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1.
Arch Virol ; 164(10): 2613-2616, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31321586

RESUMO

A new virus with a circular double-stranded DNA genome was discovered in green Sichuan pepper with vein clearing symptoms. Its complete genome of 8,014 bp contains three open reading frames (ORF) on the plus strand, which is typical of members of the genus Badnavirus in the family Caulimoviridae. Sequence comparisons revealed that the new virus has the highest nucleotide sequence identity with grapevine vein-clearing virus (GVCV). In particular, the identity of the two viruses in the ORF3 RT-RNase H region is 71.9%, which is below the species demarcation cutoff of 80% for badnaviruses. Phylogenetic analysis also placed the new virus with GVCV in a cluster. The virus was tentatively named "green Sichuan pepper vein clearing-associated virus" (GSPVCaV). The geographical distribution and genetic diversity of GSPVCaV were studied. Another isolate was found to be highly divergent.


Assuntos
Badnavirus/classificação , Badnavirus/isolamento & purificação , DNA Viral/genética , Filogeografia , Doenças das Plantas/virologia , Zanthoxylum/virologia , DNA/química , DNA/genética , DNA Circular/química , DNA Circular/genética , DNA Viral/química , Genoma Viral , Fases de Leitura Aberta , Análise de Sequência de DNA , Homologia de Sequência
2.
Arch Virol ; 164(10): 2551-2558, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31321588

RESUMO

Here, we report two novel enteroviruses, designated as SD-S67 and SD-S68, isolated from a goat farm. Their complete genome sequences were determined and found to be 7455 and 7465 nucleotides in length, respectively. Molecular characterization revealed that SD-S67 is closely related to bovine enterovirus strain 261 and that SD-S68 to caprine enterovirus strain CEV-JL14. Phylogenetic analysis showed that SD-S67 clustered with members of the species Enterovirus F, and that SD-S68 clustered with enteroviruses of goats and sheep. Recombination analysis showed that SD-S67 is likely to have undergone several recombination events in the process of its evolution. To the best of our knowledge, this is the first report of an enterovirus F isolate from a goat and of a coinfection with enteroviruses of different species in the same goat herd.


Assuntos
Infecções por Enterovirus/veterinária , Enterovirus/classificação , Enterovirus/isolamento & purificação , Doenças das Cabras/virologia , Filogenia , Animais , Análise por Conglomerados , Efeito Citopatogênico Viral , Enterovirus/genética , Infecções por Enterovirus/virologia , Genoma Viral , Cabras , Microscopia Eletrônica de Transmissão , Recombinação Genética , Análise de Sequência de DNA , Homologia de Sequência , Vírion/ultraestrutura , Cultura de Vírus
3.
Arch Virol ; 164(10): 2605-2608, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31300889

RESUMO

Porcine reproductive and respiratory syndrome virus 1 is a major cause of swine morbidity and mortality in various parts of the world, including Hungary. A national elimination programme to reduce the associated economic burden was initiated in Hungary in 2012. Using extensive laboratory surveillance, we identified and isolated an unusual PRRSV strain. The complete coding sequence of this isolate was determined and analyzed. The genome of this Hungarian PRRSV1 strain, HUN60077/16, is 15,081 nucleotides in length. Phylogenetic and recombination analysis showed a mosaic structure of the genome where a large fragment of ORF1b and the genomic region coding for ORF3 to ORF7 showed a very close genetic relationship to the vaccine virus Unistrain, while the ORF1a region, the 3' end of ORF1b, and the whole ORF2 were only distantly related to this or any other PRRSV1 strain whose genome sequence is available in the GenBank database. Genomic characterization of PRRSV strains is crucial when possible vaccine-associated cases are identified. This approach not only helps to identify genetic interactions between vaccine and wild-type PRRSV1 strains but may also be needed to prevent trust in commercial vaccines from being undermined.


