Biology subjects

Sivabalasarma, S.

Publications and source records attributed to Sivabalasarma, S..

17 recordsLinked to original sources

bioRxiv2026

Regulation of Sulfolobus acidocaldarius surface structures by the PP2A core interaction module

Protein phosphorylation is a central regulatory mechanism that enables organisms to adapt to changing environmental conditions. The hyperthermophilic archaeon Sulfolobus acidocaldar-ius encodes only two phosphatases: the dual-specificity phosphatase PTP and the ser-ine/threonine phosphatase PP2A. PP2A has previously been implicated in archaellum regula-tion, and its deletion results in a hypermotile phenotype. Under starvation conditions, PP2A associates with a stress regulatory module comprising the archaellum repressors ArnA and ArnB, the universal stress protein UspA, and a GPN-loop GTPase. Here, we investigated PP2A-associated proteins under normal growth conditions and following UV-induced DNA damage. Pulldown experiments using a genomically HA-tagged PP2A strain identified a PP2A-associated basal regulatory module consisting of ArnA, ArnB, ArnE, and PTP, distinct from the previously described starvation-associated network. In addition, several proteins involved in the biogenesis and regulation of type IV pili co-purified with PP2A. Functional analyses using thermomicroscopy and electron microscopy revealed that deletion of {triangleup}pp2a, {triangleup}arnA, or {triangleup}arnB abolishes Aap-pilus formation and twitching motility, demonstrating that the PP2A regulatory network controls both swimming and surface-associated motility. In con-trast, the same network exerted only a modulatory effect on UV-induced cell aggregation. Together, our findings establish PP2A as a central regulator coordinating multiple archaeal surface structures through phosphorylation-dependent signaling.

microbiology↗

bioRxiv2026

A conserved oscillatory system that positions the divisome in Archaea

Precise placement of the cell division machinery is essential for cell division in most organisms, yet the mechanisms responsible for this process vary substantially across the domains of life. In Eukaryotes, cell division is primarily driven by ESCRT-based systems, while many Bacteria rely on the Min system to ensure accurate positioning of the division septum. By contrast, the mechanisms by which Archaea spatially regulate divisome assembly remain largely unknown. Here, we identify a three-protein system, which we term divisome positioning proteins A, B and C (DipA, DipB and DipC) that is essential for correct divisome positioning in Haloferax volcanii. Deletion of any dip gene results in mislocalization of cell division proteins and the formation of abundant minicells, similar to Min defects in bacteria. Furthermore, we show that all three Dip proteins undergo pole-to-pole oscillation, and that DipB and DipC assemble into ring-like structures at midcell. Biochemical analyses demonstrate that DipA is a membrane binding GTPase whose association with the membrane is disrupted by DipB. Notably, Dip homologues are widely conserved across diverse archaeal lineages that employ FtsZ-based division, indicating that the Dip system is a broadly distributed key regulator of FtsZ-based cell division in Archaea. Despite its striking functional similarities to the bacterial Min system, the Dip system is entirely unrelated at the sequence level, representing a compelling example of convergent evolution of oscillatory spatial regulators in distinct domains of life.

microbiology↗

bioRxiv2026

Diversification and functional expansion of archaeal TFF machineries

Archaea have several cell surface structures that belong to the type 4 filament (TFF) superfamily. What all have in common is the presence of core minimal assembly systems consisting of an ATPase, a platform protein, a filament forming protein and a class III signal peptidase. Here we generated novel MacSyFinder2 models to identify and classify archaeal TFF systems. Our analysis revealed a vast diversity of archaeal TFF with several members harboring one or more TFF assembly machineries. Structure-based phylogenetic analyses revealed that the variable N-terminal domain of TFF-related ATPases reflects the subsystem clustering. This indicates a diversification of the core machinery components within the archaeal secretion ATPase family driven through structural innovation. Genome-wide screening of SP-III containing proteins revealed the widespread presence of substrate binding proteins with SPIII. We hypothesize that these binding proteins with canonical SPIII cleavage sites are used to functionalize TFF machineries for efficient substrate scavenging, expanding the functional repertoire of archaeal TFF systems beyond currently characterized roles. Author SummaryThe type IV filament superfamily (TFF) comprises a broad group of surface structures that are widespread across both Archaea and Bacteria. While most well-characterized members originate from bacteria, only a limited number of archaeal TFF systems have been experimentally studied so far. Here, we expand the known diversity of archaeal TFF loci through bioinformatic and comparative genomic analyses. Our results show that these systems are far more diverse and versatile than previously appreciated, often exhibiting specialized functions. The structural diversification of the ATPase machinery likely played a key role in driving the functional diversification of TFF systems in Archaea. Overall, these findings deepen our understanding of how archaea adapt and persist in diverse environments, highlighting their surface structures as essential tools for communication, adhesion, and nutrient acquisition.

