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1.
Braz J Biol ; 83: e245379, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495147

RESUMO

Population growth is increasing rapidly around the world, in these consequences we need to produce more foods to full fill the demand of increased population. The world is facing global warming due to urbanizations and industrialization and in this concerns plants exposed continuously to abiotic stresses which is a major cause of crop hammering every year. Abiotic stresses consist of Drought, Salt, Heat, Cold, Oxidative and Metal toxicity which damage the crop yield continuously. Drought and salinity stress severally affected in similar manner to plant and the leading cause of reduction in crop yield. Plants respond to various stimuli under abiotic or biotic stress condition and express certain genes either structural or regulatory genes which maintain the plant integrity. The regulatory genes primarily the transcription factors that exert their activity by binding to certain cis DNA elements and consequently either up regulated or down regulate to target expression. These transcription factors are known as masters regulators because its single transcript regulate more than one gene, in this context the regulon word is fascinating more in compass of transcription factors. Progress has been made to better understand about effect of regulons (AREB/ABF, DREB, MYB, and NAC) under abiotic stresses and a number of regulons reported for stress responsive and used as a better transgenic tool of Arabidopsis and Rice.


Assuntos
Regulação da Expressão Gênica de Plantas , Regulon , Secas , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Regulon/genética , Estresse Fisiológico/genética
2.
Curr Microbiol ; 78(10): 3696-3707, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34426858

RESUMO

We previously reported that the two-component system MacRS regulates morphogenesis and production of the blue-pigmented antibiotic actinorhodin (ACT) in Streptomyces coelicolor. In this study, the role of MacRS was further extended to include control of the production of the red-pigmented antibiotic undecylprodigiosin (RED) and the calcium-dependent antibiotic (CDA), and control of other important cellular activities. Our data indicated that disruption of the MacRS TCS reduced production not only of ACT but also of RED and CDA. RNA-Seq analysis revealed that genes involved in both secondary metabolism and primary metabolism are differentially expressed in the MacRS deletion mutant ΔmacRS. Moreover, we found that genes of the Zur regulon are also markedly downregulated in ΔmacRS, suggesting a role for macRS in zinc homeostasis. In addition to previously identified MacR sites with strong matches to the MacR consensus recognition sequence, a genome-wide search revealed over one hundred less-stringent matches, including potential sites upstream of absR1, crgA, and smeA. Electrophoretic mobility shift assays demonstrated that MacR binds some of these sites in vitro. Although there is no strong MacR site upstream of the ACT regulatory gene actII-orf4 (sco5085), we showed that an engineered MacR site enhanced ACT production, providing an approach for modulating production of useful compounds. Altogether, our work suggests an important role for MacRS in a range of cellular activities in Streptomyces and its potential application in strain engineering.


Assuntos
Streptomyces coelicolor , Antraquinonas , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulon , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
3.
Mol Oral Microbiol ; 36(5): 278-290, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34351080

RESUMO

Zinc (Zn2+ ) is an essential divalent trace metal for living cells. Intracellular zinc homeostasis is critical to the survival and virulence of bacteria. Thus, the frequent fluctuations of salivary zinc, caused by the low physiological level and the frequent exogenous zinc introduction, present a serious challenge for bacteria colonizing the oral cavity. However, the regulation strategies to keep intracellular Zn2+ homeostasis in Streptococcus mutans, an important causative pathogen of dental caries, are unknown. Because zinc uptake is primarily mediated by an ATP-binding ABC transporter AdcABC in Streptococcus strains, we examined the function of AdcABC and transcription factor AdcR in S. mutans in this study. The results demonstrated that deletion of either adcA or adcCB gene impaired the growth but enhanced the extracellular polymeric matrix production in S. mutans, both of which could be relieved after excessive Zn2+ supplementation. Using RNA sequencing analysis, quantitative reverse transcription polymerase chain reaction examination, LacZ-reporter studies, and electrophoretic mobility shift assay, we showed that a MarR (multiple antibiotic resistance regulator) family transcription factor, AdcR, negatively regulates the expression of the genes adcR, adcC, adcB, and adcA by acting on the adcRCB and adcA promoters in response to Zn2+ concentration in their environmental niches. The deletion of adcR increases the sensitivity of S. mutans to excessive Zn2+ supply. Taken together, our findings suggest that Adc regulon, which consists of a Zn2+ uptake transporter AdcCBA and a Zn2+ -responsive repressor AdcR, plays a prominent role in the maintenance of intracellular zinc homeostasis of S. mutans.


