A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2−, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses

Etude de la diversité microbienne des bassins hypersalés anoxiques profonds de la Mer Méditerranée orientale

 Since 1983, the existence of Deep Hypersaline anoxic basins (DHABs) has been revealed in the Eastern Mediterranean basin. These basins which represent extremes environments (anoxic, hypersalty, dark, high hydrostatic pressure)  have  been  under  investigation  and  increasing  interest  the  last  fifteen  years.  The  molecular  approaches revealed  the  presence  of  active  and  uncultivated  microbial  communities  in  the  DHABs  with  two  main  metabolic processes  such  as  methanogenesis  and  sulphate-reduction.  Until  now,  no  representative  strain  of  the  different uncultivated lineages thriving in the DHABs (ex : lineages MSBL, Mediteranean Sea Brine Lake) or involved in the two processes  have  been  yet  cultivated.  The  main  objectives  of  the  thesis  work  were  to  (i)  identify  the  microbial  key players in the processes of methanogenesis and sulphate-reduction, (ii) isolate new microbes adapted to hypersaline conditions and to study their physiology, (iii) to compare the genome and the physiology of the strains belonging to the genus  Methanohalophilus,  isolated  from  three  different  basins  (Thetis,  Kryos,  Tyro),  (iv)  attempt  to  cultivate  and isolate  strains  belonging  to  the  uncultivated  lineages  described  in  the  DHABs.  Our  cultivation  approach  allow  to cultivate and isolate three methanogenic methylotrophic moderately halophile strains belonging to the genus Methanohalophilus from the basins Thetis, Kryos and Tyro. A chemo-taxonomic and genomic characterization of the isolates  revealed  the  capacity  of  the  strains  to  grow  under  in  situ  conditions.  Genome  analysis  revealed  the streamlining reduction (by 10%) of the 3 genomes of our deep sea isolates compared to the terrestrial species of the genus  Methanohalophilus  and  also  an  adaptation  of  the  isolates  to  the  in  situ  conditions.  The  isolate  SLHTYRO represent a new species of the genus for which we propose the name M. profundus strain SLHTYRO. Other microbial isolates belonging to genus Marinobacter, Halomonas, and Halanaerobium obtained could be involved in a syntrophic relationship with archaeal partners of the Methanohalophilus genus in order to produce methylated compounds from betaine which in turn is used as catabolic substrates in the methanogenesis process. A new strain belonging to a new genus affiliated the the Bacteroidetes phylum and phylogenetically close to clones identified in the DHABs has been cultivated and isolated in pure culture. This hard to grow isolate represents the first cultivated members of the diverse uncultivated lineages discovered in the DHABs Depuis  1983,  l’existence  de  bassins  hypersalés  profonds  anoxiques  (DHABs  Deep  Hypersaline  Anoxic Basin) dans la Méditerranée orientale a été révélée. Ces bassins qui représentent un environnement extrême (anoxie, hypersalé, pression hydrostatique, absence de lumière)  font l’objet de nombreuses études microbiologiques qui se sont intensifiées depuis une quinzaine d’années. Les approches moléculaires ont révélé l’existence de communautés microbiennes actives mais encore incultivées (ex : lignées MSBL, Mediteranean Sea Brine Lake) dans ces DHABS, avec notamment des métabolismes microbiens tels que la méthanogénèse et la sulfato-réduction. A ce jour, aucun représentant  cultivé  affilié  à  ces  groupes  d’incultivés  ou  réalisant  les  deux  processus  microbiens  cités  n’ont  été caractérisé.  Les  objectifs  majeurs  de  ce  travail de  thèse  portaient  sur l’identification des  principaux  groupes métaboliques  microbiens  et  particulièrement  les  acteurs  microbiens  impliqués  dans  les  processus  dominants  de méthanogénèse  et  de  sulfato-réduction.  Les  approches  culturales  ont  conduit  à  l’isolement  de  3  souches  de méthanogènes  halophiles  modérées  de  trois  bassins  (Thetis,  Kryos,  Tyro)  affiliées  phylogénétiquement  au  genre Methanohalophilus.  Une  caractérisation  chimio-taxonomique  et  génomique  de  ces  souches  a  été  menée.  Les résultats  ont  démontré  la  capacité  des  souches  isolées  à  se  développer  dans  les  conditions  in  situ  (température, salinité  et  pression).  L’analyse  des  génomes  des  souches  des  bassins  hypersalés  (milieux  profonds  vs milieux  de surface) a révélé d’une part une réduction de 10% de la taille des génomes des souches isolées du milieu profond et d’autre part indique une adaptation des souches aux conditions in situ. L’isolement de microorganismes appartenant notamment    au  genre  Marinobacter,  Halomomas  et  Halanaerobium  permet  de  proposer  un  modèle  d’interaction syntrophique conduisant à la production des composés méthylés nécessaires aux souches du genre Methanohalophilus pour la réaction de méthanogénèse méthylotrophe dans ces bassins. Un nouveau genre bactérien proche de séquences issues des DHABS et appartenant au phylum des Bacteroidetes a été isolé et est maintenu en culture stable. Ce microorganisme très difficile à cultiver représente le premier isolat appartenant à  un des groupes d’incultivés mis en évidence dans les DHABs

