Cloning and characteristics of the antibacterial peptide gene abaecin in the bumblebee Bombus lantschouensis (Hymenoptera: Apidae)

Among the antimicrobial peptides, abaecin is rich in proline content and plays a vital role in insect innate immune defense. Here, the full-length gene of abaecin from the bumblebee Bombus lantschouensis was cloned, and its expression profiles for different tissues, developmental stages and reproductive statuses were analyzed by RT-qPCR. Meanwhile, the responses of abaecin to a bacterium (Escherichia coli) and a fungus (Beauveria bassiana) were tested. The full length of abaecin cDNA was 470 bp, and the open reading frame (ORF) was 258 bp, encoding a polypeptide of 85 amino acids. The abaecin gene consists of three exons and two introns. Phylogenetic analysis showed that Bombus ignitus was the closest species to B. lantschouensis base on putative Abaecin protein sequence. Expression analysis showed that abaecin was expressed broadly in different tissues, with the highest expression in fat bodies and extremely low expression in antennae. Regarding developmental stage, low expression of abacein was detected in eggs and larvae, and high expression was detected in pupal stages. The highest expression was observed at the Pw pupal stage (pupae with an unpigmented body cuticle and white eyes), and the expression then decreased from the Pp (pupae with pink eyes) to the Pdd (dark-eye pupae with a dark-pigmented cuticle) stages. In addition, the expression of abaecin was higher in egg-laying than in non-egg-laying female bumblebees. Both E. coli and B. bassiana infections induced the expression of abaecin. Our results indicated that the abaecin gene plays important roles in the development, reproduction and immune responses of bumblebees. During the artificial rearing of bumblebees, a good environment should be created to avoid infection with bacteria or fungi.     

Concurrent Capillaria and Heterakis Infections in Zoo Rock Partridges, Alectoris graeca

Two adult rock partridges raised in a city zoo were examined parasitologically and pathologically. Two distinctive eggs resembling those of Capillaria and Heterakis were detected in the feces. At necropsy, a markedly-dilated duodenum with severe catarrhal exudates, containing adult worms of Capillaria sp. and Heterakis sp. in the cecum, was observed. Male Capillaria had the cloacal aperture extended almost terminally with a small bursal lobe and an unsheathed spicule with transverse folds without spines. Female Capillaria had a vulva that was slightly prominent and slightly posterior to the union of the esophagus and intestine. The esophagus of the adult Capillaria was more than a half as long as the body in the male, but was much shorter in the female. Based on these morphological features, the capillarid nematode was identified as Capillaria obsignata. The male adult worms of Heterakis was identifiable by 2 dissimilar spicules, a unique morphological feature where the right spicule was considerably longer than the left, which is also a characteristic feature of Heterakis gallinarum. This is the first report of concurrent infections with C. obsignata and H. gallinarium in rock partridges.     

Preparation of chlorophyll a, chlorophyll b and bacteriochlorophyll a by means of column chromatography with diethylaminoethylcellulose

Chlorophyll a, chlorophyll b and bacteriochlorophyll a were prepared by means of column chromatography with Sephadex LH-20 and diethylaminoethylcellulose. This method provides purified preparations of chlorophylls in about 3 h.To prepare chlorophyll a, blue-green or red algae were used as the starting material. Chlorophyll a was extracted with 90% aqueous acetone from cells of blue-green algae, Anabaena variabilis, Anacystis nidulans and Tolypothrix tenuis, and with 90% aqueous methanol from thalli of a red alga, Porphyra yezoensis. Chlorophyll a was collected as precipitates by adding dioxane and water to the extract according to the method of Iriyama et al. [6]. The crude chlorophyll a preparation was applied to a Sephadex LH-20 column with chloroform as the eluent and then to a DEAE-cellulose column with a chloroform/methanol mixture (49 : 1, v/v) as the eluent. Analysis with thin layer chromatography revealed that the chlorophyll a preparation contained no detectable contaminants.Bacteriochlorophyll a was prepared in a similar manner from purple photosynthetic bacteria, Rhodopseudomonas spheroides and Chromatium vinosum.In order to prepare chlorophyll b, chloroplasts of spinach leaves were used as the starting material. A mixture of chlorophylls a and b was obtained in the same way as described for the preparation of chlorophyll a from the blue-green algae. To separate chlorophyll b from chlorophyll a, the mixture was applied to a diethylaminoethylcellulose column which was developed with a hexane/2-propanol mixture (5 : 2, v/v).     

