Genome-wide analysis of codon usage bias patterns in an enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> F18 strain

Enterogenic Escherichia coli (ETEC) F18 strains are the main pathogenic bacteria causing severe diarrhea in humans and domestic animals. However, the information about synonymous codon usage pattern of ETEC F18 genome remains unclear. We conducted a genome-wide analysis of synonymous codon usage patterns in the ETEC F18 strain SRA: SAMN02471895. After filtering of the complete genome sequence, 4327 coding sequences were analyzed using multivariate statistical methods to calculate synonymous codon usage patterns and to evaluate the influence of various factors in shaping the codon usage. The mean GC content was 51.38%, with a slight preference for G/C-ending codons. Twenty-two codons were determined as ‘‘optimal codons”. ENC plots showed some of the genes were on or close to the expected curve, while only points with low-ENC values were below the curve. PR2 analysis showed that GC and AT were not used proportionally, suggesting major roles for mutational pressure and natural selection in shaping usage. Neutrality plots showed a significant correlation between GC12 and GC3, suggesting that mutational pressure is responsible for nucleotide composition in shaping the strength of codon usage. Translational selection was the main factor shaping the codon usage pattern of ETEC F18 genome, while other factors such as protein length, GRAVY and ARO values also influenced codon usage to some extent. We analyzed the codon usage pattern systematically and identified the factors shaping codon usage bias in the ETEC F18 genome. Such information further elucidates the mechanisms of synonymous codon usage bias and provides the basis of molecular genetic engineering and evolutionary studies.     

Optimization of codon usage of the envelope protein E2 gene from various genotypes of hepatitis C virus to predict the expression level in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Hepatitis C virus infection (HCV) alarmingly increases worldwide; it causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, so there is urgent need of developing effective and sufficient quantity of vaccine. HCV envelope protein E2 is the main target for developing as a vaccine candidate. Presently recombinant proteins can successfully be used as a vaccine for many diseases. This concern, it is challenging to produce sufficient quantities of many recombinant proteins from their expression hosts. One of the main factors affecting the success of expression of foreign genes in heterologous hosts is the divergence of codon usage of the target gene from that used in the expression system. In this study, we optimized the various genotypes of HCV envelope protein E2 gene according to the codon usage of Pichia pastoris and predicted the expression level. Synonymous codon usage of E2 adapted to that used by P. pastoris was estimated using the relative synonymous codon usage value (RSCU), codon adaptation index (CAI) and effective number of codon (ENC). The CAI of optimized HCV E2 sequences was enhanced from 0.638 to 0.833 and %GC was decreased from 56.05 to 44.05; this was significantly (p < 0.01) different from the native sequences. Codon with RSCU value less than one was replaced with most preferred synonymous codons. The ENC values of optimized HCV E2 sequences varied from 47.00 to 47.50, with a mean value of 47.15 and an SD of 0.14. Our study suggested that, from the measured values of predicted expression level, the codon optimized HCV E2 protein could be produced in sufficient quantity in the expression host; knowledge of the codon usage patterns of E2 of various genotypes facilitate the production of a promising unique vaccine candidate for HCV.     

Analysis of synonymous codon usage bias in <Emphasis Type="Italic">helicase</Emphasis> gene from <Emphasis Type="Italic">Autographa californica</Emphasis> multiple nucleopolyhedrovirus

The helicase gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is not only involved in viral DNA replication, but also plays a role in viral host range. To identify the codon usage bias of helicase of AcMNPV, the codon usage bias of helicase was especially studies in AcMNPV and 41 reference strains of baculoviruses by calculating the codon adaptation index (CAI), effective number of codon (ENc), relative synonymous codon usage (RSCU), and other indices. The helicase of baculovirus is less biased (mean ENc?=?50.539?>?40; mean CAI?=?0.246). AcMNPV helicase has a strong bias toward the synonymous codons with G and C at the third codon position (GC3s?=?53.6%). The plot of GC3s against ENc values revealed that GC compositional constraints are the main factor that determines the codon usage bias of major of helicase. Several indicators supported that the codon usage pattern of helicase is mainly subject to mutation pressure. Analysis of variation in codon usage and amino acid composition indicated AcMNPV helicase shows the significant preference for one or more postulated codons for each amino acid. A cluster analysis based on RSCU values suggested that AcMNPV is evolutionarily closer to members of group I alphabaculovirus. Comparison of the codon usage pattern among E. coli, yeast, mouse, human and AcMNPV showed that yeast is a suitable expression system for AcMNPV helicase. AcMNPV helicase shows weak codon usage bias. This study may help in elucidating the functional mechanism of AcMNPV helicase and the evolution of baculovirus helicases.     