microbiology↗

bioRxiv2026

Distinct core minor pilin complexes prime specialized type IV filaments in cyanobacteria

The model cyanobacterium, Synechocystis sp. PCC 6803 encodes in addition to the major pilin of the Type IV pilus filament, an extensive, partially uncharacterized repertoire of minor pilins. For those cyanobacterial minor pilins that have been characterized, their roles span a surprisingly diverse range of functions. To elucidate the roles of uncharacterized minor pilins in a systematic way, we applied structural phylogenomics across 90 genomes, classifying cyanobacterial pilins into six conserved families that form two distinct putative core priming complexes. We demonstrate that these complexes initiate the assembly of two morphologically distinct Type IV pilus filaments: short, hyper-dynamic pili for natural competence, and long, adhesive pili for motility and phototaxis. Structural analysis revealed a conserved beta-solenoid domain in PilX subunits, which we propose fulfills the "tip plug" function of PilY1 homologs. Proteomic data indicate that the minor pilin PilX2 facilitates the assembly of motility pili, which is required to maintain DnaJ3 co-chaperone levels and trigger cAMP-dependent surface sensing. These findings challenge the concept of a single multipurpose pilus, establishing that cyanobacteria operate two specialized nanomachines optimized for the conflicting biophysical requirements of DNA uptake and surface motility.

microbiology↗

bioRxiv2026

Control of motility and cell shape of Haloferax volcanii is linked by a transcriptional regulator

Archaea rely on motility and morphological plasticity to navigate their environments, yet the transcriptional regulation of these processes remains poorly understood. In Haloferax volcanii, archaellum-dependent motility is transcriptionally regulated, but an EarA-like master regulator is absent. Here, we identify CsmR as a transcriptional regulator that links archaellum biogenesis and cell-shape transitions in H. volcanii. Deletion of csmR abolished detectable motility, whereas overexpression increased motility and promoted a sustained rod-like morphology. Comparative transcriptomics defined a CsmR-associated regulon that includes archaellum and chemotaxis genes as well as rod-shape determinants (e.g., Sph3 and RdfA), and upstream motif enrichment supports a direct role for CsmR in transcriptional control. Furthermore, csmR and cirA, a KaiC-like regulator, share extensive transcriptional overlap, with CirA likely fine-tuning CsmR-mediated regulation through post-translational modification. These findings establish CsmR as a key integrator of motility and cell shape regulation in Haloferax volcanii, suggesting that haloarchaea coordinate these fundamental processes through an unidentified transcriptional network. Moreover, Northern blotting and cell shape observation suggest that transcription factor RosR is involved in the regulation of an sRNA that shares extensive overlap with the cirA gene, possibly fine-tuning the effect of CirA on the regulation of the archaellum cluster and the rod shape determinants sph3 and rdfA. Understanding this interplay provides new insights into archaeal adaptability and may reveal broader regulatory principles in prokaryotic cell biology.

microbiology↗

bioRxiv2025

Filamentous growth, cell envelope architecture, and surface appendages of a member of the Chloroflexota, Litorilinea aerophila

