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1.
Nat Commun ; 11(1): 3862, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737291

RESUMO

Allostery in proteins influences various biological processes such as regulation of gene transcription and activities of enzymes and cell signaling. Computational approaches for analysis of allosteric coupling provide inexpensive opportunities to predict mutations and to design small-molecule agents to control protein function and cellular activity. We develop a computationally efficient network-based method, Ohm, to identify and characterize allosteric communication networks within proteins. Unlike previously developed simulation-based approaches, Ohm relies solely on the structure of the protein of interest. We use Ohm to map allosteric networks in a dataset composed of 20 proteins experimentally identified to be allosterically regulated. Further, the Ohm allostery prediction for the protein CheY correlates well with NMR CHESCA studies. Our webserver, Ohm.dokhlab.org, automatically determines allosteric network architecture and identifies critical coupled residues within this network.


Assuntos
Algoritmos , Proteínas Quimiotáticas Aceptoras de Metil/química , Mapeamento de Interação de Proteínas/estatística & dados numéricos , Software , Regulação Alostérica , Sítio Alostérico , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Internet , Proteínas Quimiotáticas Aceptoras de Metil/antagonistas & inibidores , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína
2.
PLoS Pathog ; 16(8): e1008776, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845938

RESUMO

Enteroaggregative Escherichia coli (EAEC) is a diarrheagenic pathotype associated with traveler's diarrhea, foodborne outbreaks and sporadic diarrhea in industrialized and developing countries. Regulation of virulence in EAEC is mediated by AggR and its negative regulator Aar. Together, they control the expression of at least 210 genes. On the other hand, we observed that about one third of Aar-regulated genes are related to metabolism and transport. In this study we show the AggR/Aar duo controls the metabolism of lipids. Accordingly, we show that AatD, encoded in the AggR-regulated aat operon (aatPABCD) is an N-acyltransferase structurally similar to the essential Apolipoprotein N-acyltransferase Lnt and is required for the acylation of Aap (anti-aggregation protein). Deletion of aatD impairs post-translational modification of Aap and causes its accumulation in the bacterial periplasm. trans-complementation of 042aatD mutant with the AatD homolog of ETEC or with the N-acyltransferase Lnt reestablished translocation of Aap. Site-directed mutagenesis of the E207 residue in the putative acyltransferase catalytic triad disrupted the activity of AatD and caused accumulation of Aap in the periplasm due to reduced translocation of Aap at the bacterial surface. Furthermore, Mass spectroscopy revealed that Aap is acylated in a putative lipobox at the N-terminal of the mature protein, implying that Aap is a lipoprotein. Lastly, deletion of aatD impairs bacterial colonization of the streptomycin-treated mouse model. Our findings unveiled a novel N-acyltransferase family associated with bacterial virulence, and that is tightly regulated by AraC/XylS regulators in the order Enterobacterales.


Assuntos
Acetiltransferases/metabolismo , Fator de Transcrição AraC/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica , Acetiltransferases/genética , Acilação , Animais , Fator de Transcrição AraC/química , Fator de Transcrição AraC/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Filogenia , Conformação Proteica , Virulência
3.
Nature ; 584(7821): 470-474, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32669712

RESUMO

The rate of cell growth is crucial for bacterial fitness and drives the allocation of bacterial resources, affecting, for example, the expression levels of proteins dedicated to metabolism and biosynthesis1,2. It is unclear, however, what ultimately determines growth rates in different environmental conditions. Moreover, increasing evidence suggests that other objectives are also important3-7, such as the rate of physiological adaptation to changing environments8,9. A common challenge for cells is that these objectives cannot be independently optimized, and maximizing one often reduces another. Many such trade-offs have indeed been hypothesized on the basis of qualitative correlative studies8-11. Here we report a trade-off between steady-state growth rate and physiological adaptability in Escherichia coli, observed when a growing culture is abruptly shifted from a preferred carbon source such as glucose to fermentation products such as acetate. These metabolic transitions, common for enteric bacteria, are often accompanied by multi-hour lags before growth resumes. Metabolomic analysis reveals that long lags result from the depletion of key metabolites that follows the sudden reversal in the central carbon flux owing to the imposed nutrient shifts. A model of sequential flux limitation not only explains the observed trade-off between growth and adaptability, but also allows quantitative predictions regarding the universal occurrence of such tradeoffs, based on the opposing enzyme requirements of glycolysis versus gluconeogenesis. We validate these predictions experimentally for many different nutrient shifts in E. coli, as well as for other respiro-fermentative microorganisms, including Bacillus subtilis and Saccharomyces cerevisiae.


