Microbial diversity in the predatory mite Metaseiulus occidentalis (Acari: Phytoseiidae) and its prey, Tetranychus urticae (Acari: Tetranychidae)

Microorganisms associated with the predatory mite Metaseiulus (=Typhlodromus or Galendromus) occidentalis (Nesbitt) and its prey, the two-spotted spider mite Tetranychus urticae (Koch), were assessed using a high-fidelity polymerase chain reaction (PCR) protocol and primers designed to identify Eubacteria, Archaeabacteria, iridoviruses, Helicosporidia, Cytophaga-like microorganisms, Wolbachia and its bacteriophage WO, fungi and yeast-like organisms. Sequences from four bacterial species related to Wolbachia (α-Proteobacteria), Cardinium, Bacteroidetes, and Enterobacter (γ-Proteobacteria) were obtained from M. occidentalis, and three sequences related to Wolbachia, Rickettsia, and Caulobacter (α-Proteobacteria) were obtained from T. urticae. No nucleotide differences were detected between the 16S rRNA, wspA or wspB Wolbachia sequences obtained from M. occidentalis and T. urticae, which suggest that horizontal transfer of Wolbachia could have occurred. Southern blot analyses of genomic DNA from both M. occidentalis and T. urticae using wspA probes were negative, indicating that this Wolbachia sequence is not integrated into the nuclear genome of either species. Two of the T. urticae colonies tested contained the WO bacteriophage, but none of the six M. occidentalis populations were infected. New M. occidentalis-specific forward and reverse 16S rRNA primers based on the Wolbachia, Cardinium, Bacteroidetes, and Enterobacter sequences obtained were designed and used to amplify PCR products from each of two laboratory and four field-collected samples of M. occidentalis females and eggs, indicating that these infections are widespread. Likewise, species-specific primers for T. urticae were designed for the Wolbachia, Rickettsia, and Caulobacter sequences obtained and used to evaluate T. urticae from strawberries, wine grapes, hops, almonds, and cherries from California, Washington, and Florida; all were positive for Wolbachia and Caulobacter but two of the six were negative for Rickettsia. None of the M. occidentalis colonies tested were positive for the microsporidium Oligosporidium occidentalis, which previously had been associated with a pathogenic condition in some of our laboratory colonies. The Gainesville colonies of M. occidentalis and T. urticae were negative for iridovirus, Archaeabacteria, fungi, Helicosporidia, and yeast-like organisms. So far, Wolbachia is the only symbiont that is shared by this predator and its prey.     

Production of Penicillin G acylase from Bacillus sp.: Effect of medium components

A Bacillus sp. producing a high level of intracellular penicillin G acylase (PAC) was isolated. The PAC production in this strain was induced by phenylacetic acid. Various carbon and nitrogen sources were evaluated for their effect on growth and PAC production at 28 °C and pH 7.0. Cells grown in medium supplemented with sucrose as carbon source and tryptone as nitrogen source produced maximum activity of 6.45 and 8.92 U mg^–1, respectively. Maximum concentration of PAC (10.1 Umg^–1) was produced by the cells grown in the medium containing sucrose and tryptone, which was twofold higher than the production in basal medium.     

Crystal structure of the jacalin-T-antigen complex and a comparative study of lectin-T-antigen complexes

Thomsen-Friedenreich antigen (Galbeta1-3GalNAc), generally known as T-antigen, is expressed in more than 85% of human carcinomas. Therefore, proteins which specifically bind T-antigen have potential diagnostic value. Jacalin, a lectin from jack fruit (Artocarpus integrifolia) seeds, is a tetramer of molecular mass 66kDa. It is one of the very few proteins which are known to bind T-antigen. The crystal structure of the jacalin-T-antigen complex has been determined at 1.62A resolution. The interactions of the disaccharide at the binding site are predominantly through the GalNAc moiety, with Gal interacting only through water molecules. They include a hydrogen bond between the anomeric oxygen of GalNAc and the pi electrons of an aromatic side-chain. Several intermolecular interactions involving the bound carbohydrate contribute to the stability of the crystal structure. The present structure, along with that of the Me-alpha-Gal complex, provides a reasonable qualitative explanation for the known affinities of jacalin to different carbohydrate ligands and a plausible model of the binding of the lectin to T-antigen O-linked to seryl or threonyl residues. Including the present one, the structures of five lectin-T-antigen complexes are available. GalNAc occupies the primary binding site in three of them, while Gal occupies the site in two. The choice appears to be related to the ability of the lectin to bind sialylated sugars. In either case, most of the lectin-disaccharide interactions are at the primary binding site. The conformation of T-antigen in the five complexes is nearly the same.     