Assuntos
Cárie Dentária , Regulon , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Homeostase , Humanos , Regulon/genética , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Zinco/metabolismo
4.
BMC Bioinformatics ; 22(1): 399, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376148

RESUMO

Numerous genomes are sequenced and made available to the community through the NCBI portal. However, and, unlike what happens for gene function annotation, annotation of promoter sequences and the underlying prediction of regulatory associations is mostly unavailable, severely limiting the ability to interpret genome sequences in a functional genomics perspective. Here we present an approach where one can download a genome of interest from NCBI in the GenBank Flat File (.gbff) format and, with a minimum set of commands, have all the information parsed, organized and made available through the platform web interface. Also, the new genomes are compared with a given genome of reference in search of homologous genes, shared regulatory elements and predicted transcription associations. We present this approach within the context of Community YEASTRACT of the YEASTRACT + portal, thus benefiting from immediate access to all the comparative genomics queries offered in the YEASTRACT + portal. Besides the yeast community, other communities can install the platform independently, without any constraints. In this work, we exemplify the usefulness of the presented tool, within Community YEASTRACT, in constructing a dedicated database and analysing the genome of the highly promising oleaginous red yeast species Rhodotorula toruloides currently poorly studied at the genome and transcriptome levels and with limited genome editing tools. Regulatory prediction is based on the conservation of promoter sequences and available regulatory networks. The case-study examined is focused on the Haa1 transcription factor-a key regulator of yeast resistance to acetic acid, an important inhibitor of industrial bioconversion of lignocellulosic hydrolysates. The new tool described here led to the prediction of a RtHaa1 regulon with expected impact in the optimization of R. toruloides robustness for lignocellulosic and pectin-rich residue biorefinery processes.


Assuntos
Regulon , Leveduras , Anotação de Sequência Molecular , Rhodotorula , Fatores de Transcrição , Leveduras/genética
5.
Appl Environ Microbiol ; 87(17): e0047321, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34160269

RESUMO

The heat shock response (HSR) is a universal cellular response that promotes survival following temperature increase. In filamentous Streptomyces, which accounts for ∼70% of commercial antibiotic production, HSR is regulated by transcriptional repressors; in particular, the widespread MerR-family regulator HspR has been identified as a key repressor. However, functions of HspR in other biological processes are unknown. The present study demonstrates that HspR pleiotropically controls avermectin production, morphological development, and heat shock and H2O2 stress responses in the industrially important species Streptomyces avermitilis. HspR directly activated ave structural genes (aveA1 and aveA2) and H2O2 stress-related genes (katA1, catR, katA3, oxyR, ahpC, and ahpD), whereas it directly repressed heat shock genes (HSGs) (the dnaK1-grpE1-dnaJ1-hspR operon, clpB1p, clpB2p, and lonAp) and developmental genes (wblB, ssgY, and ftsH). HspR interacted with PhoP (response regulator of the widespread PhoPR two-component system) at dnaK1p to corepress the important dnaK1-grpE1-dnaJ1-hspR operon. PhoP exclusively repressed target HSGs (htpG, hsp18_1, and hsp18_2) different from those of HspR (clpB1p, clpB2p, and lonAp). A consensus HspR-binding site, 5'-TTGANBBNNHNNNDSTSHN-3', was identified within HspR target promoter regions, allowing prediction of the HspR regulon involved in broad cellular functions. Taken together, our findings demonstrate a key role of HspR in the coordination of a variety of important biological processes in Streptomyces species. IMPORTANCE Our findings are significant to clarify the molecular mechanisms underlying HspR function in Streptomyces antibiotic production, development, and H2O2 stress responses through direct control of its target genes associated with these biological processes. HspR homologs described to date function as transcriptional repressors but not as activators. The results of the present study demonstrate that HspR acts as a dual repressor/activator. PhoP cross talks with HspR at dnaK1p to coregulate the heat shock response (HSR), but it also has its own specific target heat shock genes (HSGs). The novel role of PhoP in the HSR further demonstrates the importance of this regulator in Streptomyces. Overexpression of hspR strongly enhanced avermectin production in Streptomyces avermitilis wild-type and industrial strains. These findings provide new insights into the regulatory roles and mechanisms of HspR and PhoP and facilitate methods for antibiotic overproduction in Streptomyces species.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico/metabolismo , Peróxido de Hidrogênio/farmacologia , Ivermectina/análogos & derivados , Proteínas Repressoras/metabolismo , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Temperatura Alta , Ivermectina/metabolismo , Regulon , Proteínas Repressoras/genética , Streptomyces/efeitos dos fármacos , Streptomyces/genética , Estresse Fisiológico
6.
J Bacteriol ; 203(16): e0022421, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34096777

