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1.
Science ; 368(6489): 424-427, 2020 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-32217749

RESUMEN

Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2'-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and ß. Because of the transient subunit association, an atomic resolution structure of an active α2ß2 RNR complex has been elusive. We used a doubly substituted ß2, E52Q/(2,3,5)-trifluorotyrosine122-ß2, to trap wild-type α2 in a long-lived α2ß2 complex. We report the structure of this complex by means of cryo-electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.


Asunto(s)
Proteínas de Escherichia coli/química , Ribonucleótido Reductasas/química , Biocatálisis , Dominio Catalítico , Microscopía por Crioelectrón , Proteínas de Escherichia coli/genética , Holoenzimas/química , Holoenzimas/genética , Conformación Proteica , Ribonucleótido Reductasas/genética , Tirosina/química
2.
BMC Infect Dis ; 20(1): 108, 2020 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-32033541

RESUMEN

BACKGROUND: Urinary tract infection (UTI) is a common cause of morbidity worldwide. Uropathogenic Escherichia coli (UPEC) bacteria are the major cause of urinary tract infections. UPEC strains derive from different phylogenetic groups and possess an arsenal of virulence factors that contribute to their ability to overcome different defense mechanisms and cause disease. The objective of this study was to identify phylogroup and virulence genes of UPEC among urinary tract infection patients. METHODS: A cross sectional study was conducted from January 1, 2017 to October 9, 2017. E. coli bacteria were isolated from UTI patients using culture and conventional biochemical tests. Identification of phylogroup and genes that encodes for virulence factors was done using multiplex polymerase chain reaction (PCR). Data was processed and analyzed with SPSS version16.0 and Epi-info version 3.4.1 software. RESULTS: The most common urologic clinical manifestation combinations in this study were dysuria, urine urgency and urgency incontinence. The frequent UPEC virulence gene identified was fimH 164 (82%), followed by aer 109 (54.5%), hly 103 (51.5%), pap 59 (29.5%), cnf 58 (29%), sfa 50 (25%) and afa 24 (12%).There was significant association between pap gene and urine urgency (p-0.016); sfa and dysuria and urine urgency (p-0.019 and p-0.043 respectively); hly and suprapubic pain (p-0.002); aer and suprapubic pain, flank pain and fever (p-0.017, p-0.040, p-0.029 respectively). Majority of E. coli isolates were phylogroup B2 60(30%) followed by D 55(27.5%), B1 48(24%) and A 37(18.5%). There was significant association between E. coli phylogroup B2 and three virulence genes namely afa, pap, and sfa (p-0.014, p-0.002, p-0.004 respectively). CONCLUSION: In this study the most frequent E. coli virulence gene was fimH, followed by aer, hly, pap, cnf, sfa and afa respectively. There was significant association between E. coli virulence genes and clinical symptoms of UTI. The phylogenetic analysis indicates majority of uropathogenic E. coli isolates were phylogroup B2 followed by phylogroup D. Phylogroup B2 carries more virulence genes. Hence, targeting major UPEC phylogroup and virulence genes for potential vaccine candidates is essential for better management of UTI and further research has to be conducted in this area.


Asunto(s)
Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Genes Bacterianos , Filogenia , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/patogenicidad , Virulencia/genética , Adulto , Estudios Transversales , Etiopía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Encuestas y Cuestionarios , Escherichia coli Uropatógena/aislamiento & purificación , Factores de Virulencia/genética
3.
Nucleic Acids Res ; 48(5): 2199-2208, 2020 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-32009151

RESUMEN

Microorganisms use zinc-sensing regulators to alter gene expression in response to changes in the availability of zinc, an essential micronutrient. Under zinc-replete conditions, the Fur-family metalloregulator Zur binds to DNA tightly in its metallated repressor form to Zur box operator sites, repressing the transcription of zinc uptake transporters. Derepression comes from unbinding of the regulator, which, under zinc-starvation conditions, exists in its metal-deficient non-repressor forms having no significant affinity with Zur box. While the mechanism of transcription repression by Zur is well-studied, little is known on how derepression by Zur could be facilitated. Using single-molecule/single-cell measurements, we find that in live Escherichia coli cells, Zur's unbinding rate from DNA is sensitive to Zur protein concentration in a first-of-its-kind biphasic manner, initially impeded and then facilitated with increasing Zur concentration. These results challenge conventional models of protein unbinding being unimolecular processes and independent of protein concentration. The facilitated unbinding component likely occurs via a ternary complex formation mechanism. The impeded unbinding component likely results from Zur oligomerization on chromosome involving inter-protein salt-bridges. Unexpectedly, a non-repressor form of Zur is found to bind chromosome tightly, likely at non-consensus sequence sites. These unusual behaviors could provide functional advantages in Zur's facile switching between repression and derepression.


