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1.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35105804

RESUMO

Microbial communities frequently invade one another as a whole, a phenomenon known as community coalescence. Despite its potential importance for the assembly, dynamics, and stability of microbial consortia, as well as its prospective utility for microbiome engineering, our understanding of the processes that govern it is still very limited. Theory has suggested that microbial communities may exhibit cohesiveness in the face of invasions emerging from collective metabolic interactions across microbes and their environment. This cohesiveness may lead to correlated invasional outcomes, where the fate of a given taxon is determined by that of other members of its community-a hypothesis known as ecological coselection. Here, we have performed over 100 invasion and coalescence experiments with microbial communities of various origins assembled in two different synthetic environments. We show that the dominant members of the primary communities can recruit their rarer partners during coalescence (top-down coselection) and also be recruited by them (bottom-up coselection). With the aid of a consumer-resource model, we found that the emergence of top-down or bottom-up cohesiveness is modulated by the structure of the underlying cross-feeding networks that sustain the coalesced communities. The model also predicts that these two forms of ecological coselection cannot co-occur under our conditions, and we have experimentally confirmed that one can be strong only when the other is weak. Our results provide direct evidence that collective invasions can be expected to produce ecological coselection as a result of cross-feeding interactions at the community level.


Assuntos
Consórcios Microbianos/fisiologia , Modelos Biológicos
2.
PLoS Biol ; 17(12): e3000550, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830028

RESUMO

Understanding the link between community composition and function is a major challenge in microbial population biology, with implications for the management of natural microbiomes and the design of synthetic consortia. Specifically, it is poorly understood whether community functions can be quantitatively predicted from traits of species in monoculture. Inspired by the study of complex genetic interactions, we have examined how the amylolytic rate of combinatorial assemblages of six starch-degrading soil bacteria depend on the separate functional contributions from each species and their interactions. Filtering our results through the theory of biochemical kinetics, we show that this simple function is additive in the absence of interactions among community members. For about half of the combinatorially assembled consortia, the amylolytic function is dominated by pairwise and higher-order interactions. For the other half, the function is additive despite the presence of strong competitive interactions. We explain the mechanistic basis of these findings and propose a quantitative framework that allows us to separate the effect of behavioral and population dynamics interactions. Our results suggest that the functional robustness of a consortium to pairwise and higher-order interactions critically affects our ability to predict and bottom-up engineer ecosystem function in complex communities.


Assuntos
Consórcios Microbianos/fisiologia , Interações Microbianas/fisiologia , Microbiota/fisiologia , Bactérias/genética , Microbiota/genética , Solo/química , Microbiologia do Solo
3.
Int J Mol Sci ; 22(2)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440735

RESUMO

The appearance of carbapenem-resistant Klebsiella pneumoniae has increased the use of colistin as a last-resort antibiotic for treating infections by this pathogen. A consequence of its use has been the spread of colistin-resistant strains, in several cases carrying colistin resistance genes. In addition, when susceptible strains are confronted with colistin during treatment, mutation is a major cause of the acquisition of resistance. To analyze the mechanisms of resistance that might be selected during colistin treatment, an experimental evolution assay for 30 days using as a model the clinical K. pneumoniae kp52145 isolate in the presence of increasing amounts of colistin was performed. All evolved populations presented a decreased susceptibility to colistin, without showing cross-resistance to antibiotics belonging to other structural families. We did not find any common mutation in the evolved mutants, neither in already known genes, previously known to be associated with the resistance phenotype, nor in new ones. The only common genetic change observed in the strains that evolved in the presence of colistin was the amplification of a 34 Kb sequence, homologous to a prophage (Enterobacteria phage Fels-2). Our data support that gene amplification can be a driving force in the acquisition of colistin resistance by K. pneumoniae.


Assuntos
Antibacterianos/farmacologia , Cromossomos Bacterianos/genética , Colistina/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Bacteriófagos/genética , Biologia Computacional/métodos , Evolução Molecular , Humanos , Klebsiella pneumoniae/virologia
4.
J Biol Chem ; 288(37): 26625-34, 2013 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-23921390

RESUMO

Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.


Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/fisiologia , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Permeabilidade da Membrana Celular , Microscopia Crioeletrônica , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Citometria de Fluxo , Microscopia Confocal , Microscopia de Fluorescência , Fosfatidilcolinas/química , Plasmídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas
5.
bioRxiv ; 2023 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-37961608

RESUMO

When microbial communities form, their composition is shaped by selective pressures imposed by the environment. Can we predict which communities will assemble under different environmental conditions? Here, we hypothesize that quantitative similarities in metabolic traits across metabolically similar environments lead to predictable similarities in community composition. To that end, we measured the growth rate and by-product profile of a library of proteobacterial strains in a large number of single nutrient environments. We found that growth rates and secretion profiles were positively correlated across environments when the supplied substrate was metabolically similar. By analyzing hundreds of in-vitro communities experimentally assembled in an array of different synthetic environments, we then show that metabolically similar substrates select for taxonomically similar communities. These findings lead us to propose and then validate a comparative approach for quantitatively predicting the effects of novel substrates on the composition of complex microbial consortia.

6.
Cell Syst ; 13(1): 29-42.e7, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-34653368

RESUMO

For microbiome biology to become a more predictive science, we must identify which descriptive features of microbial communities are reproducible and predictable, which are not, and why. We address this question by experimentally studying parallelism and convergence in microbial community assembly in replicate glucose-limited habitats. Here, we show that the previously observed family-level convergence in these habitats reflects a reproducible metabolic organization, where the ratio of the dominant metabolic groups can be explained from a simple resource-partitioning model. In turn, taxonomic divergence among replicate communities arises from multistability in population dynamics. Multistability can also lead to alternative functional states in closed ecosystems but not in metacommunities. Our findings empirically illustrate how the evolutionary conservation of quantitative metabolic traits, multistability, and the inherent stochasticity of population dynamics, may all conspire to generate the patterns of reproducibility and variability at different levels of organization that are commonplace in microbial community assembly.


Assuntos
Microbiota , Dinâmica Populacional , Reprodutibilidade dos Testes
7.
Elife ; 102021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33877964

RESUMO

A major open question in microbial community ecology is whether we can predict how the components of a diet collectively determine the taxonomic composition of microbial communities. Motivated by this challenge, we investigate whether communities assembled in pairs of nutrients can be predicted from those assembled in every single nutrient alone. We find that although the null, naturally additive model generally predicts well the family-level community composition, there exist systematic deviations from the additive predictions that reflect generic patterns of nutrient dominance at the family level. Pairs of more-similar nutrients (e.g. two sugars) are on average more additive than pairs of more dissimilar nutrients (one sugar-one organic acid). Furthermore, sugar-acid communities are generally more similar to the sugar than the acid community, which may be explained by family-level asymmetries in nutrient benefits. Overall, our results suggest that regularities in how nutrients interact may help predict community responses to dietary changes.


Assuntos
Bactérias/metabolismo , Microbiota , Nutrientes/metabolismo , Ácidos/metabolismo , Compostos Orgânicos/metabolismo , Açúcares/metabolismo
8.
BMC Genomics ; 11: 31, 2010 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-20074347

RESUMO

BACKGROUND: The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome. RESULTS: Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings. CONCLUSIONS: The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology.


Assuntos
Perfilação da Expressão Gênica , Leishmania infantum/genética , Glicoproteínas de Membrana/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Células Cultivadas , Meios de Cultura , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Alinhamento de Sequência , Temperatura
9.
Genomics ; 93(6): 551-64, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19442635

