Molecular Cloning and Characterisation of Farnesyl Pyrophosphate Synthase from Tripterygium wilfordii

Farnesylpyrophosphate synthase (FPS) catalyzes the biosynthesis of farnesyl pyrophosphate (FPP), which is an important precursor of sesquiterpenoids such as artemisinin and wilfordine. In the present study, we report the molecular cloning and characterization of two full-length cDNAs encoding FPSs from Tripterygium wilfordii (TwFPSs). TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli. Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment. The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products.     

Gluconacetobacter maltaceti sp. nov., a novel vinegar producing acetic acid bacterium

Comparison of HaeIII- and HpaII-restriction profiles of PCR-amplified 16S-23S rDNA ITS regions of Gluconacetobacter sp. LMG 1529^T and SKU 1109 with restriction profiles of reference strains of acetic acid bacteria described by Tr?ek and Teuber [34] revealed the same but unique restriction profiles for LMG 1529^T and SKU 1109. Further analyses of nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rDNA ITS sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated both strains to a single phylogenetic cluster well separated from the other species of the genus Gluconacetobacter. DNA–DNA hybridizations confirmed their novel species identity by 73% DNA–DNA relatedness between both strains, and values below the species level (<70%) between SKU 1109 and the type strains of the closest phylogenetic neighbors. The classification of strains LMG 1529^T and SKU 1109 into a single novel species was confirmed also by AFLP and (GTG)₅-PCR DNA fingerprinting data, as well as by phenotypic data. Strains LMG 1529^T and SKU 1109 can be differentiated from their closely related Gluconacetobacter species, Gluconacetobacter entanii and Gluconacetobacter hansenii, by their ability to form 2-keto-d-gluconic acid from d-glucose, their ability to use d-mannitol, d-gluconate and glycerol as carbon source and form acid from d-fructose, and their ability to grow without acetic acid. The major fatty acid of LMG 1529^T and SKU 1109 is C_18:1ω7c (60.2–64.8%). The DNA G + C content of LMG 1529^T and SKU 1109 is 62.5 and 63.3 mol% respectively. The name Gluconacetobacter maltaceti sp. nov. is proposed. The type strain is LMG 1529^T (= NBRC 14815^T = NCIMB 8752^T).     

Significant involvement of PEP-CK in carbon assimilation of C4 eudicots

Background and Aims

C₄ eudicot species are classified into biochemical sub-types of C₄ photosynthesis based on the principal decarboxylating enzyme. Two sub-types are known, NADP-malic enzyme (ME) and NAD-ME; however, evidence for the occurrence or involvement of the third sub-type (phosphoenolpyruvate carboxykinase; PEP-CK) is emerging. In this study, the presence and activity of PEP-CK in C₄ eudicot species of Trianthema and Zaleya (Sesuvioideae, Aizoaceae) is clarified through analysis of key anatomical features and C₄ photosynthetic enzymes.

Methods

Three C₄ species (T. portulacastrum, T. sheilae and Z. pentandra) were examined with light and transmission electron microscopy for leaf structural properties. Activities and immunolocalizations of C₄ enzymes were measured for biochemical characteristics.

Key Results

Leaves of each species possess atriplicoid-type Kranz anatomy, but differ in ultrastructural features. Bundle sheath organelles are centripetal in T. portulacastrum and Z. pentandra, and centrifugal in T. sheilae. Bundle sheath chloroplasts in T. portulacastrum are almost agranal, whereas mesophyll counterparts have grana. Both T. sheilae and Z. pentandra are similar, where bundle sheath chloroplasts contain well-developed grana while mesophyll chloroplasts are grana deficient. Cell wall thickness is significantly greater in T. sheilae than in the other species. Biochemically, T. portulacastrum is NADP-ME, while T. sheilae and Z. pentandra are NAD-ME. Both T. portulacastrum and Z. pentandra exhibit considerable PEP-CK activity, and immunolocalization studies show dense and specific compartmentation of PEP-CK in these species, consistent with high PEP-CK enzyme activity.

