Design and evaluation of 16S rRNA sequence based oligonucleotide probes for the detection and quantification of Comamonas testosteroni in mixed microbial communities
© Bathe and Hausner; licensee BioMed Central Ltd. 2006
Received: 10 April 2006
Accepted: 13 June 2006
Published: 13 June 2006
The β-proteobacterial species Comamonas testosteroni is capable of biotransformation and also biodegradation of a range of chemical compounds and thus potentially useful in chemical manufacturing and bioremediation. The ability to detect and quantify members of this species in mixed microbial communities thus may be desirable.
We have designed an oligonucleotide probe for use in fluorescent in situ hybridization (FISH) and two pairs of PCR primers targeting a C. testosteroni subgroup. The FISH probe and one of the PCR primer pairs are suitable for quantification of C. testosteroni in mixed microbial communities using FISH followed by quantitative image analysis or real-time quantitative PCR, respectively. This has been shown by analysis of samples from an enrichment of activated sludge on testosterone resulting in an increase in abundance and finally isolation of C. testosteroni. Additionally, we have successfully used quantitative PCR to follow the C. testosteroni abundance during a laboratory scale wastewater bioaugmentation experiment.
The oligonucleotides presented here provide a useful tool to study C. testosteroni population dynamics in mixed microbial communities.
Comamonas testosteroni is a ubiquitously occuring β-proteobacterial species which has been isolated from aquatic as well as terrestrial environments. It has been shown to be capable of transformations of steroid compounds [1–4] and also of degradation of aromatic hydrocarbons [5–10]. C. testosteroni is thus of interest for potential biotechnological applications such as chemical transformations in fine chemical manufacturing  and bioremediation processes [7, 12], as indicated by laboratory results.
Due to the widespread environmental distribution and the potential relevance of C. testosteroni in biotransformation processes, it may be of interest to follow the population dynamics of this species over time in a mixed-species environment. Since culture-dependent methods are not sufficiently accurate to detect and quantify one particular species within a mixed bacterial community, methods targeting the 16S rRNA and/or its gene(s) need to be applied to such a problem, as has already been done in previous investigations [13–16]. Probes for species of the genera Comamonas and Delftia (COM1424 , and PPT ) as well as for species of the family Comamonadaceae (CTE ) have been described earlier, but might lack the desired specificity. The use of reporter genes such as gfp or dsRed is also possible, but only applicable to environments with intentionally introduced labelled strains and not to the investigation of uncharacterized environmental samples. Additionally, the introduction of reporter genes may impair the environmental fitness of the introduced strain , and variations in reporter expression might interfere with detection. The use of reporter gene carrying strains also means working with genetically modified organisms, which may not be possible in all cases.
The following study reports on the development of 16S rRNA sequence based oligonucleotide probes which are suitable for detection and quantification of Comamonas testosteroni in mixed cultures using PCR and/or fluorescent in situ hybridization (FISH).
16S rRNA sequence diversity of Comamonas testosteroni
PCR primers and FISH probes used in this study
Pure culture evaluation of the designed oligonucleotides
All oligonucleotides were evaluated in situ on a selection of proteobacterial pure cultures including all C. testosteroni strains available from the German Collection of Microorganisms (DSM). The tests revealed that the newly designed primers and probe were specific for the strains of this species. Primer pair CteA1 yielded a PCR product with a size of 571 bp, whereas primer pair CteA2 produced an amplicon of 167 bp length. It should be noted here that both primer pairs produced products from all examined C. testosteroni cultures, indicating that all tested cultures fell within clade A. A clade B culture was not available for testing, but since all developed oligonucleotides except primer CteA2-rev show 3 or more mismatches with the available clade B sequences, it can be expected that these strains would be excluded from detection by PCR or FISH. The CY3 labelled FISH probe CteA gave well-fluorescing signals when used with 30% of formamide in the hybridization buffer. Cells of all available C. testosteroni isolates except of strain DSM 6781 gave well-fluorescing signals when FISH was applied to paraformaldehyde fixed cells. To identify the reason for the reduced signal intensity in strain DSM 6781, its 16S rRNA sequence was determined [EMBL:AM113745]. The sequence had a central mismatch with probe CteA. Accordingly, we suggest to work with a degenerate version of the probe with the sequence CAT GAC CCG (G/A)GG ATA TTA GC. All non-target strains tested did not produce PCR products with primers CteA1 and CteA2, nor did they give a fluorescence signal when using probe CteA in FISH, although low background fluorescence was observed in some cultures.
Analysis of testosterone enrichment cultures and isolates
In order to test the developed oligonucleotides on an unknown mixed culture, we provided conditions for the enrichment of C. testosteroni from activated sludge by two successive enrichment steps in liquid media containing testosterone as the sole source of carbon and energy and finally by spreading samples of the second culture on agar plates composed of the same medium. Samples of the activated sludge, the two enrichment cultures, and of five isolates obtained from the testosterone plates were then analysed by PCR and FISH.