RESUMO

The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of vfr transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed vfr transcription under the control of the rhaBp rhamnose-inducible promoter system (designated PRha) and found that PRha promoter activity was highly dependent upon vfr. Vfr dependence was also observed for the araBp arabinose-inducible promoter (designated PBAD). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the PRha and PBAD promoters in vitro. Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in PRha and PBAD promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution. IMPORTANCE Heterologous gene expression and complementation constitute a valuable and widely utilized tool in bacterial genetics. The arabinose-inducible ParaBAD (PBAD) and rhamnose-inducible PrhaBAD (PRha) promoter systems are commonly used in P. aeruginosa genetics and prized for the tight control and dynamic expression ranges that can be achieved. In this study, we demonstrate that the activity of both promoters is dependent upon the cAMP-dependent transcription factor Vfr. While this poses an obvious problem for use in a vfr mutant background, the issue is more pervasive, considering that vfr transcription/synthesis and cAMP homeostasis are highly integrated into the cellular physiology of the organism and influenced by numerous global regulatory systems. Fortunately, the synthetic PTac promoter is not subject to Vfr regulatory control.


Assuntos
Arabinose/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/metabolismo , Ramnose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Repressão Catabólica , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Regulon , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
7.
Nat Commun ; 12(1): 2677, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976139

RESUMO

To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation. We also identify transcriptional and chromatin accessibility programs distinguishing each major cell type. Regulons, which are co-regulated gene sets sharing binding sites for a common transcription factor driver, recapitulate cell type clustering. We identify both cell type-specific and sex-specific regulons that are highly correlated with promoter accessibility, but not with methylation state, supporting the centrality of chromatin accessibility in shaping cell-defining transcriptional programs. The sn multi-omics atlas is accessible at snpituitaryatlas.princeton.edu.


Assuntos
Cromatina/genética , Metilação de DNA , Redes Reguladoras de Genes , Hipófise/metabolismo , Regulon/genética , Transcriptoma/genética , Animais , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Modelos Genéticos , Hipófise/citologia , Regiões Promotoras Genéticas/genética , Fatores Sexuais
9.
J Bacteriol ; 203(13): e0052320, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33927048