Conclusions

Involvement of PEP-CK in C₄ NADP-ME T. portulacastrum and NAD-ME Z. petandra occurs irrespective of biochemical sub-type, or the position of bundle sheath chloroplasts. Ultrastructural traits, including numbers of bundle sheath peroxisomes and mesophyll chloroplasts, and degree of grana development in bundle sheath chloroplasts, coincide more directly with PEP-CK recruitment. Discovery of high PEP-CK activity in C₄ Sesuvioideae species offers a unique opportunity for evaluating PEP-CK expression and suggests the possibility that PEP-CK recruitment may exist elsewhere in C₄ eudicots.     

Genome Sequence of Kitasatospora setae NBRC 14216T: An Evolutionary Snapshot of the Family Streptomycetaceae

Kitasatospora setae NBRC 14216^T (=KM-6054^T) is known to produce setamycin (bafilomycin B1) possessing antitrichomonal activity. The genus Kitasatospora is morphologically similar to the genus Streptomyces, although they are distinguishable from each other on the basis of cell wall composition and the 16S rDNA sequence. We have determined the complete genome sequence of K. setae NBRC 14216^T as the first Streptomycetaceae genome other than Streptomyces. The genome is a single linear chromosome of 8 783 278 bp with terminal inverted repeats of 127 148 bp, predicted to encode 7569 protein-coding genes, 9 rRNA operons, 1 tmRNA and 74 tRNA genes. Although these features resemble those of Streptomyces, genome-wide comparison of orthologous genes between K. setae and Streptomyces revealed smaller extent of synteny. Multilocus phylogenetic analysis based on amino acid sequences unequivocally placed K. setae outside the Streptomyces genus. Although many of the genes related to morphological differentiation identified in Streptomyces were highly conserved in K. setae, there were some differences such as the apparent absence of the AmfS (SapB) class of surfactant protein and differences in the copy number and variation of paralogous components involved in cell wall synthesis.     

A new cryptic species in the genus Tubariomyces (Inocybaceae, Agaricales)

A new Tubariomyces species from Italy, T. similis, is described and illustrated. It is phenotypically very close to T. hygrophoroides but, based on a combined ITS-LSU rDNA analysis, phylogenetically distinct.     

Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on multilocus sequence analysis

A pathogenic Spiroplasma penaei strain was isolated from the hemolymph of moribund Pacific white shrimp, Penaeus vannamei. The shrimp sample originated from a shrimp farm near Cartagena, Colombia, that was suffering from high mortalities in ponds with very low salinity and high temperatures. This new emerging disease in a marine crustacean in the Americas is described as a systemic infection. The multilocus phylogenetic analysis suggests that S. penaei strain has a terrestrial origin. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S rDNA and 23S-5S rDNA intergenic spacer region, were reconstructed using the distance-based Neighboring-Joining (NJ) method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborate the observations by other investigators using the 16S rDNA gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to Spiroplasma insolitum and distantly related to Spiroplasma sp. SHRIMP from China.     

Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.     

内生真菌和内生细菌接种对雷公藤生长和次生代谢产物积累的影响

为探究内生真菌与内生细菌对雷公藤(Tripterygium wilfordii)的生长和次生代谢产物积累的相互作用,用内生真菌NS33、NS6和内生细菌LG3、LY1单独或跨界联合接种雷公藤,对雷公藤的生长和雷公藤甲素、雷公藤红素合成进行了研究。结果表明,单独或混合培养的菌株具有分泌铁载体、吲哚乙酸(IAA)和溶磷能力,对种子萌发、芽伸长和根系活力有显著促进作用。接种菌株NS33、NS6、NS6-LG3和NS6-LY1均显著促进了雷公藤组培苗的生长。单独或联合接种菌株均能显著提高雷公藤组培苗雷公藤甲素和雷公藤红素的积累,其中NS33-LG3和LG3的作用最显著。菌株NS33与LG3能够协同促进IAA的分泌、小麦幼苗根系活力和雷公藤红素的积累;菌株NS6与LY1协同提高了雷公藤组培苗的高度、质量和雷公藤红素的积累。因此,内生真菌与内生细菌联合接种对雷公藤生长和次生代谢产物积累具有一定的协同效应,显示出实际应用潜力。     

