Advanced Wastewater Management Centre, The University of Queensland, St Lucia, 4072, Australia
Correspondence
Christine Yeates
cyeates{at}awmc.uq.edu.au
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are listed in Table 2.
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INTRODUCTION |
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The crux of reliable PGS probe design is the database of sequences from which probes can be designed. As more sequences become available, there is often a need to redesign probes to recognize cross-reacting groups or an altered phylogenetic group definition (e.g. Daims et al., 1999). Frequently, the information content of the 16S rRNA is a limiting factor. To overcome this, PGS probes have been designed to target other genes, most notably the 23S rRNA, such as the probes for the subphyla Gammaproteobacteria (GAM42a) and Betaproteobacteria (BET42a) in helix 42 of the 23S rRNA (Manz et al., 1992
). However, there is a paucity of sequences for 23S rDNA in comparison to those for 16S rDNA and the phylogeny of the two genes may not be congruent. These issues are particularly relevant for investigations of uncultivated organisms.
Application of FISH probes requires optimization of the stringency of binding to avoid non-specific results. This is generally achieved with the use of controls such as cultured organisms whose sequence in the rRNA probe target zone has zero or one mismatch to the designed oligonucleotide sequence. With PGS FISH probes that target uncultured bacterial groups, probe optimization is often carried out using environmental samples known to contain the organism and determined by reduction in fluorescent signal intensity of specific bacteria as observed by epifluorescence or confocal laser scanning microscopy. However, the specificity of any single probe to the target group is not guaranteed since the sample includes a large proportion of unknown organisms. A solution to this limitation is the design and application of multiple PGS probes that encompass the phylogenetic hierarchy of the uncultured target group. This has been described as the top-to-bottom approach (Amann et al., 1995). In any case, methodical re-evaluation and redesign of PGS probes should be practised (Daims et al., 1999
; Loy et al., 2002
) since the increasing and thus changing rDNA database affects the phylogenetic placement of sequences and consequent PGS FISH probe design (Amann et al., 1995
). With the top-to-bottom approach, two of the most widely used PGS probes are GAM42a and BET42a, targeting helix 42 of the 23S rRNA from the subphyla Gammaproteobacteria and Betaproteobacteria, respectively (Manz et al., 1992
). Use of these probes in FISH studies of wastewater communities has given unexpected results.
Microbial communities involved in wastewater treatment processes have been studied by FISH since 1993 (Wagner et al., 1993), and the procedure has led to the detection of several bacteria pivotal to success or failure of these processes (Crocetti et al., 2000
, 2002
; Juretschko et al., 1998
; Rosselló-Mora et al., 1995
; Schmid et al., 2000
; Wagner et al., 1994a
, b
). Glycogen-accumulating organisms (GAOs) are considered important destabilizers of enhanced biological phosphorus removal (EBPR) due to their capacity to overgrow the organisms responsible for EBPR, the polyphosphate-accumulating organisms (PAOs). The taxon Candidatus Competibacter phosphatis' (henceforth called Competibacter) was recently identified and linked to the in situ GAO phenotype (Crocetti et al., 2002
) and numerous PGS probes targeting Competibacter have been reported (Crocetti et al., 2002
; Kong et al., 2002
; Nielsen et al., 1999
). Several researchers have noted that some cells hybridizing these Competibacter-specific FISH probes did not bind GAM42a, despite the phylogenetic placement of Competibacter in the Gammaproteobacteria subphylum (Crocetti et al., 2002
; Kong et al., 2002
; Liu et al., 2001
; Nielsen et al., 1999
). Nielsen et al. (1999)
suggested this was related to the specificity of GAM42a which was originally designed based on a limited number of 23S rRNA sequences. Crocetti et al. (2002)
and Kong et al. (2002)
found some cells bound probes specific for Competibacter as well as the BET42a probe. Crocetti et al. (2002)
quantified different bacterial groups and in two separate laboratory-scale processes, 88 % and about 50 % of the cells binding GAOQ431 (for Competibacter) also bound BET42a (for Betaproteobacteria).
These results highlight the difficulties associated with the application of PGS probes for organisms without cultured representatives and the need for continual PGS probe re-evaluation. Research reported in this study investigated why the probing inconsistencies summarized above with Competibacter were occurring. This involved examining the sequence for helix 42 within the 23S rRNA gene from Competibacter. The study revealed that the probing inconsistencies were related to polymorphisms within the probe-binding site in helix 42 of the 23S rRNA.
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METHODS |
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Analysis of sequence data.
Sequences for the 16S and 23S rDNAs were compiled and preliminarily analysed by BLAST (Altschul et al., 1997). The 23S rDNA sequence components of the clones (10601085 nt) were aligned in ARB (http://www.arb-home.de) and once missing or ambiguous characters were excluded, the phylogenetically analysed datasets contained 939 nt (23S rDNA). rDNA data analysis was performed by using previously reported methods (Björnsson et al., 2002
). GenBank accession numbers for the sequences are presented in Table 2
.
