Characterization of an extracellular protease and its cDNA from the nematode-trapping fungus Monacrosporium microscaphoides

2006 ◽  
Vol 52 (2) ◽  
pp. 130-139 ◽  
Author(s):  
Miao Wang ◽  
Jinkui Yang ◽  
Ke-Qin Zhang
Keyword(s):  
Serine Protease ◽  
Catalytic Triad ◽  
Infection Process ◽  
Gene Encoding ◽  
Protein Substrates ◽  
Nematocidal Activity ◽  
Infection Mechanisms ◽  
Biocontrol Potential

To better exploit the biocontrol potential of nematophagous fungi, it is important to fully understand the molecular background of the infection process. In this paper, several nematode-trapping fungi were surveyed for nematocidal activity. From the culture filtrate of Monacrosporium microscaphoides, a neutral serine protease (designated Mlx) was purified by chromatography. This protease could immobilize the nematode Penagrellus redivivus in vitro and degrade its purified cuticle, suggesting that Mlx could serve as a virulence factor during infection. Characterization of the purified protease revealed a molecular mass of approximately 39 kDa, an isoelectric point of 6.8, and optimum activity at pH 9 at 65 °C. Mlx has broad substrate specificity, and it hydrolyzes protein substrates, including casein, skimmed milk, collagen, and bovine serum albumin. The gene encoding Mlx was also cloned and the nucleotide sequence was determined. The deduced amino acid sequence contained the conserved catalytic triad of aspartic acid – histidine – serine and showed high similarity with two cuticle-degrading proteases (PII and Aoz1), which were purified from the nematode-trapping fungus Arthrobotrys oligospora. Research on infection mechanisms of nematode-trapping fungi has thus far only focused on A. oligospora. However, little is known about other nematode-trapping fungi. Our report is among the first to describe the purification and cloning of an infectious protease from a different nematode-trapping fungus.Key words: extracellular serine protease, Monacrosporium microscaphoides, nematode-trapping fungus, nematocidal activity.

Virulence and Genetic Characterization of Six Baculovirus Strains Isolated From Different Populations of Spodoptera Frugiperda (Lepidoptera: Noctuidae)

2021 ◽  
Author(s):  
Ingrid Zanella-Saenz ◽  
Elisabeth A. Herniou ◽  
Jorge E. Ibarra ◽  
Ma.Cristina Del Rincón-Castro ◽  
Ilse Alejandra Huerta-Arredondo
Keyword(s):  
Biological Control ◽  
Cell Cultures ◽  
Spodoptera Frugiperda ◽  
Fall Armyworm ◽  
The United States ◽  
Infection Mechanisms ◽  
Different Populations

Abstract Fall armyworm (FAW), Spodoptera frugiperda (Smith, 1797), is a polyphagous, voracious, and economically important agricultural pest. Biological control of FAW is a strategy that must be further explored. This study evaluated six baculovirus strains isolated from infected FAW larvae from Mexico, Argentina, Honduras, and the United States. Five alphabaculoviruses (SfNPV-An2, SfNPV-Arg, SfNPV-Fx, SfNPV-Ho and SfNPV-Sin) and one betabaculovirus (SfGV-RV), were tested against FAW larvae, showing a wide diversity of virulence levels among strains when their estimated LC50s were compared, being SfNPVArg, SfNPV-Ho and SfNPV-Fx more virulent than SfNPV-An 2 , SfNPV-Sin and SfGV-RV. To determine any virulence difference in vitro studies of these isolates, Sf9 cell cultures were used. Interestingly, only ODVs from four of the test SfNPV strains showed infectivity on Sf9 cell cultures, and some differences in virulence were observed. Genomic restriction analyses and partial sequences of lef-8, lef-9 , and polh/granulin genes showed little variability among alphabaculoviruses, both, among them and with previously reported sequences. However, sequences from SfGV-RV were closer to previously reported sequences from the SfGVVG008 strain than the SfGV-Arg and SfGV-VG014 strains. The great difference in the in vivo virulence was not correlated with great similarity among the isolates. The characterization of these six baculoviruses isolates offers the basis for exploring their potential as biological control agents against S. frugiperda, as well the initial studies on their specific infection mechanisms, evolution, and ecology.

scholarly journals In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

2003 ◽  
Vol 77 (6) ◽  
pp. 3669-3679 ◽  
Author(s):  
Caterina Trozzi ◽  
Linda Bartholomew ◽  
Alessandra Ceccacci ◽  
Gabriella Biasiol ◽  
Laura Pacini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
In Vitro Selection ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

