Environmental Microbiology

Summary Gold ore processing uses cyanide ( CN − ), which often results in large volumes of thiocyanate‐ ( SCN − ) contaminated wastewater requiring treatment. Microbial communities can degrade SCN − and CN − , but little is known about their membership and metabolic potential. Microbial‐based remediation strategies will benefit from an ecological understanding of organisms involved in the breakdown of SCN − and CN − into sulfur, carbon and nitrogen compounds. We performed metagenomic analysis of samples from two laboratory‐scale bioreactors used to study SCN − and CN − degradation. Community analysis revealed the dominance of Thiobacillus spp., whose genomes harbour a previously unreported operon for SCN − degradation. Genome‐based metabolic predictions suggest that a large portion of each bioreactor community is autotrophic, relying not on molasses in reactor feed but using energy gained from oxidation of sulfur compounds produced during SCN − degradation. Heterotrophs, including a bacterium from a previously uncharacterized phylum, compose a smaller portion of the reactor community. Predation by phage and eukaryotes is predicted to affect community dynamics. Genes for ammonium oxidation and denitrification were detected, indicating the potential for nitrogen removal, as required for complete remediation of wastewater. These findings suggest optimization strategies for reactor design, such as improved aerobic/anaerobic partitioning and elimination of organic carbon from reactor feed.

Summary To date, six candidate genera of anaerobic ammonium‐oxidizing (anammox) bacteria have been identified, and numerous studies have been conducted to understand their ecophysiology. In this study, we examined the physiological characteristics of an anammox bacterium in the genus ‘ C andidatus   J ettenia’. Planctomycete KSU ‐1 was found to be a mesophilic (20–42.5°C) and neutrophilic ( pH 6.5–8.5) bacterium with a maximum growth rate of 0.0020 h −1 . Planctomycete KSU ‐1 cells showed typical physiological and structural features of anammox bacteria; i.e. 29 N 2 gas production by coupling of 15 NH 4 + and 14 NO 2 − , accumulation of hydrazine with the consumption of hydroxylamine and the presence of anammoxosome. In addition, the cells were capable of respiratory ammonification with oxidation of acetate. Notably, the cells contained menaquinone‐7 as a dominant respiratory quinone. Proteomic analysis was performed to examine underlying core metabolisms, and high expressions of hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, nitrite/nitrate oxidoreductase and carbon monoxide dehydrogenase/acetyl‐ CoA synthase were detected. These proteins require iron or copper as a metal cofactor, and both were dominant in planctomycete KSU ‐1 cells. On the basis of these experimental results, we proposed the name ‘ C a . J ettenia caeni’ sp. nov. for the bacterial clade of the planctomycete KSU ‐1.

Summary Two morphotypes of multicellular magnetotactic prokaryotes ( MMPs ) have been identified: spherical (several species) and ellipsoidal (previously one species). Here, we report novel ellipsoidal MMPs that are ∼ 10 × 8 μm in size, and composed of about 86 cells arranged in six to eight interlaced circles. Each MMP was composed of cells that synthesized either bullet‐shaped magnetite magnetosomes alone, or both bullet‐shaped magnetite and rectangular greigite magnetosomes. They showed north‐seeking magnetotaxis, ping‐pong motility and negative phototaxis at a velocity up to 300 μm s −1 . During reproduction, they divided along either their long‐ or short‐body axes. For genetic analysis, we sorted the ellipsoidal MMPs with micromanipulation and amplified their genomes using multiple displacement amplification. We sequenced the 16 S r RNA gene and found 6.9% sequence divergence from that of ellipsoidal MMPs , Candidatus Magnetananas tsingtaoensis and > 8.3% divergence from those of spherical MMPs . Therefore, the novel MMPs belong to different species and genus compared with the currently known ellipsoidal and spherical MMPs respectively. The novel MMPs display a morphological cell differentiation, implying a potential division of labour. These findings provide new insights into the diversity of MMPs in general, and contribute to our understanding of the evolution of multicellularity among prokaryotes.

Summary In an acetate‐fed anaerobic–aerobic membrane bioreactor with deteriorated enhanced biological phosphorus removal ( EBPR ), D efluviicoccus ‐related tetrad‐forming organisms ( DTFO ) were observed to predominate in the microbial community. Using metagenomics, a partial genome of the predominant DTFO , ‘ C andidatus   D efluviicoccus tetraformis strain TFO 71’, was successfully constructed and characterized. Examining the genome confirmed the presence of genes related to the synthesis and degradation of glycogen and polyhydroxyalkanoate ( PHA ), which function as energy and carbon storage compounds. TFO 71 and ‘ C andidatus   A ccumulibacter phosphatis’ ( CAP ) UW ‐1 and CAP UW ‐2, representative polyphosphate‐accumulating organisms ( PAO ), have PHA metabolism‐related genes with high homology, but TFO 71 has unique genes for PHA synthesis, gene regulation and granule management. We further discovered genes encoding DTFO polyphosphate ( polyP ) synthesis, suggesting that TFO 71 may synthesize polyP under untested conditions. However, TFO 71 may not activate these genes under EBPR conditions because the retrieved genome does not contain all inorganic phosphate transporters that are characteristic of PAOs ( CAP UW ‐1, CAP UW ‐2, M icrolunatus phosphovorus   NM ‐1 and T etrasphaera species). As a first step in characterizing EBPR ‐associated DTFO metabolism, this study identifies important differences between DTFO and PAO that may contribute to EBPR community competition and deterioration.