Assuntos
Genoma Viral , Filogenia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Recombinação Genética , Vacinas Virais/genética , Animais , Genótipo , Hungria , Fases de Leitura Aberta , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Análise de Sequência de DNA , Homologia de Sequência , Suínos
4.
Gene ; 713: 143974, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31301484

RESUMO

An orthologous gene of SEPALLATA1, designated as IiSEP1, was isolated from Isatis indigotica. The genomic DNA of IiSEP1 is 3.1 Kb in length. The full-length cDNA of IiSEP1 is 1481 bp and contains a 756 bp ORF encoding a 251-amino-acid protein. Sequence comparison revealed that IiSEP1 belonged to the MADS-box gene family. IiSEP1 contains 7 exons and 6 introns, showing similar exon-intron structure with Arabidopsis SEP1. Phylogenetic analysis suggested that IiSEP1 belonged to AGL2/SEP subfamily and was likely to be an I. indigotica ortholog of Arabidopsis SEP1. Quantitative real-time PCR showed that IiSEP1 was predominantly expressed in the reproductive organs. Ectopic expression of IiSEP1 in Arabidopsis resulted in early flowering, accompanied with the reduction of inflorescence number and the production of terminal flower on the top of the main stems. Moreover, IiSEP1 overexpressing flowers generated numerous variations in phenotype. The sepals were changed into petal-sepal mosaic structures or displayed carpelloid features, and transparent ovules were formed in internal surface of these sepals. In addition, some flowers were constituted by sepals and pistil, but lacked petals and stamens. Taken together, IiSEP1 might play important roles in reproductive growth of I. indigotica and could affect the morphogenesis of flowers and fruits.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Fatores de Transcrição Forkhead/genética , Isatis/crescimento & desenvolvimento , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Sequência de Aminoácidos , Arabidopsis/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Isatis/genética , Proteínas de Domínio MADS/genética , Fenótipo , Plantas Geneticamente Modificadas/genética , Homologia de Sequência
5.
Gene ; 713: 143975, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31302167

RESUMO

Hair is one of the defining characteristics of mammals. The hair shaft has a two-layer structure comprising the cortex, which is the inner layer and is composed of cortical cells, and the cuticle, which is the outermost layer. S100 calcium-binding protein A3 (S100A3) is expressed at high levels in the human hair cuticle. Arginine 51 of S100A3 protein is citrullinated specifically by peptidylarginine deiminase 3 (PAD3), and this citrullination is related to maturation of the cuticle. However, the detailed evolutionary processes of S100A3 and PAD3 during mammalian evolution are unknown. Here, we show that nonsynonymous changes in S100A3 accelerated in the common ancestral branch of mammals, probably as a result of positive selection that returned after the acquisition of hair cuticle-specific function in mammals. Later, pseudogenisation or nonfunctionalisation of S100A3 and PAD3 occurred in some species, such as the cetaceans. Our results show that positive selection and relaxation of the functional constraints of genes played important roles in the evolution of mammalian hair.


Assuntos
Evolução Molecular , Cabelo/química , Mamíferos/genética , Desiminases de Arginina em Proteínas/genética , Proteínas S100/genética , Seleção Genética , Sequência de Aminoácidos , Animais , Filogenia , Homologia de Sequência
6.
Comput Biol Chem ; 80: 498-511, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176140

RESUMO

Magnesium (Mg) is an important micronutrient for various physiological processes in plants. In this study, putative Magnesium Transporter (MGT) genes have been identified in Solanum lycopersicum, Solanum tuberosum, Brachypodium distachyon, Fagaria vesca, Brassica juncea and were classified into 5 distinct groups based on their sequence homology. MGT genes are very diverse and possess very low sequence identity within its family. However, the Gly-Met-Asn (GMN) signature motif is present in most of the genes which are believed to be essential for Mg2+ recognition. In S. lycopersicum, different physiological root growth pattern was observed in both Mg excess and deficient conditions. Quantitative RT-PCR gene expression study shows that most of the SlMGT genes were upregulated in response to Mg deficient condition.


