Publications

4526

Crotalaria spectabilis Roth. (Fabaceae), commonly known as showy rattlebox, is an herbaceous legume mainly used as a green manure crop to improve soil properties and as a source of durable fiber. However, the plant is toxic to mammals and birds because of the presence of pyrrolidizine alkaloids. A native of India and the Malay Peninsula, the species has been introduced into other areas such as the United States and Pacific Islands where the plant is an invader of cultivated lands. Fasciated rattlebox plants were sighted in fields in New Delhi in February 2010, with approximately 99% of the examined plants symptomatic. Symptoms included flattening of stems in a descending gradient of severity from the apex to the base of each affected branch. Shoots showed longitudinal undulations bearing highly reduced leaves and uneven distribution of flowers and fruits. To identify the causal agent, 10 symptomatic plants and 8 asymptomatic plants (latter sampled from a field approximately 1.5 km away) were collected for nucleic acid analysis. Total genomic DNA was extracted from flattened stems as well as the roots of symptomatic plants, and from the same tissues of asymptomatic plants, and subjected to nested-PCR using phytoplasma 16S ribosomal DNA universal primer pair P1/P7, followed by R16F2/R2 (4). A known aster yellows-infected Catharanthus roseus plant was used as a control sample. Results depicted a characteristic phytoplasma amplicon of 1.25 kb in all samples from symptomatic plants and the control plant. No amplification was observed from asymptomatic plants. To obtain a full-length sequence, a representative amplicon was purified with the QIAquick gel extraction kit (QIAGEN, Valencia, CA), cloned into pGEM-T Easy vector (Promega, Madison, WI), and sequenced. Comparison of the 1,243-bp sequence (Genbank Accession No. HM137557) using BLAST analysis of the NCBI database showed 99% homology with sequences of members of 16SrI group phytoplasmas, i.e., ‘Candidatus Phytoplasma asteris’ such as Japanese spurge yellows (AB551736.1), Mulberry yellow dwarf (GQ249410.1), and Bamboo witches'-broom (FJ853161.1) phytoplasmas. The profiles of in vitro restriction fragment length polymorphism (RFLP) analysis obtained by digestion of the nested-PCR products with HhaI, KpnI, and AluI were similar to those of in silico RFLP analyses and coincided with the pattern of the 16SrI-B subgroup. Phylogenetic analysis of phytoplasma 16S rDNA sequences based on the maximum likelihood method using MEGA Version 4.1 also placed the Crotalaria fasciation (CF) phytoplasma within the 16SrI-B cluster. In India, C. tetragona plants infected with 16SrI phytoplasma (FJ185141) causing witches'-broom symptoms (1) showed 98% similarity with the CF phytoplasma. However, the results support previous molecular investigations associating 16SrI phytoplasma with fasciation of herbaceous plants, including Lilium spp. (2) and Asparagus officinalis (3). To our knowledge, C. spectabilis represents a new host that can be fasciated as a result of phytoplasma infection. Because of the weedy nature of C. spectabilis, this host could facilitate spread of the phytoplasma. References: (1) P. Baiswar et al. Plant Pathol. 19:17, 2009. (2) A. Bertaccini et al. FEMS Microbiol. Lett. 249:79, 2005. (3) J. Franova and K. Petrzik. J. Phytopathol. 158:317, 2010.K. (4) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996.

Summary ‘ Candidatus Magnetobacterium bavaricum’ is unusual among magnetotactic bacteria (MTB) in terms of cell size (8–10 µm long, 1.5–2 µm in diameter), cell architecture, magnetotactic behaviour and its distinct phylogenetic position in the deep‐branching Nitrospira phylum. In the present study, improved magnetic enrichment techniques permitted high‐resolution scanning electron microscopy and energy dispersive X‐ray analysis, which revealed the intracellular organization of the magnetosome chains. Sulfur globule accumulation in the cytoplasm point towards a sulfur‐oxidizing metabolism of ‘ Candidatus M. bavaricum’. Detailed analysis of ‘ Candidatus M. bavaricum’ microhabitats revealed more complex distribution patterns than previously reported, with cells predominantly found in low oxygen concentration. No correlation to other geochemical parameters could be observed. In addition, the analysis of a metagenomic fosmid library revealed a 34 kb genomic fragment, which contains 33 genes, among them the complete rRNA gene operon of ‘ Candidatus M. bavaricum’ as well as a gene encoding a putative type IV RubisCO large subunit.

Two novel thermophilic and slightly acidophilic strains, Kam940Tand Kam1507b, which shared 99 % 16S rRNA gene sequence identity, were isolated from terrestrial hot springs of the Uzon caldera on the Kamchatka peninsula. Cells of both strains were non-motile, regular cocci. Growth was observed between 55 and 85 °C, with an optimum at 65–70 °C (doubling time, 6.1 h), and at pH 4.5–7.5, with optimum growth at pH 5.5–6.0. The isolates were strictly anaerobic organotrophs and grew on a narrow spectrum of energy-rich substrates, such as beef extract, gelatin, peptone, pyruvate, sucrose and yeast extract, with yields above 107cells ml−1. Sulfate, sulfite, thiosulfate and nitrate added as potential electron acceptors did not stimulate growth when tested with peptone. H2at 100 % in the gas phase inhibited growth on peptone. Glycerol dibiphytanyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl rings were present in the lipid fraction of isolate Kam940T. The G+C content of the genomic DNA of strain Kam940Twas 37 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates were archaea of the phylumCrenarchaeota, only distantly related to the cultured members of the classThermoprotei(no more than 89 % identity), and formed an independent lineage adjacent to the ordersDesulfurococcalesandAcidilobalesand clustering only with uncultured clones from hot springs of Yellowstone National Park and Iceland as the closest relatives. On the basis of their phylogenetic position and novel phenotypic features, isolates Kam940Tand Kam1507b are proposed to be assigned to a new genus and species,Fervidicoccus fontisgen. nov., sp. nov. The type strain ofFervidicoccus fontisis strain Kam940T(=DSM 19380T=VKM B-2539T). The phylogenetic data as well as phenotypic properties suggest that the novel crenarchaeotes form the basis of a new family,Fervidicoccaceaefam. nov., and order,Fervidicoccalesord. nov., within the classThermoprotei.

