RESUMO
Contrary to multicellular organisms that display segmentation during development, communities of unicellular organisms are believed to be devoid of such sophisticated patterning. Unexpectedly, we find that the gene expression underlying the nitrogen stress response of a developing Bacillus subtilis biofilm becomes organized into a ring-like pattern. Mathematical modeling and genetic probing of the underlying circuit indicate that this patterning is generated by a clock and wavefront mechanism, similar to that driving vertebrate somitogenesis. We experimentally validated this hypothesis by showing that predicted nutrient conditions can even lead to multiple concentric rings, resembling segments. We additionally confirmed that this patterning mechanism is driven by cell-autonomous oscillations. Importantly, we show that the clock and wavefront process also spatially patterns sporulation within the biofilm. Together, these findings reveal a biofilm segmentation clock that organizes cellular differentiation in space and time, thereby challenging the paradigm that such patterning mechanisms are exclusive to plant and animal development.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/genética , Biofilmes/crescimento & desenvolvimento , Padronização Corporal/genética , Bacillus subtilis/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Cinética , Modelos Biológicos , Nitrogênio/metabolismo , Transdução de Sinais/genética , Somitos/crescimento & desenvolvimento , Esporos Bacterianos/crescimento & desenvolvimento , Estresse Fisiológico/genética , Fatores de TempoRESUMO
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
Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.
Assuntos
Membrana Externa Bacteriana/metabolismo , Magnésio/metabolismo , Ribossomos/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteômica , Proteínas Ribossômicas/metabolismoRESUMO
In ribosome-associated quality control (RQC), Rqc2/NEMF closely supports the E3 ligase Ltn1/listerin in promoting ubiquitylation and degradation of aberrant nascent-chains obstructing large (60S) ribosomal subunits-products of ribosome stalling during translation. However, while Ltn1 is eukaryote-specific, Rqc2 homologs are also found in bacteria and archaea; whether prokaryotic Rqc2 has an RQC-related function has remained unknown. Here, we show that, as in eukaryotes, a bacterial Rqc2 homolog (RqcH) recognizes obstructed 50S subunits and promotes nascent-chain proteolysis. Unexpectedly, RqcH marks nascent-chains for degradation in a direct manner, by appending C-terminal poly-alanine tails that act as degrons recognized by the ClpXP protease. Furthermore, RqcH acts redundantly with tmRNA/ssrA and protects cells against translational and environmental stresses. Our results uncover a proteolytic-tagging mechanism with implications toward the function of related modifications in eukaryotes and suggest that RQC was already active in the last universal common ancestor (LUCA) to help cope with incomplete translation.
Assuntos
Alanina/metabolismo , Bacillus subtilis/metabolismo , Células Procarióticas/metabolismo , Proteólise , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Células Eucarióticas/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Riboswitches are common gene regulatory units mostly found in bacteria that are capable of altering gene expression in response to a small molecule. These structured RNA elements consist of two modular subunits: an aptamer domain that binds with high specificity and affinity to a target ligand and an expression platform that transduces ligand binding to a gene expression output. Significant progress has been made in engineering novel aptamer domains for new small molecule inducers of gene expression. Modified expression platforms have also been optimized to function when fused with both natural and synthetic aptamer domains. As this field expands, the use of these privileged scaffolds has permitted the development of tools such as RNA-based fluorescent biosensors. In this review, we summarize the methods that have been developed to engineer new riboswitches and highlight applications of natural and synthetic riboswitches in enzyme and strain engineering, in controlling gene expression and cellular physiology, and in real-time imaging of cellular metabolites and signals.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Regulação Bacteriana da Expressão Gênica , Engenharia Genética/métodos , Riboswitch , Aptâmeros de Nucleotídeos/síntese química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ligantes , Imagem Molecular/métodos , Rhodocyclaceae/genética , Rhodocyclaceae/metabolismoRESUMO
Severe changes in the environmental redox potential, and resulting alterations in the oxidation states of intracellular metabolites and enzymes, have historically been considered negative stressors, requiring responses that are strictly defensive. However, recent work in diverse organisms has revealed that more subtle changes in the intracellular redox state can act as signals, eliciting responses with benefits beyond defense and detoxification. Changes in redox state have been shown to influence or trigger chromosome segregation, sporulation, aerotaxis, and social behaviors, including luminescence as well as biofilm establishment and dispersal. Connections between redox state and complex behavior allow bacteria to link developmental choices with metabolic state and coordinate appropriate responses. Promising future directions for this area of study include metabolomic analysis of species- and condition-dependent changes in metabolite oxidation states and elucidation of the mechanisms whereby the redox state influences circadian regulation.