Fingerprinting by amplified fragment length polymorphism (AFLP) and barcoding by three plastidic markers in the genus <Emphasis Type="Italic">Wolffiella</Emphasis> Hegelm

Amplified fragment length polymorphism (AFLP) fingerprinting and three different plastidic DNA regions (rpl16, rps16, atpF-atpH) were used to investigate species identity in the genus Wolffiella. For this purpose, clones (67 in total) belonging to all ten species were selected. Almost all the species were represented by more than one clone. The fingerprinting technique, AFLP, clearly distinguished the species, W. caudata, W. gladiata, W. neotropica, W. rotunda, and W. welwitschii. Apart from confirming the molecular identity of these five species, the plastidic markers could delineate two additional species, W. hyalina and W. denticulata, although the conclusion concerning the latter is restricted by the availability of only one clone. The efficiency of the plastid-derived markers in identifying the number of species-specific clusters followed the sequence rps16 > rpl16 > atpF-atpH. The species W. lingulata, W. oblonga, and W. repanda could not be identified by any of the molecular methods presented here, but could be strictly defined on a morphological basis. In several clones, high amounts of genetic admixtures between different species were detected. Further, simulation studies demonstrated that these clones are genetic hybrids. This might be one of the obstacles in molecular identification of species in the genus Wolffiella.     

Centromere repositioning explains fundamental number variability in the New World monkey genus <Emphasis Type="Italic">Saimiri</Emphasis>

Cytogenetics has historically played a key role in research on squirrel monkey (genus Saimiri) evolutionary biology. Squirrel monkeys have a diploid number of 2n = 44, but vary in fundamental number (FN). Apparently, differences in FN have phylogenetic implications and are correlated with geographic regions. A number of hypothetical mechanisms were proposed to explain difference in FN: translocations, heterochromatin, or, most commonly, pericentric inversions. Recently, an additional mechanism, centromere repositioning, was discovered, which can alter chromosome morphology and FN. Here, we used chromosome banding, chromosome painting, and BAC-FISH to test these hypotheses. We demonstrate that centromere repositioning on chromosomes 5 and 15 is the mechanism that accounts for differences in FN. Current phylogenomic trees of platyrrhines provide a temporal framework for evolutionary new centromeres (ENC) in Saimiri. The X-chromosome ENC could be up to 15 million years (my) old that on chromosome 5 as recent as 0.3 my. The chromosome 15 ENC is intermediate, as young as 2.24 my. All ENC have abundant satellite DNAs indicating that the maturation process was fairly rapid. Callithrix jacchus was used as an outgroup for the BAC-FISH data analysis. Comparison with scaffolds from the S. boliviensis genome revealed an error in the last marmoset genome release. Future research including at the sequence level will provide better understanding of chromosome evolution in Saimiri and other platyrrhines. Probably other cases of differences in chromosome morphology and FN, both within and between taxa, will be shown to be due to centromere repositioning and not pericentric inversions.     

Codon usage <Emphasis Type="BoldItalic">vis-a-vis</Emphasis> start and stop codon context analysis of three dicot species

To understand the variation in genomic composition and its effect on codon usage, we performed the comparative analysis of codon usage and nucleotide usage in the genes of three dicots, Glycine max, Arabidopsis thaliana and Medicago truncatula. The dicot genes were found to be A/T rich and have predominantly A-ending and/or T-ending codons. GC3s directly mimic the usage pattern of global GC content. Relative synonymous codon usage analysis suggests that the high usage frequency of A/T over G/C mononucleotide containing codons in AT-rich dicot genome is due to compositional constraint as a factor of codon usage bias. Odds ratio analysis identified the dinucleotides TpG, TpC, GpA, CpA and CpT as over-represented, where, CpG and TpA as under-represented dinucleotides. The results of (NcExp?NcObs)/NcExp plot suggests that selection pressure other than mutation played a significant role in influencing the pattern of codon usage in these dicots. PR2 analysis revealed the significant role of selection pressure on codon usage. Analysis of varience on codon usage at start and stop site showed variation in codon selection in these sites. This study provides evidence that the dicot genes were subjected to compositional selection pressure.     

