Biology subjects

Pohlschroder, M.

Publications and source records attributed to Pohlschroder, M..

13 recordsLinked to original sources

bioRxiv2026

Characterization of aliA and aliB deletion mutants reveals a dominant role of AliA in Haloferax volcanii lipoprotein lipidation

Protein lipidation is a widespread strategy for anchoring proteins to cellular membranes across all domains of life, yet the mechanisms underlying this process in archaea remain poorly understood. Recently, the first archaeal enzymes involved in lipobox-containing protein (lipoprotein) biogenesis, AliA and AliB, were identified and characterized in the model archaeon Haloferax volcanii. Although these paralogs share significant sequence similarity, distinct deletion phenotypes suggest differences in their substrate specificity and function. Here, we employed large-scale Triton X-114 fractionation followed by quantitative proteomics and lipid-specific mass spectrometry to systematically analyze AliA- and AliB-dependent lipoprotein lipidation. Deletion of aliA affected substantially more lipoproteins in Hfx. volcanii than deletion of aliB, markedly diminishing their TX-114 enrichment--indicating reduced hydrophobicity--and abolishing thioether-linked archaeol modification. This establishes AliA as the primary enzyme responsible for archaeal lipoprotein lipidation. In contrast, deletion of aliB affected only a small subset of lipoproteins and did not significantly reduce thioether-linked archaeol levels. In addition to defining distinct and non-redundant roles for AliA and AliB, this study provides the first large-scale experimental validation of predicted archaeal lipoproteins and identifies candidate components of the archaeal lipoprotein biogenesis pathway, substantially advancing mechanistic understanding and enabling improved lipoprotein prediction in this previously underexplored field.

molecular biology↗

bioRxiv2025

A Two-Component Regulatory System Mediates Quorum Sensing-Dependent Morphology and Motility Transitions in the Archaeon Haloferax volcanii

Quorum sensing (QS) enables microorganisms--including bacteria, eukaryotes, and viruses--to coordinate collective behaviors in response to population density. Despite their ecological and evolutionary significance, QS mechanisms in Archaea remain poorly characterized. The halophilic archaeon Haloferax volcanii provides a model for archaeal QS, transitioning from motile rods to non-motile disks in a density-dependent response to a secreted disk-forming signal (DFS). To identify components of the DFS regulatory network, we screened for spontaneous mutants that retained motility in DFS-containing soft-agar medium. One candidate, HVO_1357, encodes a predicted response regulator located adjacent to a histidine kinase (HVO_1356) and a second response regulator (HVO_1358), consistent with an extended two-component regulatory system (TCS). Based on our results, these genes encode quorum-sensing associated regulators (Qar), therefore, we propose rename them qarA (HVO_1357), qarB (HVO_1356), and qarC (HVO_1358). Deletion of qarA enabled cells to swim on DFS-containing soft-agar plates and conferred hypermotility on standard soft-agar media; however, these phenotypes were not due to changes in motility-related parameters, but a reduced sensitivity to DFS for induction of the non-motile, disk-shaped state. In contrast, {Delta}qarB and {Delta}qarC strains were non-motile and exhibited premature disk formation during normal growth. Suppressor mutations that restored motility to {Delta}qarB and {Delta}qarC mapped exclusively to qarA, suggesting QarA is the central regulator of this system. Transcriptomic analyses revealed that qarA deletion leads to upregulation of genes involved in motility and rod-shape formation. Together, these findings reveal qarABC as a DFS-responsive regulatory module and represent the first TCS in archaea shown to control QS-dependent behavior. IMPORTANCEArchaea are ubiquitous and play key roles across diverse ecosystems--including human microbiomes--yet little is known about how they communicate with one another and with other organisms, or how these interactions shape their ecological impact. Such intercellular communication, including quorum sensing (QS), allows microorganisms to coordinate behaviors critical for survival, adaptation, and community organization. In this study, we identify the first archaeal two-component regulatory system that is involved in QS-dependent regulation, providing a foundation for understanding how organisms in this domain sense and respond to population cues. By revealing a previously unknown aspect of archaeal biology, this work represents an important step toward understanding how archaeal communication shapes both their physiology and their interactions within complex microbial communities.

