Ecology, Evolution, Behavior and Systematics

Abstract Enhanced biological phosphorus removal (EBPR) activated sludge communities enriched in ‘Candidatus Accumulibacter’ relatives are widely used in wastewater treatment, but much remains to be learned about molecular-level controls on the EBPR process. The expression of genes found in the carbon and polyphosphate metabolic pathways in Accumulibacter was investigated using reverse transcription quantitative PCR. During a normal anaerobic/aerobic EBPR cycle, gene expression exhibited a dynamic change in response to external acetate, oxygen, phosphate concentrations and probably internal chemical pools. Anaerobic acetate addition induced expression of genes associated with the methylmalonyl-CoA pathway enabling the split mode of the tricarboxylic acid (TCA) cycle. Components of the full TCA cycle were induced after the switch to aerobic conditions. The induction of a key gene in the glyoxylate shunt pathway was observed under both anaerobic and aerobic conditions, with a higher induction by aeration. Polyphosphate kinase 1 from Accumulibacter was expressed, but did not appear to be regulated by phosphate limitation. To understand how Accumulibacter responds to disturbed electron donor and acceptor conditions, we perturbed the process by adding acetate aerobically. When high concentrations of oxygen were present simultaneously with acetate, phosphate-release was almost completely inhibited, and polyphosphate kinase 1 transcript abundance decreased. Genes associated with the methylmalonyl-CoA pathway were repressed and genes associated with the aerobic TCA cycle exhibited higher expression under this perturbation, suggesting that more acetyl-CoA was metabolized through the TCA cycle. These findings suggest that several genes involved in EBPR are tightly regulated at the transcriptional level.

Five Gram-negative, facultatively anaerobic, non-spore-forming, coccoid rod-shaped bacterial isolates were obtained from infant formula and an infant formula production environment and were investigated by use of a polyphasic taxonomic study. Biochemical tests and partialrpoBgene sequence analysis of the five isolates revealed that they formed two distinct groups in the familyEnterobacteriaceae, closely related to several species of the generaPantoeaandErwinia, which indicated a phylogenetic position within the genusPantoeaor the genusErwinia. Multilocus sequence analysis of concatenated partialatpD,gyrB,infBandrpoBgene sequences of two of the isolates suggested that they represented two novel species of the genusPantoea, phylogenetically related most closely toPantoea septica. The five isolates had general characteristics consistent with those of the genusPantoea, and DNA–DNA hybridizations between two representatives and the type strains of their phylogenetically closest relatives based on comparative 16S rRNA gene sequence analysis showed that the isolates represented two novel genospecies. These two genospecies could be differentiated from each other based on fermentation of galacturonate, sorbitol and potassium 5-ketogluconate. They could be differentiated from phylogenetically relatedPantoeaspecies based on their ability to ferment lactose and to utilizeβ-gentiobiose and raffinose, their inability to ferment or utilized-arabitol, and their inability to produce indole. On the basis of the results obtained, the five isolates are considered to represent two novel species of the genusPantoea, for which the namesPantoea gaviniaesp. nov. (type strain A18/07T=LMG 25382T=DSM 22758T) andPantoea calidasp. nov. (type strain 1400/07T=LMG 25383T=DSM 22759T) are proposed.

Abstract Marine pelagic redoxclines are areas of enhanced biogeochemical cycling inhabited by distinct functional groups of prokaryotes. In this study, the diversity and abundance of archaeal and bacterial nitrifying populations throughout a pelagic redoxcline in the central Baltic Sea were examined using a suite of molecular methods. 16S rRNA/rRNA gene as well as bacterial and archaeal amoA mRNA/amoA gene fingerprints and clone libraries revealed that the putative nitrifying assemblages consisted solely of one crenarchaeotal subcluster, named GD2, which was closely related to Candidatus Nitrosopumilus maritimus. Neither distinct differences between transcript- and gene-based fingerprints nor pronounced differences in the crenarchaeotal composition throughout the whole redoxcline were detected. The abundance of this GD2 subgroup, as determined by the oligonucleotide probe Cren537 and the newly developed and more specific probe Cren679 showed that GD2 and total crenarchaeotal cell numbers were nearly identical throughout the redoxcline. The highest GD2 abundance (2.3 × 105 cells ml−1) occurred in the suboxic zone, accounting for around 26% of total prokaryotic cells. Below the chemocline, GD2 abundance was relatively stable (1.5–1.9 × 105 cells ml−1). Archaeal amoA expression was detected only in the putative nitrification zone and formed a narrow band in the suboxic layer, where ammonium, oxygen, nitrate, nitrite and phosphate concentrations were below 5 μmol l−1. To our knowledge this is the first study to show the dominance of only one crenarchaeotal nitrifying key cluster in a natural habitat. The metabolic properties and survival mechanisms present in this cluster inside and outside the nitrification zone remain to be determined.

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.

A novel halophilic actinomycete, designated strain YIM 93223T, was isolated from a salt lake in Xinjiang Province, north-west China, and was subjected to a polyphasic taxonomic study. The isolate grew at 25–45 °C, at pH 6–8 and in the presence of 1–15 % (w/v) NaCl; no growth was observed in the absence of NaCl. Strain YIM 93223T contained meso-diaminopimelic acid, glutamic acid and alanine as cell-wall amino acids, and glucose and galactose as major whole-cell-wall sugars. Major fatty acids were iso-C16 : 0, C16 : 0 and C16 : 1 ω7c/iso-C15 : 0 2-OH. MK-8(H4) was the predominant menaquinone. The genomic DNA G+C content was 66.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 93223T belongs to the genus Amycolatopsis. It shared highest 16S rRNA gene sequence similarity with Amycolatopsis palatopharyngis 1BDZT (96.6 %) and Amycolatopsis marina Ms392AT (96.4 %), but lower values (94.5–96.2 %) with the type strains of other recognized species of the genus Amycolatopsis. On the basis of the data from this polyphasic study, strain YIM 93223T is considered to represent a novel species of the genus Amycolatopsis, for which the name Amycolatopsis halophila sp. nov. is proposed. The type strain is YIM 93223T (=DSM 45216T =KCTC 19403T).

Summary Here we report the first metatranscriptomic analysis of gene expression and regulation of ‘ Candidatus Accumulibacter’‐enriched lab‐scale sludge during enhanced biological phosphorus removal (EBPR). Medium density oligonucleotide microarrays were generated with probes targeting most predicted genes hypothesized to be important for the EBPR phenotype. RNA samples were collected at the early stage of anaerobic and aerobic phases (15 min after acetate addition and switching to aeration respectively). We detected the expression of a number of genes involved in the carbon and phosphate metabolisms, as proposed by EBPR models (e.g. polyhydroxyalkanoate synthesis, a split TCA cycle through methylmalonyl‐CoA pathway, and polyphosphate formation), as well as novel genes discovered through metagenomic analysis. The comparison between the early stage anaerobic and aerobic gene expression profiles showed that expression levels of most genes were not significantly different between the two stages. The majority of upregulated genes in the aerobic sample are predicted to encode functions such as transcription, translation and protein translocation, reflecting the rapid growth phase of Accumulibacter shortly after being switched to aerobic conditions. Components of the TCA cycle and machinery involved in ATP synthesis were also upregulated during the early aerobic phase. These findings support the predictions of EBPR metabolic models that the oxidation of intracellularly stored carbon polymers through the TCA cycle provides ATP for cell growth when oxygen becomes available. Nitrous oxide reductase was among the very few Accumulibacter genes upregulated in the anaerobic sample, suggesting that its expression is likely induced by the deprivation of oxygen.