Summary Knowledge of intraspecific variation in symbioses may aid in understanding the ecology of widespread insects in different parts of their range. We investigated bacterial symbionts of A delges tsugae , a pest of hemlocks in eastern N orth A merica introduced from A sia. Amplification, cloning, and sequencing of bacterial 16 S rDNA , in situ hybridizations, and electron microscopy revealed that A . tsugae harbours up to five bacterial phylotypes, according to population. Three G ammaproteobacteria species are maternally transmitted. The first, designated ‘ C a . P seudomonas adelgestsugas’ resides in the haemocoel, and was detected in all populations except T aiwan. The second phylotype, ‘ C a . S erratia symbiotica’, resides in bacteriocytes of populations on T suga sieboldii in J apan and in E . N orth A merica. The third phylotype, designated ‘ C a . A nnandia adelgestsuga’, clustered within a lineage of several insect endosymbionts that included B uchnera aphidicola . It was detected in bacteriocytes in all populations, and in salivary glands of first instars. Two B etaproteobacteria phylotypes were detected in some J apanese T . sieboldii and eastern N orth A merica populations, and were observed only in salivary glands with no evidence of maternal transmission. Our results support the ideas that symbiont gain and loss has been volatile in adelgids, and that symbionts may help to trace the source of invasive species.

Summary Many hemipterans are associated with symbiotic bacteria, which are usually found intracellularly in specific bacteriomes. In this study, we provide the first molecular identification of the bacteriome‐associated, obligate endosymbiont in a G ondwanan relict insect taxon, the moss bugs ( H emiptera: C oleorrhyncha: P eloridiidae), which represents one of the oldest lineages within the H emiptera. Endosymbiotic associations of fifteen species of the family were analysed, covering representatives from S outh A merica, A ustralia/ T asmania and N ew Z ealand. Phylogenetic analysis based on four kilobases of 16 S –23 S r RNA gene fragments showed that the obligate endosymbiont of P eloridiidae constitute a so far unknown group of G ammaproteobacteria which is named here ‘ Candidatus Evansia muelleri’. They are related to the sternorrhynchous endosymbionts C andidatus   P ortiera and C andidatus   C arsonella. Comparison of the primary‐endosymbiont and host ( COI  + 28 S rRNA ) trees showed overall congruence indicating co‐speciation the hosts and their symbionts. The distribution of the endosymbiont within the insect body and its transmission was studied using FISH . The endosymbionts were detected endocellularly in a pair of bacteriomes as well as in the ‘symbiont ball’ of the posterior pole of each developing oocyte. Furthermore, ultrastructural analysis of the M alpighian tubules revealed that most host nuclei are infected by an endosymbiotic, intranuclear bacterium that was determined as an A lphaproteobacterium of the genus R ickettsia .

Summary A novel moderately thermophilic, facultatively anaerobic chemoorganotrophic bacterium strain P3M ‐2 T was isolated from a microbial mat developing on the wooden surface of a chute under the flow of hot water (46° C ) coming out of a 2775‐m‐deep oil exploration well ( T omsk region, R ussia). Strain P3M ‐2 T is a moderate thermophile and facultative anaerobe growing on mono‐, di‐ or polysaccharides by aerobic respiration, fermentation or by reducing diverse electron acceptors [nitrite, F e( III ), As ( V )]. Its closest cultivated relative (90.8% rRNA gene sequence identity) is I gnavibacterium album , the only chemoorganotrophic member of the phylum C hlorobi . New genus and species M elioribacter roseus are proposed for isolate P3M ‐2 T . Together with I . album , the new organism represents the class I gnavibacteria assigned to the phylum Chlorobi . The revealed group includes a variety of uncultured environmental clones, the 16S rRNA gene sequences of some of which have been previously attributed to the candidate division ZB1 . Phylogenetic analysis of M . roseus and I . album based on their 23S rRNA and RecA sequences confirmed that these two organisms could represent an even deeper, phylum‐level lineage. Hence, we propose a new phylum Ignavibacteriae within the Bacteroidetes – C hlorobi group with a sole class I gnavibacteria , two families I gnavibacteriaceae and M elioribacteraceae and two species I . album and M . roseus . This proposal correlates with chemotaxonomic data and phenotypic differences of both organisms from other cultured representatives of C hlorobi . The most essential differences, supported by the analyses of complete genomes of both organisms, are motility, facultatively anaerobic and obligately organotrophic mode of life, the absence of chlorosomes and the apparent inability to grow phototrophically.

