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1.
Nat Commun ; 11(1): 4827, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973167

RESUMO

In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure localized immediately downstream of stop codons, and provide experimental evidence for its role in governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that RTSs are abundant, being associated with 18%-65% of genes in 128 analyzed bacterial genomes representing all phyla, and are selectively depleted when translation re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious. Our results support a potentially universal role for the RTS in controlling translation termination-insulation and re-initiation across bacteria.


Assuntos
Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon/genética , RNA Mensageiro/química , RNA Mensageiro/fisiologia , Bactérias/classificação , Bactérias/genética , Códon de Terminação/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos/genética , Iniciação Traducional da Cadeia Peptídica , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Ribossomos/metabolismo
2.
PLoS Comput Biol ; 16(9): e1008159, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925923

RESUMO

Intracellular spatial heterogeneity is frequently observed in bacteria, where the chromosome occupies part of the cell's volume and a circuit's DNA often localizes within the cell. How this heterogeneity affects core processes and genetic circuits is still poorly understood. In fact, commonly used ordinary differential equation (ODE) models of genetic circuits assume a well-mixed ensemble of molecules and, as such, do not capture spatial aspects. Reaction-diffusion partial differential equation (PDE) models have been only occasionally used since they are difficult to integrate and do not provide mechanistic understanding of the effects of spatial heterogeneity. In this paper, we derive a reduced ODE model that captures spatial effects, yet has the same dimension as commonly used well-mixed models. In particular, the only difference with respect to a well-mixed ODE model is that the association rate constant of binding reactions is multiplied by a coefficient, which we refer to as the binding correction factor (BCF). The BCF depends on the size of interacting molecules and on their location when fixed in space and it is equal to unity in a well-mixed ODE model. The BCF can be used to investigate how spatial heterogeneity affects the behavior of core processes and genetic circuits. Specifically, our reduced model indicates that transcription and its regulation are more effective for genes located at the cell poles than for genes located on the chromosome. The extent of these effects depends on the value of the BCF, which we found to be close to unity. For translation, the value of the BCF is always greater than unity, it increases with mRNA size, and, with biologically relevant parameters, is substantially larger than unity. Our model has broad validity, has the same dimension as a well-mixed model, yet it incorporates spatial heterogeneity. This simple-to-use model can be used to both analyze and design genetic circuits while accounting for spatial intracellular effects.


Assuntos
Bactérias , Redes Reguladoras de Genes/genética , Genes Bacterianos/genética , Modelos Biológicos , Bactérias/química , Bactérias/citologia , Bactérias/genética , Biologia Computacional , Difusão , Espaço Intracelular/química , Espaço Intracelular/genética
3.
PLoS One ; 15(8): e0236054, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750053

RESUMO

INTRODUCTION: Multi-drug resistance is a major challenge in the control of tuberculosis. Despite newer modalities for diagnosis and treatment, people are still suffering from this disease. Understanding the common gene mutations conferring rifampicin and isoniazid resistance is crucial for the implementation of effective molecular tools at local and national levels. Hence, this study aimed to evaluate the molecular detection of rifampicin and isoniazid-resistant gene mutations in M.tuberculosis isolates in Addis Ababa, Ethiopia. METHOD: Health Center-based cross-sectional study was conducted between January and September 2017 in Addis Ababa, Ethiopia. The collected sputum samples were processed for mycobacterial isolation and Region of difference 9 based polymerase chain reaction for species identification. To characterize the rifampicin and isoniazid-resistant M. tuberculosis isolates, a molecular genetic assay (GenoType MTBDRplus) was used; the assay is based on DNA-STRIP technology. RESULT: Culture positivity was confirmed in 82.6% (190/230) of smear-positive newly diagnosed pulmonary tuberculosis cases enrolled in the study. From 190 isolates 93.2% were sensitive for both rifampicin and isoniazid, and 6.8% of the isolates were resistant to at least one of the tested anti-TB drugs. Gene mutations were observed in all studied multidrug resistance-associated gene loci (rpoB, katG, and inhA). Two isolates exhibited heteroresistance, a mutated, as well as wild type sequences, were detected in the respective strains. MDR-TB case was observed in 1.1% (2/190) of the cases. All the MDR-TB cases were positive for HIV and found to have a history of prior hospital admission. CONCLUSION: In our finding a relatively high prevalence of any drug resistance was observed and the overall prevalence of multidrug-resistant tuberculosis was 1.1%.The majority of drug-resistant isolates demonstrated common mutations. Heteroresistant strains were detected, signaling the existence of an M.tuberculosis population with variable responses to anti-tuberculosis drugs or of mixed infections.


Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Adolescente , Adulto , Antituberculosos/uso terapêutico , Estudos Transversais , Análise Mutacional de DNA , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Etiópia , Feminino , Genes Bacterianos/genética , Loci Gênicos/genética , Humanos , Isoniazida/farmacologia , Isoniazida/uso terapêutico , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase , Rifampina/farmacologia , Rifampina/uso terapêutico , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adulto Jovem
4.
J Environ Manage ; 274: 111190, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32771773

RESUMO

The objectives of this study were to clarify the distribution as well as the removal mechanism of antibiotic resistance genes (ARGs) within three sludge treatment wetlands (STWs) during a loading period of two years. Three STW units were constructed and run during the loading period: Unit 1 (U1) built with aeration tubes, Unit 2 (U2) built with aeration tubes and reeds, and Unit 3 (U3) built with reeds only. All targeted ARGs, intI1, and 16S rRNA were detected in residual sludge in the order of magnitude: 16S rRNA>sul1>intI1>sul2>tetC>tetA>ermB. The abundance of the five targeted ARGs, intI1, and 16S rRNA increased in residual sludge, during the loading period, which may be due to the increase in bacteria caused by the continuous import of exogenous nutrients. However, STWs can also remove ARGs from sewage during the loading period and the mean removal efficiency of five resistance genes was 73.0%. The removal rates of intI1 and 16S rRNA were 73.5% and 78.6%, respectively. Positive correlations were detected in abundance of most ARGs and intI1, as well as 16S rRNA (P < 0.05), indicating intI1 plays a vital part in the propagation of ARGs. The removal of bacteria harboring these genes also occurs in the STW units.


Assuntos
Esgotos , Áreas Alagadas , Animais , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos/genética , RNA Ribossômico 16S/genética , Águas Residuárias/análise
5.
Proc Natl Acad Sci U S A ; 117(29): 17418-17428, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636267

RESUMO

Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer-dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO2-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO2-fixing modules to improve photosynthesis.


Assuntos
Organelas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Synechococcus/metabolismo , Carbono/metabolismo , Ciclo do Carbono , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Fotossíntese , Subunidades Proteicas/metabolismo , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética , Synechococcus/genética , Transcriptoma
6.
Proc Natl Acad Sci U S A ; 117(29): 17249-17259, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641516

RESUMO

Control of infections caused by carbapenem-resistant Klebsiella pneumoniae continues to be challenging. The success of this pathogen is favored by its ability to acquire antimicrobial resistance and to spread and persist in both the environment and in humans. The emergence of clinically important clones, such as sequence types 11, 15, 101, and 258, has been reported worldwide. However, the mechanisms promoting the dissemination of such high-risk clones are unknown. Unraveling the factors that play a role in the pathobiology and epidemicity of K. pneumoniae is therefore important for managing infections. To address this issue, we studied a carbapenem-resistant ST-15 K. pneumoniae isolate (Kp3380) that displayed a remarkable adherent phenotype with abundant pilus-like structures. Genome sequencing enabled us to identify a chaperone-usher pili system (Kpi) in Kp3380. Analysis of a large K. pneumoniae population from 32 European countries showed that the Kpi system is associated with the ST-15 clone. Phylogenetic analysis of the operon revealed that Kpi belongs to the little-characterized γ2-fimbrial clade. We demonstrate that Kpi contributes positively to the ability of K. pneumoniae to form biofilms and adhere to different host tissues. Moreover, the in vivo intestinal colonizing capacity of the Kpi-defective mutant was significantly reduced, as was its ability to infect Galleria mellonella The findings provide information about the pathobiology and epidemicity of Kpi+ K. pneumoniae and indicate that the presence of Kpi may explain the success of the ST-15 clone. Disrupting bacterial adherence to the intestinal surface could potentially target gastrointestinal colonization.


