Galactose-Specific Fimbrial Adhesin of Enteroaggregative <Emphasis Type="Italic">Escherichia coli</Emphasis>: A Possible Aggregative Factor

A galactose-specific adhesin was isolated from the fimbriae of an enteroaggregative Escherichia coli (EAEC) strain. The adhesin was found to be a high molecular weight aggregate of the 18-kDa monomer. The dimeric (36 kDa) and tetrameric (76 kDa) forms appeared in sodium dodecyl sulphate polyacrylamide gel electrophoresis when a higher concentration of the adhesin was used. The IgG_AD (IgG against adhesin) obtained from the immune sera raised in rabbits against purified adhesin could detect all three forms of the adhesin even from the crude fimbrial preparation. The IgG_AD failed to recognize the adhesin in the presence of galactose, thereby suggesting the antibody-binding site and the sugar-binding site on the adhesin might be same or overlapping. Furthermore, the IgG_AD could localize the adhesin exclusively on the fimbriae as observed in immunogold electron microscopy. The aggregative adherence of the bacteria to HEp-2 cells was reduced to 70% in the presence of the IgG_AD. A glycoprotein (34 kDa) present in the membrane fraction of HEp-2 cells interacted with the purified adhesin in a galactose-specific manner. The IgG_AD could recognize the adhesin from the crude fimbrial preparation of 9 out of 10 clinical isolates of EAEC strains but failed to identify any protein from the crude fimbrial preparation of Salmonella typhimurium (fim +ve as well as fim −ve strain), Vibrio cholerae (WO7) or Escherichia coli DH5α.     

PCR-based specific detection of Ralstonia solanacearum by amplification of cytochrome c1 signal peptide sequences

A polymerase chain reaction (PCR)-based method was developed to detect the DNA of Ralstonia solanacearum, the causal agent of bacterial wilt in various crop plants. One pair of primers (RALSF and RALSR), designed using cytochrome c1 signal peptide sequences specific to R. solanacearum, produced a PCR product of 932 bp from 13 isolates of R. solanacearum from several countries. The primer specificity was then tested using DNA from 21 isolates of Ralstonia, Pseudomonas, Burkholderia, Xanthomonas, and Fusarium oxysporum f. sp. dianthi. The specificity of the cytochrome c1 signal peptide sequences in R. solanacearum was further confirmed by a DNA-dot blot analysis. Moreover, the primer pair was able to detect the pathogen in artificially inoculated soil and tomato plants. Therefore, the present results indicate that the primer pair can be effectively used for the detection of R. solanacearum in soil and host plants.     

Identification of two entomopathogenic bacteria from a nematode pathogenic to the Oriental beetle, Blitopertha orientalis

A pathogenic nematode, Butlerius sp., was isolated from Oriental beetle, Blitopertha orientalis. The infective juveniles exhibited dose- as well as time-dependent entomopathogenicity on the larvae of B. orientalis. Two bacterial species, Providencia vermicola (KACC 91278) and Flavobacterium sp. (KACC 91279), were isolated from the infective juveniles and identified. P. vermicola outnumbered Flavobacterium sp. in the nematode host, in which the colony density of P. vermicola was found to be 21 times higher than that of Flavobacterium sp. However, when the two bacterial species were cocultured in culture media without the nematode host, they showed similar growth rates. Both bacteria induced significant entomopathogenicity against Spodoptera exigua larvae infesting economically important vegetable crops, where P. vermicola was more potent than Flavobacterium sp.     

Ubiquinone-10 production using Agrobacterium tumefaciens dps gene in Escherichia coli by coexpression system

Ubiquinone (Coenzyme Q; abbreviation, UQ) acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system, and has been widely used in pharmaceuticals. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study we have cloned, expressed the decaprenyl diphosphate synthase, designated dps gene, from Agrobacterium tumefaciens, and succeeded in detecting UQ-10 in addition to innate UQ-8 in Escherichia coli. Furthermore, the production of UQ-10 was higher than UQ-8. To establish an efficient expression system for UQ-10 production, we used genes, including ubiC, ubiA, and ubiG involved in UQ biosynthesis in E. coli, to construct a better co-expression system. The expression coupled by dps and ubiCA was effective for increasing UQ-10 production by five times than that by expressing single dps gene in the shake flask culture. To study for a large-scale production of UQ-10 in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of 85.40 g/L and 94.58 g/L dry cell weight (DCW), and UQ-10 content of 50.29 mg/L and 45.86 mg/L was obtained after 32.5 h and 27.5 h of cultivation, subsequent to isopropyl-β-d-thiogalactopy ranoside and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout the fermentation.     