The multicopper oxidase gene family in the brown planthopper,Nilaparvata lugens

The multicopper oxidase (MCO) family of enzymes includes laccases, ascorbate oxidases, bilirubin oxidases and a subgroup of metal oxidases. On the basis of a bioinformatics investigation, we identified 7 genes encoding putative multicopper oxidase proteins in the genome of the brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae). MCO1 and MCO2 are conserved, while others diverse in insects. Analysis of developmental and tissue-specific expression patterns revealed the following: NlMCO2 was mainly expressed in the integument, and its expression peaked periodically during molting; NlMCO3 was an ovary-specific MCO gene with a high expression level only at the adult stage; NlMCO4 was a salivary gland-specific MCO gene that was expressed at all developmental stages; NlMCO5 only had short-term expression in the middle of the fourth instar stage and was expressed mainly in the gut; NlMCO6 had a developmental expression pattern similar to that of NlMCO2 and was expressed in most N. lugens tissues; and NlMCO1 was expressed in most N. lugens tissues except for the testis, whereas NlMCO7 was mainly expressed in the gut and the Malpighian tube. BPHs injected with double-stranded RNA (dsRNA) targeting NlMCO2 failed to pigment and sclerotize, were colorless and soft-bodied and subsequently died in a short time. Lethal phenotypes were also observed in insects challenged by dsRNA targeting NlMCO6. However, no observable morphological or internal structural abnormality was obtained in the insects treated with dsRNA for NlMCO1, NlMCO3, NlMCO4, NlMCO5 or NlMCO7.     

Genetic analysis of D-xylose metabolism pathways in Gluconobacter oxydans 621H

D-xylose is one of the most abundant carbohydrates in nature. This work focuses on xylose metabolism of Gluconobacter oxydans as revealed by a few studies conducted to understand xylose utilization by this strain. Interestingly, the G. oxydans 621H Δmgdh strain (deficient in membrane-bound glucose dehydrogenase) was greatly inhibited when grown on xylose and no xylonate accumulation was observed in the medium. These experimental observations suggested that the mgdh gene was responsible for the conversion of xylose to xylonate in G. oxydans, which was also verified by whole-cell biotransformation. Since 621H Δmgdh could still grow on xylose in a very small way, two seemingly important genes in the oxo-reductive pathway for xylose metabolism, a xylitol dehydrogenase-encoding gox0865 (xdh) gene and a putative xylulose kinase-encoding gox2214 (xk) gene, were knocked out to investigate the effects of both genes on xylose metabolism. The results showed that the gox2214 gene was not involved in xylose metabolism, and there might be other genes encoding xylulose kinase. Though the gox0865 gene played a less important role in xylose metabolism compared to the mgdh gene, it was significant in xylitol utilization in G. oxydans, which meant that gox0865 was a necessary gene for the oxo-reductive pathway of xylose in vivo. To sum up, when xylose was used as the carbon source, the majority of xylose was directly oxidized to xylonate for further metabolism in G. oxydans, whereas only a minor part of xylose was metabolized by the oxo-reductive pathway.     

Characterization of MATE-Type Multidrug Efflux Pumps from Klebsiella pneumoniae MGH78578

We previously described the cloning of genes related to drug resistance from Klebsiella pneumoniae MGH78578. Of these, we identified a putative gene encoding a MATE-type multidrug efflux pump, and named it ketM. Escherichia coli KAM32 possessing ketM on a plasmid showed increased minimum inhibitory concentrations for norfloxacin, ciprofloxacin, cefotaxime, acriflavine, Hoechst 33342, and 4'',6-diamidino-2-phenyl indole (DAPI). The active efflux of DAPI was observed in E. coli KAM32 possessing ketM on a plasmid. The expression of mRNA for ketM was observed in K. pneumoniae cells, and we subsequently disrupted ketM in K. pneumoniae ATCC10031. However, no significant changes were observed in drug resistance levels between the parental strain ATCC10031 and ketM disruptant, SKYM. Therefore, we concluded that KetM was a multidrug efflux pump, that did not significantly contribute to intrinsic resistance to antimicrobial chemicals in K. pneumoniae. MATE-type transporters are considered to be secondary transporters; therefore, we investigated the coupling cations of KetM. DAPI efflux by KetM was observed when lactate was added to produce a proton motive force, indicating that KetM effluxed substrates using a proton motive force. However, the weak efflux of DAPI by KetM was also noted when NaCl was added to the assay mixture without lactate. This result suggests that KetM may utilize proton and sodium motive forces.     

Viable, but non-culturable, state of a green fluorescence protein-tagged environmental isolate of Salmonella typhi in groundwater and pond water

An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescence protein (GFP) isolated from Aequorea victoria. The hybrid transposon mini-Tn5 gfp was transconjugated from E. coli to S. typhi, resulting in constitutive GFP production. The survival of S. typhi GFP155 introduced into groundwater and pond water microcosms was examined by GFP-based plate counts, total cell counts, and direct viable counts. A comparison between GFP-based direct viable counts and plate counts was a good method for verifying the viable, but non-culturable (VBNC), state of S. typhi. The entry into a VBNC state of S. typhi was shown in all microcosms. S. typhi survived longer in groundwater than in pond water as both a culturable and a VBNC state.     

