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.     

Development of <Emphasis Type="Italic">mazF</Emphasis>-based markerless genome editing system and metabolic pathway engineering in <Emphasis Type="Italic">Candida tropicalis</Emphasis> for producing long-chain dicarboxylic acids

Candida tropicalis can grow with alkanes or plant oils as the sole carbon source, and its industrial application thus has great potential. However, the choice of a suitable genetic operating system can effectively increase the speed of metabolic engineering. MazF functions as an mRNA interferase that preferentially cleaves single-stranded mRNAs at ACA sequences to inhibit protein synthesis, leading to cell growth arrest. Here, we constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C. tropicalis genes to increase the rate of conversion of oils into long-chain dicarboxylic acids. To reduce the β-oxidation of fatty acids, the carnitine acetyltransferase gene (CART) was deleted using the gene editing system, and the yield of long-chain acids from the strain was increased to 8.27 g/L. By two homologous single exchanges, the promoters of both the cytochrome P450 gene and the NADPH–cytochrome P450 reductase gene were subsequently replaced by the constitutively expressed promoter pGAP, and the production of long-chain dicarboxylic acids by the generated strain (C. tropicalis PJPP1702) reached 11.39 g/L. The results of fed-batch fermentation showed that the yield of long-chain acids from the strain was further increased to 32.84 g/L, which was 11.4 times higher than that from the original strain. The results also showed that the pPICPJ-mazF-based markerless editing system may be more suited for completing the genetic editing of C. tropicalis.     

Molecular cloning and functional characterization of a cinnamate 4-hydroxylase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Cinnamate 4-hydroxylase (C4H) catalyzes the regioselective para-hydroxylation of trans-cinnamic acid to form p-coumaric acid, the biosynthetic precursor of phenylpropanoid-based polymers. These biopolymers play an essential role in plant structure construction, development, and defense. Herein the open reading frame of CaC4H2 was cloned from Camptotheca acuminata, a deciduous camptothecin-producing tree native to China. CaC4H2 showed 94 % amino acid residues identity with those of reported CaC4H, which suggested that CaC4H2 is an isoform of C4Hs presented in C. acuminata. The intact CaC4H2 was overexpressed in Escherichia coli with its functional reaction partner cytochrome P450 reductase, CamCPR, which transfers electrons from NADPH to CaC4H2 to support the catalytic hydroxylation activity of CaC4H2. Upon incubating trans-cinnamic acid with the recombinant CaC4H2 and tCamCPR, the formation of p-coumaric acid was confirmed by the HPLC–DAD and UPLC-DAD-ESIMS analyses, which indicated the catalytic hydroxylation activity of CaC4H2. Quantitative real-time PCR analyses showed that CaC4H2 was expressed in all tissues of C. acuminata seedlings, which is consistent with the well-known conclusion that the C4H-catalyzed hydroxylation reaction is a key step within the biosynthetic pathway of phenylpropanoids. The functional characterization of CaC4H2 will be useful for molecular breeding and sustainable utilization and protection of the camptothecin-producing plant.     

Cloning and expression of a cytochrome P450 hydroxylase gene from <Emphasis Type="Italic">Amycolatopsis orientalis</Emphasis>: hydroxylation of epothilone B for the production of epothilone F

Degenerate PCR primers were used to amplify cytochrome P450 gene fragments from the high-GC gram-negative bacteria Amycolatopsis orientalis, which catalyzes the hydroxylation of epothilone B to produce epothilone F. The amplified fragments were used as hybridization probes to identify and clone two intact cytochrome P450 genes. The expression of one of the cloned genes in a Streptomyces lividans transformant resulted in the biotransformation of epothilone B to epothilone F. The conversion of epothilone B to epothilone F by the S. lividans transformant was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy.     

