RESUMO
Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , DNA , Edição de Genes , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA/metabolismo , DNA/genética , Edição de Genes/métodos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Células HEK293 , Domínios Proteicos , Genoma Humano , Modelos Moleculares , Estrutura Terciária de Proteína , Conformação de Ácido Nucleico , Biocatálise , Magnésio/química , Magnésio/metabolismoRESUMO
Biofilm formation is generally recognized as a bacterial defense mechanism against environmental threats, including antibiotics, bacteriophages, and leukocytes of the human immune system. Here, we show that for the human pathogen Vibrio cholerae, biofilm formation is not only a protective trait but also an aggressive trait to collectively predate different immune cells. We find that V. cholerae forms biofilms on the eukaryotic cell surface using an extracellular matrix comprising primarily mannose-sensitive hemagglutinin pili, toxin-coregulated pili, and the secreted colonization factor TcpF, which differs from the matrix composition of biofilms on other surfaces. These biofilms encase immune cells and establish a high local concentration of a secreted hemolysin to kill the immune cells before the biofilms disperse in a c-di-GMP-dependent manner. Together, these results uncover how bacteria employ biofilm formation as a multicellular strategy to invert the typical relationship between human immune cells as the hunters and bacteria as the hunted.
Assuntos
Vibrio cholerae , Animais , Humanos , Vibrio cholerae/metabolismo , Comportamento Predatório , Biofilmes , Fímbrias Bacterianas , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.
Assuntos
Antituberculosos , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Choque Térmico/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , ProteostaseRESUMO
Subcellular compartmentalization is a defining feature of all cells. In prokaryotes, compartmentalization is generally achieved via protein-based strategies. The two main classes of microbial protein compartments are bacterial microcompartments and encapsulin nanocompartments. Encapsulins self-assemble into proteinaceous shells with diameters between 24 and 42 nm and are defined by the viral HK97-fold of their shell protein. Encapsulins have the ability to encapsulate dedicated cargo proteins, including ferritin-like proteins, peroxidases, and desulfurases. Encapsulation is mediated by targeting sequences present in all cargo proteins. Encapsulins are found in many bacterial and archaeal phyla and have been suggested to play roles in iron storage, stress resistance, sulfur metabolism, and natural product biosynthesis. Phylogenetic analyses indicate that they share a common ancestor with viral capsid proteins. Many pathogens encode encapsulins, and recent evidence suggests that they may contribute toward pathogenicity. The existing information on encapsulin structure, biochemistry, biological function, and biomedical relevance is reviewed here.
Assuntos
Bactérias , Proteínas de Bactérias , Archaea/genética , Archaea/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , FilogeniaRESUMO
Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.
Assuntos
Proteínas de Bactérias , Chaperonas Moleculares , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , ProteóliseRESUMO
CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads.
Assuntos
Proteínas Associadas a CRISPR , Elementos de DNA Transponíveis , Elementos de DNA Transponíveis/genética , Sistemas CRISPR-Cas , Transposases/genética , Proteínas de Ligação a DNA/metabolismo , RNA , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genéticaRESUMO
Bacterial colonies composed of genetically identical individuals can diversify to yield variant cells with distinct genotypes. Variant outgrowth manifests as sectors. Here, we show that Type VI secretion system (T6SS)-driven cell death in Vibrio cholerae colonies imposes a selective pressure for the emergence of variant strains that can evade T6SS-mediated killing. T6SS-mediated cell death occurs in two distinct spatiotemporal phases, and each phase is driven by a particular T6SS toxin. The first phase is regulated by quorum sensing and drives sectoring. The second phase does not require the T6SS-injection machinery. Variant V. cholerae strains isolated from colony sectors encode mutated quorum-sensing components that confer growth advantages by suppressing T6SS-killing activity while simultaneously boosting T6SS-killing defenses. Our findings show that the T6SS can eliminate sibling cells, suggesting a role in intra-specific antagonism. We propose that quorum-sensing-controlled T6SS-driven killing promotes V. cholerae genetic diversity, including in natural habitats and during disease.
Assuntos
Sistemas de Secreção Tipo VI , Vibrio cholerae , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Variação Genética , Percepção de Quorum , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Vibrio cholerae/metabolismoRESUMO
Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.
Assuntos
Bioquímica/métodos , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Luz , Optogenética/métodos , Processos Fotoquímicos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Fitocromo/química , Fitocromo/metabolismo , Domínios Proteicos , Engenharia de Proteínas/métodos , Vitamina B 12/metabolismoRESUMO
On the occasion of the 2021 Lasker Basic Medical Research Award to Karl Deisseroth, Peter Hegemann, and Dieter Oesterhelt (for "the discovery of light-sensitive microbial proteins that can activate or deactivate individual brain cells-leading to the development of optogenetics and revolutionizing neuroscience"), Deisseroth reflects on this international collaboration, his basic mechanistic and structural discoveries regarding microbial channels that transduce photons into ion current, the causal exploration of brain cell function, and the pressing mysteries of psychiatry.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Emoções , Proteínas de Membrana/metabolismo , Bacteriorodopsinas/metabolismo , Channelrhodopsins/metabolismo , Humanos , Optogenética , Membrana Purpúrea/metabolismoRESUMO
Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.
