RESUMO
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 Euryarchaeota, using the model organism Haloferax volcanii. 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 small GTPase for EV formation in H. volcanii that is also present across other diverse evolutionary branches of Archaea. We propose the name, ArvA, for the identified family of archaeal vesiculating GTPases. Additionally, we show that two genes in the same operon with arvA (arvB and arvC) are also involved in EV formation. Both, arvB and arvC, are closely associated with arvA in the majority of other archaea encoding ArvA. Our work demonstrates that small GTPases involved in membrane deformation and vesiculation, ubiquitous in Eukaryotes, are also present in Archaea and are widely distributed across diverse archaeal phyla.
Assuntos
Euryarchaeota , Vesículas Extracelulares , Haloferax volcanii , Proteínas Monoméricas de Ligação ao GTP , Euryarchaeota/genética , Archaea/genética , RNA , Haloferax volcanii/genética , Vesículas Extracelulares/genéticaRESUMO
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.
Assuntos
Proteínas Arqueais , Clonagem Molecular , Vetores Genéticos , Haloferax volcanii , Proteínas Luminescentes , Plasmídeos , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismoRESUMO
Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.
Assuntos
Quimiocina CXCL12/metabolismo , MicroRNAs/genética , Infiltração de Neutrófilos/imunologia , Receptores CXCR4/metabolismo , Animais , Quimiocina CXCL12/imunologia , Técnicas de Silenciamento de Genes/métodos , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/metabolismo , Receptores CXCR4/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Peixe-Zebra/imunologiaRESUMO
Rod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple alternative division inhibition pathways exist during the SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism.Importance:Filamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.
RESUMO
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-phase rods, as well as the pleomorphic rods and tubules were dependent on the function of the tubulin-like cytoskeletal protein, CetZ1. Our results reveal 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.
Assuntos
Haloferax volcanii/citologia , Haloferax volcanii/crescimento & desenvolvimento , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Meios de Cultura/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Haloferax volcanii/metabolismo , Nutrientes/análise , Oligoelementos/análiseRESUMO
Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.
Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Forma Celular , Haloferax volcanii/citologia , Haloferax volcanii/metabolismo , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Divisão Celular , Membrana Celular/metabolismo , Cristalografia por Raios X , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Movimento , Tubulina (Proteína)/químicaRESUMO
Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections. These bacteria undertake a multistage infection cycle involving invasion of and proliferation within urinary tract epithelial cells, leading to the rupture of the host cell and dispersal of the bacteria, some of which have a highly filamentous morphology. Here, we established a microfluidics-based model of UPEC infection of immortalized human bladder epithelial cells that recapitulates the main stages of bacterial morphological changes during the acute infection cycle in vivo and allows the development and fate of individual cells to be monitored in real time by fluorescence microscopy. The UPEC-infected bladder cells remained alive and mobile in nonconfluent monolayers during the development of intracellular bacterial communities (IBCs). Switching from a flow of growth medium to human urine resulted in immobilization of both uninfected and infected bladder cells. Some IBCs continued to develop and then released many highly filamentous bacteria via an extrusion-like process, whereas other IBCs showed strong UPEC proliferation, and yet no filamentation was detected. The filamentation response was dependent on the weak acidity of human urine and required component(s) in a low molecular-mass (<3,000 Da) fraction from a mildly dehydrated donor. The developmental fate for bacteria therefore appears to be controlled by multiple factors that act at the level of the whole IBC, suggesting that variable local environments or stochastic differentiation pathways influence IBC developmental fates during infection.
Assuntos
Células Epiteliais/microbiologia , Técnicas Analíticas Microfluídicas , Escherichia coli Uropatogênica/patogenicidade , Escherichia coli Uropatogênica/ultraestrutura , Linhagem Celular Transformada , Movimento Celular , Proliferação de Células , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Modelos Biológicos , Reologia , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , Infecções Urinárias/microbiologia , Infecções Urinárias/patologia , Escherichia coli Uropatogênica/fisiologia , Urotélio/microbiologia , Urotélio/patologiaRESUMO
One mechanism for achieving accurate placement of the cell division machinery is via Turing patterns, where nonlinear molecular interactions spontaneously produce spatiotemporal concentration gradients. The resulting patterns are dictated by cell shape. For example, the Min system of Escherichia coli shows spatiotemporal oscillation between cell poles, leaving a mid-cell zone for division. The universality of pattern-forming mechanisms in divisome placement is currently unclear. We examined the location of the division plane in two pleomorphic archaea, Haloferax volcanii and Haloarcula japonica, and showed that it correlates with the predictions of Turing patterning. Time-lapse analysis of H. volcanii shows that divisome locations after successive rounds of division are dynamically determined by daughter cell shape. For H. volcanii, we show that the location of DNA does not influence division plane location, ruling out nucleoid occlusion. Triangular cells provide a stringent test for Turing patterning, where there is a bifurcation in division plane orientation. For the two archaea examined, most triangular cells divide as predicted by a Turing mechanism; however, in some cases multiple division planes are observed resulting in cells dividing into three viable progeny. Our results suggest that the division site placement is consistent with a Turing patterning system in these archaea.
