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1.
Proc Natl Acad Sci U S A ; 119(33): e2200061119, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35960846

RESUMO

DNA looping has emerged as a central paradigm of transcriptional regulation, as it is shared across many living systems. One core property of DNA looping-based regulation is its ability to greatly enhance repression or activation of genes with only a few copies of transcriptional regulators. However, this property based on a small number of proteins raises the question of the robustness of such a mechanism with respect to the large intracellular perturbations taking place during growth and division of the cell. Here we address the issue of sensitivity to variations of intracellular parameters of gene regulation by DNA looping. We use the lac system as a prototype to experimentally identify the key features of the robustness of DNA looping in growing Escherichia coli cells. Surprisingly, we observe time intervals of tight repression spanning across division events, which can sometimes exceed 10 generations. Remarkably, the distribution of such long time intervals exhibits memoryless statistics that is mostly insensitive to repressor concentration, cell division events, and the number of distinct loops accessible to the system. By contrast, gene regulation becomes highly sensitive to these perturbations when DNA looping is absent. Using stochastic simulations, we propose that the observed robustness to division emerges from the competition between fast, multiple rebinding events of repressors and slow initiation rate of the RNA polymerase. We argue that fast rebinding events are a direct consequence of DNA looping that ensures robust gene repression across a range of intracellular perturbations.


Assuntos
Divisão Celular , DNA Bacteriano , Óperon Lac , Divisão Celular/genética , DNA Bacteriano/química , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Repressores Lac/genética , Repressores Lac/metabolismo , Conformação de Ácido Nucleico , Análise de Célula Única
2.
Nature ; 606(7916): 953-959, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35705811

RESUMO

Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial for the maintenance of cellular integrity and enable survival in challenging environments1-5. The function of the outer membrane is dependent on outer membrane proteins (OMPs), which are inserted into the membrane by the ß-barrel assembly machine6,7 (BAM). Growing Escherichia coli cells segregate old OMPs towards the poles by a process known as binary partitioning, the basis of which is unknown8. Here we demonstrate that peptidoglycan underpins the spatiotemporal organization of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little effect on its activity, leading to preferential insertion of OMPs at division sites. The synchronization of OMP biogenesis with cell wall growth results in the binary partitioning of OMPs as cells divide. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation, a potential vulnerability that could be exploited in future antibiotic design.


Assuntos
Proteínas da Membrana Bacteriana Externa , Membrana Celular , Escherichia coli , Peptidoglicano , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Parede Celular/metabolismo , Escherichia coli/química , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Peptidoglicano/biossíntese , Peptidoglicano/metabolismo , Dobramento de Proteína
3.
Proc Natl Acad Sci U S A ; 119(20): e2201585119, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35544692

RESUMO

Many cellular activities in bacteria are organized according to their growth rate. The notion that ppGpp measures the cell's growth rate is well accepted in the field of bacterial physiology. However, despite decades of interrogation and the identification of multiple molecular interactions that connects ppGpp to some aspects of cell growth, we lack a system-level, quantitative picture of how this alleged "measurement" is performed. Through quantitative experiments, we show that the ppGpp pool responds inversely to the rate of translational elongation in Escherichia coli. Together with its roles in inhibiting ribosome biogenesis and activity, ppGpp closes a key regulatory circuit that enables the cell to perceive and control the rate of its growth across conditions. The celebrated linear growth law relating the ribosome content and growth rate emerges as a consequence of keeping a supply of ribosome reserves while maintaining elongation rate in slow growth conditions. Further analysis suggests the elongation rate itself is detected by sensing the ratio of dwelling and translocating ribosomes, a strategy employed to collapse the complex, high-dimensional dynamics of the molecular processes underlying cell growth to perceive the physiological state of the whole.


