Biology subjects

Duggin, I. G.

Publications and source records attributed to Duggin, I. G..

At least 19 recordsLinked to original sources

bioRxiv2025

An Asgard archaeon from a modern analog of ancient microbial mats

It has been proposed that eukaryotic cells evolved via symbiosis between sulfate-reducing bacteria and hydrogen-producing archaea. Here we describe a highly enriched culture of a novel Asgard archaeon, Nerearchaeum marumarumayae, with a bacterium Stromatodesulfovibrio nilemahensis from a stromatolite-associated microbial mat. The N. marumarumayae genome indicates it produces H2, acetate, formate, and sulfite, while S. nilemahensis synthesizes amino acids and vitamins, which can be exchanged in a syntrophic partnership. Electron cryotomography revealed N. marumarumayae cells produce chains of budded envelope vesicles attached to the coccoid cell body by extracellular fibers, and intracellular tube- and cage-like structures. Furthermore, the two species were observed interacting via intercellular nanotubes assembled by the bacterium. These characteristics and interactions may reflect an early step in the symbiotic evolution of eukaryotic cells.

microbiology↗

bioRxiv2024

A model, mixed-species urinary catheter biofilm derived from spinal cord injury patients

Complex multispecies biofilms consistently colonise the interior of indwelling urinary catheters, causing persistent asymptomatic bacteriuria and frequent symptomatic episodes in long-term catheterized individuals. Simple single-species models often fail to capture the complexities of mixed-species interactions, leading to limited conclusions about microbial behaviour and treatment efficacy. Additionally, using lab-based organisms can obscure the genomic diversity found in real-world infections. The primary objective of this study was to establish a stable and reproducible in vitro biofilm model derived from the multi-species clinical flora associated with catheter-related infections, reflecting the dynamics of in vivo infections. Biofilm samples from clinical catheters of spinal cord injury (SCI) participants were used to establish polymicrobial macro-fluidic models within catheters. Metagenomic techniques using short-read Illumina and long-read Oxford Nanopore sequencing was used to assess the community composition, produce metagenome-assembled genomes (MAGs), analyse strain-level phylogeny diversity and single nucleotide polymorphisms (SNP) of isolates. Antibiotic resistance tests using our models highlighted the drastic differences between planktonic bacteria, single-species, and multispecies biofilms. In silico analysis of antibiotic resistance further revealed a high number of varied resistance genes present in these communities. The models developed and characterised in this study are expected to facilitate more effective strategies to prevent and treat catheter-associated infections.

microbiology↗

bioRxiv2024

Tubulin-like proteins CetZ1 and CetZ2 coordinate cellular morphogenesis in the archaeon Haloferax volcanii

CetZs are archaeal tubulin superfamily cytoskeletal proteins implicated in the control of cell shape and motility. In the pleomorphic archaeon Haloferax volcanii, CetZ1 is required for the transformation of a discoid, or plate-like, cell morphology to the rod shape during early-mid log phase cultures and for the development of swimming motility. In this study, we found that the paralog CetZ2 is not required for rod development or reversion to plates in log phase but was strongly upregulated later in stationary phase, where it promotes the maintenance of plate cell shape. CetZ2 cytoskeletal structures visualized through the production of a functional fluorescent tagged CetZ2 were most dynamic specifically in mid-stationary phase, where they showed directional movement around the cell edge and other complex cytomotive-like behaviours. Furthermore, in mid-stationary phase, the dynamics of CetZ1 and CetZ2 cytoskeletal structures were specifically dependent on the presence of one another and their GTPase activities that control the polymerization-depolymerization cycle. Together, the results suggest that CetZ2 counteracts the CetZ1-based rod development pathway to maintain plate shape, and they imply that additional stationary-phase factors are also involved. CetZ1 and CetZ2 are co-conserved in many haloarchaea, suggesting that their coordinated functions at different stages of the growth cycle are widely utilized to control cell shapeshifting. More generally, our findings show that CetZ paralogues can have distinct and non-redundant functions, indicating that diversification and specialisation within the tubulin superfamily has occurred multiple times in archaeal evolution. SignificanceThis study describes the first defined function of a CetZ2 protein, which represents a distinct haloarchaeal protein family within the near-universal tubulin superfamily of cytoskeletal proteins. Using the model archaeal organism Haloferax volcanii, we found that CetZ2 maintains the plate or disk-like cell morphology specifically in the stationary phase of the growth cycle, by counteracting CetZ1-based rod development. We also showed for the first time that CetZ2 can form dynamic cytoskeletal filaments that show directional movement around the cell in mid-stationary phase. The CetZ1-CetZ2 interplay we detected specifically at this stage may represent a paradigm for understanding the evolution of antagonistic cytoskeletal functions, such as those that led to a reliance on active and inactive subunits during the early evolution of microtubules in eukaryotes.

