization by Trichoderma Reesei with the Perspective of Developing Fungi-Mediated Self-Healing Concrete.

Concrete, a widely used building material, often suffers from cracks that lead to corrosion and degradation. A promising solution to enhance its durability is the use of fungi as self-healing agents, specifically by harnessing their ability to promote calcium carbonate (CaCO3) precipitation on their cell walls. However, the ideal conditions for CaCO3 precipitation by the filamentous fungal species Trichoderma reesei are still unclear. In this study, the biomineralization properties of T. reesei in liquid media are investigated. Two different calcium sources, calcium chloride (CaCl2) and calcium lactate are tested, at varying concentrations and in the presence of different nutritional sources that support growth of T. reesei. This study also explores the effects on fungal growth upon adding cement to the medium. Calcium lactate promotes greater fungal biomass production, although less crystals are formed as compared to samples with CaCl2. An increasing calcium concentration positively influences fungal growth and precipitation, but this effect is hindered upon the addition of cement. The highest amounts of biomass and calcium carbonate precipitation are achieved with potato dextrose broth as a nutritional source. By identifying the optimal conditions for CaCO3 precipitation by T. reesei, this study highlights its potential as a self-healing agent in concrete.

The genome-wide binding profile of the Sulfolobus solfataricus transcription factor Ss-LrpB shows binding events beyond direct transcription regulation.

BACKGROUND: Gene regulatory processes are largely resulting from binding of transcription factors to specific genomic targets. Leucine-responsive Regulatory Protein (Lrp) is a prevalent transcription factor family in prokaryotes, however, little information is available on biological functions of these proteins in archaea. Here, we study genome-wide binding of the Lrp-like transcription factor Ss-LrpB from Sulfolobus solfataricus. RESULTS: Chromatin immunoprecipitation in combination with DNA microarray analysis (ChIP-chip) has revealed that Ss-LrpB interacts with 36 additional loci besides the four previously identified local targets. Only a subset of the newly identified binding targets, concentrated in a highly variable IS-dense genomic region, is also bound in vitro by pure Ss-LrpB. There is no clear relationship between the in vitro measured DNA-binding specificity of Ss-LrpB and the in vivo association suggesting a limited permissivity of the crenarchaeal chromatin for transcription factor binding. Of 37 identified binding regions, 29 are co-bound by LysM, another Lrp-like transcription factor in S. solfataricus. Comparative gene expression analysis in an Ss-lrpB mutant strain shows no significant Ss-LrpB-mediated regulation for most targeted genes, with exception of the CRISPR B cluster, which is activated by Ss-LrpB through binding to a specific motif in the leader region. CONCLUSIONS: The genome-wide binding profile presented here implies that Ss-LrpB is associated at additional genomic binding sites besides the local gene targets, but acts as a specific transcription regulator in the tested growth conditions. Moreover, we have provided evidence that two Lrp-like transcription factors in S. solfataricus, Ss-LrpB and LysM, interact in vivo.

Cis-regulatory logic in archaeal transcription.

For cellular fitness and survival, gene expression levels need to be regulated in response to a wealth of cellular and environmental signals. TFs (transcription factors) execute a large part of this regulation by interacting with the basal transcription machinery at promoter regions. Archaea are characterized by a simplified eukaryote-like basal transcription machinery and bacteria-type TFs, which convert sequence information into a gene expression output according to cis-regulatory rules. In the present review, we discuss the current state of knowledge about these rules in archaeal systems, ranging from DNA-binding specificities and operator architecture to regulatory mechanisms.

Recent technological innovations in mycelium materials as leather substitutes: a patent review.

Leathery mycelium materials, made from the vegetative part of filamentous fungi, have garnered significant interest in recent years due to their great potential of providing environmentally sustainable alternatives to animal- and plastic-based leathers. In this systematic patent review, we provide an in-depth overview of the fabrication methods for mycelium materials as leather substitutes recently described in patents. This overview includes strategies for fungal biomass generation and industrial developments in the sector. We discuss the use of various fungal species, plasticizers, crosslinking agents, and post-processing techniques, thereby highlighting potential gaps in scientific knowledge and identifying opportunities, challenges, and concerns in the field. Our analysis suggests that mycelium materials have significant potential for commercialization, with a growing number of companies betting on this new class of biomaterials. However, we also reveal the need for further scientific research to fully understand the properties of these materials and to unlock potential applications. Overall, this patent review delineates the current state of the art in leathery mycelium materials.