In order to assess the suitability of the oligonucleotides for quantification of C. testosteroni in mixed cultures, primer pair CteA2 was used in real-time quantitative PCR and probe CteA was used for quantitative image analysis. The primer pair Eub341F/534R and the FISH probes Eub338 mix were used in parallel as a measure of total cell abundance. The ratios between C. testosteroni specific signals and the eubacterial specific signals will produce a fractional number representing the C. testosteroni abundance in a given sample. This approach has been applied to cells labelled by FISH  or fluorescent proteins  and has also been used in real-time PCR quantification .
Determination of C. testosteroni abundance in a reactor bioaugmentation experiment via quantitative PCR
A further test of the qPCR primers CteA2 in mixed culture experiments was done on DNA samples extracted from 3 laboratory scale biofilm reactors in an experiment to introduce degradation of 3-chloroaniline (3CA) via conjugal transfer of the 3CA-degradative plasmid pNB2. A full description of the experiment can be found elsewhere . Briefly, all reactors where initially inoculated with an activated sludge-derived mixed bacterial culture. Reactor PB then received a Pseudomonas putida strain and reactor P received a Comamonas testosteroni strain, both carrying plasmid pNB2, whereas control reactor N did not receive additional bacteria. The P. putida strain was not able to degrade 3CA, whereas the C. testosteroni strain was. All reactors received a feed containing 3CA and four easily degradable carbon sources (glucose, gluconate, acetate, citrate). Reactor PB started to degrade 3CA after an initial lag phase, and finally, a number of C. testosteroni pNB2-transconjugants capable of 3CA-degradation could be isolated from this reactor. Reactor P showed 3CA-degradation from the beginning of the experiment on, whereas no degradation could be observed in reactor N.
In our study, we present two sets of PCR primers and an rRNA directed oligonucleotide probe for use in FISH which can be applied for detection and quantification of a Comamonas testosteroni subgroup in mixed bacterial populations.
Relative quantifications (target group vs. a measure of whole eubacterial cell abundance) conducted by real-time PCR and FISH followed by image analysis demonstrate the same development of C. testosteroni populations during an enrichment process using testosterone as a carbon source. However, since neither of the two methods measure cell number, but rRNA gene copy number (PCR) and cell area (FISH), a direct numerical comparison is not possible here. Both quantification methods may display a sample-inherent bias, when the average 16S rRNA gene copy number per cell differs from the number of 16S rRNA gene copies per C. testosteroni cell in PCR or the average cell area in a sample differs from the average area of a C. testosteroni cell in FISH. Additionally, we applied the real-time PCR approach presented here to successfully determine abundance of C. testosteroni in a set of laboratory-scale wastewater treatment reactors inoculated with activated sludge bacteria and additionally bioaugmented with different bacteria of which one was a C. testosteroni strain.
Comparison of the target groups of the new probe set with the previously described probes targeting Comamonas species reveals that our oligonucleotides exclusively match sequences of Comamonas testosteroni strains. Primer CteA2-rev is somewhat less specific, but the combination with primer CteA2-for ensures specificity of the primer pair. In contrast, probe CTE matches with most sequences of the genera Comamonas, Acidovorax and Hydrogenophaga, but has mismatches with the sequences of C. koreensis, Comamonas sp. 12022, C. testosteroni SMCC B329 (clade B), and of the Delftia species among the sequences shown in Fig. 1. Probe PPT targets mainly Comamonas and Acidovorax, but both PPT and CTE also target a range of sequences from other genera of the Comamonadaceae . Among the previously published probes, COM1424 has the narrowest range targeting predominantly Comamonas and Delftia .
The oligonucleotides presented here are a useful tool to study C. testosteroni population dynamics in mixed microbial communities, when considering the method-inherent bias disussed above.
Bacterial strains and culture conditions
The following strains of Comamonas testosteroni were used: DSM 38, 1455, 1622, 6781, 11414, 12678, 50241, 50242, 50244, LMG 19554, SB3  and SB4 . In addition, C. aquatica ATCC 11330, C. nitrativorans DSM 13191, C. koreensis TK17 , C. terrigena TK41 , Delftia acidovorans ATCC 15668, Delftia tsuruhatensis SB5 , Pseudomonas putida DSM 291, Pseudomonas aeruginosa PAO1 , Novosphingobium capsulatum DSM 30196, Aeromonas hydrophila DSM 30187, Escherichia coli DH5α, Serratia ficaria ATCC 33105, Acinetobacter calcoaceticus BD413 , Ralstonia eutropha DSM 531, and Burkholderia cepacia H111  were included as non-target organisms. All cultures were maintained on nutrient broth at 30°C.