microbiology↗

bioRxiv2024

Prediction of symptomatic and asymptomatic bacteriuria in spinal cord injury patients using machine learning

BackgroundIndividuals with spinal cord injuries (SCI) frequently rely on urinary catheters to drain urine from the bladder, making them susceptible to asymptomatic and symptomatic catheter-associated bacteriuria and urinary tract infections (UTI). Proper identification of these conditions lacks precision, leading to inappropriate antibiotic use which promotes selection for drug-resistant bacteria. Since infection often leads to dysbiosis in the microbiome and correlates with health status, this study aimed to develop a machine learning-based diagnostic framework to predict potential UTI by monitoring urine and/or catheter microbiome data, thereby minimising unnecessary antibiotic use and improving patient health. ResultsMicrobial communities in 609 samples (309 catheter and 300 urine) with asymptomatic and symptomatic bacteriuria status were analysed using 16S rRNA gene sequencing from 27 participants over 18 months. Microbial community compositions were significantly different between asymptomatic and symptomatic bacteriuria, suggesting microbial community signatures have potential application as a diagnostic tool. A significant decrease in local (alpha) diversity was noted in symptomatic bacteriuria compared to the asymptomatic bacteriuria (P < 0.01). Beta diversity measured in weighted unifrac also showed a significant difference (P < 0.05) between groups. Supervised machine learning models trained on amplicon sequence variant (ASVs) counts and bacterial taxonomic abundances (Taxa) to classify symptomatic and asymptomatic bacteriuria with a 10-fold cross-validation approach. Combining urine and catheter microbiome data improved the model performance during cross-validation, yielding a mean area under the receiver operating characteristic curve (AUROC) of 0.91-0.98 (Interquartile range, IQR 0.93-0.96) and 0.78-0.91 (IQR 0.86-0.88) for ASVs and taxonomic features, respectively. ASVs and taxa features achieve a mean AUROC of 0.85-1 (IQR 0.93-0.98) and 0.69-0.99 (IQR 0.78-0.88) in the independent held-out test set, respectively, signifying their potential in differentiating symptomatic and asymptomatic bacteriuria states. ConclusionsOur findings demonstrate that signatures within catheter and urine microbiota could serve as tools to monitor the health status of SCI patients. Establishing an early warning system based on these microbial signatures could equip physicians with alternative management strategies, potentially reducing UTI episodes and associated hospital costs, thus significantly improving patient quality of life while mitigating the impact of drug-resistant UTI.

microbiology↗

bioRxiv2024

MinD proteins regulate CetZ1 localisation in Haloferax volcanii

CetZ proteins are archaea-specific homologues of the cytoskeletal proteins FtsZ and tubulin. In the pleomorphic archaeon Haloferax volcanii, CetZ1 contributes to the development of rod shape and motility, and has been implicated in the proper assembly and positioning of the archaellum and chemotaxis motility proteins. CetZ1 shows complex subcellular localization, including irregular midcell structures and filaments along the long axis of developing rods and patches at the cell poles of the motile rod cell type. The polar localizations of archaellum and chemotaxis proteins are also influenced by MinD4, the only previously characterized archaeal member of the MinD family of ATPases, which are better known for their roles in the positioning of the division ring in bacteria. Using minD mutant strains and CetZ1 subcellular localization studies, we show here that a second minD homolog, minD2, has a strong influence on motility and the localization of CetZ1. Knockout of the minD2 gene altered the distribution of a fluorescent CetZ1-mTq2 fusion protein in a broad midcell zone and along the edges of rod cells, and inhibited the localization of CetZ1-mTq2 at the cell poles. MinD4 had a similar but weaker influence on motility and CetZ1-mTq2 localization. The MinD2/4 mutant strains formed rod cell shapes like the wildtype at an early log stage of growth. Our results are consistent with distinct roles for CetZ1 in rod shape formation and at the poles of mature rods, that are positioned through the action of the MinD proteins and contribute to the development of swimming motility in multiple ways. They represent the first report of MinD proteins controlling the positioning of tubulin superfamily proteins in archaea.