Litorilinea aerophila, a filamentous bacterium of the phylum Chloroflexota (class Caldilineae), exhibits unique morphological and cell envelope features that challenge traditional bacterial models. Initially described as Gram-negative, Chloroflexota are increasingly considered as monoderm, lacking a true outer membrane. In this study, we investigated the growth behaviour, cell morphology, and cell appendages of Litorilinea aerophila using light microscopy, cryo-electron tomography, and structural biology. Dry weight-based growth assays revealed a prolonged lag phase (~40 h) followed by exponential and stationary phases. Light and fluorescence microscopy revealed irregular indentations along cell filaments, accompanied by a diffuse distribution of DNA, indicating a multicellular organization. Thin-section electron microscopy confirmed septa formation, and in late growth stages, filaments became shorter with more defined indentations and membrane vesicle release. Next to the already characterised bacterial archaellum of Litorilinea aerophila, two additional types of surface appendages were identified: (i) pilus-like structures consistent with Tad pili, and (ii) grappling hook like structures. These findings contribute to the understanding of cell envelope diversity, growth, and surface structures in filamentous Chloroflexota.

microbiology↗

bioRxiv2025

Regulation of YAP activity by nuclear G-actin binding

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/667612v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1d109c2org.highwire.dtl.DTLVardef@d7d6daorg.highwire.dtl.DTLVardef@5ab7fcorg.highwire.dtl.DTLVardef@1e51b37_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO YAP functions as a G-actin binding protein. Three high affinity actin binding sites, L65, L68 and W199 are identified in YAP that when mutated into YAPDDY attenuate YAP/actin interaction as well as YAP activity. Thus, nuclear actin binding to YAP is necessary for its transcriptional activity as a co-activator of TEAD. Incorporation of G-actin into the YAP/TEAD complex is predicted to induce TEAD conformational change involving a 4 interface. Therefore, the formation of a dynamic TEAD/YAP/actin ternary complex necessary for transcription is proposed. C_FIG The Yes-associated protein YAP belongs to the TEAD (TEA/ATTS domain) transcriptional co-activators that shuttle between cytoplasm and nuclear compartment. YAP and its paralog TAZ (transcriptional co-activator with PDZ-binding motif) play essential roles in the Hippo pathway to control tissue and organ size. In addition, YAP is critically involved in numerous cellular processes such as differentiation, proliferation, cell migration and cancer metastasis as well as mechanotransduction and cytoskeletal dynamics. The actin cytoskeleton controls YAP activity in multiple ways via tensile forces, cell density and cell-cell adhesion as well as shear stress or other biomechanical cues. Here we discover YAP as a novel G-actin binding protein. We identify three high affinity YAP actin binding sites involving critical residues within the N-terminus of YAP. Moreover, actin binding to YAP is necessary for its transcriptional activity and function such as during cell density control. Mutation of the three actin binding residues results in a loss of nuclear YAP co-activator function towards TEAD while actin binding to YAP is required for TEAD target gene regulation. Our data point towards the formation of a dynamic TEAD/YAP/actin ternary complex necessary for transcription.

cell biology↗

bioRxiv2025

Horizontal gene transfer of the functional archaellum machinery to Bacteria

Motility in Archaea is driven by a nanomachinery called the archaellum. So far, archaella have been exclusively described for the archaeal domain; however, a recent study reported the presence of archaellum gene clusters in bacterial strains of the SAR202 clade (Chloroflexota). Here, we show that bona fide archaellum gene clusters are widespread in several members of the Chloroflexota, which in turn lack any bacterial flagellar components. Analysis of archaellum encoding loci and predicted structures show remarkable similarity to the archaellum machinery. Moreover, using cryoEM single particle analysis, we solved the structure of the bacterial archaellum from Litorilinea aerophila, demonstrating the successful expression and assembly of this machinery in Bacteria and its function in swimming motility. Finally, a phylogenomic analysis revealed two horizontal gene transfer events from euryarchaeal members to Chloroflexota. In summary, our study demonstrates that a functional and assembled archaellum machinery can be successfully exchanged between the two prokaryotic domains.

microbiology↗

bioRxiv2024

New components of the community based DNA-repair mechanism in Sulfolobales

After exposure to UV light, Sulfolobus acidocaldarius cells aggregate in a species-specific manner to exchange DNA and repair double-strand breaks via homologous recombination. The formation of cell-cell interactions is mediated by Ups pili. DNA exchange subsequently occurs through the Ced system, which imports DNA. To identify novel players in these processes, we investigated several genes upregulated after UV exposure by creating in-frame deletion mutants and performing cell aggregation and DNA exchange assays. This led to the identification of two novel components involved in the Ups and Ced systems: UpsC, a minor pilin of the Ups pili, and CedD, a VirD4-like ATPase essential for DNA import. Altogether, these findings provide new insights into the fascinating DNA damage response of Sulfolobales.