A somatic cell-derived system for studying both early and late mitotic events in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 449-462
Author(s):  
J. Nakagawa ◽  
G.T. Kitten ◽  
E.A. Nigg
Keyword(s):  
Chromatin Condensation ◽  
Nuclear Lamina ◽  
Free System ◽  
Cell Free System ◽  
Protein Substrates ◽  
Nuclear Envelope Breakdown ◽  
Biochemical Fractionation ◽  
A Cell

We describe a cell-free system for studying mitotic reorganization of nuclear structure. The system utilizes soluble extracts prepared from metaphase-arrested somatic chicken cells and supports both the disassembly and subsequent partial reassembly of exogenous nuclei. By fluorescence microscopy, biochemical fractionation, protein phosphorylation assays and electron microscopy, we show that chicken embryonic nuclei incubated in extracts prepared from metaphase-arrested chicken hepatoma cells undergo nuclear envelope breakdown, lamina depolymerization and chromatin condensation. These prophase-like events are strictly dependent on ATP and do not occur when nuclei are incubated in interphase extracts. Compared to interphase extracts, metaphase extracts show increased kinase activities toward a number of nuclear protein substrates, including lamins and histone H1; moreover, they specifically contain four soluble phosphoproteins of Mr 38,000, 75,000, 95,000 and 165,000. Following disassembly of exogenous nuclei in metaphase extracts, telophase-like reassembly of a nuclear lamina and re-formation of nuclear membranes around condensed chromatin can be induced by depletion of ATP from the extract. We anticipate that this reversible cell-free system will contribute to the identification and characterization of factors involved in regulatory and mechanistic aspects of mitosis.

scholarly journals Characterization of the tyramine-producing pathway in Sporolactobacillus sp. P3J

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1841-1849 ◽  
Author(s):  
Monika Coton ◽  
María Fernández ◽  
Hein Trip ◽  
Victor Ladero ◽  
Niels L. Mulder ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Cloning And Expression ◽  
Clear Correlation ◽  
Tyrosine Decarboxylase ◽  
Gene Encoding ◽  
Polycistronic Mrna ◽  
Tyrosine Concentration ◽  
Putative Phage

A sporulated lactic acid bacterium (LAB) isolated from cider must was shown to harbour the tdc gene encoding tyrosine decarboxylase. The isolate belonged to the Sporolactobacillus genus and may correspond to a novel species. The ability of the tdc-positive strain, Sporolactobacillus sp. strain P3J, to produce tyramine in vitro was demonstrated by using HPLC. A 7535 bp nucleotide sequence harbouring the putative tdc gene was determined. Analysis of the obtained sequence showed that four tyramine production-associated genes [tyrosyl-tRNA synthetase (tyrS), tyrosine decarboxylase (tdc), tyrosine permease (tyrP) and Na+/H+ antiporter (nhaC)] were present and were organized as already described in other tyramine-producing LAB. This operon was surrounded by genes showing the highest identities with mobile elements: a putative phage terminase and a putative transposase (downstream and upstream, respectively), suggesting that the tyramine-forming trait was acquired through horizontal gene transfer. Transcription analyses of the tdc gene cluster suggested that tyrS and nhaC are expressed as monocistronic genes while tdc would be part of a polycistronic mRNA together with tyrP. The presence of tyrosine in the culture medium induced the expression of all genes except for tyrS. A clear correlation was observed between initial tyrosine concentration and tyramine production combined with an increase in the final pH reached by the culture. Finally, cloning and expression of the tyrP gene in Lactococcus lactis demonstrated that its product catalyses the exchange of tyrosine and tyramine.

scholarly journals Characterization of the Exoglucanase-Encoding Gene PaEXG2 and Study of Its Role in the Biocontrol Activity of Pichia anomala Strain K

2003 ◽  
Vol 93 (9) ◽  
pp. 1145-1152 ◽  
Author(s):  
Cathy Grevesse ◽  
Philippe Lepoivre ◽  
Mohamed Haïssam Jijakli
Keyword(s):  
Marker Gene ◽  
Glycosylation Site ◽  
Pichia Anomala ◽  
Gene Encoding ◽  
Biocontrol Activity ◽  
Monophosphate Decarboxylase ◽  
Uracil Auxotroph

