A Gene Transfer Agent and a Dynamic Repertoire of Secretion Systems Hold the Keys to the Explosive Radiation of the Emerging Pathogen Bartonella

Gene transfer agents (GTAs) randomly transfer short fragments of a bacterial genome. A novel putative GTA was recently discovered in the mouse-infecting bacterium Bartonella grahamii. Although GTAs are widespread in phylogenetically diverse bacteria, their role in evolution is largely unknown. Here, we present a comparative analysis of 16 Bartonella genomes ranging from 1.4 to 2.6 Mb in size, including six novel genomes from Bartonella isolated from a cow, two moose, two dogs, and a kangaroo. A phylogenetic tree inferred from 428 orthologous core genes indicates that the deadly human pathogen B. bacilliformis is related to the ruminant-adapted clade, rather than being the earliest diverging species in the genus as previously thought. A gene flux analysis identified 12 genes for a GTA and a phage-derived origin of replication as the most conserved innovations. These are located in a region of a few hundred kb that also contains 8 insertions of gene clusters for type III, IV, and V secretion systems, and genes for putatively secreted molecules such as cholera-like toxins. The phylogenies indicate a recent transfer of seven genes in the virB gene cluster for a type IV secretion system from a cat-adapted B. henselae to a dog-adapted B. vinsonii strain. We show that the B. henselae GTA is functional and can transfer genes in vitro. We suggest that the maintenance of the GTA is driven by selection to increase the likelihood of horizontal gene transfer and argue that this process is beneficial at the population level, by facilitating adaptive evolution of the host-adaptation systems and thereby expansion of the host range size. The process counters gene loss and forces all cells to contribute to the production of the GTA and the secreted molecules. The results advance our understanding of the role that GTAs play for the evolution of bacterial genomes.     

Clostridium taeniosporum is a close relative of the Clostridium botulinum Group II

Clostridium taeniosporum is a Gram-positive, anaerobic, rod-shaped non-toxigenic organism isolated from Crimean lake silt. It is unique in forming spores from which about twelve large, flat, ribbon-like appendages emanate. These ribbon-like structures, about 4.5 μm long and 0.45 μm wide, are assembled from smaller fibrils with 5 nm diameter spherical heads attached to thin tails about 1–2 nm in diameter and about 40 nm in length. The appendages have four major components, a glycoprotein with a collagen-like region, two proteins each of which contains two conserved domains of unknown function, and an ortholog of the Bacillus subtilis spore morphogenetic protein SpoVM. Genes for three of these and other, possibly related proteins, cluster on two chromosome fragments. Here we report that C. taeniosporum is saccharolytic, non-proteolytic, and produces both acetic and butyric acid fermentation products. It synthesizes α-d-glucosidase and N-acetyl-β,d-glucoseaminidase constitutively. These physiological properties are similar to those of the C. botulinum Group II. Genotypically, C. taeniosporum is also closely related to the same Group II, based on 16S rDNA sequences. C. taeniosporum differs from typical C. botulinum Group II strains because it is non-toxigenic and in forming the ribbon-like spore appendages. These major differences among otherwise closely related organisms suggest lateral transfer of genes for appendage synthesis and for toxigenicity.     

Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis

The family B DNA polymerase gene from the euryarchaeon Thermococcus waiotapuensis (Twa) contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. The gene is split by two intein-coding sequences that forms a continuous open reading frame with the three polymerase exteins. Twa DNA polymerase genes with (whole gene) and without (genetically intein-spliced) inteins were expressed in Escherichia coli Rosetta(DE3)pLysS. The inteins of the expressed whole gene were easily spliced during purification. The molecular mass of the purified Twa DNA polymerase was about 90 kDa, as estimated by SDS-PAGE. The optimal pH for Twa DNA polymerase activity was 6.0 and the optimal temperature was 75 °C. The enzyme was activated by magnesium ions. The half-life of the enzyme at 99 °C was about 4 h. The optimal buffer for PCR with Twa DNA polymerase was 50 mM Tris–HCl (pH 8.2), 2.0 mM MgCl₂, 30 mM KCl, 2.0 mM (NH₄)₂SO₄, 0.01% Triton X-100, and 0.005% BSA. The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR.     