Assuntos
Escherichia coli , Guanosina Tetrafosfato , Elongação Traducional da Cadeia Peptídica , Ribossomos , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Guanosina Tetrafosfato/metabolismo , Ribossomos/metabolismo
4.
Proc Natl Acad Sci U S A ; 119(14): e2115032119, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35344432

RESUMO

Cell-to-cell heterogeneity in gene expression and growth can have critical functional consequences, such as determining whether individual bacteria survive or die following stress. Although phenotypic variability is well documented, the dynamics that underlie it are often unknown. This information is important because dramatically different outcomes can arise from gradual versus rapid changes in expression and growth. Using single-cell time-lapse microscopy, we measured the temporal expression of a suite of stress-response reporters in Escherichia coli, while simultaneously monitoring growth rate. In conditions without stress, we found several examples of pulsatile expression. Single-cell growth rates were often anticorrelated with reporter levels, with changes in growth preceding changes in expression. These dynamics have functional consequences, which we demonstrate by measuring survival after challenging cells with the antibiotic ciprofloxacin. Our results suggest that fluctuations in both gene expression and growth dynamics in stress-response networks have direct consequences on survival.


Assuntos
Escherichia coli , Regulação Bacteriana da Expressão Gênica , Estresse Fisiológico , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Fenótipo , Análise de Célula Única , Estresse Fisiológico/genética
5.
Mar Drugs ; 20(3)2022 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-35323468

RESUMO

Volatile compounds from the marine cyanolichen Lichina pygmaea, collected from the Moroccan Atlantic coast, were extracted by hydrodistillation and their putative chemical composition was investigated by gas chromatography coupled to mass spectrometry (GC/MS). Based on the obtained results, Lichina pygmaea volatile compounds (LPVCs) were mainly dominated by sesquiterpenes compounds, where γ-himachalene, ß-himachalene, (2E,4E)-2,4 decadienal and α-himachalene were assumed to be the most abundant constituents, with percentage of 37.51%, 11.71%, 8.59% and 7.62%, respectively. LPVCs depicted significant antimicrobial activity against all tested strains (Staphylococcus aureus CCMM B3, Pseudomonas aeruginosa DSM 50090, Escherichia coli ATCC 8739 and Candida albicans CCMM-L4) with minimum inhibitory concentration (MIC) values within the range of 1.69-13.5 mg/mL. Moreover, this LPVC showed interesting scavenging effects on the 2,2-diphenyl-1-picrylhydrazyl radical with an IC50 of 0.21 mg/mL. LPVCs could be an approving resource with moderate antimicrobial potential and interesting antioxidant activity for cosmetics and pharmaceutical applications.


Assuntos
Anti-Infecciosos , Antioxidantes , Ascomicetos/química , Sesquiterpenos , Compostos Orgânicos Voláteis , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Compostos de Bifenilo/química , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Picratos/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Sesquiterpenos/análise , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/isolamento & purificação , Compostos Orgânicos Voláteis/farmacologia
6.
Proc Natl Acad Sci U S A ; 119(10): e2117930119, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35239434

RESUMO

SignificanceWhile most small, regulatory RNAs are thought to be "noncoding," a few have been found to also encode a small protein. Here we describe a 164-nucleotide RNA that encodes a 28-amino acid, amphipathic protein, which interacts with aerobic glycerol-3-phosphate dehydrogenase and increases dehydrogenase activity but also base pairs with two mRNAs to reduce expression. The coding and base-pairing sequences overlap, and the two regulatory functions compete.


Assuntos
Carbono/metabolismo , Escherichia coli/metabolismo , RNA Bacteriano/fisiologia , Meios de Cultura , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Galactose/metabolismo , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo
7.
J Microbiol ; 60(2): 192-206, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35102526

