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1.
Artigo em Inglês | MEDLINE | ID: mdl-32086301

RESUMO

The four regulatory genes fscR1-fscR4 in Streptomyces sp. FR-008 form a genetic arrangement widely distributed in macrolide-producing bacteria. Our previous work has demonstrated that fscR1 and fscR4 are critical for production of the polyene antibiotic candicidin. In this study, we further characterized the role of the other two regulatory genes, fscR2 and fscR3, focusing on the relationship between these four regulatory genes. Disruption of a single or multiple regulatory genes did not affect bacterial growth, but transcription of genes in the candicidin biosynthetic gene cluster decreased, and candicidin production was abolished, indicating a critical role for each of the four regulatory genes, including fscR2 and fscR3, in candicidin biosynthesis. We found that fscR1-fscR4, although differentially expressed throughout the growth phase, displayed a similar temporal expression pattern, with an abrupt increase in the early exponential phase, coincident with initial detection of antibiotic production in the same phase. Our data suggest that the four regulatory genes fscR1-fscR4 have varying degrees of control over structural genes in the biosynthetic cluster, under the conditions examined. Extensive transcriptional analysis indicated that complex regulation exists between these four regulatory genes, forming a regulatory network, with FscR1 and FscR4 at a lower level. Comprehensive cross-complementation analysis indicates that functional complementation is restricted among the four regulators and unidirectional, with fscR1 complementing the loss of fscR3/R4 and fscR4 complementing loss of fscR2 Our study provides more insights into the role of, and the regulatory network formed by, these four regulatory genes controlling production of an important pharmaceutical compound.ImportanceThe regulation of antibiotic biosynthesis by Streptomyces species is complex, especially for biosynthetic gene clusters with multiple regulatory genes. The biosynthetic gene cluster for the polyene antibiotic candicidin contains four consecutive regulatory genes, which encode regulatory proteins from different families and which form a subcluster within the larger biosynthetic gene cluster in Streptomyces sp. FR-008. Syntenic arrangements of these regulatory genes are widely distributed in polyene gene clusters, such as the amphotericin and nystatin gene clusters, suggesting a conserved regulatory mechanism controlling production of these clinically important medicines. However, the relationships between these multiple regulatory genes are unknown. In this study, we determined that each of these four regulatory genes is critical for candicidin production. Additionally, using transcriptional analyses, bioassays, HPLC analysis, and genetic cross-complementation, we showed that FscR1-FscR4 comprise a hierarchical regulatory network that controls candicidin production and is likely representative of how expression of other polyene biosynthetic gene clusters is controlled.

2.
FEMS Microbiol Ecol ; 96(3)2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31917409

RESUMO

Many endogenous plasmids carry no noticeable benefits for their bacterial hosts, and the persistence of these 'cryptic plasmids' and their functional impacts are mostly unclear. In this study, we investigated these uncertainties using the social bacterium Myxococcus fulvus 124B02 and its endogenous plasmid pMF1. pMF1 possesses diverse genes that originated from myxobacteria, suggesting a longstanding co-existence of the plasmid with various myxobacterial species. The curing of pMF1 from 124B02 had almost no phenotypic effects on the host. Laboratory evolution experiments showed that the 124B02 strain retained pMF1 when subcultured on dead Escherichia coli cells but lost pMF1 when subcultured on living E. coli cells or on casitone medium; these results indicated that the persistence of pMF1 in 124B02 was environment-dependent. Curing pMF1 caused the mutant to lose the ability to predate and develop fruiting bodies more quickly than the pMF1-containing strain after they were subcultured on dead E. coli cells, which indicated that the presence of pMF1 in M. fulvus 124B02 has some long-term effects on its host. The results provide some new insights into the persistence and impacts of cryptic plasmids in their natural bacterial cells.

