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1.
Biomed. environ. sci ; Biomed. environ. sci;(12): 949-958, 2023.
Article in English | WPRIM | ID: wpr-1007869

ABSTRACT

OBJECTIVE@#This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase (DGC) in Vibrio cholerae and how its transcription is regulated by Fur and HapR.@*METHODS@#The roles of VCA0560 was investigated by utilizing various phenotypic assays, including colony morphological characterization, crystal violet staining, Cyclic di-GMP (c-di-GMP) quantification, and swimming motility assay. The regulation of the VCA0560 gene by Fur and HapR was analyzed by luminescence assay, electrophoretic mobility shift assay, and DNase I footprinting.@*RESULTS@#VCA0560 gene mutation did not affect biofilm formation, motility, and c-di-GMP synthesis in V. cholerae, and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity. The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator HapR.@*CONCLUSION@#Overexpressed VCA0560 functions as an active DGC in V. cholerae, and its transcription is repressed by Fur and HapR.


Subject(s)
Vibrio cholerae/genetics , Biofilms , Quorum Sensing , Mutation , Gene Expression Regulation, Bacterial , Bacterial Proteins/genetics
2.
Chinese Journal of Biotechnology ; (12): 4719-4730, 2022.
Article in Chinese | WPRIM | ID: wpr-970343

ABSTRACT

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.


Subject(s)
Vibrio parahaemolyticus/metabolism , Escherichia coli/metabolism , Bacterial Proteins/metabolism , Transcription Factors/genetics , Gene Expression Regulation, Bacterial
3.
Biol. Res ; 55: 8-8, 2022. ilus
Article in English | LILACS | ID: biblio-1383912

ABSTRACT

BACKGROUND: Salmonella Typhimurium is a Gram negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S . Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild type strain, suggesting that ompX mRNA is also regulated at a post transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2 induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.


Subject(s)
Animals , Mice , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism , Bacterial Outer Membrane Proteins/genetics , Porins/genetics , Porins/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology
4.
Biol. Res ; 55: 7-7, 2022. ilus, graf
Article in English | LILACS | ID: biblio-1383911

ABSTRACT

BACKGROUND: Aerobic metabolism generates reactive oxygen species that may cause critical harm to the cell. The aim of this study is the characterization of the stress responses in the model aromatic degrading bacterium Paraburkholderia xenovorans LB400 to the oxidizing agents paraquat and H 2 O2. METHODS: Antioxidant genes were identified by bioinformatic methods in the genome of P. xenovorans LB400, and the phylogeny of its OxyR and SoxR transcriptional regulators were studied. Functionality of the transcriptional regulators from strain LB400 was assessed by complementation with LB400 SoxR of null mutant P. aeruginosa ΔsoxR, and the construction of P. xenovorans pIZ oxyR that overexpresses OxyR. The effects of oxidizing agents on P. xenovorans were studied measuring bacterial susceptibility, survival and ROS formation after exposure to paraquat and H 2 O2. The effects of these oxidants on gene expression (qRT PCR) and the proteome (LC-MS/MS) were quantified. RESULTS: P. xenovorans LB400 possesses a wide repertoire of genes for the antioxidant defense including the oxyR , ahpC , ahpF , kat , trxB , dpsA and gorA genes, whose orthologous genes are regulated by the transcriptional regulator OxyR in E. coli . The LB400 genome also harbors the soxR, fumC , acnA , sodB , fpr and fldX genes, whose orthologous genes are regulated by the transcriptional regulator SoxR in E. coli . The functionality of the LB400 soxR gene was confirmed by complementation of null mutant P. aeruginosa Δ soxR . Growth, susceptibility, and ROS formation assays revealed that LB400 cells were more susceptible to paraquat than H2O2. Transcriptional analyses indicated the upregulation of the oxyR , ahpC1 , katE and ohrB genes in LB400 cells after exposure to H2O2, whereas the oxyR , fumC , ahpC1 , sodB1 and ohrB genes were induced in presence of paraquat. Proteome analysis revealed that paraquat induced the oxidative stress response proteins AhpCF and DpsA, the universal stress protein UspA and the RNA chaperone CspA. Both oxidizing agents induced the Ohr protein, which is involved in organic peroxide resistance. Notably, the overexpression of the LB400 oxyR gene in P. xenovorans significantly decreased the ROS formation and the susceptibility to paraquat, suggesting a broad OxyR regulated antioxidant response. CONCLUSIONS: This study showed that P. xenovorans LB400 possess a broad range oxidative stress response, which explain the high resistance of this strain to the oxidizing compounds paraquat and H2O2.