Genetic factors in individual radiation sensitivity

Cancer risk and radiation sensitivity are often associated with alterations in DNA repair, cell cycle, or apoptotic pathways. Interindividual variability in mutagen or radiation sensitivity and in cancer susceptibility may also be traced back to polymorphisms of genes affecting e.g. DNA repair capacity. We studied possible associations between 70 polymorphisms of 12 DNA repair genes with basal and initial DNA damage and with repair thereof. We investigated DNA damage induced by ionizing radiation in lymphocytes isolated from 177 young lung cancer patients and 169 cancer-free controls. We also sought replication of our findings in an independent sample of 175 families (in total 798 individuals). DNA damage was assessed by the Olive tail moment (OTM) of the comet assay. DNA repair capacity (DRC) was determined for 10, 30 and, 60 min of repair.Genes involved in the single-strand-repair pathway (SSR; like XRCC1 and MSH2) as well as genes involved in the double-strand-repair pathway (DSR; like RAD50, XRCC4, MRE11 and ATM) were found to be associated with DNA damage. The most significant association was observed for marker rs3213334 (p = 0.005) of XRCC1 with basal DNA damage (B), in both cases and controls. A clear additive effect on the logarithm of OTM was identified for the marker rs1001581 of the same LD-block (p = 0.039): B_CC = −1.06 (95%-CI: −1.16 to −0.96), B_CT = −1.02 (95%-CI: −1.11 to −0.93) and B_TT = −0.85 (95%-CI: −1.01 to −0.68). In both cases and controls, we observed significantly higher DNA basal damage (p = 0.007) for carriers of the genotype AA of marker rs2237060 of RAD50 (involved in DSR). However, this could not be replicated in the sample of families (p = 0.781). An alteration to DRC after 30 min of repair with respect to cases was observed as borderline significant for marker rs611646 of ATM (involved in DSR; p = 0.055), but was the most significant finding in the sample of families (p = 0.009).Our data indicate that gene variation impacts measurably on DNA damage and repair, suggesting at least a partial contribution to radiation sensitivity and lung cancer susceptibility.     

Characterization and PCR optimization of the thermostable family B DNA polymerase from Thermococcus guaymasensis

The gene encoding Thermococcus guaymasensis DNA polymerase (Tgu DNA polymerase) was cloned and sequenced. The 2328 bp Tgu DNA polymerase gene encoded a 775 amino acid residue protein. Alignment of the entire amino acid sequence revealed a high degree of sequence homology between Tgu DNA polymerase and other archaeal family B DNA polymerases. The Tgu DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was then purified by heat treatment followed by two steps of chromatography. The optimum pH and temperature were 7.5 and 80 °C, respectively. The optimal buffer for PCR with Tgu DNA polymerase consisted of 50 mM Tris–HCl (pH 8.2), 4 mM MgCl₂, 50 mM KCl, and 0.02% Triton X-100. Tgu DNA polymerase revealed 4-fold higher fidelity (3.17 × 10^?6) than Taq DNA polymerase (12.13 × 10^?6) and a faster amplification rate than Taq and Pfu DNA polymerases. Tgu DNA polymerase had an extension rate of 30 bases/s and a processivity of 150 nucleotides (nt). Thus, Tgu DNA polymerase has some faster elongation rate and a higher processivity than Pfu DNA polymerase. Use of different ratios of Taq and Tgu DNA polymerases determined that a ratio of 4:1 efficiently facilitated long PCR (approximately 15 kb) and a 3-fold lower error rate (4.44 × 10^?6) than Taq DNA polymerase.     

Synthesis and characterization of a fluorescent probe for α-tocopherol suitable for fluorescence microscopy

Previously prepared fluorescent derivatives of α-tocopherol have shown tremendous utility in both in vitro exploration of the mechanism of ligand transfer by the α-tocopherol transfer protein (α-TTP) and the intracellular transport of α-tocopherol in cells and tissues. We report here the synthesis of a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) containing α-tocopherol analog having extended conjugation with an alkenyl thiophene group that extends the absorption and emission maxima to longer wavelengths (λ_ex = 571 nm and λ_em = 583 nm). The final fluorophore thienyl-ene-BODIPY-α-tocopherol, 2, binds to recombinant human α-TTP with a K_d = 8.7 ± 1.1 nM and is a suitable probe for monitoring the secretion of α-tocopherol from cultured Mcf7#189 cells.     