Assuntos
Adaptação Fisiológica , Meio Ambiente , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Acetatos/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Divisão Celular , Escherichia coli/enzimologia , Escherichia coli/genética , Fermentação , Gluconeogênese , Glucose/metabolismo , Glicólise , Metabolômica , Modelos Biológicos , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(31): 18540-18549, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675239

RESUMO

Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imagem Individual de Molécula , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(26): 15006-15017, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554497

RESUMO

Cytochrome bo 3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F1Fo-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo 3 oxidase in giant unilamellar vesicles made of PDMS-g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min-1 To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS-g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.


Assuntos
Membrana Celular/enzimologia , Grupo dos Citocromos b/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Membrana Celular/química , Membrana Celular/genética , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fosfatidilcolinas/metabolismo , Polímeros/química , Prótons
6.
PLoS One ; 15(5): e0227725, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469888

RESUMO

The surge in the prevalence of drug-resistant bacteria in poultry is a global concern as it may pose an extended threat to humans and animal health. The present study aimed to investigate the colonization proportion of extended-spectrum ß-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae (EPE and CPE, respectively) in the gut of healthy poultry, Gallus gallus domesticus in Kaski district of Western Nepal. Total, 113 pooled rectal swab specimens from 66 private household farms and 47 commercial poultry farms were collected by systematic random sampling from the Kaski district in western Nepal. Out of 113 pooled samples, 19 (28.8%) samples from 66 backyard farms, and 15 (31.9%) from 47 commercial broiler farms were positive for EPE. Of the 38 EPE strains isolated from 34 ESBL positive rectal swabs, 31(81.6%) were identified as Escherichia coli, five as Klebsiella pneumoniae (13.2%), and one each isolate of Enterobacter species and Citrobacter species (2.6%). Based on genotyping, 35/38 examined EPE strains (92.1%) were phylogroup-1 positive, and all these 35 strains (100%) had the CTX-M-15 gene and strains from phylogroup-2, and 9 were of CTX-M-2 and CTX-M-14, respectively. Among 38 ESBL positive isolates, 9 (23.7%) were Ambler class C (Amp C) co-producers, predominant were of DHA, followed by CIT genes. Two (6.5%) E. coli strains of ST131 belonged to clade C, rest 29/31 (93.5%) were non-ST131 E. coli. None of the isolates produced carbapenemase. Twenty isolates (52.6%) were in-vitro biofilm producers. Univariate analysis showed that the odd of ESBL carriage among commercial broilers were 1.160 times (95% CI 0.515, 2.613) higher than organically fed backyard flocks. This is the first study in Nepal, demonstrating the EPE colonization proportion, genotypes, and prevalence of high-risk clone E. coli ST131 among gut flora of healthy poultry. Our data indicated that CTX-M-15 was the most prevalent ESBL enzyme, mainly associated with E. coli belonging to non-ST131clones and the absence of carbapenemases.


Assuntos
Galinhas/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Microbioma Gastrointestinal/genética , beta-Lactamases/genética , Animais , Biofilmes/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/patogenicidade , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Humanos , Nepal/epidemiologia , Aves Domésticas/microbiologia , beta-Lactamases/isolamento & purificação
7.
Mol Cell ; 79(1): 140-154.e7, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32464091

RESUMO

Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.


Assuntos
DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DnaB Helicases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Holoenzimas/química , DNA Primase/genética , DNA Bacteriano , DNA Polimerase Dirigida por DNA/genética , DnaB Helicases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Holoenzimas/genética , Holoenzimas/metabolismo , Conformação Molecular , Ligação Proteica , Conformação Proteica
8.
Nat Chem Biol ; 16(7): 776-782, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32367018

RESUMO

In type II polyketide synthases (PKSs), the ketosynthase-chain length factor (KS-CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here, we report the crystal structures of the Iga11-Iga12 (KS-CLF) heterodimer and the covalently cross-linked Iga10=Iga11-Iga12 (ACP=KS-CLF) tripartite complex. The latter structure revealed the molecular basis of the interaction between Iga10 and Iga11-Iga12, which differs from that between the ACP and KS of Escherichia coli fatty acid synthase. Furthermore, the reaction pocket structure and site-directed mutagenesis revealed that the negative charge of Asp 113 of Iga11 prevents further condensation using a ß-ketoacyl product as a substrate, which distinguishes IgaPKS from typical type II PKSs. This work will facilitate the future rational design of PKSs.


