Characterization of a Gemella-like organism isolated from an abscess of a rabbit: description of Gemella cunicula sp. nov.

An unknown Gram-positive, catalase-negative, ovoid-shaped bacterium isolated from the submandibular abscess of a rabbit was subjected to a polyphasic taxonomic analysis. Comparative 16S rRNA gene sequencing demonstrated the unknown coccus represents a new subline within the genus Gemella. The unknown isolate was readily distinguished from other recognized members of the genus Gemella, namely Gemella haemolysans, Gemella bergeri, Gemella morbillorum, Gemella palaticanis and Gemella sanguinis, by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium is classified in the genus Gemella as Gemella cuniculi sp. nov. The type strain is CCUG 42726T.

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.

Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines

Introgression in Festulolium is a potentially powerful tool to isolate genes for a large number of traits which differ between Festuca pratensis Huds. and Lolium perenne L. Not only are hybrids between the two species fertile, but the two genomes can be distinguished by genomic in situ hybridisation and a high frequency of recombination occurs between homoeologous chromosomes and chromosome segments. By a programme of introgression and a series of backcrosses, L. perenne lines have been produced which contain small F. pratensis substitutions. This material is a rich source of polymorphic markers targeted towards any trait carried on the F. pratensis substitution not observed in the L. perenne background. We describe here the construction of an F. pratensis BAC library, which establishes the basis of a map-based cloning strategy in L. perenne. The library contains 49,152 clones, with an average insert size of 112 kbp, providing coverage of 2.5 haploid genome equivalents. We have screened the library for eight amplified fragment length polymorphism (AFLP) derived markers known to be linked to an F. pratensis gene introgressed into L. perenne and conferring a staygreen phenotype as a consequence of a mutation in primary chlorophyll catabolism. While for four of the markers it was possible to identify bacterial artificial chromosome (BAC) clones, the other four AFLPs were too repetitive to enable reliable identification of locus-specific BACs. Moreover, when the four BACs were partially sequenced, no obvious coding regions could be identified. This contrasted to BACs identified using cDNA sequences, when multiple genes were identified on the same BAC.

Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel

Chen LM, Zhao J, Musa-Aziz R, Pelletier MF, Drummond IA, Boron WF. Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water and gas channel. Am J Physiol Regul Integr Comp Physiol 299: R1163-R1174, 2010. First published August 25, 2010; doi:10.1152/ajpregu.00319.2010.-The mammalian aquaporins AQP1, AQP4, and AQP5 have been shown to function not only as water channels but also as gas channels. Zebrafish have two genes encoding an AQP1 homologue, aqp1a and aqp1b. In the present study, we cloned the cDNA that encodes the zebrafish protein Aqp1a from the 72-h postfertilization (hpf) embryo of Danio rerio, as well as from the swim bladder of the adult. The deduced amino-acid sequence of aqp1a consists of 260 amino acids and is 59% identical to human AQP1. By analyzing the genomic DNA sequence, we identified four exons in the aqp1a gene. By in situ hybridization, aqp1a is expressed transiently in the developing vasculature and in erythrocytes from 16 to 48 h of development. Later, at 72 hpf, aqp1a is expressed in dermal ionocytes and in the swim bladder. Western blot analysis of adult tissues reveals that Aqp1a is most highly expressed in the eye and swim bladder. Xenopus oocytes expressing aqp1a have a channel-dependent (*) osmotic water permeability (P(f)*) that is indistinguishable from that of human AQP1. On the basis of the magnitude of the transient change in surface pH (Delta pHS) that were recorded as the oocytes were exposed to either CO(2) or NH(3), we conclude that zebrafish Aqp1a is permeable to both CO(2) and NH(3). The ratio (Delta pHS*)CO2/P(f)* is about half that of human AQP1, and the ratio (Delta pHS*)NH3/P(f)* is about one-quarter that of human AQP1. Thus, compared with human AQP1, zebrafish Aqp1a has about twice the selectivity for CO(2) over NH(3).