Superantigen profiles of emm and emm-like typeable and nontypeable pharyngeal streptococcal isolates of South India

Background

Toxins derived from jellyfishes have been exploited as a model for the development of new drug promising applications to treat neurodegenerative diseases. The present work is aimed to evaluate the acute toxicity of crude venom of Pelagia noctiluca and then to screen the analgesic and antibutyrylcholinestrasic (anti-BuChE) activities of the crude venom and its fractions.

Methods

Sephadex G75 gel was used to separate crude venom of Pelagia noctiluca, which led to some fractions. In addition, in vivo analgesic and in vitro plasma antibutyrylcholinestrasic activities were carried out with Pelagia crude venom and its fractions respectively.

Results

The crude venom and its fractions displayed analgesic and anti-BuChE activities at different doses without inducing acute toxicity. Fraction 2 possesses the highest analgesic and antibutyrylcholinestrasic properties. The crude venom and fraction 1 had shown to possess less significant inhibitory activity against analgesic and antibutyrylcholinestrasic models.

Conclusions

Based on this study, the crude venom of Pelagia noctiluca is found to be a useful tool for probing pharmacological activity. The purification and the determination of chemical structures of compounds of active fractions of the venom are under investigation.     

Aurora B controls kinetochore-microtubule attachments by inhibiting Ska complex-KMN network interaction

The KMN network (named according to the acronym for KNL1, Mis12, and Ndc80) and the more recently identified Ska complex (Ska1-3) have been shown to mediate kinetochore (KT)-microtubule (MT) attachments. How these two complexes cooperate to achieve stable end-on attachments remains unknown. In this paper, we show that Aurora B negatively regulates the localization of the Ska complex to KTs and that recruitment of the Ska complex to KTs depends on the KMN network. We identified interactions between members of the KMN and Ska complexes and demonstrated that these interactions are regulated by Aurora B. Aurora B directly phosphorylated Ska1 and Ska3 in vitro, and expression of phosphomimetic mutants of Ska1 and Ska3 impaired Ska KT recruitment and formation of stable KT-MT fibers (K-fibers), disrupting mitotic progression. We propose that Aurora B phosphorylation antagonizes the interaction between the Ska complex and the KMN network, thereby controlling Ska recruitment to KTs and stabilization of KT-MT attachments.     

Application of cross-linked enzyme aggregates of Bacillus badius penicillin G acylase for the production of 6-aminopenicillanic acid

AIMS: Optimization of 6-aminopenicillanic acid (6-APA) production using cross-linked enzyme aggregates (CLEA) of Bacillus badius penicillin G acylase (PAC). METHODS AND RESULTS: CLEA-PAC was prepared using purified/partially purified PAC with phenylacetic acid as active-site blocking agent and glutaraldehyde as cross-linker. Conversion of penicillin G to 6-APA by CLEA-PAC was optimized using response surface methodology (RSM) (central composite rotatable design) consisting of a three-factor-two-level pattern with 20 experimental runs. CONCLUSION: Nearly, 80% of immobilization yield was obtained when partially purified enzyme was used for the preparation of CLEA-PAC. Quantitative conversion of penicillin G to 6-APA was observed within 60 min and the CLEA-PAC was reusable for 20 repeated cycles with 100% retention of enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The faster conversion of penicillin G to 6-APA by CLEA-PAC and efficient reusability holds a strong potential for the industrial application.     