Subject(s)
Gene Expression Regulation, Bacterial , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Oxidation-Reduction , Repressor Proteins/genetics , Repressor Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromatography, Liquid , Oxidative Stress , Burkholderiaceae , Escherichia coli/genetics , Tandem Mass Spectrometry , Hydrogen Peroxide/pharmacology
5.
Braz. j. biol ; 82: e228009, 2022. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1249287

ABSTRACT

Background: Pseudomonas aeruginosa is a common opportunistic pathogenic bacterium with the ability to develop a strong communication pathway by quorum sensing system and different virulent factors. Among the various important secretions of P. aeruginosa rhamnolipid is important biological detergent, believed to be involved in the development of the biofilm and intercellular communication. It readily dissolves the lung surfactants that are then easily catalyzed by the phospholipases and in this way is involved in the acute pulmonary infection. Objective: research work was designed to investigate virulence and gene associated with virulence in P. aeruginosa responsible for pulmonary infections. Methods: In current study polymerase chain reaction (PCR) was used for the detection of the rhlR (rhamnolipid encoding) gene of isolated strains. A number of assays were performed that ensured its virulent behavior. Disc diffusion method was used to check its antibiotic resistance. Isolated strains were resistant to a number of antibiotics applied. Result: It was found that males are more prone to respiratory infections as compared to females. Male members with age of 44-58 and 59-73 are at a higher risk, while females with age of 44-58 are also at a risk of pulmonary infections. Antibiotic resistance was observed by measuring zone of inhibition in strains GCU-SG-M4, GCU-SG-M3, GCU-SG-M5, GCU-SG-M2, GCU-SG-M1 and GCU-SG-M6. GCU-SG-M2 was resistant to fluconazole (FLU), clarithromycin (CLR), cefixime (CFM) and Penicillin (P10). No zone of inhibition was observed. But it showed unusual diffused zone around the Ak and MEM antibiotic discs. rhl R gene and 16s rRNA gene were characterized and analyzed. Conclusion: Findings from current study would help in raising awareness about antibiotic resistance of P. aeruginosa, and also the sequence of rhl R gene can be used as the diagnostic marker sequence to identify the virulent rhl R gene sequence from the samples when isolated from sputum of Pneumonia patients.


Antecedentes: Pseudomonas aeruginosa é uma bactéria patogênica oportunista comum, com a capacidade de desenvolver uma forte via de comunicação pelo sistema de detecção de quorum e diferentes fatores virulentos. Entre as várias secreções importantes de P. aeruginosa rhamnolipid, há um importante detergente biológico, que se acredita estar envolvido no desenvolvimento do biofilme e na comunicação intercelular. Dissolve rapidamente os surfactantes pulmonares que são facilmente catalisados pelas fosfolipases e, dessa maneira, estão envolvidos na infecção pulmonar aguda. Objetivo: O trabalho de pesquisa foi desenhado para investigar a virulência e o gene associado à virulência em P. aeruginosa responsável por infecções pulmonares. Métodos: No presente estudo, a reação em cadeia da polimerase (PCR) foi utilizada para a detecção do gene rhlR (codificação ramnolipídeo) de cepas isoladas. Foram realizados vários ensaios que garantiram seu comportamento virulento. O método de difusão em disco foi utilizado para verificar sua resistência a antibióticos. As estirpes isoladas foram resistentes a vários antibióticos aplicados. Resultado: Verificou-se que os homens são mais propensos a infecções respiratórias em comparação às mulheres. Membros do sexo masculino com idade entre 44 e 58 e 59 e 73 anos correm maior risco, enquanto mulheres com idade entre 44 e 58 anos também correm risco de infecções pulmonares. A resistência aos antibióticos foi observada medindo a zona de inibição nas cepas GCU-SG-M4, GCU-SG-M3, GCU-SG-M5, GCUSG-M2, GCU-SG-M1 e GCU-SG-M6. O GCU-SG-M2 foi resistente ao fluconazol (FLU), claritromicina (CLR), cefixima (CFM) e penicilina (P10). Nenhuma zona de inibição foi observada. Mas se notou uma zona difusa incomum ao redor dos discos antibióticos Ak e MEM. Os genes rhl R e 16s rRNA foram caracterizados e analisados. Conclusão: As conclusões do presente estudo ajudariam a aumentar a conscientização sobre a resistência a antibióticos de P. aeruginosa e, também, a sequência do gene rhl R pode ser usada como sequência de diagnóstico para identificar a sequência virulenta do gene rhl R das amostras quando isoladas do escarro de pacientes com pneumonia.