Assuntos
Fímbrias Bacterianas/genética , Klebsiella pneumoniae/genética , Chaperonas Moleculares/genética , Células A549 , Animais , Antibacterianos , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Carbapenêmicos/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Células Epiteliais/microbiologia , Europa (Continente) , Feminino , Deleção de Genes , Genes Bacterianos/genética , Humanos , Infecções por Klebsiella , Klebsiella pneumoniae/citologia , Klebsiella pneumoniae/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Tipagem de Sequências Multilocus , Óperon , Filogenia
7.
BMC Infect Dis ; 20(1): 544, 2020 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-32711470

RESUMO

BACKGROUND: This study aimed to identify ten different 16S rRNA methyltransferase genes (rmtA, rmtB, rmtC, rmtD, armA, rmtF, npmA, rmtH, rmtE and rmtG) and their coexisting ESBL and carbapenemase with the emergence of three E.coli clones within a single study centre. METHODS: A total of 329 non-duplicate E.coli isolates were studied to detect the presence of 16S rRNA methyltransferases along with ß-lactamases (TEM, SHV, OXA, VEB, GES, PER,CTX-M types, NDM, OXA-48,VIM, IMP and KPC) using PCR assay. Horizontal transferability were validated by transformation and conjugation analysis. Plasmid incompatibility typing and MLST analysis was also performed. RESULTS: A total of 117 isolates were found to be resistant to at least one of the aminoglycoside antibiotics. It was observed that 77 (65.8%) were positive for 16S rRNA methyltransferases. Among them thirty nine isolates were found to harbour only blaCTX-M-15, whereas combination of genes were observed in three isolates (blaVEB+ blaCTX-M-15 in 2 isolates and blaPER + blaCTX-M-15 in 1 isolate). blaNDM and blaOXA-48 like genes were found in 23 and 9 isolates, respectively. All the resistance genes were conjugatively transferable, and incompatibility typing showed multiple 16S rRNA methyltransferase genes were originated from a single Inc. I1 group. MLST analysis detected 3 clones of E.coliST4410, ST1341 and ST3906. CONCLUSION: The present study identified emergence of three clones of E.coli, resistant to aminoglycoside -cephalosporin- carbapenem. This warrants immediate measures to trace their transmission dynamics in order to slow down their spread in clinical setting.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Metiltransferases/genética , beta-Lactamases/genética , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Genes Bacterianos/genética , Humanos , Índia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus
8.
Gene ; 758: 144951, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32683080