3.
Microb Cell Fact ; 18(1): 123, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291955

RESUMO

BACKGROUND: Myxococcus xanthus DK1622 is a model system for studying multicellular development, predation, cellular differentiation, and evolution. Furthermore, it is a rich source of novel secondary metabolites and is widely used as heterologous expression host of exogenous biosynthetic gene clusters. For decades, genetic modification of M. xanthus DK1622 has mainly relied on kanamycin and tetracycline selection systems. RESULTS: Here, we introduce an alternative selection system based on chloramphenicol (Cm) to broaden the spectrum of available molecular tools. A chloramphenicol-resistant growth phase and a chloramphenicol-susceptible growth phase before and after chloramphenicol-induction were prepared, and later sequenced to identify specific genes related to chloramphenicol-repercussion and drug-resistance. A total of 481 differentially expressed genes were revealed in chloramphenicol-resistant Cm5_36h and 1920 differentially expressed genes in chloramphenicol-dormant Cm_8h. Moreover, the gene expression profile in the chloramphenicol-dormant strain Cm_8h was quite different from that of Cm5_36 which had completely adapted to Cm, and 1513 differentially expression genes were identified between these two phenotypes. Besides upregulated acetyltransferases, several transporter encoding genes, including ABC transporters, major facilitator superfamily transporters (MFS), resistance-nodulation-cell division (RND) super family transporters and multidrug and toxic compound extrusion family transporters (MATE) were found to be involved in Cm resistance. After the knockout of the most highly upregulated MXAN_2566 MFS family gene, mutant strain DK-2566 was proved to be sensitive to Cm by measuring the growth curve in the Cm-added condition. A plasmid with a Cm resistance marker was constructed and integrated into chromosomes via homologous recombination and Cm screening. The integration efficiency was about 20% at different concentrations of Cm. CONCLUSIONS: This study provides a new antibiotic-based selection system, and will help to understand antibiotic resistance mechanisms in M. xanthus DK1622.


Assuntos
Resistência ao Cloranfenicol/genética , Deleção de Genes , Perfilação da Expressão Gênica , Recombinação Homóloga , Myxococcus xanthus/genética , Antibacterianos/farmacologia , Edição de Genes , Família Multigênica , Myxococcus xanthus/efeitos dos fármacos , Transcriptoma
4.
Microb Biotechnol ; 12(4): 763-774, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31069998

RESUMO

Glycosylation of natural products can influence their pharmacological properties, and efficient glycosyltransferases (GTs) are critical for this purpose. The polyketide epothilones are potent anti-tumour compounds, and YjiC is the only reported GT for the glycosylation of epothilone. In this study, we phylogenetically analysed 8261 GTs deposited in CAZy database and revealed that YjiC locates in a subbranch of the Macrolide I group, forming the YjiC-subbranch with 160 GT sequences. We demonstrated that the YjiC-subbranch GTs are normally efficient in epothilone glycosylation, but some showed low glycosylation activities. Sequence alignment of YjiC-subbranch showed that the 66th and 77th amino acid residues, which were close to the catalytic cavity in molecular docking model, were conserved in five high-active GTs (Q66 and P77) but changed in two low-efficient GTs. Site-directed residues swapping at the two positions in the two low-active GTs (BssGT and BamGT) and the high-active GT BsGT-1 demonstrated that the two amino acid residues played an important role in the catalytic efficiency of epothilone glycosylation. This study highlights that the potent GTs for appointed compounds are phylogenetically grouped with conserved residues for the catalytic efficiency.

5.
Mol Microbiol ; 112(1): 29-46, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30927282

RESUMO

In Streptomyces, GlnR is an activator protein that activates nitrogen-assimilation genes under nitrogen-limiting conditions. However, less is known regarding the regulation of these genes under nitrogen-rich conditions. We determined that the developmental regulator MtrA represses nitrogen-assimilation genes in nitrogen-rich media and that it competes with GlnR for binding to GlnR boxes. The GlnR boxes upstream of multiple nitrogen genes, such as amtB, were confirmed as MtrA binding sites in vitro by electrophoretic mobility shift assays and in vivo by ChIP-qPCR analysis. Transcriptional analysis indicated that, on nutrient-rich medium, MtrA profoundly repressed expression of nitrogen-associated genes, indicating opposing roles for MtrA and GlnR in the control of nitrogen metabolism. Using in vitro and in vivo analysis, we also showed that glnR is itself a direct target of MtrA and that MtrA represses glnR transcription. We further demonstrated functional conservation of MtrA homologues in the recognition of GlnR boxes upstream of nitrogen genes from different actinobacterial species. As mtrA and glnR are widespread among actinomycetes, this mechanism of potential competitive control over nitrogen metabolism genes may be common in this group, adding a major new layer of complexity to the known regulatory network for nitrogen metabolism in Streptomyces and related species.