Exploring Bacterial Diversity of Endodontic Microbiota by Cloning and Sequencing 16S rRNA

Introduction: The characterization of microbial communities infecting the endodontic system in each clinical condition may help on the establishment of a correct prognosis and distinct strategies of treatment. The purpose of this study was to determine the bacterial diversity in primary endodontic infections by 16S ribosomal-RNA (rRNA) sequence analysis. Methods: Samples from root canals of untreated asymptomatic teeth (n = 12) exhibiting periapical lesions were obtained, 165 rRNA bacterial genomic libraries were constructed and sequenced, and bacterial diversity was estimated. Results: A total of 489 clones were analyzed (mean, 40.7 +/- 8.0 clones per sample). Seventy phylotypes were identified of which six were novel phylotypes belonging to the family Ruminococcaceae. The mean number of taxa per canal was 10.0, ranging from 3 to 21 per sample; 65.7% of the cloned sequences represented phylotypes for which no cultivated isolates have been reported. The most prevalent taxa were Atopobium rimae (50.0%), Dialister invisus, Pre-votella oris, Pseudoramibacter alactolyticus, and Tannerella forsythia (33.3%). Conclusions: Although several key species predominate in endodontic samples of asymptomatic cases with periapical lesions, the primary endodontic infection is characterized by a wide bacterial diversity, which is mostly represented by members of the phylum Firmicutes belonging to the class Clostridia followed by the phylum Bacteroidetes. (J Ended 2011;37:922-926)

Cloning and overexpression of the xylose isomerase gene from Burkholderia sacchari and production of polyhydroxybutyrate from xylose

A different organization for the xyl operon was found in different genomes of Burkholderia and Pseudomomas species. Degenerated primers were designed based on Burkholderia genomes and used to amplify the xylose isomerase gene (xylA) from Burkholderia sacchari IPT101 The gene encoded a protein of 329 amino acids, which showed the highest similarity (90%) to the homologous gene of Burkholderia dolosa. It was cloned in the broad host range plasmid pBBR1MCS-2, which partially restored growth and polyhydroxybutyrate production capability in xylose to a B. sacchari xyl(-) mutant. When xylA was overexpressed in the wild-type strain, it was not able to increase growth and polyhydroxybutyrate production, suggesting that XylA activity is not limiting for xylose utilization in B. sacchari.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO(R) vector which contains thioredoxin and polyhistidine tags at the C-and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 mu g/mL growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Indol-butyric acid levels on cashew cloning by air-layering process

Um estudo foi conduzido com a finalidade de determinar a possibilidade de clonagem do cajueiro (Anacardium occidentale) por alporquia e a influência do AIB (ácido indolbutírico) nesse processo. Adotou-se delineamento experimental inteiramente casualizado, com 4 tratamentos, 10 alporques por parcela, repetidos por 4 vezes, num total de 160 alporques. Os tratamentos constaram das concentrações de AIB: 0 (testemunha), 1.000, 3.000 e 5.000 mg.kg-1. Foram avaliadas as percentagens de sobrevivência, calejamento e enraizamento, bem como número e comprimento médio de raízes. A maior percentagem de sobrevivência (67,5%) foi observada para a testemunha e concentração de 1.000 mg.kg-1, enquanto a melhor percentagem de enraizamento (82%) foi relacionada com o nível de 1.000 mg.kg-1. Para o número e comprimento médio de raízes, os melhores resultados foram concernentes à dose de 5.000 mg.kg-1. Não houve influência do AIB na clonagem do cajueiro por alporquia.

Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor), Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa.

Identification of a gene encoding adaptin-like protein in the Paracoccidioides brasiliensis genome by random amplified polymorphic DNA analysis

Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pbl 8) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pbl Ba by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein beta-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.

Cloning and molecular characterization of Trypanosoma cruzi U2, U4, U5, and U6 small nuclear RNAs

Small nuclear RNAs (snRNAs) are important factors in the functioning of eukaryotic cells that form several small complexes with proteins; these ribonucleoprotein particles (U snRNPs) have an essential role in the pre-mRNA processing, particularly in splicing, catalyzed by spliceosomes, large RNA-protein complexes composed of various snRNPs. Even though they are well defined in mammals, snRNPs are still not totally characterized in certain trypanosomatids as Trypanosoma cruzi. For this reason we subjected snRNAs (U2, U4, U5, and U6) from T. cruzi epimastigotes to molecular characterization by polymerase chain reaction (PCR) and reverse transcription-PCR. These amplified sequences were cloned, sequenced, and compared with those other of trypanosomatids. Among these snRNAs, U5 was less conserved and U6 the most conserved. Their respective secondary structures were predicted and compared with known T. brucei structures. In addition, the copy number of each snRNA in the T. cruzi genome was characterized by Southern blotting.