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The GAM42a, BET42a and GAM42_C1033 probes all have only one base difference between them (Table 1). To increase the differentiation between probe and target due to the single base mismatch, unlabelled competitor probes were added (Manz et al., 1992
). FISH using GAM42a, BET42a or GAM42_C1033 at all times included the labelled probe with the two others unlabelled and functioning as competitors at the same concentration.
The formamide concentration for optimum stringency of GAM42_C1033 was determined by performing FISH at formamide concentrations from 25 to 50 % (5 % increments) with paraformaldehyde-fixed pure cultures of a gammaproteobacterium and a betaproteobacterium isolated from activated sludge. The isolates were confirmed as members of these subphyla by analysis of their 16S rDNA sequences. Additionally, their 23S rDNA sequence at the GAM42a/BET42a probe target in helix 42 confirmed they possessed the correct probe target string and thus they were suitable as negative controls for probe GAM42_C1033. When the competitor probes were present, GAM42_C1033 did not bind to the control cultures at any formamide concentration. Therefore, the optimal stringency was determined to be at the formamide concentration where GAM42_C1033 no longer bound to the negative control cultures without the unlabelled competitor probes.
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RESULTS |
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The complete amplicon encompassing the 16S rDNA, IGS region and 23S rDNA was sequenced from two Q sludge clones and ten T sludge clones. Partial sequences only of the 16S rDNA portion and the 23S rDNA portion were generated from four Q sludge clones and three T sludge clones. Sequence analysis of the clones showed that for the T sludge the large number of OTUs did not indicate high diversity at the 16S rDNA or 23S rDNA level, because the clone sequences were 96100 % and 9299 % identical to each other, respectively. Examination of the 10 full-length sequences of the amplicons generated from the T sludge revealed that many of the restriction enzyme sites existed within the IGS (data not shown) and were likely to be responsible for the generation of the relatively large number of OTUs. The IGS regions from the clones of both sludges were found to contain genes for transfer RNAs (data not shown).
The DNA fragments sequenced here were derived from cloned PCR products that are assumed to come from the same cells. The 16S rDNA portion of each cloned fragment showed 96100 % identity to previously reported Competibacter sequences (Crocetti et al., 2002), identifying the source cells for the cloned fragments as Competibacter. BLAST searches using the 23S rDNA portions of the sequences showed the closest relatives of Competibacter to be Gammaproteobacteria at 8893 % and these sequences were included in the phylogenetic analysis shown in Fig. 1
. Phylogenetic analysis of the 23S rDNA sequences placed Competibacter as a monophyletic group within the Gammaproteobacteria and outside the Betaproteobacteria (Fig. 1
).
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Application of GAM42_C1033 probe
No binding of GAM42_C1033 to either of the negative control cultures was observed at any formamide concentration when unlabelled competitor probes were present.
The sludge from a laboratory-scale anaerobicaerobic cycling SBR demonstrated to have the GAO phenotype was examined by FISH with GAM42a, BET42a, GAM42_C1033 and GAOQMIX. Fig. 2(ae) confirms the presence of numerous Competibacter cells (GAOQMIX binding) in this SBR. Most of these cells also bound either GAM42a or GAM42_C1033 (Fig. 2a
, arrowed) which is consistent with the cloning results from the Q and T sludges (see above). However, there are some GAOQMIX-binding cells that also bound BET42a (Fig. 2b
, arrowed) and some that bound none of the 23S rRNA-targeted probes (Fig. 2c
, arrowed, and Fig. 2d
). To examine the in situ arrangements of these cells, a non-homogenized sample from the laboratory-scale SBR was examined (Fig. 2e
). Cells binding both GAM42_C1033 and GAOQMIX probes, or GAM42_C1033 alone or BET42a alone tended to exist as discrete clusters.
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DISCUSSION |
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Phylogenetic analysis of 16S and 23S rDNA sequences from Competibacter
The substantial 16S rDNA sequence database for Competibacter was initiated with partial sequences from denaturing gradient gel electrophoresis studies (Liu et al., 2000; Nielsen et al., 1999
) which were complemented by near-complete sequences (Crocetti et al., 2002
; Dabert et al., 2001
) and additional partial sequences (Kong et al., 2002
) from cloning studies. The 16S rDNA sequences described in our study were highly identical to other Competibacter sequences (Crocetti et al., 2002
), confirming that Competibacter was the source for the linked 23S rDNA sequence information. The 23S rDNA sequence information therefore reflects both the GAM42a target site and phylogeny according to 23S rDNA for this organism.