Chromosome number variability in central european members of the<Emphasis Type="Italic">Festuca ovina</Emphasis> and<Emphasis Type="Italic">F. pallens</Emphasis> groups (sect.<Emphasis Type="Italic">Festuca</Emphasis>)

Chromosome numbers for 98 plants ofF. pallens, 19 ofF. psammophila, F. belensis andF. vaginata, and 44 ofF. ovina (originating from Austria, the Czech Republic, Germany, Slovakia and Latvia) are given. In addition to theF. ovina andF. pallens groups, chromosome counts for the following taxa are also reported:F. alpestris (2n=14) reported for the first time in this work,F. amethystina subsp.amethystina (2n=28),F. brevipila (2n=42),F. cinerea (2n=28),F. rupicola subsp.rupicola (2n=42) andF. versicolor subsp.versicolor (2n=14).InF. pallens, two ploidy levels (2n=2x=14+0-1B, 2n=4x=28+0-1B) as well as two natural triploid plants (2n=21+0-1B), were found. In addition to the fourF. pallens types that have been distinguished in Austria, one new tetraploid type (F. pallens “scabrifolia”) from the Czech Republic and Germany is reported and its taxonomy is discussed. The distributions of the Oberösterreich-Niederösterreich and Pannonisches-HügellandF. pallens types outside of Austria are documented.Only the diploid chromosome number (2n=14) was found inF. psammophila andF. vaginata. Chromosome numbers forF. psammophila subsp.muellerstollii andF. belensis (both 2n=14) were determined here for the first time. Two ploidy levels, 2n=14+0-5B corresponding toF. ovina subsp.ovina and 2n=28 corresponding toF. ovina subsp.guestphalica andF. cf.duernsteinensis were confirmed inF. ovina. Differences in chromosome structure (simple and multiple secondary constrictions) betweenF. pallens as opposed toF. psammophila andF. vaginata are discussed. A complete survey of published chromosome counts for Central European species from theF. ovina andF. pallens groups is included.     

Development and relations of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> and <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> in soil

The development of Fusarium culmorum and Pseudomonas fluorescens in soil, and the relations between them, were studied using membrane filters containing the fungus, the bacterium, or both microorganisms; the filters were incubated in soil. F. culmorum was identified by indirect immunofluorescence; the GUS-labeled strain was used to visualize P. fluorescens. It was found that F. culmorum introduced in soil can develop as a saprotroph, with the formation of mycelium, macroconidia, and a small amount of chlamydospores. Introduction of glucose and cellulose resulted in increased density of the F. culmorum mycelium and macroconidia. P. fluorescens suppressed the development of the F. culmorum mycelium in soil, but stimulated chlamydospore formation. Decreased mycelial density in the presence of P. fluorescens was more pronounced in soil without additions and less pronounced in the case of introduction of glucose or cellulose. F. culmorum had no effect on P. fluorescens growth in soil.     

Diversity and antifungal activity of endophytic bacteria associated with <Emphasis Type="Italic">Panax ginseng</Emphasis> seedlings

Ginseng (Panax ginseng C.A. Meyer) is a medicinal crop that requires a long culture time before it is ready to harvest, thus generating high economic and environmental costs. Symbiotic bacteria that live within the plant provide the host plant with many advantages in terms of metabolism and disease resistance. Here, we isolated endophytic bacteria from various tissues of P. ginseng seedlings using a culture-dependent method and we compared their tissue distribution. In addition, their antimicrobial activity against two fungal pathogens was investigated. Based on 16S rRNA sequencing, we identified 21 bacterial strains from ginseng seedlings. Leaves and rhizomes showed higher bacterial species diversity than root bodies and tails. While Bacillus strains were detected in all tissues, Xanthomonas and Micrococcaceae strains were specifically isolated from rhizome and leaf tissues, respectively. Fourteen bacterial strains showed antimicrobial activities against Cylindrocarpon destructans and/or Botrytis cinerea, with different activities. Among them, two strains (PgKB29 and PgKB35) showed strong antimicrobial activities against both fungi. Taken together, these results provide a better understanding of endophytic bacteria in P. ginseng seedlings and suggest the possibility of biological control of fungal pathogens using endophytic bacteria.     