Construction of a high-resolution linkage map of a rice brown planthopper (Nilaparvata lugens Stål) resistance gene bph2

The brown planthopper (BPH), Nilaparvata lugens Stål, is a significant insect pest of rice (Oryza sa-tiva L.). bph2 is one of the 12 major BPH resistance genes so far identified in several indica cultivars and two wild relatives. We have constructed a high-resolution linkage map as a foundation for map-based cloning of the bph2 locus. An advanced mapping population derived from a cross of ’Tsukushibare’ (a susceptible japonica cultivar) with ’Norin-PL4’ (an authentic bph2-introgression line) was used. Segregation analysis by the mass seedling test showed that bph2 behaved as a single dominant gene. Through bulked segregant analysis and linkage analysis, bph2 was located within a 3.2-cM region containing eight AFLP markers. One marker (KAM4) showed complete co-segregation with bph2, and bph2 was mapped within a 1.0-cM region delimited by KAM3 and KAM5, two flanking markers. KAM4 was converted into a PCR-based sequence-tagged-site (STS) marker and its co-segregation with bph2 was validated.     

Cellulosic ethanol production by Zymomonas mobilis harboring an endoglucanase gene from Enterobacter cloacae

Enterobactercloacae was isolated from the gut of the wood feeding termite, Heterotermesindicola, and a 2.25-kb fragment conferring cellulase activity was cloned in Escherichiacoli. The cloned fragment contained a 1083-bp ORF which could encode a protein belonging to glycosyl hydrolase family 8. The cellulase gene was introduced into Zymomonasmobilis strain Microbial Type Culture Collection centre (MTCC) on a plasmid and 0.134 filter paper activity unit (FPU)/ml units of cellulase activity was observed with the recombinant bacterium. Using carboxymethyl cellulose and 4% NaOH pretreated bagasse as substrates, the recombinant strain produced 5.5% and 4% (V/V) ethanol respectively, which was threefold higher than the amount obtained with the original E.cloacae isolate. The recombinant Z. mobilis strain could be improved further by simultaneous expression of cellulase cocktails before utilizing it for industrial level ethanol production.     

Inhibition of protein synthesis by products of lipid peroxidation

Effects of lipid peroxidation products on in vivo and in vitro protein synthesis have been studied. Malondialdehyde (MDA), a product, and a routinely used index of lipid peroxidation, inhibits in vivo protein synthesis in the two mosses, Tortula ruralis and Cratoneuron filicinum, and in pea (Pisum sativum) leaf discs. When wheat germ supernatant or poly(A)-rich mRNA of T. ruralis was incubated with MDA its subsequent activity in a cell-free protein-synthesizing system was reduced. When MDA was added directly to the in vitro protein-synthesizing mixture containing moss polyribosomes, the inhibition of amino acid incorporation was small. However, when simultaneous lipid peroxidation was allowed to occur along with in vitro protein synthesis there was a strong inhibition of amino acid incorporation and MDA accumulated in the reaction mixture indicating that products of lipid peroxidation other than, and apparently more toxic than, MDA were involved. It was concluded that lipid peroxidation inhibits protein synthesis probably by releasing toxic products which may react with and inactivate some components of the protein-synthesizing complex.     

Relationships between Bambusa species (Poaceae,Bambusoideae) revealed by random amplified polymorphic DNA

RAPD (random amplified polymorphic DNA) molecular markers were used to investigate relationships between a sample of Bambusa species from South Eastern China that have been placed in Bambusa or in several segregate genera, Dendrocalamopsis, Leleba, Lingnania, Neosinocalamus and Sinocalamus by different authors. On the resultant neighbor-joining tree, a thorny core Bambusa cluster was distinguished, as was a Lingnania group, and a cluster of Dendrocalamus species with more capitate inflorescences. However, Leleba was found to be a polyphyletic group in the present study.     

Preferential formation of (5S,6R)-thymine glycol for oligodeoxyribonucleotide synthesis and analysis of drug binding to thymine glycol-containing DNA

We previously reported the chemical synthesis of oligonucleotides containing thymine glycol, a major form of oxidative DNA damage. In the preparation of the phosphoramidite building block, the predominant product of the osmium tetroxide oxidation of protected thymidine was (5R,6S)-thymidine glycol. To obtain the building block of the other isomer, (5S,6R)-thymidine glycol, in an amount sufficient for oligonucleotide synthesis, the Sharpless asymmetric dihydroxylation (AD) reaction was examined. Although the reaction was very slow, (5S,6R)-thymidine glycol was obtained in preference to the (5R,6S) isomer. The ratio of (5S,6R)- and (5R,6S)-thymidine glycols was 2:1, and a trans isomer was also formed. When an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate, was used as a co-solvent, the reaction became faster, and the yield was improved without changing the preference. The phosphoramidite building block of (5S,6R)-thymidine glycol was prepared, and oligonucleotides containing 5S-thymine glycol were synthesized. One of the oligonucleotides was used to analyze the binding of distamycin A to thymine glycol-containing DNA by Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements. Distamycin A bound to a duplex containing either isomer of thymine glycol within the AATT target site, and its binding was observed even when the thymine glycol was placed opposite cytosine.     