microbiology↗

bioRxiv2024

Cell division protein A (CdpA) assembles and anchors the two FtsZ rings at midcell in haloarchaea

Many archaea appear to divide through the coordinated activities of two FtsZ homologues (FtsZ1 and FtsZ2) and another bacterial cell division homologue (SepF), which are part of the midcell division ring. Here, we identify an additional protein (HVO_0739, renamed CdpA) that is involved in cell division in Haloferax volcanii, with homologues in other Haloarchaea. CdpA localizes at the mid-cell division ring, and this requires the presence of the ring-assembly protein FtsZ1. The division constriction protein FtsZ2 also influenced the proper midcell assembly and structure of CdpA. In the absence of CdpA, cells frequently failed to divide properly, and FtsZ1 formed poorly condensed pseudo-helical structures spanning across a broad region of the cell, whereas FtsZ2 showed mispositioned foci, nano-rings, and filaments. The rate of directional movement of FtsZ1 and FtsZ2 structures around the division ring appeared minimally affected by loss of CdpA, which resulted in continual repositioning of the aberrant FtsZ structures in the cells. In contrast to the FtsZ proteins, CdpA formed relatively immobile foci around the ring. Protein domain function studies, pull-down assays, and multimer structure predictions suggest that CdpA is part of a membrane complex that tethers FtsZ2 and other division proteins to the midcell membrane. Our discovery of an archaeal FtsZ organisation and midcell anchor protein offers new insights into cell division mechanisms that are similar across the tree of life.

microbiology↗

bioRxiv2024

CetZ1-dependent assembly and positioning of the motility machinery in haloarchaea

The archaeal tubulin-like cytoskeletal protein CetZ1 is required for rod-cell morphogenesis during the development of motility in Haloferax volcanii. This is expected to improve swimming speed and directionality. Here, we found that deletion of cetZ1 or expression of a GTPase-defective mutant caused a substantial defect in the assembly of the motility machinery, including the archaellum base marker protein ArlD1, the chemotaxis sensory array adapter CheW1, and signal transducer CheY. Furthermore, overexpression of cetZ1 reduced the assembly and polar placement of the motility machinery without detectably affecting the rod shape of motile cells. In contrast, deletion of the conserved paralog cetZ2 caused no defects in swimming or rod shape, although expression of the cetZ2 GTPase-defective mutant reduced motility whereas cetZ2 overexpression caused mild hyper-motility; these effects were dependent on the presence of cetZ1. A functional CetZ1-mTq2 fusion strongly localized at the poles of mature motile cells, where it partially co-localized with the motility machinery markers. These results suggest that CetZ1 has another role in the organisation or structure of the cell poles that promotes the assembly of the motility machinery. The multiple roles and locations of CetZ1 during motile cell development are reminiscent of the multiple functions of eukaryotic cytoskeletal proteins.

microbiology↗

bioRxiv2024

Dynamic self-association of archaeal tubulin-like protein CetZ1 drives Haloferax volcanii morphogenesis

Tubulin superfamily (TSF) proteins include the well-known eukaryotic tubulin and bacterial FtsZ families, and lesser-known archaeal CetZ family. In eukaryotes and bacteria, GTP-dependent polymerization and self-association of tubulin and FtsZ protofilaments are integral to the formation of cytoskeletal structures with essential roles in cell division, growth, and morphology. Archaeal CetZs are implicated in the control of cell shape and motility through unknown mechanisms. Here, we reveal a sequence of subcellular localization patterns of CetZ1, the prototypical member of the CetZ family, during stages of Haloferax volcanii rod cell development, in which it plays an essential role. Like tubulin and FtsZ, we found that CetZ1 formed GTP-dependent polymers in vitro, which appear to associate laterally as irregular polymer bundles. Mutations targeting regions predicted to mediate self-association and dynamic turnover of CetZ1, including the longitudinal (GTPase T7 and T4 loops) and lateral assembly interfaces, perturbed or altered rod shape development and subcellular assembly and dynamics, and caused corresponding effects on polymerization in vitro. Remarkably, a conspicuous amphipathic protrusion in the large microtubule (M-) loop, a characteristic of the CetZ1 subfamily, also strongly influenced function and assembly. Our findings reveal the importance of dynamic CetZ1 self-association in cellular morphogenesis involving multiple regions of the TSF fold, including tubulin- and FtsZ-like structural characteristics and CetZ1-specific features. Furthermore, they support a mechanism involving CetZ1 dynamic guidance of cell envelope-associated structures that reshape the cell during morphogenesis.