RESUMO

Antibiotic resistance is one of the major health concerns of the present century. The direct discharge of urban sewage, hospital effluents, and pharmaceutical wastes increases the concentration of antibiotics in riverine ecosystems. This provides selection pressure for the development of novel antibiotic-resistant strains. In this study, metagenomics approach was employed a for constructing a comprehensive profile of the Antibiotic Resistance Genes (ARGs) identified in the sediments of the Yamuna River. A total of 139 ARGs were identified from 39 microbial species. Abundance analysis revealed that, aminoglycoside, beta-lactam, macrolide, and tetracycline resistance genes were highly abundant in the sediment samples obtained from the Yamuna River. The evolutionary relationships among the ARGs were studied by phylogenetic analyses, which revealed that, the identified resistome comprised eight clusters. Network analysis was performed for investigating the broad-spectrum profiles of the ARGs and their enrichment in different biological functions and pathways. Protein-protein interaction (PPI) analyses revealed that, 76, 36, 18, and 5 Gene Ontology (GO)-terms were significantly enriched in Biological process, Molecular Function, Cellular Component, and KEGG Pathways analysis, respectively. The present study elucidates the ecology of microbial antibiotic resistance in the riverine ecosystem of the Yamuna River and provides novel insights into the environmental hotspots that are amenable to the emergence of ARGs in the contaminated riverine hydrosphere.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Sedimentos Geológicos/microbiologia , Poluentes Químicos da Água/análise , Agricultura , Bactérias/genética , Bactérias/isolamento & purificação , Ecossistema , Genes Bacterianos/genética , Índia , Metagenoma/genética , Metagenômica , Testes de Sensibilidade Microbiana , Filogenia , Uso Excessivo de Medicamentos Prescritos/efeitos adversos , Rios/microbiologia
9.
Arch Microbiol ; 202(8): 2117-2125, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32506149

RESUMO

Plastics composed of polyethylene are non-biodegradable and are mostly harmful to the environment. Literature studies documented that the extent of microbial degradation of low-density polyethylene (LDPE) seems to be insufficient and the underlying mechanisms of such degradation remain unexplored. In the present study, efforts were given to degrade LDPE by a recently isolated bacteria Enterobacter cloacae AKS7. Scanning electron microscopic (SEM) image, tensile strength, and weight loss analysis confirmed the efficient degradation of LDPE by AKS7. To investigate the mechanism, it was observed that with the progression of time, the extent of microbial colonization got increased considerably over the LDPE surface. It was also observed that the organism (AKS7) gradually increased the secretion of extracellular polymeric substances (EPS) suggesting the formation of efficient biofilm over the LDPE surface. Furthermore, to comprehend the role of cell-surface hydrophobicity towards biofilm formation, two mutants of AKS7 were screened that showed a considerable reduction in cell-surface hydrophobicity in contrast to its wild type. The result showed that the mutants revealed compromised LDPE degradation than wild-type cells of AKS7. Further investigation revealed that the mutant cells of AKS7 were incapable of adhering to LDPE in contrast to wild-type cells. Thus, the results demonstrated that the cell-surface hydrophobicity of AKS7 favors the development of microbial biofilm over LDPE that leads to the enhanced degradation of LDPE by AKS7. Therefore, the organism holds the assurance to be considered as a promising bio-remediating agent for the sustainable degradation of polythene-based hazardous waste.


Assuntos
Enterobacter cloacae/genética , Enterobacter cloacae/metabolismo , Recuperação e Remediação Ambiental , Polietileno/metabolismo , Aderência Bacteriana/genética , Biodegradação Ambiental , Biofilmes , Genes Bacterianos/genética , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Varredura , Mutação
10.
Nat Commun ; 11(1): 3105, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561723

RESUMO

Genetic perturbations that affect bacterial resistance to antibiotics have been characterized genome-wide, but how do such perturbations interact with subsequent evolutionary adaptation to the drug? Here, we show that strong epistasis between resistance mutations and systematically identified genes can be exploited to control spontaneous resistance evolution. We evolved hundreds of Escherichia coli K-12 mutant populations in parallel, using a robotic platform that tightly controls population size and selection pressure. We find a global diminishing-returns epistasis pattern: strains that are initially more sensitive generally undergo larger resistance gains. However, some gene deletion strains deviate from this general trend and curtail the evolvability of resistance, including deletions of genes for membrane transport, LPS biosynthesis, and chaperones. Deletions of efflux pump genes force evolution on inferior mutational paths, not explored in the wild type, and some of these essentially block resistance evolution. This effect is due to strong negative epistasis with resistance mutations. The identified genes and cellular functions provide potential targets for development of adjuvants that may block spontaneous resistance evolution when combined with antibiotics.