Assuntos
Divisão Celular , Haloferax volcanii/citologia , Haloferax volcanii/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Forma Celular , Haloferax/citologia , Haloferax/genética , Haloferax/metabolismo , Haloferax volcanii/genéticaRESUMO
Proper protein anchoring is key to the biogenesis of prokaryotic cell surfaces, dynamic, resilient structures that play crucial roles in various cell processes. A novel surface protein anchoring mechanism in Haloferax volcanii depends upon the peptidase archaeosortase A (ArtA) processing C-termini of substrates containing C-terminal tripartite structures and anchoring mature substrates to the cell membrane via intercalation of lipid-modified C-terminal amino acid residues. While this membrane protein lacks clear homology to soluble sortase transpeptidases of Gram-positive bacteria, which also process C-termini of substrates whose C-terminal tripartite structures resemble those of ArtA substrates, archaeosortases do contain conserved cysteine, arginine and arginine/histidine/asparagine residues, reminiscent of His-Cys-Arg residues of sortase catalytic sites. The study presented here shows that ArtAWT -GFP expressed in trans complements ΔartA growth and motility phenotypes, while alanine substitution mutants, Cys173 (C173A), Arg214 (R214A) or Arg253 (R253A), and the serine substitution mutant for Cys173 (C173S), fail to complement these phenotypes. Consistent with sortase active site replacement mutants, ArtAC173A -GFP, ArtAC173S -GFP and ArtAR214A -GFP cannot process substrates, while replacement of the third residue, ArtAR253A -GFP retains some processing activity. These findings support the view that similarities between certain aspects of the structures and functions of the sortases and archaeosortases are the result of convergent evolution.
Assuntos
Aminoaciltransferases/metabolismo , Cisteína Endopeptidases/metabolismo , Haloferax volcanii/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Archaea/genética , Proteínas Arqueais/metabolismo , Arginina/metabolismo , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Evolução Biológica , Catálise , Domínio Catalítico , Sequência Conservada/genética , Cisteína/metabolismo , Cisteína Endopeptidases/genética , Evolução Molecular , Histidina/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Bacterial filamentation occurs when rod-shaped bacteria grow without dividing. To identify genetically encoded inhibitors of division that promote filamentation, we used cell sorting flow cytometry to enrich filamentous clones from an inducible expression library, and then identified the cloned DNA with high-throughput DNA sequencing. We applied the method to an expression library made from fragmented genomic DNA of uropathogenic E. coli UTI89, which undergoes extensive reversible filamentation in urinary tract infections and might encode additional regulators of division. RESULTS: We identified 55 genomic regions that reproducibly caused filamentation when expressed from the plasmid vector, and then further localized the cause of filamentation in several of these to specific genes or sub-fragments. Many of the identified genomic fragments encode genes that are known to participate in cell division or its regulation, and others may play previously-unknown roles. Some of the prophage genes identified were previously implicated in cell division arrest. A number of the other fragments encoded potential short transcripts or peptides. CONCLUSIONS: The results provided evidence of potential new links between cell division and distinct cellular processes including central carbon metabolism and gene regulation. Candidate regulators of the UTI-associated filamentation response or others were identified amongst the results. In addition, some genomic fragments that caused filamentation may not have evolved to control cell division, but may have applications as artificial inhibitors. Our approach offers the opportunity to carry out in depth surveys of diverse DNA libraries to identify new genes or sequences encoding the capacity to inhibit division and cause filamentation.
Assuntos
Bactérias/genética , Divisão Celular/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Escherichia coli/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Fases de Leitura Aberta , Fenótipo , Escherichia coli Uropatogênica/genéticaRESUMO
In comparison with bacteria and eukaryotes, the large and diverse group of microorganisms known as archaea possess a great diversity of cytoskeletal proteins, including members of the tubulin superfamily. Many species contain FtsZ, CetZ and even possible tubulins; however, some major taxonomic groups do not contain any member of the tubulin superfamily. Studies using the model archaeon, Halferax volcanii have recently been instrumental in defining the fundamental roles of FtsZ and CetZ in archaeal cell division and cell shape regulation. Structural studies of archaeal tubulin superfamily proteins provide a definitive contribution to the cytoskeletal field, showing which protein-types must have developed prior to the divergence of archaea and eukaryotes. Several regions of the globular core domain - the "signature" motifs - combine in the 3D structure of the common molecular fold to form the GTP-binding site. They are the most conserved sequence elements and provide the primary basis for identification of new superfamily members through homology searches. The currently well-characterised proteins also all share a common mechanism of GTP-dependent polymerisation, in which GTP molecules are sandwiched between successive subunits that are arranged in a head-to-tail manner. However, some poorly-characterised archaeal protein families retain only some of the signature motifs and are unlikely to be capable of dynamic polymerisation, since the promotion of depolymerisation by hydrolysis to GDP depends on contributions from both subunits that sandwich the nucleotide in the polymer.
