New Strategy for Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains

ABSTRACT We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.

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Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase

Journal of Clinical Microbiology ◽

10.1128/jcm.36.1.41-47.1998 ◽

1998 ◽

Vol 36 (1) ◽

pp. 41-47 ◽

Cited By ~ 201

Author(s):

Claire Poyart ◽

Gilles Quesne ◽

Stephane Coulon ◽

Patrick Berche ◽

Patrick Trieu-Cuot

Keyword(s):

Superoxide Dismutase ◽

Pcr Assay ◽

Degenerate Primers ◽

Gene Encoding ◽

Genes Encoding ◽

Type Strains ◽

Internal Fragment ◽

Rrna Sequences ◽

16S Rrna Sequences

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodAint ) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus,S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus,S. canis, S. cricetus, S. downei,S. dysgalactiae, S. equi subsp.equi, S. equi subsp. zooepidemicus,S. equinus, S. gordonii, S. iniae,S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis,S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis,S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodAint fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of theirsodAint fragments on the phylogenetic tree of the sodAint fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

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Molecular cloning and characterization of two genes encoding sigma factors that direct transcription from a Bacillus thuringiensis crystal protein gene promoter.

Journal of Bacteriology ◽

10.1128/jb.173.12.3846-3854.1991 ◽

1991 ◽

Vol 173 (12) ◽

pp. 3846-3854 ◽

Cited By ~ 53

Author(s):

L F Adams ◽

K L Brown ◽

H R Whiteley

Keyword(s):

Bacillus Thuringiensis ◽

Molecular Cloning ◽

Protein Gene ◽

Gene Promoter ◽

Sigma Factors ◽

Crystal Protein ◽

Direct Transcription ◽

Genes Encoding

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Characterization of cry Genes in a Mexican Bacillus thuringiensis Strain Collection

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4965-4972.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4965-4972 ◽

Cited By ~ 182

Author(s):

Alejandra Bravo ◽

Sergio Sarabia ◽

Lorena Lopez ◽

Hernesto Ontiveros ◽

Carolina Abarca ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Specific Primers ◽

Cry Proteins ◽

Pcr Product ◽

Strain Collection ◽

Lepidopteran Insects ◽

Ecological Patterns ◽

Biogeographical Regions ◽

Selection Of

ABSTRACT Mexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity. A total of 496 Bacillus thuringiensis strains were isolated from 503 soil samples collected from the five macroregions of the country. The characterization of the strain collection provided useful information on the ecological patterns of distribution of B. thuringiensis and opportunities for the selection of strains to develop novel bioinsecticidal products. The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect the cry1,cry3, cry5, cry7, cry8,cry9, cry11, cry12,cry13, cry14, cry21, andcyt genes. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Six pairs of general primers are used in this method. Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers. Strains containingcry1 genes were the most abundant in our collection (49.5%). Thirty-three different cry1-type profiles were identified. B. thuringiensis strains harboringcry3 genes represented 21.5% of the strains, and 7.9% of the strains contained cry11 and cyt genes.cry7, cry8, and cry9 genes were found in 0.6, 2.4, and 2.6% of the strains, respectively. No strains carrying cry5, cry12, cry13,cry14, or cry21 genes were found. Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera. Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observed cry genes.

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Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis

MGG Molecular & General Genetics ◽

10.1007/bf00338072 ◽

1986 ◽

Vol 205 (3) ◽

pp. 390-397 ◽

Cited By ~ 46

Author(s):

Catherine Bourgouin ◽

André Klier ◽

Georges Rapoport

Keyword(s):

Bacillus Thuringiensis ◽

Bacillus Thuringiensis Israelensis ◽

Delta Endotoxin ◽

Genes Encoding

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Protein O-Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans

Eukaryotic Cell ◽

10.1128/ec.00371-08 ◽

2009 ◽

Vol 8 (10) ◽

pp. 1465-1474 ◽

Cited By ~ 34

Author(s):

Masatoshi Goto ◽

Yuka Harada ◽

Takuji Oka ◽

Sho Matsumoto ◽

Kaoru Takegawa ◽

...

Keyword(s):

Aspergillus Nidulans ◽

Morphological Characteristics ◽

Normal Growth ◽

Growth Conditions ◽

Osmotic Stabilizer ◽

Genes Encoding ◽

Development And Differentiation ◽

Encoding Protein ◽

Slow Growing

ABSTRACT Aspergillus nidulans possesses three pmt genes encoding protein O-d-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology 150:1973-1982, 2004). In the present paper, we describe the characterization of the pmtA paralogues pmtB and pmtC. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A pmtB disruptant showed wild-type (wt) colony formation at 30°C but slightly repressed growth at 42°C. Conidiation of the pmtB disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. A pmtA and pmtB double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single pmt mutants, the pmtC disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42°C further enabled the pmtC disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three pmt disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth.

