Flow cytometric sorting and RFLP analysis of phosphate accumulating bacteria in an enhanced biological phosphorus removal system

Kawaharasaki, Mamoru; Manome, Akira; Kanagawa, Takahiro; Nakamura, Kazunori

doi:10.2166/wst.2002.0469

Cited by 22 publications

(10 citation statements)

References 13 publications

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“…A biomass sample sent to this laboratory from a badly bulking activated sludge plant in Whyalla, South Australia, was dominated by a Gram-negative filament in distinctive bundles, which was identified from its morphological features (Eikelboom, 2000;Seviour and Nielsen, 2010) as type 0914. This morphotype did not stain positively for either PHA (Ostle and Holt, 1982) or polyP (Kawaharasaki et al, 2002), nor were any sulfur granules seen in their cells under phase-contrast microscopy (Jenkins et al, 2004). The Whyalla plant was configured as a pair of sequencing batch reactors, which could be run either in parallel or sequentially, and removed both phosphorus and nitrogen microbiologically.…”

Section: Resultsmentioning

confidence: 99%

The activated sludge bulking filament Eikelboom morphotype 0914 is a member of the Chloroflexi

Speirs

McIlroy

Petrovski

et al. 2011

Environ Microbiol Rep

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The filamentous bacterium Eikelboom morphotype type 0914 responsible for bulking in activated sludge plants is identified here for the first time as a member of the phylum Chloroflexi subgroup 1. Two FISH probes, CFX67a and CFX67b, targeting the 16S rRNA sequences of this filament morphotype were designed, validated and used successfully for its in situ identification. A survey of plants in eastern Australia with the CFX67a probe showed it targeted only the type 0914 morphotype that was common especially in long sludge age plants designed to remove phosphorus and nitrogen microbiologically, although being in very low abundance in many samples. Filaments responding to the CFX67b probe also exhibited the type 0914 morphology but were less frequent, although again occurring in similarly configured plants. All these filaments showed an uneven FISH signal suggesting their ribosomes are localized at the ends of their cells. Furthermore, some generated distinctive FISH signals in all biomass samples containing them, where only certain cells within any single trichome fluoresced with probes designed against different target sites. Helper probes for each of these were required before all cells fluoresced above the visual detection limits of FISH.

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Section: Resultsmentioning

confidence: 99%

The activated sludge bulking filament Eikelboom morphotype 0914 is a member of the Chloroflexi

Speirs

McIlroy

Petrovski

et al. 2011

Environ Microbiol Rep

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“…In this analysis, we used a flow cytometry method to determine relative cellular polyP accumulation in different populations of human bone marrow mononuclear cells. Several authors have described similar approaches for detecting polyP in bacteria populations [34][35][36][37] but, to our knowledge, this is the first time that this technique has been applied in human cells to detect polyP.…”

Section: Discussionmentioning

confidence: 99%

Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription

Jimenez-Nuñez¹,

Moreno-Sanchez²,

Hernández-Ruiz³

et al. 2012

Haematologica

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The online version of this article has a Supplementary Appendix. BackgroundIn hematology there has recently been increasing interest in inorganic polyphosphate. This polymer accumulates in platelet granules and its functions include modulating various stages of blood coagulation, inducing angiogenesis, and provoking apoptosis of plasma cells. In this study we evaluated the characteristics of intracellular polyphosphate in myeloma cell lines, in primary myeloma cells from patients, and in other human B-cell populations from healthy donors. Design and MethodsWe have developed a novel flow cytometric method for detecting levels of polyphosphate in cell populations. We also used confocal microscopy and enzymatic analysis to study polyphosphate localization and characteristics. ResultsWe found that myeloma plasma cells contain higher levels of intracellular polyphosphate than normal plasma cells and other B-cell populations. Localization experiments indicated that high levels of polyphosphate accumulate in the nucleolus of myeloma cells. As the principal function of the nucleolus involves transcription of ribosomal DNA genes, we found changes in the cellular distribution of polyphosphate after the inhibition of nucleolar transcription. In addition, we found that RNA polymerase I activity, responsible for transcription in the nucleolus, is also modulated by polyphosphate, in a dose-dependent manner. ConclusionsOur results show an unusually high accumulation of polyphosphate in the nucleoli of myeloma cells and a functional relationship of this polymer with nucleolar transcription. Haematologica 2012;97(8):1264-1271. doi:10.3324/haematol.2011 This is an open-access paper. Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n

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“…To detect polyphosphate (polyP) and poly-␤-hydroxyalkanoates (PHA) in cells, DAPI (4Ј,6Ј-diamidino-2-phenylindole) (23) and Nile blue A (42), respectively, were used as detailed in Ahn et al (1).…”

Section: Methodsmentioning

confidence: 99%

Filamentous Bacterium Eikelboom Type 0092 in Activated Sludge Plants in Australia Is a Member of the Phylum Chloroflexi

Speirs

Nittami

McIlroy

et al. 2009

Appl Environ Microbiol

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Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum Chloroflexi. Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removalactivated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-␤-hydroxyalkanoates or polyphosphate.Most activated sludge plants suffer from the operational disorders of bulking and foaming, both of which are caused by excessive growth of certain filamentous bacteria. Several different filament morphotypes have been described from systems treating domestic and industrial wastes (17, 18) but, in the absence of pure cultures, many of these have never been characterized sufficiently to resolve their taxonomy or provide them with valid names. Hence, they are often still referred to as numerical types persisting from the study of Eikelboom (18). Success has been achieved with some cultured and uncultured filaments in elucidating their phylogeny from 16S rRNA sequence analyses (6, 9, 10, 31, 50) and providing them with valid names (37, 49). Furthermore, with such sequence information, rRNA targeted oligonucleotide probes have been designed for their in situ identification and, together with microautoradiography (MAR) and other techniques (29,40), their ecophysiology may be elucidated (25,27,28). Type 0092, originally described by Eikelboom (18), appears prominently in many filament surveys carried out on plants around the world, where microscopy was used to identify them (35, 48). These morphotypes have been associated especially with long sludge age (Ͼ15-day) operational conditions (22) and thus frequently appear in enhanced biological phosphate removal (EBPR) systems (see, for example, reference 11), where the biomass is recycled repeatedly through anaerobic: aerobic zones. Consequently, this filament morphotype was classified as an "all-zone" grower by Wanner and Grau (52), able in their view to grow under aerobic, anoxic, and anaerobic conditions. However, its physiology from pure culture studies was described as being strictly aerobic (13, 21). These isolates were never deposited in recognized culture collections, and so confirmation of their identity is difficult. Similarly, the precise identification of the type 0092 filaments claimed to have been cultured by Ramothokang et al. (43) is unclear.Type 0092 has ver...

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Flow cytometric sorting and RFLP analysis of phosphate accumulating bacteria in an enhanced biological phosphorus removal system

Cited by 22 publications

References 13 publications

The activated sludge bulking filament Eikelboom morphotype 0914 is a member of the Chloroflexi

The activated sludge bulking filament Eikelboom morphotype 0914 is a member of the Chloroflexi

Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription

Filamentous Bacterium Eikelboom Type 0092 in Activated Sludge Plants in Australia Is a Member of the Phylum Chloroflexi

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