Structural analysis of the lipopolysaccharide derived core oligosaccharides of Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and the genome strain 5b

The structures of the core oligosaccharides of the lipopolysaccharides (LPS) from Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and 5b were elucidated. The LPS's were subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the core oligosaccharides were determined on the basis of the combined data from these experiments. [carbohydrate formula see text] For serotype 1: R is (1S)-GalaNAc-(1-->4,6)-alpha-Gal II-(1-->3)-beta-Gal I-(1-->, and R' is H For serotype 2: R is beta-Glc III-(1-->, and R' is D-alpha-D-Hep V-(1--> For serotypes 5a and 5b: R is H and R' is D-alpha-D-Hep V-(1--> All oligosaccharides elaborated a conserved inner core structure, as illustrated. All sugars were in the pyranose ring form apart from the open-chain N-acetylgalactosamine, the identification of which in the serotype 1 LPS was of interest.     

Structural characterization of the antigenic O-polysaccharide in the lipopolysaccharide produced by Actinobacillus pleuropneumoniae serotype 14

The antigenic O-polysaccharide of the lipopolysaccharide produced by Actinobacillus pleuropneumoniae serotype 14 was shown by chemical analysis and 1D and 2D nuclear magnetic resonance methods to be a high-molecular-mass polymer of a repeating disaccharide unit composed of a chain of (1-->5)-linked beta-D-galactofuranose (beta-D-Galf) residues substituted at their O-2 positions by alpha-D-galactopyranose residues (D-Galp) (1:1): [formula: see text].     

血清7型胸膜肺炎放线杆菌apxⅡC-/apxⅠA+基因缺失重组菌株的构建与鉴定

通过接合转移和SacB负向筛选方法,成功构建了一株apxⅡC缺失的血清7型胸膜肺炎放线杆菌重组菌株。首先构建重组转移质粒pEHA1。将pEHA1转化供体菌大肠杆菌(E.coliβ2155),并将其与野生型APP血清7型亲本菌混合培养约5h,然后涂到含氯霉素抗性的培养基培养,挑取阳性克隆,接种到无抗性液体培养基,培养后涂于含有蔗糖的的固体培养基,培养一定时间后挑取蔗糖抗性的克隆,即可得到目的突变株。通过PCR、遗传稳定性、外毒素分泌、重组位点序列分析证明重组菌构建成功。通过对重组菌生物学特性进行初步研究,表明突变株生长能力未受影响,对小鼠毒力显著降低。该突变株构建体系的建立为猪传染性胸膜肺炎减毒活疫苗的开发及对胸膜肺炎放线杆菌新基因的功能研究奠定了良好基础。     

Agrobacterium-mediated transformation in chickpea (Cicer arietinum L.) with an insecticidal protein gene: optimisation of different factors

Agrobacterium-mediated transformation in chickpea was developed using strain LBA4404 carrying nptII, uidA and cryIAc genes and transformants selected on Murashige and Skoog’s basal medium supplemented with benzyladenine, kinetin and kanamycin. Integration of transgenes was demonstrated using polymerase chain reaction and Southern blot hybridization of T₀ plants. The expression of CryIAc delta endotoxin and GUS enzyme was shown by enzyme linked immunosorbent assay and histochemical assay respectively. The transgenic plants (T₀) showed more tolerance to infection by Helicoverpa armigera compared to control plants. Various factors such as explant source, cultivar type, different preculture treatment period of explants, co-cultivation period, acetosyringone supplementation, Agrobacterium harboring different plasmids, vacuum infiltration and sonication treatment were tested to study the influence on transformation frequency. The results indicated that use of epicotyl as explant, cultivar ICCC37, Agrobacterium harboring plasmid pHS102 as vector, preculture of explant for 48 h, co-cultivation period of 2 days at 25°C and vacuum infiltration for 15 min produced the best transformation results. Sonication treatment of explants with Agrobacteria for 80 s was found to increase the frequency of transformation.     