The Proteobacteria, a much-studied and well-recognized phylum in the bacterial domain, is phylogenetically divided into several subphyla, including Betaproteobacteria and Gammaproteobacteria. Initially, the subdivisions were created with 16S rRNA data from pure-cultured representatives, but currently the 16S rRNA database is overwhelmingly composed of sequences obtained from cloning studies of 16S rDNA from diverse samples. In contemporary phylogenetic analyses, the Betaproteobacteria subphylum has been found to be an infrataxon of the Gammaproteobacteria, not a sister taxon (Ludwig & Klenk, 2001). The FISH probes used to identify these two subphyla are in the exact same part of the 23S rRNA and differ from each other by only one nucleotide.
The reason for probing inconsistencies
The research described in this paper was motivated by several reported probing inconsistencies where many Competibacter (a member of the Gammaproteobacteria) cells did not bind GAM42a (for Gammaproteobacteria) (Crocetti et al., 2002; Kong et al., 2002
; Liu et al., 2001
; Nielsen et al., 1999
) and many Competibacter cells did bind BET42a (for Betaproteobacteria) (Crocetti et al., 2002
; Kong et al., 2002
). The 23S rDNA portion of the clones was used to determine the sequence of the probe region (positions 10271043) and specifically the only variable nucleotide in the probe region at position 1033. Of the 19 Competibacter clones, 8 had the GAM42a probe target, but 11 had G at position 1033. Due to non-canonical base pairing, G-A or G-T hybrids can exist, and therefore GAM42a or BET42a could bind to Competibacter cells with G at position 1033. It is known that G-T and G-A hybrids are stronger than many other mismatches such as A-A, T-T, C-T or C-A (Lathe, 1990
). The addition of an unlabelled competitor probe is required to differentiate the relatively weak A-A or T-T mismatch at position 1033 between GAM42a-Betaproteobacteria cells and BET42a-Gammaproteobacteria cells, respectively. So therefore, the absence of a competitor probe in FISH experiments studying organisms like Competibacter with the previously unknown G at position 1033 in their 23S rRNA, could readily lead to hybridization between them and GAM42a (G-A) or BET42a (G-T). The Competibacter cells with the perfect GAM42a probe target would bind GAM42a, but the Competibacter cells with G at position 1033 were likely to be responsible for the reported probing inconsistencies (Crocetti et al., 2002
; Kong et al., 2002
; Liu et al., 2001
; Nielsen et al., 1999
).
Helix 42 of the 23S rRNA a good choice for probe design?
At the time GAM42a and BET42a were designed (Manz et al., 1992) their future comprehensiveness could not be guaranteed. Position 10271043 was the best region found to discriminate the Gammaproteobacteria and Betaproteobacteria, but even so the probes only vary by 1 nt at position 1033, while sequences from all other bacteria varied by at least 2 nt within this region. Manz et al. (1992)
couched this probe design by recommending that the probes be used in combination with other more specific probes. The GAM42a and BET42a probes were recently re-evaluated using the current database of 23S rRNA sequences (Loy et al., 2002
). The coverage of Gammaproteobacteria by GAM42a was found to be 90·8 % and the coverage of Betaproteobacteria by BET42a was 92·6 %. It therefore appears, based on the current sequences in the 23S rRNA database, that these probes are still suitable. However, the current study reveals that there are Gammaproteobacteria that have a different nucleotide at the differentiating position for these subphylum-specific probes. The secondary structure of the probe-binding region shows that the nucleotide at position 1033 does not pair with another nucleotide (Ludwig et al., 1995
). Therefore, the selective constraints are likely to be different for this position. It appears that this is the case with some of the Competibacter cells seen in this study, but has not been reported for any other available bacterial sequences in the current 23S rRNA database. Since some cells in the full-scale wastewater treatment plant bound GAM42_C1033 but not GAOQMIX (Fig. 2f
), bacteria besides Competibacter have G at position 1033, but are yet to be described. The presence of organisms with the other possible base (C) at this position should also be explored.
This research has demonstrated some limitations of BET42a and GAM42a for highlighting Competibacter which belong in the Gammaproteobacteria radiation sensu Ludwig & Klenk (2001). Bacteria other than Competibacter were also targeted by GAM42_C1033 in full-scale sludge samples, but their phylogenetic placement is not known. The sequence of the bacterial 23S rDNA from positions 10271043 should be comprehensively studied, especially in Betaproteobacteria and Gammaproteobacteria, and also in bacteria from other Proteobacteria lineages. The approach used in this study could be employed to address this issue with mixed microbial communities. It could be extended to the study of larger amplicons encompassing the complete 16S rDNA and more of the 23S rDNA. The ubiquity of Proteobacteria and the ecological significance of many of their members, has made GAM42a and BET42a two of the most widely used of all probes. Microbiologists should re-evaluate how these probes are used in the top-to-bottom approaches to community structure determination.
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ACKNOWLEDGEMENTS |
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Received 8 November 2002;
revised 2 February 2003;
accepted 13 February 2003.