An overlooked hybrid between the two diploid <Emphasis Type="Italic">Chenopodium</Emphasis> species in Central Europe determined by microsatellite and morphological analysis

The presence and extent of hybridization within the Chenopodium album aggregate (Amaranthaceae) is still unclear. Although many hybrid combinations have been described, their existence in the field has never been systematically studied and verified. The main aim of this study was to ascertain the extent of interspecific hybridization between the diploid species C. ficifolium and C. suecicum using highly variable nuclear microsatellite markers. Due to the absence of such kind of molecular markers for the whole C. album group, we divided the analysis into two steps: (1) Eleven microsatellite loci designed for the closely related species C. quinoa were cross-amplified in five Eurasian species of the C. album diploid–polyploid complex, i.e. C. album s.s. (6x), C. striatiforme (4x), C. strictum (4x), C. ficifolium (2x) and C. suecicum (2x); (2) For the detection of interspecific hybridization between C. ficifolium and C. suecicum, we sampled 480 individuals from five localities in Central Europe. We also investigated morphological differences between the parental taxa and their hybrid and devised a key for their determination. Analysis of variation in microsatellite loci using Bayesian methods, PCoA and Neighbour-joining tree identified 32 F1 hybrids. These F1 hybrids, described here as C. paradoxum Mandák, formed a cluster between well-differentiated parental species, combining the morphological characters of both their parents. Moreover, genetic analyses also recognized several F2 or backcross hybrids, whose delimitation, mainly from C. suecicum and F1 hybrids, based on morphological characters, is problematic.     

Reduction of soil-borne pathogen <Emphasis Type="Italic">Fusarium solani</Emphasis> reproduction in soil enriched with phenolic acids by inoculation of endophytic fungus <Emphasis Type="Italic">Phomopsis liquidambari</Emphasis>

The increase of soil-borne pathogens induced by phenolic acids that accumulate in continuous cropping soil reduces the yield and quality of crops. The aims of this study were to investigate (i) the biological control of Fusarium solani, in soil enriched with phenolic acids, by the inoculation of the endophytic fungus Phomopsis liquidambari, and (ii) the biocontrol mechanisms involved. Inoculation of P. liquidambari significantly inhibited the reproduction of F. solani. The prompt degradation of soil phenolic acids by P. liquidambari was determined, but no direct antagonism relationship was observed between P. liquidambari and F. solani, implying the alleviated stimulation of phenolic acids was a major factor in controlling F. solani. Moreover, the presence of glucose did not significantly impact the biocontrol function of P. liquidambari, and P. liquidambari inoculation significantly alleviated disease severity of peanut. Therefore, P. liquidambari could be an effective means to control F. solani in phenolic acids-rich continuous cropping soils.     

Sequencing of the plastome in the leafless green mycoheterotroph <Emphasis Type="Italic">Cymbidium macrorhizon</Emphasis> helps us to understand an early stage of fully mycoheterotrophic plastome structure

To date, plastome studies of mycoheterotrophic orchids have focused on nongreen mycoheterotrophic or partially mycoheterotrophic species. Cymbidium macrorhizon is a fully mycoheterotrophic orchid that lacks leaves and roots, although its inflorescence rachis is pale green. It has degraded stomata, specific fungal partners, and high concentrations of heavy stable nitrogen and carbon isotopes. Therefore, the plastome of this species is expected to represent an early stage of a fully mycoheterotrophic plastome. In this study, we sequenced the plastomes of C. macrorhizon and closely related species (C. ensifolium, C. kanran, and C. lancifolium). Plastomes of the four Cymbidium species were almost identical structurally, but differed somewhat from those of previously studied species. The genes for the photosynthetic subunits of NADH dehydrogenase, ndhF and ndhH, were absent from all four newly sequenced plastomes, whereas only ndhJ was absent from C. ensifolium. In section Pachyrhizanthe (C. lancifolium and C. macrorhizon), ndhE, ndhI, and ndhJ were pseudogenized. With the exception of ndh and ycf, 64 protein-coding genes in C. macrorhizon were apparently functional. Most of them were highly conserved and under purifying selection. Therefore, no direct evidence is available to suggest that genes related to photosynthesis have lost their functions in C. macrorhizon. This discordance between molecular and physiological features for the trophic status of C. macrorhizon might result from a lag between photosynthetic function loss and relaxed purifying selection.     

Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> GS1

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450_cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads’ superior resilience compared to E. coli. Whole-cell P. putida harboring P450_cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450_cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield Y_P/S of 79 %. To the authors’ knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.     