Subject(s)
Humans , Male , Female , Pneumonia , Pseudomonas aeruginosa/genetics , RNA, Ribosomal, 16S , Glycolipids , Gene Expression Regulation, Bacterial
6.
Chinese Journal of Biotechnology ; (12): 2379-2392, 2021.
Article in Chinese | WPRIM | ID: wpr-887804

ABSTRACT

TetR family transcriptional regulators (TFRs) are widely distributed in bacteria and archaea, and the first discovered TFR was confirmed to control the expression of tetracycline efflux pump in Escherichia coli. TFRs can bind DNAs and ligands. Small molecule ligands can induce conformational changes of TFRs, inhibiting or promoting TFRs to control target gene expression. Currently, TFRs have a wide variety of ligands, including carbohydrates, proteins, fatty acids and their derivatives, metal ions, and so on. Due to the diversity of ligands, TFRs regulate a wide range of physiological processes, from basic carbon metabolism and nitrogen metabolism to quorum sensing and antibiotic biosynthesis. On the basis of the recent studies in our laboratory and the literature, we review here the regulatory mechanism mediated by ligands of TFRs in primary and secondary metabolism, as well as the application of ligands for TFRs in the development of gene route and the activation of antibiotic biosynthesis.


Subject(s)
Anti-Bacterial Agents , Bacteria/metabolism , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Ligands , Quorum Sensing
7.
Biomed. environ. sci ; Biomed. environ. sci;(12): 299-308, 2021.
Article in English | WPRIM | ID: wpr-878362

ABSTRACT

Objective@#To investigate reciprocal regulation between Fur and two RyhB homologs in @*Methods@#Regulatory relationships were assessed by a combination of colony morphology assay, primer extension, electrophoretic mobility shift assay and DNase I footprinting.@*Results@#Fur bound to the promoter-proximal DNA regions of @*Conclusion@#Fur and the two RyhB homologs exert negative reciprocal regulation, and RyhB homologs have a positive regulatory effect on biofilm formation in


Subject(s)
Bacterial Proteins/metabolism , Biofilms , Gene Expression Regulation, Bacterial/physiology , Yersinia pestis/physiology
8.
Article in English | WPRIM | ID: wpr-1010472

ABSTRACT

Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. RsbR, an upstream regulator of the sigma B (SigB) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion (ΔrsbR), complementation (C-ΔrsbR), and phosphorylation site mutations in the rsbR (RsbR-T175A, RsbR-T209A, and RsbR-T175A-T209A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsbR reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175A disabled RsbR complementation, while RsbR-T209A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of RsbR are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.