Genome sequence and description of Timonella senegalensis gen. nov., sp. nov., a new member of the suborder Micrococcinae

Timonella senegalensis strain JC301^T gen. nov., sp. nov. is the type strain of T. senegalensis gen. nov., sp. nov., a new species within the newly proposed genus Timonella. This bacterial strain was isolated from the fecal flora of a healthy Senegalese patient. In this report, we detail the features of this organism, together with the complete genome sequence and annotation. Timonella senegalensis strain JC301^T exhibits the highest 16S rRNA similarity (95%) with Sanguibacter marinus, the closest validly published bacterial species. The genome of T. senegalensis strain JC301^T is 3,010,102-bp long, with one chromosome and no plasmid. The genome contains 2,721 protein-coding genes and 72 RNA genes, including 5 rRNA genes. The genomic annotation revealed that T. senegalensis strain JC301^T possesses the complete complement of enzymes necessary for the de novo biosynthesis of amino acids and vitamins (except for riboflavin and biotin), as well as the enzymes involved in the metabolism of various carbon sources, chaperone genes, and genes involved in the regulation of polyphosphate and glycogen levels.     

Stereochemistry of 2-aminopimelic acid and related amino acids in three species of asplenium

We previously reported two free D-amino acids, D-2-aminopimelic acid (D-APA) and trans-3,4-dehydro-D-2-aminopimelic acid (D-Δ-APA), from Asplenium unilaterale. In the present work we isolated 4-hydroxy-2-aminopimelic acid (OH-APA) from the same plant and determined it to be the α-L-form. We also investigated the configurations of these amino acids isolated from A. prolongatum and A. wilfordii which are morphologically distinct from A. unilaterale. In A. prolongatum, APA was the D- and OH-APA was the L-isomer. In contrast, APA from A. wilfordii was partially racemized and the degree of racemization was significantly different in plant material collected in July and November, L:D = 3:2 and 3:7, respectively. In A. wilfordii OH-APA was almost pure L- and Δ-APA was mostly the D-isomer.     

Definition of a novel symbiovar (sv. retamae) within Bradyrhizobium retamae sp. nov., nodulating Retama sphaerocarpa and Retama monosperma

In this paper we analyze through a polyphasic approach several Bradyrhizobium strains isolated in Spain and Morocco from root nodules of Retama sphaerocarpa and Retama monosperma. All the strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium lablabi CCBAU 23086^T, with 99.41% identity with respect to the strain Ro19^T. Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII were divergent in Ro19^T and B. lablabi CCBAU 23086^T, with identity values of 95.71%, 93.75% and 93.11%, respectively. These differences were congruent with DNA–DNA hybridization analysis that revealed an average of 35% relatedness between the novel species and B. lablabi CCBAU 23086^T. Also, differential phenotypic characteristics of the new species were found with respect to the already described species of Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose to classify the group of strains isolated from R. sphaerocarpa and R. monosperma as a novel species named Bradyrhizobium retamae sp. nov. (type strain Ro19^T = LMG 27393^T = CECT 8261^T). The analysis of symbiotic genes revealed that some of these strains constitute a new symbiovar within genus Bradyrhizobium for which we propose the name “retamae”, that mainly contains nodulating strains isolated from Retama species in different continents.     

