Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.850
Filtrar
1.
Arch Microbiol ; 202(1): 105-114, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31485711

RESUMO

Escherichia coli MazF is a toxin protein that cleaves RNA at ACA sequences. Its activation has been thought to cause growth inhibition, primarily through indiscriminate cleavage of RNA. To investigate responses following MazF activation, transcriptomic profiles of mazF-overexpressing and non-overexpressing E. coli K12 cells were compared. Analyses of differentially expressed genes demonstrated that the presence and the number of ACA trimers in RNA was unrelated to cellular RNA levels. Mapping differentially expressed genes onto the chromosome identified two chromosomal segments in which upregulated genes formed clusters, and these segments were absent in the chromosomes of E. coli strains other than K12. These results suggest that MazF regulates selective, rather than indiscriminate, categories of genes, and is involved in the regulation of horizontally acquired genes. We conclude that the primary role of MazF is not only cleaving RNA indiscriminately but also generating a specific cellular state.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , RNA/genética , Proteínas de Ligação a DNA/genética , Endorribonucleases/genética , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , RNA/química
2.
World J Microbiol Biotechnol ; 35(12): 189, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31748890

RESUMO

Fossil fuels consumption impacts the greenhouse gas emissions. Biofuels are considered as alternative renewable energy sources to reduce the fossil fuels dependency. Bioethanol produced by recombinant microorganisms is a widely suggested alternative to increase the yield in fermentation processes. However, ethanol and acetate accumulation under the fermentation process had been described as important stressors for the metabolic capabilities of the microorganisms, stopping the fermentation process and affecting the ethanol yield. Ethanol tolerance is a determining factor in the improvement of fermentative properties of microorganisms; however understanding of ethanol tolerance is limited. The engineered Escherichia coli KO11 strain has been studied in detail and used as an ethanologenic bacteria model. The strain is capable of using glucose and xylose for an efficient ethanol yield. In the current work, the effect of the iron-sulfur cluster (ISC) over-expression in the KO11 strain, on its tolerance and ethanol yield, was evaluated. Fatty acids profiles of membrane phospholipids in the E. coli KO11 were modified under ethanol addition, but not due to the hscA mutation. The hscA mutation provoked a decrease in ethanol tolerance in the Kmp strain when was grown with 2% ethanol, in comparison to KO11 parent strain. Ethanol tolerance was improved in the mutant Kmp complemented with the recombinant isc gene cluster (pJC10 plasmid) from LD50 2.16% to LD50 3.8% ethanol. In batch fermentation on 1 L bioreactor using mineral medium with glucose (120 g/L), the KO11 strain showed ethanol production efficiencies of ~ 76.9%, while the hscA mutant (Kmp) ~ 75.4% and the transformed strain Kmp(pJC10) showed ~ 92.4% efficiency. Ethanol amount increase in the engineered Kmp(pJC10) strain was correlated with less organic acids (such as acetate and lactate) production in the fermentation medium (2.3 g/L), compared to that in the KO11 (17.05 g/L) and the Kmp (16.62 g/L). Alcohol dehydrogenase (ADH) activity was increased ~ 350% in the transformed Kmp(pJC10) strain, whereas in the Kmp mutant, the phosphoglycerate kinase (PGK), pyruvate kinase (PYK), and ADH activities were diminished, comparing to KO11. The results suggest that the isc system over-expression in the ethanologenic E. coli KO11 strain, increases ethanol yield mainly by improving ethanol tolerance and ADH activity, and by redirecting the metabolic flux from acetate synthesis to ethanol.


Assuntos
Ácidos/metabolismo , Tolerância a Medicamentos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Família Multigênica/genética , Álcool Desidrogenase/genética , Técnicas de Cultura Celular por Lotes , Biocombustíveis , Reatores Biológicos , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Etanol/toxicidade , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Fermentação , Engenharia Genética , Glucose/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas com Ferro-Enxofre/genética , Cinética , Redes e Vias Metabólicas/genética , Mutação , Xilose/metabolismo
3.
World J Microbiol Biotechnol ; 35(11): 169, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31654140