Assuntos
Biofilmes/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Esporos Bacterianos/metabolismo , Aliivibrio fischeri/genética , Aliivibrio fischeri/crescimento & desenvolvimento , Aliivibrio fischeri/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/crescimento & desenvolvimento , Caulobacter crescentus/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Glutationa/metabolismo , Proteínas de Membrana/genética , Oxirredução , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismoRESUMO
Bacteriophages (phages) typically exhibit a narrow host range, yet they tremendously impact horizontal gene transfer (HGT). Here, we investigate phage dynamics in communities harboring phage-resistant (R) and sensitive (S) bacteria, a common scenario in nature. Using Bacillus subtilis and its lytic phage SPP1, we demonstrate that R cells, lacking SPP1 receptor, can be lysed by SPP1 when co-cultured with S cells. This unanticipated lysis was triggered in part by phage lytic enzymes released from nearby infected cells. Strikingly, we discovered that occasionally phages can invade R cells, a phenomenon we termed acquisition of sensitivity (ASEN). We found that ASEN is mediated by R cells transiently gaining phage attachment molecules from neighboring S cells and provide evidence that this molecular exchange is driven by membrane vesicles. Exchange of phage attachment molecules could even occur in an interspecies fashion, enabling phage adsorption to non-host species, providing an unexplored route for HGT. VIDEO ABSTRACT.
Assuntos
Fagos Bacilares/fisiologia , Bacillus subtilis/virologia , Bacteriólise , Receptores Virais/metabolismo , Bacillus/virologia , Fagos Bacilares/enzimologia , Bacillus subtilis/metabolismo , Especificidade de Hospedeiro , Staphylococcus aureus/virologia , Transdução GenéticaRESUMO
Germination is the process by which spores emerge from dormancy. Although spores can remain dormant for decades, the study of germination is an active field of research. In this issue of Genes & Development, Gao and colleagues (pp. 31-45) address a perplexing question: How can a dormant spore initiate germination in response to environmental cues? Three distinct complexes are involved: GerA, a germinant-gated ion channel; 5AF/FigP, a second ion channel required for amplification; and SpoVA, a channel for dipicolinic acid (DPA). DPA release is followed by rehydration of the spore core, thus allowing the resumption of metabolic activity.
Assuntos
Proteínas de Bactérias , Esporos Bacterianos , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Esporos/metabolismo , Canais Iônicos/metabolismo , Bacillus subtilis/metabolismoRESUMO
Bacterial spores can remain dormant for decades yet rapidly germinate and resume growth in response to nutrients. GerA family receptors that sense and respond to these signals have recently been shown to oligomerize into nutrient-gated ion channels. Ion release initiates exit from dormancy. Here, we report that a distinct ion channel, composed of SpoVAF (5AF) and its newly discovered partner protein, YqhR (FigP), amplifies the response. At high germinant concentrations, 5AF/FigP accelerate germination; at low concentrations, this complex becomes critical for exit from dormancy. 5AF is homologous to the channel-forming subunit of GerA family receptors and is predicted to oligomerize around a central pore. 5AF mutations predicted to widen the channel cause constitutive germination during spore formation and membrane depolarization in vegetative cells. Narrow-channel mutants are impaired in germination. A screen for suppressors of a constitutively germinating 5AF mutant identified FigP as an essential cofactor of 5AF activity. We demonstrate that 5AF and FigP interact and colocalize with GerA family receptors in spores. Finally, we show that 5AF/FigP accelerate germination in B. subtilis spores that have nutrient receptors from another species. Our data support a model in which nutrient-triggered ion release by GerA family receptors activates 5AF/FigP ion release, amplifying the response to germinant signals.
