Advanced Wastewater Management Centre, Department of Microbiology and Parasitology, The University of Queensland, Brisbane 4072, Australia1
ComBinE group, Advanced Computational Modelling Centre, The University of Queensland, Brisbane 4072, Australia2
Author for correspondence: Linda L. Blackall. Tel: +61 7 3365 4645. Fax: +61 7 3365 4620. e-mail: blackall{at}biosci.uq.edu.au
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ABSTRACT |
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Keywords: activated sludge, filamentous bacteria, fluorescence in situ hybridization (FISH), phylogeny, microbial ecology
Abbreviations: BNR, biological nutrient removal; FISH, fluorescent in situ hybridization
c The EMBL accession numbers for the sequences reported in this paper are X84472 (strain SBR1029 16S rDNA), X84474 (strain SBR1031 16S rDNA), X84498 (strain SBR1064 16S rDNA), X84565 (strain SBR2022 16S rDNA), X84576 (strain SBR2037 16S rDNA) and X84607 (strain SBR2076 16S rDNA).
a Present address: Department of Biotechnology, Centre of Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-22100 Lund, Sweden.
b Present address: Department of Environmental Science, Policy and Management, Hilgard Hall, University of California Berkeley, Berkeley CA 94720, USA.
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INTRODUCTION |
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Over the last decade, molecular biological methods have been used to identify and monitor filamentous micro-organisms (Blackall, 1994 ; Bradford et al., 1996
; Erhart et al., 1997
; Kanagawa et al., 2000
; Wagner et al., 1994
). In particular, fluorescence in situ hybridization (FISH) using 16S rRNA-targeting oligonucleotide probes (DeLong et al., 1989
) is an invaluable technique for directly identifying micro-organisms in their natural settings (Amann et al., 2001
). Thus far, only a minority of filamentous bacteria in BNR systems have been identified using phylogenetic stains (Seviour & Blackall, 1999
). However, these comprise a wide diversity of organisms including members of well-studied bacterial phyla such as Proteobacteria (e.g. Thiothrix spp.; Howarth et al., 1999
; Kanagawa et al., 2000
), Actinobacteria (e.g. Microthrix parvicella; Blackall et al., 1994
), Firmicutes (e.g. some Nostocoida limicola I; Liu et al., 2000
) and Bacteroidetes (e.g. Type 0092, Bradford et al., 1996
). Recently, less studied bacterial phyla such as Chloroflexi (Beer et al., 2002
; Bradford et al., 1996
), Planctomycetes (Liu et al., 2001
) and candidate phylum TM7 (Hugenholtz et al., 2001a
) have also been shown by molecular methods to have filamentous representatives in sludge. Phylum-level FISH probes exist for the Planctomycetes (Neef et al., 1998
) and TM7 group (Hugenholtz et al., 2001a
), but only species-specific FISH probes have been published for the Chloroflexi phylum (Beer et al., 2002
; Sekiguchi et al., 2001
). Therefore, the extent of representatives belonging to this latter phylum in BNR sludges is unknown.
The aim of the present study was to design phylum- and subdivision-specific oligonucleotide probes for the Chloroflexi and to evaluate them on sludge samples using FISH, to determine the abundance, morphology and spatial distribution of Chloroflexi in activated sludges.
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METHODS |
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Phylogenetic analysis and probe design.
Chloroflexi 16S rDNA sequences determined in the present study and available from the public databases were imported and aligned in the ARB software package (http://www.arb-home.de). Phylogenetic trees were inferred from the alignment as previously described (Klein et al., 2001 ). The dataset was checked for chimaeric sequences by inferring independent trees from the 5' and 3' halves of the alignment and looking for branching incongruencies (partial treeing analysis). Chloroflexi-specific oligonucleotide probes were designed as described previously (Hugenholtz et al., 2001a
, b
). Selected parameters of the probes are detailed in Table 2
. Additionally, an oligonucleotide primer designed for Chloroflexi-specific PCR (Gich et al., 2001
) called GNSB-941f was evaluated unmodified as a FISH probe.