RESUMO

In the two-component system of NisRK from Lactococcus lactis, the production of nisin is affected by transmembrane NisK and activation of intracellular NisR. The transcription of nisin structural genes can be induced by derivatives of nisin. NisR activation leads to the activation of nisA/Z transcription, which encodes the nisin maturation machinery, nisin regulation and activation of the nisFEG operon to confer immunity. The aim of this study was to express the Lactococcus lactis histidine phosphokinase NisK and response regulator NisR in E. coli, and to perform activity assays and in silico analysis. In silico methods were applied to study the properties and structures of the NisK and NisR proteins, including prediction of physicochemical characteristics, secondary and tertiary structure, stability and ligand-receptor interactions.pET32a and pET28a vectors containing synthetic nisK and nisR genes were transformed into E. coli followed by IPTG induction. SDS-PAGE and western blotting methods were applied to confirm the presence and identity of the amplified proteins. Following purification, the proteins were dialyzed and then prepared for activity assay. The CAI index showed that the genes was compatible with the E. coli host and that the proteins have effective expression. Also, the mRNA prediction results suggest that there is enough mRNA stability for efficient translation in the new host. NisK and NisR recombinant proteins were expressed in E. coli with half - lives of around 10 h and were confirmed with molecular weights of 27 kDa and 69 kDa, respectively, by SDS-PAGE and western blotting. The secondary structure of the recombinant proteins as predicted by circular dichroism spectroscopy was similar to the in silico protein structures. Activity assay of recombinant NisK was performed by measuring the amount of consumed ATP according to the light produced by luciferase. Because NisK and NisR have a direct impact on each other, they have an essential role in increasing the production of nisin and they can be used in different research fields. Our results demonstrated that recombinant proteins NisK and NisR preserved their structure and function after expression.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Histidina Quinase/genética , Lactococcus lactis/genética , Proteínas Recombinantes/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Simulação por Computador , Ensaios Enzimáticos , Escherichia coli/genética , Instabilidade Genômica , Histidina Quinase/química , Histidina Quinase/isolamento & purificação , Histidina Quinase/metabolismo , Lactococcus lactis/enzimologia , Simulação de Acoplamento Molecular , Peso Molecular , Nisina/metabolismo , Conformação de Ácido Nucleico , Óperon , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Análise de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Transformação Genética
4.
PLoS Genet ; 15(10): e1008362, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658256

RESUMO

Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes.


Assuntos
Cólera/genética , Proteínas de Ligação a DNA/genética , Transativadores/genética , Vibrio cholerae/genética , Biofilmes/crescimento & desenvolvimento , Cólera/microbiologia , Regulação Bacteriana da Expressão Gênica/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Regiões Promotoras Genéticas/genética , Percepção de Quorum/genética , Fatores de Transcrição/genética , Vibrio cholerae/patogenicidade
5.
PLoS Genet ; 15(10): e1008232, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31622331

RESUMO

In nature, most bacteria live in biofilms where they compete with their siblings and other species for space and nutrients. Some bacteria produce antibiotics in biofilms; however, since the diffusion of antibiotics is generally hindered in biofilms by extracellular polymeric substances, i.e., the biofilm matrix, their function remains unclear. The Bacillus subtilis yitPOM operon is a paralog of the sdpABC operon, which produces the secreted peptide toxin SDP. Unlike sdpABC, yitPOM is induced in biofilms by the DegS-DegU two-component regulatory system. High yitPOM expression leads to the production of a secreted toxin called YIT. Expression of yitQ, which lies upstream of yitPOM, confers resistance to the YIT toxin, suggesting that YitQ is an anti-toxin protein for the YIT toxin. The alternative sigma factor SigW also contributes to YIT toxin resistance. In a mutant lacking yitQ and sigW, the YIT toxin specifically inhibits biofilm formation, and the extracellular neutral protease NprB is required for this inhibition. The requirement for NprB is eliminated by Δeps and ΔbslA mutations, either of which impairs production of biofilm matrix polymers. Overexpression of biofilm matrix polymers prevents the action of the SDP toxin but not the YIT toxin. These results indicate that, unlike the SDP toxin and many conventional antibiotics, the YIT toxin can pass through layers of biofilm matrix polymers to attack cells within biofilms with assistance from NprB. When the wild-type strain and the YIT-sensitive mutant were grown together on a solid medium, the wild-type strain formed biofilms that excluded the YIT-sensitive mutant. This observation suggests that the YIT toxin protects B. subtilis biofilms against competitors. Several bacteria are known to produce antibiotics in biofilms. We propose that some bacteria including B. subtilis may have evolved specialized antibiotics that can function within biofilms.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Antibacterianos/biossíntese , Bacillus subtilis/crescimento & desenvolvimento , Endopeptidases/genética , Regulação Bacteriana da Expressão Gênica/genética , Mutação , Óperon/genética
6.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31591167