Evaluation of Nostoc Strain ATCC 53789 as a Potential Source of Natural Pesticides

The cyanobacterium Nostoc strain ATCC 53789, a known cryptophycin producer, was tested for its potential as a source of natural pesticides. The antibacterial, antifungal, insecticidal, nematocidal, and cytotoxic activities of methanolic extracts of the cyanobacterium were evaluated. Among the target organisms, nine fungi (Armillaria sp., Fusarium oxysporum f. sp. melonis, Penicillium expansum, Phytophthora cambivora, P. cinnamomi, Rhizoctonia solani, Rosellinia, sp., Sclerotinia sclerotiorum, and Verticillium albo-atrum) were growth inhibited and one insect (Helicoverpa armigera) was killed by the extract, as well as the two model organisms for nematocidal (Caenorhabditis elegans) and cytotoxic (Artemia salina) activity. No antibacterial activity was detected. The antifungal activity against S. sclerotiorum was further studied with both extracts and biomass of the cyanobacterium in a system involving tomato as a host plant. Finally, the herbicidal activity of Nostoc strain ATCC 53789 was evaluated against a grass mixture. To fully exploit the potential of this cyanobacterium in agriculture as a source of pesticides, suitable application methods to overcome its toxicity toward plants and nontarget organisms must be developed.     

Sister Chromatid Exchange Frequencies in Escherichia coli Analyzed by Recombination at the dif Resolvase Site

Sister chromatid exchange (SCE) in Escherichia coli results in the formation of circular dimer chromosomes, which are converted back to monomers by a compensating exchange at the dif resolvase site. Recombination at dif is site specific and can be monitored by utilizing a density label assay that we recently described. To characterize factors affecting SCE frequency, we analyzed dimer resolution at the dif site in a variety of genetic backgrounds and conditions. Recombination at dif was increased by known hyperrecombinogenic mutations such as polA, dut, and uvrD. It was also increased by a fur mutation, which increased oxidative DNA damage. Recombination at dif was eliminated by a recA mutation, reflecting the role of RecA in SCE and virtually all homologous recombination in E. coli. Interestingly, recombination at dif was reduced to approximately half of the wild-type levels by single mutations in either recB or recF, and it was virtually eliminated when both mutations were present. This result demonstrates the importance of both RecBCD and RecF to chromosomal recombination events in wild-type cells.     

Evaluation of a Brucella melitensis mutant deficient in O-polysaccharide export system ATP-binding protein as a rough vaccine candidate

Rough Brucella mutants have been sought as vaccine candidates that do not interfere with the conventional serological diagnosis of brucellosis. In this study, a rough mutant of Brucella melitensis was generated by the disruption of the wzt gene, which encodes the O-polysaccharide (O-PS) export system ATP-binding protein. In vivo, the mutant 16MΔwzt was attenuated and conferred a level of protection against B. melitensis 16M challenge similar to that conferred by the vaccine strain B. melitensis M5 in mice. In pregnant sheep, the mutant 16MΔwzt did not induce abortion. In vitro, 16MΔwzt was more susceptible to polymyxin B and complement-mediated killing than B. melitensis 16M was. Most importantly, although 16MΔwzt had a rough phenotype, it was able to synthesize O-PS and did not induce detectable specific antibodies in sheep. These results suggested that 16MΔwzt deserved to further systematic evaluation as a vaccine for target animal hosts due to its promising features.     

Purification and properties of a protease inhibitor from crayfish hemolymph

A protease inhibitor from the hemolymph of crayfish, Astacus astacus, has been purified by differential centrifugation, acid precipitation and preparative isoelectric focusing. The inhibitor was apparent homogenous in SDS-electrophoresis and had a molecular weight of 23,000. pI was determined to be 4.7 by isoelectric focusing. No inhibitory activity was lost when the inhibitor was incubated in a pH range of 1–11.5. The purified inhibitor was heat stable. Urea (6 m) had no effect upon the inhibitor. The inhibitor was active against subtilisin and a partly purified protease from the fungus Aphanomyces astaci. Pronase was slightly inhibited whereas trypsin, chymotrypsin, papain, Arthrobacter protease, and extracellular proteases from the fungi Aphanomyces stellatus and A. laevis were unaffected. The importance of protease inhibitors in pathogenesis between the parasitic fungus, A. astaci, and its crayfish host, A. astacus is discussed.