Assuntos
Proteína de Transporte de Acila/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Ácido Graxo Sintases/química , Policetídeo Sintases/química , Policetídeos/química , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Biocatálise , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por Substrato
9.
Proc Natl Acad Sci U S A ; 117(21): 11597-11607, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385156

RESUMO

The distribution of fitness effects of mutation plays a central role in constraining protein evolution. The underlying mechanisms by which mutations lead to fitness effects are typically attributed to changes in protein specific activity or abundance. Here, we reveal the importance of a mutation's collateral fitness effects, which we define as effects that do not derive from changes in the protein's ability to perform its physiological function. We comprehensively measured the collateral fitness effects of missense mutations in the Escherichia coli TEM-1 ß-lactamase antibiotic resistance gene using growth competition experiments in the absence of antibiotic. At least 42% of missense mutations in TEM-1 were deleterious, indicating that for some proteins collateral fitness effects occur as frequently as effects on protein activity and abundance. Deleterious mutations caused improper posttranslational processing, incorrect disulfide-bond formation, protein aggregation, changes in gene expression, and pleiotropic effects on cell phenotype. Deleterious collateral fitness effects occurred more frequently in TEM-1 than deleterious effects on antibiotic resistance in environments with low concentrations of the antibiotic. The surprising prevalence of deleterious collateral fitness effects suggests they may play a role in constraining protein evolution, particularly for highly expressed proteins, for proteins under intermittent selection for their physiological function, and for proteins whose contribution to fitness is buffered against deleterious effects on protein activity and protein abundance.


Assuntos
Evolução Molecular , Aptidão Genética/genética , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(21): 11692-11702, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32393643

RESUMO

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., ß-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.


Assuntos
Proteínas de Bactérias , Endopeptidases , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Moleculares , Mutação/genética , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/genética , Conformação Proteica , Vibrio cholerae/enzimologia , Vibrio cholerae/genética
11.
PLoS Genet ; 16(4): e1008750, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32348296

RESUMO

Plasmids, when transferred by conjugation in natural environments, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida. Expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. ardC was also required if the hsdRMS system was present in E. coli recipient cells. Thus, ArdC has antirestriction activity against the HsdRMS system and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity was not needed for the antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggered an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies and settle into new hosts.


Assuntos
Conjugação Genética , Proteínas Virais/química , Domínio Catalítico , Cristalografia por Raios X , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Especificidade de Hospedeiro , Magnésio/química , Metaloproteases/química , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
12.
Phys Chem Chem Phys ; 22(17): 9518-9533, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32319475

RESUMO

Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally reduced by thioredoxin. Purified EcTPx as dithiol and disulfide behaves as a monomer under near physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation of EcTPx. To explore this process, the FF → LU and LU → FF transitions were studied using conventional molecular dynamics simulations and an enhanced conformational sampling technique for different oxidation and protonation states of the active site cysteine residues CP and CR. Our results suggest that the FF → LU transition has a higher associated energy barrier than the refolding LU → FF process in agreement with the relatively low experimental turnover number of EcTPx. Furthermore, in silico designed single-point mutants of αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionarily optimized to fully speed-up the conformational transition of wild-type EcTPx.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , Proteínas Periplásmicas/química , Peroxidases/química , Dobramento de Proteína , Simulação por Computador , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação/genética , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Conformação Proteica
13.
Nucleic Acids Res ; 48(10): 5397-5406, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32338761

RESUMO

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction-modification systems, is also active against BREX. In contrast to the wild-type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R-M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.


Assuntos
Bacteriófago T7/fisiologia , Proteínas Virais/metabolismo , Bacteriófago T7/genética , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/virologia , Plasmídeos , Proteínas Virais/genética
14.
BMC Infect Dis ; 20(1): 312, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345218