Regulating the regulators: responses of four plant growth regulators during clonal propagation of <Emphasis Type="Italic">Lachenalia montana</Emphasis>

Geraniol, the precursor of terpenoid indole alkaloids can be converted to the 10-hydroxy geraniol by the function of geraniol 10-hydroxylase. In our study, for the first time, a full-length cytochrome P450 monooxygenase (P450) geraniol 10-hydroxylase (PgCYP76C9) cDNA was isolated and characterized from Panax ginseng Meyer. The gene has an open reading frame (ORF) of 1503 base pairs and encodes a precursor protein of 501 amino acids residues. The calculated molecular mass of the protein is approximately 56.3 kDa with a predicated isoelectric point of 8.45. Amino acid identities between PgG10H and other P450s of the CYP76 family in the database had revealed that the deduced amino acid of PgG10H sharing a higher sequence homology with geraniol 10-hydroxylase-like proteins encoded by Cinchona calisaya and Lonicera japonica. We implemented a molecular modeling method to evaluate the possible interaction of geraniol with PgG10H active site. Our finding showed that the geraniol was the potential ligand for PgG10H in P. ginseng. Expression of PgG10H gene was tissue-regulated and showed high expression in 3-year-old ginseng flowers and roots. Expression of PgG10H was differentially induced in ginseng, not only during Pseudomonas syringae infection and wounding but also after exposure to methyl jasmonate and salt stress. Furthermore, overexpression of the newly identified ginseng geraniol 10-hydroxylase P450 gene in Arabidopsis caused terpenoid indole alkaloid dihydrositsirikine production and also conferred enhanced resistance to P. syringae.     

Reassessment of hydrogen tolerance in <Emphasis Type="Italic">Caldicellulosiruptor saccharolyticus</Emphasis>

Background

Caldicellulosiruptor saccharolyticus has the ability to produce hydrogen (H₂) at high yields from a wide spectrum of carbon sources, and has therefore gained industrial interest. For a cost-effective biohydrogen process, the ability of an organism to tolerate high partial pressures of H₂ (P_H2) is a critical aspect to eliminate the need for continuous stripping of the produced H₂ from the bioreactor.

Results

Herein, we demonstrate that, under given conditions, growth and H₂ production in C. saccharolyticus can be sustained at P_H2 up to 67 kPa in a chemostat. At this P_H2, 38% and 16% of the pyruvate flux was redirected to lactate and ethanol, respectively, to maintain a relatively low cytosolic NADH/NAD ratio (0.12 mol/mol). To investigate the effect of the redox ratio on the glycolytic flux, a kinetic model describing the activity of the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was developed. Indeed, at NADH/NAD ratios of 0.12 mol/mol (K i of NADH = 0.03 ± 0.01 mM) GAPDH activity was inhibited by only 50% allowing still a high glycolytic flux (3.2 ± 0.4 mM/h). Even at high NADH/NAD ratios up to 1 mol/mol the enzyme was not completely inhibited. During batch cultivations, hydrogen tolerance of C. saccharolyticus was dependent on the growth phase of the organism as well as the carbon and energy source used. The obtained results were analyzed, based on thermodynamic and enzyme kinetic considerations, to gain insight in the mechanism underlying the unique ability of C. saccharolyticus to grow and produce H₂ under relatively high P_H2.

Conclusion

C. saccharolyticus is able to grow and produce hydrogen at high P_H2, hence eliminating the need of gas sparging in its cultures. Under this condition, it has a unique ability to fine tune its metabolism by maintaining the glycolytic flux through regulating GAPDH activity and redistribution of pyruvate flux. Concerning the later, xylose-rich feedstock should be preferred over the sucrose-rich one for better H₂ yield.

     

Molecular analyses of <Emphasis Type="Italic">Ficus erecta</Emphasis> and its allies within the subsection <Emphasis Type="Italic">Frutescentiae</Emphasis> (Moraceae)