Assuntos
Antibacterianos/farmacologia , Evolução Molecular Direcionada/métodos , Resistência Microbiana a Medicamentos/genética , Epistasia Genética , Escherichia coli K12/genética , Escherichia coli K12/efeitos dos fármacos , Deleção de Genes , Genes Bacterianos/genética , Seleção Genética/efeitos dos fármacos
11.
Parasitol Res ; 119(8): 2713-2717, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32506253

RESUMO

Here, we provide the first mass molecular screening of medically important mosquitoes for Bartonella species using multiple genetic markers. We examined a total of 72,115 mosquito specimens, morphologically attributed to Aedes vexans (61,050 individuals), Culex pipiens (10,484 individuals) and species of the Anopheles maculipennis complex (581 individuals) for Bartonella spp. The initial screening yielded 63 Bartonella-positive A. vexans mosquitoes (mean prevalence 0.1%), 34 Bartonella-positive C. pipiens mosquitoes (mean prevalence 0.3%) and 158 Bartonella-positive A. maculipennis group mosquitoes (mean prevalence 27.2%). Several different Bartonella ITS sequences were recovered. This study highlights the need for molecular screening of mosquitoes, the most important vectors of arthropod-borne pathogens, for potential bacterial agents.


Assuntos
Infecções por Bartonella/transmissão , Bartonella/isolamento & purificação , Culicidae/microbiologia , Mosquitos Vetores/microbiologia , Animais , Bartonella/classificação , Bartonella/genética , Infecções por Bartonella/epidemiologia , Culicidae/classificação , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Monitoramento Epidemiológico , Europa (Continente)/epidemiologia , Genes Bacterianos/genética , Mosquitos Vetores/classificação
13.
PLoS Negl Trop Dis ; 14(6): e0008342, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32497120

RESUMO

Tick-borne rickettsioses are world-spreading infectious zoonoses. Ticks serve as reservoirs and vectors for Rickettsia and play a key role in transmission of rickettsioses. Most of the Chinese rickettsiosis patients are reported from Northeastern China but the distribution of tick and tick-borne Rickettsia species in Northeastern China remain poorly studied. In this study, a total of 1,286 ticks were captured from the seven counties of Harbin, an area in Northeastern China, and the tick-borne Rickettsia species were identified by PCR and sequencing of rrs, gltA, groEL, ompA and 17-kDa antigen-encoding genes. Of the 5 identified tick species, Haemaphysalis longicornis and Ixodes persulcatus were the predominant tick species in the livestock and vegetation, respectively. Rickettsia raoultii and "Candidatus Rickettsia tarasevichiae" were the two detectable Rickettsia species in the ticks with a 28.8% positive rate but no rickettsiae were found in ticks of Haemaphysalis concinna. R. raoultii detected in 37.6% of the Dermacentor nuttalli, Dermacentor silvarum and H. longicornis ticks while "Ca. R. tarasevichiae" was only present in 22.8% of the I. persulcatus ticks. In particular, the positive rate of both R. raoultii and "Ca. R. tarasevichiae" in ticks from the livestock (40.7%) was significantly higher than that from the vegetation (19.5%). The results indicate that the tick and tick-borne Rickettsia species are diverse in different regions of Harbin due to geographic difference and the ticks from livestock may play a more important role in transmission of rickettsioses to human.


Assuntos
Genes Bacterianos/genética , Rickettsia/classificação , Rickettsia/genética , Carrapatos/microbiologia , Animais , China , Humanos , Ixodidae/microbiologia , Filogenia , Reação em Cadeia da Polimerase , Infecções por Rickettsia/microbiologia , Análise de Sequência , Doenças Transmitidas por Carrapatos/microbiologia
14.
PLoS One ; 15(4): e0232244, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353000