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FISH was carried out on paraformaldehyde-fixed samples with methods detailed by Amann (1995) , using a 1·5 h hybridization time and published or determined formamide concentrations. Following FISH, samples were observed with a Bio-Rad Radiance 2000 confocal laser scanning microscope using a Nikon 60x oil immersion objective. FITC, Cy3 and Cy5 were excited with an Ar laser (488 nm), HeNe laser (543 nm) and red diode laser (637 nm) and collected with 500530 nm BP, 550625 nm BP and 660 LP emission filters, respectively. Images were collected and final image evaluation was done in Adobe Photoshop.
For the activated sludge screening, paraformaldehyde-fixed samples were triple hybridized with two Chloroflexi phylum or subdivision targeting probes and EUBMIX probe suite targeting most Bacteria (Daims et al., 1999 ). CFXMIX was also used and this was composed of equal amounts of the Chloroflexi phylum probes GNSB-941 and CFX1223 labelled with the same fluorochrome.
Subjective scoring of abundance of filamentous bacteria and Chloroflexi.
The abundance of all filamentous organisms and filamentous Chloroflexi in the samples was measured according to the subjective scoring method of Jenkins et al. (1993) where the observations are rated on a scale from 0 (none) to 6 (excessive) (Table 1
). Phase-contrast images were captured on an Olympus BH2 microscope. Final image evaluation was done in Adobe Photoshop. Chloroflexi-specific measurements were made using Cy5 labelled CFXMIX (Table 1
). Images were captured as previously mentioned. The proportions of filaments in Chloroflexi subdivisions 1 and 3 were determined using Cy3-labelled CFX784 or CFX109, respectively, in combination with Cy5-labelled CFXMIX. Filament abundances were again subjectively scored whereby the proportions of Chloroflexi 1 and 3 were ranked in relation to all Chloroflexi. Observations were rated from none to all observed (Table 1
). All estimations were based on a mean of 710 independent hybridizations and subjective scorings.
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RESULTS |
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As with all broad-specificity probes it was difficult to design probes which hit all sequences in the target group. Compromises had to be made to obtain as broad a coverage of the Chloroflexi as possible while minimizing coverage of non-target organisms. This is highlighted in Fig. 1 where the specificity of the Chloroflexi probes and of the general bacterial probe, EUBMIX (Daims et al., 1999
), is summarized in the columns to the right of the dendrogram. Probes with no mismatches to a given sequence in the dendrogram (likely to hybridize successfully to that sequence) are indicated by black squares and probes with one or more mismatches to a given sequence (unlikely to hybridize successfully to that sequence) are indicated by white squares. Grey squares indicate that the probe will likely hybridize to the target sequence, but that an unresolved base was present in the sequence. A grey square was also used in the case of Herpetosiphon and EUBMIX where there is one or more base mismatches between the probes and target string (Daims et al., 1999
). Nevertheless, we found Herpetosiphon species successfully bound EUBMIX in FISH. The sequences targeted by the two phylum-level probes differed slightly (Fig. 1
) and we recommend using the probes together as CFXMIX to improve overall coverage of the Chloroflexi phylum. The coverage of subdivision 1 by CFX784 was patchy; mainly 1a and 1b were targeted, including about half of the sludge-clone sequences. Subdivision 3 was well covered by CFX109 with the exception of Roseiflexus and very recently published Roseiflexus-like environmental sequences (Boomer et al., 2002
).
BNR sludge survey
All full-scale sludge samples investigated had common to excessive general filamentous bacterial populations (Table 1). In the two laboratory-scale sludges the number of filaments was lower than in the full-scale sludges (Table 1
). Chloroflexi were ubiquitous in the samples examined by FISH (Table 1
) and the predominant morphotype observed was filamentous, suggesting that this morphotype is common and widespread in the Chloroflexi. In six of 12 full-scale plant samples examined, Chloroflexi were ranked as very common to abundant and in only one full-scale plant were Chloroflexi ranked at few (Table 1
). The fraction of subdivision 1 and 3 Chloroflexi filaments relative to all Chloroflexi filaments was subjectively scored and the results from many observations are recorded in Table 1
. Images representing particular results are shown in Fig. 2
.