molecular biology↗

bioRxiv2024

E. coli division machinery drives cocci development inside host cells

Escherichia coli is arguably one of the most studied bacterial model systems in modern biology. Under normal laboratory conditions E. coli adopts its characteristic rod-shape. However, during stress conditions E. coli has been shown to undergo conditional morphology changes to inhibit division and grow into highly elongated forms. Here, on the other end of the morphology spectra, using an in-vitro infection model system combined with advanced imaging we show uropathogenic E. coli rods dividing to form and proliferate as cocci inside human bladder epithelial cells. In these intracellular bacterial communities, the frequency of cell division outpaced the rate of cell growth, resulting in smaller cocci cells. This mechanism was guided by an active FtsZ-governed division machinery, directed to midcell by division-site placement systems. These results show how a previously uncharacterised level of morphological plasticity occurs in bacteria with traditionally well-defined rod shape.

microbiology↗

bioRxiv2024

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

microbiology↗

bioRxiv2023

Genetic requirements for uropathogenic E. coli proliferation in the bladder cell infection cycle

Uropathogenic Escherichia coli (UPEC) requires an adaptable physiology to survive the wide range of environments experienced in the host, including gut and urinary tract surfaces. To identify UPEC genes required during intracellular infection, we developed a transposon-directed insertion-site sequencing (TraDIS) approach for cellular infection models and searched for genes in a library of [~]20,000 E. coli UTI89 transposon-insertion mutants that are specifically required for growth in M9-glycerol minimal medium, and at the distinct stages of infection of cultured bladder epithelial cells. Some of the functional requirements apparent for growth in M9-glycerol overlapped with those for the intracellular stage of infection, notably nutrient utilization, polysaccharide and macromolecule precursor biosynthesis, and cell envelope stress tolerance. Two genes implicated in both conditions were confirmed through independent gene deletion studies: neuC (sialic acid capsule biosynthesis) and hisF (histidine biosynthesis). Distinct sets of UPEC genes were also implicated in bacterial dispersal, where UPEC erupt from bladder cells in highly filamentous or motile forms upon exposure to human urine, and during recovery from infection in rich (LB) medium. Genes linked to septal peptidoglycan processes, ytfB and dedD, appeared to play roles in dispersal and may help stabilize cell division or the envelope during envelope stress created during infection. Our findings support a view that the host intracellular environment and infection cycle are multi-nutrient limited and create stress that demand an array of biosynthetic, cell envelope integrity and biofilm-related functions of UPEC. IMPORTANCEUrinary tract infections (UTIs) are one of the most frequent infections worldwide. Uropathogenic Escherichia coli (UPEC), which accounts for [~]80 % of UTIs, must rapidly adapt to highly variable host environments, such as the gut, bladder sub-surface and urine. In this study, we searched for UPEC genes required for bacterial growth and survival throughout the cellular infection cycle. Genes required for de novo synthesis of biomolecules and cell envelope integrity appeared to be important, and other genes were also implicated in bacterial dispersal and recovery from infection of cultured bladder cells. With further studies of individual gene function, their potential as therapeutic targets may be realized. This study expands knowledge of the UTI infection cycle and establishes an approach to genome-wide functional analyses of stage-resolved microbial infections.

microbiology↗

bioRxiv2023

Extracellular vesicles of Euryarchaeida: precursor to eukaryotic membrane trafficking

Since their discovery, extracellular vesicles (EVs) have changed our view on how organisms interact with their extracellular world. EVs are able to traffic a diverse array of molecules across different species and even domains, facilitating numerous functions. In this study, we investigate EV production in Haloferax volcanii, as representative for Euryarchaeida. We uncover that EVs enclose RNA, with specific transcripts preferentially enriched, including those with regulatory potential, and conclude that EVs can act as an RNA communication system between haloarchaea. We demonstrate the key role of an EV-associated Ras superfamily GTPase for EV formation in H. volcanii that is also present across other diverse evolutionary branches of Archaea. Ras superfamily GTPases are key players in eukaryotic intracellular vesicle formation and trafficking mechanisms that have been crucial for the emergence of Eukaryotes. Therefore, we propose that archaeal EV formation could reveal insights into the origin of the compartmentalized eukaryotic cell.