RESUMO

BACKGROUND: Microorganisms living in saline environments are forced to regulate turgor via the synthesis of organic osmoprotective compounds. Microbial adaptation to fluctuations in external salinity includes degradation of compatible solutes. Here we have examined the biochemical pathway of degradation of the cyclic imino acid ectoine, the major osmoprotector in halotolerant methane-utilizing bacteria. METHODS: The BLAST search of the genes involved in ectoine degradation in the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z was performed with the reference sequences of Halomonas elongata. The genes for the key enzymes of the pathway were disrupted by insertion mutagenesis and the cellular metabolites in the methanol extracts of mutant cells were analyzed by HPLC. The doeA gene from Mm. alcaliphilum 20Z was heterologously expressed in Escherichia coli to identify the product of ectoine hydrolysis catalyzed by ectoine hydrolase DoeA. RESULTS: We have shown that the halotolerant methanotroph Mm. alcaliphilum 20Z possesses the doeBDAC gene cluster coding for putative ectoine hydrolase (DoeA), Nα-acetyl-L-2,4-diaminobutyrate deacetylase (DoeB), diaminobutyrate transaminase (DoeD) and aspartate-semialdehyde dehydrogenase (DoeC). The deletion of the doeA gene resulted in accumulation of the higher level of ectoine compared to the wild type strain. Nγ-acetyl-L-2,4-diaminobutyrate (Nγ-acetyl-DAB), a substrate for ectoine synthase, was found in the cytoplasm of the wild type strain. Nα-acetyl-L-2,4-diaminobutyrate (Nα-acetyl-DAB), a substrate for the DoeB enzyme, appeared in the cells as a result of exposure of the doeB mutant to low osmotic pressure. The genes for the enzymes involved in ectoine degradation were found in all aerobic methylotrophs capable of ectoine biosynthesis. These results provide the first evidence for the in vivo operation of the ectoine degradation pathway in methanotrophs and thus expand our understanding of the regulation mechanisms of bacterial osmoadaptation. CONCLUSIONS: During adaptation to the changes in external osmolarity, halophilic and halotolerant methylotrophs cleave ectoine, thereby entering the carbon and nitrogen of the compatible solute to the central metabolic pathways. The biochemical route of ectoine degradation in the halotolerant methanotroph Mm. alcaliphilum 20Z is similar to that in heterotrophic halophiles. We have shown that ectoine hydrolase DoeA in this methanotroph hydrolyzes ectoine with the formation of the only isomer: Nα-acetyl-DAB. All aerobic methylotrophs capable of ectoine biosynthesis harbor the genetic determinants for ectoine degradation.


Assuntos
Diamino Aminoácidos/metabolismo , Redes e Vias Metabólicas/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Halomonas/genética , Halomonas/metabolismo , Redes e Vias Metabólicas/genética , Methylococcaceae/genética , Methylococcaceae/metabolismo , Família Multigênica/genética , Pressão Osmótica/fisiologia , Salinidade
15.
Nucleic Acids Res ; 48(11): 5967-5985, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32406921

RESUMO

During infection of a host, Pseudomonas aeruginosa orchestrates global gene expression to adapt to the host environment and counter the immune attacks. P. aeruginosa harbours hundreds of regulatory genes that play essential roles in controlling gene expression. However, their contributions to the bacterial pathogenesis remain largely unknown. In this study, we analysed the transcriptomic profile of P. aeruginosa cells isolated from lungs of infected mice and examined the roles of upregulated regulatory genes in bacterial virulence. Mutation of a novel regulatory gene pvrA (PA2957) attenuated the bacterial virulence in an acute pneumonia model. Chromatin immunoprecipitation (ChIP)-Seq and genetic analyses revealed that PvrA directly regulates genes involved in phosphatidylcholine utilization and fatty acid catabolism. Mutation of the pvrA resulted in defective bacterial growth when phosphatidylcholine or palmitic acid was used as the sole carbon source. We further demonstrated that palmitoyl coenzyme A is a ligand for the PvrA, enhancing the binding affinity of PvrA to its target promoters. An arginine residue at position 136 was found to be essential for PvrA to bind palmitoyl coenzyme A. Overall, our results revealed a novel regulatory pathway that controls genes involved in phosphatidylcholine and fatty acid utilization and contributes to the bacterial virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Genes Bacterianos/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Animais , Arginina/metabolismo , Sequência de Bases , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Ligantes , Camundongos , Modelos Moleculares , Mutação , Ácido Palmítico/metabolismo , Palmitoil Coenzima A/metabolismo , Fosfatidilcolinas/metabolismo , Pneumonia Bacteriana/microbiologia , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Transcriptoma , Virulência/genética
16.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(2): 132-139, 2020 Apr 08.
Artigo em Chinês | MEDLINE | ID: mdl-32458601