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The maximum intended target group could be observed by the use of the two phylum-specific probes (GNSB-941 and CFX1223) in a mixture called CFXMIX. FISH using CFXMIX and EUBMIX showed that the presence of filamentous Chloroflexi was in general very high (e.g. Fig. 2d). As reflected in the selection of images shown in Fig. 2
, the filamentous Chloroflexi commonly occurred inside flocs.
Of the subdivisions investigated, Chloroflexi-1 was less abundant than Chloroflexi-3 (Table 1). This is demonstrated by the relatively low abundance of white filaments in Fig. 2(e)
(Chloroflexi-1) and greater abundance of white filaments in Fig. 2(f)
(Chloroflexi-3). Chloroflexi-1 were generally thin (<1 µm), medium length (1050 µm), smooth filaments mainly found inside bacterial flocs but were occasionally thick (>1 µm), short (<10 µm), segmented filaments bridging flocs. There were several different morphotypes of Chloroflexi-3 including thin, short and long (>50 µm) intrafloc filaments; thick curved filaments; straight, thick (>2 µm) filaments; segmented, thin and thick (>2 µm), long, interfloc-bridging filaments; and thin filaments arranged in bundles and composed of clearly demarcated cells giving the impression of beads.
In the case where a full-scale plant (Noosa) was sampled on several different occasions, the total number of filamentous bacteria and Chloroflexi observed was fairly constant. However, between different Noosa samples, there was great variation in Chloroflexi-3 abundance, ranging from most of the Chloroflexi to only a few (Noosa 1-3, Table 1).
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DISCUSSION |
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Bergeys Manual of Systematic Bacteriology has formally proposed the name Chloroflexi (Garrity & Holt, 2001 ) to supersede the previous common name green non-sulfur (Woese, 1987
) for this phylum of bacteria. Chloroflexi comprises four well-represented subdivisions labelled 1 to 4 in Fig. 1
in accordance with a previous classification of this phylum (Hugenholtz et al., 1998
). Chloroflexi-1 has recently undergone significant expansion due to the addition of many environmental clone sequences, and within this subdivision, there are two large monophyletic groups we have called a and b (Fig. 1
) which are relatively well-targeted by CFX784. The environmental clone sequences of Chloroflexi-1 largely come from pollutant-contaminated habitats, while the only pure culture, UNI-1, is from a recently described upflow anaerobic sludge blanket reactor (Sekiguchi et al., 2001
). Chloroflexi-2 contains the well-known tetrachloroethene dechlorinator Dehalococcoides ethenogenes (Maymó-Gatell et al., 1997
) along with clone sequences (Fig. 1
). No target sites suitable for a Chloroflexi-2 probe were found. Chloroflexi-3 contains most of the pure-cultured representatives of Chloroflexi including Chloroflexus spp., Oscillochloris spp., Roseiflexus castenholzii, Herpetosiphon spp. and Heliothrix oregonensis, the last of which is not shown in Fig. 1
due to only a partial 16S rDNA sequence (871 nt) being available in the public databases. The sequences in Chloroflexi-3 are relatively well targeted by CFX109. This subdivision is formally proposed as the class Chloroflexi (Garrity & Holt, 2001
). Chloroflexi-4 is composed of clone sequences from marine and lake-water environments and, as was the case for subdivision 2, no suitable probe sites were found for this subdivision. In addition to the four well-represented subdivisions, there are at least five further subdivision-level lineages as evidenced by one or a few 16S rDNA sequences for each lineage. These include Thermomicrobium roseum and Sphaerobacter thermophilus which form a monophyletic subdivision in the Chloroflexi (Thermomicrobia in Fig. 1
). These organisms were originally included in subdivision 3 (Hugenholtz et al., 1998
), but this relationship has not held up with the inclusion of additional sequences (Fig. 1
). Furthermore, T. roseum is classified in a separate phylum, the Thermomicrobia, in Bergeys Manual (Garrity & Holt, 2001
) and S. thermophilus as a member of the Actinobacteria (Demharter et al., 1989
; Garrity & Holt, 2001
). However, present phylogenetic evidence indicates that both are members of the Chloroflexi phylum (Fig. 1
).