Assuntos
Proteínas de Transporte de Cátions/genética , Genoma , Magnoliopsida/genética , Proteínas de Plantas/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Magnésio/metabolismo , Magnoliopsida/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Conformação Proteica em alfa-Hélice , Reação em Cadeia da Polimerase em Tempo Real , Sementes/genética , Homologia de Sequência , Estresse Fisiológico/genética , Regulação para Cima
7.
BMC Genomics ; 20(1): 335, 2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053062

RESUMO

BACKGROUND: The recently published complete mitogenome of the European lobster (Homarus gammarus) that was generated using long-range PCR exhibits unusual gene composition (missing nad2) and gene rearrangements among decapod crustaceans with strong implications in crustacean phylogenetics. Such atypical mitochondrial features will benefit greatly from validation with emerging long read sequencing technologies such as Oxford Nanopore that can more accurately identify structural variation. RESULTS: We re-sequenced the H. gammarus mitogenome on an Oxford Nanopore Minion flowcell and performed a long-read only assembly, generating a complete mitogenome assembly for H. gammarus. In contrast to previous reporting, we found an intact mitochondrial nad2 gene in the H. gammarus mitogenome and showed that its gene organization is broadly similar to that of the American lobster (H. americanus) except for the presence of a large tandemly duplicated region with evidence of pseudogenization in one of each duplicated protein-coding genes. CONCLUSIONS: Using the European lobster as an example, we demonstrate the value of Oxford Nanopore long read technology in resolving problematic mitogenome assemblies. The increasing accessibility of Oxford Nanopore technology will make it an attractive and useful tool for evolutionary biologists to verify new and existing unusual mitochondrial gene rearrangements recovered using first and second generation sequencing technologies, particularly those used to make phylogenetic inferences of evolutionary scenarios.


Assuntos
Evolução Biológica , Biologia Computacional/métodos , Duplicação Gênica , Genoma Mitocondrial , Proteínas Mitocondriais/genética , Nanoporos , Nephropidae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala , Família Multigênica , Nephropidae/metabolismo , Filogenia , Análise de Sequência de DNA , Homologia de Sequência
8.
Acta Crystallogr D Struct Biol ; 75(Pt 5): 498-504, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31063152

RESUMO

Bovine meat and milk factors (BMMFs) are circular, single-stranded episomal DNAs that have been detected in bovine meat and milk products. BMMFs are thought to have roles in human malignant and degenerative diseases. BMMFs encode a replication initiator protein (Rep) that is actively transcribed and translated in human cells. In this study, a Rep WH1 domain encoded on a BMMF (MSBI1.176) isolated from a multiple sclerosis human brain sample was determined to 1.53 Šresolution using X-ray crystallography. The overall structure of the MSBI1.176 WH1 domain was remarkably similar to other Rep structures, despite having a low (28%) amino-acid sequence identity. The MSBI1.176 WH1 domain contained elements common to other Reps, including five α-helices, five ß-strands and a hydrophobic pocket. These new findings suggest that the MSBI1.176 Rep might have comparable roles and functions to other known Reps of different origins.


Assuntos
Encéfalo/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Esclerose Múltipla/metabolismo , Plasmídeos/isolamento & purificação , Plasmídeos/metabolismo , Transativadores/química , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Homologia de Sequência
9.
BMC Genomics ; 20(1): 388, 2019 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-31103028

