Publications

4839

Abstract Background The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia. Results We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C1-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a major upward shift in the isoelectric points of proteins. Conclusion The results of genome analysis of M. infernorum support the monophyly of the PVC superphylum. M. infernorum possesses a streamlined genome but seems to have acquired numerous genes including those for enzymes of methylotrophic pathways via horizontal gene transfer, in particular, from Proteobacteria. Reviewers This article was reviewed by John A. Fuerst, Ludmila Chistoserdova, and Radhey S. Gupta.

Termites harbor diverse symbiotic gut microorganisms, the majority of which are as yet uncultivable and their interrelationships unclear. Here, we present the complete genome sequence of the uncultured Bacteroidales endosymbiont of the cellulolytic protist Pseudotrichonympha grassii, which accounts for 70% of the bacterial cells in the gut of the termite Coptotermes formosanus . Functional annotation of the chromosome (1,114,206 base pairs) unveiled its ability to fix dinitrogen and recycle putative host nitrogen wastes for biosynthesis of diverse amino acids and cofactors, and import glucose and xylose as energy and carbon sources. Thus, nitrogen fixation and cellulolysis are coupled within the protist's cells. This highly evolved symbiotic system probably underlies the ability of the worldwide pest termites Coptotermes to use wood as their sole food.

A new ‘Candidatus Liberibacter’ species was recently identified in tomato, capsicum, and potato in New Zealand. The tomato/potato psyllid, Bactericera cockerelli, is thought to be the vector of this species of liberibacter. During studies to determine additional host plants of the pathogen, leaves of Solanum betaceum (tamarillo, also known as tree tomato) and leaves and stems of Physalis peruviana (cape gooseberry) were collected from a home garden in South Auckland, New Zealand in July of 2008. These plants were not showing any obvious disease symptoms. They were located close to a commercial glasshouse site containing known liberibacter-infected tomatoes, and many psyllids were observed on the tamarillo tree over the summer and until late autumn. Total DNA was extracted from four tamarillo and two cape gooseberry samples with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Samples from tamarillo that were used for the extraction were taken from the midveins of old and young leaves and from young petioles. For cape gooseberry, samples were from the leaf midveins and the stems. The samples were tested by PCR using primers OA2 (GenBank Accession No. EU834130) and OI2c (1). These primers amplify a 1,160-bp fragment of the 16S rRNA gene of the new liberibacter species. Amplicons of the expected size were obtained from all four tamarillo samples, with no amplification from negative control tamarillo plants grown from seed in an insect-proof glasshouse. Almost the entire length of the 16S rRNA gene was amplified using primer pairs fD2 (3)/OI2c and OA2/rP1 (3), and the 16S-23S rRNA intergenic spacer was amplified with primer pair OI2/23S1 (2). These amplicons, along with that from the OA2/OI2c primer pair, were directly sequenced, and overlapping fragments were assembled using the SeqMan software of the LaserGene package (DNASTAR, Inc., Madison, WI) (GenBank Accession No. EU935004). A 650-bp fragment of the β operon was also amplified and sequenced directly (GenBank Accession No. EU935005). BLAST analysis showed 100% nt identity to the liberibacter of tomato (GenBank Accession Nos. EU834130 and EU834131) and potato (GenBank Accession Nos. EU849020 and EU919514). The two cape gooseberry samples produced amplicons of the expected size with the 16S rRNA and β operon primers and the origin of the fragments were confirmed by direct sequencing with BLAST analysis showing 100% nt identity to isolates from tomato, potato, and tamarillo. To determine the distribution of disease, 53 samples of 10 leaves each (representing two leaves from five plants) were collected randomly from a commercial tamarillo crop in South Auckland. Small sections of the midveins were removed from each of the 10 leaves, bulked, and DNA was extracted as described above. The samples were tested by PCR using primer pair OA2/OI2c. Amplicons of the expected size were obtained from 2 of the 53 samples. To our knowledge, this is the first report of a liberibacter in tamarillo and cape gooseberry. It is unknown if the liberibacter induces symptoms in these species or if they act as symptomless reservoirs of the pathogen. The infected plants will be observed for symptom development over the course of a growing season. References: (1) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (2) S. Jagoueix et al. Int. J. Syst. Bacteriol. 47:224, 1997. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