Chloroflexi-1 contains the majority of 16S rDNA sequences from molecular phylogenetic surveys including 16 of the 17 clones obtained from activated sludge studies shown in Fig. 1. The majority of activated sludge clones in Chloroflexi-1 (11 clones of 16) were generated from full-scale activated sludge biomass (Juretschko et al., 2002
; Snaidr et al., 1997
). However, using FISH, Chloroflexi-3 filaments in full-scale activated sludge processes were more abundant than Chloroflexi-1 filaments (Table 1
). This discrepancy is most likely explained by the non-quantitative nature of PCR-clone libraries which can give skewed representations of the relative abundance of organisms present in a sample largely due to the PCR step (Hugenholtz & Goebel, 2001
). Chloroflexi-3 is best known from hotspring and hypersaline isolates or clones but does contain isolates such as Herpetosiphon species obtained from full-scale activated sludges (Bradford et al., 1996
; Senghas & Lingens, 1985
; Trick & Lingens, 1984
) and a clone from a laboratory-scale process (SBR2022, Bond et al., 1995
). Herpetosiphon was found to be responsible for bulking in this environment and its role in degradation of macromolecules from influent sewage was speculated upon (Reichenbach, 1992
). Recently, an isolate of another filamentous bulking sludge organism, Type 1851 was phylogenetically placed within Chloroflexi-3, most closely related to R. castenholzii but with only 84% identity (Beer et al., 2002
). In our survey, some of the sludge filaments binding the Chloroflexi-3 probe CFX109 could have been Herpetosiphon sp. or Type 1851. However, their common location buried within sludge flocs, precluded us from using their morphology as observed by phase-contrast microscopy or by staining and bright-field microscopy to identify them. By FISH, the filamentous bacteria were highly visible even when present in the centre of flocs, and clearly identifiable due to the phylogenetic basis of the oligonucleotide probe design (see Fig. 2
). This demonstrates the advantages of FISH over the traditional in situ identification method. The application of FISH would also reduce underestimation of filamentous bacteria when they occur within sludge flocs.
Molecular phylogenetic surveys indicate that members of the Chloroflexi are found in numerous diverse habitats apart from activated sludges, such as geothermal springs (Boomer et al., 2002 ); hypersaline mats (Nübel et al., 2001
); the deep subsurface (Chandler et al., 1998
); and aerobic/anoxic (Juretschko et al., 2002
), anaerobic (Sekiguchi et al., 2001
) and dechlorinating enrichments (Maymó-Gatell et al., 1997
). Isolated representatives of the Chloroflexi display a wide range of phenotypes (Hugenholtz et al., 1998
). However, from our study, we cannot deduce the physiological traits of the Chloroflexi filaments in sludge. For example, most of the isolated bacteria belonging to Chloroflexi-3 are phototrophic, but we cannot infer phototrophy for filaments binding the Chloroflexi-3 probe (CFX109) because the target group is too broad and likely contains representatives with a wide range of physiologies. Chloroflexi-specific FISH using the probes described in this study could be used in concert with in situ microautoradiography to determine specific aspects of their phenotype (Lee et al., 1999
) or be used to direct cultivation attempts.