RESUMO

Toxin-antitoxin (TA) systems are growth-controlling genetic elements consisting of an intracellular toxin protein and its cognate antitoxin. TA systems have been spread among microbial genomes through horizontal gene transfer and are now prevalent in most bacterial and archaeal genomes. Under normal growth conditions, antitoxins tightly counteract the activity of the toxins. Upon stresses, antitoxins are inactivated, releasing activated toxins, which induce growth arrest or cell death. In this study, among nine functional TA modules in Bosea sp. PAMC 26642 living in Arctic lichen, we investigated the functionality of BoHigBA2. BohigBA2 is located close to a genomic island and adjacent to flagellar gene clusters. The expression of BohigB2 induced the inhibition of E. coli growth at 37°C, which was more manifest at 18°C, and this growth defect was reversed when BohigA2 was co-expressed, suggesting that this BoHigBA2 module might be an active TA module in Bosea sp. PAMC 26642. Live/dead staining and viable count analyses revealed that the BoHigB2 toxin had a bactericidal effect, causing cell death. Furthermore, we demonstrated that BoHigB2 possessed mRNA-specific ribonuclease activity on various mRNAs and cleaved only mRNAs being translated, which might impede overall translation and consequently lead to cell death. Our study provides the insight to understand the cold adaptation of Bosea sp. PAMC 26642 living in the Arctic.


Assuntos
Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Bradyrhizobiaceae/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Sistemas Toxina-Antitoxina , Antitoxinas/genética , Regiões Árticas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Família Multigênica , RNA Mensageiro/metabolismo
8.
Microbiol Spectr ; 10(1): e0225321, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35196813

RESUMO

Persister cells are a small subpopulation of phenotypic variants that survive high concentrations of bactericidal antibiotics. Their survival mechanisms are not heritable and can be formed stochastically or triggered by environmental stresses such as antibiotic treatment. In this study, high-throughput screening of an Escherichia coli promoter library and subsequent validation experiments identified several genes whose expression was upregulated by antibiotic treatment. Among the identified genes, waaG, guaA, and guaB were found to be important in persister cell formation in E. coli as their deletion significantly enhanced the sensitivity of cells to various antibiotics. The GuaA and GuaB enzymes form the upstream reactions of ppGpp (a global persister molecule) biosynthesis, and the deletion of guaA and guaB drastically perturbs the ppGpp regulon in E. coli. WaaG, a lipopolysaccharide glucosyltransferase, plays an important role in shaping the outer membrane structure, and the deletion of waaG dissipates the proton gradient (ΔpH) component of cellular proton motive force (PMF), perturbs cellular ATP production, and reduces type I persister formation in stationary phase. Active respiration in the stationary phase, which drives the PMF, was previously shown to play a critical role in type I persister formation, and our results associated with the waaG deficient strain further corroborate these findings. IMPORTANCE Persistence is a nonheritable trait by which normal growing cells switch phenotypically to antibiotic tolerant persister cells. This transient state enables persister cells to recover and grow into an antibiotic-sensitive population. Persister cells have been observed in many pathogenic and nonpathogenic bacteria. Previous studies highlight the complexity and diversity of bacterial persister-cell mechanisms, many of which still remain to be elucidated. Here, using promoter and knockout cell libraries in Escherichia coli, we have identified genes that reveal novel persister mechanisms. As persistence is a critical survival strategy that evolved in many bacteria, our study will enhance the current molecular-level understanding of this conserved mechanism.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Regiões Promotoras Genéticas , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana
9.
Viruses ; 14(2)2022 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-35215879