RESUMO

Virulence genes are regulated by a complex regulatory network in Staphylococcus aureus Some of the regulators are global in nature and affect many downstream genes. MgrA is a multiple-gene regulator that has been shown to activate genes involved in capsule biosynthesis and repress surface protein genes. The goal of this study was to demonstrate the biological significance of MgrA regulation of capsule and surface proteins. We found that strain Becker possessed one fibronectin-binding protein, FnbA, and that FnbA was the predominant protein involved in invasion of nonphagocytic HeLa cells. By genetic analysis of strains with different amounts of capsule, we demonstrated that capsule impeded invasion of HeLa cells by masking the bacterial cell wall-anchored protein FnbA. Using variants with different levels of mgrA transcription, we further demonstrated that MgrA negatively impacted invasion by activating the cap genes involved in capsule biosynthesis and repressing the fnbA gene. Thus, we conclude that MgrA negatively impacts cell invasion of S. aureus Becker by promoting capsule and repressing FnbA.


Assuntos
Adesinas Bacterianas/metabolismo , Cápsulas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade , Adesinas Bacterianas/genética , Cápsulas Bacterianas/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Polissacarídeos Bacterianos/metabolismo , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Virulência/genética
7.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527124

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium whose type III secretion system (T3SS) plays a critical role in acute infections. Translocation of the T3SS effectors into host cells induces cytotoxicity. In addition, the T3SS promotes the intracellular growth of P. aeruginosa during host infections. The T3SS regulon genes are regulated by an AraC-type regulator, ExsA. In this study, we found that an extracellular metalloprotease encoded by impA (PA0572) is under the regulation of ExsA. An ExsA consensus binding sequence was identified upstream of the impA gene, and direct binding of the site by ExsA was demonstrated via an electrophoretic mobility shift assay. We further demonstrate that secreted ImpA cleaves the macrophage surface protein CD44, which inhibits the phagocytosis of the bacterial cells by macrophages. Combined, our results reveal a novel ExsA-regulated virulence factor that cooperatively inhibits the functions of macrophages with the T3SS.


Assuntos
Proteínas de Bactérias/metabolismo , Macrófagos/imunologia , Metaloproteases/metabolismo , Fagocitose/imunologia , Pseudomonas aeruginosa/imunologia , Serina Endopeptidases/metabolismo , Transativadores/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação Bacteriana da Expressão Gênica/genética , Células HeLa , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Pseudomonas aeruginosa/genética , Transativadores/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
8.
Nucleic Acids Res ; 47(18): 9871-9887, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31504767

RESUMO

The human opportunistic pathogen Staphylococcus aureus produces numerous small regulatory RNAs (sRNAs) for which functions are still poorly understood. Here, we focused on an atypical and large sRNA called RsaC. Its length varies between different isolates due to the presence of repeated sequences at the 5' end while its 3' part is structurally independent and highly conserved. Using MS2-affinity purification coupled with RNA sequencing (MAPS) and quantitative differential proteomics, sodA mRNA was identified as a primary target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase involved in oxidative stress response. Remarkably, rsaC gene is co-transcribed with the major manganese ABC transporter MntABC and, consequently, RsaC is mainly produced in response to Mn starvation. This 3'UTR-derived sRNA is released from mntABC-RsaC precursor after cleavage by RNase III. The mature and stable form of RsaC inhibits the synthesis of the Mn-containing enzyme SodA synthesis and favors the oxidative stress response mediated by SodM, an alternative SOD enzyme using either Mn or Fe as co-factor. In addition, other putative targets of RsaC are involved in oxidative stress (ROS and NOS) and metal homeostasis (Fe and Zn). Consequently, RsaC may balance two interconnected defensive responses, i.e. oxidative stress and metal-dependent nutritional immunity.