Subject(s)
Alanine/genetics , Bacillus subtilis , Bacterial Proteins/metabolism , Binding Sites , Gene Deletion , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Homeostasis , Hydrogen-Ion Concentration , Listeria monocytogenes/metabolism , Listeriosis/microbiology , Mutation , Phenotype , Phosphoproteins/metabolism , Phosphorylation , Sigma Factor/metabolism , Stress, Physiological
9.
Chinese Journal of Biotechnology ; (12): 558-566, 2019.
Article in Chinese | WPRIM | ID: wpr-771353

ABSTRACT

Bacterial biofilm refers to a tunicate-like biological group composed of polysaccharide, protein and nucleic acid secreted by bacteria on the surface of the mucous membrane or biological material. The biofilm formation is a major cause of chronic infections. Bacteria could produce some secondary metabolites during the growth and reproduction. Some of them act as signaling molecules allowing bacteria to communicate and regulate many important physiological behaviors at multiple-cell level, such as bioluminescence, biofilm formation, motility and lifestyles. Usually, these signal molecules play an important role in the formation of bacterial biofilm. We review here the effects of related signal molecules of Quorum Sensing, cyclic diguanylate, Two-Component Systems and sRNA on the biofilm formation. Focusing on these regulation mechanism of signal molecules in the process of biofilm formation is necessary for the prevention and treatment of some chronic diseases.


Subject(s)
Bacterial Proteins , Biofilms , Cyclic GMP , Gene Expression Regulation, Bacterial , Protein Binding , Quorum Sensing
10.
Chinese Journal of Biotechnology ; (12): 1925-1941, 2019.
Article in Chinese | WPRIM | ID: wpr-771742

ABSTRACT

Harnessing industrial microorganisms to utilize renewable feedstocks and meanwhile produce biofuels, bulk chemicals, food ingredients, nutraceuticals, pharmaceuticals, industrial enzymes, etc. is the basis for successful biological industries. Robust traits of industrial microorganisms including high yield and productivity as well as stress tolerance are controlled by sophisticated genetic regulatory networks. Engineering robustness of industrial microorganisms requires systematic and global perturbations at the genome-wide scale to accelerate the accumulation of diversified genotypic mutations, thus generating desirable phenotypes. We review heve the mechanisms of genetic regulation and stress response in robust industrial organisms, the global perturbations and multiplex accelerated evolution at the genome-wide scale, as well as the global perturbation of cellular redox balance. In the future, based on system biology and synthetic biology, more efforts should be further devoted to understanding the mechanisms behind robust traits in industrial microorganisms under industrial niches for modeling and prediction as well as systematic engineering.


Subject(s)
Environment , Gene Expression Regulation, Bacterial , Gene Regulatory Networks , Genetics , Industrial Microbiology , Metabolic Engineering , Synthetic Biology
11.
Chinese Journal of Biotechnology ; (12): 1500-1510, 2019.
Article in Chinese | WPRIM | ID: wpr-771779

ABSTRACT

MarR family transcription regulators are ubiquitous among bacteria and archaea. They extensively control multiple cellular processes and elaborately regulate the expression of genes involved in virulence, stress response and antibiotics at translational level. In Xanthomonas campestris pv. campestris, insertional inactivation of MarR family transcription regulator HpaR (XC2827) resulted in significantly decrease in virulence and increase in the production of the extracellular proteases. Here, we reported that the genome of Xcc 8004 encodes nine MarR family transcription regulators. The MarR family transcription regulators, HpaR (XC2827) and XC0449, were heterologous expressed and purified. In vitro MST and Pull-down assay confirmed the physical interaction between HpaR and XC0449. Phenotypical assay determined that deletion of XC0449 resulted in substantial virulence attenuation. In vitro EMSA, in vivo qRT-PCR and GUS activity assay identified that HpaR and XC0449 coordinately act as the transcriptional activator to regulate the expression of the virulence-associated gene XC0705, and eventually control the bacterial virulence and the production of extracellular proteases.