RESUMO

Salmonella and Escherichia coli (E. coli) food contamination could lead to serious foodborne diseases. The gradual increase in the incidence of foodborne disease invokes new and efficient methods to limit food pathogenic microorganism contamination. In this study, a polyvalent broad-spectrum Escherichia phage named Tequatrovirus EP01 was isolated from pig farm sewage. It could lyse both Salmonella Enteritidis (S. Enteritidis) and E. coli and exhibited broad host range. EP01 possessed a short latent period (10 min), a large burst size (80 PFU/cell), and moderate pH stability (4-10) and appropriate thermal tolerance (30-80 °C). Electron microscopy and genome sequence revealed that EP01 belonged to T4-like viruses genus, Myoviridae family. EP01 harbored 12 CDSs associated with receptor-binding proteins and lacked virulence genes and drug resistance genes. We tested the inhibitory effect of EP01 on S. Enteritidis, E. coli O157:H7, E. coli O114:K90 (B90), and E. coli O142:K86 (B) in liquid broth medium (LB). EP01 could significantly reduce the counts of all tested strains compared with phage-free groups. We further examined the effectiveness of EP01 in controlling bacterial contamination in two kinds of foods (meat and milk) contaminated with S. Enteritidis, E. coli O157:H7, E. coli O114:K90 (B90), and E. coli O142:K86 (B), respectively. EP01 significantly reduced the viable counts of all the tested bacteria (2.18-6.55 log10 CFU/sample, p < 0.05). A significant reduction of 6.55 log10 CFU/cm2 (p < 0.001) in bacterial counts on the surface of meat was observed with EP01 treatment. Addition of EP01 at MOI of 1 decreased the counts of bacteria by 4.3 log10 CFU/mL (p < 0.001) in milk. Generally, the inhibitory effect exhibited more stable at 4 °C than that at 28 °C, whereas the opposite results were observed in milk. The antibacterial effects were better at MOI of 1 than that at MOI of 0.001. These results suggests that phage EP01-based method is a promising strategy of controlling Salmonella and Escherichia coli pathogens to limit microbial food contamination.


Assuntos
Escherichia coli/virologia , Contaminação de Alimentos/prevenção & controle , Myoviridae/fisiologia , Salmonella enteritidis/virologia , Animais , Bacteriólise , Escherichia coli/crescimento & desenvolvimento , Microbiologia de Alimentos , Genoma Viral , Especificidade de Hospedeiro , Carne/microbiologia , Leite/microbiologia , Myoviridae/classificação , Myoviridae/genética , Myoviridae/isolamento & purificação , Filogenia , Salmonella enteritidis/crescimento & desenvolvimento , Esgotos/virologia , Suínos
10.
Int J Mol Sci ; 23(3)2022 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-35163069

RESUMO

Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising a combination of promoters and terminators enabling the expression of nCas9-based BE and sgRNA to nontoxic levels, eventually leading to successful base editing. We demonstrate the use of IRI-CCE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e) for bacteria, exhibiting that each independent BE has its specific editing pattern for a given target site depending on protospacer length. In summary, CRISPR-BE components expressed without lethal effects on cell survival in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and sgRNAs.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/crescimento & desenvolvimento , Edição de Genes/métodos , Sistemas CRISPR-Cas , Citidina Desaminase/genética , Escherichia coli/genética , Glicoproteínas/genética , Humanos , Proteínas Nucleares/genética , Proteínas/genética
11.
Int J Mol Sci ; 23(3)2022 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-35163759

RESUMO

Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Técnicas Bacteriológicas/instrumentação , Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Membrana Transportadoras/genética , Técnicas Bacteriológicas/métodos , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Glicólise , Glioxilatos/metabolismo , Fenômenos Magnéticos , Oxigênio/metabolismo , Aldeído Pirúvico/metabolismo , Voo Espacial , Ausência de Peso
12.
Microbiol Spectr ; 10(1): e0170621, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35171014