Assuntos
Bacillus subtilis , Proteínas de Membrana , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Membrana/genética , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismoRESUMO
Small proteins, here defined as proteins of 50 amino acids or fewer in the absence of processing, have traditionally been overlooked due to challenges in their annotation and biochemical detection. In the past several years, however, increasing numbers of small proteins have been identified either through the realization that mutations in intergenic regions are actually within unannotated small protein genes or through the discovery that some small, regulatory RNAs encode small proteins. These insights, together with comparative sequence analysis, indicate that tens if not hundreds of small proteins are synthesized in a given organism. This review summarizes what has been learned about the functions of several of these bacterial small proteins, most of which act at the membrane, illustrating the astonishing range of processes in which these small proteins act and suggesting several general conclusions. Important questions for future studies of these overlooked proteins are also discussed.
Assuntos
Proteínas/química , RNA/química , Aminoácidos/química , Animais , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Bacteriófagos/metabolismo , Divisão Celular , Códon , Biologia Computacional , Drosophila , Escherichia coli/metabolismo , Fases de Leitura Aberta , Proteínas Quinases/metabolismo , Ribossomos/química , Transdução de SinaisRESUMO
In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth in response to nutrients. The small molecule dipicolinic acid (DPA) plays a central role in both the stress resistance of the dormant spore and its exit from dormancy during germination. The spoVA locus is required for DPA import during sporulation and has been implicated in its export during germination, but the molecular bases are unclear. Here, we define the minimal set of proteins encoded in the Bacillus subtilis spoVA operon required for DPA import and demonstrate that these proteins form a membrane complex. Structural modeling of these components combined with mutagenesis and in vivo analysis reveal that the C and Eb subunits form a membrane channel, while the D subunit functions as a cytoplasmic plug. We show that point mutations that impair the interactions between D and the C-Eb membrane complex reduce the efficiency of DPA import during sporulation and reciprocally accelerate DPA release during germination. Our data support a model in which DPA transport into spores involves cycles of unplugging and then replugging the C-Eb membrane channel, while nutrient detection during germination triggers DPA release by unplugging it.
Assuntos
Proteínas de Bactérias , Esporos Bacterianos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Ácidos Picolínicos/metabolismo , Esporos Bacterianos/genéticaRESUMO
Intrinsically disordered protein regions (IDRs) have been implicated in diverse nuclear and cytoplasmic functions in eukaryotes, but their roles in bacteria are less clear. Here, we report that extracytoplasmic IDRs in Bacillus subtilis are required for cell wall homeostasis. The B. subtilis σI transcription factor is activated in response to envelope stress through regulated intramembrane proteolysis (RIP) of its membrane-anchored anti-σ factor, RsgI. Unlike canonical RIP pathways, we show that ectodomain (site-1) cleavage of RsgI is constitutive, but the two cleavage products remain stably associated, preventing intramembrane (site-2) proteolysis. The regulated step in this pathway is their dissociation, which is triggered by impaired cell wall synthesis and requires RsgI's extracytoplasmic IDR. Intriguingly, the major peptidoglycan polymerase PBP1 also contains an extracytoplasmic IDR, and we show that this region is important for its function. Disparate IDRs can replace the native IDRs on both RsgI and PBP1, arguing that these unstructured regions function similarly. Our data support a model in which the RsgI-σI signaling system and PBP1 represent complementary pathways to repair gaps in the PG meshwork. The IDR on RsgI senses these gaps and activates σI, while the IDR on PBP1 directs the synthase to these sites to fortify them.