Subject(s)
Bacterial Proteins , Gene Expression Regulation, Bacterial , Transcription Factors , Virulence , Xanthomonas campestris
12.
Braz. j. microbiol ; Braz. j. microbiol;49(2): 429-442, Apr.-June 2018. tab, graf
Article in English | LILACS | ID: biblio-889226

ABSTRACT

Abstract Bacteria are important sources of cellulases with various industrial and biotechnological applications. In view of this, a non-hemolytic bacterial strain, tolerant to various environmental pollutants (heavy metals and organic solvents), showing high cellulolytic index (7.89) was isolated from cattle shed soil and identified as Bacillus sp. SV1 (99.27% pairwise similarity with Bacillus korlensis). Extracellular cellulases showed the presence of endoglucanase, total cellulase and β-glucosidase activities. Cellulase production was induced in presence of cellulose (3.3 times CMCase, 2.9 times FPase and 2.1 times β-glucosidase), and enhanced (115.1% CMCase) by low-cost corn steep solids. An in silico investigation of endoglucanase (EC 3.2.1.4) protein sequences of three Bacillus spp. as query, revealed their similarities with members of nine bacterial phyla and to Eukaryota (represented by Arthropoda and Nematoda), and also highlighted of a convergent and divergent evolution from other enzymes of different substrate [(1,3)-linked beta-d-glucans, xylan and chitosan] specificities. Characteristic conserved signature indels were observed among members of Actinobacteria (7 aa insert) and Firmicutes (9 aa insert) that served as a potential tool in support of their relatedness in phylogenetic trees.


Subject(s)
Animals , Cattle , Bacillus/enzymology , Cellulase/genetics , Cellulase/metabolism , Evolution, Molecular , Bacillus/growth & development , Bacillus/isolation & purification , Cellulose/metabolism , Computational Biology , Feces/microbiology , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , INDEL Mutation , Sequence Analysis, DNA , Sequence Homology , Substrate Specificity , Zea mays/metabolism
13.
Braz. j. microbiol ; Braz. j. microbiol;49(1): 200-206, Jan.-Mar. 2018. tab, graf
Article in English | LILACS | ID: biblio-889189

ABSTRACT

ABSTRACT Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.


Subject(s)
Humans , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacteroides fragilis/drug effects , Bacteroides fragilis/genetics , Bacteroides Infections/microbiology , Repressor Proteins/genetics , Bacterial Proteins/metabolism , Bacteroides fragilis/isolation & purification , Bacteroides fragilis/metabolism , Gene Expression Regulation, Bacterial/drug effects , Gene Silencing , Microbial Sensitivity Tests , Repressor Proteins/metabolism
14.
Braz. j. microbiol ; Braz. j. microbiol;49(1): 97-103, Jan.-Mar. 2018. graf
Article in English | LILACS | ID: biblio-889210

ABSTRACT

ABSTRACT Freezing temperatures are a major challenge for life at the poles. Decreased membrane fluidity, uninvited secondary structure formation in nucleic acids, and protein cold-denaturation all occur at cold temperatures. Organisms adapted to polar regions possess distinct mechanisms that enable them to survive in extremely cold environments. Among the cold-induced proteins, cold shock protein (Csp) family proteins are the most prominent. A gene coding for a Csp-family protein, cspB, was cloned from an arctic bacterium, Polaribacter irgensii KOPRI 22228, and overexpression of cspB greatly increased the freeze-survival rates of Escherichia coli hosts, to a greater level than any previously reported Csp. It also suppressed the cold-sensitivity of an E. coli csp-quadruple deletion strain, BX04. Sequence analysis showed that this protein consists of a unique domain at its N-terminal end and a well conserved cold shock domain at its C-terminal end. The most common mechanism of Csp function in cold adaption is melting of the secondary structures in RNA and DNA molecules, thus facilitating transcription and translation at low temperatures. P. irgensii CspB bound to oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. The unprecedented level of freeze-tolerance conferred by P. irgensii CspB suggests a crucial role for this protein in survival in polar environments.