Codon optimization of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> mating factor alpha prepro-leader to improve recombinant protein production in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To evaluate different codon optimization parameters on the Saccharomyces cerevisiae-derived mating factor α prepro-leader sequence (MFLS) to improve Candida antarctica lipase B (CAL-B) secretory production in Pichia pastoris.

Results

Codon optimization based on the individual codon usage (ICU) and codon context (CC) design parameters enhanced secretory production of CAL-B to 7 U/ml and 12 U/ml, respectively. Only 3 U/ml was obtained with the wild type sequence while the sequence optimized using both ICU and CC objectives showed intermediate performance of 10 U/ml. These results clearly show that CC is the most relevant parameter for the codon optimization of MFLS in P. pastoris, and there is no synergistic effect achieved by considering both ICU and CC together.

Conclusion

The CC optimized MFLS increased secretory protein production of CAL-B in P. pastoris by fourfold.

     

Molecular evidence for hybridization between invasive <Emphasis Type="Italic">Solidago canadensis</Emphasis> and native <Emphasis Type="Italic">S</Emphasis>. <Emphasis Type="Italic">virgaurea</Emphasis>

Hybridization between alien and native species is biologically very important and could lead to genetic erosion of native taxa. Solidago × niederederi was discovered over a century ago in Austria and described by Khek as a natural hybrid between the alien (nowadays regarded also as invasive) S. canadensis and native S. virgaurea. Although interspecific hybridization in the genus Solidago is considered to be relatively common, hybrid nature of S. × niederederi has not been independently proven using molecular tools, to date. Because proper identification of the parentage for the hybrid Solidago individuals solely based on morphological features can be misleading, in this paper we report an additive polymorphism pattern expressed in the ITS sequences obtained from individuals representing S. × niederederi, and confirm the previous hypothesis that the parental species of this hybrid are S. canadensis and S. virgaurea. Additionally, based on variability at the cpDNA rpl32-trnL locus, we showed that in natural populations hybridization occurs in both directions.     

Comparative evolutionary genomics of <Emphasis Type="Italic">Corynebacterium</Emphasis> with special reference to codon and amino acid usage diversities

The present study has been aimed to the comparative analysis of high GC composition containing Corynebacterium genomes and their evolutionary study by exploring codon and amino acid usage patterns. Phylogenetic study by MLSA approach, indel analysis and BLAST matrix differentiated Corynebacterium species in pathogenic and non-pathogenic clusters. Correspondence analysis on synonymous codon usage reveals that, gene length, optimal codon frequencies and tRNA abundance affect the gene expression of Corynebacterium. Most of the optimal codons as well as translationally optimal codons are C ending i.e. RNY (R-purine, N-any nucleotide base, and Y-pyrimidine) and reveal translational selection pressure on codon bias of Corynebacterium. Amino acid usage is affected by hydrophobicity, aromaticity, protein energy cost, etc. Highly expressed genes followed the cost minimization hypothesis and are less diverged at their synonymous positions of codons. Functional analysis of core genes shows significant difference in pathogenic and non-pathogenic Corynebacterium. The study reveals close relationship between non-pathogenic and opportunistic pathogenic Corynebaterium as well as between molecular evolution and survival niches of the organism.     

Early characterization of somatic hybrids from symmetric protoplast electrofusion of <Emphasis Type="Italic">Solanum pinnatisectum</Emphasis> Dun. and <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Variability of 31 somatic hybrids of Solanum pinnatisectum Dun. with Solanum tuberosum L. for leaf morphology, plant vigor, resistance to Phytophthora infestans, ploidy level, and cytoplasm type was evaluated in vitro. The composition of these somatic hybrids was as follows: [S. pinnatisectum Dun. (2n = 2x = 24; cytoplasmic type Wγ) + S. tuberosum L. (2n = 4x = 48; cytoplasmic type Tß)]. Based on leaf morphology and plant growth vigor, plants were divided into three groups, including plants close to tbr parent with unlobed leaves, small plants with scarcely dissected leaves, and vigorous plants with asymmetrically and pinnately lobed leaves. Nine of the somatic hybrids were found to be highly resistant to P. infestans. Somatic hybrids were either tetraploid or hexaploid, with hexaploids being predominant. The cytoplasm of somatic hybrids was either Tßγ or Wßγ, with Tßγ being more common. Overall, in contrast to leaf morphology and growth vigor, level of resistance to P. infestans was not related to either ploidy level or type of cytoplasm. These findings demonstrate that early in vitro selection of promising hybrids can be useful in breeding programs.     