RESUMO

BACKGROUND: Hydrothermal vents and cold seeps are typical deep-sea chemosynthetically-driven ecosystems that allow high abundance of specialized macro-benthos. To gather knowledge about the genetic basis of adaptation to these extreme environments, species shared between different habitats, especially for the dominant species, are of particular interest. The galatheid squat lobster, Shinkaia crosnieri Baba and Williams, 1998, is one of the few dominant species inhabiting both deep-sea hydrothermal vents and cold seeps. In this study, we performed transcriptome analyses of S. crosnieri collected from the Iheya North hydrothermal vent (HV) and a cold seep in the South China Sea (CS) to provide insights into how this species has evolved to thrive in different deep-sea chemosynthetic ecosystems. RESULTS: We analyzed 5347 orthologs between HV and CS to identify genes under positive selection through the maximum likelihood approach. A total of 82 genes were identified to be positively selected and covered diverse functional categories, potentially indicating their importance for S. crosnieri to cope with environmental heterogeneity between deep-sea vents and seeps. Among 39,806 annotated unigenes, a large number of differentially expressed genes (DEGs) were identified between HV and CS, including 339 and 206 genes significantly up-regulated in HV and CS, respectively. Most of the DEGs associated with stress response and immunity were up-regulated in HV, possibly allowing S. crosnieri to increase its capability to manage more environmental stresses in the hydrothermal vents. CONCLUSIONS: We provide the first comprehensive transcriptomic resource for the deep-sea squat lobster, S. crosnieri, inhabiting both hydrothermal vents and cold seeps. A number of stress response and immune-related genes were positively selected and/or differentially expressed, potentially indicating their important roles for S. crosnieri to thrive in both deep-sea vents and cold seeps. Our results indicated that genetic adaptation of S. crosnieri to different deep-sea chemosynthetic environments might be mediated by adaptive evolution of functional genes related to stress response and immunity, and alterations in their gene expression that lead to different stress resistance. However, further work is required to test these proposed hypotheses. All results can constitute important baseline data for further studies towards elucidating the adaptive mechanisms in deep-sea crustaceans.


Assuntos
Adaptação Fisiológica , Anomuros/genética , Biodiversidade , Temperatura Baixa , Fontes Hidrotermais , Transcriptoma , Sequência de Aminoácidos , Animais , Anomuros/crescimento & desenvolvimento , Anomuros/metabolismo , Ecossistema , Filogenia , Homologia de Sequência
10.
J Plant Res ; 132(4): 521-529, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115708

RESUMO

Shoots of the aquatic eudicot family, Podostemaceae, exhibit unusual organogenesis with mixed leaf and stem identities. New shoots arise at the base of the older shoot with shoot apical meristem (SAM) identity but the entire SAM differentiates into a "leaf" as it develops in the Podostemoideae subfamily. The "leaves" are tightly arranged in a zigzag manner to form an apparent distichous shoot as a whole. Although previous studies have suggested that Podostemoideae shoots have evolved by modifying the ancestral sympodial branching system in the basal Tristichoideae subfamily, this evolutionary scenario requires elucidation at the molecular level. To confirm that the shoots arise as axillary shoots, in the present study, we examined gene expression patterns in plumular shoots of Zeylanidium tailichenoides using CUP-SHAPED COTYLEDON 3 (CUC3) and SHOOT MERISTEMLESS (STM) orthologs, which are involved in the determination of axils and meristem formation in model plants. Expression of the CUC3 ortholog was detected at the adaxial base of cotyledons and parental shoots where the new shoots are initiated, while STM ortholog was expressed at the initiation site and in the young shoot primordia throughout early shoot development. The results demonstrate that each Z. tailichenoides shoot arises as an axillary bud in a manner similar to axillary meristem formation in model plants involving CUC3 and STM genes. Considering that each of the two cotyledons produces an axillary bud that in turn continues to form its own axillary bud independently, the apparent distichous shoot in Z.tailichenoides is not a single shoot, but a composite of two sympodially branched shoots.


Assuntos
Proteínas de Arabidopsis/fisiologia , Malpighiales/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/metabolismo , Fatores de Transcrição/fisiologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Hibridização In Situ , Malpighiales/crescimento & desenvolvimento , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Brotos de Planta/crescimento & desenvolvimento , Homologia de Sequência , Fatores de Transcrição/genética
11.
PLoS Negl Trop Dis ; 13(4): e0007335, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31002713