Assuntos
Proteínas de Bactérias/genética , Estresse Oxidativo/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica/genética , Homeostase/genética , Humanos , Manganês/química , Oxirredução , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Inanição , Superóxido Dismutase/química , Superóxido Dismutase/genética
9.
Nucleic Acids Res ; 47(18): 9934-9949, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31504787

RESUMO

The pathogenicity of Mycobacterium tuberculosis depends upon its ability to catabolize host cholesterol. Upregulation of the methylcitrate cycle (MCC) is required to assimilate and detoxify propionyl-CoA, a cholesterol degradation product. The transcription of key genes prpC and prpD in MCC is activated by MtPrpR, a member of a family of prokaryotic transcription factors whose structures and modes of action have not been clearly defined. We show that MtPrpR has a novel overall structure and directly binds to CoA or short-chain acyl-CoA derivatives to form a homotetramer that covers the binding cavity and locks CoA tightly inside the protein. The regulation of this process involves a [4Fe4S] cluster located close to the CoA-binding cavity on a neighboring chain. Mutations in the [4Fe4S] cluster binding residues rendered MtPrpR incapable of regulating MCC gene transcription. The structure of MtPrpR without the [4Fe4S] cluster-binding region shows a conformational change that prohibits CoA binding. The stability of this cluster means it is unlikely a redox sensor but may function by sensing ambient iron levels. These results provide mechanistic insights into this family of critical transcription factors who share similar structures and regulate gene transcription using a combination of acyl-CoAs and [4Fe4S] cluster.


Assuntos
Acil Coenzima A/química , Proteínas de Bactérias/química , Mycobacterium tuberculosis/genética , Fatores de Transcrição/química , Acil Coenzima A/genética , Proteínas de Bactérias/genética , Colesterol/genética , Regulação Bacteriana da Expressão Gênica/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/patogenicidade , Proteínas PrPC/química , Proteínas PrPC/genética , Fatores de Transcrição/genética , Tuberculose/genética , Tuberculose/microbiologia
10.
Nucleic Acids Res ; 47(18): 9925-9933, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31410466

RESUMO

DdrO is an XRE family transcription repressor that, in coordination with the metalloprotease PprI, is critical in the DNA damage response of Deinococcus species. Here, we report the crystal structure of Deinococcus geothermalis DdrO. Biochemical and structural studies revealed the conserved recognizing α-helix and extended dimeric interaction of the DdrO protein, which are essential for promoter DNA binding. Two conserved oppositely charged residues in the HTH motif of XRE family proteins form salt bridge interactions that are essential for promoter DNA binding. Notably, the C-terminal domain is stabilized by hydrophobic interactions of leucine/isoleucine-rich helices, which is critical for DdrO dimerization. Our findings suggest that DdrO is a novel XRE family transcriptional regulator that forms a distinctive dimer. The structure also provides insight into the mechanism of DdrO-PprI-mediated DNA damage response in Deinococcus.


Assuntos
Proteínas de Bactérias/genética , Dano ao DNA/genética , Sequências Hélice-Volta-Hélice/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos/genética , Deinococcus/química , Deinococcus/genética , Regulação Bacteriana da Expressão Gênica/genética , Metaloproteases/química , Metaloproteases/genética , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Secundária de Proteína , Fatores de Transcrição/química
11.
Microbiol Res ; 228: 126306, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31422233

RESUMO

The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mCherry, gfp, luxAB, lacZ, sacBR, and PBAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 105 distinct mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_3474, annotated as fabR and involved in fatty acid metabolism, was screened out and identified as a novel regulator mediating T3SS and T6SS expression. In addition, the fabR mutants displayed critical virulence attenuation in turbot. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be applied for functional genomics studies in various bacteria.