Comparative genome analysis of rice-pathogenic Burkholderia provides insight into capacity to adapt to different environments and hosts

Background

In addition to human and animal diseases, bacteria of the genus Burkholderia can cause plant diseases. The representative species of rice-pathogenic Burkholderia are Burkholderia glumae, B. gladioli, and B. plantarii, which primarily cause grain rot, sheath rot, and seedling blight, respectively, resulting in severe reductions in rice production. Though Burkholderia rice pathogens cause problems in rice-growing countries, comprehensive studies of these rice-pathogenic species aiming to control Burkholderia-mediated diseases are only in the early stages.

Results

We first sequenced the complete genome of B. plantarii ATCC 43733^T. Second, we conducted comparative analysis of the newly sequenced B. plantarii ATCC 43733^T genome with eleven complete or draft genomes of B. glumae and B. gladioli strains. Furthermore, we compared the genome of three rice Burkholderia pathogens with those of other Burkholderia species such as those found in environmental habitats and those known as animal/human pathogens. These B. glumae, B. gladioli, and B. plantarii strains have unique genes involved in toxoflavin or tropolone toxin production and the clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bacterial immune system. Although the genome of B. plantarii ATCC 43733^T has many common features with those of B. glumae and B. gladioli, this B. plantarii strain has several unique features, including quorum sensing and CRISPR/CRISPR-associated protein (Cas) systems.

Conclusions

The complete genome sequence of B. plantarii ATCC 43733^T and publicly available genomes of B. glumae BGR1 and B. gladioli BSR3 enabled comprehensive comparative genome analyses among three rice-pathogenic Burkholderia species responsible for tissue rotting and seedling blight. Our results suggest that B. glumae has evolved rapidly, or has undergone rapid genome rearrangements or deletions, in response to the hosts. It also, clarifies the unique features of rice pathogenic Burkholderia species relative to other animal and human Burkholderia species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1558-5) contains supplementary material, which is available to authorized users.     

A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker

Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn903 kanamycin resistance determinant (km(r)) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae. The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive. A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A. pleuropneumoniae mutants that had lost the km(r)-sacB cassette.     

A new tarkadectine primate from the Eocene of Inner Mongolia,China: phylogenetic and biogeographic implications

Tarka and Tarkadectes are Middle Eocene mammals known only from the Rocky Mountains region of North America. Previous work has suggested that they are members of the Plagiomenidae, an extinct family often included in the order Dermoptera. Here we describe a new primate, Tarkops mckennai gen. et sp. nov., from the early Middle Eocene Irdinmanha Formation of Inner Mongolia, China. The new taxon is particularly similar to Tarka and Tarkadectes, but it also displays many features observed in omomyids. A phylogenetic analysis based on a data matrix including 59 taxa and 444 dental characters suggests that Tarkops, Tarka and Tarkadectes form a monophyletic group—the Tarkadectinae—that is nested within the omomyid clade. Within Omomyidae, tarkadectines appear to be closely related to Macrotarsius. Dermoptera, including extant and extinct flying lemurs and plagiomenids, is recognized as a clade nesting within the polyphyletic group of plesiadapiforms, therefore supporting the previous suggestion that the relationship between dermopterans and primates is as close as that between plesiadapiforms and primates. The distribution of tarkadectine primates on both sides of the Pacific Ocean basin suggests that palaeoenvironmental conditions appropriate to sustain primates occurred across a vast expanse of Asia and North America during the Middle Eocene.     

The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway

The C2 domain is a targeting domain that responds to intracellular Ca²⁺ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P₂ specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca²⁺ specifically binds to the Ca²⁺-binding region, facilitating PtdIns(4,5)P₂ access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P₂ binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P₂ binding are essential for the plasma membrane localization of PKCα when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P₂ and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P₂ is hydrolyzed to generate diacylglycerol and IP₃, an important amount still remains in the membrane where it is available to activate PKCα. These findings entail revision of the currently accepted model of PKCα recruitment to the membrane and its activation.     