RESUMO

Serratia marcescens is an enteric bacterium that can function as an opportunistic pathogen with increasing incidence in clinical settings. This is mainly due to the ability to express a wide range of virulence factors and the acquisition of antibiotic resistance mechanisms. For these reasons, S. marcescens has been declared by the World Health Organization (WHO) as a research priority to develop alternative antimicrobial strategies. In this study, we found a PhoP-binding motif in the promoter region of transcriptional regulator RamA of S. marcescens RM66262. We demonstrated that the expression of ramA is autoregulated and that ramA is also part of the PhoP/PhoQ regulon. We have also shown that PhoP binds directly and specifically to ramA, mgtE1, mgtE2, lpxO1, and lpxO2 promoter regions and that RamA binds to ramA and lpxO1 but not to mgtE1 and lpxO2, suggesting an indirect control for the latter genes. Finally, we have demonstrated that in S. marcescens, RamA overexpression induces the AcrAB-TolC efflux pump, required to reduce the susceptibility of the bacteria to tetracycline and nalidixic acid. In sum, we here provide the first report describing the regulation of ramA under the control of the PhoP/PhoQ regulon and the regulatory role of RamA in S. marcescens. IMPORTANCE We demonstrate that in S. marcescens, the transcriptional regulator RamA is autoregulated and also controlled by the PhoP/PhoQ signal transduction system. We show that PhoP is able to directly and specifically bind to ramA, mgtE1, mgtE2, lpxO1, and lpxO2 promoter regions. In addition, RamA is able to directly interact with the promoter regions of ramA and lpxO1 but indirectly regulates mgtE1 and lpxO2. Finally, we found that in S. marcescens, RamA overexpression induces the AcrAB-TolC efflux pump, required to reduce susceptibility to tetracycline and nalidixic acid. Collectively, these results further our understanding of the PhoP/PhoQ regulon in S. marcescens and demonstrate the involvement of RamA in the protection against antibiotic challenges.


Assuntos
Proteínas de Bactérias/metabolismo , Resistência Microbiana a Medicamentos/genética , Serratia marcescens/genética , Serratia marcescens/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Antibacterianos , Proteínas de Bactérias/genética , Cloranfenicol , Microbioma Gastrointestinal , Regulação Bacteriana da Expressão Gênica , Homeostase , Lipídeo A , Ácido Nalidíxico , Fenótipo , Regulon , Transdução de Sinais , Tetraciclina , Fatores de Virulência
10.
Nucleic Acids Res ; 49(6): 3274-3293, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33693882

RESUMO

LuxR is a TetR family master quorum sensing (QS) regulator activating or repressing expression of hundreds of genes that control collective behaviors in Vibrios with underlying mechanism unknown. To illuminate how this regulator controls expression of various target genes, we applied ChIP-seq and DNase I-seq technologies. Vibrio alginolyticus LuxR controls expression of ∼280 genes that contain either symmetric palindrome (repDNA) or asymmetric (actDNA) binding motifs with different binding profiles. The median number of LuxR binding sites for activated genes are nearly double for that of repressed genes. Crystal structures of LuxR in complex with the respective repDNA and actDNA motifs revealed a new mode of LuxR DNA binding that involves contacts of its N-terminal extension to the minor groove. The N-terminal contacts mediated by Arginine-9 and Arginine-11 differ when LuxR binds to repDNA vs actDNA, leading to higher binding affinity at repressed targets. Moreover, modification of LuxR binding sites, binding profiles, and N-terminal extension have important consequences on QS-regulated phenotypes. These results facilitate fundamental understanding of the high flexibility of mechanisms of LuxR control of gene activation and repression in Vibrio QS, which may facilitate to design QS inhibiting chemicals that interfere with LuxR regulation to effectively control pathogens.


Assuntos
Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum/genética , Proteínas Repressoras/química , Transativadores/química , Ativação Transcricional , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Regulon , Vibrio alginolyticus/genética
11.
Food Microbiol ; 97: 103755, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33653528