RESUMO

Enteroviruses (EVs) are among the most common viruses infecting humans worldwide but only a few Non-Polio Enterovirus (NPEV) isolates have been characterized in the Democratic Republic of Congo (DR Congo). Moreover, circulating vaccine-derived polioviruses (PVs) [cVDPVs] isolated during multiple outbreaks in DR Congo from 2004 to 2018 have been characterized so far only by the sequences of their VP1 capsid coding gene. This study was carried to i) investigate the circulation and genetic diversity of NPEV and polio vaccine isolates recovered from healthy children and Acute Flaccid Paralysis (AFP) patients, ii) evaluate the occurrence of genetic recombination among EVs belonging to the Enterovirus C species (including PVs) and iii) identify the virological factors favoring multiple emergences of cVDPVs in DR Congo. The biological material considered in this study included i) a collection of 91 Sabin-like PVs, 54 cVDPVs and 150 NPEVs isolated from AFP patients between 2008 and 2012 in DR Congo and iii) a collection of 330 stool specimens collected from healthy children in 2013 in the Kasai Oriental and Maniema provinces of DR Congo. Studied virus isolates were sequenced in four distinct sub-genomic regions 5'-UTR, VP1, 2CATPase and 3Dpol. Resulting sequences were compared through comparative phylogenetic analyses. Virus isolation showed that 19.1% (63/330) healthy children were infected by EVs including 17.9% (59/330) of NPEVs and 1.2% (4/330) of type 3 Sabin-like PVs. Only one EV-C type, EV-C99 was identified among the NPEV collection from AFP patients whereas 27.5% of the 69 NPEV isolates typed in healthy children belonged to the EV-C species: CV-A13 (13/69), A20 (5/69) and A17 (1/69). Interestingly, 50 of the 54 cVDPVs featured recombinant genomes containing exogenous sequences in at least one of the targeted non-structural regions of their genomes: 5'UTR, 2CATPase and 3Dpol. Some of these non-vaccine sequences of the recombinant cVDPVs were strikingly related to homologous sequences from co-circulating CV-A17 and A20 in the 2CATPase region as well as to those from co-circulating CV-A13, A17 and A20 in the 3Dpol region. This study provided the first evidence uncovering CV-A20 strains as major recombination partners of PVs. High quality AFP surveillance, sensitive environmental surveillance and efficient vaccination activities remain essential to ensure timely detection and efficient response to recombinant cVDPVs outbreaks in DR Congo. Such needs are valid for any epidemiological setting where high frequency and genetic diversity of Coxsackieviruses A13, A17 and A20 provide a conducive viral ecosystem for the emergence of virulent recombinant cVDPVs.


Assuntos
Enterovirus Humano C/genética , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Evolução Molecular , Poliovirus/genética , Recombinação Genética , Linhagem Celular , Criança , República Democrática do Congo/epidemiologia , Humanos , Filogenia , Vacina Antipólio Oral , Estudos Prospectivos , RNA Viral/genética , RNA Viral/isolamento & purificação , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência
12.
Acta Crystallogr D Struct Biol ; 75(Pt 4): 437-450, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30988260

RESUMO

Although the HcpR regulator plays a vital step in initiation of the nitrosative stress response in many Gram-negative anaerobic bacteria, the molecular mechanisms that it uses to mediate gas sensing are not well understood. Here, a 2.6 Šresolution crystal structure of the N-terminal sensing domain of the anaerobic periodontopathogen Porphyromonas gingivalis HcpR is presented. The protein has classical features of the regulators belonging to the FNR-CRP family and contains a hydrophobic pocket in its N-terminal sensing domain. It is shown that heme bound to HcpR exhibits heme iron as a hexacoordinate system in the absence of nitric oxide (NO) and that upon nitrosylation it transitions to a pentacoordinate system. Finally, small-angle X-ray scattering experiments on full-length HcpR reveal that the C-terminal DNA-binding domain of HcpR has a high degree of interdomain flexibility.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Heme/metabolismo , Óxido Nítrico/metabolismo , Estresse Nitrosativo , Porphyromonas gingivalis/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Cristalografia por Raios X/métodos , Modelos Moleculares , Estrutura Molecular , Porphyromonas gingivalis/fisiologia , Conformação Proteica , Homologia de Sequência
13.
BMC Genomics ; 20(1): 272, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30952207