Assuntos
Bacillus subtilis , Proteínas Intrinsicamente Desordenadas , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , HomeostaseRESUMO
Peptidoglycan and almost all surface glycopolymers in bacteria are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP)1-4. These UndP-linked precursors are transported across the membrane and polymerized or directly transferred to surface polymers, lipids or proteins. UndP is then flipped to regenerate the pool of cytoplasmic-facing UndP. The identity of the flippase that catalyses transport has remained unknown. Here, using the antibiotic amphomycin that targets UndP5-7, we identified two broadly conserved protein families that affect UndP recycling. One (UptA) is a member of the DedA superfamily8; the other (PopT) contains the domain DUF368. Genetic, cytological and syntenic analyses indicate that these proteins are UndP transporters. Notably, homologues from Gram-positive and Gram-negative bacteria promote UndP transport in Bacillus subtilis, indicating that recycling activity is broadly conserved among family members. Inhibitors of these flippases could potentiate the activity of antibiotics targeting the cell envelope.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte , Sequência Conservada , Evolução Molecular , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Fosfatos de Poli-Isoprenil , Antibacterianos/farmacologia , Bacillus subtilis/citologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/citologia , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Sintenia , Peptidoglicano/metabolismo , Parede Celular/química , Parede Celular/metabolismoRESUMO
In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal "tails." How such tailing can occur without the regular suite of translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) of native complexes, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with RqcP, an Hsp15 family protein. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/ultraestruturaRESUMO
Aborted translation produces large ribosomal subunits obstructed with tRNA-linked nascent chains, which are substrates of ribosome-associated quality control (RQC). Bacterial RqcH, a widely conserved RQC factor, senses the obstruction and recruits tRNAAla(UGC) to modify nascent-chain C termini with a polyalanine degron. However, how RqcH and its eukaryotic homologs (Rqc2 and NEMF), despite their relatively simple architecture, synthesize such C-terminal tails in the absence of a small ribosomal subunit and mRNA has remained unknown. Here, we present cryoelectron microscopy (cryo-EM) structures of Bacillus subtilis RQC complexes representing different Ala tail synthesis steps. The structures explain how tRNAAla is selected via anticodon reading during recruitment to the A-site and uncover striking hinge-like movements in RqcH leading tRNAAla into a hybrid A/P-state associated with peptidyl-transfer. Finally, we provide structural, biochemical, and molecular genetic evidence identifying the Hsp15 homolog (encoded by rqcP) as a novel RQC component that completes the cycle by stabilizing the P-site tRNA conformation. Ala tailing thus follows mechanistic principles surprisingly similar to canonical translation elongation.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Elongação Traducional da Cadeia Peptídica , RNA Bacteriano/metabolismo , RNA de Transferência de Alanina/metabolismo , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , RNA Bacteriano/genética , RNA de Transferência de Alanina/genéticaRESUMO
ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.
Assuntos
Bacillus subtilis/citologia , Citidina Trifosfato/metabolismo , Pirofosfatases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Membrana Celular/metabolismo , Cromossomos Bacterianos/genética , Citidina Trifosfato/fisiologia , Proteínas do Citoesqueleto/genética , Pirofosfatases/fisiologiaRESUMO
Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.
Assuntos
Regulação Alostérica/genética , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/genética , Guanosina Pentafosfato/genética , Pirofosfatases/genética , Aminoácidos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Domínio Catalítico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolases/genética , Ribossomos/genética , Ribossomos/metabolismo , Inanição/genética , Inanição/metabolismoRESUMO
Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in diverse bacteria.
Assuntos
Bacillus subtilis , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ribossomos/metabolismo , Peptídeos/metabolismoRESUMO
We propose a concept for the folding and self-assembly of the pore-forming TatA complex from the Twin-arginine translocase and of other membrane proteins based on electrostatic "charge zippers." Each subunit of TatA consists of a transmembrane segment, an amphiphilic helix (APH), and a C-terminal densely charged region (DCR). The sequence of charges in the DCR is complementary to the charge pattern on the APH, suggesting that the protein can be "zipped up" by a ladder of seven salt bridges. The length of the resulting hairpin matches the lipid bilayer thickness, hence a transmembrane pore could self-assemble via intra- and intermolecular salt bridges. The steric feasibility was rationalized by molecular dynamics simulations, and experimental evidence was obtained by monitoring the monomer-oligomer equilibrium of specific charge mutants. Similar "charge zippers" are proposed for other membrane-associated proteins, e.g., the biofilm-inducing peptide TisB, the human antimicrobial peptide dermcidin, and the pestiviral E(RNS) protein.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/metabolismo , Toxinas Bacterianas/química , Proteínas de Escherichia coli/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/química , Peptídeos/metabolismo , Dobramento de Proteína , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The peptidoglycan cell wall is a hallmark of the bacterial subkingdom. Surprisingly, many modern bacteria retain the ability to switch into a wall-free state called the L-form. L-form proliferation is remarkable in being independent of the normally essential FtsZ-based division machinery and in occurring by membrane blebbing and tubulation. We show that mutations leading to excess membrane synthesis are sufficient to drive L-form division in Bacillus subtilis. Artificially increasing the cell surface area to volume ratio in wild-type protoplasts generates similar shape changes and cell division. Our findings show that simple biophysical processes could have supported efficient cell proliferation during the evolution of early cells and provide an extant biological model for studying this problem.