Thermodynamic Characterization of the Interaction between Prefoldin and Group II Chaperonin

Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1). In this study, we determined the crystal structure of the T. KS-1 PFDβ2 subunit and characterized the interactions between T. KS-1 CPNs (CPNα and CPNβ) and T. KS-1 PFDs (PFDα1-β1 and PFDα2-β2). As predicted from its amino acid sequence, the PFDβ2 subunit conforms to a structure similar to those of the PFDβ1 subunit and the Pyrococcus horikoshii OT3 PFDβ subunit, with the exception of the tip of its coiled-coil domain, which is thought to be the CPN interaction site. The interactions between T. KS-1 CPNs and PFDs (CPNα and PFDα1-β1; CPNα and PFDα2-β2; CPNβ and PFDα1-β1; and CPNβ and PFDα2-β2) were analyzed using the Biacore T100 system at various temperatures ranging from 20 to 45 ºC. The affinities between PFDs and CPNs increased with an increase in temperature. The thermodynamic parameters calculated from association constants showed that the interaction between PFD and CPN is entropy driven. Among the four combinations of PFD-CPN interactions, the entropy difference in binding between CPNβ and PFDα2-β2 was the largest, and affinity significantly increased at higher temperatures. Considering that expression of PFDα2-β2 and CPNβ subunit is induced upon heat shock, our results suggest that PFDα1-β1 is a general PFD for T. KS-1 CPNs, whereas PFDα2-β2 is specific for CPNβ.     

Contrasting genetic structuring in the closely related basidiomycetes Trichaptum abietinum and Trichaptum fuscoviolaceum (Hymenochaetales)

Trichaptum abietinum and Trichaptum fuscoviolaceum (Hymenochaetales, Basidiomycota) are closely related saprotrophic fungi, widely distributed on coniferous wood in temperate regions worldwide. Three intersterility groups have previously been detected in T. abietinum, while no prezygotic barriers have been proven within T. fuscoviolaceum. The aim of this study was to reveal the phylogeography and genetic relationship between these two closely related species and to explore whether the previously observed intersterility groups in T. abietinum are reflected in the genetic data. We assembled worldwide fruit body collections of both species (N = 314) and generated DNA sequences from three nuclear (ITS2, LSU, IGS) and one mitochondrial rDNA region (mtLSU). The two species are genetically well separated in all analyses. In correspondence with observations from earlier mating studies, our results revealed that T. fuscoviolaceum is genetically more uniform than T. abietinum. Multiple genetic sub-groups exist in T. abietinum that may correspond to the previously observed intersterility groups. However, there is low consistency across the investigated loci in delimiting the different sub-groups, except for a consistent North American group. As for many other widespread fungi, a complex phylogeographic pattern is found in T. abietinum which may have been formed by geographic, as well as multiple genetic intersterility barriers.     

Detection of Quorum Sensing Signal Molecules in Edwardsiella ictaluri Ei-151

Edwardsiella ictaluri is a Gram-negative pathogenic bacterium in the family Enterobacteriaceae that causes enteric septicemia of catfish, which has become a significant problem in the aquaculture of striped catfish (Pangasianodon hypophthalmus) in Vietnam. In this study, a bacterium designated as Ei-151 was isolated from diseased striped catfish and proved to be virulent. Based on 16S rDNA sequencing and phenotypic tests, the pathogenic bacterium was identified as Edw. ictaluri. The presence of quorum sensing signal molecules in Edw. ictaluri Ei-151 was detected with different biosensor strains. The results showed that Ei-151 produced at least three kinds of acylated homoserine lactone (AHL) signal molecules as detected with the biosensor Agrobacterium tumefaciens KYC55, and the AHLs fingerprint was similar to that of Edw. tarda. During its entire growth, the levels of AHLs and autoinducer-2 produced by Ei-151 peaked at the stationary phase (OD₆₀₀ 1.8), which suggested that both of them may function at the stationary phase. No Cholerae autoinducer-1-like activity (including Edw. ictaluri LMG7860^T) was detected.     

