Apy Gene Research Articles

Whole-genome resequencing using next-generation and Nanopore sequencing for molecular characterization of T-DNA integration in transgenic poplar 741

BackgroundThe molecular characterization information of T-DNA integration is not only required by public risk assessors and regulators, but is also closely related to the expression of exogenous and endogenous genes. At present, with the development of sequencing technology, whole-genome resequencing has become an attractive approach to identify unknown genetically modified events and characterise T-DNA integration events.ResultsIn this study, we performed genome resequencing of Pb29, a transgenic high-resistance poplar 741 line that has been commercialized, using next-generation and Nanopore sequencing. The results revealed that there are two T-DNA insertion sites, located at 9,283,905–9,283,937 bp on chromosome 3 (Chr03) and 10,868,777–10,868,803 bp on Chr10. The accuracy of the T-DNA insertion locations and directions was verified using polymerase chain reaction amplification. Through sequence alignment, different degrees of base deletions were detected on the T-DNA left and right border sequences, and in the flanking sequences of the insertion sites. An unknown fragment was inserted between the Chr03 insertion site and the right flanking sequence, but the Pb29 genome did not undergo chromosomal rearrangement. It is worth noting that we did not detect the API gene in the Pb29 genome, indicating that Pb29 is a transgenic line containing only the BtCry1AC gene. On Chr03, the insertion of T-DNA disrupted a gene encoding TAF12 protein, but the transcriptional abundance of this gene did not change significantly in the leaves of Pb29. Additionally, except for the gene located closest to the T-DNA integration site, the expression levels of four other neighboring genes did not change significantly in the leaves of Pb29.ConclusionsThis study provides molecular characterization information of T-DNA integration in transgenic poplar 741 line Pb29, which contribute to safety supervision and further extensive commercial planting of transgenic poplar 741.

Charting the sequence-activity landscape of peptide inhibitors of translation termination

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api's activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.

Characterization of a novel aspartyl protease inhibitor from Haemonchus contortus

BackgroundAspartyl protease inhibitor (API) was thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. Studies on Ostertagia ostertagi, Ascaris suum and Brugia malayi indicated that aspins might play roles in nematode infection. In a recent study, proteins differentially expressed between free-living third-stage larvae (L3) and activated L3 (xL3) of Haemonchus contortus were identified by 2D-DIGE. API was found downregulated in xL3 when compared with L3. However, there was no report about the functions of H. contortus API in the parasite-host interaction. In this study, the gene encoding API from H. contortus was cloned, expressed, and part of its biological characteristics were studied.ResultsA DNA fragment of 681 bp was amplified by RT-PCR. Ninety one percent of the amino acid sequence was similar with that for aspin from O. ostertagi. The recombinant API protein was fusion-expressed with a molecular weight of 48 × 103. Results of Western blot showed that the recombinant API could be recognized by serum from goat infected with H. contortus. It was found that API was localized exclusively in the subcutaneous tissue and epithelial cells of the gastrointestinal tract in adult H. contortus. qRT-PCR suggested that the API gene was differentially transcribed in different life-cycle stages, with the lowest level in female adults and the highest in free-living L3 larvae. Enzyme inhibition assay indicated that the recombinant API can inhibit the activity of pepsin significantly, and the optimal reaction pH and temperature were 4.0 and 37–50 °C respectively. In vitro study showed that the recombinant API could induce goat PBMCs to express IFN-γ, IL-4 and IL-10.ConclusionsA new aspartyl protease inhibitor was cloned from H. contortus and its characteristics were studied for the first time. The results indicate that API may regulate the immune response of the host and play roles in the infection.

Activation of futile cycles as an approach to increase ethanol yield during glucose fermentation in Saccharomyces cerevisiae

ABSTRACTAn increase in ethanol yield by yeast from the fermentation of conventional sugars such as glucose and sucrose is possible by reducing the production of a key byproduct such as cellular biomass. Previously we have reported that overexpression of PHO8 gene encoding non-specific ATP-hydrolyzing alkaline phosphatase can lead to a decrease in cellular ATP content and to an increase in ethanol yield during glucose fermentation by Saccharomyces cerevisiae. In this work we further report on 2 new successful approaches to reduce cellular levels of ATP that increase ethanol yield and productivity. The first approach is based on the overexpression of the heterologous Escherichia coli apy gene encoding apyrase or SSB1 part of the chaperon that exhibit ATPase activity in yeast. In the second approach we constructed a futile cycle by the overexpression of S. cerevisiae genes encoding pyruvate carboxylase and phosphoenolpyruvate carboxykinase in S. cerevisiae. These genetically engineered strains accumulated more ethanol compared to the wild-type strain during alcoholic fermentation.