RESUMO

BACKGROUND: The interferon-induced transmembrane (IFITM) protein family comprises a class of restriction factors widely characterised in humans for their potent antiviral activity. Their biological activity is well documented in several animal species, but their genetic variation and biological mechanism is less well understood, particularly in avian species. RESULTS: Here we report the complete sequence of the domestic chicken Gallus gallus IFITM locus from a wide variety of chicken breeds to examine the detailed pattern of genetic variation of the locus on chromosome 5, including the flanking genes ATHL1 and B4GALNT4. We have generated chIFITM sequences from commercial breeds (supermarket-derived chicken breasts), indigenous chickens from Nigeria (Nsukka) and Ethiopia, European breeds and inbred chicken lines from the Pirbright Institute, totalling of 206 chickens. Through mapping of genetic variants to the latest chIFITM consensus sequence our data reveal that the chIFITM locus does not show structural variation in the locus across the populations analysed, despite spanning diverse breeds from different geographic locations. However, single nucleotide variants (SNVs) in functionally important regions of the proteins within certain groups of chickens were detected, in particular the European breeds and indigenous birds from Ethiopia and Nigeria. In addition, we also found that two out of four SNVs located in the chIFITM1 (Ser36 and Arg77) and chIFITM3 (Val103) proteins were simultaneously under positive selection. CONCLUSIONS: Together these data suggest that IFITM genetic variation may contribute to the capacities of different chicken populations to resist virus infection.


Assuntos
Antígenos de Diferenciação/genética , Evolução Molecular , Loci Gênicos , Marcadores Genéticos , Polimorfismo de Nucleotídeo Único , Seleção Genética , Sequência de Aminoácidos , Animais , Galinhas , Mapeamento Cromossômico , Variações do Número de Cópias de DNA , Genoma , Análise de Sequência de DNA , Homologia de Sequência
14.
Enzyme Microb Technol ; 126: 32-40, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31000162

RESUMO

Functionally uncharacterized gene Rv3627c is predicted to encode a carboxypeptidase in the pathogen of Mycobacterium tuberculosis (M. tuberculosis), which remains a major threat to human health. Here, we sought to reveal the function of Rv3627c and to elucidate its effects on mycobacterial growth. Rv3627c was purified from E. coli using Ni2+-NTA affinity chromatography, and its identity was confirmed with a monoclonal anti-polyhistidine antibody. An enzyme activity assay involving a d-amino acid oxidase-peroxidase coupled colorimetric reaction and high-performance thin layer chromatography was performed. A pull-down assay and MS-MS were also employed to identify putative interaction partners of Rv3627c. Scanning electron microscopy and transmission electron microscopy were performed to observe any morphological alterations to Mycobacterium smegmatis (M. smegmatis). We successfully obtained soluble expressed Rv3627c and identified it as carboxypeptidase using prepared peptidoglycan. Four proteins were identified as potential interaction partners with Rv3627c based on results obtained from both a pull-down assay and MS/MS analysis. Rv3627c over-expression induced M. smegmatis cells to become elongated, and promoted the formation of increased numbers of Z-rings. Rv3627c, a novel carboxypeptidase in M. tuberculosis identified in this study, exerts important effects on mycobacterial cell morphology and cell division. This functional information provides a promising insight into anti-mycobacterial target designs.


Assuntos
Proteínas de Bactérias/metabolismo , Carboxipeptidases/metabolismo , Divisão Celular , Mycobacterium smegmatis/citologia , Mycobacterium smegmatis/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Carboxipeptidases/genética , Clonagem Molecular , Dipeptídeos/metabolismo , Humanos , Mapas de Interação de Proteínas , Homologia de Sequência
15.
Enzyme Microb Technol ; 126: 50-61, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31000164