Characterization of Bovidae sex-determining gene SRY

In mammals, testis determination is under the control of the sex-determining gene SRY. This Y-linked gene encodes a protein with a DNA binding domain similar to those found in high-mobility-group proteins. Here we report the cloning and sequences of the SRY genes of yak and Chinese native cattle. Our data show that SRY genes in Bovidae are less divergent, especially in the coding and 3'' regions.     

Viral clones from the GOS expedition with an unusual photosystem-I gene cassette organization

Cyanobacteria have a key role in marine photosynthesis, which contributes to the global carbon cycle and to the world oxygen supply. Genes encoding for photosystem-II (PSII) and photosystem-I (PSI) reaction centers are found in different cyanophage genomes, and it was suggested that the horizontal transfer of these genes might be involved in increasing phage fitness. We have further analyzed a rare viral Global Ocean Sampling (GOS) clone containing PSI genes. This clone contains the unusual PSI gene organization psaD->C->A, as opposed to the more frequently observed viral psaJF->C->A->B->K->E->D organization, and was detected only once in the GOS metagenome. Our analyses identified more occurrences with similar arrangement and indicate that this PSI viral gene organization (now psaD->C->A->B), although rare, is authentic and represents a new PSI gene arrangement.     

Molecular Characterization of Aldolase from Heterodera glycines and Globodera rostochiensis

Fructose-bisphosphate aldolase (EC 4.1.2.13) is a key enzyme in glycolysis. We have characterized full-length coding sequences for aldolase genes from the cyst nematodes Heterodera glycines and Globodera rostochiensis, the first for any plant-parasitic nematode. Nucleotide homology is high (83% identity), and the respective sequences encode 40 kDa proteins with 89% amino acid identity. Genomic sequences contain six introns located at identical positions in both genes. Intron 4 in the H. glycines gene is >500 bp. Partial genomic sequences determined for seven other cyst nematode species reveal that the large fourth intron is characteristic of Heterodera but not Globodera aldolase genes. Total aldolase-like specific activity in homogenates from H. glycines was 2-fold lower than in either Caenorhabditis elegans or Panagrellus redivivus (P = 0.001). Activity in H. glycines samples was higher in juvenile stages than in adults (P = 0.003). Heterodera glycines aldolase has Km = 41 µM and is inhibited by treatment with carboxypeptidase A or sodium borohydride.     

A parasitic selfish gene that affects host promiscuity

Selfish genes demonstrate transmission bias and invade sexual populations despite conferring no benefit to their hosts. While the molecular genetics and evolutionary dynamics of selfish genes are reasonably well characterized, their effects on hosts are not. Homing endonuclease genes (HEGs) are one well-studied family of selfish genes that are assumed to be benign. However, we show that carrying HEGs is costly for Saccharomyces cerevisiae, demonstrating that these genetic elements are not necessarily benign but maybe parasitic. We estimate a selective load of approximately 1–2% in ‘natural’ niches. The second aspect we examine is the ability of HEGs to affect hosts'' sexual behaviour. As all selfish genes critically rely on sex for spread, then any selfish gene correlated with increased host sexuality will enjoy a transmission advantage. While classic parasites are known to manipulate host behaviour, we are not aware of any evidence showing a selfish gene is capable of affecting host promiscuity. The data presented here show a selfish element may increase the propensity of its eukaryote host to undergo sex and along with increased rates of non-Mendelian inheritance, this may counterbalance mitotic selective load and promote spread. Demonstration that selfish genes are correlated with increased promiscuity in eukaryotes connects with ideas suggesting that selfish genes promoted the evolution of sex initially.     