RESUMO

OBJECTIVE: To evaluate the effects of water body environments on the microbial community of Oncomelania hupensis snails in marshlands of the eastern Dongting Lake where natural extinction of O. hupensis snails are found, so as to explore the correlation between the natural extinction of O. hupensis snails and the microbial community in snails. METHODS: Snails were caged water bodies in the Qianliang Lake marshland (Qianliang Lake regions) where natural extinction of snails was found and in the Junshan Park marshland (Junshan Park regions) in the eastern Dongting Lake for 30 days, and then all snails were collected and identified for survival or death. DNA sequencing of the fungi and bacteria was performed in snails before and after immersion in waters, and the biodiversity and abundance were analyzed. RESULTS: The survival rates of O. hupensis snails were 28.0% (70/250) and 64.8% (162/250) in Qianliang Lake regions and Junshan Park regions 30 days after immersion in waters, respectively (χ2 = 81.365, P < 0.01). The number of the fungal community and the biodiversity of the bacterial community were both greater in snails caged in Qianliang Lake regions post-immersion than pre-immersion, and there was a significant difference in the structure of the fungal and bacterial communities. The microbial community with a significant difference included Flavobacteriaceae,which was harmful to O. hupensis snails. CONCLUSIONS: The water body environment affects the composition of the microbial community in O. hupensis snails in marshlands with natural snail distinction around the eastern Dongting Lake; however, further studies are required to investigate whether the natural distinction of snails is caused by water body environments-induced changes of the microbial spectrum in O. hupensis snails.


Assuntos
Biodiversidade , Lagos , Microbiota , Caramujos , Animais , China , Genes Bacterianos/genética , Microbiota/genética , Caramujos/microbiologia , Caramujos/fisiologia , Áreas Alagadas
17.
PLoS Negl Trop Dis ; 14(4): e0008128, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32236098

RESUMO

Cholera continues to be an important public health concern in developing countries where proper hygiene and sanitation are compromised. This severe diarrheal disease is caused by the Gram-negative pathogen Vibrio cholerae belonging to serogroups O1 and O139. Cholera toxin (CT) is the prime virulence factor and is directly responsible for the disease manifestation. The ctxB gene encodes cholera toxin B subunit (CTB) whereas the A subunit (CTA) is the product of ctxA gene. Enzymatic action of CT depends on binding of B pentamers to the lipid-based receptor ganglioside GM1. In recent years, emergence of V. cholerae Haitian variant strains with ctxB7 allele and their rapid spread throughout the globe has been linked to various cholera outbreaks in Africa and Asia. These strains produce classical type (WT) CTB except for an additional mutation in the signal sequence region where an asparagine (N) residue replaces a histidine (H) at the 20th amino acid position (H20N) of CTB precursor (pre-CTB). Here we report that Haitian variant V. cholerae O1 strains isolated in Kolkata produced higher amount of CT compared to contemporary O1 El Tor variant strains under in vitro virulence inducing conditions. We observed that the ctxB7 allele, itself plays a pivotal role in higher CT production. Based on our in silico analysis, we hypothesized that higher accumulation of toxin subunits from ctxB7 allele might be attributed to the structural alteration at the CTB signal peptide region of pre-H20N CTB. Overall, this study provides plausible explanation regarding the hypertoxigenic phenotype of the Haitian variant strains which have spread globally, possibly through positive selection for increased pathogenic traits.