RESUMO

The biocontrol activity of some soil strains of Chromobacterium sp. against pathogenic fungi has been attributed to secreted chitinases. The aim of this work was to characterize biochemically a recombinant chitinase (CvChi47) from C. violaceum ATCC 12472 and to investigate its effects on phytopathogenic fungi. CvChi47 is a modular enzyme with 450 amino acid residues, containing a type I signal peptide at the N-terminal region, followed by one catalytic domain belonging to family 18 of the glycoside hydrolases, and two type-3 chitin-binding domains at the C-terminal end. The recombinant enzyme was expressed in Escherichia coli as a His-tagged protein and purified to homogeneity. The native signal peptide of CvChi47 was used to direct its secretion into the culture medium, from where the recombinant product was purified by affinity chromatography on chitin and immobilized metal. The purified protein showed an apparent molecular mass of 46 kDa, as estimated by denaturing polyacrylamide gel electrophoresis, indicating the removal of the signal peptide. CvChi47 was a thermostable protein, retaining approximately 53.7% of its activity when heated at 100 °C for 1 h. The optimum hydrolytic activity was observed at 60 °C and pH 5. The recombinant chitinase inhibited the conidia germination of the phytopathogenic fungi Fusarium oxysporum and F. guttiforme, hence preventing mycelial growth. Furthermore, atomic force microscopy experiments revealed a pronounced morphological alteration of the cell surface of conidia incubated with CvChi47 in comparison to untreated cells. Taken together, these results show the potential of CvChi47 as a molecular tool to control plant diseases caused by these Fusarium species.


Assuntos
Antifúngicos/farmacologia , Quitinases/metabolismo , Chromobacterium/enzimologia , Fusarium/crescimento & desenvolvimento , Doenças das Plantas/prevenção & controle , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Quitinases/química , Quitinases/genética , Clonagem Molecular , Estabilidade Enzimática , Fusarium/efeitos dos fármacos , Doenças das Plantas/microbiologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento , Temperatura Ambiente
16.
Enzyme Microb Technol ; 126: 69-76, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31000166

RESUMO

The beta-propeller phytase (BPP) is an enzyme that hydrolyzes phyate to release inorganic phosphorus. The BPP produced by Pseudomonas sp. FB15 (PSphy) possesses an additional N-terminal domain that is not present in BPP produced by other Bacillus species. In this study, BPP produced by Bacillus sp. SJ-10 (SJ-10phy) was fused with the N-terminal domain of PSphy and the enzymatic properties of the resulting fusion protein (FUSJ-10phy) were investigated. FUSJ-10phy exhibited an optimal temperature that was 10 °C lower than that of the wild-type enzyme. A comparison of kinetic parameters showed that the turnover rate of FUSJ-10phy was 2.39-fold higher than that of SJ-10phy, representing a 1.79-fold increase in catalytic efficiency. In addition, BPP produced by Bacillus sp. SJ-48 has relatively low sequence similarity with SJ-10phy, was fused with N-terminal domain (FUSJ-48phy). FUSJ-48phy also increased catalytic efficiency and changed the optimal temperature. These results indicate that, when fused to other BPPs, the N-terminal domain of PSphy increases catalytic efficiency and enzyme activity at lower temperatures.


Assuntos
6-Fitase/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Pseudomonas/enzimologia , Proteínas Recombinantes de Fusão/metabolismo , Temperatura Ambiente , 6-Fitase/química , 6-Fitase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência
17.
J Genet ; 982019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30945676

RESUMO

Calmodulin-binding transcription activators (CAMTAs) are a family of transcription factors that play an important role in plants' response to the various biotic and abiotic stresses. The common bean (Phaseolus vulgaris L.) is one of the most important crops in the world and plays a pivotal role in sustainable agriculture. To date, the composition of CAMTA genes in genomes of Phaseolus species and their role in resistance to drought stress are not known. In this study, five PhavuCAMTA genes were characterized in common bean genome through bioinformatics analysis, the morphological and biochemical response of 23 Ph.vulgaris genotypes to different levels of drought stress were evaluated and the expression patterns of PhCAMTA1 in the leaf tissues of sensitive and tolerant genotypes were analysed. Gene structure, protein domain organization and phylogenetic analyses showed that the CAMTAs of Phaseolus were structurally similar and clustered into three groups as other plant CAMTAs. Genotypes showeda differential response to drought stress. Thus, the plant height, number of nodes and flower, total chlorophyll and total protein content, and activity of antioxidant enzymes (ascorbate peroxidase and catalase) in plants significantly influenced by genotype and drought stress interaction. Moreover, the resistant and susceptible genotypes were identified according to three-dimensional plots and the expression patterns of PhavuCAMTA1 gene were studied using real-time quantitative polymerase chain reaction. The results of the present study serve as the basis for future functional studies on the Phaseolus CAMTA family.