Complete genome sequence and whole-genome phylogeny of Kosmotoga pacifica type strain SLHLJ1T from an East Pacific hydrothermal sediment

Kosmotoga pacifica strain SLHLJ1T is a thermophilic chemoorganoheterotrophic bacterium isolated from a deep-sea hydrothermal sediment. It belongs to the physiologically homogeneous Thermotogaceae family. Here, we describe the phenotypic features of K. pacifica together with its genome sequence and annotation. The chromosome has 2,169,170 bp, organized in one contig. A total of 1897 candidate protein-encoding genes and 177 RNA genes were identified. The 16S rRNA gene sequence of this strain is distantly related to sequences of some relatives classified in the same genus (K. olearia 7.02% and K. shengliensis 7.83%), with dissimilarity percentages close to the threshold generally described for genus delineation. Nevertheless, the percentage of conserved proteins (POCP), which is much higher than 50% (around 70%), together with phenotypic features of the isolates, confirm the affiliation all Kosmotoga species described so far to the same genus

Methanocaldococcus lauensis sp. nov., a novel deep-sea hydrothermal vent hyperthermophilic methanogen

Three hyperthermophilic methanogens, designated strain SG7T, strain SG1 and strain SLH, were isolated from the ABE and Tu’i Malila deep-sea hydrothermal vent fields along the Eastern Lau Spreading Center. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strains SG7T, SG1 and SLH were affiliated with the genus Methanocaldococcus within the family Methanocaldococcaceae , order Methanococcales . They shared 95.5–99.48 % 16S rRNA gene sequence similarity to other Methanocaldococcus species and were most closely related to Methanocaldococcus bathoardescens . Cells of strains SG7T, SG1 and SLH were cocci, with a diameter of 1.0–2.2 µm. The three strains grew between 45 and 93 °C (optimum, 80–85 °C), at pH 5.0–7.1 (optimum pH 6.2) and with 10–50 g l−1 NaCl (optimum 20–25 g l−1). Genome analysis revealed the presence of a 5.1 kbp plasmid in strain SG7T. Based on the results of average nucleotide identity and digital DNA–DNA hybridization analyses, we propose that strains SG1 and SG7T are representatives of a novel species, for which the name Methanocaldococcus lauensis sp. nov. is proposed; the type strain is SG7T (=DSM 109608T=JCM 39049T)

Persephonella atlantica sp. nov.: How to adapt to physico-chemical gradients in high temperature hydrothermal habitats

A novel thermophilic, microaerophilic and anaerobic, hydrogen- sulphur- and thiosulphate-oxidising bacterium, designated MO1340T, was isolated from a deep-sea hydrothermal chimney collected from the Lucky Strike hydrothermal vent field on the Mid-Atlantic Ridge. Cells were short, motile rods of 1.4 - 2.2 µm length and 0.5 - 0.8 µm width. Optimal growth was observed for a NaCl concentration of 2.5 % (w/v) at pH 6.5. As for other members of the genus Persephonella, strain MO1340T was strictly chemolithoautotrophic and could oxidise hydrogen, elemental sulphur or thiosulphate using oxygen as electron acceptor. Anaerobic nitrate reduction using hydrogen could also be performed. Each catabolic reaction had a different optimal growth temperature (65 to 75 °C) and an optimal dissolved oxygen concentration (11.4 to 119.7 µM at 70 °C for aerobic reactions) that varied according to the electron donors utilised. These experimental results are consistent with the distribution of these catabolic substrates along the temperature gradient observed in active hydrothermal systems. They strongly suggest that this adaptive strategy could confer a selective advantage for strain MO1340T in the dynamic part of the ecosystem where hot, reduced hydrothermal fluid mixes with cold, oxygenated seawater. Phylogenetic analysis indicated that strain MO1340T was a member of the genus Persephonella within the order Hydrogenothermales as it shared a 16S rRNA gene sequence similarity <95.5 % and ANI respectively 75.66 % with closest described Persephonella (P. hydrogeniphila 29WT). On the basis of the physiological and genomic properties of the new isolate, the name Persephonella atlantica sp. nov. is proposed. The type strain is MO1340T (=UBOCC-M-3359T =JCM 34026T)

Kosmotoga pacifica sp. nov., a thermophilic chemoorganoheterotrophic bacterium isolated from an East Pacific hydrothermal sediment