microbiology↗

bioRxiv2024

MinD2 modulates cell shape and motility in the archaeon Haloferax volcanii

In bacteria and archaea, proteins of the ParA/MinD family of ATPases regulate the spatiotemporal organization of various cellular cargoes, including cell division proteins, motility structures, chemotaxis systems, and chromosomes. In bacteria, such as Escherichia coli, MinD proteins are crucial for the correct placement of the Z-ring at mid-cell during cell division. However, previous studies have shown that none of the 4 MinD homologs present in the archaeon Haloferax volcanii have a role in cell division, suggesting that these proteins regulate different cellular processes in haloarchaea. Here, we show that while deletion of MinD2 in H. volcanii ({Delta}minD2) does not affect cell growth or division, it impacts cell shape and motility by mispositioning the chemotaxis arrays and archaellum motors. Finally, we explore the links between MinD2 and MinD4, which has been previously shown to modulate the localization of chemosensory arrays and archaella in H. volcanii, finding that the two MinD homologues have synergistic effects in regulating the positioning of the motility machinery. Collectively, our findings identify MinD2 as an important link between cell shape and motility in H. volcanii and further our understanding of the mechanisms by which multiple MinD proteins regulate cellular functions in haloarchaea.

microbiology↗

bioRxiv2024

Archaeal type six secretion system mediates contact-dependent antagonism

Microbial communities are shaped by cell-cell interactions. Even though archaea are often found in associations with other microorganisms, the mechanisms structuring these communities are poorly understood. Here we report the structure and function of haloarchaeal contractile injection systems (CISs). Using a combination of functional assays and time lapse imaging, we show that Halogeometricum borinquense exhibits antagonism towards Haloferax volcanii by inducing cell lysis and inhibiting proliferation. This antagonism is contact-dependent and requires a functional CIS, which is encoded by a gene cluster that is associated with toxin-immunity pairs. Cryo-focused ion beam milling and imaging by cryo-electron tomography revealed CISs bound to the cytoplasmic membrane, resembling bacterial type six secretion systems (T6SSs). We show that related T6SS gene clusters are conserved and expressed in other haloarchaeal strains with antagonistic behavior. Our data provides a mechanistic framework for understanding how archaea may shape microbial communities and impact the food webs they inhabit. TeaserT6SSs are widespread in the archaeal domain and used to kill other archaea.

microbiology↗

bioRxiv2023

The second messenger c-di-AMP controls natural competence via ComFB signaling protein

Natural competence requires a contractile pilus system. Here, we provide evidence that the pilus biogenesis and natural competence in cyanobacteria are regulated by the second messenger c-di-AMP. Furthermore, we show that the ComFB signaling protein is a novel c-di-AMP-receptor protein, widespread in bacterial phyla, and required for pilus biogenesis and DNA uptake.

microbiology↗

bioRxiv2023

CryoEM reveals the structure of an archaeal pilus involved in twitching motility

Amongst the major archaeal filament types, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). Within Sulfolobales, member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus (Uvp) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Uvp in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility. All previously solved Aap appear to have almost identical helical structures. Here, we present a cryoEM structure of the Aap of the archaeal model organism Sulfolobus acidocaldarius. We identify the component subunit as AapB and find that while its structure follows the canonical T4P blueprint, it adopts three distinct conformations within the pilus. The tri-conformer Aap structure that we describe challenges our current understanding of pilus structure and sheds new light on the principles of twitching motility.

microbiology↗

bioRxiv2023

Archaeal self-activating GPN-loop GTPases involve a lock-switch-rock mechanism for GTP hydrolysis

