Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Bacteriol ; 201(24)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31570530

RESUMO

The glpD (MSMEG_6761) gene encoding glycerol-3-phosphate dehydrogenase was shown to be crucial for M. smegmatis to utilize glycerol as the sole carbon source. The glpD gene likely forms the glpFKD operon together with glpF and glpK, encoding a glycerol facilitator and glycerol kinase, respectively. The gylR (MSMEG_6757) gene, whose product belongs to the IclR family of transcriptional regulators, was identified 182 bp upstream of glpF It was demonstrated that GylR serves as a transcriptional activator and is involved in the induction of glpFKD expression in the presence of glycerol. Three GylR-binding sites with the consensus sequence (GKTCGRC-N3-GYCGAMC) were identified in the upstream region of glpF by DNase I footprinting analysis. The presence of glycerol-3-phosphate was shown to decrease the binding affinity of GylR to the glpF upstream region with changes in the quaternary structure of GylR from tetramer to dimer. Besides GylR, cAMP receptor protein (Crp) and an alternative sigma factor, SigF, are also implicated in the regulation of glpFKD expression. Crp functions as a repressor, while SigF induces expression of glpFKD under energy-limiting conditions. In conclusion, we suggest here that the glpFKD operon is under the tripartite control of GylR, SigF, and Crp, which enables M. smegmatis to integrate the availability of glycerol, cellular energy state, and cellular levels of cAMP to exquisitely control expression of the glpFKD operon involved in glycerol metabolism.IMPORTANCE Using genetic approaches, we first revealed that glycerol is catabolized through the glycolytic pathway after conversion to dihydroxyacetone phosphate in two sequential reactions catalyzed by glycerol kinase (GlpK) and flavin adenine dinucleotide (FAD)-containing glycerol-3-phosphate dehydrogenase (GlpD) in M. smegmatis Our study also revealed that in addition to the GylR transcriptional activator that mediates the induction of the glpFKD operon by glycerol, the operon is regulated by SigF and Crp, which reflect the cellular energy state and cAMP level, respectively.


Assuntos
Proteínas de Bactérias/fisiologia , Proteína Receptora de AMP Cíclico/fisiologia , Regulação Bacteriana da Expressão Gênica , Glicerol Quinase/fisiologia , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/fisiologia , Mycobacterium smegmatis/metabolismo , Óperon , Fator sigma/fisiologia , Fatores de Transcrição/fisiologia , Ácidos Glicéricos/farmacologia , Mycobacterium smegmatis/genética
2.
Mol Cells ; 40(9): 632-642, 2017 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-28843272

RESUMO

The DevSR (DosSR) two-component system, which is a major regulatory system involved in oxygen sensing in mycobacteria, plays an important role in hypoxic induction of many genes in mycobacteria. We demonstrated that overexpression of the kinase domain of Mycobacterium tuberculosis (Mtb) PknB inhibited transcriptional activity of the DevR response regulator in Mycobacterium smegmatis and that this inhibitory effect was exerted through phosphorylation of DevR on Thr180 within its DNA-binding domain. Moreover, the purified kinase domain of Mtb PknB significantly phosphorylated RegX3, NarL, KdpE, TrcR, DosR, and MtrA response regulators of Mtb that contain the Thr residues corresponding to Thr180 of DevR in their DNA-binding domains, implying that transcriptional activities of these response regulators might also be inhibited when the kinase domain of PknB is overexpressed.


Assuntos
Hipóxia Celular/genética , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Oxigênio/química , Oxigênio/metabolismo , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tuberculose/genética , Tuberculose/microbiologia
3.
PLoS One ; 9(11): e111680, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25365321

RESUMO

The ahpC (MSMEG_4891) gene encodes alkyl hydroperoxide reductase C in Mycobacterium smegmatis mc2155 and its expression is induced under oxidative stress conditions. Two well-defined inverted repeat sequences (IR1 and IR2) were identified in the upstream region of ahpC. Using a crp (cAMP receptor protein: MSMEG_6189) mutant and in vitro DNA-binding assay, it was demonstrated that the IR1 sequence serves as a Crp-binding site and that Crp functions as an activator in the regulation of ahpC expression. The expression level of ahpC was shown to be proportional to intracellular cAMP levels. Intracellular levels of cAMP were increased in M. smegmatis, when it was treated with oxidative stress inducers. The IR2 sequence is very similar to the known consensus sequence of FurA-binding sites and involved in the negative regulation of ahpC expression. Taken together, these results suggest that the induction of ahpC expression under oxidative stress conditions probably results from a combinatory effect of both inactivation of FurA by oxidative stress and activation of Crp in response to increased levels of cAMP.