Assuntos
Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Edwardsiella/genética , Regulação Bacteriana da Expressão Gênica/genética , Testes Genéticos/métodos , Genoma Bacteriano/genética , Animais , Mapeamento Cromossômico , Farmacorresistência Bacteriana/genética , Ácidos Graxos/metabolismo , Doenças dos Peixes/microbiologia , Biblioteca Gênica , Genes Reporter/genética , Genômica/métodos , Mutagênese Insercional/métodos , Fatores de Transcrição/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo VI/genética , Virulência , Fatores de Virulência/genética
12.
Res Microbiol ; 170(8): 338-344, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31376485

RESUMO

The transport of peptides in microorganisms plays an important role in their physiology and behavior, both as a nutrient source and as a proxy to sense their environment. This latter function is evidenced in Gram-positive bacteria where cell-cell communication is mediated by small peptides. Here, we highlight the importance of the oligopeptide permease (Opp) systems in the various major processes controlled by signaling peptides, such as sporulation, virulence and conjugation. We underline that the functioning of these communication systems is tightly linked to the developmental status of the bacteria via the regulation of opp gene expression by transition phase regulators.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Bactérias Gram-Positivas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico/fisiologia , Fatores de Terminação de Peptídeos/metabolismo , Percepção de Quorum/fisiologia , Transdução de Sinais/fisiologia
13.
Arch Microbiol ; 201(10): 1459-1464, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31363787

RESUMO

Streptomyces is currently the main producer of microbial pharmaceuticals from its secondary metabolites as natural products. It will be more beneficial if the promoters, which are particularly strong during the secondary metabolism of Streptomyces, are used to drive the efficient production of desired natural products with the coordination of bacterial growth. Here, in an industrial natamycin producer Streptomyces chattanoogensis L10, a strong promoter groESp was identified for this purpose based on the comparative proteomic analysis of the primary and secondary metabolism. With a constitutive promoter ermEp* as a control, the activity of groESp was weak in the primary metabolism, but about sixfold higher than ermEp* in the secondary metabolism, when the representative antibiotic natamycin was highly produced. Furthermore, when ScnRII, a pathway-specific positive regulator in natamycin biosynthesis, was expressed under groESp, the productivity of natamycin was about 20% higher in the secondary metabolism than that from ermEp*, but had no discrimination in the early 2 days. Thus, we showed that proteomics is an effective alternative way to identify promoters for the high yield of natamycin in S. chattanoogensis, and this strategy can be widely adaptable to other Streptomyces species for the full development of secondary metabolites with promising bioactivities.


Assuntos
Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial/métodos , Natamicina/biossíntese , Regiões Promotoras Genéticas/genética , Proteômica , Streptomyces/genética , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Metabolismo Secundário
14.
Mol Genet Genomics ; 294(5): 1359-1371, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31363904

RESUMO

Previous studies revealed important roles of small RNAs (sRNAs) in regulation of bacterial metabolism, stress responses and virulence. However, only a minor fraction of sRNAs is well characterized with respect to the spectra of their targets, conditional expression profiles and actual mechanisms they use to regulate gene expression to control particular biological pathways. To learn more about the specific contribution of sRNAs to the global regulatory network controlling the Escherichia coli central carbon metabolism (CCM), we employed microarray analysis and compared transcriptome profiles of E. coli cells grown on two alternative minimal media supplemented with either pyruvate or glucose, respectively. Microarray analysis revealed that utilization of these alternative carbon sources led to profound differences in gene expression affecting all major gene clusters associated with CCM as well as expression of several known (CyaR, RyhB, GcvB and RyeA) and putative (C0652) sRNAs. To assess the impact of transcriptional reprogramming of gene expression on E. coli protein abundance, we also employed two-dimensional protein gel electrophoresis. Our experimental data made it possible to determine the major pathways for pyruvate assimilation when it is used as a sole carbon source and reveal the impact of other key processes (i.e., energy production, molecular transport and cell resistance to stress) associated with the CCM in E. coli. Moreover, some of these processes were apparently controlled by GcvB, RyhB and CyaR at the post-transcriptional level, thus indicating the complexity and interconnection of the regulatory networks that control CCM in bacteria.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Glucose/metabolismo , Ácido Pirúvico/metabolismo , Proteínas de Escherichia coli/genética , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Transcrição Genética/genética , Transcriptoma/genética
15.
Res Microbiol ; 170(6-7): 296-299, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31279087