ERCC1 haplotypes modify bladder cancer risk: A case–control study

Bladder cancer risk is highly influenced by environmental and/or predisposing genetic factors. In the last decades growing evidence of the major role played by DNA repair systems in the developing of bladder cancer has been provided. To better investigate the involvement of DNA repair genes previously reported to be significantly associated with bladder cancer risk, we examined in a case–control study (456 cases and 376 hospital controls) 36 single nucleotide polymorphisms (SNPs) in 10 DNA repair genes, through a better gene coverage and a deep investigation of the haplotype role. A single SNP analysis showed a significantly increased risk given by XRCC1-rs915927 G allele (OR = 1.55, CI 95% 1.02–2.37 for dominant model) and a protective effect of the rare alleles of 3 ERCC1 SNPs: rs967591 (OR = 0.66, CI 95% 0.46–0.95), rs735482 (OR = 0.62, CI 95% 0.42–0.90) and rs2336219 (OR = 0.63, CI 95% 0.43–0.93). Haplotype analysis revealed that cases had a statistically significant excess of XRCC3-TAGT and ERCC1-GAT haplotypes, whereas ERCC1-AAC, MGMT-TA, XRCC1-TGCC and ERCC2-TGAA haplotypes were significantly underrepresented. Together with other published data on large case–control studies, our findings provide epidemiological evidence supporting a link between DNA repair gene variants and bladder cancer development, and suggest that the effects of high-order interactions should be taken into account as modulating factors affecting bladder cancer risk. A detailed characterization of DNA repair genetic variation is warranted and might ultimately help to identify multiple susceptibility variants that could be responsible for joint effects on the risk.     

Folate deprivation induces cell cycle arrest at G0/G1 phase and apoptosis in hippocampal neuron cells through down-regulation of IGF-1 signaling pathway

Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (P < 0.01) down-regulated, while SLC19A1 was up-regulated (P < 0.01) in FD group. FD cells exhibited significantly (P < 0.05) higher protein content of BHMT, MAT2b and DNMT3a, as well as increased SAM/SAH concentrations and global DNA hypermethylation. The expression of the total and all the 3 classes of IGF-1 mRNA variants was significantly (P < 0.01) down-regulated and IGF-1 concentration was decreased (P < 0.05) in the culture media. IGF-1 signaling pathway was also compromised with diminished activation (P < 0.05) of STAT3, AKT and mTOR. CpG hypermethylation was detected in the promoter regions of IGF-1 and FOLR1 genes, while higher SLC19A1 mRNA corresponded to hypomethylation of its promoter. IGF-1 supplementation in FD media significantly abolished FD-induced decrease in cell viability. However, IGF-1 had limited effect in rescuing the cell phenotype when added 24 h after FD. Taken together, down-regulation of IGF-1 expression and signaling is involved in FD-induced cell cycle arrest and apoptosis in HT-22 hippocampal neuron cells, which is associated with an abnormal activation of methyl transfer pathway and hypermethylation of IGF-1 gene promoter.     

Sex steroid-induced DNA methylation changes and inflammation response in prostate cancer