Subject(s)
Bacterial Proteins/metabolism , Flavobacteriaceae/physiology , Cold Shock Proteins and Peptides/metabolism , Arctic Regions , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Cold Temperature , Ecosystem , Flavobacteriaceae/isolation & purification , Flavobacteriaceae/genetics , Cold Shock Proteins and Peptides/genetics
15.
Chinese Journal of Biotechnology ; (12): 1895-1905, 2018.
Article in Chinese | WPRIM | ID: wpr-771419

ABSTRACT

The exogenous gene expression and its impacts on the bacterial population are important to study quorum sensing systems and synthetic biology industry. However, the behavior of exogenous protein expressing bacteria remains poorly understood. To find out which factors are playing a critical role in the growth of population and exogenous gene expression, we measured Lux-type receptor-regulated exogenous gene expression under the induction of N-acyl homoserine lactone (N-AHL) signaling molecules and impacts on the bacterial population dynamics after such stimulation. To analyze the cause of fitness burden of bacteria, we set up a hypothetical mathematical model. Previous studies often arrogate this phenomenon to the synthesis cost and the toxicity of N-AHL signaling molecule. However, we suggested another possible cause of the fitness burden.


Subject(s)
Bacteria , Gene Expression , Gene Expression Regulation, Bacterial , Quorum Sensing
16.
Chinese Journal of Biotechnology ; (12): 1760-1771, 2018.
Article in Chinese | WPRIM | ID: wpr-776292

ABSTRACT

Promoter, an essential regulatory element, is widely used for metabolic engineering of industrial strains. Corynebacterium glutamicum is an important industrial workhorse to produce various amino acids. However, strong constitutive promoters that are applicable to C. glutamicum are rarely reported. In this study, we first performed a time-series transcriptome analysis of a glutamate hyper-producing strain C. glutamicum SL4 by using RNA-Seq. Overall, we picked 10 samples at different time during the fermentation process. By analyzing the time-series transcriptome data, we selected 10 candidate genes with the highest transcriptional level. These genes were all transcribed stably during the fermentation process. We subsequently cloned the promoter sequences and evaluated the promoters' strength in strain SL4 using a red fluorescent protein reporter system. To evaluate the universality of the promoters in different C. glutamicum strains, we further tested the performance of some promoters in wild type C. glutamicum strains, including ATCC 13869 and ATCC 13032. The strongest promoter was further characterized using LacZ as a reporter in all the three C. glutamicum strains. Finally, we successfully obtained three constitutive promoters with universality, PcysK, PgapA and PfumC. PcysK is the most efficient promoter among the three C. glutamicum strains. In strains SL4 and ATCC 13869, the strength of PcysK is 2-fold of the strong inducible promoter Ptac using the red fluorescent protein as a reporter and 4-fold of Ptac using LacZ as a reporter. Moreover, the strength of PcysK reaches 30%-40% of Ptac in strain ATCC 13032. The promoter PcysK is identified as a strong promoter for the first time, which can be used as an efficient biobrick for metabolic engineering of synthesis pathways in C. glutamicum.


Subject(s)
Corynebacterium glutamicum , Genetics , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Metabolic Engineering , Promoter Regions, Genetic , Transcriptome
17.
Biomed. environ. sci ; Biomed. environ. sci;(12): 290-299, 2018.
Article in English | WPRIM | ID: wpr-690658

ABSTRACT

<p><b>OBJECTIVE</b>Macrolide susceptibility and drug resistance mechanisms of clinical non-tuberculous mycobacteria (NTM) isolates were preliminarily investigated for more accurate diagnosis and treatment of the infection in China.</p><p><b>METHODS</b>Four macrolides, including clarithromycin (CLAR), azithromycin (AZM), roxithromycin (ROX), and erythromycin (ERY), were used to test the drug susceptibility of 310 clinical NTM isolates from six provinces of China with the broth microdilution method. Two resistance mechanisms, 23S rRNA and erm, were analyzed with nucleotide sequence analysis.</p><p><b>RESULTS</b>Varied effectiveness of macrolides and species-specific resistance patterns were observed. Most Mycobacterium abscessus subsp. massiliense were susceptible and all M. fortuitum were highly resistant to macrolides. All the drugs, except for erythromycin, exhibited excellent activities against slow-growing mycobacteria, and drug resistance rates were below 22.2%. Only four highly resistant strains harbored 2,058/2,059 substitutions on rrl and none of other mutations were related to macrolide resistance. G2191A and T2221C on rrl were specific for the M. abscessus complex (MABC). Seven sites, G2140A, G2210C, C2217G, T2238C, T2322C, T2404C, and A2406G, were specifically carried by M. avium and M. intracellulare. Three sites, A2192G, T2358G, and A2636G, were observed only in M. fortuitum and one site G2152A was specific for M. gordonae. The genes erm(39) and erm(41) were detected in M. fortuitum and M. abscessus and inducible resistance was observed in relevant sequevar.</p><p><b>CONCLUSION</b>The susceptibility profile of macrolides against NTM was demonstrated. The well-known macrolide resistance mechanisms, 23S rRNA and erm, failed to account for all resistant NTM isolates, and further studies are warranted to investigate macrolide resistance mechanisms in various NTM species.</p>


Subject(s)
Humans , Anti-Bacterial Agents , Pharmacology , Bacterial Proteins , Genetics , Metabolism , China , Drug Resistance, Bacterial , Gene Expression Regulation, Bacterial , Macrolides , Pharmacology , Mycobacterium , Genetics , Polymorphism, Genetic
18.
Rev. bras. pesqui. méd. biol ; Braz. j. med. biol. res;51(8): e7044, 2018. graf
Article in English | LILACS | ID: biblio-951748

ABSTRACT

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.


Subject(s)
Animals , Bacterial Proteins/genetics , Clostridium perfringens/genetics , Sequence Analysis, RNA/methods , Genes, MDR , Drug Resistance, Multiple, Bacterial/genetics , Mass Spectrometry/methods , Bacterial Proteins/metabolism , Electrophoresis, Gel, Two-Dimensional/methods , Gene Expression Regulation, Bacterial/genetics , Genome, Bacterial/genetics , Clostridium perfringens/classification , Clostridium perfringens/drug effects , Clostridium perfringens/metabolism , DNA, Complementary , Proteome/genetics , Transcriptome/genetics , Gene Ontology
19.
Braz. j. infect. dis ; Braz. j. infect. dis;21(4): 386-390, July-Aug. 2017. graf
Article in English | LILACS | ID: biblio-888886

ABSTRACT

Abstract Mycobacterium tuberculosis (MTB) adopts a special survival strategy to overcome the killing mechanism(s) of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3) of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ) and recombinant murine interleukin-10 (rmIL-10) on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50 ng/ml) of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50 ng/ml) significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Interferon-gamma/pharmacology , Interleukin-10/pharmacology , Macrophages/microbiology , Mycobacterium tuberculosis/genetics , Antigens, Bacterial/metabolism , Time Factors , Bacterial Proteins/genetics , Recombinant Proteins/pharmacology , Down-Regulation/drug effects , Dose-Response Relationship, Drug , Antigens, Bacterial/genetics
20.
Mem. Inst. Oswaldo Cruz ; 112(7): 499-503, July 2017. graf
Article in English | LILACS | ID: biblio-1040573

ABSTRACT

ABSTRACT Staphylococcus aureus pandemic clone USA300 has, in addition to its constitutive arginine catabolism (arc) gene cluster, an arginine catabolism mobile element (ACME) carrying another such cluster, which gives this clone advantages in colonisation and infection. Gene arcR, which encodes an oxygen-sensitive transcriptional regulator, is inside ACME and downstream of the constitutive arc gene cluster, and this situation may have an impact on its activation. Different relative expression behaviours are proven here for arcRACME and the arcACME operon compared to the constitutive ones. We also show that the artificially expressed recombinant ArcRACME protein binds to the promoter region of the arcACME operon; this mechanism can be related to a positive feedback model, which may be responsible for increased anaerobic survival of the USA300 clone during infection-related processes.


Subject(s)
Humans , Operon/genetics , Arginine/genetics , Staphylococcus aureus/genetics , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Arginine/metabolism , Staphylococcus aureus/metabolism , Gene Expression Regulation, Bacterial/genetics , Interspersed Repetitive Sequences/genetics , Genes, Bacterial/genetics
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