Summary A combination of microscopic, molecular and biogeochemical methods was used to study the structure, phylogenetics and vertical distribution of spherical multicellular magnetotactic prokaryotes ( MMPs ) of intertidal sediments in the Y ellow S ea. These MMPs were 5.5 μm in diameter and composed of approximately 15–30 cells. They synthesized bullet‐shaped magnetites in chains or clusters. Phylogenetic analysis of 16 S rRNA gene sequences suggested that these MMPs represent a novel species affiliated to the D eltaproteobacteria. To study their vertical distribution and the relationship to geochemical parameters, sediment cores were collected after the redox potential was measured in situ . The sediments were composed of yellow, grey and black layers from the surface to depth. The spherical MMPs were concentrated near the grey‐black layer transition at a depth of 8–12 cm, while coccoid‐shaped magnetotactic bacteria near the yellow‐grey layer transition at a depth of 3–5 cm. The intertidal MMPs showed a deeper distribution at more reduced environments than coccoid‐shaped magnetotactic bacteria, and MMPs in lagoon sediments. Additionally the MMPs were concentrated significantly in layers with high proportion of fine sand and total organic carbon, rich in leachable iron but poor in nitrate. These results show an adaptation of spherical MMPs to the peculiar intertidal sediment habitat.

Summary Anaerobic ammonium‐oxidizing (anammox) bacteria are responsible for a significant portion of the loss of fixed nitrogen from the oceans, making them important players in the global nitrogen cycle. To date, marine anammox bacteria found in marine water columns and sediments worldwide belong almost exclusively to the ‘ Candidatus Scalindua’ species, but the molecular basis of their metabolism and competitive fitness is presently unknown. We applied community sequencing of a marine anammox enrichment culture dominated by ‘ Candidatus Scalindua profunda’ to construct a genome assembly, which was subsequently used to analyse the most abundant gene transcripts and proteins. In the S. profunda assembly, 4756 genes were annotated, and only about half of them showed the highest identity to the only other anammox bacterium of which a metagenome assembly had been constructed so far, the freshwater ‘ Candidatus Kuenenia stuttgartiensis’. In total, 2016 genes of S. profunda could not be matched to the K. stuttgartiensis metagenome assembly at all, and a similar number of genes in K. stuttgartiensis could not be found in S. profunda . Most of these genes did not have a known function but 98 expressed genes could be attributed to oligopeptide transport, amino acid metabolism, use of organic acids and electron transport. On the basis of the S. profunda metagenome, and environmental metagenome data, we observed pronounced differences in the gene organization and expression of important anammox enzymes, such as hydrazine synthase (HzsAB), nitrite reductase (NirS) and inorganic nitrogen transport proteins. Adaptations of Scalindua to the substrate limitation of the ocean may include highly expressed ammonium, nitrite and oligopeptide transport systems and pathways for the transport, oxidation, and assimilation of small organic compounds that may allow a more versatile lifestyle contributing to the competitive fitness of Scalindua in the marine realm.

Summary Microautoradiography combined with fluorescence in situ hybridization (MAR‐FISH) was used to screen for potential polyphosphate‐accumulating organisms (PAOs) in full‐scale enhanced biological phosphorus removal (EBPR) plants. Clone library analyses and application of MAR‐FISH using newly designed probes revealed that small rods related to uncultured Halomonas within the gammaproteobacterial family Halomonadaceae were actively involved in uptake of orthophosphate. Although deeply branched in the Gammaproteobacteria , they were not targeted by the gammaproteobacterial probe (GAM42a). A part of them were also not targeted with the general bacterial probes (EUBmix). They could take up short‐chain fatty acids (e.g. acetate and propionate) and ethanol under both anaerobic and aerobic conditions. Polyhydroxyalkanoate storage was observed under anaerobic conditions. There was no indication of a denitrifying capability. A survey of the occurrence of these Halomonas ‐PAOs in 23 full‐scale EBPR plants revealed that they made up 0.5–5.7% of all bacteria in the plants, and were often in higher abundance than the well‐described PAOs ‘ Candidatus Accumulibacter phosphatis’. This indicates a potentially important role for these uncultured Halomonas bacteria in the EBPR process in full‐scale plants and we propose to name them ‘ Candidatus Halomonas phosphatis’.