RESUMO

BACKGROUND: While there is increasing knowledge about the gut microbiome, the factors influencing and the significance of the gut resistome are still not well understood. Infant gut commensals risk transferring multidrug-resistant antibiotic resistance genes (ARGs) to pathogenic bacteria. The rapid spread of multidrug-resistant pathogenic bacteria is a worldwide public health concern. Better understanding of the naïve infant gut resistome may build the evidence base for antimicrobial stewardship in both humans and in the food industry. Given the high carriage rate of extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Asia, we aimed to evaluate community prevalence, dynamics, and longitudinal changes in antibiotic resistance gene (ARG) profiles and prevalence of ESBL-producing E. coli and K. pneumoniae in the intestinal microbiome of infants participating in the Growing Up in Singapore Towards Healthy Outcomes (GUSTO) study, a longitudinal cohort study of pregnant women and their infants. METHODS: We analysed ARGs in the first year of life among 75 infants at risk of eczema who had stool samples collected at multiple timepoints using metagenomics. RESULTS: The mean number of ARGs per infant increased with age. The most common ARGs identified confer resistance to aminoglycoside, beta-lactam, macrolide and tetracycline antibiotics; all infants harboured these antibiotic resistance genes at some point in the first year of life. Few ARGs persisted throughout the first year of life. Beta-lactam resistant Escherichia coli and Klebsiella pneumoniae were detected in 4 (5.3%) and 32 (42.7%) of subjects respectively. CONCLUSION: In this longitudinal cohort study of infants living in a region with high endemic antibacterial resistance, we demonstrate that majority of the infants harboured several antibiotic resistance genes in their gut and showed that the infant gut resistome is diverse and dynamic over the first year of life.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Eczema/diagnóstico , Microbioma Gastrointestinal/efeitos dos fármacos , Aminoglicosídeos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eczema/etiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Feminino , Humanos , Lactente , Recém-Nascido , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Estudos Longitudinais , Masculino , Risco , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia
15.
Proc Natl Acad Sci U S A ; 117(15): 8462-8467, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32238560

RESUMO

In bacteria, the first two steps of gene expression-transcription and translation-are spatially and temporally coupled. Uncoupling may lead to the arrest of transcription through RNA polymerase backtracking, which interferes with replication forks, leading to DNA double-stranded breaks and genomic instability. How transcription-translation coupling mitigates these conflicts is unknown. Here we show that, unlike replication, translation is not inhibited by arrested transcription elongation complexes. Instead, the translating ribosome actively pushes RNA polymerase out of the backtracked state, thereby reactivating transcription. We show that the distance between the two machineries upon their contact on mRNA is smaller than previously thought, suggesting intimate interactions between them. However, this does not lead to the formation of a stable functional complex between the enzymes, as was once proposed. Our results reveal an active, energy-driven mechanism that reactivates backtracked elongation complexes and thus helps suppress their interference with replication.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , Sequência de Bases , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , RNA Mensageiro/genética , Ribossomos/genética , Fatores de Elongação da Transcrição/genética
16.
Nat Commun ; 11(1): 1515, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251291

RESUMO

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.


Assuntos
Evolução Molecular Direcionada/métodos , Escherichia coli/enzimologia , Depuradores de Radicais Livres/metabolismo , Engenharia Metabólica , Álcool Feniletílico/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Vias Biossintéticas/genética , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Estudos de Viabilidade , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Álcool Feniletílico/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tirosina/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Ácido Vanílico/metabolismo
17.
Acta Trop ; 207: 105485, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32277927

RESUMO

Carbapenems are traditionally recognized to be the last resort drugs to treat infections due to MDR organisms such as E. coli. As such, the emergence of New Delhi metallo-ß-lactamase-producing E. coli strains have become a challenging threat to the public health. In this regard, we examined the molecular characteristics of carbapenem-resistant E. coli (CRE) isolated from waterfowls in China's tropical island, Hainan. A total of 311 single E. coli strains were obtained from 20 various farms of healthy ducks and geese in 2 districts of Hainan island. The CRE strains were initially identified via phenotypic resistance and modified Hodge test. PCR assay and subsequent nucleotide sequencing were used to detect different types of carbapenemase encoding genes (blaNDM, blaVIM, blaIMP, blaOXA and blaKPC). In addition, MLST and PFGE analyses were also performed. Among the 311 E. coli strains, 8 strains were detected to produce a single type of carbapenemase i.e. NDM-1 (2.6%). A total of 5 sequence types (STs) were observed, of which ST10 was the most prevalent accounting for 37.5% (3/8). Moreover, these 8 isolates yielded 6 different PFGE clusters but showed approximately related PFGE types, suggesting the propagation of similar clone between the farms. This is the first report on the identification of NDM-1-producing E. coli from waterfowls in Hainan island, China. Our results emphasize the need for better efforts to control the further spread of NDM-1-producing E. coli strains in this tropical island.