6.
Biomolecules ; 8(4)2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30404219

RESUMO

Myxococcus xanthus DK1622 is a rich source of novel secondary metabolites, and it is often used as an expression host of exogenous biosynthetic gene clusters. However, the frequency of obtaining large genome-deletion variants by using traditional strategies is low, and progenies generated by homologous recombination contain irregular deletions. The present study aims to develop an efficient genome-engineering system for this bacterium based on the Cre/loxP system. We first verified the functionality of the native cre system that was integrated into the chromosome with an inducible promoter PcuoA. Then we assayed the deletion frequency of 8-bp-spacer-sequence mutants in loxP by Cre recombinase which was expressed by suicide vector pBJ113 or self-replicative vector pZJY41. It was found that higher guanine content in a spacer sequence had higher deletion frequency, and the self-replicative vector was more suitable for the Cre/loxP system, probably due to the leaky expression of inducible promoter PcuoA. We also inspected the effects of different antibiotics and the native or synthetic cre gene. Polymerase chain reaction (PCR) and sequencing of new genome joints confirmed that the Cre/loxP system was able to delete a 466 kb fragment in M. xanthus. This Cre/loxP-mediated recombination could serve as an alternative genetic manipulation method.


Assuntos
Edição de Genes , Genoma Bacteriano , Integrases/metabolismo , Myxococcus xanthus/genética , Recombinação Genética/genética , Antibacterianos/farmacologia , Sequência de Bases , Cromossomos Bacterianos/genética , Deleção de Genes , Família Multigênica , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Recombinases/metabolismo , Sideróforos/metabolismo
7.
Biochim Biophys Acta Gene Regul Mech ; 1861(10): 928-937, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30496038

RESUMO

Chaperonin groEL genes are duplicated in approximately 20% of bacteria, and the duplicates are differentially transcribed due to their divergent functions. The coordinated regulation of this differential transcription is as yet undetermined. In this study, we reported that the controlling inverted repeat of chaperone expression (CIRCE) element (the HrcA-binding site located upstream of the promoter) evolved for the transcriptional regulation of duplicate groELs. CIRCE composition and locations were found to be phylogenetically conserved in bacterial taxa. Myxococcus xanthus DK1622 has two CIRCE elements (CIRCE1groESL1 and CIRCE2groESL1) in the promoter region of groESL1 and one CIRCE element (CIRCEgroEL2) before groEL2. We also found that negative HrcA and positive ?32 regulators coordinated the transcription of duplicate groELs, and that the double deletion in DK1622 eliminated transcriptional differences and reduced the heat-shock responses of groELs. In vitro binding assays showed that HrcA protein binding was biased towards CIRCE1groESL1, followed by CIRCEgroEL2, but that HrcA proteins failed to bind with CIRCE2groESL1. Mutation experiments revealed that single-nucleotide mutations in the inverted repeat regions changed the HrcA-binding abilities of CIRCEs. We constructed an in vivo transcription-regulation system in Escherichia coli to pair each of the regulators with a groEL promoter. The results indicated that the transcriptional regulation performed by HrcA and ?32 was biased towards the groEL2 and groEL1 promoters, respectively. Based on promoter-sequence characteristics, we proposed a model of the coordinated regulation of the transcription of duplicate groELs in M. xanthus DK1622.