Assuntos
Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Mycobacterium smegmatis/enzimologia , Estresse Oxidativo , Peroxirredoxinas/biossíntese , Proteínas de Bactérias/genética , Mycobacterium smegmatis/genética , Peroxirredoxinas/genética
4.
FEMS Microbiol Lett ; 343(1): 26-33, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23480849

RESUMO

The DevSR two-component system in Mycobacterium smegmatis consists of the DevS histidine kinase and the DevR response regulator. It is a regulatory system that is involved in the adaptation of mycobacteria to hypoxic and NO stresses. Using the yeast two-hybrid assay and pull-down assay, it was demonstrated that the phosphoaccepting Asp (Asp54) of DevR is important for protein-protein interactions between DevR and DevS. The negative charge of Asp54 of DevR was shown to play an important role in protein-protein interactions between DevR and DevS. When the Lys104 residue, which is involved in transmission of conformational changes induced by phosphorylation of the response regulator, was replaced with Ala, the mutant form of DevR was not phosphorylated by DevS and functionally inactive in vivo. However, the K104A mutation in DevR only slightly affected protein-protein interactions between DevR and DevS.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/fisiologia , Protamina Quinase/metabolismo , Mapeamento de Interação de Proteínas , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Centrifugação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mycobacterium smegmatis/genética , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
5.
J Microbiol ; 50(2): 270-7, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22538656

RESUMO

Using yeast two-hybrid assay, we investigated protein-protein interactions between all orthologous histidine kinase (HK)/response regulator (RR) pairs of M. tuberculosis H37Rv and identified potential protein-protein interactions between a noncognate HK/RR pair, DosT/NarL. The protein interaction between DosT and NarL was verified by phosphotransfer reaction from DosT to NarL. Furthermore, we found that the DosT and DosS HKs, which share considerable sequence similarities to each other and form a two-component system with the DosR RR, have different cross-interaction capabilities with NarL: DosT interacted with NarL, while DosS did not. The dimerization domains of DosT and DosS were shown to be sufficient to confer specificity for DosR, and the different cross-interaction abilities of DosS and DosT with NarL were demonstrated to be attributable to variations in the amino acid sequences of the α2-helices of their dimerization domains.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/enzimologia , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Histidina Quinase , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Ligação Proteica , Proteínas Quinases/genética , Alinhamento de Sequência
6.
J Bacteriol ; 192(19): 4868-75, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20675480

RESUMO

The DosS (DevS) and DosT histidine kinases form a two-component system together with the DosR (DevR) response regulator in Mycobacterium tuberculosis. DosS and DosT, which have high sequence similarity to each other over the length of their amino acid sequences, contain two GAF domains (GAF-A and GAF-B) in their N-terminal sensory domains. Complementation tests in conjunction with phylogenetic analysis showed that DevS of Mycobacterium smegmatis is more closely related to DosT than DosS. We also demonstrated in vivo that DosS and DosT of M. tuberculosis play a differential role in hypoxic adaptation. DosT responds to a decrease in oxygen tension more sensitively and strongly than DosS, which might be attributable to their different autooxidation rates. The different responsiveness of DosS and DosT to hypoxia is due to the difference in their GAF-A domains accommodating the hemes. Multiple alignment analysis of the GAF-A domains of mycobacterial DosS (DosT) homologs and subsequent site-directed mutagenesis revealed that just one substitution of E87, D90, H97, L118, or T169 of DosS with the corresponding residue of DosT is sufficient to convert DosS to DosT with regard to the responsiveness to changes in oxygen tension.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Protamina Quinase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Teste de Complementação Genética , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Mycobacterium smegmatis/genética , Filogenia , Protamina Quinase/classificação , Protamina Quinase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
7.
J Bacteriol ; 192(15): 3925-33, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20511503

RESUMO

The cutR gene was identified 314 bp upstream of the divergently oriented cutB1C1A1 operon encoding carbon monoxide (CO) dehydrogenase in Mycobacterium sp. strain JC1. Its deduced product was composed of 320 amino acid residues with a calculated molecular mass of 34.1 kDa and exhibits a basal sequence similarity to the regulatory proteins belonging to the LysR family. Using a cutR deletion mutant, it was demonstrated that CutR is required for the efficient utilization of CO by Mycobacterium sp. strain JC1 growing with CO as the sole source of carbon and energy. CutR served as a transcriptional activator for expression of the duplicated cutBCA operons (cutB1C1A1 and cutB2C2A2) and was involved in the induction of the cutBCA operons by CO. The cutBCA operons were also subjected to catabolite repression. An inverted repeat sequence (TGTGA-N(6)-TCACA) with a perfect match with the binding motif of cyclic AMP receptor protein was identified immediately upstream of and overlapping with the translational start codons of cutB1 and cutB2. This palindrome sequence was shown to be involved in catabolite repression of the cutBCA operons. The transcription start point of cutR was determined to be the nucleotide G located 36 bp upstream of the start codon of cutR. Expression of cutR was higher in Mycobacterium sp. strain JC1 grown with glucose than that grown with CO.