Assuntos
Alelos , Toxina da Cólera/genética , Cólera/microbiologia , Genes Bacterianos/genética , Vibrio cholerae O1/genética , Técnicas de Tipagem Bacteriana , Cólera/epidemiologia , Toxina da Cólera/química , Toxina da Cólera/metabolismo , Surtos de Doenças , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Haiti/epidemiologia , Humanos , RNA Bacteriano , Sorogrupo , Virulência/genética , Fatores de Virulência/genética
18.
Nat Commun ; 11(1): 1614, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32235841

RESUMO

The heterocycle 1,2,3-triazole is among the most versatile chemical scaffolds and has been widely used in diverse fields. However, how nature creates this nitrogen-rich ring system remains unknown. Here, we report the biosynthetic route to the triazole-bearing antimetabolite 8-azaguanine. We reveal that its triazole moiety can be assembled through an enzymatic and non-enzymatic cascade, in which nitric oxide is used as a building block. These results expand our knowledge of the physiological role of nitric oxide synthase in building natural products with a nitrogen-nitrogen bond, and should also inspire the development of synthetic biology approaches for triazole production.


Assuntos
Bactérias/metabolismo , Óxido Nítrico/metabolismo , Triazóis/metabolismo , Azaguanina/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produtos Biológicos , Vias Biossintéticas/genética , Genes Bacterianos/genética , Óxido Nítrico Sintase/metabolismo , Nitrogênio , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo , Biologia Sintética
19.
Nat Commun ; 11(1): 1618, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238808

RESUMO

In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we lack predictive rules for designing effective gRNA target sites. Here, we identify multiple features of bacterial promoters that impose stringent requirements on CRISPRa target sites. Notably, we observe narrow, 2-4 base windows of effective sites with a periodicity corresponding to one helical turn of DNA, spanning ~40 bases and centered ~80 bases upstream of the TSS. However, we also identify two features suggesting the potential for broad scope: CRISPRa is effective at a broad range of σ70-family promoters, and an expanded PAM dCas9 allows the activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.


Assuntos
Bactérias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulação Bacteriana da Expressão Gênica , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Escherichia coli/genética , Proteínas de Escherichia coli , Genes Bacterianos/genética , Regiões Promotoras Genéticas , RNA Guia/genética , Transativadores , Ativação Transcricional
20.
Nat Microbiol ; 5(5): 735-745, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341568

RESUMO

The multidrug-resistant Staphylococcus capitis NRCS-A clone is responsible for sepsis in preterm infants in neonatal intensive care units (NICUs) worldwide. Here, to retrace the spread of this clone and to identify drivers of its specific success, we investigated a representative collection of 250 S. capitis isolates from adults and newborns. Bayesian analyses confirmed the spread of the NRCS-A clone and enabled us to date its emergence in the late 1960s and its expansion during the 1980s, coinciding with the establishment of NICUs and the increasing use of vancomycin in these units, respectively. This dynamic was accompanied by the acquisition of mutations in antimicrobial resistance- and bacteriocin-encoding genes. Furthermore, combined statistical tools and a genome-wide association study convergently point to vancomycin resistance as a major driver of NRCS-A success. We also identified another S. capitis subclade (alpha clade) that emerged independently, showing parallel evolution towards NICU specialization and non-susceptibility to vancomycin, indicating convergent evolution in NICU-associated pathogens. These findings illustrate how the broad use of antibiotics can repeatedly lead initially commensal drug-susceptible bacteria to evolve into multidrug-resistant clones that are able to successfully spread worldwide and become pathogenic for highly vulnerable patients.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Sepse Neonatal/microbiologia , Staphylococcus capitis/efeitos dos fármacos , Staphylococcus capitis/genética , Adulto , Teorema de Bayes , França , Genes Bacterianos/genética , Genoma Bacteriano , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Infecções Estafilocócicas/microbiologia , Staphylococcus capitis/isolamento & purificação , Staphylococcus capitis/patogenicidade , Vancomicina/uso terapêutico
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