Ficus (Moraceae) is a keystone group in tropical and subtropical forests with remarkable diversity of species and taxonomical challenges as a consequence of fig–pollinator coevolution. Ficus subsect. Frutescentiae includes about 30 species that are predominantly shrubs or small trees with Terminalia branching. Many of these species are difficult to delimit morphologically, and the group includes a tangle of uncertain taxa and incorrectly applied names. We conducted a phylogenetic analysis with internal and external transcribed spacer data (ITS and ETS) and data from 18 polymorphic microsatellite loci to evaluate the species status of the most perplexing members of this subsection. The results confirm the monophyly of subsect. Frutescentiae, with F. pedunculosa as sister to the rest. The F. erecta complex comprises approximately 17 taxa: F. erecta, F. abelii, F. boninsimae, F. nishimurae, F. iidaiana, F. gasparriniana var. laceratifolia, F. gasparriniana var. viridescens, F. pyriformis, F. stenophylla, F. fusuiensis, F. fengkaiensis, F. sinociliata, F. tannoensis, F. vaccinioides, F. formosana, F. pandurata, and F. periptera. The last five of these were supported as good species, while the others were not well supported by the present evidence. Evidence also supported the status of the non-F. erecta complex species including. F. pedunculosa, F. ischnopoda, F. heteromorpha, and F. variolosa. Ficus filicauda and F. neriifolia are possibly conspecific. The species status of F. potingensis should be restored and it should be treated as a member of section Eriosycea. Identification of the remaining taxa (F. gasparriniana var. esquirolii, F. ruyuanensis, F. daimingshanensis, F. chapaensis, F. changii, F. trivia, and F. tuphapensis) and their relationships to the F. erecta complex were not clarified. As a whole, only ten species in this subsection are confirmed, one is excluded, one is synonymous, and the others are either unresolved or short of samples. There appears to be a consistent genetic background among these unresolved groups, which suggests that repeated hybridization (as a result of pollinator host shifts) has filled up the interspecific gaps during the fig–pollinator coevolution process.     

Expression on roots and contribution to maize phytostimulation of 1-aminocyclopropane-1-decarboxylate deaminase gene <Emphasis Type="Italic">acdS</Emphasis> in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> F113

Aims

The plant-beneficial bacterium Pseudomonas fluorescens F113 harbours an acdS gene, which enables deamination of 1-aminocyclopropane-1-carboxylate. The impact of abiotic and biotic factors on the expression of this gene was assessed, as well as the plant-beneficial properties of F113 under different soil moistures.

Methods

An acdS-egfp biosensor was constructed in F113, validated in vitro and used to analyse, by microscopy, its expression on roots of Zea mays comparatively to Beta vulgaris. An acdS mutant was constructed and compared with the wild-type to characterize plant-beneficial effects of F113 on maize lines EP1 and FV2, under well-watered and water deficit conditions.

Results

Different patterns of root colonization and acdS expression were observed according to plant genotype. acdS rhizoplane expression was higher on Beta vulgaris, and on maize line FV2 and hybrid PR37Y15 than on maize line EP1 and teosinte. Strain F113 but not its acdS mutant promoted root growth of EP1 under well-watered conditions and germination of FV2 under water deficit conditions.

Conclusions

Maize lines differed in their ability to induce acdS expression and to respond to P. fluorescens F113. The maize line leading to higher acdS expression, FV2, was the one benefiting from inoculation under water deficit.

     

An NADPH-dependent <Emphasis Type="Italic">Lactobacillus composti</Emphasis> short-chain dehydrogenase/reductase: characterization and application to (<Emphasis Type="Italic">R</Emphasis>)-1-phenylethanol synthesis

A new anti-Prelog short-chain dehydrogenase/reductase (SDR) encoding gene lcsdr was cloned from Lactobacillus composti DSM 18527, and heterologously expressed in Escherichia coli. LcSDR is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and has a molecular weight of approximately 30 kDa. The optimal pH and temperature were 6.5 and 30?°C, respectively. The maximal reaction rate V_max was 133.9 U mg^?1; the Michaelis–Menten constant K _m of LcSDR were 0.345 mM for acetophenone (1a), and 0.085 mM for NADPH. Through introducing an EsGDH-catalyzed NADPH regeneration system, a biocatalytic process for (R)-1-phenylethanol ((R)-1b) was developed with outstanding time–space yield. Under the optimized conditions, 50 g l^?1 1a was converted to (R)-1b in 2 h with a yield of 93.8%, enantiomeric excess of product (e.e._p) above 99% and space–time yield of 562.8 g l^?1 d^?1.     