RESUMO

Quorum sensing (QS) is a molecular communication system that bacteria use to harmonize the regulation of genes in a cell density-dependent manner. In proteobacteria, QS is involved, among others, in virulence, biofilm formation or CRISPR-Cas gene regulation. Here, we report for the first time the effect of a QS-interfering enzyme to alter the regulation of CRISPR-Cas systems in model and clinical strains of Pseudomonas aeruginosa, as well as in the marine bacterium Chromobacterium violaceum CV12472. The expression of CRISPR-Cas genes decreased in most cases suggesting that enzymatic disruption of QS is promising for modulating phage-bacteria interactions.


Assuntos
Acil-Butirolactonas/metabolismo , Sistemas CRISPR-Cas/genética , Chromobacterium/genética , Regulação Bacteriana da Expressão Gênica/genética , Pseudomonas aeruginosa/genética , Percepção de Quorum/genética , Bacteriófagos/genética , Bacteriófagos/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Chromobacterium/isolamento & purificação , Chromobacterium/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo
16.
Res Microbiol ; 170(6-7): 263-271, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31279088

RESUMO

In bacteria, phosphate (Pi) stress response is governed by the two-component regulatory system, sensor kinase PhoR and its cognate response regulatory protein PhoB. The arsenite [As(III)]-oxidizing bacterium Agrobacterium tumefaciens GW4 contains two phoB genes, phoB1 and phoB2. phoB1 is adjacent to As(III)-oxidizing genes, however, the functions of PhoB1 and PhoB2 remain unclear. Here, phoB1 and phoB2 were each deleted in-frame, and proteomics, qRT-PCR and protein-DNA interaction were performed. We found that (1) phoB1 and phoB2 were both upregulated under low Pi conditions and phoB1 was induced by As(III), but phoB2 was not; (2) deletion of phoB1 reduced As(III)-oxidizing efficiency and protein-DNA interaction analysis showed PhoB1 could interact with aioXSR promoter to regulate As(III) oxidation; (3) deletions of phoB1 or phoB2 both reduced exopolysaccharides (EPS) synthesis; and (4) PhoB1 influenced Pi uptake, As(III) oxidation, EPS synthesis, TCA cycle, energy production and stress response with As(III), and PhoB2 was associated with Pi uptake and EPS synthesis in low Pi conditions. These results showed PhoB1 and PhoB2 were both involved in Pi acquisition, PhoB1 was more important with As(III) and PhoB2 played a major role without As(III). Strain GW4 uses these two regulators to survive under low Pi and arsenic-rich environments.


Assuntos
Agrobacterium tumefaciens/genética , Arsenitos/metabolismo , Proteínas de Bactérias/genética , Fosfatos/metabolismo , Polissacarídeos Bacterianos/metabolismo , Agrobacterium tumefaciens/metabolismo , Ciclo do Ácido Cítrico/genética , Proteínas de Ligação a DNA/genética , Metabolismo Energético/genética , Regulação Bacteriana da Expressão Gênica/genética , Oxirredução , Polissacarídeos Bacterianos/biossíntese
17.
Mol Cell ; 75(3): 498-510.e5, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31256988

RESUMO

In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Francisella/genética , Virulência/genética , DNA/genética , Clivagem do DNA , Regulação Bacteriana da Expressão Gênica/genética , Lipoproteínas/biossíntese , Lipoproteínas/genética , RNA/genética , Transcrição Genética
18.
Microbes Environ ; 34(3): 260-267, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31257307

RESUMO

The soybean symbiont Bradyrhizobium diazoefficiens grows anaerobically in the presence of nitrate using the denitrification pathway, which involves the nap, nir, nor, and nos genes. We previously showed that NasT acts as a transcription antitermination regulator for nap and nos gene expression. In the present study, we investigated the targets of NasT in B. diazoefficiens during denitrifying growth by performing transcription profiling with RNA-seq and quantitative reverse-transcription PCR. Most of the genes with altered expression in the absence of NasT were related to nitrogen metabolism, specifically several systems for branched-chain amino acid transport. The present results suggest that the reduced expression of genes involved in nitrogen acquisition leads to the induction of alternative sets of genes with similar functions. The ΔnasT mutant of B. diazoefficiens grew better than the wild type under denitrifying conditions. However, this enhanced growth was completely abolished by an additional loss of the narK or bjgb genes, which encode cytoplasmic systems for nitrite and nitric oxide detoxification, respectively. Since the expression of narK and bjgb was increased in the ΔnasT mutant, the growth of the ΔnasT mutant may be promoted by increased detoxification activity.