Revision of the subfamily Opiinae (Hymenoptera,Braconidae) from Hunan (China), including thirty-six new species and two new genera

The species of the subfamily Opiinae (Hymenoptera: Braconidae) from Hunan (Oriental China) are revised and illustrated. Thirty-six new species are described: Apodesmia bruniclypealis Li & van Achterberg, sp. n., Apodesmia melliclypealis Li & van Achterberg, sp. n., Areotetes albiferus Li & van Achterberg, sp. n., Areotetes carinuliferus Li & van Achterberg, sp. n., Areotetes striatiferus Li & van Achterberg, sp. n., Coleopioides diversinotum Li & van Achterberg, sp. n., Coleopioides postpectalis Li & van Achterberg, sp. n., Fopius dorsopiferus Li, van Achterberg & Tan, sp. n., Indiopius chenae Li & van Achterberg, sp. n., Opiognathus aulaciferus Li & van Achterberg, sp. n., Opiognathus brevibasalis Li & van Achterberg, sp. n., Opius crenuliferus Li & van Achterberg, sp. n., Opius malarator Li, van Achterberg & Tan, sp. n., Opius monilipalpis Li & van Achterberg, sp. n., Opius pachymerus Li & van Achterberg, sp. n., Opius songi Li & van Achterberg, sp. n., Opius youi Li & van Achterberg, sp. n., Opius zengi Li & van Achterberg, sp. n., Phaedrotoma acuticlypeata Li & van Achterberg, sp. n., Phaedrotoma angiclypeata Li & van Achterberg, sp. n., Phaedrotoma antenervalis Li & van Achterberg, sp. n., Phaedrotoma depressiclypealis Li & van Achterberg, sp. n., Phaedrotoma flavisoma Li & van Achterberg, sp. n., Phaedrotoma nigrisoma Li & van Achterberg, sp. n., Phaedrotoma protuberator Li & van Achterberg, sp. n., Phaedrotoma rugulifera Li & van Achterberg, sp. n., Li & van Achterberg,Phaedrotoma striatinota Li & van Achterberg, sp. n., Phaedrotoma vermiculifera Li & van Achterberg, sp. n., Rhogadopsis latipennis Li & van Achterberg, sp. n., Rhogadopsis longicaudifera Li & van Achterberg, sp. n., Rhogadopsis maculosa Li, van Achterberg & Tan, sp. n., Rhogadopsis obliqua Li & van Achterberg, sp. n., Rhogadopsis sculpturator Li & van Achterberg, sp. n., Utetes longicarinatus Li & van Achterberg, sp. n. and Xynobius notauliferus Li & van Achterberg, sp. n. Areotetes van Achterberg & Li, gen. n. (type species: Areotetes carinuliferus sp. n.) and Coleopioides van Achterberg & Li, gen. n. (type species: Coleopioides postpectalis sp. n. are described. All species are illustrated and keyed. In total 30 species of Opiinae are sequenced and the cladograms are presented. Neopius Gahan, 1917, Opiognathus Fischer, 1972, Opiostomus Fischer, 1972, and Rhogadopsis Brèthes, 1913, are treated as a valid genera based on molecular and morphological differences. Opius vittata Chen & Weng, 2005 (not Opius vittatus Ruschka, 1915), Opius ambiguus Weng & Chen, 2005 (not Wesmael, 1835) and Opius mitis Chen & Weng, 2005 (not Fischer, 1963) are primary homonymsandarerenamed into Phaedrotoma depressa Li & van Achterberg, nom. n., Opius cheni Li & van Achterberg, nom. n. andOpius wengi Li & van Achterberg, nom. n., respectively. Phaedrotoma terga (Chen & Weng, 2005) comb. n.,Diachasmimorpha longicaudata (Ashmead, 1905) and Biosteres pavitita Chen & Weng, 2005, are reported new for Hunan, Opiostomus aureliae (Fischer, 1957) comb. n. is new for China and Hunan; Xynobius maculipennis(Enderlein, 1912) comb. n. is new for Hunan and continental China and Rhogadopsis longuria (Chen & Weng, 2005) comb. n. is new for Hunan. The following new combinations are given: Apodesmia puncta (Weng & Chen, 2005) comb. n., Apodesmia tracta (Weng & Chen, 2005) comb. n., Areotetes laevigatus (Weng & Chen, 2005) comb. n., Phaedrotoma dimidia (Chen & Weng, 2005) comb. n., Phaedrotoma improcera (Weng & Chen, 2005) comb. n., Phaedrotoma amputata (Weng & Chen, 2005) comb. n., Phaedrotoma larga (Weng & Chen, 2005) comb. n., Phaedrotoma osculas (Weng & Chen, 2005) comb. n., Phaedrotoma postuma (Chen & Weng, 2005) comb. n., Phaedrotoma rugulosa (Chen & Weng, 2005) comb. n., Phaedrotoma tabularis (Weng & Chen, 2005) comb. n., Rhogadopsis apii (Chen & Weng, 2005) comb. n., Rhogadopsis dimidia (Chen & Weng, 2005) comb. n., Rhogadopsis diutia (Chen & Weng, 2005) comb. n., Rhogadopsis longuria (Chen & Weng, 2005) comb. n., Rhogadopsis pratellae(Weng & Chen, 2005) comb. n., Rhogadopsis pratensis (Weng & Chen, 2005) comb. n., Rhogadopsis sculpta (Chen & Weng, 2005) comb. n., Rhogadopsis sulcifer (Fischer, 1975) comb. n., Rhogadopsis tabidula(Weng & Chen, 2005) comb. n., Xynobius complexus (Weng & Chen, 2005) comb. n., Xynobius indagatrix (Weng & Chen, 2005) comb. n., Xynobius multiarculatus (Chen & Weng, 2005) comb. n.The following (sub)genera are synonymised: Snoflakopius Fischer, 1972, Jucundopius Fischer, 1984, Opiotenes Fischer, 1998, and Oetztalotenes Fischer, 1998, with Opiostomus Fischer, 1971; Xynobiotenes Fischer, 1998, with Xynobius Foerster, 1862; Allotypus Foerster, 1862, Lemnaphilopius Fischer, 1972, Agnopius Fischer, 1982, and Cryptognathopius Fischer, 1984, with Apodesmia Foerster, 1862; Nosopoea Foerster, 1862, Tolbia Cameron, 1907, Brachycentrus Szépligeti, 1907, Baeocentrum Schulz, 1911, Hexaulax Cameron, 1910, Coeloreuteus Roman, 1910, Neodiospilus Szépligeti, 1911, Euopius Fischer, 1967, Gerius Fischer, 1972, Grimnirus Fischer, 1972, Hoenirus Fischer, 1972, Mimirus Fischer, 1972, Gastrosema Fischer, 1972, Merotrachys Fischer, 1972, Phlebosema Fischer, 1972, Neoephedrus Samanta, Tamili, Saha & Raychaudhuri, 1983, Adontopius Fischer, 1984, Kainopaeopius Fischer, 1986, Millenniopius Fischer, 1996, and Neotropopius Fischer, 1999, with Phaedrotoma Foerster, 1862.     