BackgroundSex steroid hormones have been reported to induce inflammation causing dysregulation of cytokines in prostate cancer cells. However, the underlying epigenetic mechanism has not well been studied. The objective of this study was to evaluate the effect of sex steroid hormones on epigenetic DNA methylation changes in prostate cancer cells using a signature PCR methylation array panel that correspond to 96 genes with biological function in the human inflammatory and autoimmune signals in prostate cancer. Of the 96-gene panel, 32 genes showed at least 10% differentially methylation level in response to hormonal treatment when compared to untreated cells. Genes that were hypomethylated included CXCL12, CXCL5, CCL25, IL1F8, IL13RAI, STAT5A, CXCR4 and TLR5; and genes that were hypermethylated included ELA2, TOLLIP, LAG3, CD276 and MALT1. Quantitative RT-PCR analysis of select genes represented in a cytokine expression array panel showed inverse association between DNA methylation and gene expression for TOLLIP, CXCL5, CCL18 and IL5 genes and treatment of prostate cancer cells with 5′-aza-2′-deoxycytidine with or without trichostatin A induced up-regulation of TOLLIP expression. Further analysis of relative gene expression of matched prostate cancer tissues when compared to benign tissues from individual patients with prostate cancer showed increased and significant expression for CCL18 (2.6-fold; p < 0.001), a modest yet significant increase in IL5 expression (1.17-fold; p = 0.015), and a modest increase in CXCL5 expression (1.4-fold; p = 0.25). In conclusion, our studies demonstrate that sex steroid hormones can induce aberrant gene expression via differential methylation changes in prostate carcinogenesis.     

Studies on the genotoxic effect of chromium oxide (Cr VI): Interaction with deoxyribonucleic acid in solution

Chromium is a toxic and carcinogenic compound widely distributed in environment. In the present study we have investigated the interaction of chromium oxide with DNA employing UV/vis and fluorescence spectroscopy as well as Circular dichroism, thermal denaturation, retardation polyacrylamide gel electrophoresis and DNA-cellulose affinity techniques. The results showed that the binding of chromium oxide to DNA is concentration dependent; at low concentration shows a little effect but ant higher concentrations (>100 μg/ml) reduced the absorbance at 260 and 210 nm producing hypochromicity. Also λ_max of the metal at 210, 260 and 350 nm was reduced. DNA chromophores quenched with the chromium oxide and decreased fluorescence emission intensity. Upon binding of the metal to DNA the elliplicity at positive extreme was decreased (275 nm) and increased the ellipticity of the DNA at negative extreme 245 nm. Thermal denaturation profile of DNA shifted to higher degrees upon chromium oxide binding which accompanied by hypochromicity. Also, affinity of chromium oxide to double stranded DNA was higher than single stranded DNA. From the result it is concluded that chromium oxide interacts with DNA via two modes of interaction inducing structural changes and DNA compaction evidence providing chromium oxide genotoxicity.     

Genomic damages in peripheral blood lymphocytes and association with polymorphisms of three glutathione S-transferases in workers exposed to formaldehyde

DNA and chromosome damages in peripheral blood lymphocytes were evaluated in 151 workers occupationally exposed to formaldehyde (FA) and 112 non-FA exposed controls. The effects of polymorphisms in three glutathione-S-transferase (GSTs) genes on the DNA and chromosome damages were assessed as well. Alkaline comet assay and cytokinesis-block micronucleus (CBMN) assay were used to determine DNA and chromosome damages, respectively. The genotypes of GSTP1 (Ile105Val), GSTT1, and GSTM1 were assayed. The mean 8-h time-weighted average (TWA) concentrations of FA in two plywood factories were 0.83 ppm (range: 0.08–6.30 ppm). FA-exposed workers had higher olive tail moment (TM) and CBMN frequency compared with controls (Olive TM, 3.54, 95%CI = 3.19–3.93 vs. 0.93, 95%CI = 0.78–1.10, P < 0.01; CBMN frequency, 5.51 ± 3.37 vs. 2.67 ± 1.32, P < 0.01). Olive TM and the CBMN frequency also had a dose-dependent relation with the personal FA exposure. Significant association between FA exposure history and olive TM and CBMN frequency were also identified. The level of olive TM was slightly higher in FA-exposed workers with GSTM1 null genotype than those with non-null genotype (3.86, 95%CI = 3.31–4.50 vs. 3.27, 95%CI = 2.83–3.78, P = 0.07) with adjustment of covariates. We also found that FA-exposed workers carrying GSTP1 Val allele had a slightly higher CBMN frequency compared with workers carrying only the wild-type allele (6.32 ± 3.78 vs. 5.01 ± 2.98, P = 0.05). Our results suggest that the FA exposure in this occupational population increased DNA and chromosome damages and polymorphisms in GSTs genes may modulate the genotoxic effects of FA exposure.     