Reclassification of Saccharomycodes sinensis,Proposal of Yueomyces sinensis gen. nov., comb. nov. within Saccharomycetaceae (Saccharomycetales,Saccharomycotina)

The phylogenetic position of Saccharomycodes sinensis has been debated by yeast taxonomists. In this study, a multigene phylogenetic analysis based on four regions, namely the 18S ribosomal DNA (rDNA), the D1/D2 domains of the 26S rDNA, the second largest subunit of RNA polymerase II gene (RPB2) and translation elongation factor 1-α gene (EF1-α), were performed to address the phylogenetic placement of S. sinensis. Our result indicated that S. sinensis belongs to Saccharomycetaceae instead of Saccharomycodaceae, and forms a single species lineage divergent from the other genera within Saccharomycetaceae. Yueomyces gen. nov. (MycoBank No. MB 811648) is proposed in the Saccharomycetaceae with Y. sinensis comb. nov. (MycoBank No. MB 811649, type strain CGMCC 2.01395^T = IFO 10111^T = CBS 7075^T) as the type species.     

Detection of the Quarantine Species Thrips palmi by Loop-Mediated Isothermal Amplification

Thrips palmi (from the order Thysanoptera) is a serious insect pest of various crops, including vegetables, fruits and ornamental plants, causing significant economic losses. Its presence constitutes a double threat; not only does T. palmi feed on the plants, it is also a vector for several plant viruses. T. palmi originated in Asia, but has spread to North and Central America, Africa, Oceania and the Caribbean in recent decades. This species has been sporadically noted in Europe and is under quarantine regulation in the European Union. For non-specialists its larval stages are indistinguishable morphologically from another widespread and serious insect pest Frankliniella occidentalis (a non-quarantine species in the European Union) as well as other frequently occurring thrips. In this study, we have developed a loop-mediated isothermal amplification protocol to amplify rDNA regions of T. palmi. The results were consistent whether isolated DNA or crushed insects were used as template, indicating that the DNA isolation step could be omitted. The described method is species-specific and sensitive and provides a rapid diagnostic tool to detect T. palmi in the field.     

Ribosomal DNA Comparisons of Globodera from Two Continents

Ribosomal DNA (rDNA) sequence data were compared for five species of Globodera, including G. rostochiensis, G. pallida, G. virginiae, and two undescribed Globodera isolates from Mexico collected from weed species and maintained on Solanum dulcamara. The rDNA comparisons included both internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene, and small portions of the 3'' end of the 18S gene and the 5'' end of the 28S gene. Phylogenetic analysis of the rDNA sequence data indicated that the two potato cyst nematodes, G. pallida and especially G. rostochiensis, are closely related to the Mexican isolates, whereas G. virginiae is relatively dissimilar to the others and more distantly related. The data are consistent with the thesis that Mexico is the center of origin for the potato cyst nematodes.     

R2 dynamics in Triops cancriformis (Bosc, 1801) (Crustacea,Branchiopoda, Notostraca): turnover rate and 28S concerted evolution

The R2 retrotransposon is here characterized in bisexual populations of the European crustacean Triops cancriformis. The isolated element matches well with the general aspects of the R2 family and it is highly differentiated from that of the congeneric North American Triops longicaudatus. The analysis of 5′ truncations indicates that R2 dynamics in T. cancriformis populations show a high turnover rate as observed in Drosophila simulans. For the first time in the literature, though, individuals harboring truncation variants, but lacking the complete element, are found. Present results suggest that transposition-mediated deletion mechanisms, possibly involving genomic turnover processes acting on rDNAs, can dramatically decrease the copy number or even delete R2 from the ribosomal locus. The presence of R2 does not seem to impact on the nucleotide variation of inserted 28S rDNA with respect to the uninserted genes. On the other hand, a low level of polymorphism characterizes rDNA units because new 28S variants continuously spread across the ribosomal array. Again, the interplay between transposition-mediated deletion and molecular drive may explain this pattern.