Alteration in apyrase enzyme attenuated virulence of Shigella flexneri

Virulence of Shigella is attributed to the genes presence in chromosome or in the megaplasmid. The apy gene which is located in the megaplasmid of Shigella species encodes for apyrase enzyme, a pathogenesis-associated enzyme causing mitochondrial damage and host cell death. In this study we constructed an apy mutant of Shigella flexneri by insertional activation using a kanamycin resistant gene cassette. The wild type apy gene of S. flexneri 2a was PCR amplified, cloned and mutated with insertion of kanamycin resistant gene cassette (aphA). The mutated construct (apy: aphA) was subcloned into a conjugative suicidal vector (pWM91) at the unique Sma1 and Sac1 sites. The mutation of the wild apy gene in the construct was confirmed by DNA sequencing. The mutated construct was introduced into wild type S. flexneri 2a by conjugation with Escherichia coli. After undergoing homologous recombination, the wild apy gene was deleted from the construct using the sucrose selection method. Non-functional activity of the apyrase enzyme in the constructed strain by colorimetric test indicated the successful mutation of the apyrase enzyme. This strain with mutated apy gene was evaluated for its protective efficacy using the guinea pig keratoconjunctivitis model. The strain was Sereny negative and it elicited a significant protection following challenge with wild S. flexneri strain. This apy mutant strain will form a base for the development of a vaccine target for shigellosis.

Biodegradation of pyrene by a newly isolated Proteus vulgaris

This work was aimed at identifying a new strain which can degrade pyrene effectively and quickly. Strain 4Bi was identified as Proteus vulgaris based on several morphological, biochemical characters and 16S rRNA phylogeny by using conventional methods, API and 16S rRNA gene sequence analysis. 16S rRNA gene sequence of the bacterium has been deposited in GenBank database under the accession number FJ799903. Carbon free mineral medium (CFMM plus pyrene) used for P. vulgaris 4Bi was excessively sufficient for positive improvement of bacterial growth and pyrene removal. The growth rates and pyrene degradation rates were increased depending on the time in CFMM. At a biodegradation rate with an initial pyrene concentration of 0.5 mg /ml, 71.5% of pyrene was degraded in 7 days. Pyrene-degradation metabolites were detected by HPLC and TLC analyses. Pyrene is a highly stable compound containing four fused aromatic rings and is one of the representative higher molecular weight-polycyclic aromatic hydrocarbons that are recalcitrant to biodegradation. Whereas, our bacterium could degrad pyrene effectively and quickly. With our study, the strain of P. vulgaris was firstly reported among pyrene-degrading bacteria. The ability of this strain is very useful for the remediation of pyrene one of PAH pollutants. Also, degradation metabolites detected in the study are not toxic or nonpersistent compounds in nature. These results indicated that this novel pyrene-degrading strain has a strong potential for further bioremediation processes without undesirable metabolites. Key words: Polycyclic aromatic hydrocarbons, pyrene, Proteus vulgaris,identification, biodegradation, metabolites.

Api, A Novel Medicago truncatula Symbiotic Mutant Impaired in Nodule Primordium Invasion

Genetic approaches have proved to be extremely useful in dissecting the complex nitrogen-fixing Rhizobium-legume endosymbiotic association. Here we describe a novel Medicago truncatula mutant called api, whose primary phenotype is the blockage of rhizobial infection just prior to nodule primordium invasion, leading to the formation of large infection pockets within the cortex of noninvaded root outgrowths. The mutant api originally was identified as a double symbiotic mutant associated with a new allele (nip-3) of the NIP/LATD gene, following the screening of an ethylmethane sulphonate-mutagenized population. Detailed characterization of the segregating single api mutant showed that rhizobial infection is also defective at the earlier stage of infection thread (IT) initiation in root hairs, as well as later during IT growth in the small percentage of nodules which overcome the primordium invasion block. Neither modulating ethylene biosynthesis (with L-alpha-(2-aminoethoxyvinylglycine or 1-aminocyclopropane-1-carboxylic acid) nor reducing ethylene sensitivity in a skl genetic background alters the basic api phenotype, suggesting that API function is not closely linked to ethylene metabolism or signaling. Genetic mapping places the API gene on the upper arm of the M. truncatula linkage group 4, and epistasis analyses show that API functions downstream of BIT1/ERN1 and LIN and upstream of NIP/LATD and the DNF genes.

Survival and escape of Agrobacterium tumefaciens in triploid hybrid lines of Chinese white poplar transformed with two insect-resistant genes

Two partly modified insect-resistant genes ( BtCryI Ac gene [Bt gene toxin against Lepidopterean insects] and API gene [arrowhead proteinase inhibitor]) were transferred to the triploid hybrid of Chinese white poplar (( Populus tomentosa Carr. × Populus bolleana Louche) × Populus tomentosa Carr.) mediated by A. tumefaciens. The survival of Agrobacterium in transgenic plants was examined during the processes of transplanting and subculturing on the nutrient medium. The results suggested that 80% of the plants, which were obtained by repeated selection on media added with 50 mg/L kanamycin and 300 mg/l carbenicillin, showed positive reactions after examination using molecular methods. The ELISA test indicated that the Bt toxoprotein was expressed in seven of the transgenic sub-clones. Leaves, stems, and roots of all the 28 transgenic plants were cultured on the YEB medium added with 50 mg/L kanamycin, and it was found that Agrobacterium survived in three sub-clones (33, 37, 5) and could have existed for 24 months in the bottle. These three transgenic sub-clones were transplanted and cultivated for one month in the room, and then the target Agrobacterium was found in rhizosphere of the sub-clone 33.