A novel strictly anaerobic thermophilic heterotrophic bacterium, strain SLHLJ1T, was isolated from a Pacific hydrothermal sediment. Cells were Gram-negative coccobacilli (approximately 1.0 × 0.6 μm) with a toga. It grew at temperatures between 33 and 78 °C (optimum 70 °C). Elemental sulphur and l-cystine stimulated its growth. It contained C16:0, C16:1 ω11c, C18:0 and C18:1 ω9c as major fatty acids (>5 %), 3 phospholipids and 2 glycolipids as polar lipids. Its DNA G+C content was 43.7 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain SLHLJ1T within the family Thermotogaceae. The novel isolate was most closely related to Kosmotoga arenicorallina (97.93 % 16S rRNA gene sequence similarity), K. olearia (92.43 %) and K. shengliensis (92.17 %). On the basis of phenotypic, chemotaxonomic and phylogenetic comparisons with its closest relatives, we propose its assignment to a novel species of the genus Kosmotoga. The name Kosmotoga pacifica sp. nov. is proposed with strain SLHLJ1T (=DSM 26965T = JCM 19180T = UBOCC 3254T) as the type species

Methanohalophilus profundi sp. nov., a methylotrophic halophilic piezophilic methanogen isolated from a deep hypersaline anoxic basin

A novel anaerobic methylotrophic halophilic methanogen strain SLHTYROT was isolated from a deep hypersaline anoxic basin called “Tyro” located in the Eastern Mediterranean Sea. Cells of SLHTYROT were motile cocci. The strain SLHTYROT grew between 12 and 37 °C (optimum 30 °C), at pH between 6.5 and 8.2 (optimum pH 7.5) and salinity from 45 to 240 g L−1 NaCl (optimum 135 g L−1). Strain SLHTYROT was methylotrophic methanogen able to use methylated compounds (trimethylamine, dimethylamine, monomethylamine and methanol). Strain SLHTYROT was able to grow at in situ hydrostatic pressure and temperature conditions (35 MPa, 14 °C). Phylogenetic analysis based on 16S rRNA gene and mcrA gene sequences indicated that strain SLHTYROT was affiliated to genus Methanohalophilus within the order Methanosarcinales. It shared >99.16% of the 16S rRNA gene sequence similarity with strains of other Methanohalophilus species. Based on ANIb, AAI and dDDH measurements, and the physiological properties of the novel isolate, we propose that strain SLHTYROT should be classified as a representative of a novel species, for which the name Methanohalophilus profundi sp. nov. is proposed; the type strain is SLHTYROT (=DSM 108854 = JCM 32768 = UBOCC-M-3308)

Thermococcus piezophilus sp. nov., a novel hyperthermophilic and piezophilic archaeon with a broad pressure range for growth, isolated from a deepest hydrothermal vent at the Mid-Cayman Rise

A novel strictly anaerobic, hyperthermophilic archaeon, designated strain CDGST, was isolated from a deep-sea hydrothermal vent in the Cayman Trough at 4964 m water depth. The novel isolate is obligate anaerobe and grows chemoorganoheterotrophically with stimulation of growth by sulphur containing compounds. Its growth is optimal at 75 °C, pH 6.0 and under a pressure of 50 MPa. It possesses the broadest hydrostatic pressure range for growth that has ever been described for a microorganism. Its genomic DNA G + C content is 51.11 mol%. The novel isolate belongs to the genus Thermococcus. Phylogenetic analyses indicated that it is most closely related to Thermococcus barossii DSM17882T based on its 16S rRNA gene sequence, and to ‘Thermococcus onnurineus’ NA1 based on its whole genome sequence. The average nucleotide identity scores with these strains are 77.66% for T. barossii and 84.84% for ‘T. onnurineus’, respectively. Based on the draft whole genome sequence and phenotypic characteristics, strain CDGST is suggested to be separated into a novel species within the genus Thermococcus, with proposed name Thermococcus piezophilus (type strain CDGST = ATCC TSD-33T = UBOCC 3296T)

Proposed minimal standards for description of methanogenic archaea

Methanogenic archaea are a diverse, polyphyletic group of strictly anaerobic prokaryotes capable of producing methane as their primary metabolic product. It has been over three decades since minimal standards for their taxonomic description have been proposed. In light of advancements in technology and amendments in systematic microbiology, revision of the older criteria for taxonomic description is essential. Most of the previously recommended minimum standards regarding phenotypic characterization of pure cultures are maintained. Electron microscopy and chemotaxonomic methods like whole-cell protein and lipid analysis are desirable but not required. Because of advancements in DNA sequencing technologies, obtaining a complete or draft whole genome sequence for type strains and its deposition in a public database are now mandatory. Genomic data should be used for rigorous comparison to close relatives using overall genome related indices such as average nucleotide identity and digital DNA–DNA hybridization. Phylogenetic analysis of the 16S rRNA gene is also required and can be supplemented by phylogenies of the mcrA gene and phylogenomic analysis using multiple conserved, single-copy marker genes. Additionally, it is now established that culture purity is not essential for studying prokaryotes, and description of Candidatus methanogenic taxa using single-cell or metagenomics along with other appropriate criteria is a viable alternative. The revisions to the minimal criteria proposed here by the members of the Subcommittee on the Taxonomy of Methanogenic Archaea of the International Committee on Systematics of Prokaryotes should allow for rigorous yet practical taxonomic description of these important and diverse microbes