Properties,Microstructure and Heat Seal Ability of Bilayer Films Based on Fish Gelatin and Emulsified Gelatin Films

Bilayer films prepared by the lamination of fish gelatin film (GF) and its corresponding emulsified film (EF) with different thickness ratios (7:3, 5:5 and 3:7) were characterized. Bilayer films had the similar tensile strength (TS) to EF (p > 0.05) but showed lower elongation at break (EAB) (p < 0.05). All bilayer films showed the lower water vapor permeability (WVP) but higher oxygen permeability (OP) than GF. Bilayer films had varying ΔE* (total color different), where the highest value was observed in that laminated with higher thickness ratio of EF (p < 0.05). Lower light transmission and higher transparency value were obtained for bilayer films, compared to GF (p < 0.05). Based on scanning electron microscopic (SEM) cross-section micrographs, all bilayer films consisted of two layers. Differential scanning calorimetric (DSC) analysis revealed that the bilayer films had the higher glass transition temperature (T _g) than GF but lower than EF. All bilayer films were heat sealable, however their seal strength and seal efficiency were lower than those of GF (p < 0.05). Therefore, the thickness ratios of GF and EF had a marked effect on the mechanical and barrier properties as well as heat sealing ability of resulting bilayer films.     

<Emphasis Type="Italic">Braf</Emphasis>, <Emphasis Type="Italic">Kras</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis> epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p < 0.001 as well as in patients with age above 50 years with a p value = 0.008. By applying logistic regression analysis it was reported that the H. pylori qPCR is a significant predictor to the adenocarcinoma with OR = 1.025 (95 % CI: 1. 002–1.048), with sensitivity of 90 % and specificity of 100 %. Adenocarcinoma patients had a significantly higher mean age and levels of H. Pylori, Braf, K-ras, methylated MGMT and methylated MLH1 than those of gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. Conclusion: H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.     

The chloroplast genome sequence from <Emphasis Type="Italic">Eugenia uniflora</Emphasis>, a Myrtaceae from Neotropics

Eugenia uniflora is a plant native to tropical America that holds great ecological and economic importance. The complete chloroplast (cp) genome sequence of Eugenia uniflora, a member of the Neotropical Myrtaceae family, is reported here. The genome is 158,445 bp in length and exhibits a typical quadripartite structure of the large (LSC, 87,459 bp) and small (SSC, 18,318 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 26,334 bp). It contains 111 unique genes, including 77 protein-coding genes, 30 tRNAs and 4 rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Comparison of the entire cp genomes of E. uniflora L. and three other Myrtaceae revealed an expansion of 43 bp in the intergenic spacer located between the IRA/large single-copy (LSC) border and the first gene of LSC region. Simple sequence repeat (SSR) analysis revealed that most SSRs are AT rich, which contribute to the overall AT richness of the cp genome. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the noncoding regions. Phylogenetic analysis among 58 species based on 57 cp genes demonstrated a closer relationship between E. uniflora L. and Syzygium cumini (L). Skeels compared to the Eucalyptus clade in the Myrtaceae family. The complete cp genome sequence of E. uniflora reported here has importance for population genetics, as well as phylogenetic and evolutionary studies in this species and other Myrtaceae species from Neotropical regions.     

Two new complete mitochondrial genomes of <Emphasis Type="Italic">Dorcus</Emphasis> stag beetles (Coleoptera,Lucanidae)