Assuntos
Proteínas de Bactérias/genética , Chaperonina 60/genética , Regulação Bacteriana da Expressão Gênica , Genes Duplicados , Regiões Promotoras Genéticas , Proteínas de Bactérias/biossíntese , Chaperonina 60/biossíntese , Proteínas de Choque Térmico/metabolismo , Myxococcus xanthus/genética , Filogenia , Proteínas Repressoras/metabolismo , Fator sigma/metabolismo , Transcrição Genética
8.
FEMS Microbiol Lett ; 365(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29931327

RESUMO

The genome of Streptomyces coelicolor encodes hundreds of putative regulatory proteins, most of which are of unknown function, including SCO5351. In this study, we determined that deletion of sco5351 largely abrogates production of actinorhodin (ACT) and reduces production of the calcium-dependent antibiotic (CDA). Comprehensive transcriptional analyses indicated that transcription of genes of the ACT pathway, including the pathway-specific regulator actII-orf4 and those involved in the building of the chemical compound, was markedly lower in Δsco5351 in the late growth phase. However, transcription of genes in the CDA cluster was notably reduced in Δsco5351 only in the early growth phase, suggesting that SCO5351 has a regulatory role throughout growth. Similar to the observations with Δsco5351, ACT production was blocked by mutagenesis of three conserved amino acids potentially involved in dimerization of SCO5351, indicating that protein dimerization is critical to the function of SCO5351. In addition, disruption of sco5351 delayed the formation of aerial mycelium and spores under the conditions tested and, consistent with this, transcription of developmental genes associated with spore formation was reduced in Δsco5351, implying that SCO5351 is involved in developmental control. Our findings reveal SCO5351 as a pleiotropic regulator with roles in both secondary metabolism and morphological development in S. coelicolor.


Assuntos
Antraquinonas/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Streptomyces coelicolor/crescimento & desenvolvimento , Streptomyces coelicolor/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Deleção de Genes , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Metabolismo Secundário , Streptomyces coelicolor/genética
9.
Org Biomol Chem ; 16(21): 3952-3960, 2018 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-29749412

RESUMO

Ferroptosis is a non-apoptotic, iron dependent form of regulated cell death that is characterized by the accumulation of lipid hydroperoxides. It has drawn considerable attention owing to its putative involvement in diverse neurodegenerative diseases. Ferrostatins are the first identified inhibitors of ferroptosis and they inhibit ferroptosis by efficiently scavenging free radicals in lipid bilayers. However, their further medicinal application has been limited due to the deficient knowledge of the lipid peroxyl radical-trapping mechanism. In this study, experimental and theoretical methods were performed to illustrate the possible lipid hydroperoxide inhibition mechanism of ferrostatins. The results show that an ortho-amine (-NH) moiety from ferrostatins can simultaneously interact with lipid radicals, and then form a planar seven-membered ring in the transition state, and finally present greater reactivity. NBO analysis shows that the formed planar seven-membered ring forces ortho-amines into better alignment with the aromatic π-system. It significantly increases the magnitudes of amine conjugation and improves spin delocalization in the transition state. Additionally, a classical H-bond type interaction was discovered between a radical and an o-NH group as another transition state stabilizing effect. This type of radical-trapping mechanism is novel and has not been found in diphenylamine or traditional polyphenol antioxidants. It can be said that o-phenylenediamine is a privileged pharmacophore for the design and development of ferroptosis inhibitors.


Assuntos
Morte Celular/efeitos dos fármacos , Cicloexilaminas/farmacologia , Desenho de Drogas , Fenilenodiaminas/farmacologia , Antioxidantes/farmacologia , Depuradores de Radicais Livres , Humanos , Peróxidos Lipídicos/metabolismo , Peróxidos Lipídicos/farmacologia , Relação Estrutura-Atividade
10.
Microb Cell Fact ; 17(1): 15, 2018 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-29378572

RESUMO

BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. CONCLUSIONS: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.


Assuntos
Sistemas CRISPR-Cas/genética , Epotilonas/biossíntese , Família Multigênica , Myxococcus xanthus/genética , Proteínas Recombinantes/genética , Transcrição Genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Epotilonas/genética , Myxococcus xanthus/metabolismo , Regiões Promotoras Genéticas , RNA Guia/genética , Metabolismo Secundário , Ativação Transcricional
11.
Microb Cell Fact ; 16(1): 142, 2017 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-28814300

RESUMO

BACKGROUND: The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. RESULTS: In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA-sgRNA-tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. CONCLUSIONS: By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.