RESUMO

Two diverse conjugative plasmids can interact within bacterial cells. However, to the best of our knowledge, the interaction between blaCTX-M-bearing IncFII plasmid and mcr-1-carrying IncI2 plasmid colocated on the same bacterial host has not been reported. This study was initiated to explore the interaction and to analyze the reasons that these two plasmids are often coresident in multidrug-resistant Escherichia coli. To assess the interactions on plasmid stabilities, fitness costs, and transfer rates, we constructed two groups of isogenic derivatives, C600FII, C600I2, and C600FII+I2 of E. coli C600 and J53FII, J53I2, and J53FII+I2 of E. coli J53, respectively. We found that carriage of FII and I2 plasmids, independently and together, had not impaired the growth of the bacterial host. It was difficult for the single plasmid FII or I2 in E. coli C600 to reach stable persistence for a long time in an antibiotic-free environment, while the stability would be striking improved when they coresided. Meanwhile, plasmids FII and I2, whether together or apart, could notably enhance the fitness advantage of the host; moreover, E. coli coharboring plasmids FII and I2 presented more obvious fitness advantage than that carrying single plasmid FII. Coresident plasmids FII and I2 could accelerate horizontal cotransfer by conjugation. The transfer rates from a strain carrying coresident FII and I2 plasmids increased significantly when it mated with a recipient cell carrying one of them. Our findings highlight the advantages of coinhabitant FII and I2 plasmids in E. coli to drive the persistence and spread of plasmid-carried blaCTX-M and mcr-1 genes, although the molecular mechanisms of their coresidence warrant further study. IMPORTANCE More and more Enterobacteriaceae carry both blaCTX-M and mcr-1, which are usually located on IncFII-type and IncI2-type plasmids in the same bacterial host, respectively. However, the study on advantages of coresident plasmids in bacterial host is still sparse. Here, we investigated the stability, fitness cost, and cotransfer traits associated with coresident IncFII-type and IncI2-type plasmids in E. coli. Our results show that coinhabitant plasmids in E. coli are more stable, confer more fitness advantages, and are easier to transfer and cotransfer than a single plasmid IncFII or IncI2. Our findings confirm the advantages of coresident plasmids of blaCTX-M-bearing IncFII and mcr-1-bearing IncI2 in clinical E. coli, which will pose a serious threat to clinical therapy and public health.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Plasmídeos/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Enterobacteriaceae/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia
13.
PLoS One ; 17(2): e0264094, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35213576

RESUMO

Lactoferrin (LF) is a multifunctional protein with a broad spectrum of antimicrobial activities. In this study, we investigated the antimicrobial activity of LF against the potato common scab pathogen Streptomyces scabiei, which causes severe damage to potato tubers. LF derived from bovine (bLF) had much higher activity against S. scabiei than human LF. The minimal inhibitory concentration of bLF was 3.9 µM. The effects of both apo-bLF (iron-free) and holo-bLF (iron-saturated) on S. scabiei were not different. Bovine lactoferricin (LFcinB), a short peptide with a length of 25 amino acid residues located in the N-terminal region of bLF, showed antimicrobial activity against S. scabiei, similar to that of bLF. These results indicated that the antimicrobial activity of bLF against S. scabiei cannot be attributed to its iron-chelating effect but to the bioactivity of its peptides. When S. scabiei was treated with the fusion protein of mCherry-LFcinB (red fluorescent protein) expressed in Escherichia coli, the pseudohyphal cells instantly glowed, indicating that the peptide electrostatically binds to the surface of S. scabiei. An assay of synthetic peptides, with modified number of arginine (Arg) and tryptophan (Trp) residues based on the antimicrobial center (RRWQWR) of LFcinB showed that Trp residues are implicated in the antimicrobial activity against S. scabiei; however, Arg residues are also necessary to carry Trp residues to the cell surface to fully exert its activity. Although the single amino acid effect of Trp had low activity, Trp derivatives showed much higher activity against S. scabiei, suggesting that the derivatives effectively bind to the cell surface (cell membrane) by themselves without a carrier. Thus, amino acid derivatives might be considered effective and alternative antimicrobial substances.


Assuntos
Antibacterianos/farmacologia , Lactoferrina/farmacologia , Solanum tuberosum/microbiologia , Streptomyces/crescimento & desenvolvimento , Animais , Antibacterianos/química , Bovinos , Escherichia coli/crescimento & desenvolvimento , Humanos , Lactoferrina/química
14.
Int J Mol Sci ; 23(3)2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35163108