The complete mitogenome of Bombyx mori strain Dazao (Lepidoptera: Bombycidae) and comparison with other lepidopteran insects

The complete mitochondrial genome (mitogenome) of Bombyx mori strain Dazao (Lepidoptera: Bombycidae) was determined to be 15,653 bp, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a A + T-rich region. It has the typical gene organization and order of mitogenomes from lepidopteran insects. The AT skew of this mitogenome was slightly positive and the nucleotide composition was also biased toward A + T nucleotides (81.31%). All PCGs were initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which was initiated by CGA. The cox1 and cox2 genes had incomplete stop codons consisting of just a T. All the tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA. The A + T-rich region of the mitogenome was 495 bp in length and consisted of several features common to the lepidopteras. Phylogenetic analysis showed that the B. mori Dazao was close to Bombycidae.     

Environmental Factors Influencing Gene Transfer Agent (GTA) Mediated Transduction in the Subtropical Ocean

Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the marine environment with the strains examined is favored during times of elevated bacterial and GTA abundance as well as in areas of higher salinity.     

Shigella flexneri type-specific antigen V: cloning,sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV

With lysogeny by bacteriophage SfV, Shigella flexneri serotype Y is converted to serotype 5a. The glucosyl transferase gene (gtr) from bacteriophage SfV of S. flexneri, involved in serotype-specific conversion, was cloned and characterized. The DNA sequence of a 3.7 kb EcoRI–BamHI fragment of bacteriophage SfV which includes the gtr gene was determined. This gene, encoding a polypeptide of 417 aa with 47.67 kDa molecular mass, caused partial serotype conversion of S. flexneri from serotype Y to type V antigen as demonstrated by Western blotting and the sensitivity of the hybrid strain to phage Sf6. The deduced protein of the partially sequenced open reading frame upstream of the gtr showed similarity to various glycosyl transferases of other bacteria. Orf3, separated from the gtr by a non-coding region and transcribed convergently, codes for a 167 aa (18.8 kDa) protein found to have homology with tail fibre genes of phage lambda and P2.     

A novel MODIS algorithm to estimate chlorophyll a concentration in eutrophic turbid lakes

A novel approach was developed to estimate phytoplankton biomass in eutrophic turbid lakes, using MODIS bands designed for land and atmospheric studies. The Baseline Normalized Difference Bloom Index (BNDBI) uses the difference of remote-sensing reflectance (Rrs, sr⁻¹) at 555 nm (band 4) and 645 nm (band 1) after baseline correction using bands at 469 nm and 859 nm: (Rrs′(555) − Rrs′(645))/(Rrs′(555) + Rrs′(645)). BNDBI takes advantage of the Chl-a’s absorption minimum near 572 nm and absorption maximum near 667 nm. Using data from Lake Chaohu, the index showed a strong relationship with Chl-a concentrations in conditions that would normally saturate more sensitive ocean-color sensors. Extensive field measurements were used to calibrate and validate the algorithm with unbiased root-mean-square-error (URMSE) of 47.9% when compared to in situ Rrs data. A reduced sensitivity to atmospheric effects was accomplished by using a baseline correction approach, anchored at 469 nm and 859 nm to correct the radiances at 555 nm and 645 nm. Radiative transfer simulations showed that the algorithm can be applied directly to MODIS Rayleigh-corrected reflectance (Rrc) after adjusting algorithm coefficients (URMSE uncertainty of 56.4% for MODIS Rrc data) for Chl-a concentrations <1000 μg L⁻¹. Comparative analyses showed that the index was resistant to changes in turbidity and organic matter concentrations. Theoretical simulations, image comparisons and spectral analyses demonstrated that the index was robust in a range of complex atmospheric and surface conditions, with different aerosol types, optical thickness (τ_a555), solar/viewing geometry, sun glint and thin clouds. A comparison with other MODIS and MERIS Chl-a algorithms for turbid waters showed that BNDBI provided consistent results with the advantage of using MODIS wavebands that remain unsaturated in high turbidity conditions. The BNDBI opens new possibilities to explore bio-optical dynamics in turbid eutrophic lakes using data from a range of satellite sources.     