Genetic variability of the banded murex (<Emphasis Type="Italic">Hexaplex trunculus</Emphasis>) revealed by <Emphasis Type="Italic">ND2</Emphasis> and <Emphasis Type="Italic">ITS2</Emphasis> sequences

The gastropod Hexaplex trunculus is widely distributed in a relatively large range of habitats, but has no dispersal stage. We investigated its genetic structure across its distribution range, from Mediterranean Sea to adjacent Atlantic coasts, by sequencing mitochondrial DNA portions of the NADH dehydrogenase gene ND2 (420 pb) and the internal transcribed spacer ITS2 (450 pb). Our results suggested a significant genetic variability of ND2 (π = 0.009 and Hd = 0.629) and low variability of the ITS2 sequences. A strong phylogeographic break, separated the Aegean populations from those of Western/Eastern Mediterranean and the Atlantic ones, was founded. The tow lineages may have been separated by vicariance events due to the Peloponnese break that separates the Aegean populations from other populations and was maintained until now by the quasi-circular anticyclonic front associated to the straits of Cretan Arc of the Peloponnesian Peninsula. Tunisian coasts appear particularly diverse since the two divergent lineages co-occured. These results may have management consequences since H. trunculus is a high commercial value harvested species.     

The NADPH oxidase AoNoxA in <Emphasis Type="Italic">Arthrobotrys oligospora</Emphasis> functions as an initial factor in the infection of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Reactive oxygen species (ROS) produced by NADPH oxidases can serve as signaling molecules to regulate a variety of physiological processes in multi-cellular organisms. In the nematophagous fungus Arthrobotrys oligospora, we found that ROS were produced during conidial germination, hyphal extension, and trap formation in the presence of nematodes. Generation of an AoNoxA knockout strain demonstrated the crucial role of NADPH oxidase in the production of ROS in A. oligospora, with trap formation impaired in the AoNoxA mutant, even in the presence of the nematode host. In addition, the expression of virulence factor serine protease P186 was up-regulated in the wild-type strain, but not in the mutant strain, in the presence of Caenorhabditis elegans. These results indicate that ROS derived from AoNoxA are essential for full virulence of A. oligospora in nematodes.     

Enhanced succinic acid production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> with increasing the available NADH supply and glucose consumption rate by decreasing H<Superscript>+</Superscript>-ATPase activity

Objectives

To enhance succinic acid production in Corynebacterium glutamicum by increasing the supply of NADH and the rate of glucose consumption by decreasing H⁺-ATPase activity.

Results

A mutant of C. glutamicum NC-3-1 with decreased H⁺-ATPase activity was constructed. This increased the rate of glycolysis and the supply of NADH. Fermentation of C. glutamicum NC-3-1 gave 39 % higher succinic acid production (113 and 81 g/l), a 29 % higher succinic acid yield (0.94 and 0.73 g succinic acid/g glucose) and decreased by-products formation compared to that of C. glutamicum NC-3 in 5 l bioreactor.

Conclusion

The point mutation in C. glutamicum NC-3-1 increased the rate of glycolysis and resulted in higher succinic acid production, higher succinic acid yield and significantly decreased formation of by-products.

     

Isolation of <Emphasis Type="Italic">Campylobacter</Emphasis> spp. from Three Species of Antarctic Penguins in Different Geographic Locations

The presence of Campylobacter species was studied in three Antarctic penguin species, Adélie (Pygoscelis adeliae), chinstrap (Pygoscelis antarctica) and gentoo (Pygoscelis papua). A total of 390 penguins were captured in 12 different rookeries along the Antarctic Peninsula with differences in the amount of human visitation: six colonies were highly visited [Stranger Point, King George Island (P. papua and P. adeliae); Hannah Point, Livingston Island (P. papua and P. antarctica); Deception Island (P. antarctica); and Paradise Bay, Antarctic Peninsula (P. papua)], and six colonies were rarely visited [Devil’s Point, Byers Peninsula, Livingston Island (P. papua); Cierva Cove, Antarctic Peninsula (P. papua); Rongé Island (P. papua and P. antarctica); Yalour Island (P. adeliae); and Avian Island (P. adeliae)]. A total of 23 strains were isolated from penguins from nine different rookeries. Campylobacter lari subsp. lari was isolated from eight samples (seven from P. papua and one from P. adeliae); C. lari subsp. concheus from 13 (ten from P. adeliae and three from P. antarctica) and C. volucris from two samples (both from P. papua). We did not find any significant differences in the prevalence of Campylobacter spp. between the populations in highly and rarely visited areas. This is the first report of C. lari subsp. concheus and C. volucris isolation from penguins in the Antarctic region.