RESUMO

The biodiversity of microorganisms is maintained by intricate nets of interactions between competing species. Impaired functionality of human microbiomes correlates with their reduced biodiversity originating from aseptic environmental conditions and antibiotic use. Microbiomes of wild animals are free of these selective pressures. Microbiota provides a protecting shield from invasion by pathogens in the wild, outcompeting their growth in specific ecological niches. We applied ultrahigh-throughput microfluidic technologies for functional profiling of microbiomes of wild animals, including the skin beetle, Siberian lynx, common raccoon dog, and East Siberian brown bear. Single-cell screening of the most efficient killers of the common human pathogen Staphylococcus aureus resulted in repeated isolation of Bacillus pumilus strains. While isolated strains had different phenotypes, all of them displayed a similar set of biosynthetic gene clusters (BGCs) encoding antibiotic amicoumacin, siderophore bacillibactin, and putative analogs of antimicrobials including bacilysin, surfactin, desferrioxamine, and class IId cyclical bacteriocin. Amicoumacin A (Ami) was identified as a major antibacterial metabolite of these strains mediating their antagonistic activity. Genome mining indicates that Ami BGCs with this architecture subdivide into three distinct families, characteristic of the B. pumilus, B. subtilis, and Paenibacillus species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated B. pumilus strains efficiently inhibit the growth of both Gram-positive S. aureus and Gram-negative E. coli in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing B. pumilus can be attributed to the metabolomic profile predetermined by their biosynthetic fingerprint. Ultrahigh-throughput isolation of natural probiotic strains from wild animal microbiomes, as well as their metabolic reprogramming, opens up a new avenue for pathogen control and microbiome remodeling in the food industry, agriculture, and healthcare.


Assuntos
Animais Selvagens/microbiologia , Antibacterianos/administração & dosagem , Bacillus pumilus/química , Escherichia coli/crescimento & desenvolvimento , Microbiota , Probióticos/administração & dosagem , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , Genoma Bacteriano , Metaboloma , Família Multigênica , Probióticos/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos
15.
Int J Mol Sci ; 23(3)2022 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-35163814

RESUMO

Combining multiple drugs or biologically active substances for wound healing could not only resist the formation of multidrug resistant pathogens, but also achieve better therapeutic effects. Herein, the hydrophobic fluoroquinolone antibiotic ciprofloxacin (CIP) and the hydrophilic broad-spectrum antibiotic tetracycline hydrochloride (TH) were introduced into the coaxial polycaprolactone/gelatin (PCL/GEL) nanofiber mat with CIP loaded into the PCL (core layer) and TH loaded into the GEL (shell layer), developing antibacterial wound dressing with the co-delivering of the two antibiotics (PCL-CIP/GEL-TH). The nanostructure, physical properties, drug release, antibacterial property, and in vitro cytotoxicity were investigated accordingly. The results revealed that the CIP shows a long-lasting release of five days, reaching the releasing rate of 80.71%, while the cumulative drug release of TH reached 83.51% with a rapid release behavior of 12 h. The in vitro antibacterial activity demonstrated that the coaxial nanofiber mesh possesses strong antibacterial activity against E. coli and S. aureus. In addition, the coaxial mats showed superior biocompatibility toward human skin fibroblast cells (hSFCs). This study indicates that the developed PCL-CIP/GEL-TH nanofiber membranes hold enormous potential as wound dressing materials.


Assuntos
Ciprofloxacina/administração & dosagem , Escherichia coli/crescimento & desenvolvimento , Pele/citologia , Staphylococcus aureus/crescimento & desenvolvimento , Tetraciclina/administração & dosagem , Cicatrização , Animais , Bandagens , Linhagem Celular , Ciprofloxacina/química , Ciprofloxacina/farmacologia , Modelos Animais de Doenças , Composição de Medicamentos , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Gelatina/química , Humanos , Viabilidade Microbiana , Nanofibras , Poliésteres/química , Pele/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Tetraciclina/química , Tetraciclina/farmacologia
16.
Molecules ; 27(4)2022 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-35208949