Summary The ammonium‐oxidizing microbial community was investigated in a granular sludge anaerobic ammonium‐oxidizing (anammox) reactor that was operated for about 1 year with high anaerobic ammonium oxidation activity (up to 0.8 kg NH 4 + ‐N m −3  day −1 ). A Planctomycetales ‐specific 16S rRNA gene library was constructed to analyse the diversity of the anaerobic ammonium‐oxidizing bacteria (AnAOB). Most of the specifically amplified sequences (15/16) were similar to each other (> 99%) but were distantly related to all of the previously recognized sequences (< 94%), with the exception of an unclassified anammox‐related clone, KSU‐1 (98%). An ammonia monooxygenase ( amoA ) gene library was also analysed to investigate the diversity of ‘aerobic’ ammonium‐oxidizing bacteria (AAOB) from the β‐ Proteobacteria . Most of the amoA gene fragments (53/55) clustered in the Nitrosomonas europaea–Nitrosococcus mobilis group which has been reported to prevail under oxygen‐limiting conditions. The quantitative results from real‐time polymerase chain reaction (PCR) amplification showed that the dominant AnAOB comprised approximately 50% of the total bacterial 16S rRNA genes in the reactor, whereas the AAOB of β‐ Proteobacteria represented only about 3%. A large fragment (4008 bp) of the rRNA gene cluster of the dominant AnAOB (AS‐1) in this reactor sludge was sequenced and compared with sequences of other Planctomycetales including four anammox‐related candidate genera. The partial sequence of hydrazine‐oxidizing enzyme ( hzo ) of dominant AnAOB was also identified using new designed primers. Based on this analysis, we propose to tentatively name this new AnAOB Candidatus ‘Jettenia asiatica’.

Summary The anaerobic oxidation of ammonium (anammox) contributes significantly to the global loss of fixed nitrogen and is carried out by a deep branching monophyletic group of bacteria within the phylum Planctomycetes . Various studies have implicated anammox to be the most important process responsible for the nitrogen loss in the marine oxygen minimum zones (OMZs) with a low diversity of marine anammox bacteria. This comprehensive study investigated the anammox bacteria in the suboxic zone of the Black Sea and in three major OMZs (off Namibia, Peru and in the Arabian Sea). The diversity and population composition of anammox bacteria were investigated by both, the 16S rRNA gene sequences and the 16S‐23S rRNA internal transcribed spacer (ITS). Our results showed that the anammox bacterial sequences of the investigated samples were all closely related to the Candidatus Scalindua genus. However, a greater microdiversity of marine anammox bacteria than previously assumed was observed. Both phylogenetic markers supported the classification of all sequences in two distinct anammox bacterial phylotypes: Candidatus Scalindua clades 1 and 2. Scalindua 1 could be further divided into four distinct clusters, all comprised of sequences from either the Namibian or the Peruvian OMZ. Scalindua 2 consisted of sequences from the Arabian Sea and the Peruvian OMZ and included one previously published 16S rRNA gene sequence from Lake Tanganyika and one from South China Sea sediment (97.9–99.4% sequence identity). This cluster showed only ≤ 97% sequence identity to other known Candidatus Scalindua species. Based on 16S rRNA gene and ITS sequences we propose that the anammox bacteria of Scalindua clade 2 represent a novel anammox bacterial species, for which the name Candidatus Scalindua arabica is proposed. As sequences of this new cluster were found in the Arabian Sea, the Peruvian OMZ, in Lake Tanganyika and in South China sediment, we assume a global distribution of Candidatus Scalindua arabica as it is observed for Candidatus Scalindua sorokinii/brodae (or Scalindua clade 1).

DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator , composes >99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earth's crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.