Assuntos
Secas , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Phaseolus/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Estresse Fisiológico , Sequência de Aminoácidos , Perfilação da Expressão Gênica , Filogenia , Homologia de Sequência
18.
J Genet ; 982019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30945693

RESUMO

Genes encoding structurally independent phosphotriesterases (PTEs) are identified in soil bacteria. These pte genes, often identified on mobilizable and self-transmissible plasmids are organized as mobile genetic elements. Their dissemination through lateral gene transfer is evident due to the detection of identical organophosphate degradation genes among soil bacteria with little orno taxonomic relationship. Convergent evolution of PTEs provided selective advantages to the bacterial strain as they convert toxic phosphotriesters (PTs) into a source of phosphate. The residues of organophosphate (OP) compounds that accumulate in a soil are proposed to contribute to the evolution of PTEs through substrate-assisted gain-of-function. This review provides comprehensive information on lateral transfer of pte genes and critically examines proposed hypotheses on their evolution in the light of the short half-life of OPs in the environment. The review also proposes alternate factors that have possibly contributed to the evolution and lateral mobility of PTEs by taking into account their biology and analyses of pte genes in genomic and metagenomic databases.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/genética , Transferência Genética Horizontal , Organofosfatos/metabolismo , Hidrolases de Triester Fosfórico/genética , Solo/química , Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Evolução Molecular , Hidrolases de Triester Fosfórico/metabolismo , Plasmídeos , Homologia de Sequência
19.
Fungal Biol ; 123(4): 318-329, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30928040

RESUMO

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast' polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast' polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes.


Assuntos
Ascomicetos/enzimologia , Ascomicetos/metabolismo , Líquens/microbiologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Polímeros/metabolismo , Ascomicetos/genética , Sequência Conservada , Filogenia , Homologia de Sequência
20.
PLoS Pathog ; 15(4): e1007666, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30934025

RESUMO

Fusarium Head Blight (FHB) is the number one floral disease of cereals and poses a serious health hazard by contaminating grain with the harmful mycotoxin deoxynivalenol (DON). Fungi adapt to fluctuations in their environment, coordinating development and metabolism accordingly. G-protein coupled receptors (GPCRs) communicate changes in the environment to intracellular G-proteins that direct the appropriate biological response, suggesting that fungal GPCR signalling may be key to virulence. Here we describe the expansion of non-classical GPCRs in the FHB causing pathogen, Fusarium graminearum, and show that class X receptors are highly expressed during wheat infection. We identify class X receptors that are required for FHB disease on wheat, and show that the absence of a GPCR can cause an enhanced host response that restricts the progression of infection. Specific receptor sub-domains are required for virulence. These non-classical receptors physically interact with intracellular G-proteins and are therefore bona fide GPCRs. Disrupting a class X receptor is shown to dysregulate the transcriptional coordination of virulence traits during infection. This amounts to enhanced wheat defensive responses, including chitinase and plant cell wall biosynthesis, resulting in apoplastic and vascular occlusions that impede infection. Our results show that GPCR signalling is important to FHB disease establishment.


Assuntos
Resistência à Doença/imunologia , Fusarium/patogenicidade , Interações Hospedeiro-Patógeno , Doenças das Plantas/imunologia , Receptores Acoplados a Proteínas-G/metabolismo , Triticum/microbiologia , Sequência de Aminoácidos , Parede Celular/metabolismo , Doenças das Plantas/microbiologia , Conformação Proteica , Receptores Acoplados a Proteínas-G/química , Homologia de Sequência , Virulência
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