Ala160 and His116 residues are involved in activity and specificity of apyrase, an ATP-hydrolysing enzyme produced by enteroinvasive Escherichia coli

The virulence plasmid-carried apy (phoN2) gene of Shigella and related enteroinvasive Escherichia coli (EIEC) encodes apyrase, an ATP-diphosphohydrolase belonging to class A of the non-specific acid phosphatases (A-NSAPs). Apyrase and A-NSAPs share three domains of conserved amino acids (domains D1-D3) containing residues forming the putative active site of apyrase. In spite of their similarity, apyrase and A-NSAPs show different substrate specificity, apyrase being able to hydrolyse nucleotide tri- and diphosphates, but not monophosphates, as well as p-nitrophenyl phosphate (pNPP), while A-NSAPs are also active towards monophosphates and pNPP. In this paper, to get further insights into the structure-function relationship of apyrase, a random and site-directed mutagenesis of the apy gene of EIEC strain HN280 was conducted. Results indicate that amino acids located within the D2 and D3 conserved domains (Ser157 and Arg192, respectively) as well as residues located in the N-terminal (Ser97) and C-terminal (Glu233) domains are required for enzyme activity. Surprisingly, Ala160, located near the D2 domain and considered to be important for enzyme specificity, is required for enzyme activity, as its substitution with Thr led to the inactivation of enzyme activity. Furthermore, residue His116 is involved in apyrase specificity, since the H116L apyrase mutant shows substrate specificity resembling that of A-NSAPs.

Enteroinvasive Escherichia coli virulence-plasmid-carried apyrase (apy) and ospB genes are organized as a bicistronic operon and are subject to differential expression.

In Shigella flexneri and enteroinvasive Escherichia coli (EIEC) the expression of the virulence-plasmid(pINV)-carried potential pathogenesis-associated apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated by the same regulators that govern the expression of virulence genes. To understand the transcriptional organization of the apy gene, the authors sequenced an 8023 bp PstI fragment of the pINV of EIEC strain HN280, which encompasses apy as well as its adjacent genes. The PstI fragment displays 99% identity with the corresponding fragment of pWR100, the pINV of S. flexneri strain M90T, and contains four genes. One of these genes, ospB, encodes a secreted protein of unknown activity and is located immediately upstream of apy. Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that ospB and apy are co-transcribed as a 2 kb bicistronic, temperature-regulated mRNA from an upstream promoter that precedes ospB. The 2 kb mRNA is post-transcriptionally processed in the intercistronic ospB-apy region, leading to the considerable accumulation of a more stable 1 kb apy-specific mRNA (half-life of 2.2+/-0.3 min, versus 27+/-4 s for the 2 kb transcript). Upon temperature induction, peak expression of the ospB-apy operon occurs when bacteria enter into the late phases of bacterial growth, where the apy-specific transcript was found to be much more prevalent if compared to the ospB-apy transcript.

Expression of the virulence plasmid-carried apyrase gene (apy) of enteroinvasive Escherichia coli and Shigella flexneri is under the control of H-NS and the VirF and VirB regulatory cascade.

The transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneri and enteroinvasive Escherichia coli (EIEC) is induced at 37 degreesC and repressed at 30 degreesC. In this work, we report that the O135: K-:H- EIEC strain HN280 and S. flexneri SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the apy gene. In addition, the S. flexneri strains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the phoN-Sf gene. Both apy and phoN-Sf are pINV-carried loci whose contribution to the pathogenicity of enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both the apy and the phoN-Sf genes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of virB transcription) expressed the apy gene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Deltahns deletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of virB transcription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb-) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of virF or virB and by the presence of the intact apy locus or intact apy and phoN-Sf loci were detected among Crb- mutants of HN280 and SFZM53, respectively. While all Crb- apy+ mutants of HN280 failed to produce apyrase, Crb- apy+ phoN-Sf+ mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying cloned virF (pMYSH6504) or virB (pBN1) into Crb- mutants of HN280 and SFZM53 lacking virF or virB, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes, apy is another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of enteroinvasive bacteria.

The Apyrase Gene of the Vector Mosquito, Aedes eegypti, Is Expressed Specifically in the Adult Female Salivary Glands

The yellow fever mosquito, Aedes aegypti, expresses a gene, Apyrase (Apy), that encodes an ATP-diphosphohydrolase The product of this gene is a secreted enzyme that facilitates hematophagy by preventing platelet aggregation in the host. Apy gene expression is limited to the cells of the distal-lateral and medial lobes of the adult female salivary glands. Apyrase protein levels, detectable by antibodies, peak in the salivary glands about 4 days after adult emergence and remain high after a blood meal. Primary sequence analysis of a genomic clone encoding apyrase reveals a unique TAAATA sequence and seven introns, as well as other conserved features of eukaryotic genes. The temporal, sex- and tissue-specific expression of the Apy gene is consistent with its role as encoding a platelet anti-aggregation factor that functions to facilitate hematophagy and decrease probing time.