RESUMO

Shewanella baltica is a typical specific spoilage organism causing the deterioration of seafood, but the exact regulation of its adaptive and competitive dominance in diverse environments remains undefined. In this study, the regulatory function of two sigma factors, RpoS and RpoN, in environmental adaptation and spoilage potential were evaluated in S. baltica SB02. Two in-frame deletion mutants, ΔrpoS and ΔrpoN, were constructed to explore the roles in their motility, biofilm formation, stress response and spoilage potential, as well as antibiotics by comparing the phenotypes and transcription with those of wild type (WT) strain. Compared with WT strain, the ΔrpoN showed the slower growth and weaker motility due to loss of flagella, while swimming of the ΔrpoS was increased. Deletion of rpoN significantly decreased biofilm biomass, and production of exopolysaccharide and pellicle, resulting in a thinner biofilm structure, while ΔrpoS formed the looser aggregation in biofilm. Resistance of S. baltica to NaCl, heat, ethanol and three oxidizing disinfectants apparently declined in the two mutants compared to WT strain. The ΔrpoN mutant decreased sensory score, accumulation of trimethylamine, putrescine and TVB-N and protease activity, while a weaker effect was observed in ΔrpoS. The two mutants had significantly higher susceptibility to antibiotics than WT strain, especially ΔrpoN. Deficiency of rpoN and rpoS significantly repressed the activities of two diketopiperazines related to quorum sensing (QS). Furthermore, transcriptome analyses revealed that RpoN was involved in the regulation of the expression of 143 genes, mostly including flagellar assembly, nitrogen and amino acid metabolism, ABC transporters. Transcript changes of seven differentially expressed coding sequences were in agreement with the phenotypes observed in the two mutants. Our findings reveal that RpoN, as a central regulator, controls the fitness and bacterial spoilage in S. baltica, while RpoS is a key regulatory factor of stress response. Characterization of these two sigma regulons in Shewanella has expanded current understanding of a possible co-regulatory mechanism with QS for adaptation and spoilage potential.


Assuntos
Proteínas de Bactérias/metabolismo , Perciformes/microbiologia , Shewanella/fisiologia , Fator sigma/metabolismo , Adaptação Fisiológica , Animais , Proteínas de Bactérias/genética , Biofilmes , Contaminação de Alimentos/análise , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Regulon , Shewanella/genética , Fator sigma/genética
12.
mBio ; 12(1)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531387

RESUMO

CsrA is a posttranscriptional global regulator in Vibrio cholerae Although CsrA is critical for V. cholerae survival within the mammalian host, the regulatory targets of CsrA remain mostly unknown. To identify pathways controlled by CsrA, RNA-seq transcriptome analysis was carried out by comparing the wild type and the csrA mutant grown to early exponential, mid-exponential, and stationary phases of growth. This enabled us to identify the global effects of CsrA-mediated regulation throughout the V. cholerae growth cycle. We found that CsrA regulates 22% of the V. cholerae transcriptome, with significant regulation within the gene ontology (GO) processes that involve amino acid transport and metabolism, central carbon metabolism, lipid metabolism, iron uptake, and flagellum-dependent motility. Through CsrA-RNA coimmunoprecipitation experiments, we found that CsrA binds to multiple mRNAs that encode regulatory proteins. These include transcripts encoding the major sigma factors RpoS and RpoE, which may explain how CsrA regulation affects such a large proportion of the V. cholerae transcriptome. Other direct targets include flrC, encoding a central regulator in flagellar gene expression, and aphA, encoding the virulence gene transcription factor AphA. We found that CsrA binds to the aphA mRNA both in vivo and in vitro, and CsrA significantly increases AphA protein synthesis. The increase in AphA was due to increased translation, not transcription, in the presence of CsrA, consistent with CsrA binding to the aphA transcript and enhancing its translation. CsrA is required for the virulence of V. cholerae and this study illustrates the central role of CsrA in virulence gene regulation.IMPORTANCE Vibrio cholerae, a Gram-negative bacterium, is a natural inhabitant of the aqueous environment. However, once ingested, this bacterium can colonize the human host and cause the disease cholera. In order to successfully transition between its aqueous habitat and the human host, the bacterium must sense changes in its environment and rapidly alter gene expression. Global regulators, including CsrA, play an integral role in altering the expression of a large number of genes to promote adaptation and survival, which is required for intestinal colonization. We used transcriptomics and a directed CsrA-RNA coimmunoprecipitation to characterize the CsrA regulon and found that CsrA alters the expression of more than 800 transcripts in V. cholerae Processes regulated by CsrA include motility, the rugose phenotype, and virulence pathways. CsrA directly binds to the aphA transcript and positively regulates the production of the virulence regulator AphA. Thus, CsrA regulates multiple processes that have been linked to pathogenesis.


Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Ligação a RNA/fisiologia , Vibrio cholerae/genética , Regulação Bacteriana da Expressão Gênica , Regulon , Transativadores/genética , Transcriptoma , Vibrio cholerae/patogenicidade , Virulência
13.
Nucleic Acids Res ; 49(4): 2357-2374, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33638994

RESUMO

RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription.


Assuntos
Proteínas de Bactérias/química , Regiões Promotoras Genéticas , Regulon , Salmonella typhimurium/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Modelos Moleculares , Mutação , Fosforilação , Conformação Proteica , Salmonella typhimurium/metabolismo , Transcrição Genética
14.
Biochem Biophys Res Commun ; 540: 16-21, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33429195

RESUMO

Polyphosphate, which is ubiquitous in cells in nature, is involved in a myriad of cellular functions, and has been recently focused on its metabolism related with microbial acclimation to phosphorus-source fluctuation. In view of the ecological importance of cyanobacteria as the primary producers, this study investigated the responsibility of polyphosphate metabolism for cellular acclimation to phosphorus starvation in a cyanobacterium, Synechocystis sp. PCC 6803, with the use of a disruptant (Δppx) as to the gene of exopolyphosphatase that is responsible for polyphosphate degradation. Δppx was similar to the wild type in the cellular content of polyphosphate to show no defect in cell growth under phosphorus-replete conditions. However, under phosphorus-starved conditions, Δppx cells were defective in a phosphorus-starvation dependent decrease of polyphosphate to show deleterious phenotypes as to their survival and the stabilization of the photosystem complexes. These results demonstrated some crucial role of exopolyphosphatase to degrade polyP in the acclimation of cyanobacterial cells to phosphorus-starved conditions. Besides, it was found that ppx expression is induced in Synechocystis cells in response to phosphorus starvation through the action of the two-component system, SphS and SphR, in the phosphate regulon. The information will be a foundation for a fuller understanding of the process of cyanobacterial acclimation to phosphorus fluctuation.


Assuntos
Hidrolases Anidrido Ácido/genética , Fósforo/deficiência , Fósforo/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Aclimatação , Proteínas de Bactérias/genética , Viabilidade Microbiana , Polifosfatos/metabolismo , Regulon , Synechocystis/citologia , Synechocystis/enzimologia
15.
Proc Natl Acad Sci U S A ; 118(5)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33495331

RESUMO

After the Great Oxidation Event (GOE), iron availability was greatly decreased, and photosynthetic organisms evolved several alternative proteins and mechanisms. One of these proteins, plastocyanin, is a type I blue-copper protein that can replace cytochrome c 6 as a soluble electron carrier between cytochrome b 6 f and photosystem I. In most cyanobacteria, expression of these two alternative proteins is regulated by copper availability, but the regulatory system remains unknown. Herein, we provide evidence that the regulatory system is composed of a BlaI/CopY-family transcription factor (PetR) and a BlaR-membrane protease (PetP). PetR represses petE (plastocyanin) expression and activates petJ (cytochrome c 6), while PetP controls PetR levels in vivo. Using whole-cell extracts, we demonstrated that PetR degradation requires both PetP and copper. Transcriptomic analysis revealed that the PetRP system regulates only four genes (petE, petJ, slr0601, and slr0602), highlighting its specificity. Furthermore, the presence of petE and petRP in early branching cyanobacteria indicates that acquisition of these genes could represent an early adaptation to decreased iron bioavailability following the GOE.