The investigated sludges were not suffering from bulking and the general intrafloc location of the Chloroflexi would not support a potential role in bulking. Bossier & Verstraete (1996) suggest filamentous organisms in activated sludge provide a stabilizing backbone for the three-dimensional microbial aggregates called flocs. The intrafloc location of the Chloroflexi and their relative abundance support this hypothesis which likely explains one important role for these organisms in the activated sludge ecosystem. The potential role of Chloroflexi in macromolecule degradation (Reichenbach, 1992
) should also be evaluated. Therefore, collectively, there appear numerous important roles for Chloroflexi in activated sludge and these roles have not been well studied in relation to Chloroflexi microbial ecology.
Although only sludges from Queensland and New South Wales were inspected for Chloroflexi in the present study, we anticipate that representatives of this bacterial phylum will be ubiquitous in activated sludges because the microbial communities in activated sludge demonstrate a remarkable consistency on a global scale (Seviour & Blackall, 1999 ). For example, the important bulking filament M. parvicella was initially phylogenetically characterized from an Australian isolate (Blackall et al., 1994
) which subsequently proved to be representative of these organisms in activated sludge globally. The probes designed and optimized in this study will likely be useful in the study of wastewater treatment and in microbial ecology studies in general.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Amann, R., Fuchs, B. M. & Behrens, S. (2001). The identification of microorganisms by fluorescence in situ hybridisation. Curr Opinion Biotechnol 12, 231-236.[Medline]
Beer, M., Seviour, E. M., Kong, Y., Cunningham, M. A., Blackall, L. L. & Seviour, R. J. (2002). Phylogeny of the filamentous bacterium Eikelboom type 1851, and design and application of a 16S rRNA targeted oligonucleotide for its in situ identification in activated sludge. FEMS Microbiol Lett 207, 179-183.[Medline]
Blackall, L. L. (1994). Molecular identification of activated sludge foaming bacteria. Water Sci Technol 29, 35-42.
Blackall, L. L., Seviour, E. M., Cunningham, M. A., Seviour, R. J. & Hugenholtz, P. (1994). Microthrix parvicella is a novel, deep branching member of the actinomycetes subphylum. Syst Appl Microbiol 17, 513-518.
Bond, P. L., Hugenholtz, P., Keller, J. & Blackall, L. L. (1995). Bacterial community structures of phosphate-removing and non-phosphate-removing activated sludges from sequencing batch reactors. Appl Environ Microbiol 61, 1910-1916.[Abstract]
Boomer, S. M., Lodge, D. P., Dutton, B. E. & Pierson, B. (2002). Molecular characterization of novel red green nonsulfur bacteria from five distinct hot spring communities in Yellowstone National Park. Appl Environ Microbiol 68, 346-355.
Bossier, P. & Verstraete, W. (1996). Triggers for microbial aggregation in activated sludge. Appl Microbiol Biotechnol 45, 1-6.
Bradford, D., Hugenholtz, P., Seviour, E. M., Cunningham, M., Stratton, H., Seviour, R. J. & Blackall, L. L. (1996). 16S rRNA analysis of isolates obtained from Gram-negative, filamentous bacteria micromanipulated from activated sludge. Syst Appl Microbiol 19, 334-343.
Chandler, D. P., Brockman, F. J., Bailey, T. J. & Fredrickson, J. K. (1998). Phylogenetic diversity of Archaea and Bacteria in a deep subsurface paleosol. Microb Ecol 36, 37-50.[Medline]
Crocetti, G. R., Hugenholtz, P., Bond, P. L., Schuler, A., Keller, J., Jenkins, D. & Blackall, L. L. (2000). Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation. Appl Environ Microbiol 66, 1175-1182.