RESUMO

Metacyclic promastigotes are transmitted during bloodmeals after development inside the gut of the sandfly vector. The isolation from axenic cultures of procyclic and metacyclic promastigotes by peanut lectin agglutination followed by differential centrifugation is controversial in Leishmania infantum. The purpose of this study has been to isolate both fractions simultaneously from the same population in stationary phase of axenic culture and compare their expression profiles by whole-genome shotgun DNA microarrays. The 317 genes found with meaningful values of stage-specific regulation demonstrate that negative selection of metacyclic promastigotes by PNA agglutination is feasible in L. infantum and both fractions can be isolated. This subpopulation up-regulates a cysteine peptidase A and several genes involved in lipophosphoglycan, proteophosphoglycan and glycoprotein biosynthesis, all related with infectivity. In fact, we have confirmed the increased infection rate of PNA(-) promastigotes by U937 human cell line infection experiments. These data support that metacyclic promastigotes are related with infectivity and the lack of agglutination with PNA is a phenotypic marker for this subpopulation.


Assuntos
Regulação da Expressão Gênica/genética , Genoma de Protozoário/genética , Leishmania infantum/genética , Aglutinação , Aminoácidos/metabolismo , Animais , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Ácidos Graxos/metabolismo , Perfilação da Expressão Gênica , Humanos , Leishmania infantum/patogenicidade , Estágios do Ciclo de Vida/genética , Análise de Sequência com Séries de Oligonucleotídeos , Aglutinina de Amendoim/metabolismo , Virulência
10.
Mol Cell Biol ; 25(19): 8755-61, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16166653

RESUMO

Schizosaccharomyces pombe rRNA genes contain three replication fork barriers (RFB1-3) located in the nontranscribed spacer. RFB2 and RFB3 require binding of the transcription terminator factor Reb1p to two identical recognition sequences that colocalize with these barriers. RFB1, which is the strongest of the three barriers, functions in a Reb1p-independent manner, and cognate DNA-binding proteins for this barrier have not been identified yet. Here we functionally define RFB1 within a 78-bp sequence located near the 3' end of the rRNA coding region. A protein that specifically binds to this sequence was purified by affinity chromatography and identified as Sap1p by mass spectrometry. Specific binding to RFB1 was confirmed by using Sap1p expressed in Escherichia coli. Sap1p is essential for viability and is required for efficient mating-type switching. Mutations in RFB1 that precluded formation of the Sap1p-RFB1 complex systematically abolished replication barrier function, indicating that Sap1p is required for replication fork blockage at RFB1.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Genes Fúngicos , RNA Ribossômico/química , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Sequência de Bases , Sítios de Ligação , Cromatografia de Afinidade , DNA/química , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel Bidimensional , Escherichia coli/metabolismo , Espectrometria de Massas , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Oligonucleotídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , RNA Ribossômico/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição
11.
J Biosci Bioeng ; 125(1): 15-22, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28821380

RESUMO

Non-dividing persisters, bacteria that can survive in the presence of antibiotics by pausing their metabolic activity, are among the many causes of the refractory nature of bacterial infections. Here we constructed a recombinant Escherichia coli strain that enables to distinguish non-dividing from dividing cell based on Z-ring during cell division. Then, non-dividing cells and dividing cells were successfully separated using a fluorescence activated cell sorter. The sorted non-dividing cells showed significantly higher tolerance toward ofloxacin than dividing cells, which indicates that persisters were concentrated with the methodology. Transcriptional analysis revealed that genes involved in guanosine tetraphosphate synthesis are upregulated in persisters, which represses transcription and DNA replication and leads to ofloxacin tolerance. Lactate dehydrogenase and several ATP-binding cassette transporters were upregulated in persisters to adapt to anaerobic metabolism. In addition, nitrite and dimethyl sulfoxide (DMSO) may be used as reducible substrates for alternative energy generation pathways. Our methodology revealed a unique transcriptional profile of E. coli persisters.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Perfilação da Expressão Gênica , Ofloxacino/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/biossíntese , Anaerobiose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Dimetil Sulfóxido/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Guanosina Tetrafosfato/biossíntese , L-Lactato Desidrogenase/biossíntese , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Nitritos/metabolismo
12.
Science ; 361(6401): 469-474, 2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-30072533