Molecular mechanisms of regulation by a ß-alanine-responsive Lrp-type transcription factor from Acidianus hospitalis.

The leucine-responsive regulatory protein (Lrp) family of transcriptional regulators is widespread among prokaryotes and especially well-represented in archaea. It harbors members with diverse functional mechanisms and physiological roles, often linked to the regulation of amino acid metabolism. BarR is an Lrp-type regulator that is conserved in thermoacidophilic Thermoprotei belonging to the order Sulfolobales and is responsive to the non-proteinogenic amino acid ß-alanine. In this work, we unravel molecular mechanisms of the Acidianus hospitalis BarR homolog, Ah-BarR. Using a heterologous reporter gene system in Escherichia coli, we demonstrate that Ah-BarR is a dual-function transcription regulator that is capable of repressing transcription of its own gene and activating transcription of an aminotransferase gene, which is divergently transcribed from a common intergenic region. Atomic force microscopy (AFM) visualization reveals a conformation in which the intergenic region appears wrapped around an octameric Ah-BarR protein. ß-alanine causes small conformational changes without affecting the oligomeric state of the protein, resulting in a relief of regulation while the regulator remains bound to the DNA. This regulatory and ligand response is different from the orthologous regulators in Sulfolobus acidocaldarius and Sulfurisphaera tokodaii, which is possibly explained by a distinct binding site organization and/or by the presence of an additional C-terminal tail in Ah-BarR. By performing site-directed mutagenesis, this tail is shown to be involved in ligand-binding response.

Screening fungal strains isolated from a limestone cave on their ability to grow and precipitate CaCO 3 in an environment relevant to concrete.

Fungi-mediated self-healing concrete is a novel approach that promotes the precipitation of calcium carbonate (CaCO 3 ) on fungal hyphae to heal the cracks in concrete. In this study, we set out to explore the potential of fungal species isolated from a limestone cave by investigating their ability to precipitate CaCO 3 and to survive and grow in conditions relevant to concrete. Isolated strains belonging to the genera Botryotrichum sp. , Trichoderma sp. and Mortierella sp. proved to be promising candidates for fungi-mediated self-healing concrete attributed to their growth properties and CaCO 3 precipitation capabilities in the presence of cement.

Transcriptional and translational dynamics underlying heat shock response in the thermophilic crenarchaeon Sulfolobus acidocaldarius.

IMPORTANCE: Heat shock response is the ability to respond adequately to sudden temperature increases that could be harmful for cellular survival and fitness. It is crucial for microorganisms living in volcanic hot springs that are characterized by high temperatures and large temperature fluctuations. In this study, we investigated how S. acidocaldarius, which grows optimally at 75°C, responds to heat shock by altering its gene expression and protein production processes. We shed light on which cellular processes are affected by heat shock and propose a hypothesis on underlying regulatory mechanisms. This work is not only relevant for the organism's lifestyle, but also with regard to its evolutionary status. Indeed, S. acidocaldarius belongs to the archaea, an ancient group of microbes that is more closely related to eukaryotes than to bacteria. Our study thus also contributes to a better understanding of the early evolution of heat shock response.

Foundation of the Belgian Society for Viruses of Microbes and Meeting Report of Its Inaugural Symposium.

The Belgian Society for Viruses of Microbes (BSVoM) was founded on 9 June 2022 to capture and enhance the collaborative spirit among the expanding community of microbial virus researchers in Belgium. The sixteen founders are affiliated to fourteen different research entities across academia, industry and government. Its inaugural symposium was held on 23 September 2022 in the Thermotechnical Institute at KU Leuven. The meeting program covered three thematic sessions launched by international keynote speakers: (1) virus-host interactions, (2) viral ecology, evolution and diversity and (3) present and future applications. During the one-day symposium, four invited keynote lectures, ten selected talks and eight student pitches were given along with 41 presented posters. The meeting hosted 155 participants from twelve countries.