Assuntos
Patos/microbiologia , Escherichia coli/isolamento & purificação , Gansos/microbiologia , beta-Lactamases/biossíntese , Animais , Escherichia coli/enzimologia , Ilhas
18.
Nucleic Acids Res ; 48(10): 5616-5623, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32343306

RESUMO

Bacterial ribosomal RNAs (rRNAs) are transcribed as precursors and require processing by Ribonucleases (RNases) to generate mature and functional rRNAs. Although the initial steps of rRNA processing in Escherichia coli (E. coli) were described several decades ago, the enzymes responsible for the final steps of 5S and 23S rRNA 5'-end maturation have remained unknown. Here, I show that RNase AM, a recently identified 5' to 3' exonuclease, performs the last step of 5S rRNA 5'-end maturation. RNase AM was also found to generate the mature 5' end of 23S rRNA, subsequent to a newly identified prior processing step. Additionally, RNase AM was found to mature the 5' end of 16S rRNA, a reaction previously attributed to RNase G. These findings indicate a major role for RNase AM in cellular RNA metabolism and establish a biological role for the first 5' to 3' RNA exonuclease identified in E. coli.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Exorribonucleases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/metabolismo , RNA Ribossômico 5S/metabolismo , Escherichia coli/genética
19.
Biochim Biophys Acta Mol Cell Res ; 1867(8): 118719, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32302670

RESUMO

The bacterial twin-arginine (Tat) pathway serves in the exclusive secretion of folded proteins with bound cofactors. While Tat pathways in Gram-negative bacteria and chloroplast thylakoids consist of conserved TatA, TatB and TatC subunits, the Tat pathways of Bacillus species and many other Gram-positive bacteria stand out for their minimalist nature with the core translocase being composed of essential TatA and TatC subunits only. Here we addressed the question whether the minimal TatAyCy translocase of Bacillus subtilis recruits additional cellular components that modulate its activity. To this end, TatAyCy was purified by affinity- and size exclusion chromatography, and interacting co-purified proteins were identified by mass spectrometry. This uncovered the cell envelope stress responsive LiaH protein as an accessory subunit of the TatAyCy complex. Importantly, our functional studies show that Tat expression is tightly trailed by LiaH induction, and that LiaH itself determines the capacity and quality of TatAyCy-dependent protein translocation. In contrast, LiaH has no role in high-level protein secretion via the general secretion (Sec) pathway. Altogether, our observations show that protein translocation by the minimal Tat translocase TatAyCy is tightly intertwined with an adequate bacterial response to cell envelope stress. This is consistent with a critical need to maintain cellular homeostasis, especially when the membrane is widely opened to permit passage of large fully-folded proteins via Tat.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Estresse Fisiológico/fisiologia , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Dobramento de Proteína , Transporte Proteico/fisiologia , Especificidade por Substrato
20.
Biochim Biophys Acta Bioenerg ; 1861(8): 148203, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32305411

RESUMO

The quinol oxidation site QD in E. coli respiratory nitrate reductase A (EcNarGHI) reacts with the three isoprenoid quinones naturally synthesized by the bacterium, i.e. ubiquinones (UQ), menaquinones (MK) and demethylmenaquinones (DMK). The binding mode of the demethylmenasemiquinone (DMSK) intermediate to the EcNarGHI QD quinol oxidation site is analyzed in detail using 1,2H hyperfine (hf) spectroscopy in combination with H2O/D2O exchange experiments and DFT modeling, and compared to the menasemiquinone one bound to the QD site (MSKD) previously studied by us. DMSKD and MSKD are shown to bind in a similar and strongly asymmetric manner through a short (~1.7 Å) H-bond. The origin of the specific hf pattern resolved on the DMSKD field-swept EPR spectrum is unambiguously ascribed to slightly inequivalent contributions from two ß-methylene protons of the isoprenoid side chain. DFT calculations show that their large isotropic hf coupling constants (Aiso ~12 and 15 MHz) are consistent with both (i) a specific highly asymmetric binding mode of DMSKD and (ii) a near in-plane orientation of its isoprenyl chain at Cß relative to the aromatic ring, which differs by ~90° to that predicted for free or NarGHI-bound MSK. Our results provide new insights into how the conformation and the redox properties of different natural quinones are selectively fine-tuned by the protein environment at a single Q site. Such a fine-tuning most likely contributes to render NarGHI as an efficient and flexible respiratory enzyme to be used upon rapid variations of the Q-pool content.


Assuntos
Teoria da Densidade Funcional , Escherichia coli/enzimologia , Nitrato Redutase/metabolismo , Quinonas/metabolismo , Análise Espectral , Modelos Moleculares , Nitrato Redutase/química , Ligação Proteica , Conformação Proteica
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