Daims, H., Bruhl, A., Amann, R., Schleifer, K. H. & Wagner, M. (1999). The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22, 434-444.[Medline]
Dalevi, D., Hugenholtz, P. & Blackall, L. L. (2001). A multiple-outgroup approach to resolving division-level phylogenetic relationships using 16S rDNA data. Int J Syst Evol Microbiol 51, 385-391.[Abstract]
DeLong, E. F., Wickham, G. S. & Pace, N. R. (1989). Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243, 1360-1363.[Medline]
Demharter, W., Hensel, R., Smida, J. & Stackebrandt, E. (1989). Sphaerobacter thermophilus gen. nov., sp. nov., a deeply rooting member of the actinomycetes subdivision isolated from thermophilically treated sewage sludge. Syst Appl Microbiol 11, 261-266.
Eikelboom, D. H. & van Buijsen, H. J. J. (1983). Microscopic Sludge Investigation Manual, 2nd edn. Delft: TNO Research Institute for Environmental Hygiene, Water and Soil Division.
Eikelboom, D., Andreadakis, A. & Andreason, K. (1998). Survey of filamentous populations in nutrient removal plants in four European countries. Water Sci Technol 37, 281-289.
Erhart, R., Bradford, D., Seviour, R. J., Amann, R. I. & Blackall, L. L. (1997). Development and use of fluorescent in situ hybridization probes for the detection and identification of Microthrix parvicella in activated sludge. Syst Appl Microbiol 20, 310-318.
Garrity, G. M. & Holt, J. G. (2001). Chloroflexi phy. nov. In Bergeys Manual of Systematic Bacteriology, 2nd edn, vol. 1, The Archaea and the Deeply Branching and Phototrophic Bacteria, pp. 427446. Edited by D. R. Boone & R. W. Castenholz. New York: Springer.
Gich, F., Garcia-Gil, J. & Overmann, J. (2001). Previously unknown and phylogenetically diverse members of the green nonsulfur bacteria are indigenous to freshwater lakes. Arch Microbiol 177, 1-10.[Medline]
Howarth, R., Unz, R. F., Seviour, E. M., Seviour, R. J., Blackall, L. L., Pickup, R. W., Gwyn Jones, J., Yaguchi, J. & Head, I. M. (1999). Phylogenetic relationships of filamentous sulfur bacteria (Thiothrix spp. and Eikelboom type 021N bacteria) isolated from wastewater-treatment plants and description of Thiothrix eikelboomii sp. nov., Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov and Thiothrix defluvii sp. nov. Int J Syst Bacteriol 49, 1817-1827.[Abstract]
Hugenholtz, P. & Goebel, B. M. (2001). The polymerase chain reaction as a tool to investigate microbial diversity in environmental samples. In Environmental Molecular Microbiology: Protocols and Applications , pp. 31-42. Edited by P. A. Rochelle. New York:Horizon Scientific.
Hugenholtz, P., Goebel, B. M. & Pace, N. R. (1998). Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180, 4765-4774.
Hugenholtz, P., Tyson, G. W., Webb, R. I., Wagner, A. M. & Blackall, L. L. (2001a). Investigation of candidate division TM7, a recently recognized major lineage of the domain bacteria with no known pure-culture representatives. Appl Environ Microbiol 67, 411-419.
Hugenholtz, P., Tyson, G. W. & Blackall, L. L. (2001b). Design and evaluation of 16S rRNA-targeted oligonucleotide probes for fluorescence in situ hybridisation. In Gene Probes: Principles and Protocols , pp. 29-42. Edited by M. Aquino de Muro & R. Rapley. London:Humana.
Jenkins, D., Richard, M. G. & Daigger, G. T. (1993). Manual on the Causes and Control of Activated Sludge Bulking and Foaming. New York: Lewis.
Juretschko, S., Loy, A., Lehner, A. & Wagner, M. (2002). The microbial community composition of a nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analyzed by the full-cycle rRNA approach. Syst Appl Microbiol 25, 84-99.[Medline]
Kanagawa, T., Kamagata, Y., Aruga, S., Kohno, T., Horn, M. & Wagner, M. (2000). Phylogenetic analysis of and oligonucleotide probe development for Eikelboom Type 021N filamentous bacteria isolated from bulking activated sludge. Appl Environ Microbiol 66, 5043-5052.