Assuntos
Aldeído Oxirredutases/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Complexos Multienzimáticos/metabolismo , Mycobacterium/enzimologia , Mycobacterium/genética , Aldeído Oxirredutases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Óperon , Sítio de Iniciação de Transcrição
8.
J Bacteriol ; 190(20): 6795-804, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18708494

RESUMO

The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O(2). The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis. The binding of O(2) to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O(2) and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/fisiologia , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Protamina Quinase/química , Protamina Quinase/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Cristalografia por Raios X , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Compostos Ferrosos/metabolismo , Heme/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Estrutura Terciária de Proteína , Alinhamento de Sequência , Ubiquinona/metabolismo , Vitamina K 2/metabolismo
9.
J Bacteriol ; 189(15): 5617-25, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17557830

RESUMO

In this study, the H303A mutant form of the cbb(3) oxidase (H303A oxidase), which has the H303A mutation in its catalytic subunit (CcoN), was purified from Rhodobacter sphaeroides. The H303A oxidase showed the same catalytic activity as did the wild-type form of the oxidase (WT oxidase). The heme contents of the mutant and WT forms of the cbb(3) oxidase were also comparable. However, the puf and puc operons, which are under the control of the PrrBA two-component system, were shown to be derepressed aerobically in the R. sphaeroides strain expressing the H303A oxidase. Since the strain harboring the H303A oxidase exhibited the same cytochrome c oxidase activity as the stain harboring the WT oxidase did, the aerobic derepression of photosynthesis gene expression observed in the H303A mutant appears to be the result of a defective signaling function of the H303A oxidase rather than reflecting any redox changes in the ubiquinone/ubiquinol pool. It was also demonstrated that ubiquinone inhibits not only the autokinase activity of full-length PrrB but also that of the truncated form of PrrB lacking its transmembrane domain, including the proposed quinone binding sequence. These results imply that the suggested ubiquinone binding site within the PrrB transmembrane domain is not necessary for the inhibition of PrrB kinase activity by ubiquinone. Instead, it is probable that signaling through H303 of the CcoN subunit of the cbb(3) oxidase is part of the pathway through which the cbb(3) oxidase affects the relative kinase/phosphatase activity of the membrane-bound PrrB.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , Regulação Bacteriana da Expressão Gênica , Fotossíntese/fisiologia , Proteínas Quinases/metabolismo , Rhodobacter sphaeroides/fisiologia , Substituição de Aminoácidos/genética , Sítios de Ligação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Histidina Quinase , Mutação de Sentido Incorreto , Fosforilação , Fotossíntese/genética , Rhodobacter sphaeroides/genética , Transdução de Sinais/genética , Ubiquinona/metabolismo
10.
Biochemistry ; 43(24): 7915-23, 2004 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-15196036

RESUMO

The PrrBA two-component system in Rhodobacter sphaeroides 2.4.1, which is composed of the PrrB histidine kinase and the PrrA response regulator, controls the expression of all of the photosynthesis genes, either directly or indirectly, in response to changes in oxygen tension. In vivo under aerobic conditions it is the cbb(3) cytochrome c oxidase which generates an inhibitory signal preventing the accumulation of activated PrrA. Using purified cbb(3) cytochrome c oxidase, PrrB, and PrrA, we demonstrate in vitro that the cbb(3) oxidase inhibits PrrB activity by apparently increasing the intrinsic PrrB phosphatase activity, which dephosphorylates phosphorylated PrrA without alteration of the PrrB kinase activity. The transmembrane domain of PrrB is required for the enhancement of PrrB phosphatase activity by the cbb(3) oxidase. Full-length PrrB has a significantly greater ability to phosphorylate PrrA than does truncated PrrB lacking the transmembrane domain. This is at least in part due to the lower autophosphorylation rate of the truncated PrrB relative to the full-length PrrB. This finding provides evidence that the sensing domain (transmembrane domain) of PrrB plays an important role not only in optimally sensing the state of the cbb(3) oxidase but also in maintaining the correct conformation of PrrB, providing optimal autokinase activity.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Quinases/metabolismo , Rhodobacter sphaeroides/metabolismo , Transdução de Sinais , Sequência de Bases , Primers do DNA , Histidina Quinase , Fosforilação
11.
Microbiology (Reading) ; 149(Pt 4): 949-960, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12686637

RESUMO

A new genetic locus was identified in Rhodobacter sphaeroides which is required for optimal synthesis of the light-harvesting spectral complexes as well as for optimal growth under anaerobic conditions with dimethyl sulfoxide (DMSO) as a terminal electron acceptor. The primary structure of the deduced osp gene product shows significant homology to the receiver domain of known response regulators common to bacterial two-component systems. However, site-directed mutagenesis revealed that the Osp protein appears not to be involved in a phospho-relay signal transduction pathway. Paradoxically, the effect of the Osp protein upon spectral complex levels is exerted at the transcriptional level of photosynthesis gene expression. The absence of the Osp protein does not appear to have a general effect on house-keeping metabolism. In cells lacking Osp, the levels of DMSO reductase appear to be normal. The quaternary structure of the Osp protein was determined to be a homodimer and it was directly demonstrated that Osp does not bind to the promoter region of photosynthesis genes as judged by mobility-shift experiments and primary structure analysis.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/genética , Sequência de Aminoácidos , Anaerobiose , Proteínas de Bactérias/metabolismo , Dimetil Sulfóxido/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Rhodobacter sphaeroides/crescimento & desenvolvimento
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...