Assuntos
Citocromos c/metabolismo , Peptídeo Hidrolases/metabolismo , Plastocianina/metabolismo , Synechocystis/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Cobre/farmacologia , Epistasia Genética/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Regulon/genética , Synechocystis/efeitos dos fármacos
16.
Artigo em Inglês | MEDLINE | ID: mdl-33361299

RESUMO

Bacteria have evolved distinct molecular mechanisms as a defense against oxidative stress. The foremost regulator of the oxidative stress response has been found to be OxyR. However, the molecular details of regulation upstream of OxyR remain largely unknown and need further investigation. Here, we characterize an oxidative stress and antibiotic tolerance regulator, OsaR (PA0056), produced by Pseudomonas aeruginosa Knocking out of osaR increased bacterial tolerance to aminoglycoside and ß-lactam antibiotics, as well as to hydrogen peroxide. Expression of the oxyR regulon genes oxyR, katAB, and ahpBCF was increased in the osaR mutant. However, the OsaR protein does not regulate the oxyR regulon genes through direct binding to their promoters. PA0055, osaR, PA0057, and dsbM are in the same gene cluster, and we provide evidence that expression of those genes involved in oxidant tolerance is controlled by the binding of OsaR to the intergenic region between osaR and PA0057, which contain two divergent promoters. The gene cluster is also regulated by PA0055 via an indirect effect. We further discovered that OsaR formed intramolecular disulfide bonds when exposed to oxidative stress, resulting in a change of its DNA binding affinity. Taken together, our results indicate that OsaR is inactivated by oxidative stress and plays a role in the tolerance of P. aeruginosa to aminoglycoside and ß-lactam antibiotics.


Assuntos
Pseudomonas aeruginosa , Regulon , Aminoglicosídeos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Oxirredução , Estresse Oxidativo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Regulon/genética , Transativadores
17.
ACS Synth Biol ; 10(1): 173-182, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33375785

RESUMO

Fungal natural products (NPs) comprise a vast number of bioactive molecules with diverse activities, and among them are many important drugs. However, the yields of fungal NPs from native producers are usually low, and total synthesis of structurally complex NPs is challenging. As such, downstream derivatization and optimization of lead fungal NPs can be impeded by the high cost of obtaining sufficient starting material. In recent years, reconstitution of NP biosynthetic pathways in heterologous hosts has become an attractive alternative approach to produce complex NPs. Here, we present an efficient, cloning-free strategy for the cluster refactoring and total biosynthesis of fungal NPs in Aspergillus nidulans. Our platform places our genes of interest (GOIs) under the regulation of the robust asperfuranone afo biosynthesis gene machinery, allowing for their concerted activation upon induction. We demonstrated the utility of our system by creating strains that can synthesize high-value NPs, citreoviridin (1), mutilin (2), and pleuromutilin (3), with good to high yield and purity. This platform can be used not only for producing NPs of interests (i.e., total biosynthesis) but also for elucidating cryptic biosynthesis pathways.


Assuntos
Aspergillus nidulans/metabolismo , Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , Aspergillus nidulans/genética , Aurovertinas/química , Aurovertinas/metabolismo , Benzofuranos/química , Benzofuranos/metabolismo , Produtos Biológicos/química , Diterpenos/química , Diterpenos/metabolismo , Genes Fúngicos , Recombinação Homóloga , Cetonas/química , Cetonas/metabolismo , Família Multigênica , Plasmídeos/genética , Plasmídeos/metabolismo , Compostos Policíclicos/química , Compostos Policíclicos/metabolismo , Regulon/genética
18.
FEBS Lett ; 594(24): 4189-4200, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33270910

RESUMO

Pathway analysis methods are frequently applied to cancer gene expression data to identify dysregulated pathways. These methods often infer pathway activity based on the expression of genes belonging to a given pathway, even though the proteins ultimately determine the activity of a given pathway. Furthermore, the association between gene expression levels and protein activities is not well-characterized. Here, we posit that pathway-based methods are effective not because of the correlation between expression and activity of members of a given pathway, but because pathway gene sets overlap with the genes regulated by transcription factors (TFs). Thus, pathway-based methods do not inform about the activity of the pathway of interest but rather reflect changes in TF activities.