The systematics of Dorcus MacLeay has been a long-standing debate. Mitochondrial genomes were widely used to deeply understand the phylogeny of problematic taxa in virtue of their genetic importance and comprehensiveness. To provide more useful genetic data for resolving the systematic disputation of Dorcus stag beetles. The complete mitochondrial genomes of Dorcus hopei and Dorcus seguyi were obtained using the next generation sequencing. Characteristics of the two genomes are explicated through comparing their genome organization and base composition, protein-coding genes and codon usage, intergenic spacers and non-coding region, transfer and ribosomal RNA genes and control region. Phylogenetic relationships were reconstructed using Maximum likelihood and Bayesian inference analyses based on the concatenated nucleotide sequences of 13 PCGs from 9 stag beetles and 3 scarab beetles. The complete mitogenomes of D. hopei and D. seguyi was 16,026 bp/17,955 bp long, respectively. A tandem repeat with the length of 940 bp was presented in the A+T-rich region in D. hopei. An unexpected non-coding region of 332 bp was located between nad2 and trnW in D. seguyi. The phylogenetic analyses robustly supported that D. hopei formed a branch with the generic type of D. parallelipipedus. Whereas D. seguyi was not covered in the branch of (D. hopei?+?D. parallelipipedus), but was sister to them. The results indicated that D. hopei should be a good member of Dorcus MacLeay. The taxonomic status of D. seguyi remained to be studied furtherly.     

Genetic polymorphisms of patients on stable warfarin maintenance therapy in a Ghanaian population

Background

Warfarin is a widely prescribed anticoagulant with narrow therapeutic window for thromboembolic events. Warfarin displays large individual variability in dose requirements. The purpose of this study is to assess the contribution of patient-specific and genetic risk factors to dose requirements of patients on either high or low warfarin maintenance dose in Ghana. Blood samples were collected from 141 (62 males, 79 females) Ghanaian patients on stable warfarin dose to determine their INR. Influence of patient specific factors and gene variations within VKORC1, CYP2C9 and CYP4F2 were determined in patients on either high or low warfarin maintenance dose.

Results

One hundred and forty-one patients took part in the study with 79 (56%) participants being Female. The median age of the study participants was 48 years [IQR: 34–58]. The median duration for patients to be on warfarin therapy was 24 months [IQR: 10–72]. Majority of the study participants (80.9%, n = 114) did not have any side effects to warfarin. CYP2C9*2 and CYP2C9*3 variant alleles were not detected. VKORC1 variant allele was observed at 6% and CYP4F2 variant allele was observed at 41%. Duration of patients on warfarin therapy was marginally associated with high warfarin dose (adjusted OR = 1.01 [95% CI 1.00–1.02], p = 0.033) while the odds of heterozygous individuals (G/A) for VKORC1 gene to have high warfarin dose compared to persons with homozygous (G/G) (adjusted OR = 0.06 [95% CI 0.01–0.63], p = 0.019). Age, gender, diagnosis, presence of side effects and other medications were not associated with warfarin dose (p = 0.05).

Conclusion

This study provides data on VKORC1 and CYP4F2 variants among an indigenous African population. Duration of patients on warfarin therapy was marginally associated with high warfarin dose. CYP2C9*2 and *3 variants were not detected and may not be the most important genetic factor for warfarin maintenance dose among Ghanaians.

     

<Emphasis Type="SmallCaps">d</Emphasis>-Xylose fermentation,xylitol production and xylanase activities by seven new species of <Emphasis Type="Italic">Sugiyamaella</Emphasis>

Sixteen yeast isolates identified as belonging to the genus Sugiyamaella were studied in relation to D-xylose fermentation, xylitol production, and xylanase activities. The yeasts were recovered from rotting wood and sugarcane bagasse samples in different Brazilian regions. Sequence analyses of the internal transcribed spacer (ITS) region and the D1/D2 domains of large subunit rRNA gene showed that these isolates belong to seven new species. The species are described here as Sugiyamaella ayubii f.a., sp. nov. (UFMG-CM-Y607^T = CBS 14108^T), Sugiyamaella bahiana f.a., sp. nov. (UFMG-CM-Y304^T = CBS 13474^T), Sugiyamaella bonitensis f.a., sp. nov. (UFMG-CM-Y608^T = CBS 14270^T), Sugiyamaella carassensis f.a., sp. nov. (UFMG-CM-Y606^T = CBS 14107^T), Sugiyamaella ligni f.a., sp. nov. (UFMG-CM-Y295^T = CBS 13482^T), Sugiyamaella valenteae f.a., sp. nov. (UFMG-CM-Y609^T = CBS 14109^T) and Sugiyamaella xylolytica f.a., sp. nov. (UFMG-CM-Y348^T = CBS 13493^T). Strains of the described species S. boreocaroliniensis, S. lignohabitans, S. novakii and S. xylanicola, isolated from rotting wood of Brazilian ecosystems, were also compared for traits relevant to xylose metabolism. S. valenteae sp. nov., S. xylolytica sp. nov., S. bahiana sp. nov., S. bonitensis sp. nov., S. boreocarolinensis, S. lignohabitans and S. xylanicola were able to ferment d-xylose to ethanol. Xylitol production was observed for all Sugiyamaella species studied, except for S. ayubii sp. nov. All species studied showed xylanolytic activity, with S. xylanicola, S. lignohabitans and S. valenteae sp. nov. having the highest values. Our results suggest these Sugiyamaella species have good potential for biotechnological applications.     