RESUMO

A major unresolved question in microbiome research is whether the complex taxonomic architectures observed in surveys of natural communities can be explained and predicted by fundamental, quantitative principles. Bridging theory and experiment is hampered by the multiplicity of ecological processes that simultaneously affect community assembly in natural ecosystems. We addressed this challenge by monitoring the assembly of hundreds of soil- and plant-derived microbiomes in well-controlled minimal synthetic media. Both the community-level function and the coarse-grained taxonomy of the resulting communities are highly predictable and governed by nutrient availability, despite substantial species variability. By generalizing classical ecological models to include widespread nonspecific cross-feeding, we show that these features are all emergent properties of the assembly of large microbial communities, explaining their ubiquity in natural microbiomes.


Assuntos
Bactérias/classificação , Bactérias/metabolismo , Consórcios Microbianos , Plantas/microbiologia , Microbiologia do Solo , Bactérias/isolamento & purificação
13.
Mol Cell Biol ; 24(1): 398-406, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14673172

RESUMO

Polar replication fork barriers (RFBs) near the 3' end of the rRNA transcriptional unit are a conserved feature of ribosomal DNA (rDNA) replication in eukaryotes. In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage. On the contrary, in the budding yeast Saccharomyces cerevisiae, the rRNA transcription termination factors are not required for RFBs. Here we characterized the rDNA RFBs in the fission yeast Schizosaccharomyces pombe. S. pombe rDNA contains three closely spaced polar replication barriers named RFB1, RFB2, and RFB3 in the 3' to 5' order. The transcription termination protein reb1 and its two binding sites, present at the 3' end of the coding region, were required for fork arrest at RFB2 and RFB3 in vivo. On the other hand, fork arrest at the strongest RFB1 barrier was independent of the above transcription termination factors. Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs. These results suggest that during evolution, cis- and trans-acting factors required for rRNA transcription termination became involved in replication fork blockage also. S. pombe is suggested to be a transitional species in which both mechanisms coexist.


Assuntos
Replicação do DNA , DNA Ribossômico , Proteínas de Ligação a DNA/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição
14.
Front Microbiol ; 8: 539, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28421043

RESUMO

The synthetic bacterial prionoid RepA-WH1 causes a vertically transmissible amyloid proteinopathy in Escherichia coli that inhibits growth and eventually kills the cells. Recent in vitro studies show that RepA-WH1 builds pores through model lipid membranes, suggesting a possible mechanism for bacterial cell death. By comparing acutely (A31V) and mildly (ΔN37) cytotoxic mutant variants of the protein, we report here that RepA-WH1(A31V) expression decreases the intracellular osmotic pressure and compromise bacterial viability under either aerobic or anaerobic conditions. Both are effects expected from threatening membrane integrity and are in agreement with findings on the impairment by RepA-WH1(A31V) of the proton motive force (PMF)-dependent transport of ions (Fe3+) and ATP synthesis. Systems approaches reveal that, in aerobiosis, the PMF-independent respiratory dehydrogenase NdhII is induced in response to the reduction in intracellular levels of iron. While NdhII is known to generate H2O2 as a by-product of the autoxidation of its FAD cofactor, key proteins in the defense against oxidative stress (OxyR, KatE), together with other stress-resistance factors, are sequestered by co-aggregation with the RepA-WH1(A31V) amyloid. Our findings suggest a route for RepA-WH1 toxicity in bacteria: a primary hit of damage to the membrane, compromising bionergetics, triggers a stroke of oxidative stress, which is exacerbated due to the aggregation-dependent inactivation of enzymes and transcription factors that enable the cellular response to such injury. The proteinopathy caused by the prion-like protein RepA-WH1 in bacteria recapitulates some of the core hallmarks of human amyloid diseases.