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The Isolation and Characterization of nrc-1 and nrc-2, Two Genes Encoding Protein Kinases That Control Growth and Development in Neurospora crassa

Genetics ◽

10.1093/genetics/149.1.117 ◽

1998 ◽

Vol 149 (1) ◽

pp. 117-130 ◽

Cited By ~ 4

Author(s):

Gregory O Kothe ◽

Stephen J Free

Keyword(s):

Neurospora Crassa ◽

Insertional Mutagenesis ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Developmental Program ◽

Isolation And Characterization ◽

Control Growth ◽

Genes Encoding ◽

Encoding Protein

Abstract Using an insertional mutagenesis approach, a series of Neurospora crassa mutants affected in the ability to control entry into the conidiation developmental program were isolated. One such mutant, GTH16-T4, was found to lack normal vegetative hyphae and to undergo constitutive conidiation. The affected gene has been named nrc-1, for nonrepressible conidiation gene #1. The nrc-1 gene was cloned from the mutant genomic DNA by plasmid rescue, and was found to encode a protein closely related to the protein products of the Saccharomyces cerevisiae STE11 and Schizosaccharomyces pombe byr2 genes. Both of these genes encode MAPKK kinases that are necessary for sexual development in these organisms. We conclude the nrc-1 gene encodes a MAPKK kinase that functions to repress the onset of conidiation in N. crassa. A second mutant, GTH16-T17, was found to lack normal vegetative hyphae and to constitutively enter, but not complete, the conidiation program. The affected locus is referred to as nrc-2 (nonrepressible conidiation gene #2). The nrc-2 gene was cloned and found to encode a serine-threonine protein kinase. The kinase is closely related to the predicted protein products of the S. pombe kad5, and the S. cerevisiae YNRO47w and KIN82 genes, three genes that have been identified in genome sequencing projects. The N. crassa nrc-2 gene is the first member of this group of kinases for which a phenotype has been defined. We conclude a functional nrc-2-encoded serine/threonine kinase is required to repress entry into the conidiation program.

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Characterization of a mitochondrion-like organelle inCryptosporidium parvum

Parasitology ◽

10.1017/s003118200400527x ◽

2004 ◽

Vol 129 (1) ◽

pp. 1-18 ◽

Cited By ~ 58

Author(s):

L. PUTIGNANI ◽

A. TAIT ◽

H. V. SMITH ◽

D. HORNER ◽

J. TOVAR ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Protozoan Parasite ◽

Diarrhoeal Disease ◽

Morphological Description ◽

Detailed Knowledge ◽

Degenerate Primers ◽

Energy Filtering ◽

Partial Cdna Sequence ◽

Genes Encoding

Cryptosporidium parvumis a protozoan parasite that causes widespread diarrhoeal disease in humans and other animals and is responsible for large waterborne outbreaks of cryptosporidiosis. Unlike many organisms belonging to the phylum Apicomplexa, such asPlasmodiumspp. andToxoplasma gondii, there is no clinically proven drug treatment against this parasite. Aspects of the basic biology ofC. parvumremain poorly understood, including a detailed knowledge of key metabolic pathways, its genome organization and organellar complement. Previous studies have proposed thatC. parvumlacks a relic plastid organelle, or ‘apicoplast’, but that it may possess a mitochondrion. Here we characterize a mitochondrion-like organelle inC. parvumby (i) ultrastructural and morphological description (ii) localization of heterologous mitochondrial chaperonin antibody probes (iii) phylogenetic analysis of genes encoding mitochondrial transport proteins (iv) identification and analysis of mitochondrion-associated gene sequences. Our descriptive morphological analysis was performed by energy-filtering transmission electron microscopy (EFTEM) ofC. hominisandC. parvum. The ‘mitochondrion-like’ organelle was characterized by labelling the structure with a heterologous mitochondrial chaperonin probe (hsp60) both in immunoelectron microscopy (IMEM) and immunofluorescence (IMF). Phylogenetic analysis of the mitochondrial import system and housekeeping components (hsp60 and hsp70-dnaK) suggested that theC. parvummitochondrion-like organelle is likely to have descended from a common ancestral apicomplexan mitochondrion. We also identified a partial cDNA sequence coding for an alternative oxidase (AOX) gene, a component of the electron transport chain which can act as an alternative to the terminal mitochondrial respiratory complexes III and IV, which has not yet been reported in any other member of this phylum. Degenerate primers developed to identify selected mitochondrial genes failed to identify either cytochrome oxidase subunit I, or cytochrome b. Taken together, our data aim to provide new insights into the characterization of thisCryptosporidiumorganelle and a logical framework for future functional investigation.

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Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis

Parasitology ◽

10.1017/s0031182016002195 ◽

2016 ◽

Vol 144 (5) ◽

pp. 571-582 ◽

Cited By ~ 2

Author(s):

ERICK AMADOR ◽

KARLA LÓPEZ-PACHECO ◽

NATALY MORALES ◽

ROBERTO CORIA ◽

IMELDA LÓPEZ-VILLASEÑOR

Keyword(s):

Cell Cycle ◽

Recombinant Proteins ◽

Trichomonas Vaginalis ◽

Cell Cycle Control ◽

Cyclin Dependent Kinases ◽

Genes Encoding ◽

Cdc28 Kinase ◽

Cycle Regulation ◽

Encoding Protein

SUMMARYCyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

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