Asunto(s)
ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Transcripción Genética , Sitios de Unión , Cromosomas Bacterianos/química , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Unión Proteica , Multimerización de Proteína , Análisis de la Célula Individual , Zinc/metabolismo
4.
Can J Microbiol ; 66(4): 328-336, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32017602

RESUMEN

This study examined the biofilm-forming ability of six non-O157 Shiga-toxin-producing Escherichia coli (STEC) strains: O116:H21, wzx-Onovel5:H19, O129:H21, O129:H23, O26:H11, and O154:H10 on stainless steel coupons after 24, 48, and 72 h of incubation at 22 °C and after 168 h at 10 °C. The results of crystal violet staining revealed that strains O129:H23 and O154:H10 were able to form biofilms on both the submerged surface and the air-liquid interface of coupons, whereas strains O116:H21, wzx-Onovel5:H19, O129:H21, and O26:H11 formed biofilm only at the air-liquid interface. Viable cell counts and scanning electron microscopy showed that biofilm formation increased (p < 0.05) over time. The biofilm-forming ability of non-O157 STEC was strongest (p < 0.05) at 22 °C after 48 h of incubation. The strongest biofilm former regardless of temperature was O129:H23. Generally, at 10 °C, weak to no biofilm was observed for isolates O154:H10, O116:H21, wzx-Onovel5:H19, O26:H11, and O129:H21 after 168 h. This study found that temperature affected the biofilm-forming ability of non-O157 STEC strains. Overall, our data indicate a high potential for biofilm formation by the isolates at 22 °C, suggesting that non-O157 STEC strains could colonize stainless steel within food-processing facilities. This could serve as a potential source of adulteration and promote the dissemination of these potential pathogens in food.


Asunto(s)
Biopelículas , Manipulación de Alimentos/instrumentación , Escherichia coli Shiga-Toxigénica/fisiología , Contaminación de Equipos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/crecimiento & desarrollo , Acero Inoxidable/química
5.
Poult Sci ; 99(2): 1117-1123, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-32029147

RESUMEN

Colibacillosis, caused by Escherichia coli, is one of the most common bacterial diseases of chickens. The high incidence and considerable economic losses associated with colibacillosis make it a significant concern worldwide. In recent years, the efficacy of colistin has been severely impacted by the emergence of plasmid-mediated colistin resistance genes, especially mcr-1. Therefore, monitoring of antibiotic resistance, particularly colistin resistance, amongst E. coli strains is vitally important to the future growth and sustainability of the poultry industry. In this study, a total of 130 E. coli strains were isolated from the livers of chickens displaying symptoms of colibacillosis in Tai'an, China. Isolates were screened for their susceptibility to various antibiotics and for the presence of mobile colistin resistance genes and other antibiotic resistance genes. Overall, 75 (57.7%) isolates showed resistance to colistin and were positive for mcr-1. The mobile colistin resistance genes, mcr-2, -3, and -4, were not detected in this study. Of the 75 mcr-1-positive isolates, all (100%) also carried tetracycline resistance genes, 71 (94.7%) also contained genes associated with ß-lactam resistance, 59 (78.7%) contained aminoglycoside resistance genes, and 57 (76%) contained sulfonamide resistance genes. This high prevalence of multidrug resistance among mcr-1-positive E. coli isolates, including the production of extended-spectrum ß-lactamases, is highly concerning. The surveillance findings presented here will be conducive to our understanding of the prevalence and characteristics of multidrug-resistance in E. coli in the Tai'an area and will provide a better scientific basis for the clinical treatment of colibacillosis in chickens.