Assuntos
Bactérias/genética , Biofilmes , DNA Bacteriano/química , Matriz Extracelular/metabolismo , Espaço Extracelular/química , Animais , Especificidade de Anticorpos , Proteínas de Bactérias/metabolismo , Linhagem Celular , Chinchila , DNA Cruciforme , Desoxirribonucleases/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Gasdermin B (GSDMB) belongs to a large family of pore-forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of natural killer cells by suppressing granzyme-A-mediated activation of GSDMB. In contrast to the canonical function of most gasdermin family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveal a mechanism employed by pathogens to counteract this host defense system.
Assuntos
Biomarcadores Tumorais/metabolismo , Interações Hospedeiro-Patógeno , Células Matadoras Naturais/imunologia , Proteínas de Neoplasias/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Shigella flexneri/fisiologia , Ubiquitinação , Animais , Proteínas de Bactérias/metabolismo , Cardiolipinas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Feminino , Granzimas/metabolismo , Humanos , Lipídeo A/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Viabilidade Microbiana , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Especificidade por SubstratoRESUMO
By following up the gut microbiome, 51 human phenotypes and plasma levels of 1,183 metabolites in 338 individuals after 4 years, we characterize microbial stability and variation in relation to host physiology. Using these individual-specific and temporally stable microbial profiles, including bacterial SNPs and structural variations, we develop a microbial fingerprinting method that shows up to 85% accuracy in classifying metagenomic samples taken 4 years apart. Application of our fingerprinting method to the independent HMP cohort results in 95% accuracy for samples taken 1 year apart. We further observe temporal changes in the abundance of multiple bacterial species, metabolic pathways, and structural variation, as well as strain replacement. We report 190 longitudinal microbial associations with host phenotypes and 519 associations with plasma metabolites. These associations are enriched for cardiometabolic traits, vitamin B, and uremic toxins. Finally, mediation analysis suggests that the gut microbiome may influence cardiometabolic health through its metabolites.
Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Microbioma Gastrointestinal , Metaboloma , Metagenoma , Microbiota , Adulto , Idoso , Idoso de 80 Anos ou mais , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/metabolismo , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos , Fezes/microbiologia , Feminino , Instabilidade Genômica , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Adulto JovemRESUMO
Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Família Multigênica , Nostoc/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Galinhas , Microscopia Crioeletrônica , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Evolução Molecular , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestrutura , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
Tn7-like transposons have co-opted CRISPR systems, including class 1 type I-F, I-B, and class 2 type V-K. Intriguingly, although these CRISPR-associated transposases (CASTs) undergo robust CRISPR RNA (crRNA)-guided transposition, they are almost never found in sites targeted by the crRNAs encoded by the cognate CRISPR array. To understand this paradox, we investigated CAST V-K and I-B systems and found two distinct modes of transposition: (1) crRNA-guided transposition and (2) CRISPR array-independent homing. We show distinct CAST systems utilize different molecular mechanisms to target their homing site. Type V-K CAST systems use a short, delocalized crRNA for RNA-guided homing, whereas type I-B CAST systems, which contain two distinct target selector proteins, use TniQ for RNA-guided DNA transposition and TnsD for homing to an attachment site. These observations illuminate a key step in the life cycle of CAST systems and highlight the diversity of molecular mechanisms mediating transposon homing.
Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Elementos de DNA Transponíveis/fisiologia , DNA Bacteriano/metabolismo , RNA Guia de Cinetoplastídeos , Transposases/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/genética , Edição de Genes , Recombinação Genética , Transposases/genéticaRESUMO
Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chlamydomonas/metabolismo , Multimerização Proteica , Synechocystis/metabolismo , Tilacoides/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Membrana Celular/metabolismo , Chlamydomonas/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Fluorescência Verde/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Luz , Lipídeos/química , Modelos Moleculares , Nucleotídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estresse Fisiológico/efeitos da radiação , Synechocystis/ultraestrutura , Tilacoides/ultraestruturaRESUMO
PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestrutura , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Lipossomas Unilamelares/metabolismoRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.
Assuntos
Sistemas CRISPR-Cas/efeitos dos fármacos , Edição de Genes/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais/genética , Vírus/genética , Archaea/genética , Archaea/imunologia , Archaea/virologia , Bactérias/genética , Bactérias/imunologia , Bactérias/virologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coevolução Biológica , Proteínas Associadas a CRISPR/antagonistas & inibidores , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , DNA/antagonistas & inibidores , DNA/química , DNA/genética , DNA/metabolismo , Clivagem do DNA/efeitos dos fármacos , Endodesoxirribonucleases/antagonistas & inibidores , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Família Multigênica , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia , Vírus/metabolismo , Vírus/patogenicidadeRESUMO
Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/biossíntese , Antígenos O/biossíntese , Poliprenois/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Carbono-Oxigênio Ligases/química , Carbono-Oxigênio Ligases/genética , Carbono-Oxigênio Ligases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Ácidos Teicoicos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport, and how these changes power rotation of the flagellum remain unknown. Here, we present ~3 Å-resolution cryoelectron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a detailed mechanistic model for motor function and switching of rotational direction.
Assuntos
Proteínas de Bactérias/ultraestrutura , Flagelos/ultraestrutura , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica/métodos , Flagelos/metabolismo , Conformação Proteica , TorqueRESUMO
Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.