Assuntos
Archaea/metabolismo , Proteínas Arqueais/metabolismo , Tubulina (Proteína)/classificação , Tubulina (Proteína)/metabolismoRESUMO
Archaea of the genus Sulfolobus have a single-circular chromosome with three replication origins. All three origins fire in every cell in every cell cycle. Thus, three pairs of replication forks converge and terminate in each replication cycle. Here, we report 2D gel analyses of the replication fork fusion zones located between origins. These indicate that replication termination involves stochastic fork collision. In bacteria, replication termination is linked to chromosome dimer resolution, a process that requires the XerC and D recombinases, FtsK and the chromosomal dif site. Sulfolobus encodes a single-Xer homologue and its deletion gave rise to cells with aberrant DNA contents and increased volumes. Identification of the chromosomal dif site that binds Xer in vivo, and biochemical characterization of Xer/dif recombination revealed that, in contrast to bacteria, dif is located outside the fork fusion zones. Therefore, it appears that replication termination and dimer resolution are temporally and spatially distinct processes in Sulfolobus.
Assuntos
Cromossomos de Archaea , Replicação do DNA , Sulfolobus solfataricus/genética , DNA Arqueal/genética , Fenótipo , Recombinação GenéticaRESUMO
In prokaryotes the genome is organized in a dynamic structure called the nucleoid, which is embedded in the cytoplasm. We show here that in the archaeon Haloferax volcanii, compaction and reorganization of the nucleoid is induced by stresses that damage the genome or interfere with its replication. The fraction of cells exhibiting nucleoid compaction was proportional to the dose of the DNA damaging agent, and results obtained in cells defective for nucleotide excision repair suggest that breakage of DNA strands triggers reorganization of the nucleoid. We observed that compaction depends on the Mre11-Rad50 complex, suggesting a link to DNA double-strand break repair. However, compaction was observed in a radA mutant, indicating that the role of Mre11-Rad50 in nucleoid reorganisation is independent of homologous recombination. We therefore propose that nucleoid compaction is part of a DNA damage response that accelerates cell recovery by helping DNA repair proteins to locate their targets, and facilitating the search for intact DNA sequences during homologous recombination.
Assuntos
Proteínas Arqueais/metabolismo , DNA Arqueal/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Genoma Arqueal , Haloferax volcanii/fisiologia , Anisomicina/farmacologia , Quebras de DNA , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Replicação do DNARESUMO
Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.
Assuntos
Replicação do DNA , DNA Arqueal/biossíntese , Sulfolobus acidocaldarius/genética , Sulfolobus solfataricus/genética , Bromodesoxiuridina/análise , Ciclo Celular , DNA Arqueal/análise , DNA Arqueal/química , DNA Polimerase Dirigida por DNA/análise , Desoxiuridina/análogos & derivados , Desoxiuridina/análise , Complexos Multienzimáticos/análise , Sulfolobus acidocaldarius/metabolismo , Sulfolobus solfataricus/metabolismoRESUMO
Urinary tract infection (UTI), one of the most common bacterial infections worldwide, is a typical example of an infection that is often polymicrobial in nature. 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, using clinically relevant bacteria, human epithelial bladder cells and human urine, 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.
Assuntos
Enterococcus faecalis , Células Epiteliais , Klebsiella pneumoniae , Infecções Urinárias , Escherichia coli Uropatogênica , Humanos , Infecções Urinárias/microbiologia , Enterococcus faecalis/fisiologia , Células Epiteliais/microbiologia , Escherichia coli Uropatogênica/fisiologia , Klebsiella pneumoniae/fisiologia , Bexiga Urinária/microbiologia , Bexiga Urinária/citologia , Coinfecção/microbiologia , Linhagem Celular , Interações Hospedeiro-PatógenoRESUMO
DPANN archaea are a diverse group of microorganisms 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 growth and survival. However, these interactions and their impact on the host species are poorly understood. Here, we show that a DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. During these interactions, the nanohaloarchaeon appears to enter, or be engulfed by, the host cell. Our results provide experimental evidence for a predatory-like lifestyle of an archaeon, suggesting that at least some DPANN archaea may have roles in controlling host populations and their ecology.