The PaEXG2 gene, encoding an exo-β-1,3-glucanase, was isolated from the biocontrol agent Pichia anomala strain K. PaEXG2 has the capacity for coding an acidic protein of 427 amino acids with a predicted molecular weight of 45.7 kDa, a calculated pI of 4.7, and one potential N-glycosylation site. PaEXG2 was disrupted by the insertion of the URA3 marker gene, encoding orotidine monophosphate decarboxylase in strain KU1, a uracil auxotroph derived from strain K. Strain KU1 showed inferior biocontrol activity and colonization of wounds on apples, compared to the prototrophic strain. Antagonism and colonization were recovered after the restoration of prototrophy by transformation with the URA3 gene. Integrative transformation was shown to be mostly ectopic in strain K descendants (only 4% of integration by homologous recombination). PaEXG2 disruption abolished all detectable extracellular exo-β-1,3-glucanase activity in vitro and in situ but did not affect biocontrol of Botrytis cinerea on wounded apples.

scholarly journals Role of CpSUB1, a Subtilisin-Like Protease, in Cryptosporidium parvum Infection In Vitro

2009 ◽  
Vol 8 (4) ◽  
pp. 470-477 ◽  
Author(s):  
Jane W. Wanyiri ◽  
Patsharaporn Techasintana ◽  
Roberta M. O'Connor ◽  
Michael J. Blackman ◽  
Kami Kim ◽  
...  
Keyword(s):  
Serine Protease ◽  
Cryptosporidium Parvum ◽  
Protease Activity ◽  
Diarrheal Disease ◽  
Genomic Sequence ◽  
Host Cells ◽  
Pcr Analysis ◽  
Apical Region ◽  
Gene Encoding

ABSTRACTThe apicomplexan parasiteCryptosporidiumis a significant cause of diarrheal disease worldwide. Previously, we reported that aCryptosporidium parvumsubtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity inC. parvumlysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in theC. parvumgenome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA fromC. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of ∼64 kDa and ∼48 kDa, forC. parvumlysates and proteins “shed” during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 byC. parvumlysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity inC. parvumand for processing of gp40/15. Importantly, the recombinant prodomain inhibitedC. parvuminfection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.

scholarly journals Release of Nonstop Ribosomes Is Essential

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Heather A. Feaga ◽  
Patrick H. Viollier ◽  
Kenneth C. Keiler
Keyword(s):  
Messenger Rna ◽  
Caulobacter Crescentus ◽  
Stop Codon ◽  
Selective Advantage ◽  
Gene Encoding ◽  
Content Type ◽  
Bacterial Phyla ◽  
Almost All

ABSTRACTBacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at a stop codon. Almost all bacteria use the transfer-messenger RNA (tmRNA)-basedtrans-translation pathway to release these “nonstop” ribosomes and maintain protein synthesis capacity.trans-translation is essential in some species, but in others, such asCaulobacter crescentus,trans-translation can be inactivated. To determine whytrans-translation is dispensable inC. crescentus, a Tn-seq screen was used to identify genes that specifically alter growth in cells lackingssrA, the gene encoding tmRNA. One of these genes,CC1214, was essential in ΔssrAcells. Purified CC1214 protein could release nonstop ribosomesin vitro. CC1214 is a homolog of theEscherichia coliArfB protein, and using the CC1214 sequence, ArfB homologs were identified in the majority of bacterial phyla. Most species in whichssrAhas been deleted contain an ArfB homolog, suggesting that release of nonstop ribosomes may be essential in most or all bacteria.IMPORTANCEGenes that are conserved across large phylogenetic distances are expected to confer a selective advantage. The genes required fortrans-translation,ssrAandsmpB, have been found in >99% of sequenced bacterial genomes, suggesting that they are broadly important. However, these genes can be deleted in some species without loss of viability. The identification and characterization ofC. crescentusArfB reveals whytrans-translation is not essential inC. crescentusand suggests that many other bacteria are likely to use ArfB to survive whentrans-translation is compromised.

scholarly journals Identification and Characterization of the Bacillus thuringiensis phaZ Gene, Encoding New Intracellular Poly-3-Hydroxybutyrate Depolymerase

2006 ◽  
Vol 188 (21) ◽  
pp. 7592-7599 ◽  
Author(s):  
Chi-Ling Tseng ◽  
Hui-Ju Chen ◽  
Gwo-Chyuan Shaw
Keyword(s):  
Bacillus Thuringiensis ◽  
Wild Type ◽  
Gene Encoding ◽  
Significant Similarity ◽  
Phb Depolymerase ◽  
New Type ◽  
Identification And Characterization

ABSTRACTA gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome ofBacillus thuringiensissubsp.israelensisATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designatedphaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD ofPseudomonas putidawas unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. TheB. thuringiensisPhaZ was inactive againstp-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-typeB. thuringiensiscells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from aphaZmutant lost this activity. ThephaZmutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S102-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate thatB. thuringiensisharbors a new type of intracellular PHB depolymerase.

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