Assuntos
Sistemas CRISPR-Cas/genética , Genes Bacterianos , Myxococcus xanthus/genética , Sequência de Bases , Família Multigênica , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , RNA de Transferência/genética , Deleção de Sequência
12.
Water Sci Technol ; 66(11): 2385-92, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23032769

RESUMO

Aquatic environments are known reservoirs of antibiotic-resistant bacteria, but little information is known about the role of Escherichia coli strain subgroups, integrons, and integron-associated gene cassettes in the prevalence of antimicrobial resistance. To address these knowledge gaps, the diversity and distribution of drug-resistant E. coli strains and their integrons in hospital wastewater (HWW) and XiaoQing River water (XQRW) in Jinan, China were compared. Phylogenetic assays showed that the isolates were distributed in every E. coli subgroup. The prevalence of antibiotic resistance in each E. coli subgroup from HWW was higher than in subgroups from XQRW, except for phylogenetic subgroup A(0). Classes 1 and 2 integrons were found in 327 strains (78.2% of the total 418 isolates) with a prevalence of 85.6% among the 209 isolates from HWW. Among 15 gene cassette arrays, dfrA17-aadA5 and dfrA12-orfF-aadA2 were the most prevalent. The prevalence of drug-resistance gene cassettes and diversity of arrays further proved that integrons were important contributors to the widespread occurrence of antibiotic resistance in E. coli among Jinan aquatic environments.


Assuntos
Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Integrons , Microbiologia da Água , Genes Bacterianos , Hospitais , Filogenia
13.
Sci China Life Sci ; 55(3): 261-7, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22437993

RESUMO

Rad51/RadA paralogs found in eukaryotes and euryarchaea play important roles during recombination and repair, and mutations in one of the human Rad51 paralogs, Rad51C, are associated with breast and ovarian cancers. The hyperthermophilic crenarchaeon Sulfolobus tokodaii encodes four putative RadA paralogs and studies on these proteins may assist in understanding the functions of human Rad51 paralogs. Here, we report the biochemical characterization of stRadC2, a S. tokodaii RadA paralog. Pull-down assays revealed that the protein was able to interact with the recombinase, RadA, and the Holliday junction endonuclease, Hjc. stRadC2 inhibited the strand exchange activity of RadA and facilitated Hjc-mediated Holliday junction DNA cleavage in vitro. RT-PCR analysis revealed that stRadC2 transcription was immediately reduced after UV irradiation, but was restored to normal levels at the late stages of DNA repair. Our results suggest that stRadC2 may act as an anti-recombination factor in DNA recombinational repair in S. tokodaii.


Assuntos
Proteínas Arqueais/química , Proteínas de Ligação a DNA/metabolismo , DNA/genética , Recombinação Genética , Sulfolobus/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Proteínas Arqueais/fisiologia , Reparo do DNA , DNA Cruciforme , DNA de Cadeia Simples/genética , Genoma Arqueal , Humanos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Rad51 Recombinase/química , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Fatores de Tempo
14.
DNA Repair (Amst) ; 11(2): 102-11, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22062475

RESUMO

Helicases and nucleases form complexes that play very important roles in DNA repair pathways some of which interact with each other at Holliday junctions. In this study, we present in vitro and in vivo analysis of Hjm and its interaction with Hjc in Sulfolobus. In vitro studies employed Hjm from the hyperthermophilic archaeon Sulfolobus tokodaii (StoHjm) and its truncated derivatives, and characterization of the StoHjm proteins revealed that the N-terminal module (residues 1-431) alone was capable of ATP hydrolysis and DNA binding, while the C-terminal one (residues 415-704) was responsible for regulating the helicase activity. The region involved in StoHjm-StoHjc (Hjc from S. tokodaii) interaction was identified as part of domain II, domain III (Winged Helix motif), and domain IV (residues 366-645) for StoHjm. We present evidence supporting that StoHjc regulates the helicase activity of StoHjm by inducing conformation change of the enzyme. Furthermore, StoHjm is able to prevent the formation of Hjc/HJ high complex, suggesting a regulation mechanism of Hjm to the activity of Hjc. We show that Hjm is essential for cell viability using recently developed genetic system and mutant propagation assay, suggesting that Hjm/Hjc mediated resolution of stalled replication forks is of crucial importance in archaea. A tentative pathway with which Hjm/Hjc interaction could have occurred at stalled replication forks is discussed.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , DNA Cruciforme/metabolismo , Sulfolobus/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Sobrevivência Celular , DNA Helicases/genética , DNA Cruciforme/genética , Resolvases de Junção Holliday/metabolismo , Hidrólise , Estrutura Terciária de Proteína , Deleção de Sequência , Sulfolobus/citologia , Sulfolobus/metabolismo
15.
Extremophiles ; 15(2): 227-34, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21197557