microbiology↗

bioRxiv2025

Quorum sensing mediates morphology and motility transitions in the model archaeon Haloferax volcanii

Quorum sensing (QS) is a mechanism of intercellular communication that enables microbes to alter gene expression and adapt to the environment. This cell-cell signaling is necessary for intra- and interspecies behaviors such as virulence and biofilm formation. While QS has been extensively studied in bacteria, little is known about cell-cell communication in archaea. Here we established an archaeal model system to study QS. We showed that for Haloferax volcanii, the transition from motile rods to non-motile disks is dependent on a possibly novel, secreted small molecule present in cell-free conditioned medium (CM). Moreover, we determined that this putative QS molecule fails to induce the morphology transition in mutants lacking the regulatory factors, DdfA and CirA. Using quantitative proteomics of wild-type cells, we detected significant differential abundances of 236 proteins in the presence of CM. Conversely, in the {Delta}ddfA mutant, addition of CM resulted in only 110 proteins of significant differential abundances. These results confirm that DdfA is involved in CM-dependent regulation. CirA, along with other proteins involved in morphology and swimming motility transitions, is among the proteins regulated by DdfA. These discoveries significantly advance our understanding of microbial communication within archaeal species.

microbiology↗

bioRxiv2024

Beyond bacterial paradigms: uncovering the functional significance and first biogenesis machinery of archaeal lipoproteins

Lipoproteins are major constituents of prokaryotic cell surfaces. In bacteria, lipoprotein attachment to membrane lipids is catalyzed by prolipoprotein diacylglyceryl transferase (Lgt). However, no Lgt homologs have been identified in archaea, suggesting the unique archaeal membrane lipids require distinct enzymes for lipoprotein lipidation. Here, we performed in silico predictions for all major archaeal lineages and revealed a high prevalence of lipoproteins across the domain Archaea. Using comparative genomics, we identified the first set of candidates for archaeal lipoprotein biogenesis components (Ali). Genetic and biochemical characterization confirmed two paralogous genes, aliA and aliB, are important for lipoprotein lipidation in the archaeon Haloferax volcanii. Disruption of AliA- and AliB-mediated lipoprotein lipidation results in severe growth defects, decreased motility, and cell-shape alterations, underscoring the importance of lipoproteins in archaeal cell physiology. AliA and AliB also exhibit different enzymatic activities, including potential substrate selectivity, uncovering a new layer of regulation for prokaryotic lipoprotein lipidation.

microbiology↗

bioRxiv2024

Involvement of ArlI, ArlJ, and CirA in Archaeal Type-IV Pilin-Mediated Motility Regulation

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established, however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used EMS mutagenesis and a motility assay to identify motile suppressors of the {Delta}pilA[1-6] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. Overexpression of these arlI and arlJ mutant constructs in the respective multi-deletion strains {Delta}pilA[1-6]{Delta}arlI and {Delta}pilA[1-6]{Delta}arlJ confirmed their role in suppressing the {Delta}pilA[1-6] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA, a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA overexpression in wild-type cells led to decreased motility. Moreover, qRT-PCR analysis revealed that in wild-type cells, higher expression levels of arlI, arlJ, and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells. Conversely, {Delta}cirA cells, which form rods during both early and mid-log phases, exhibited similar expression levels of arl genes in both growth phases. Our findings contribute to a deeper understanding of the mechanisms governing archaeal motility, highlighting the involvement of ArlI, ArlJ, and CirA in pilin- mediated motility regulation. ImportanceArchaea are close relatives of eukaryotes and play crucial ecological roles. Certain behaviors, such as swimming motility, are thought to be important for archaeal environmental adaptation. Archaella, the archaeal motility appendages, are evolutionarily distinct from bacterial flagella, and the regulatory mechanisms driving archaeal motility are largely unknown. Previous research has linked the loss of type IV pili subunits to archaeal motility suppression. This study reveals three Haloferax volcanii proteins involved in pilin-mediated motility regulation, offering a deeper understanding of motility regulation in this understudied domain while also paving the way for uncovering novel mechanisms that govern archaeal motility. Understanding archaeal cellular processes will help elucidate the ecological roles of archaea as well as the evolution of these processes across domains.