Identification of bacteria associated with underground parts of Crocus sativus by 16S rRNA gene targeted metagenomic approach

Saffron (Crocus sativus L), an autumn-flowering perennial sterile plant, reproduces vegetatively by underground corms. Saffron has biannual corm–root cycle that makes it an interesting candidate to study microbial dynamics in its rhizosphere and cormosphere (area under influence of corm). Culture independent 16S rRNA gene metagenomic study of rhizosphere and cormosphere of Saffron during flowering stage revealed presence of 22 genera but none of the genus was common in all the three samples. Bulk soil bacterial community was represented by 13 genera with Acidobacteria being dominant. In rhizosphere, out of eight different genera identified, Pseudomonas was the most dominant genus. Cormosphere bacteria comprised of six different genera, dominated by the genus Pantoea. This study revealed that the bacterial composition of all the three samples is significantly different (P < 0.05) from each other. This is the first report on the identification of bacteria associated with rhizosphere, cormosphere and bulk soil of Saffron, using cultivation independent 16S rRNA gene targeted metagenomic approach.     

Elevated Levels of Circulating DNA in Cardiovascular Disease Patients: Metagenomic Profiling of Microbiome in the Circulation

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. An expanding body of evidence supports the role of human microbiome in the establishment of CVDs and, this has gained much attention recently. This work was aimed to study the circulating human microbiome in CVD patients and healthy subjects. The levels of circulating cell free DNA (circDNA) was higher in CVD patients (n = 80) than in healthy controls (n = 40). More specifically, the relative levels of circulating bacterial DNA and the ratio of 16S rRNA/β-globin gene copy numbers were higher in the circulation of CVD patients than healthy individuals. In addition, we found a higher circulating microbial diversity in CVD patients (n = 3) in comparison to healthy individuals (n = 3) by deep shotgun sequencing. At the phylum level, we observed a dominance of Actinobacteria in CVD patients, followed by Proteobacteria, in contrast to that in healthy controls, where Proteobacteria was predominantly enriched, followed by Actinobacteria. The circulating virome in CVD patients was enriched with bacteriophages with a preponderance of Propionibacterium phages, followed by Pseudomonas phages and Rhizobium phages in contrast to that in healthy individuals, where a relatively greater abundance of eukaryotic viruses dominated by Lymphocystis virus (LCV) and Torque Teno viruses (TTV) was observed. Thus, the release of bacterial and viral DNA elements in the circulation could play a major role leading to elevated circDNA levels in CVD patients. The increased circDNA levels could be either the cause or consequence of CVD incidence, which needs to be explored further.     

The nuclear genome of the phytoseiid <Emphasis Type="Italic">Metaseiulus occidentalis</Emphasis> (Acari: Phytoseiidae) is among the smallest known in arthropods

The genome size of the phytoseiid Metaseiulus (=Typhlodromus or Galendromus) occidentalis (Nesbitt) needs to be estimated before the whole nuclear genome can be sequenced. Two different procedures were used to estimate the genome size of M. occidentalis; (1) flow cytometry (Marescalchi et al. in Genome 33:789–793, 1990) and (2) quantitative real-time PCR (qRT-PCR) (Wilhelm et al. in Nucleic Acids Res 31:e56, 2003). Fluorescence intensity of propidium iodide-stained nuclei of M. occidentalis was measured by flow cytometry using females, males, and eggs. Only the eggs yielded peaks, which ranged in size from 35 to 160 Mb, with a tall peak of 140 Mb in 1-day-old eggs and 65 Mb in 2-day-old eggs, respectively. However, the peaks are broad and do not provide an accurate estimate. The qRT-PCR procedure required single-copy nuclear gene sequences from this phytoseiid. This was accomplished by designing degenerate primers, amplifying the Actin and EF1α sequences from M. occidentalis, and then designing M. occidentalis-specific primers that amplified a unique sequence. The standard qRT-PCR protocol was inefficient and amplification failed frequently, so we developed a high-fidelity qRT-PCR protocol, which utilizes a mix of two DNA polymerases (Taq and a proof-reading Tgo or ACCUZYME) to consistently amplify sequences. This allowed us to estimate the nuclear genome size of M. occidentalis as 88–90 ± 5 Mb. When compared to other arthropod genomes, this appears to be very small.