15.
mBio ; 7(5)2016 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-27729511

RESUMO

When deprived of FtsZ, Escherichia coli cells (VIP205) grown in liquid form long nonseptated filaments due to their inability to assemble an FtsZ ring and their failure to recruit subsequent divisome components. These filaments fail to produce colonies on solid medium, in which synthesis of FtsZ is induced, upon being diluted by a factor greater than 4. However, once the initial FtsZ levels are recovered in liquid culture, they resume division, and their plating efficiency returns to normal. The potential septation sites generated in the FtsZ-deprived filaments are not annihilated, and once sufficient FtsZ is accumulated, they all become active and divide to produce cells of normal length. FtsZ-deprived cells accumulate defects in their physiology, including an abnormally high number of unsegregated nucleoids that may result from the misplacement of FtsK. Their membrane integrity becomes compromised and the amount of membrane proteins, such as FtsK and ZipA, increases. FtsZ-deprived cells also show an altered expression pattern, namely, transcription of several genes responding to DNA damage increases, whereas transcription of some ribosomal or global transcriptional regulators decreases. We propose that the changes caused by the depletion of FtsZ, besides stopping division, weaken the cell, diminishing its resiliency to minor challenges, such as dilution stress. IMPORTANCE: Our results suggest a role for FtsZ, in addition to its already known effect in the constriction of E. coli, in protecting the nondividing cells against minor stress. This protection can even be exerted when an inactive FtsZ is produced, but it is lost when the protein is altogether absent. These results have implications in fields like synthetic biology or antimicrobial discovery. The construction of synthetic divisomes in the test tube may need to preserve unsuspected roles, such as this newly found FtsZ property, to guarantee the stability of artificial containers. Whereas the effects on viability caused by inhibiting the activity of FtsZ may be partly overcome by filamentation, the absence of FtsZ is not tolerated by E. coli, an observation that may help in the design of effective antimicrobial compounds.


Assuntos
Proteínas do Citoesqueleto/deficiência , Escherichia coli/citologia , Escherichia coli/fisiologia , Proteínas de Bactérias , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Cromossomos Bacterianos/metabolismo , Escherichia coli/genética , Proteínas de Membrana/metabolismo
16.
PLoS One ; 10(5): e0126434, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25950808

RESUMO

Division site selection is achieved in bacteria by different mechanisms, one of them being nucleoid occlusion, which prevents Z-ring assembly nearby the chromosome. Nucleoid occlusion in E. coli is mediated by SlmA, a sequence specific DNA binding protein that antagonizes FtsZ assembly. Here we show that, when bound to its specific target DNA sequences (SBS), SlmA reduces the lifetime of the FtsZ protofilaments in solution and of the FtsZ bundles when located inside permeable giant vesicles. This effect appears to be essentially uncoupled from the GTPase activity of the FtsZ protofilaments, which is insensitive to the presence of SlmA·SBS. The interaction of SlmA·SBS with either FtsZ protofilaments containing GTP or FtsZ oligomers containing GDP results in the disassembly of FtsZ polymers. We propose that SlmA·SBS complexes control the polymerization state of FtsZ by accelerating the disassembly of the FtsZ polymers leading to their fragmentation into shorter species that are still able to hydrolyze GTP at the same rate. SlmA defines therefore a new class of inhibitors of the FtsZ ring different from the SOS response regulator SulA and from the moonlighting enzyme OpgH, inhibitors of the GTPase activity. SlmA also shows differences compared with MinC, the inhibitor of the division site selection Min system, which shortens FtsZ protofilaments by interacting with the GDP form of FtsZ.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/citologia , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Mapas de Interação de Proteínas
17.
J Mol Biol ; 403(1): 24-39, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20732327

RESUMO

Hsp70 chaperones, besides their role in assisting protein folding, are key modulators of protein disaggregation, being consistently found as components of most macromolecular assemblies isolated in proteome-wide affinity purifications. A wealth of structural information has been recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA replication initiators and consists of six distinct protein subunits (ScOrc1p-ScOrc 6p). Here, we report that a hydrophobic sequence (IL(4)) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p(2)-DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL(4) motif in one Orc4p molecule, with the substrate-binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co-expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of IL(4) selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL(4) results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL(4) motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Imageamento Tridimensional , Viabilidade Microbiana , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína
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