Assuntos
Halorubrum , Simbiose , Halorubrum/genética , Halorubrum/fisiologia , Archaea/genética , Archaea/fisiologia , Nanoarchaeota/genética , Nanoarchaeota/fisiologia , Genoma Arqueal , FilogeniaRESUMO
Tubulin superfamily (TSF) proteins are widespread, and are known for their multifaceted roles as cytoskeletal proteins underpinning many basic cellular functions, including morphogenesis, division, and motility. In eukaryotes, tubulin assembles into microtubules, a major component of the dynamic cytoskeletal network of fibres, whereas the bacterial homolog FtsZ assembles the division ring at midcell. The functions of the lesser-known archaeal TSF proteins are beginning to be identified and show surprising diversity, including homologs of tubulin and FtsZ as well as a third archaea-specific family, CetZ, implicated in the regulation of cell shape and possibly other unknown functions. In this study, we define sequence and structural characteristics of the CetZ family and CetZ1 and CetZ2 subfamilies, identify CetZ groups and diversity amongst archaea, and identify potential functional relationships through analysis of the genomic neighbourhoods of cetZ genes. We identified at least three subfamilies of orthologous CetZ proteins in the archaeal class Halobacteria, including CetZ1 and CetZ2 as well as a novel uncharacterized subfamily. CetZ1 and CetZ2 were correlated to one another as well as to cell shape and motility phenotypes across diverse Halobacteria. Among other known CetZ clusters in orders Archaeoglobales, Methanomicrobiales, Methanosarcinales, and Thermococcales, an additional uncharacterized group from Archaeoglobales and Methanomicrobiales is affiliated strongly with Halobacteria CetZs, suggesting that they originated via horizontal transfer. Subgroups of Halobacteria CetZ2 and Thermococcales CetZ genes were found adjacent to different type IV pili regulons, suggesting potential utilization of CetZs by type IV systems. More broadly conserved cetZ gene neighbourhoods include nucleotide and cofactor biosynthesis (e.g., F420) and predicted cell surface sugar epimerase genes. These findings imply that CetZ subfamilies are involved in multiple functions linked to the cell surface, biosynthesis, and motility.
Assuntos
Proteínas Arqueais , Proteínas do Citoesqueleto , Proteínas do Citoesqueleto/metabolismo , Archaea/genética , Archaea/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas de Bactérias/metabolismo , Bactérias/metabolismo , Proteínas Arqueais/metabolismoRESUMO
Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, Antarctica. Hrr. lacusprofundi is commonly used to study adaptation to cold environments and thereby a potential source for biotechnological products. Additionally, in contrast to other haloarchaeal model organisms, Hrr. lacusprofundi is also susceptible to a range of different viruses and virus-like elements, making it a great model to study virus-host interactions in a cold-adapted organism. A genetic system has previously been reported for Hrr. lacusprofundi; however, it does not allow in-frame deletions and multiple gene knockouts. Here, we report the successful generation of uracil auxotrophic (pyrE2) mutants of two strains of Hrr. lacusprofundi. Subsequently, we attempted to generate knockout mutants using the auxotrophic marker for selection. However, surprisingly, only the combination of the auxotrophic marker and antibiotic selection allowed the timely and clean in-frame deletion of a target gene. Finally, we show that vectors established for the model organism Haloferax volcanii are deployable for genetic manipulation of Hrr. lacusprofundi, allowing the use of the portfolio of genetic tools available for H. volcanii in Hrr. lacusprofundi.
RESUMO
Haloferax volcanii and other Haloarchaea can be pleomorphic, adopting different shapes, which vary with growth stages. Several studies have shown that H. volcanii cell shape is sensitive to various external factors including growth media and physical environment. In addition, several studies have noticed that the presence of a recombinant plasmid in the cells is also a factor impacting H. volcanii cell shape, notably by favoring the development of rods in early stages of growth. Here we investigated the reasons for this phenomenon by first studying the impact of auxotrophic mutations on cell shape in strains that are commonly used as genetic backgrounds for selection during strain engineering (namely: H26, H53, H77, H98, and H729) and secondly, by studying the effect of the presence of different plasmids containing selection markers on the cell shape of these strains. Our study showed that most of these auxotrophic strains have variation in cell shape parameters including length, aspect ratio, area and circularity and that the plasmid presence is impacting these parameters too. Our results indicated that ΔhdrB strains and hdrB selection markers have the most influence on H. volcanii cell shape, in addition to the sole presence of a plasmid. Finally, we discuss limitations in studying cell shape in H. volcanii and make recommendations based on our results for improving reproducibility of such studies.