microbiology↗

bioRxiv2023

The intracellular lifestyle of an archaeal symbiont

DPANN Archaea are a diverse group of organisms typically characterised by small cells and reduced genomes. To date, all cultivated DPANN Archaea are ectosymbionts that require direct cell contact with an archaeal host species for proliferation. However, the dynamics of DPANN - host interactions and the impacts of these interactions on the host species are poorly understood. Here, we show that one DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. Our data also suggest that these interactions involve invasion of the host cell by the nanohaloarchaeon. This is the first reported instance of such a predatory-like lifestyle amongst Archaea and indicates that some DPANN Archaea may interact with host populations in a manner similar to viruses.

microbiology↗

bioRxiv2023

Direct visualization of bacterial multispecies infection and symbiotic interactions within human bladder epithelial cells

Urinary tract infections (UTI) are one of the most common bacterial infections worldwide. While the overall infection course is known on a macroscale, bacterial behavior is not fully understood at the cellular level and bacterial pathophysiology during multispecies infection is not well characterized. Here we establish co-infection models combined with high resolution imaging to compare single- and multi-species bladder cell invasion events in three common uropathogens: uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae and Enterococcus faecalis. While all three species invaded the bladder cells, under flow conditions the Gram-positive E. faecalis was significantly less invasive compared to the Gram-negative UPEC and K. pneumoniae. When introduced simultaneously during an infection experiment, all three bacterial species sometimes invaded the same bladder cell, at differing frequencies suggesting complex interactions between bacterial species and bladder cells. Inside host cells, we observed encasement of E. faecalis colonies specifically by UPEC. During subsequent dispersal from the host cells, only the Gram-negative bacteria underwent infection-related filamentation (IRF). Taken together, our data suggest that bacterial multispecies invasions of single bladder cells are frequent and support earlier studies showing intraspecies cooperation on a biochemical level during UTI.

microbiology↗

bioRxiv2022

The role of bacterial size, shape and surface in macrophage engulfment of uropathogenic E. coli cells

Uropathogenic Escherichia coli (UPEC) can undergo extensive filamentation in the host during acute urinary tract infections (UTIs). It has been hypothesised that this morphological plasticity allows bacteria to avoid host immune responses such as macrophage engulfment. However, it is still unclear what properties of filaments are important in macrophage-bacteria interactions. The aim of this work was to investigate the contribution of bacterial biophysical parameters, such as cell size and shape, and physiological parameters, such as cell surface and the environment, to macrophage engulfment efficiency. Viable, reversible filaments of known lengths and volumes were produced in the UPEC strain UTI89 using a variety of methods, including exposure to cell-wall targeting antibiotics, genetic manipulation and isolation from an in vitro human bladder cell model. Quantification of the engulfment ability of macrophages using gentamicin-protection assays and fluorescence microscopy demonstrated that the ability of filaments to avoid macrophage engulfment is dependent on a combination of size (length and volume), shape, surface and external environmental factors. UTI89 filamentation was also found to occur independently of the SOS-inducible filamentation genes, sulA and ymfM, demonstrating the non-essential requirement of these genes for UTI89 filamentation and their ability to avoid macrophage engulfment. With several strains of UPEC now resistant to current antibiotics, our work identifies the importance of bacterial morphology during infection and may provide new ways to prevent or treat these infections via immune modulation or antimicrobials. Author SummaryUrinary tract infections (UTIs) are one of the most common bacterial infections worldwide with 50% of women suffering from a UTI during their lifetime. Escherichia coli is the primary bacteria responsible for UTIs and is usually found in short rod forms. However, during UTIs E. coli can elongate into extremely long thin shapes called filaments. Filaments are thought to be advantageous during infections because they are too long to be engulfed and killed by immune cells called macrophages. Due to increasing antibiotic resistance in bacteria there is a strong need for the discovery of new ways to treat infections and this is only possible once we thoroughly understand the mechanisms bacteria employ to overcome our immune response. Therefore, we investigated the effect of E. coli filamentation on macrophage engulfment along with other aspects of bacteria reported to influence engulfment. We found that the ability of filaments to avoid macrophage engulfment is dependent on a combination of size (length and volume), shape, surface and external environmental factors. Our research has highlighted the importance of bacterial shape changes during infections and provided a foundational understanding of macrophage engulfment of filaments. Eventually, this knowledge may reveal new targets for treatment of infections.