A possible novel function of dominance behaviour in queen-less colonies of the primitively eusocial wasp Ropalidia marginata

Unlike the queens of other primitively eusocial species, Ropalidia marginata queens are strikingly docile and non-aggressive individuals, never at the top of the behavioural dominance hierarchy of their colonies. Nevertheless, these queens are completely successful at suppressing worker reproduction, suggesting that they do not use aggression but employ some other mechanism (e.g. pheromones) to do so. Upon removal of the queen from a colony, a single worker, the 'potential queen', immediately begins to display highly elevated levels of aggression towards her nest mates. This individual becomes the next docile queen if the original queen is not returned. We attempt to understand the function of the temporary and amplified dominance behaviour displayed by the potential queen. We find that the dominance behaviour shown by the potential queen is unrelated to the number of her nest mates, their dominance ranks or ovarian condition. This suggests that aggression may not be used to actively suppress other workers and counter threat. Instead we find evidence that dominance behaviour is required for the potential queen's rapid ovarian development, facilitating her speedy establishment as the sole reproductive individual in the colony.     

Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis

Protoplasts isolated from cell suspension culture of Phalaenopsis “Wataboushi” were cultured by (a) embedding in gellan gum-solidified hormone-free 1/2 New Dogashima medium (1/2 NDM) containing 0.44 M sorbitol, 0.06 M sucrose and 0.1 g/l l-glutamine (standard method) and (b) beads method using beads of gellan gum or sodium alginate as the gelling agents which were surrounded by liquid NDM. Although, the two beads methods gave less frequency of initial protoplast division than the standard method, the former finally resulted in higher frequency of microcolony formation than the latter. The highest frequency of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free NDM supplemented with 10 g/l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The PLBs were regenerated into plantlets after changing the carbon source to 10 g/l sorbitol and successfully acclimatized to greenhouse conditions.     

Affordable uniform isotope labeling with <Superscript>2</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N in insect cells

For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80 % can be achieved for ¹⁵N and ¹³C with yields comparable to expression in full media. For ²H,¹⁵N and ²H,¹³C,¹⁵N labeling, incorporation is only slightly lower with 75 and 73 %, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins.     

AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

The phosphoinositide 3-kinase–Akt signaling pathway is essential to many biological processes, including cell proliferation, survival, metabolism, and angiogenesis, under pathophysiological conditions. Although 3-phosphoinositide–dependent kinase 1 (PDK1) is a primary activator of Akt at the plasma membrane, the optimal activation mechanism remains unclear. We report that adhesion molecule with IgG-like domain 2 (AMIGO2) is a novel scaffold protein that regulates PDK1 membrane localization and Akt activation. Loss of AMIGO2 in endothelial cells (ECs) led to apoptosis and inhibition of angiogenesis with Akt inactivation. Amino acid residues 465–474 in AMIGO2 directly bind to the PDK1 pleckstrin homology domain. A synthetic peptide containing the AMIGO2 465–474 residues abrogated the AMIGO2–PDK1 interaction and Akt activation. Moreover, it effectively suppressed pathological angiogenesis in murine tumor and oxygen-induced retinopathy models. These results demonstrate that AMIGO2 is an important regulator of the PDK1–Akt pathway in ECs and suggest that interference of the PDK1–AMIGO2 interaction might be a novel pharmaceutical target for designing an Akt pathway inhibitor.     

Projecting future expansion of invasive species: comparing and improving methodologies for species distribution modeling

Modeling the distributions of species, especially of invasive species in non‐native ranges, involves multiple challenges. Here, we developed some novel approaches to species distribution modeling aimed at reducing the influences of such challenges and improving the realism of projections. We estimated species–environment relationships for Parthenium hysterophorus L. (Asteraceae) with four modeling methods run with multiple scenarios of (i) sources of occurrences and geographically isolated background ranges for absences, (ii) approaches to drawing background (absence) points, and (iii) alternate sets of predictor variables. We further tested various quantitative metrics of model evaluation against biological insight. Model projections were very sensitive to the choice of training dataset. Model accuracy was much improved using a global dataset for model training, rather than restricting data input to the species’ native range. AUC score was a poor metric for model evaluation and, if used alone, was not a useful criterion for assessing model performance. Projections away from the sampled space (i.e., into areas of potential future invasion) were very different depending on the modeling methods used, raising questions about the reliability of ensemble projections. Generalized linear models gave very unrealistic projections far away from the training region. Models that efficiently fit the dominant pattern, but exclude highly local patterns in the dataset and capture interactions as they appear in data (e.g., boosted regression trees), improved generalization of the models. Biological knowledge of the species and its distribution was important in refining choices about the best set of projections. A post hoc test conducted on a new Parthenium dataset from Nepal validated excellent predictive performance of our ‘best’ model. We showed that vast stretches of currently uninvaded geographic areas on multiple continents harbor highly suitable habitats for parthenium. However, discrepancies between model predictions and parthenium invasion in Australia indicate successful management for this globally significant weed.