Three GPN-loop GTPases, GPN1-GPN3, are central to the maturation and trafficking of eukaryotic RNA polymerase II. This GTPase family is widely represented in archaea but typically occurs as single paralogs. Structural analysis of the GTP- and GDP-bound states of the Sulfolobus acidocaldarius GPN enzyme (SaGPN) showed that this central GPN-loop GTPase adopts two distinct quaternary structures. In the GTP-bound form the {gamma}-phosphate induces a tensed dimeric arrangement by interacting with the GPN region that is relaxed upon hydrolysis to GDP. Consequently, a rocking-like motion of the two protomers causes a major allosteric structural change towards the roof-like helices. Using a lock-switch-rock (LSR) mechanism, homo- and heterodimeric GPN-like GTPases are locked in the GTP-bound state and undergo large conformational changes upon GTP hydrolysis. A{Delta} saGPN strain of S. acidocaldarius was characterized by impaired motility and major changes in the proteome underscoring its functional relevance for S. acidocaldarius in vivo. Significance StatementGPN-loop GTPases have been found to be crucial for eukaryotic RNA polymerase II assembly and nuclear trafficking. Despite their ubiquitous occurrence in eukaryotes and archaea the mechanism by which these self-activating GTPases mediate their function is unknown. Our study on an archaeal representative from Sulfolobus acidocaldarius showed that these dimeric GTPases undergo large-scale conformational changes upon GTP hydrolysis, which can be summarized as a lock-switch-rock mechanism. The observed requirement of SaGPN for motility appears to be due to its large footprint on the archaeal proteome.

biochemistry↗

bioRxiv2023

Archaeal type IV pili stabilize Haloferax volcanii biofilms in flow

Biofilms represent a prevalent lifestyle of unicellular organism that confers protection to external challenges. The mechanisms by which archaea form biofilms are however not entirely clear. H. volcanii is an extremely halophilic euryarchaeon that commonly colonizes salt crust surfaces. H. volcanii produces long and thin appendages called type IV pili that are known to play a function in surface attachment and biofilm formation in archaea and bacteria. Here, we used biophysical experiments to identify critical function of type IV pili in the mechanical integrity of H. volcanii biofilms. Using interferometric scattering microscopy (iSCAT) to non-invasively visualize T4P in live cells, we find that piliation varies across mutants expressing single pilin isoforms. Using microfluidic experiments, we found that the adhesive strength of these mutants correlates with their extent of piliation. We found that in flow, H. volcanii forms clonal biofilms that extend in three dimensions. Expression of PilA2, a single pilin isoform, is sufficient to maintain normal levels of piliation and form biofilms with a structure indistinguishable from WT. Furthermore, we found that fluid flow is a crucial determinant of biofilm integrity: in the absence of flow, biofilms lose cohesion and tend to disperse in a density-dependent manner. Overall, our results demonstrate that T4P-surface and possibly T4P-T4P interactions promote biofilm formation and integrity, and that flow is a crucial ingredient regulating archaeal biofilm formation.

microbiology↗

bioRxiv2022

Donor strand complementation, isopeptide bonds and glycosylation stabilise highly resilient archaeal thread filaments

Pili are ubiquitous filamentous surface extensions that play crucial roles for bacterial and archaeal cellular processes such as adhesion, biofilm formation, motility, cell-cell communication, DNA uptake and horizontal gene transfer to name a few. Here we report on the discovery and structure of the archaeal thread - a remarkably stable archaeal pilus that belongs to a so-far largely unknown class of protein filaments. We find that the filament is highly glycosylated and interconnected via donor strand complementation, as well as isopeptide bonds, reminiscent of bacterial type I pili. Despite striking structural similarity with bacterial type-1 pili, archaeal threads appear to have evolved independently and are likely assembled by a markedly distinct mechanism.

microbiology↗

bioRxiv2020

Analysis of cell-cell bridges in Haloferax volcanii using Electron cryo-tomography reveal a continuous cytoplasm and S-layer

Halophilic archaea exchange DNA and proteins using a fusion-based mating mechanism. Scanning electron microscopy previously suggested that mating involves an intermediate state, where cells are connected by an intercellular bridge. To better understand this process, we used electron cryotomography and fluorescence microscopy to visualize cells forming these intercellular bridges. Electron cryo-tomography showed that the observed bridges were enveloped by an S-layer and connected mating cells via a continuous cytoplasm. Macromolecular complexes like ribosomes and unknown thin filamentous helical structures were visualized in the cytoplasm inside the bridges, demonstrating that these bridges can facilitate exchange of cellular components. We followed formation of a cell-cell bridge by fluorescence time-lapse microscopy between cells at a distance of 1.5 {micro}m. These results shed light on the process of haloarchaeal mating and highlight further mechanistic questions.

microbiology↗