Asunto(s)
Pollos , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Escherichia coli/veterinaria , Escherichia coli/efectos de los fármacos , Enfermedades de las Aves de Corral/epidemiología , Animales , China/epidemiología , Escherichia coli/genética , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Pruebas de Sensibilidad Microbiana/veterinaria , Enfermedades de las Aves de Corral/microbiología , Prevalencia
6.
Nat Commun ; 11(1): 904, 2020 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-32060271

RESUMEN

The generation of a chemical system capable of replication and evolution is a key objective of synthetic biology. This could be achieved by in vitro reconstitution of a minimal self-sustaining central dogma consisting of DNA replication, transcription and translation. Here, we present an in vitro translation system, which enables self-encoded replication and expression of large DNA genomes under well-defined, cell-free conditions. In particular, we demonstrate self-replication of a multipartite genome of more than 116 kb encompassing the full set of Escherichia coli translation factors, all three ribosomal RNAs, an energy regeneration system, as well as RNA and DNA polymerases. Parallel to DNA replication, our system enables synthesis of at least 30 encoded translation factors, half of which are expressed in amounts equal to or greater than their respective input levels. Our optimized cell-free expression platform could provide a chassis for the generation of a partially self-replicating in vitro translation system.


Asunto(s)
Replicación del ADN , Escherichia coli/genética , Genoma Bacteriano , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biosíntesis de Proteínas , Biología Sintética , Transcripción Genética
7.
Proc Natl Acad Sci U S A ; 117(8): 4152-4157, 2020 02 25.
Artículo en Inglés | MEDLINE | ID: mdl-32029596

RESUMEN

Whenever a genetically homogenous population of bacterial cells is exposed to antibiotics, a tiny fraction of cells survives the treatment, the phenomenon known as bacterial persistence [G.L. Hobby et al., Exp. Biol. Med. 50, 281-285 (1942); J. Bigger, The Lancet 244, 497-500 (1944)]. Despite its biomedical relevance, the origin of the phenomenon is still unknown, and as a rare, phenotypically resistant subpopulation, persisters are notoriously hard to study and define. Using computerized tracking we show that persisters are small at birth and slowly replicating. We also determine that the high-persister mutant strain of Escherichia coli, HipQ, is associated with the phenotype of reduced phenotypic inheritance (RPI). We identify the gene responsible for RPI, ydcI, which encodes a transcription factor, and propose a mechanism whereby loss of phenotypic inheritance causes increased frequency of persisters. These results provide insight into the generation and maintenance of phenotypic variation and provide potential targets for the development of therapeutic strategies that tackle persistence in bacterial infections.


Asunto(s)
Farmacorresistencia Bacteriana/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Ampicilina/farmacología , Antibacterianos/farmacología , Escherichia coli/genética , Escherichia coli/fisiología , Proteínas de Escherichia coli/genética , Microfluídica , Modelos Biológicos , Mutación
8.
Proc Natl Acad Sci U S A ; 117(7): 3528-3534, 2020 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-32015130

RESUMEN

In the cell, proteins are synthesized from N to C terminus and begin to fold during translation. Cotranslational folding mechanisms are therefore linked to elongation rate, which varies as a function of synonymous codon usage. However, synonymous codon substitutions can affect many distinct cellular processes, which has complicated attempts to deconvolve the extent to which synonymous codon usage can promote or frustrate proper protein folding in vivo. Although previous studies have shown that some synonymous changes can lead to different final structures, other substitutions will likely be more subtle, perturbing predominantly the protein folding pathway without radically altering the final structure. Here we show that synonymous codon substitutions encoding a single essential enzyme lead to dramatically slower cell growth. These mutations do not prevent active enzyme formation; instead, they predominantly alter the protein folding mechanism, leading to enhanced degradation in vivo. These results support a model in which synonymous codon substitutions can impair cell fitness by significantly perturbing cotranslational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.


Asunto(s)
Cloranfenicol O-Acetiltransferasa/química , Cloranfenicol O-Acetiltransferasa/genética , Codón/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , Mutación Silenciosa , Cloranfenicol O-Acetiltransferasa/metabolismo , Uso de Codones , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/metabolismo , Biosíntesis de Proteínas , Pliegue de Proteína
9.
Proc Natl Acad Sci U S A ; 117(7): 3610-3620, 2020 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-32024753

RESUMEN

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Factor Tu de Elongación Peptídica/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Guanosina Trifosfato/metabolismo , Cinética , Factor Tu de Elongación Peptídica/genética , Biosíntesis de Proteínas , ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/genética , Subunidades Ribosómicas Grandes/genética , Subunidades Ribosómicas Grandes/metabolismo , Ribosomas/genética
10.
Nat Commun ; 11(1): 743, 2020 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-32029744

RESUMEN

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array.


Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas Quimiotácticas Aceptoras de Metilo/química , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Escherichia coli/genética , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestructura , Histidina Quinasa/química , Histidina Quinasa/genética , Histidina Quinasa/ultraestructura , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Proteínas Quimiotácticas Aceptoras de Metilo/ultraestructura , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura
11.
PLoS Comput Biol ; 16(1): e1007643, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-32004314

RESUMEN

For a genetically identical microbial population, multi-gene expression in various environments requires effective allocation of limited resources and precise control of heterogeneity among individual cells. However, it is unclear how resource allocation and cell-to-cell variation jointly shape the overall performance. Here we demonstrate a Simpson's paradox during overexpression of multiple genes: two competing proteins in single cells correlated positively for every induction condition, but the overall correlation was negative. Yet this phenomenon was not observed between two competing mRNAs in single cells. Our analytical framework shows that the phenomenon arises from competition for translational resource, with the correlation modulated by both mRNA and ribosome variability. Thus, heterogeneity plays a key role in single-cell multi-gene expression and provides the population with an evolutionary advantage, as demonstrated in this study.


Asunto(s)
Regulación Bacteriana de la Expresión Génica/genética , Expresión Génica/genética , Genes Bacterianos/genética , Biología Computacional , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fenómenos Genéticos/genética , Modelos Estadísticos , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
12.
PLoS One ; 15(2): e0228959, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32084148

RESUMEN

Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77, O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis.


Asunto(s)
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Toxinas Bacterianas/toxicidad , Células CACO-2 , Citotoxinas/metabolismo , Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/toxicidad , Humanos , Serina Endopeptidasas/metabolismo , Sistemas de Secreción Tipo V/metabolismo , Factores de Virulencia/metabolismo
13.
PLoS One ; 15(1): e0225202, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31940351

RESUMEN

Plasmids are potent vehicles for spread of antibiotic resistance genes in bacterial populations and often persist in the absence of selection due to efficient maintenance mechanisms. We previously constructed non-conjugative high copy number plasmid vectors that efficiently displace stable plasmids from enteric bacteria in a laboratory context by blocking their replication and neutralising their addiction systems. Here we assess a low copy number broad-host-range self-transmissible IncP-1 plasmid as a vector for such curing cassettes to displace IncF and IncK plasmids. The wild type plasmid carrying the curing cassette displaces target plasmids poorly but derivatives with deletions near the IncP-1 replication origin that elevate copy number about two-fold are efficient. Verification of this in mini IncP-1 plasmids showed that elevated copy number was not sufficient and that the parB gene, korB, that is central to its partitioning and gene control system, also needs to be included. The resulting vector can displace target plasmids from a laboratory population without selection and demonstrated activity in a mouse model although spread is less efficient and requires additional selection pressure.


Asunto(s)
Infecciones Bacterianas/genética , Variaciones en el Número de Copia de ADN/genética , Farmacorresistencia Bacteriana/genética , Plásmidos/genética , Animales , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Conjugación Genética/genética , ADN Primasa/genética , Modelos Animales de Enfermedad , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/genética , Especificidad del Huésped/genética , Humanos , Ratones , Plásmidos/efectos de los fármacos
14.
BMC Bioinformatics ; 21(1): 19, 2020 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-31937255

RESUMEN

BACKGROUND: In order to improve the accuracy of constraint-based metabolic models, several approaches have been developed which intend to integrate additional biological information. Two of these methods, MOMENT and GECKO, incorporate enzymatic (kcat) parameters and enzyme mass constraints to further constrain the space of feasible metabolic flux distributions. While both methods have been proven to deliver useful extensions of metabolic models, they may considerably increase size and complexity of the models and there is currently no tool available to fully automate generation and calibration of such enzyme-constrained models from given stoichiometric models. RESULTS: In this work we present three major developments. We first conceived short MOMENT (sMOMENT), a simplified version of the MOMENT approach, which yields the same predictions as MOMENT but requires significantly fewer variables and enables direct inclusion of the relevant enzyme constraints in the standard representation of a constraint-based model. When measurements of enzyme concentrations are available, these can be included as well leading in the extreme case, where all enzyme concentrations are known, to a model representation that is analogous to the GECKO approach. Second, we developed the AutoPACMEN toolbox which allows an almost fully automated creation of sMOMENT-enhanced stoichiometric metabolic models. In particular, this includes the automatic read-out and processing of relevant enzymatic data from different databases and the reconfiguration of the stoichiometric model with embedded enzymatic constraints. Additionally, tools have been developed to adjust (kcat and enzyme pool) parameters of sMOMENT models based on given flux data. We finally applied the new sMOMENT approach and the AutoPACMEN toolbox to generate an enzyme-constrained version of the E. coli genome-scale model iJO1366 and analyze its key properties and differences with the standard model. In particular, we show that the enzyme constraints improve flux predictions (e.g., explaining overflow metabolism and other metabolic switches) and demonstrate, for the first time, that these constraints can markedly change the spectrum of metabolic engineering strategies for different target products. CONCLUSIONS: The methodological and tool developments presented herein pave the way for a simplified and routine construction and analysis of enzyme-constrained metabolic models.