Conservation genetics and geographic patterns of genetic variation of endangered shrub <Emphasis Type="Italic">Ammopiptanthus</Emphasis> (Fabaceae) in northwestern China

Phylogeographic patterns of Ammopiptanthus in northwestern China were examined with internal transcribed spacer (ITS) and three chloroplast intergenic spacers (trnH–psbA, trnL–trnF, and trnS–trnG). Two ITS genotypes (a–b) and 8 chloroplast haplotypes (A–H) were detected. Both ITS genotypes and chloroplast lineages were split in two geographic regions: western Xinjiang and the Alxa Desert. This lineage split was also supported by AMOVA analysis and the Mantel test. AMOVA showed that 89.81 % of variance in Ammopiptanthus occurred between the two geographic regions, and correlation between genetic distances and geographical distances was significant (r = 0.757, p < 0.0001). All populations in western Xinjiang shared haplotype A with high frequency, and range expansion was strongly supported by negative Fu’s F_S value, and mismatch distribution analysis, whereas populations in the Alxa Desert had higher genetic diversity and structure. We speculate that the cold and dry climate during the early Quaternary fragmented habitats of the species, limiting gene flow between regions, and interglacial periods most likely led to the range expansion in western Xinjiang. The low genetic diversity of Ammopiptanthus indicate a significant extinction risk, and protective measures should be taken immediately.     

Anthropogenic signature in the incidence and distribution of an emerging pathogen of poplars

The introduction and establishment of non-native plant pathogens into new areas can result in severe outbreaks. Septoria leaf spot and canker caused by Sphaerulina musiva is one of the most damaging poplar diseases in northeastern and north-central North America. Stem and branch cankers can be devastating on susceptible trees, leading to tree death and reduced biomass in commercial plantations. In the Pacific Northwest region of North America, the first report of the disease was made in 2006 in the Fraser Valley of British Columbia (BC), Canada. To investigate the incidence and distribution of S. musiva from its point of introduction into BC, five plantations of Populus trichocarpa (black cottonwood), 500 P. trichocarpa trees from natural populations, and 23 plantations of hybrid poplars were surveyed by using real-time PCR assays targeting S. musiva and its native sister species, S. populicola. Our survey suggests a strong anthropogenic signature to the emergence of the non-native S. musiva. Detection frequency of S. musiva was high in hybrid poplar plantations (116 trees infected, 54.2 % of the sampled trees), while detection of the native S. populicola was limited to 13.1 % (22 trees infected). By contrast, in natural stands of P. trichocarpa, less than 2 % of the trees were positive for S. musiva (7 trees) while ~75 % were positive for S. populicola (433 trees). All the S. musiva detections in natural stands of the native P. trichocarpa were from trees located in the vicinity (<2.5 km) of hybrid poplar plantations. Identification of the genotypes found in the hybrid poplar plantations revealed that they are in majority F1 progeny from P. trichocarpa × P. deltoides (T × D) (82 %) and P. nigra × P. maximowiczii (N × M) (7.8 %) crosses, which are generally susceptible (intermediate level of susceptibility between the two parental species) to the canker disease. Our results suggest that the emergence of S. musiva in BC is related to the planting of susceptible hybrid poplars. Even if the disease has not yet established itself in natural poplar populations outside of the Fraser Valley, infected plantations could act as a reservoir that could promote its spread into nearby native P. trichocarpa populations.     

A novel NADPH-dependent reductase of <Emphasis Type="Italic">Sulfobacillus acidophilus</Emphasis> TPY phenol hydroxylase: expression,characterization, and functional analysis

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X ₄ -Cys-X ₂ -Cys-X _29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.