A refined set of rRNA-targeted oligonucleotide probes for in situ detection and quantification of ammonia-oxidizing bacteria

Ammonia-oxidizing bacteria (AOB) of the betaproteobacterial genera Nitrosomonas and Nitrosospira are key nitrifying microorganisms in many natural and engineered ecosystems. Since many AOB remain uncultured, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been one of the most widely used approaches to study the community composition, abundance, and other features of AOB directly in environmental samples. However, the established and widely used AOB-specific 16S rRNA-targeted FISH probes were designed up to two decades ago, based on much smaller rRNA gene sequence datasets than available today. Several of these probes cover their target AOB lineages incompletely and suffer from a weak target specificity, which causes cross-hybridization of probes that should detect different AOB lineages. Here, a set of new highly specific 16S rRNA-targeted oligonucleotide probes was developed and experimentally evaluated that complements the existing probes and enables the specific detection and differentiation of the known, major phylogenetic clusters of betaproteobacterial AOB. The new probes were successfully applied to visualize and quantify AOB in activated sludge and biofilm samples from seven pilot- and full-scale wastewater treatment systems. Based on its improved target group coverage and specificity, the refined probe set will facilitate future in situ analyses of AOB.


Introduction
Nitrification, a key process in the biogeochemical nitrogen cycle, is the microbially mediated 42 oxidation of ammonia to nitrite and subsequently to nitrate. For many decades nitrification was 43 perceived as a process always performed by two functional groups of aerobic, FISH has been applied since 1995 (Wagner et al., 1995) in numerous studies to investigate β-90 AOB in aquatic systems, especially WWTPs, and has proven to be of immense value in this 91 context. For example, Nitrosomonas (formerly "Nitrosococcus") mobilis was identified as the were studied (Okabe et al., 1999;Schramm et al., 1998). Application of this approach to 99 calculate volumetric reaction rates even revealed the in situ whole-cell kinetics of uncultured 100 β-AOB (Nitrosospira spp.) (Schramm et al., 1999). In another study, FISH and quantitative 101 PCR (qPCR) were used to detect β-AOB in granular activated sludge. These data were the basis 102 for two ecophysiological models, which address the observed (and unexpected) higher in situ    hybridized to a β-AOB specific probe mix and to the EUB338 I-III probe mix (Table 1) (Table 3). In particular, the broad-range probe Nso1225 still covers the vast majority 258 of β-AOB, whereas probe Nso190 (originally also designed to target all β-AOB) has a highly sequences with 1-2 weak nucleotide mismatches to the probes are taken into account (Table 3).  (Table 1). Probe Ncom1025 would also potentially detect the 273 majority of the N. mobilis lineage and could thus bind to the same organisms as probe NEU 274 (Table 3). In our study, unspecific probe hybridization was confirmed in tests using biofilm  that were arranged as tetrads within loose aggregates ( Fig. 2A). The morphology of these 280 organisms was very dissimilar from the usual size and shape of the β-AOB cells and cell 281 clusters, which were also present in this sludge ( Fig. 2A). Moreover, a test hybridization revealed that the tetrad-shaped cells were also labelled by probe Gam42a (Fig. S1), suggesting 283 that these organisms were Gammaproteobacteria and unspecifically labelled by the previously 284 published AOB probes. In summary, the in silico evaluation and test hybridizations 285 demonstrated that several of the previously published FISH probes for β-AOB suffer from an 286 insufficient target group coverage and specificity. As a consequence, further use of the affected 287 probes for the in situ identification and quantification of the respective β-AOB groups should 288 be performed with caution by taking into account their actual specificities (Table 3).  cluster 8 (Fig. 3). In addition, the old probe Nsv443, which offered good coverage of the target 310 clade but unsatisfactory target specificity, was replaced with the new probe Nsp441 for the 311 genus Nitrosospira (comprising clusters 0-4) (Fig. 3). For most of the new probes, we designed  Table 1. According to an in silico analysis, the new probes in combination with the 317 designated competitors (Table 1) display a very good coverage and specificity for their target 318 β-AOB clusters, including cultured isolates as well as environmental sequences from 319 uncultured β-AOB (Table 3). Notably, the newly designed probes (with competitors) also 320 display a very high specificity with respect to non-target matches outside of the β-AOB (in 321 silico evaluation based on the SILVA SSU NR release 138 and the ARB "probe match" tool 322 with 0-2 weighted mismatches as search criterion). Merely for probe Nm_143_1010, less than 323 ten non-target betaproteobacterial sequences were found that are not covered by the competitor 324 for this probe (Table 1), and probe Nsp441 (Table 1)   However, the number for Nsp441 is low compared to the previously published probe Nsv443 327 that would potentially detect more than 500 non-target organisms. Considering that any 328 environmental sample likely contains non-target organisms, which are not present in the current 329 sequence databases, we recommend to use the newly designed β-AOB cluster-specific probes 330 in combination with the broad-range probe Nso1225 (Table 3)

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The applicability of the new probe set was tested in FISH experiments with nitrifying activated 350 sludge samples from different WWTPs in Austria, Germany, and Denmark (Table 2). In 351 particular, the newly designed probes targeting Nitrosomonas cluster 6 (Nm_OL_703) and 352 cluster 7 (Nm_EU_136) were applied to the same sludge samples that we had already used to 353 confirm the cross-hybridization of the previously published probes Cl6a192, NEU, and observed, and the hybridization patterns appeared to be completely consistent (Fig. 1B, D).

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This result is in agreement with the in silico analysis, which predicted that probes Nm_OL_703 357 and Nm_EU_136 do not target the same sequences in the database (Table 3). Furthermore, 358 when the "new AOB probe mix" (Table 1)  the novel uncultured lineage DK-WWTP (Fig. 3), was applied to activated sludge from WWTP 365 Esbjerg East where it showed specific signals with a morphology similar to that typically 366 portrayed by β-AOB (Fig. 4). We did not have access to environmental samples that were

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A quantitative comparison of the "old" and "new" AOB probe mixes (Table 1) was carried out 376 with activated sludge samples from seven WWTPs (Table 4). For six of these sludge samples, 377 highly similar biovolume fractions of β-AOB were measured by quantitative FISH with either 378 probe set (Table 4). Only for the CP Kelco sludge we obtained a much lower biovolume 379 fraction of β-AOB with the newly designed probe set than with the previously published probes (Table 4). This difference is most likely explained by the better specificity of the newly 381 designed probes, which did not stain the abundant tetrad-shaped cells in this sludge (Fig. 2).

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For all samples and experiments, high "congruency" values >90% were obtained (Table 4). 383 This value indicates that the fluorescence signals in the β-AOB probe mix images occupied  WWTPs, and, importantly, the data for CP Kelco show that the new mix also offers a better 391 specificity for β-AOB than the previously published probes (Fig. 2, Fig. S1).

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The newly designed 16S rRNA-targeted oligonucleotide probes offer a similar or better 395 coverage of different β-AOB lineages than the established probes (Table 3), and they do not 396 show the unspecific hybridization patterns that were observed for some of the previously 397 designed probes when applied to complex microbial communities ( Fig. 1 and 2). However,  (Table 3), which is why no new broad-range probe for β-AOB was designed in this study. Declaration of competing interest 415 The authors declare that they have no known competing financial interests or personal 416 relationships that could have appeared to influence the work reported in this paper.  Table 1. rRNA-targeted oligonucleotide probes used in this study. Probe Gam42a binds to the 23S rRNA, whereas all other listed probes target 702 binding sites on the 16S rRNA.