microbiology↗

bioRxiv2024

Halofilins as Emerging Bactofilin Families of Archaeal Cell Shape Plasticity Orchestrators

Bactofilins are rigid, non-polar bacterial cytoskeletal filaments that link cellular processes to specific curvatures of the cytoplasmic membrane. Although homologs of bactofilins have been identified in archaea and eukaryotes, functional studies have remained confined to bacterial systems. Here, we characterize representatives of two new families of archaeal bactofilins from the pleomorphic archaeon Haloferax volcanii, halofilin A (HalA) and halofilin B (HalB). HalA and HalB polymerize in vitro, assembling into straight bundles. HalA polymers are highly dynamic and accumulate at positive membrane curvatures in vivo, whereas HalB forms more static foci that localize in areas of local negative curvatures on the outer cell surface. Gene deletions and live-cell imaging show that halofilins are critical in maintaining morphological integrity during shape transition from disk (sessile) to rod (motile). Morphological defects in {Delta}halA result in accumulation of highly positive curvatures in rods but not in disks. Conversely, disk-shaped cells are exclusively affected by halB deletion, resulting in flatter cells. Furthermore, while {Delta}halA and {Delta}halB cells imprecisely determine the future division plane, defects arise predominantly during the disk-to-rod shape remodeling. In fact, the deletion of halA in the haloarchaeon Halobacterium salinarum, whose cells are consistently rod-shaped, impacted morphogenesis but not cell division. Increased levels of halofilins enforced drastic deformations in cells devoid of S-layer, suggesting that HalB polymers are more stable at defective S-layer lattice regions. Our results set halofilins apart from their bacterial correlate, where they provide mechanical scaffolding instead of directing envelope synthesis.

microbiology↗

bioRxiv2023

Identification and characterization of structural and regulatory cell-shape determinants in Haloferax volcanii

Archaea play indispensable roles in global biogeochemical cycles, yet many critical cellular processes, including cell-shape determination, are poorly understood. Haloferax volcanii, a model haloarchaeon, forms rods and disks, depending on growth conditions. Here, we used a combination of iterative proteomics, genetics, and live-cell imaging to identify distinct mutants that only form rods or disks. We compared the proteomes of the mutants with wild-type cells across growth phases, thereby distinguishing between protein abundance changes specific to cell shape and those related to growth phases. The corresponding results indicated a diverse set of proteins, including transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. We also identified structural proteins, including a previously unknown cytoskeletal element, the Hfx. volcanii actin homolog volactin, which plays a role in disk-shape morphogenesis. In summary, we gleaned important insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains.

microbiology↗

bioRxiv2021

Advanced understanding of prokaryotic biofilm formation using a cost-effective and versatile multi-panel adhesion (mPAD) mount

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that Pseudomonas aeruginosa wild type and a phenazine deletion mutant ({Delta}phz) form similar biofilms. Extending this analysis to anoxic conditions, we reveal that microcolony and biofilm formation can only be observed under shaking conditions and are decreased in the {Delta}phz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is not available. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for attachment to surfaces under static conditions, we demonstrate that H. volcanii mutants that lack archaella are negatively affected in their early stages of biofilm formation under shaking conditions. ImportanceDue to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.

microbiology↗

bioRxiv2021

Glycoproteomics of Haloferax volcanii reveals an extensive glycoproteome and concurrence of different N-glycosylation pathways

Glycosylation is one of the most complex post-translational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here we extend the phenotypic characterisation of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project, we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provides new insights into the roles of N-glycosylation in archaeal cell biology.

biochemistry↗

bioRxiv2020

SugarPy facilitates the universal, discovery-driven analysis of intact glycopeptides

MotivationProtein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. ResultsTherefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii. SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae. AvailabilityThe source code is freely available on GitHub (https://github.com/SugarPy/SugarPy), and its implementation in Python ensures support for all operating systems. Contactmhippler@uni-muenster.de and pohlschr@uni-muenster.de Supplementary informationSupplementary data are available online.

bioinformatics↗

bioRxiv2020

Haloferax volcanii immersed liquid biofilms develop independently of known biofilm machineries and exhibit rapid honeycomb pattern formation