Phylogenetic utility of the nuclear genes AGAMOUS 1 and PHYTOCHROME B in palms (Arecaceae): an example within Bactridinae

Background and Aims

Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics.

Methods

New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated.

Key Results

The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics.

Conclusions

AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.     

Insights from paleomicrobiology into the indigenous peoples of pre-colonial America - A Review

This review investigates ancient infectious diseases in the Americas dated to thepre-colonial period and considers what these findings can tell us about the historyof the indigenous peoples of the Americas. It gives an overview, but focuses on fourmicrobial pathogens from this period: Helicobacter pylori,Mycobacterium tuberculosis, Trypanosoma cruziand Coccidioides immitis, which cause stomach ulceration and gastriccancer, tuberculosis, Chagas disease and valley fever, respectively. These pathogenswere selected as H. pylori can give insight into ancient humanmigrations into the Americas, M. tuberculosis is associated withpopulation density and urban development, T. cruzi can elucidatehuman living conditions and C. immitis can indicate agriculturaldevelopment. A range of methods are used to diagnose infectious disease in ancienthuman remains, with DNA analysis by polymerase chain reaction one of the mostreliable, provided strict precautions are taken against cross contamination. Thereview concludes with a brief summary of the changes that took place after Europeanexploration and colonisation.     

Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.