Klein, M., Friedrich, M., Fishbain, S., Hugenholtz, P., Abicht, H., Rogers, A., Blackall, L. L., Stahl, D. A. & Wagner, M. (2001). Multiple lateral transfer events of dissimilatory sulfite reductase genes between major lineages of Bacteria. J Bacteriol 183, 6028-6035.
Lee, N., Nielsen, P. H., Andreasen, K. H., Juretschko, S., Nielsen, J. L., Schleifer, K. H. & Wagner, M. (1999). Combination of fluorescent in situ hybridization and microautoradiography a new tool for structure-function analyses in microbial ecology. Appl Environ Microbiol 65, 1289-1297.
Liu, J. R., Burrell, P., Seviour, E. M., Soddell, J. A., Blackall, L. L. & Seviour, R. J. (2000). The filamentous bacterial morphotype Nostocoida limicola I contains at least two previously described genera in the low G+C gram positive bacteria. Syst Appl Microbiol 23, 528-534.[Medline]
Liu, J.-R., McKenzie, C. A., Seviour, E. M., Webb, R. I., Blackall, L. L., Saint, C. P. & Seviour, R. J. (2001). Phylogeny of the filamentous bacterium Nostocoida limicola III from activated sludge. Int J Syst Evol Microbiol 51, 195-202.[Abstract]
Maymó-Gatell, X., Chien, Y., Gossett, J. M. & Zinder, S. H. (1997). Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science 276, 1568-1571.
Neef, A., Amann, R., Schlesner, H. & Schleifer, K.-H. (1998). Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes. Microbiology 144, 3257-3266.[Abstract]
Nübel, U., Bateson, M. M., Madigan, M. T., Kühl, M. & Ward, D. M. (2001). Diversity and distribution in hypersaline microbial mats of bacteria related to Chloroflexus spp. Appl Environ Microbiol 67, 4365-4371.
Reichenbach, H. (1992). The genus Herpetosiphon. In The Prokaryotes A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications , pp. 3785-3805. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York:Springer.
Sekiguchi, Y., Takahashi, H., Kamagata, Y., Ohashi, A. & Harada, H. (2001). In situ detection, isolation, and physiological properties of a thin filamentous microorganism abundant in methanogenic granular sludges: a novel isolate affiliated with a clone cluster, the green non-sulfur bacteria, subdivision I. Appl Environ Microbiol 67, 5740-5749.
Senghas, E. & Lingens, F. (1985). Characterization of a new gram-negative filamentous bacterium isolated from bulking sludge. Appl Microbiol Biotechnol 21, 118-124.
Seviour, R. J. & Blackall, L. L. (1999). The Microbiology of Activated Sludge. London: Kluwer.
Snaidr, J., Amann, R., Huber, I., Ludwig, W. & Schleifer, K.-H. (1997). Phylogenetic analysis and in situ identification of bacteria in activated sludge. Appl Environ Microbiol 63, 2884-2896.[Abstract]
Stahl, D. A. & Amann, R. (1991). Development and application of nucleic acid probes. In Nucleic Acid Techniques in Bacterial Systematics , pp. 205-248. Edited by E. Stackebrandt & M. Goodfellow. Chichester:Academic Press.
Trick, I. & Lingens, F. (1984). Characterization of Herpetosiphon spec. a gliding filamentous bacterium from bulking sludge. Appl Microbiol Biotechnol 19, 191-198.
Wagner, M., Amann, R., Kämpfer, P., Assmus, B., Hartmann, A., Hutzler, P., Springer, N. & Schleifer, K.-H. (1994). Identification and in situ detection of Gram-negative filamentous bacteria in activated sludge. Syst Appl Microbiol 17, 405-417.
Woese, C. R. (1987). Bacterial evolution. Microbiol Rev 51, 221-271.
Received 9 February 2002;
revised 21 March 2002;
accepted 8 May 2002.