Assuntos
Redes Reguladoras de Genes/genética , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Humanos , Regulon/genética
19.
Sci Rep ; 10(1): 21019, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273546

RESUMO

Sphingopyxis granuli strain TFA is able to grow on the organic solvent tetralin as the only carbon and energy source. The aerobic catabolic pathway for tetralin, the genes involved and their regulation have been fully characterised. Unlike most of the bacteria belonging to the sphingomonads group, this strain is able to grow in anoxic conditions by respiring nitrate, though not nitrite, as the alternative electron acceptor. In this work, two fnr-like genes, fnrN and fixK, have been identified in strain TFA. Both genes are functional in E. coli and Sphingopyxis granuli although fixK, whose expression is apparently activated by FnrN, seems to be much less effective than fnrN in supporting anaerobic growth. Global transcriptomic analysis of a ΔfnrN ΔfixK double mutant and identification of Fnr boxes have defined a minimal Fnr regulon in this bacterium. However, expression of a substantial number of anaerobically regulated genes was not affected in the double mutant. Additional regulators such regBA, whose expression is also activated by Fnr, might also be involved in the anaerobic response. Anaerobically induced stress response genes were not regulated by Fnr but apparently induced by stress conditions inherent to anaerobic growth, probably due to accumulation of nitrite and nitric oxide.


Assuntos
Proteínas de Bactérias/genética , Proteínas Ferro-Enxofre/genética , Sphingomonadaceae/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Ferro-Enxofre/metabolismo , Regulon , Sphingomonadaceae/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma
20.
mSphere ; 5(6)2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33361129

RESUMO

Two-component systems control periplasmic Cu+ homeostasis in Gram-negative bacteria. In characterized systems such as Escherichia coli CusRS, upon Cu+ binding to the periplasmic sensing region of CusS, a cytoplasmic phosphotransfer domain of the sensor phosphorylates the response regulator CusR. This drives the expression of efflux transporters, chaperones, and redox enzymes to ameliorate metal toxic effects. Here, we show that the Pseudomonas aeruginosa two-component sensor histidine kinase CopS exhibits a Cu-dependent phosphatase activity that maintains CopR in a nonphosphorylated state when the periplasmic Cu levels are below the activation threshold of CopS. Upon Cu+ binding to the sensor, the phosphatase activity is blocked and the phosphorylated CopR activates transcription of the CopRS regulon. Supporting the model, mutagenesis experiments revealed that the ΔcopS strain exhibits maximal expression of the CopRS regulon, lower intracellular Cu+ levels, and increased Cu tolerance compared to wild-type cells. The invariant phosphoacceptor residue His235 of CopS was not required for the phosphatase activity itself but was necessary for its Cu dependency. To sense the metal, the periplasmic domain of CopS binds two Cu+ ions at its dimeric interface. Homology modeling of CopS based on CusS structure (four Ag+ binding sites) clearly supports the different binding stoichiometries in the two systems. Interestingly, CopS binds Cu+/2+ with 3 × 10-14 M affinity, pointing to the absence of free (hydrated) Cu+/2+ in the periplasm.IMPORTANCE Copper is a micronutrient required as cofactor in redox enzymes. When free, copper is toxic, mismetallating proteins and generating damaging free radicals. Consequently, copper overload is a strategy that eukaryotic cells use to combat pathogens. Bacteria have developed copper-sensing transcription factors to control copper homeostasis. The cell envelope is the first compartment that has to cope with copper stress. Dedicated two-component systems control the periplasmic response to metal overload. This paper shows that the sensor kinase of the copper-sensing two-component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of its cognate response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the system is activated by copper levels compatible with the absence of free copper in the cell periplasm. These observations emphasize the diversity of molecular mechanisms that have evolved in bacteria to manage the copper cellular distribution.


Assuntos
Cobre/química , Cobre/metabolismo , Escherichia coli/enzimologia , Periplasma/enzimologia , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Escherichia coli/genética , Histidina Quinase/metabolismo , Homeostase , Periplasma/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Pseudomonas aeruginosa/genética , Regulon/genética
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