RESUMO

Nanotechnology has become a dire need of the current era and the green synthesis of nanoparticles offers several advantages over other methods. Nanobiotechnology is an emerging field that contributes to many domains of human life, such as the formulation of nanoscale drug systems or nanomedicine for the diagnosis and treatment of diseases. Medicinal plants are the main sources of lead compounds, drug candidates and drugs. This work reports the green synthesis of Ag nanoparticles (AgNPs) using the aqueous bark extract of Zanthozylum armatum, which was confirmed by a UV absorption at 457 nm. XRD analysis revealed an average size of 18.27 nm and SEM showed the particles' spherical shape, with few irregularly shaped particles due to the aggregation of the AgNPs. FT-IR revealed the critical functional groups of phytochemicals which acted as reducing and stabilizing agents. The bark extract showed rich flavonoids (333 mg RE/g) and phenolic contents (82 mg GAE/g), which were plausibly responsible for its high antioxidant potency (IC50 = 14.61 µg/mL). Extract-loaded AgNPs exhibited the highest but equal inhibition against E. coli and P. aeruginosa (Z.I. 11.0 mm), whereas methanolic bark extract inhibited to a lesser extent, but equally to both pathogens (Z.I. 6.0 mm). The aqueous bark extract inhibited P. aeruginosa (Z.I. 9.0 mm) and (Z.I. 6.0 mm) E. coli. These findings-especially the biosynthesis of spherical AgNPs of 18.27 nm-provide promise for further investigation and for the development of commercializable biomedical products.


Assuntos
Antibacterianos , Escherichia coli/crescimento & desenvolvimento , Nanopartículas Metálicas/química , Extratos Vegetais/química , Pseudomonas aeruginosa/crescimento & desenvolvimento , Prata , Zanthoxylum/química , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Prata/química , Prata/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier
17.
Molecules ; 27(3)2022 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-35163885

RESUMO

Several strategies, including inducer addition and biosensor use, have been developed for dynamical regulation. However, the toxicity, cost, and inflexibility of existing strategies have created a demand for superior technology. In this study, we designed an optogenetic dual-switch system and applied it to increase polyhydroxybutyrate (PHB) production. First, an optimized chromatic acclimation sensor/regulator (RBS10-CcaS#10-CcaR) system (comprising an optimized ribosomal binding site (RBS), light sensory protein CcaS, and response regulator CcaR) was selected for a wide sensing range of approximately 10-fold between green-light activation and red-light repression. The RBS10-CcaS#10-CcaR system was combined with a blue light-activated YF1-FixJ-PhlF system (containing histidine kinase YF1, response regulator FixJ, and repressor PhlF) engineered with reduced crosstalk. Finally, the optogenetic dual-switch system was used to rewire the metabolic flux for PHB production by regulating the sequences and intervals of the citrate synthase gene (gltA) and PHB synthesis gene (phbCAB) expression. Consequently, the strain RBS34, which has high gltA expression and a time lag of 3 h, achieved the highest PHB content of 16.6 wt%, which was approximately 3-fold that of F34 (expressed at 0 h). The results indicate that the optogenetic dual-switch system was verified as a practical and convenient tool for increasing PHB production.


Assuntos
Proteínas de Bactérias/metabolismo , Butiratos/metabolismo , Citrato (si)-Sintase/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/metabolismo , Optogenética , Proteínas de Bactérias/genética , Citrato (si)-Sintase/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Histidina Quinase/genética , Luz , Regiões Promotoras Genéticas
18.
Gene ; 821: 146295, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35181503