Asunto(s)
Enzimas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Automatización , Enzimas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Ingeniería Metabólica , Redes y Vías Metabólicas , Modelos Biológicos
15.
Nucleic Acids Res ; 48(3): 1583-1598, 2020 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-31956908

RESUMEN

Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.


Asunto(s)
Proteínas Bacterianas/genética , Bambermicinas/biosíntesis , Productos Biológicos/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Proteínas Bacterianas/antagonistas & inhibidores , GMP Cíclico/genética , GMP Cíclico/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/genética , Nucleótidos/genética , Peptidoglicano Glicosiltransferasa/antagonistas & inhibidores , Liasas de Fósforo-Oxígeno/genética , Sistemas de Mensajero Secundario/genética , Streptomycetaceae/genética , Streptomycetaceae/metabolismo , Factores de Transcripción/antagonistas & inhibidores
16.
J Zoo Wildl Med ; 50(4): 813-821, 2020 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-31926511

RESUMEN

Accredited zoos and animal parks play an important role in animal health research and conservation, providing important insights on matters of public health including zoonotic infectious diseases and antimicrobial resistance (AMR). The emergence and spread of AMR is a complex phenomenon that jeopardizes human and animal health and also threatens the long-term survival of endangered species. The presence of ß-lactamases in clinical isolates is particularly significant as they can jeopardize the efficacy of critically important antimicrobials. Although the presence of ß-lactamases and extended-spectrum ß-lactamases (ESBLs) producing Enterobacteriaceae in zoo animals has been reported, data are not available for northern European countries. In addition, few data are available on phylogenetic grouping of Escherichia coli isolated from zoo animals that can provide additional information on the host-bacterium relationship and on the pathogenicity of isolates. This study aimed to characterize fecal E. coli isolated from 33 healthy zoo animals from 22 species in Ireland, using conventional and molecular microbiological methods. All E. coli isolates were ampicillin resistant, but combined resistance to amoxicillin and clavulanic acid was not detected. Three E. coli isolates sampled from one Amur tiger, one Bornean orangutan, and one Southern white rhino were multidrug resistant, and blaTEM was detected in E. coli recovered from the Amur tiger and the Bornean orangutan. Other ß-lactamases, including ESBLs and AmpCs and plasmid-mediated mcr-1 and mcr-2, were not detected. Overall, E. coli isolates investigated were susceptible to the majority of the antimicrobials tested, and only two animals shed E. coli carrying ß-lactamase-encoding genes. The majority of isolates belonged to phylogenetic group B1. The screening of the AMR phenotype and genotype of zoo animal E. coli provides useful data that is relevant to antimicrobial stewardship in the zoo veterinary services and relevant to the bank of knowledge needed for tackling AMR.


Asunto(s)
Animales de Zoológico , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Heces/microbiología , Animales , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Filogenia
17.
PLoS One ; 15(1): e0227249, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31961890

RESUMEN

Gene expression is an intrinsically stochastic process. Fluctuations in transcription and translation lead to cell-to-cell variations in mRNA and protein levels affecting cellular function and cell fate. Here, using fluorescence time-lapse microscopy, we quantify noise dynamics in an artificial operon in Escherichia coli, which is based on the native operon of ColicinE2, a toxin. In the natural system, toxin expression is controlled by a complex regulatory network; upon induction of the bacterial SOS response, ColicinE2 is produced (cea gene) and released (cel gene) by cell lysis. Using this ColicinE2-based operon, we demonstrate that upon induction of the SOS response noise of cells expressing the operon is significantly lower for the (mainly) transcriptionally regulated gene cea compared to the additionally post-transcriptionally regulated gene cel. Likewise, we find that mutations affecting the transcriptional regulation by the repressor LexA do not significantly alter the population noise, whereas specific mutations to post-transcriptionally regulating units, strongly influence noise levels of both genes. Furthermore, our data indicate that global factors, such as the plasmid copy number of the operon encoding plasmid, affect gene expression noise of the entire operon. Taken together, our results provide insights on how noise in a native toxin-producing operon is controlled and underline the importance of post-transcriptional regulation for noise control in this system.


Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Colicinas/genética , Proteínas de Escherichia coli/genética , Operón , ARN Mensajero/genética , Respuesta SOS en Genética , Procesos Estocásticos
18.
Res Vet Sci ; 128: 308-314, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31901569

RESUMEN

As most pathogens invade the bodies through the mucosa, it is crucial to develop vaccines that induce mucosal immunity. To this end, we generated a safe and effective vaccine candidate that displayed fimbrial protein 987P of enterotoxigenic Escherichia coli (ETEC) on the surface of Lactobacillus casei (L.casei) CICC 6105 by using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. After gavage inoculation of the recombinant strain pLA-987P/L.casei into specific-pathogen-free (SPF) BALB/c mice, high levels of mucosal immunoglobulin A (IgA) were induced in fecal samples, intestine and lung lavage fluids and systemic immunoglobulin G of IgG subclasses (IgG1, IgG2b, and IgG2a) was produced in serum. T-cell proliferation assays showed the stimulation index (SI) of the groups immunized with pLA-987P/L.casei to be significantly higher than that of the control group. The recombinant L.casei promoted T cells to produce both Th1 and Th2 cytokines, while the number of splenic IL-4 Spot forming cells (SFC) exceeded the number of IFN-γ SFC by 2.26-fold (P < .01). >83.3% of the vaccinated mice were protected from challenge with a lethal dose of virulent strain C83916. These results indicate that the recombinant L.casei expressing ETEC 987P fimbrial protein could elicit a protective immune response against ETEC 987P infection effectively.


Asunto(s)
Adhesinas de Escherichia coli/inmunología , Escherichia coli Enterotoxigénica/inmunología , Vacunas contra Escherichia coli/biosíntesis , Proteínas Fimbrias/inmunología , Lactobacillus casei/inmunología , Microorganismos Modificados Genéticamente/inmunología , Adhesinas de Escherichia coli/genética , Administración Oral , Animales , Antígenos Heterófilos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Proteínas Fimbrias/genética , Inmunidad Humoral , Inmunidad Mucosa , Inmunogenicidad Vacunal , Lactobacillus casei/genética , Ratones , Ratones Endogámicos BALB C , Transformación Bacteriana/genética , Transformación Bacteriana/inmunología , Vacunación/métodos
19.
Nat Commun ; 11(1): 547, 2020 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-31992706

RESUMEN

TrkH is a bacterial ion channel implicated in K+ uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Potenciales de la Membrana/fisiología , Canales de Potasio/química , Canales de Potasio/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Difosfato , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transporte Biológico/fisiología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutagénesis , Potasio/metabolismo , Canales de Potasio/genética , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Eliminación de Secuencia , Vibrio parahaemolyticus/genética , Difracción de Rayos X
20.
Nucleic Acids Res ; 48(5): 2348-2356, 2020 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-31960057

RESUMEN

Gene gain by horizontal gene transfer is a major pathway of genome innovation in bacteria. The current view posits that acquired genes initially need to be silenced and that a bacterial chromatin protein, H-NS, plays a role in this silencing. However, we lack direct observation of the early fate of a horizontally transferred gene to prove this theory. We combine sequencing, flow cytometry and sorting, followed by microscopy to monitor gene expression and its variability after large-scale random insertions of a reporter gene in a population of Escherichia coli bacteria. We find that inserted promoters have a wide range of gene-expression variability related to their location. We find that high-expression clones carry insertions that are not correlated with H-NS binding. Conversely, binding of H-NS correlates with silencing. Finally, while most promoters show a common level of extrinsic noise, some insertions show higher noise levels. Analysis of these high-noise clones supports a scenario of switching due to transcriptional interference from divergent ribosomal promoters. Altogether, our findings point to evolutionary pathways where newly-acquired genes are not necessarily silenced, but may immediately explore a wide range of expression levels to probe the optimal ones.


Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas Fimbrias/genética , Regulación Bacteriana de la Expresión Génica , Mutagénesis Insercional , Regiones Promotoras Genéticas , Cromatina/química , Cromatina/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Fimbrias/metabolismo , Dosificación de Gen , Silenciador del Gen , Transferencia de Gen Horizontal , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Transgenes
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