VCA1008: An Anion-Selective Porin of Vibrio Cholerae

A putative porin function has been assigned to VCA1008 of Vibrio cholerae. Its coding gene, vca1008, is expressed upon colonization of the small intestine in infant mice and human volunteers, and is essential for infection. In vitro, vca1008 is expressed under inorganic phosphate limitation and, in this condition, VCA1008 is the major outer membrane protein of the bacterium. Here, we provide the first functional characterization of VCA1008 reconstituted into planar lipid bilayers. Our main findings were: 1) VCA1008 forms an ion channel that, at high voltage (~ ± 100mV), presents a voltage-dependent activity and displays closures typical of trimeric porins, with a conductance of 4.28 ± 0.04 nS (n = 164) in 1M KCl; 2) It has a preferred selectivity for anions over cations; 3) Its conductance saturates with increasing inorganic phosphate concentration, suggesting VCA1008 contains binding site(s) for this anion; 4) Its ion selectivity is controlled by both fixed charged residues within the channel and diffusion along the pore; 5) Partitioning of poly (ethylene glycol)s (PEGs) of different molecular mass suggests that VCA1008 channel has a pore exclusion limit of 0.9 nm.     

Analysis of digestion of rice planthopper by Pardosa pseudoannulata based on CO-I gene

In order to systematically study the predatory behavior and digestion regularity of spiders, real-time fluorescence quantification PCR technique was used to detect the number of CO-I genes in Pardosa pseudoannulata after it preyed on rice planthoppers in different temperatures within different periods. At 28 °C, 0, 1, 2, 4, 8, 16, and 24 h after P. pseudoannulata preyed on rich planthopper, DNA was extracted from cephalothorax and abdomen of P. pseudoannulata. Routine PCR and real-time fluorescence PCR techniques were employed for CO-I gene amplification. The results show that: The prey liquid was temporarily stored in the sucking stomach of the spider head within 2 h after prey, and gradually transferred to the midgut of the abdomen with the prolongation of time. After 4 h, CO-I gene residues of rice planthopper in the cephalothorax gradually decreased. The CO-I gene of rice planthopper was basically transferred to the abdomen after 16 h. During 0–1 h, food contained in abdominal midgut and other digestive organs was very small, CO-I gene detection was not obvious. Over time, food entered into the midgut from the sucking stomach for digestion. During 2–4 h, CO-I gene amount increased, at 2–4 h, detected CO-I gene residue reached the peak; but rapidly declined after 8, 16, and 24 h, even it is still detectable. The results at different temperatures reveal that: As the temperature increased from 26 °C to 32 °C, CO-I gene residues of rich planthopper in cephalothorax and abdomen of P. pseudoannulata gradually decreased, which indicated that the digestion rate increased with the increase of temperature with some range. However, when the temperature continued to increase to 34 °C, the digestion rate decreased.     

Production and characterization of a thermostable endo-type β-xylanase produced by a newly-isolated Streptomyces thermocarboxydus subspecies MW8 strain from Jeju Island

A xylanase-producing, Gram-positive, aerobic, and spore-forming bacterium was isolated from a soil sample collected from Jeju Island and was classified as a novel subspecies of Streptomyces thermocarboxydus on the basis of 16S rRNA gene sequence similarity, the results of DNA–DNA hybridization analysis, and phenotypic characteristics. The novel strain was named as S. thermocarboxydus subsp. MW8 (=KCTC29013 = DSM52054). This strain produced extracellular xylanase. Xylanase from the strain was purified to homogeneity and had an apparent molecular weight of 52 kDa. The NH₂-terminal sequence (Ala-Glu-Ile-Arg-Leu) was distinct from those of previously reported xylanases. The purified xylanase produced xylobiose as the end-product of birchwood xylan hydrolysis. The K_m and V_max values of the purified xylanase on birchwood xylan were 1.71 mg/ml and 357.14 U/mg, respectively. The optimum pH and temperature for the enzyme were found to be 7.0 and 50 °C, respectively, and the enzyme exhibited significant heat stability. In addition, the enzyme was active over broad pH ranges: 84% of the maximum activity at pH 5.0, 84–88% at pH 6.0, 88% at pH 8.0, and 75–81% (pH 9.0). These enzymatic properties may be very useful for use in bio-industrial applications.