Enrichment of Comamonas testosteroni from activated sludge
Activated sludge was obtained from the aeration basin of the municipal wastewater treatment plant Garching (Germany). A sample representing approximately 1 mg of dry weight was inoculated into 25 ml of MMO medium  containing 0.05% testosterone (added as a 10% solution in DMSO) as the sole carbon source and incubated for 4 d (30°C, 150 rpm), until the disappearance of the testosterone crystals indicated degradation of the compound. 250 μl of this culture was inoculated into 25 ml of fresh medium and again incubated for 2 d, and a sample was spread onto MMO plates with testosterone which were incubated for 7 d at 30°C.
Primer and probe design and evaluation
A multiple sequence alignment and calculation of a phylogenetic tree of a selection of 16S rRNA gene sequences of the genus Comamonas was done using ClustalX 1.81  (see Fig. 1 for the sequences included in the analysis). Sequence stretches showing identities within C. testosteroni clade A sequences, but having mismatches to the remaining sequences, were used to design species-specific oligonucleotides. The sequences of the oligonucleotides which were finally used in this study are shown in Table 1.
The specificities of the primers and probes developed in this study as well as those of the probes COM1424, CTE, and PPT were checked using BLAST within the NCBI website  as well as the ProbeMatch tool in the RDP-II .
For PCR from pure cultures, cells suspended in water were used as template DNA. DNA from activated sludge and the enrichment cultures was isolated as described previously .
Conventional PCR was carried out using a Mastercycler gradient (Eppendorf, Hamburg, Germany) and Qiagen HotStarTay DNA Polymerase (Qiagen, Hilden, Germany). A single reaction contained 0.2 μM of each primer CteA1-for and CteA1-rev, 0.2 mM of each dNTP, 1.5 mM MgCl2, 0.02 U/μl of Taq Polymerase in a volume of 20 μl. Either 1 μl of cell suspension or 10 ng of DNA was added as template DNA. The PCR protocol consisted of an initial denaturation of 15 min at 95°C followed by 30 cycles of 30 sec at 95°C, 1 min at 58°C and 2 min at 72°C and was concluded by a final extension of 8 min at 72°C. To exclude the possibility of PCR inhibition, parallel positive control reactions using the general eubacterial primers 341F and 534R were conducted for each template. The 16S rRNA genes of isolates were partially amplified using primers 27F and 1492R  and custom sequenced using primer 341F by MWG Biotech (Ebersberg, Germany). PCR products were analysed on 2% TAE agarose gels.
Real-time PCR was carried out in an ABI GeneAmp 5700 device (Applied Biosystems, Foster City, USA) using the Qiagen Quantitect SYBR Green PCR Kit. Each reaction was performed in triplicate and contained 12.5 μl of PCR-Mastermix, 0.2 μM of either each primer CteA2-for and CteA2-rev or 341f and 534r, 5 mM MgCl2, and 5 μl of template DNA in a final volume of 25 μl. The thermal protocol consisted of 15 min at 95°C followed by 40 cycles of 15 sec at 95°C and 60 sec at 60°C. The same serial 10fold dilutions of genomic DNA of Comamonas testosteroni DSM 50244T were used to generate standard curves for both primer pairs.
FISH and image analysis
FISH was performed as described , using paraformaldehyde as a fixative. For elucidation of the hybridization conditions for the probe CteA, hybridization temperature was kept constant at 46°C. The formamide concentration in the hybridization buffer was varied by 10% increments. C. testosteroni LMG 19554 and C. aquatica ATCC 11330 were used as positive and negative controls, respectively. The formamide concentration which resulted in clear strong signals for C. testosteroni and no signals for C. aquatica was used in subsequent hybridizations. Probe CteA was labelled with the cyanine dye CY3, whereas the EUB probe mix (an equimolar mixture of probes EUB338, EUB338-II, and EUB338-III) was labelled with the cyanine dye CY5. All hybridizations were performed as simultaneous dual colour hybridizations where the EUB mix served the role of a general counter stain for total bacterial cell number. Images were recorded using a Zeiss LSM 510 Meta (Carl Zeiss, Jena, Germany) confocal laser scanning microscope. For quantitative analyses, the percentages of area coverage of signals from the CteA probe and the EUB mix were calculated using the Quantimet Q500W (Leica, Cambridge, England) image analysis system. The abundance of C. testosteroni was calculated as the ratio of the area covered by biomass stained simultaneously with both CteA and EUB probes to the area covered by EUB-stained biomass only. For each sample, 20 microscopic fields (92 × 92 μm) were analysed.
The 16S rRNA sequences determined in this study have been deposited in the EMBL nucleotide sequence database  under the accession numbers [EMBL:AM113739–AM113745].
This work was supported by the German Research Foundation (DFG, grant Ha3164/2-2 to MH).
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