The ability to form biofilms is shared by many microorganisms, including archaea. Cells in a biofilm are encased in extracellular polymeric substances that typically include polysaccharides, proteins, and extracellular DNA, conferring protection while providing a structure that allows for optimal nutrient flow. In many bacteria, flagella and evolutionarily conserved type IV pili are required for the formation of biofilms on solid surfaces or floating at the air-liquid interface of liquid media. Similarly, in many archaea it has been demonstrated that type IV pili and, in a subset of these species, archaella are required for biofilm formation on solid surfaces. Additionally, in the model archaeon Haloferax volcanii, chemotaxis and AglB-dependent glycosylation play important roles in this process. H. volcanii also forms immersed biofilms in liquid cultures poured into Petri dishes. This study reveals that mutants of this haloarchaeon that interfere with the biosynthesis of type IV pili or archaella, as well as a chemotaxis-targeting transposon and aglB-deletion mutants, lack obvious defects in biofilms formed in liquid cultures. Strikingly, we have observed that these liquid-based biofilms are capable of rearrangement into honeycomb-like patterns that rapidly form upon removal of the Petri dish lid, a phenomenon that is not dependent on changes in light or oxygen concentration but can be induced by controlled reduction of humidity. Taken together, this study demonstrates that H. volcanii requires novel, as-yet-unidentified strategies for immersed liquid biofilm formation and also exhibits rapid structural rearrangements. ImportanceThis first molecular biological study of archaeal immersed liquid biofilms advances our basic-biological understanding of the model archaeon Haloferax volcanii. Data gleaned from this study also provide an invaluable foundation for future studies to uncover components required for immersed liquid biofilms in this haloarchaeon and also potentially for liquid biofilm formation in general, which is poorly understood compared to the formation of biofilms on surfaces. Moreover, this first description of rapid honeycomb pattern formation is likely to yield novel insights into the underlying structural architecture of extracellular polymeric substances and cells within immersed liquid biofilms.

microbiology↗

bioRxiv2020

Improved growth and morphological plasticity of Haloferax volcanii

Some microbes display pleomorphism, showing variable cell shapes in a single culture, whereas others differentiate to adapt to changed environmental conditions. The pleomorphic archaeon Haloferax volcanii commonly forms discoid-shaped ( plate) cells in culture, but may also be present as rods, and can develop into motile rods in soft agar, or longer filaments in certain biofilms. Here we report improvement of H. volcanii growth in both semi-defined and complex media by supplementing with eight trace-element micronutrients. With these supplemented media, transient development of plate cells into uniformly-shaped rods was clearly observed during the early log phase of growth; cells then reverted to plates for the late log and stationary phases. In media prepared with high-purity water and reagents, without supplemental trace elements, rods and other complex elongated morphologies ( pleomorphic rods) were observed at all growth stages of the culture; the highly-elongated cells sometimes displayed a substantial tubule at one or less frequently both poles, as well as unusual tapered and highly-curved forms. Polar tubules were observed forming by initial mid-cell narrowing or tubulation, causing a dumbbell-like shape, followed by cell division towards one end. Formation of the uniform early-log rods, as well as the pleomorphic rods and tubules were dependent on the function of the tubulin-like cytoskeletal protein, CetZ1. Our results have revealed the remarkable morphological plasticity of H. volcanii cells in response to multiple culture conditions, and should facilitate the use of this species in further studies of archaeal biology.

microbiology↗

bioRxiv2019

LIPID ANCHORING OF ARCHAEOSORTASE SUBSTRATES AND MID-CELL GROWTH IN HALOARCHAEA

The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA) and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii. ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and mor-phogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phos-phatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. H. volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth and morphological phenotypes similar to those of {triangleup}artA. Moreover, we showed the loss of covalent lipid attachment to SLG in the {triangleup}hvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the {triangleup}hvpssA and {triangleup}hvpssD strains. Strikingly, ArtA, HvPssA, and HvPssD GFP-fusions co-localized to the mid position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the mid cell prior to being secreted and lipid-anchored at the cell outer surface. Collectively, our data suggest haloarchaea use the mid cell as the main surface processing hotspot for cell elongation, division and shape determination.

cell biology↗