ABSTRACT “ Candidatus Accumulibacter” and total bacterial community dynamics were studied in two lab-scale enhanced biological phosphorus removal (EBPR) reactors by using a community fingerprint technique, automated ribosomal intergenic spacer analysis (ARISA). We first evaluated the quantitative capability of ARISA compared to quantitative real-time PCR (qPCR). ARISA and qPCR provided comparable relative quantification of the two dominant “ Ca . Accumulibacter” clades (IA and IIA) detected in our reactors. The quantification of total “ Ca . Accumulibacter” 16S rRNA genes relative to that from the total bacterial community was highly correlated, with ARISA systematically underestimating “ Ca . Accumulibacter” abundance, probably due to the different normalization techniques applied. During 6 months of normal (undisturbed) operation, the distribution of the two clades within the total “ Ca . Accumulibacter” population was quite stable in one reactor while comparatively dynamic in the other reactor. However, the variance in the clade distribution did not appear to affect reactor performance. Instead, good EBPR activity was positively associated with the abundance of total “ Ca . Accumulibacter.” Therefore, we concluded that the different clades in the system provided functional redundancy. We disturbed the reactor operation by adding nitrate together with acetate feeding in the anaerobic phase to reach initial reactor concentrations of 10 mg/liter NO 3 -N for 35 days. The reactor performance deteriorated with a concomitant decrease in the total “ Ca . Accumulibacter” population, suggesting that a population shift was the cause of performance upset after a long exposure to nitrate in the anaerobic phase.

SummaryAnaerobic ammonium‐oxidizing (anammox) bacteria are divided into three compartments by bilayer membranes (from out‐ to inside): paryphoplasm, riboplasm and anammoxosome. It is proposed that the anammox reaction is performed by proteins located in the anammoxosome and on its membrane giving rise to a proton‐motive‐force and subsequent ATP synthesis by membrane‐bound ATPases. To test this hypothesis, we investigated the location of membrane‐bound ATPases in the anammox bacterium ‘Candidatus Kuenenia stuttgartiensis’. Four ATPase gene clusters were identified in the K. stuttgartiensis genome: one typical F‐ATPase, two atypical F‐ATPases and a prokaryotic V‐ATPase. K. stuttgartiensis transcriptomic and proteomic analysis and immunoblotting using antisera directed at catalytic subunits of the ATPase gene clusters indicated that only the typical F‐ATPase gene cluster most likely encoded a functional ATPase under these cultivation conditions. Immunogold localization showed that the typical F‐ATPase was predominantly located on both the outermost and anammoxosome membrane and to a lesser extent on the middle membrane. This is consistent with the anammox physiology model, and confirms the status of the outermost cell membrane as cytoplasmic membrane. The occurrence of ATPase in the anammoxosome membrane suggests that anammox bacteria have evolved a prokaryotic organelle; a membrane‐bounded compartment with a specific cellular function: energy metabolism.

AbstractIntracellular inclusions containing chlamydia‐like organisms are frequently observed in the gill epithelial cells of Atlantic salmon, Salmo salar L., cultured in fresh water in Ireland. In this study, the causative agent was identified in four separate freshwater sites, using 16s rRNA sequencing, as ‘Candidatus Clavochlamydia salmonicola’. Histopathology and real‐time (RT) PCR were used to further assess infections. The prevalence of infection ranged from 75–100% between sites and infection intensity was highly variable. No significant lesions were associated with these infections. As a diagnostic tool, RT‐PCR proved marginally more sensitive than histopathology. The fate of ‘Candidatus Clavochlamydia salmonicola’ in Atlantic salmon post‐seawater transfer was investigated in a 12‐week marine longitudinal study. Both RT‐PCR and histopathological examination indicate that the organism disappears from the gills 4–6 weeks post‐transfer.

AbstractTwo symptomatic tomato plants exhibiting dwarfing, twisting of shoots and leaves, virescence and phyllody of flowers were collected, respectively, from a greenhouse (Soly07fi) or the field (Soly06gh) in the western region of Poland. Direct and nested polymerase chain reactions (PCR) were performed using universal phytoplasma primers P1/P7 and R16F2n/R16R2. Restriction fragment length polymorphism (RFLP) analysis of the PCR products showed that the RFLP profiles of both tested phytoplasma isolates are the same and that they belong to the phytoplasma 16S rRNA I‐C subgroup. The homology between the two strains was 99%. Phylogenetic analysis of the 16S rRNA gene sequences of the phytoplasma isolates and other phytoplasma sequences available in the GenBank database indicated that the Polish phytoplasma isolates are most closely related to the phytoplasma 16S rRNA I‐C subgroup.