RESUMO

The nuclease NurA is present in all known thermophilic archaea and has been implicated to facilitate efficient DNA double-strand break end processing in Mre11/Rad50-mediated homologous recombinational repair. To understand the structural and functional relationship of this enzyme, we constructed five site-directed mutants of NurA from Sulfolobus tokodaii (StoNurA), D56A, E114A, D131A, Y291A, and H299A, at the conserved motifs, and four terminal deletion mutants, StoNurAΔN (19-331), StoNurAΔNΔC (19-303), StoNurAΔC (1-281), and StoNurAΔC (1-303), and characterized the proteins biochemically. We found that mutation at the acidic residue, D56, E114, D131, or at the basic residue, H299, abolishes the nuclease activity, while mutation at the aromatic residue Y291 only impairs the activity. Interestingly, by chemical cross-linking assay, we found that the mutant Y291A is unable to form stable dimer. Additionally, we demonstrated that deletion of the C-terminal amino acid residues 304-331 of StoNurA results in loss of the physical and functional interaction with the single-stranded DNA-binding protein (StoSSB). These results established that the C-terminal conserved aromatic residue Y291 is involved in dimer formation and the C-terminal residues 304-331 of NurA are involved in the interaction with single-stranded DNA-binding protein.


Assuntos
Proteínas Arqueais/química , Endodesoxirribonucleases/química , Exodesoxirribonucleases/química , Mutação , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Arqueais/genética , Reagentes para Ligações Cruzadas/farmacologia , Reparo do DNA , DNA de Cadeia Simples/genética , Dimerização , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Formaldeído/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Sulfolobus/enzimologia
16.
Extremophiles ; 15(1): 67-76, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21132514

RESUMO

XPB helicase is the largest subunit of transcription factor IIH (TFIIH), a ten-subunit protein complex essential for transcription initiation and nucleotide excision repair (NER) in Eukarya. Two XPB homologues (XPBI and XPBII) are present in the genome of most crenarchaeota, one of the two major phyla of archaea; however, the biochemical properties have not been fully characterized and their cellular roles have not been clearly defined. Here, we report that XPBI from the hyperthermophilic crenarchaeon Sulfolobus tokodaii (StoXPBI) is able to destabilize double-stranded DNA (dsDNA) helix independent of ATP (designated as dsDNA melting activity). This activity is inhibited by single-stranded DNA (ssDNA) and relies on the unique N-terminal domain of StoXPBI, which is also likely responsible for the intrinsic strong ssDNA binding activity of StoXPBI as revealed by deletion analysis. We demonstrate that the ATPase activity of StoXPBII is remarkably stimulated by StoBax1, a nuclease partner of StoXPBII. The role of the unique dsDNA melting activity of XPBI in NER in archaea was discussed.


Assuntos
Proteínas Arqueais/metabolismo , DNA Helicases/metabolismo , DNA Arqueal/metabolismo , DNA de Cadeia Simples/metabolismo , Sulfolobus/enzimologia
17.
J Bacteriol ; 192(13): 3540-4, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20418393

RESUMO

Besides inhibiting RecA activity at the protein level, Deinococcus radiodurans RecX can suppress RecA induction at the transcriptional level. The regulation of RecX on recA induction is independent of RecA activity, and its N terminus is involved in this process.


