Biology subjects

Sivabalasarma, S.

Publications and source records attributed to Sivabalasarma, S..

10 recordsLinked to original sources

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↗