microbiology↗

bioRxiv2022

PinR as a Stress Inducible Inhibitory Transcription Factor in Stopping Stress-induced Mutagenesis Bacterial Evolution of Antibiotic Resistance

A recent work reports that the single treatment of {beta}-lactams can cause a SOS-independent superfast evolution of multi-drug resistance in the DNA repair deficiency Escherichia coli (E. coli), but the mechanism is not yet clear. Here, we find that the induction of PinR, a lambdoid prophage Rac, is involved in this process and facilitates the evolution of antibiotic resistance in DNA repair deficiency bacteria through the repression on the transcription of antioxidative genes and the thereafter ROS burst in cells. It is highlighted that the bacteriophage PinR can orchestrate the mutagenesis induced by the overaccumulation of ROS in cells. More importantly, we for the first time demonstrate that the deletion of pinR can avoid the rapid evolution of antibiotic resistance induced by either the single or long-term exposure to antibiotics, while strategies to target RecA, e.g., the inactivation on RecA, can be safely implemented to disarm the bacterial resistance to other antibiotics. Therefore, from a drug development perspective, our work suggests future studies on the "evolutionary potentiators" towards a safe and more effective strategy to be developed for infectious disease treatment.

microbiology↗

bioRxiv2021

Dynamic localisation of DamX regulates bacterial filamentation and division during UPEC dispersal from host cells

Uropathogenic Escherichia coli (UPEC) cells can grow into highly filamentous forms during infection of bladder epithelial cells, but this process is poorly understood. Herein we found that some UPEC filaments released from infected bladder cells in vitro grew very rapidly and by more than 100 {micro}m before initiating division, whereas others did not survive, suggesting that filamentation is a stress response that promotes dispersal. The DamX bifunctional division protein, which is essential for UPEC filamentation, was initially non-localized but then assembled at multiple division sites in the filaments prior to division. DamX rings maintained consistent thickness during constriction and remained at the septum until after membrane fusion was completed, like in rod cell division. Our findings suggest a mechanism involving regulated dissipation of DamX, leading to division arrest and filamentation, followed by its reassembly into division rings to promote UPEC dispersal and survival during infection.

microbiology↗

bioRxiv2021

CdrS is a global transcriptional regulator influencing cell division in Haloferax volcanii

Transcriptional regulators that integrate cellular and environmental signals to control cell division are well known in bacteria and eukaryotes, but their existence is poorly understood in archaea. We identified a conserved gene (cdrS) that encodes a small protein and is highly transcribed in the model archaeon Haloferax volcanii. The cdrS gene could not be deleted, but CRISPRi-mediated repression of the cdrS gene caused slow growth, cell division defects, and changed the expression of multiple genes and their products associated with cell division, protein degradation and metabolism. Consistent with this complex regulatory network, overexpression of cdrS inhibited cell division, whereas overexpression of the operon encoding both CdrS and a tubulin-like cell division protein (FtsZ2) stimulated division. ChIP-Seq identified 18 DNA-binding sites of the CdrS protein including one upstream of the promoter for diadenylate cyclase, which is an essential gene involved in c-di-AMP signalling implicated in the regulation of cell division. These findings suggest that CdrS is a transcription factor that plays a central role in a regulatory network coordinating metabolism and cell division. ImportanceCell division is a central mechanism of life, and is essential for growth and development. Bacteria and Eukarya have different mechanisms for cell division, which have been studied in detail. In contrast, cell division in Archaea is still understudied, and its regulation is poorly understood. Interestingly, different cell division machineries appear in the Archaea, with the Euryarchaeota using a cell division apparatus based on the tubulin-like cytoskeletal protein FtsZ, as in bacteria. Here we identify the small protein CdrS as essential for survival and a central regulator of cell division in the Euryarchaeon Haloferax volcanii. CdrS also appears to coordinate other cellular pathways including synthesis of signalling molecules and protein degradation. Our results show that CdrS plays a sophisticated role in cell division, including regulation of numerous associated genes. These findings are expected to initiate investigations into conditional regulation of division in archaea.

microbiology↗