RESUMO

Response to acid stress is critical for Escherichia coli to successfully complete its life-cycle. Acid resistance is an indispensable mechanism that allows neutralophilic bacteria, such as E. coli, to survive in the gastrointestinal tract. Escherichia coli acid tolerance has been extensively studied over the past decades, and most studies have focused on mechanisms of gene regulation. Bacterial two-component signal transduction systems sense and respond to external environmental changes through regulating genes expression. However, there has been little research on the mechanism of the TorR/TorS system in acid resistance, and how TorR/TorS regulate the expression ofacid-resistantgenes is still unclear. We found that TorR/TorS deletion in E. coli cells led to a growth defect in extreme acid conditions,andthis defectmightdepend on the nutritional conditionsand growth phase.TorS/TorR sensed an extremely acidic environment, and this TorR phosphorylation process might not be entirely dependent on TorS.RNA-seqand RT-qPCR results suggested that TorR regulated expressions of gadB, gadC, hdeA, gadE, mdtE, mdtF, gadX, and slp acid-resistant genes. Compared with wild-type cells, the stress response factor RpoSlevels and itsexpressions were significantly decreased in Δ torR cellsstimulated by extreme acid. And under these circumstances, the expression of iraM was significantly reduced to 0.6-fold inΔ torR cells. Electrophoreticmobility shift assay showed that TorR-His6 could interact with the rpoS promoter sequence in vitro. ß-galactosidase activity assayresultsapprovedthat TorR might bind the rpoS promoter region in vivo. After the mutation of the TorR-box in the rpoS promoter region, these interactions were no longer observed. Taken together, we propose thatTorS and potential Hanks model Ser/Thr kinase received an external acid stress signal and then phosphorylated TorR, which guided the expressions of a variety of acid resistance genes. Moreover,TorRcoped with extreme acid environmentsthroughRpoS, levels of which might be maintained byIraM. Finally,TorR may confer E. coli with the abilityto resist gastric acid, allowing the bacterium to reach the surface of the terminal ileum and large intestine mucosal epithelial cells through the gastric acid barrier, andestablishcolonization and pathogenicity.


Assuntos
Ácidos/efeitos adversos , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fosfotransferases/genética , Fator sigma/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácido Gástrico , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mutagênese Sítio-Dirigida , Fosforilação , Fosfotransferases/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de RNA , Fator sigma/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo
19.
Int J Mol Sci ; 23(3)2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-35163624

RESUMO

An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell's receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E. coli production of active multi-disulfide-bonded RBD.


Assuntos
Anticorpos Antivirais/sangue , Escherichia coli/crescimento & desenvolvimento , Peptídeos/administração & dosagem , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Clonagem Molecular , Dissulfetos/metabolismo , Escherichia coli/genética , Feminino , Humanos , Soros Imunes/metabolismo , Imunização , Camundongos , Camundongos Endogâmicos ICR , Peptídeos/genética , Peptídeos/imunologia , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Th2/metabolismo
20.
Molecules ; 27(3)2022 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-35164333

RESUMO

BACKGROUND: Infectious diseases represent a significant global strain on public health security and impact on socio-economic stability all over the world. The increasing resistance to the current antimicrobial treatment has resulted in the crucial need for the discovery and development of novel entities for the infectious treatment with different modes of action that could target both sensitive and resistant strains. METHODS: Compounds were synthesized using the classical organic chemistry methods. Prediction of biological activity spectra was carried out using PASS and PASS-based web applications. Pharmacophore modeling in LigandScout software was used for quantitative modeling of the antibacterial activity. Antimicrobial activity was evaluated using the microdilution method. AutoDock 4.2® software was used to elucidate probable bacterial and fungal molecular targets of the studied compounds. RESULTS: All compounds exhibited better antibacterial potency than ampicillin against all bacteria tested. Three compounds were tested against resistant strains MRSA, P. aeruginosa and E. coli and were found to be more potent than MRSA than reference drugs. All compounds demonstrated a higher degree of antifungal activity than the reference drugs bifonazole (6-17-fold) and ketoconazole (13-52-fold). Three of the most active compounds could be considered for further development of the new, more potent antimicrobial agents. CONCLUSION: Compounds 5b (Z)-3-(3-hydroxyphenyl)-5-((1-methyl-1H-indol-3-yl)methylene)-2-thioxothiazolidin-4-one and 5g (Z)-3-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxo-thiazolidin-3-yl]-benzoic acid as well as 5h (Z)-3-(5-((5-methoxy-1H-indol-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)benzoic acid can be considered as lead compounds for further development of more potent and safe antibacterial and antifungal agents.


Assuntos
Antibacterianos/síntese química , Antifúngicos/síntese química , Fungos/crescimento & desenvolvimento , Tiazolidinas/síntese química , Ampicilina/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fungos/efeitos dos fármacos , Imidazóis/farmacologia , Cetoconazol/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Relação Estrutura-Atividade , Tiazolidinas/química , Tiazolidinas/farmacologia
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