Assuntos
Bradyrhizobium/genética , Bradyrhizobium/metabolismo , Desnitrificação/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bradyrhizobium/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Modelos Genéticos , Mutação , Nitrito Redutases/genética , Nitrogênio/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional/genética
19.
Res Microbiol ; 170(6-7): 243-255, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31325485

RESUMO

Bacteria usually produce, release and detect quorum sensing (QS)-based signal molecules, and successively orchestrate gene expression in respond to environmental changes. Pseudoalteromonas are typical marine bacteria, but knowledge on their QS systems is extremely fragmentary. In this study, genome sequencing of Pseudoalteromonas sp. R3 was performed. Accordingly, a QS working model including three sets of hierarchically organized QS systems was proposed in strain R3. Among them, the typical LuxI/R-type QS system using acyl-homoserine lactones (AHLs) as signal molecules was characterized. Sequence similarity analysis indicated luxI encoding AHLs synthase is novel. The luxR encoding AHLs receptor is directly adjacent to luxI downstream. Notably, mutagenesis demonstrated LuxI and LuxR affect each other at transcriptional level, and both control the AHLs formation. Interestingly, it was found that LuxI/R-type QS system positively involves resistance to streptomycin. Thin-layer chromatography analysis showed strain R3 can produce 3-OH-C6-HSL and C8-HSL, which was supported by heterologous expression of LuxI in Escherichia coli. Sequence alignment analysis indicated that the N-terminal region of LuxI is more conservative than the C-terminal region, revealing the importance of N-terminal region in AHLs synthesis. The obtained findings enrich our knowledge on LuxI/R-type QS system in Pseudoalteromonas and its regulation on adaptation to marine environments.


Assuntos
Acil-Butirolactonas/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Pseudoalteromonas/genética , Percepção de Quorum/genética , Proteínas Repressoras/genética , Transativadores/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Antibacterianos/farmacologia , Sequência de Bases , Regulação Bacteriana da Expressão Gênica/genética , Genoma Bacteriano/genética , Pseudoalteromonas/efeitos dos fármacos , Pseudoalteromonas/fisiologia , Alinhamento de Sequência , Transdução de Sinais/genética , Estreptomicina/farmacologia , Sequenciamento Completo do Genoma
20.
Future Microbiol ; 14: 671-689, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31161792

RESUMO

Aim: To propose newer combinations of antibiotics effective against NDM-1-producing bacterial strains. Materials & methods: Antibiotics combinations were tested by checkerboard assay. NDM-1 protein/enzyme was expressed and purified to perform enzyme kinetics, circular dichroism and fluorescence spectroscopic studies. Results: Doripenem-cefoxitin combination and doripenem-tetracycline combination showed synergistic effect toward NDM-1-producing strains. The catalytic efficiency of NDM-1 enzyme was decreased drastically by 96.6% upon doripenem-cefoxitin treatment and by 35.54% after doripenem-tetracycline treatment. Conformational changes were observed in NDM-1 upon combination treatment. Conclusion: NDM-1-producing bacterial strains show resistance to multiple antibiotics but the combination of doripenem-cefoxitin and doripenem-tetracycline are effective against them. The combination of a carbapenem and cephamycin antibiotic is proposed for future treatment options against bacteria-producing NDM-1.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Cefoxitina/farmacologia , Doripenem/farmacologia , Tetraciclina/farmacologia , beta-Lactamases/efeitos dos fármacos , Bactérias/enzimologia , Bactérias/metabolismo , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Ensaios Enzimáticos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Cinética , Testes de Sensibilidade Microbiana , Inibidores da Síntese de Proteínas/farmacologia , Termodinâmica , beta-Lactamases/análise
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA