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1.
Chinese Journal of Biotechnology ; (12): 1578-1595, 2023.
Article in Chinese | WPRIM | ID: wpr-981155

ABSTRACT

Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.


Subject(s)
Humans , Clostridioides/metabolism , Clostridioides difficile/metabolism , Bacterial Proteins/metabolism , Virulence , Anti-Bacterial Agents/metabolism
2.
Biomedical and Environmental Sciences ; (12): 604-613, 2023.
Article in English | WPRIM | ID: wpr-981093

ABSTRACT

OBJECTIVE@#Campylobacter jejuni NCTC11168 is commonly used as a standard strain for flagellar biosynthesis research. In this report, two distinguished phenotypic isolates (CJ1Z, flhA mutant strain, lawn; CJ2S, flhA complemented strain, normal colony) appeared during laboratory passages for NCTC11168.@*METHODS@#Phenotypic assessments, including motility plates, transmission electron microscopy, biofilm formation assay, autoagglutination assay, and genome re-sequencing for these two isolates (CJ1Z, flhA mutant strain; CJ2S, flhA complemented strain) were carried out in this study.@*RESULTS@#Transmission electron microscopy revealed that the flagellum was lost in CJ1Z. Phenotypic assessments and genome sequencing of the two isolates were performed in this study. The capacity for biofilm formation, colony auto-agglutination, and isolate motility was reduced in the mutant CJ1Z. Comparative genomic analysis indicated a unique native nucleotide insertion in flhA (nt, 2154) that caused the I719Y and I720Y mutations and early truncation in flhA.@*CONCLUSION@#FlhA has been found to influence the expression of flagella in C. jejuni. To the best of our knowledge, this is the first study to describe the function of the C-terminal of this protein.


Subject(s)
Campylobacter jejuni/genetics , Bacterial Proteins/metabolism , Mutation , Biological Variation, Population
3.
Chinese Journal of Biotechnology ; (12): 4939-4949, 2023.
Article in Chinese | WPRIM | ID: wpr-1008070

ABSTRACT

Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.


Subject(s)
Cattle , Animals , Mice , Bacterial Proteins/metabolism , Bacillus cereus/metabolism , Hemolysin Proteins/metabolism , Virulence Factors/metabolism , Enterotoxins/metabolism
4.
Chinese Journal of Biotechnology ; (12): 1809-1823, 2022.
Article in Chinese | WPRIM | ID: wpr-927819

ABSTRACT

Bacillus thuringiensis is widely used as an insecticide which is safe and environmentally friendly to humans and animals. One of the important insecticidal mechanisms is the binding of Bt toxins to specific toxin receptors in insect midgut and forming a toxin perforation which eventually leads to insect death. The resistance of target pests to Bt toxins is an important factor hampering the long-term effective cultivation of Bt crops and the continuous use of Bt toxins. This review summarizes the mechanism of insect resistance to Bt toxins from the perspective of important Bt toxin receptors in midgut cells of Lepidopteran insects, which may facilitate the in-depth study of Bt resistance mechanism and pest control.


Subject(s)
Animals , Bacillus thuringiensis/genetics , Bacillus thuringiensis Toxins , Bacterial Proteins/metabolism , Endotoxins/metabolism , Hemolysin Proteins/metabolism , Insecta/metabolism , Insecticide Resistance/genetics , Insecticides/pharmacology , Pest Control, Biological
5.
Chinese Journal of Biotechnology ; (12): 1050-1060, 2022.
Article in Chinese | WPRIM | ID: wpr-927762

ABSTRACT

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.


Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/metabolism , Drug Resistance , Mycobacterium smegmatis/metabolism , Peptide Elongation Factor G/pharmacology
6.
Chinese Journal of Preventive Medicine ; (12): 601-608, 2022.
Article in Chinese | WPRIM | ID: wpr-935331

ABSTRACT

Objective: Comparative analyses of wild-type Clostridioides difficile 630 (Cd630) strain and pathogenicity locus (PaLoc) knockout mutant (ΔPaLoc) by using RNA-seq technology. Analysis of differential expression of Cd630 wild-type strain and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioides difficile. Methods: Analysis of Cd630 and ΔPaLoc mutant strains using high-throughput sequencing (RNA-seq). Clustering differentially expressed genes and screening differentially expressed genes by DESeq software. Further analysis of differential genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, cytotoxicity assays of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines. Results: The transcriptome data showed that the ΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed. Compared to the wild-type strain, CD630_36010, CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold, respectively. Whereas the hom2 (high serine dehydrogenase), the CD630_15810 (spore-forming protein), CD630_23230 (zinc-binding dehydrogenase) and CD630_23240 (galactitol 1-phosphate 5-dehydrogenase) genes were down-regulated by 0.06, 0.075, 0.133 and 0.183 fold, respectively. The GO and KEGG enrichment analyses showed that the differentially transcribed genes in ΔPaLoc were enriched in the density-sensing system, ABC transport system, two-component system, phosphotransferase (PTS) system, and sugar metabolism pathway, as well as vancomycin resistance-related pathways. Cytotoxicity assays showed that the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain. Conclusion: Transcriptional sequencing analysis of the Cd630 and ΔPaLoc mutant strains showed that the toxin genes were not transcribed. Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.


Subject(s)
Humans , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Caco-2 Cells , Clostridioides , Clostridioides difficile/genetics , Oxidoreductases/metabolism , Transcriptome , Vaccines, Attenuated
7.
Chinese Journal of Preventive Medicine ; (12): 192-196, 2022.
Article in Chinese | WPRIM | ID: wpr-935269

ABSTRACT

To explore the biofilm inhibitory efficacy of perifosine against Pseudomonas aeruginosa (P. aeruginos) and its mechanisms. Twenty-fourwell plate was used to form biofilms at the bottom and crystal violet staining was used to determine the biofilm inhibitory effects of perifosine against P. aeruginosa, the wells without perifosine was set as control group. Glass tubes combined with crystal violet staining was used to detect the gas-liqud interface related bioiflm inhibitory effects of perifosine, the wells without perifosine was set as control group. Time-growth curved was used to detect the effects of perifosine on the bacteial planktonic cells growth of P. aeruginosa, the wells without perifosine was set as control group. The interaction model between perifosine and PqsE was assessed by molecular docking assay. The inhibitory effects of perifosine on the catalytic activity of PqsE was determined by detection the production of thiols, the wells without perifosine was set as control group. Binding affinity between perifosine and PqsE was detected by plasma surface resonance. The biofims at the bottom of the microplates and air-liquid interface were effectively inhibited by perifosine at the concentration of 4-8 μg/ml. There was no influence of perifosine on the cells growth of P. aeruginosa. The resuts of molecular docking assay indicates that perifosine could interacted with PqsE with the docking score of -10.67 kcal/mol. Perifosine could inhibit the catalytic activity of PqsE in a dose-dependent manner. The binding affinity between perifosine and PqsE was comfirmed by plasma surface resonance with KD of 6.65×10-5mol/L. Perifosine could inhibited the biofilm formation of P. aeruginosa by interacting with PqsE.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Biofilms , Molecular Docking Simulation , Phosphorylcholine/analogs & derivatives , Pseudomonas aeruginosa/metabolism , Quorum Sensing
8.
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
9.
Biol. Res ; 55: 19-19, 2022. ilus, tab, graf
Article in English | LILACS | ID: biblio-1383921

ABSTRACT

BACKGROUND: Acidophilic microorganisms like Leptospirillum sp. CF 1 thrive in environments with extremely low pH and high concentrations of dissolved heavy metals that can induce the generation of reactive oxygen species (ROS). Several hypothetical genes and proteins from Leptospirillum sp. CF 1 are known to be up regulated under oxidative stress conditions. RESULTS: In the present work, the function of hypothetical gene ABH19_09590 from Leptospirillum sp. CF 1 was studied. Heterologous expression of this gene in Escherichia coli led to an increase in the ability to grow under oxidant conditions with 5 mM K2CrO4 or 5 mM H2O2. Similarly, a significant reduction in ROS production in E. coli transformed with a plasmid carrying ABH19_09590 was observed after exposure to these oxidative stress elicitors for 30 min, compared to a strain complemented with the empty vector. A co transcriptional study using RT PCR showed that ABH19_09590 is contained in an operon, here named the "och" operon, that also contains ABH19_09585, ABH19_09595 and ABH19_09600 genes. The expression of the och operon was significantly up regulated in Leptospirillum sp. CF 1 exposed to 5 mM K2CrO4 for 15 and 30 min. Genes of this operon potentially encode a NADH:ubiquinone oxidoreductase, a CXXC motif containing protein likely involved in thiol/disulfide exchange, a hypothetical protein, and a di hydroxy acid dehydratase. A comparative genomic analysis revealed that the och operon is a characteristic genetic determinant of the Leptospirillum genus that is not present in other acidophiles. CONCLUSIONS: Altogether, these results suggest that the och operon plays a protective role against chromate and hydrogen peroxide and is an important mechanism required to face polyextremophilic conditions in acid environments.


Subject(s)
Chromates/metabolism , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Operon , Bacteria/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Reactive Oxygen Species/metabolism , Oxidative Stress/genetics , Escherichia coli
10.
Biol. Res ; 55: 9-9, 2022. ilus
Article in English | LILACS | ID: biblio-1383913

ABSTRACT

BACKGROUND: Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans. This pathogen activates multiple regulatory mechanisms in response to stress, and cobalamin biosynthesis might have a potential role in bacterial protection. Low temperature is a strategy used in the food industry to control bacteria proliferation; however, L. monocytogenes can grow in cold temperatures and overcome different stress conditions. In this study we selected L. monocytogenes List2-2, a strain with high tolerance to the combination of low temperature +copper, to understand whether the cobalamin biosynthesis pathway is part of the tolerance mechanism to this stress condition. For this, we characterized the transcription level of three cobalamin biosynthesis related genes ( cbiP , cbiB, and cysG ) and the eutV gene, a transcriptional regulator encoding gene involved in ethanolamine metabolism, in L. monocytogenes strain List2-2 growing simultaneously under two environmental stressors: low temperature (8 °C) +copper (0.5 mM of CuSO4 ×5H2O). In addition, the gene cbiP , which encodes an essential cobyric acid synthase required in the cobalamin pathway, was deleted by homologous recombination to evaluate the impact of this gene in L. monocytogenes tolerance to a low temperature (8 °C) +different copper concentrations. RESULTS: By analyzing the KEGG pathway database, twenty-two genes were involved in the cobalamin biosynthesis pathway in L. monocytogenes List2-2. The expression of genes cbiP , cbiB, and cysG, and eutV increased 6 h after the exposure to low temperature +copper. The cobalamin cbiP mutant strain List2-2Δ cbiP showed less tolerance to low temperature +copper (3 mM) than the wild type L. monocytogenes List2-2. The addition of cyanocobalamin (5 nM) to the medium reverted the phenotype observed in List2-2Δ cbiP . CONCLUSION: These results indicate that cobalamin biosynthesis is necessary for L. monocytogenes growth under stress and that the cbiP gene may play a role in the survival and growth of L. monocytogenes List2-2 at low temperature +copper.


Subject(s)
Humans , Listeria monocytogenes/genetics , Temperature , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Vitamin B 12/genetics , Vitamin B 12/metabolism , Cold Temperature , Copper
11.
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
12.
Biomedical and Environmental Sciences ; (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
13.
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
14.
Genomics, Proteomics & Bioinformatics ; (4): 289-304, 2020.
Article in English | WPRIM | ID: wpr-880483

ABSTRACT

Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyltransferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate aminotransferase Sll0480 both in vivo and in vitro and regulate the enzyme activity of Sll0480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.


Subject(s)
Bacterial Proteins/metabolism , Lysine/metabolism , Methyltransferases/metabolism , Photosynthesis , Protein Processing, Post-Translational , Synechocystis/growth & development
15.
Rev. argent. microbiol ; 51(3): 201-207, set. 2019. graf
Article in English | LILACS | ID: biblio-1041825

ABSTRACT

The consumption of soybean isoflavones (IS) is associated with several beneficial properties on human health. Some lactic acid bacteria possess ß-glucosidase enzyme, that allows to obtain the active form of IS (aglycone). The solid state fermentation (SSF) has received great attention in the last years in order to obtain several valuable compounds. SSF, using soybean as substrate and Lactobacillus rhamnosus CRL 981 as starter, was studied in the present work. Sucrose was added into soybean paste to study the effect on the behavior of the selected strain. The development of L. rhamnosus CRL 981 through pH and recount measures, sugar intake, organic acid production, ß-glucosidase activity and IS conversion were analyzed. No significant differences in growth and acidity were observed between soybean pastes with and without sucrose added, but the production of lactic acid was higher in the latter paste. The ß-glucosidase activity was detected in both pastes and the complete hydrolysis of IS at 12 h of fermentation was observed. Also, this strain was able to increase the free amino acids in soybean paste. SSF, using soybean as substrate and L. rhamnosus CRL 981 as starter culture, is an alternative process to obtain a soybean product bio-enriched in active IS with attractive nutritional characteristics.


El consumo de isoflavonas de soja (IS) está asociado a diversos beneficios para la salud humana. Ciertas bacterias lácticas poseen la enzima ß-glucosidasa, que permite obtener la forma bioactiva (agliconas) de las IS. La fermentación en sustrato sólido (FSS) ha recibido gran atención en los últimos anos debido a sus numerosas ventajas, y permite la obtención de productos con valor agregado. En el presente trabajo se estudió la FSS utilizando soja como sustrato y Lactobacillus rhamnosus CRL981 como cultivo iniciador. Con el fin de estudiar el efecto de una fuente de carbono externa sobre el comportamiento de la cepa seleccionada, se adicionó sacarosa a la pasta de soja. Se evaluó el crecimiento de L. rhamnosus CRL 981 a través de medidas de pH y recuento en placa. Además, se analizó el consumo de azúcares, producción de ácidos orgánicos, actividad ß-glucosidasa y conversión de IS. No se observaron diferencias significativas en el crecimiento y acidez entre las pastas de soja sin adición de sacarosa y con ella, sin embargo, la producción de ácido láctico fue mayor en esta última. La actividad de ß-glucosidasa se detectó en ambas pastas y se observó la hidrólisis completa de IS a las 12 h de fermentación. Además, esta cepa fue capaz de aumentar los aminoácidos libres en la pasta de soja. La FSS, utilizando soja como sustrato y L. rhamnosus CRL 981 como cultivo iniciador, es un proceso alternativo para obtener un producto de soja bioenriquecido en IS bioactivas con características nutricionales atractivas.


Subject(s)
Glycine max/metabolism , Lacticaseibacillus rhamnosus/metabolism , Fermentation , Vegetable Products/analysis , Isoflavones/biosynthesis , Sucrose/pharmacology , Bacterial Proteins/metabolism , beta-Glucosidase/metabolism , Lactic Acid/biosynthesis , Food Microbiology , Amino Acids/metabolism , Hydrolysis
16.
Rev. bras. enferm ; 72(3): 760-766, May.-Jun. 2019. tab
Article in English | BDENF, LILACS | ID: biblio-1013564

ABSTRACT

ABSTRACT Objective: To evaluate the risk factors related to Klebsiella pneumoniae carbapenemase infection after renal transplantation. Methods: This was a retrospective epidemiological (case-control) study, conducted from October 2011 to march 2016. Transplanted patients with infection by this bacteria during hospitalization were selected as cases. The controls were paired by age, sex, type of donor and transplant time. The proportion of cases and controls was 1:2. Results: Thirty hundred and five patients were included in the study (45 cases and 90 controls). The risk factors found for infection by KPC were: time of hospitalization after the transplant (OR: 4.82; CI95% 2.46-9.44), delayed kidney function (OR: 5.60; CI95% 1.91-11.01) and previous infectious for another microorganism ( OR: 34.13 CI95% 3.52-132.00). Conclusion: The risk of acquisition of this bacterium was directly related to invasive procedures and exposure to the hospital environment. The findings reinforce the importance of prevention measures and control of infection by this microorganism.


RESUMEN Objetivo: Evaluar los factores de riesgo relacionados con la infección por Klebsiella pneumoniae carbapenemasa después del trasplante renal. Método: Estudio retrospectivo epidemiológico (caso-control), realizado de octubre de 2011 a marzo de 2016. Pacientes transplantados con infección por esa bacteria durante la internación fueron seleccionados como casos. Los controles se parearon por edad, sexo, tipo de donante y tiempo de trasplante. La proporción de casos y controles fue de 1: 2. Resultados: Treinta y cinco pacientes fueron incluidos en el estudio (45 casos y 90 controles). Los factores de riesgo para la infección encontrados por KPC fueron: tiempo de hospitalización después del trasplante (OR: 4,82, IC95% 2,46-9,44), función renal retardada (OR: 5,60, IC95% 1, 91-11,01) y anterior infecciosa para otro microorganismo (OR: 34,13 IC95% 3,52-132,00). Conclusión: El riesgo de adquisición de esta bacteria estuvo directamente relacionado a procedimientos invasivos y exposición al ambiente hospitalario. Los hallazgos refuerzan la importancia de medidas de prevención y control de la infección por ese microorganismo.


Subject(s)
Humans , Male , Female , Adult , Pneumonia/ethnology , Bacterial Proteins/adverse effects , beta-Lactamases/adverse effects , Klebsiella Infections/etiology , Kidney Transplantation/adverse effects , Pneumonia/chemically induced , Pneumonia/epidemiology , Bacterial Proteins/metabolism , beta-Lactamases/metabolism , Brazil/epidemiology , Klebsiella Infections/metabolism , Klebsiella Infections/epidemiology , Case-Control Studies , Retrospective Studies , Risk Factors , Kidney Transplantation/methods , Klebsiella pneumoniae/metabolism , Klebsiella pneumoniae/pathogenicity , Middle Aged
17.
Journal of Zhejiang University. Science. B ; (12): 660-669, 2019.
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
18.
Journal of Zhejiang University. Science. B ; (12): 84-94, 2019.
Article in English | WPRIM | ID: wpr-1010445

ABSTRACT

Peach brown rot, caused by Monilinia fructicola, is one of the most serious peach diseases. A strain belonging to the Actinomycetales, named Streptomyces blastmyceticus JZB130180, was found to have a strong inhibitory effect on M. fructicola in confrontation culture. Following the inoculation of peaches in vitro, it was revealed that the fermentation broth of S. blastmyceticus JZB130180 had a significant inhibitory effect on disease development by M. fructicola. The fermentation broth of S. blastmyceticus JZB130180 had an EC50 (concentration for 50% of maximal effect) of 38.3 µg/mL against M. fructicola, as determined in an indoor toxicity test. Analysis of the physicochemical properties of the fermentation broth revealed that it was tolerant of acid and alkaline conditions, temperature, and ultraviolet radiation. In addition, chitinase, cellulase, and protease were also found to be secreted by the strain. The results of this study suggest that S. blastmyceticus JZB130180 may be used for the biocontrol of peach brown rot.


Subject(s)
Ascomycota/pathogenicity , Bacterial Proteins/metabolism , Cell Wall/metabolism , Cellulase/metabolism , Chitinases/metabolism , Fermentation , Fruit/microbiology , Pest Control, Biological/methods , Phylogeny , Plant Diseases/prevention & control , Prunus persica/microbiology , Siderophores/metabolism , Streptomyces/physiology
19.
Braz. j. microbiol ; 49(4): 914-918, Oct.-Dec. 2018. tab
Article in English | LILACS | ID: biblio-974286

ABSTRACT

ABSTRACT The global emergence of carbapenemases led to the need of developing new methods for their rapid detection. The aim of this study was to evaluate the performance of the rapid tests for carbapenemase-producing and non-producing Enterobacteriaceae. Carbapenem non-susceptible Enterobacteriaceae from a surveillance study submitted to a multiplex real time PCR for carbapenemase detection were included in this study. The isolates were subjected to the rapid phenotypic tests Carba NP, Blue-Carba and Carbapenem Inactivation Method (CIM). A total of 83 carbapenemase-producing (43) and non-producing (40) isolates were included in the study. The sensitivity/specificity were 62.7%/97.5%, 95.3%/100%, and 74.4%/97.5% for Carba NP, Blue-Carba and CIM, respectively. Both Carba NP and Blue-Carba presented their final results after 75 min of incubation; the final results for CIM were obtained only after 8 h. Failure to detect OXA-370 carbapenemase was the main problem for Carba NP and CIM assays. As the Blue-Carba presented the highest sensitivity, it can be considered the best screening test. Conversely, CIM might be the easiest to perform, as it does not require special reagents. The early detection of carbapenemases aids to establish infection control measures and prevent carbapenemases to spread reducing the risk of healthcare associated infections and therapeutic failure.


Subject(s)
Humans , Bacterial Proteins/analysis , beta-Lactamases/analysis , Enterobacteriaceae/enzymology , Enterobacteriaceae Infections/microbiology , Enzyme Assays/methods , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , beta-Lactamases/genetics , beta-Lactamases/metabolism , Brazil , Carbapenems/pharmacology , Polymerase Chain Reaction , Sensitivity and Specificity , Enterobacteriaceae/isolation & purification , Enterobacteriaceae/drug effects , Enterobacteriaceae/genetics , Enterobacteriaceae Infections/diagnosis , Anti-Bacterial Agents/pharmacology
20.
Braz. j. microbiol ; 49(4): 731-741, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-974291

ABSTRACT

ABSTRACT A bacterium isolated from Sterkfontein dam was confirmed to produce bioflocculant with excellent flocculation activity. The 16S rDNA nucleotide sequence analyses revealed the bacteria to have 99% similarity to Streptomyces platensis strain HBUM174787 and the sequence was deposited in the Genbank as Streptomyces platensis with accession number FJ 486385.1. Culture conditions for optimal production of the bioflocculant included glucose as a sole carbon source, resulting in flocculating activity of 90%. Other optimal conditions included: peptone as nitrogen source; presence of Mg2+ as cations and inoculum size of 1.0% (v/v) at neutral pH of 7. Optimum dose of the purified bioflocculant for the clarification of 4 g/L kaolin clay suspension at neutral pH was 0.2 mg/mL. Energy Dispersive X-ray analysis confirmed elemental composition of the purified bioflocculant in mass proportion (%w/w): carbon (21.41), oxygen (35.59), sulphur (26.16), nitrogen (0.62) and potassium (7.48). Fourier Transform Infrared Spectroscopy (FTIR) indicated the presence of hydroxyl, carboxyl, methoxyl and amino group in the bioflocculant. The bioflocculant produced by S. platensis removed chemical oxygen demand (COD) in river water and meat processing wastewater at efficiencies of 63.1 and 46.6% respectively and reduced their turbidity by 84.3 and 75.6% respectively. The high flocculating rate and removal efficiencies displayed by S. platensis suggests its industrial application in wastewater treatment.


Subject(s)
Streptomyces/chemistry , Bacterial Proteins/metabolism , Wastewater/chemistry , Streptomyces/isolation & purification , Streptomyces/genetics , Streptomyces/metabolism , Bacterial Proteins/genetics , Water Microbiology , Carbon/metabolism , Water Purification , Rivers/chemistry , Flocculation , Nitrogen/metabolism
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