Publications

4742

Citrus huanglongbing, putatively caused by the associated bacterium ‘Candidatus Liberibacter asiaticus’, is the greatest threat to the world citrus industry today. The bacterium is spread locally and regionally by the citrus psyllid Diaphorina citri, and also can be disseminated by propagation of contaminated scion budwood that is grafted to the appropriate rootstock. The planting of ‘Ca. Liberibacter asiaticus’-free trees is a component of a comprehensive strategy to manage huanglongbing. In contrast to the scion budwood, the rootstocks used to produce these trees are grown from seed. This research was undertaken to provide evidence as to whether or not ‘Ca. L. asiaticus’ can be transmitted through seed. Two groups of 360 or more seedlings each of various citrus species were grown from seed removed from fruit on trees that were symptomatic for huanglongbing and confirmed to be infected with ‘Ca. L. asiaticus’ by polymerase chain reaction (PCR) tests. These seedlings were tested multiple times over periods of up to 3 years. No symptoms typical of huanglongbing, such as blotchy leaf mottle, chlorotic shoots, or dieback of branches, were observed in these seedlings, and none of these 723 seedlings tested positive for the presence of ‘Ca. L. asiaticus’ even after repeated testing by sensitive quantitative PCR assays. Some sour orange seedlings did have quite pronounced and atypical growth, including stunting and mild to severe leaf malformation. These atypical growth habits were limited to seedlings that arose from zygotic embryos as determined by expressed-sequence tag simple-sequence repeat analyses. Thus, no evidence of transmission of ‘Ca. L. asiaticus’ via seed was obtained, and an earlier report of transmission of the pathogen through seed was not confirmed.

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.

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.

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.