Assuntos
Proteínas de Bactérias/metabolismo , Deinococcus/metabolismo , Recombinases Rec A/metabolismo , Proteínas de Bactérias/genética , Deinococcus/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Recombinases Rec A/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Biotechnol Lett ; 32(1): 107-12, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19763406

RESUMO

A thermophilic lipase (lipGRD) from Geobacillus sp. RD-2, isolated from a hot spring in Yunnan, China, was cloned and over-expressed in Escherichia coli. The function of the conserved residue, Tyr224, near the presumed temperature switch site was analyzed by site-directed saturation mutagenesis. The activity of the wild type lipGRD was optimal at 55 degrees C and pH 7.5, but that from mutant Y224C was optimally active at 35 degrees C, whereas Y224P lipase was optimally active at 65 degrees C. Furthermore, the latter lipase retained 60% of its activity after incubation at 65 degrees C for 5 h. The conserved residue Tyr224, which is close to the lid helix, is the key amino acid residue determining the thermostability of the thermostable lipase.


Assuntos
Estabilidade Enzimática/fisiologia , Geobacillus stearothermophilus/enzimologia , Lipase/química , Lipase/metabolismo , Tirosina/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias , Clonagem Molecular , Estabilidade Enzimática/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Geobacillus stearothermophilus/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Lipase/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Tirosina/genética
19.
J Biochem ; 146(3): 337-42, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19443693

RESUMO

Apart from inhibiting RecA activity through protein-protein interactions, Deinococcus radiodurans RecX inhibits the expression of RecA and two other anti-oxidant proteins. To identify the repertoire of proteins regulated by RecX, comparative proteomic studies were undertaken on a wild-type strain (R1) and recX null mutant (RecX(-)). Two-dimensional electrophoresis followed by MALDI-TOF identification revealed 35 differentially expressed proteins, including 12 up-regulated and 23 down-regulated proteins in the mutant. The 12 up-regulated proteins are DNA repair proteins, stress response proteins, and metabolism-related proteins. Most of these have been previously characterized as ionizing radiation-induced proteins. The 23 down-regulated proteins are mainly involved in cellular metabolism, and some of these are key enzymes in the metabolic pathway. Thus, RecX is suggested to be involved in the switch between DNA damage response and normal metabolism in D. radiodurans.


Assuntos
Proteínas de Bactérias/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Deinococcus/metabolismo , Proteínas de Bactérias/genética , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deinococcus/genética , Regulação para Baixo , Eletroforese em Gel Bidimensional , Regulação Bacteriana da Expressão Gênica , Focalização Isoelétrica , Proteômica , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Regulação para Cima
20.
Biochem Biophys Res Commun ; 376(2): 369-74, 2008 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-18782564

RESUMO

The relationships among three PCNA (proliferating cell nuclear antigen) subunits in the hyperthermophilic archaeon Sulfolobus tokodaii (StoPCNAs) were analyzed and the effects of two PCNA complexes on the activities of the DNA helicase Hjm, DNA Ligase I, and Holliday junction specific endonuclease Hjc were tested. There was no strong self-interaction of each StoPCNA. StoPCNA1 and StoPCNA3 interacted with each other, so did StoPCNA2 and StoPCNA3, but no interaction between StoPCNA1 and StoPCNA2 was observed. Two trimeric complexes (designed StoPCNA123 and StoPCNA323) were formed in vitro and it was determined that StoPCNA323 was composed of one StoPCNA2 and two StoPCNA3 subunits, with StoPCNA2 bridging the two StoPCNA3 subunits. Both complexes inhibited the unwinding activity of Hjm and the ligation activity of DNA Ligase I. In contrast, both stimulated the Holliday junction cleavage activity of Hjc. Our results provide further evidence that in crenarchaea, the PCNAs exhibit diversity in subunit interaction and complex formation.


Assuntos
Proteínas Arqueais/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Sulfolobus/metabolismo , Proteínas Arqueais/genética , Sequência de Bases , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/antagonistas & inibidores , DNA Ligases/metabolismo , Dados de Sequência Molecular , Antígeno Nuclear de Célula em Proliferação/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Técnicas do Sistema de Duplo-Híbrido
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