The objective of this study was to generate a map of the accessibility of the 16S rRNA of E. coli
for fluorescently labeled oligonucleotide probes. It was therefore critical that the probe-mediated fluorescence was not affected by other parameters, such as differences in the probe quality or dissociation temperature, to name only the two most important ones. We consequently performed a rigid quality control, accepting for this study only probes purified by high-pressure liquid chromatography and labeled with carboxyfluorescein by the highly effective solid-phase synthesis with A260
ratios that were close to the ratio (260 versus 496 nm) of theoretical extinction coefficients. On the other hand, we could not determine optimal hybridization conditions for each individual probe. Even with the unsurpassed speed of flow cytometric quantification of fluorescence intensities, such an analysis of all 200 probes was beyond the reach of this study. We consequently applied all probes under standardized conditions. This also means that the quality of our data relies on two assumptions: (i) a sigmoidal behavior of probe binding over stringency and (ii) the accuracy of the 4+2 formula in estimating the dissociation temperature. In order to evaluate the plausibility of our assumptions, we tested nine probes at different hybridization temperatures and stringencies in an early phase of the experiment. Seven probes had optimal signals at 46°C, but two probes showed maximum fluorescence at 41°C despite estimated melting points of about 50°C. Nevertheless, we did not change the standard hybridization conditions, since the assumption of a roughly sigmoidal shape of the stringency-binding curve also did not hold for some probes. As reported before (5
), binding at close to but below the temperature of dissociation can be higher than that at lower stringencies. It has been speculated that this might be due to changes in the accessibility of target sites under different hybridization conditions. Since hybridization at 46°C yielded at least 80% of the maximum signal, we would like to argue here that even though we clearly cannot rule out an influence of probe-to-probe differences in optimal hybridization conditions, this effect is secondary and would change the classification of probes by at most one brightness class. Considering all factors, the fluorescent signals reported in this study for more than 200 probes should be interpreted with some care, but with the controls described above they should be reliable within ± 10% of the fluorescence of probe Eco1482 and should reflect mainly differences in 16S rRNA accessibility.
Changes in the accessibility as shown in Fig. are often steady along the primary structure of the 16S rRNA but can also be rapid and are therefore quite unpredictable. For each of the three domains of the 16S rRNA, probes of all brightness classes are present. Probes targeting domain I (Eco1 to Eco541) are, with an average of 42% of the maximum fluorescence of probe Eco1482, significantly brighter than probes targeting domains II (Eco548 to Eco917) and III (Eco926 to Eco1526), with averages of 32 and 30%, respectively. Interestingly, the almost inaccessible target sites of class VI probes are frequently in the periphery of the secondary structure model (Fig. ), including many loops, whereas regions in the center of this model seem to be more readily accessible. Since we opted at the outset of the study to use two sets of adjacent oligonucleotides of quite invariant lengths, no special care was taken to keep probes on one side of the target helix in order to avoid self-complementarity. Consequently, a lack of binding of certain of these loop-associated probes could be due to internal backfolding rather than to inaccessibility of target sites. This might apply to three probes of class VI, i.e., Eco1006, Eco1310, and Eco1506, targeting helices 37, 47, and 50, respectively. Self-complementarity of probes, however, cannot explain the clusters of class VI probes in helices 18, 22, and 26, where target site inaccessibility is found also for probes that have no self-complementarity at all.
In Fig. we compare probe-conferred fluorescence values with evolutionary conservation of the respective regions of the 16S rRNA molecule. By referring to Fig. , it should be possible to more easily select target sites that yield both highly specific and bright probes for fluorescent in situ hybridization. Clearly, some of the most variable regions also show narrow but strong minima of in situ accessibility. This might explain the failure of many fluorescent in situ hybridization experiments. Frequently, probes were intended to be as specific as possible, targeting particular species or genera or even just particular 16S rDNA sequences that were retrieved from the environment. Those probes necessarily target the most variable regions of the 16S rRNA molecule, such as, e.g., helices 6, 18, and 22. Even though these probes might have worked nicely with extracted rRNA or as primers for PCR, no binding to whole fixed cells could be detected. The comparison of the in situ accessibility map of E. coli and evolutionary conservation also shows that the regions with high variability are usually wider than the zones of low accessibility. This means that a highly variable region which had appeared to be unsuitable for in situ hybridization with one peculiar probe might still be useful if the target site is shifted by several nucleotides. The effect of shifting the target sites by a few nucleotides was therefore studied in greater detail for the three evolutionarily less conserved helices, helices 6, 18, and 23.
FIG. 4 Comparison of relative fluorescence (solid line) and average conservation (gray) of all probes targeted to E. coli. Conservation values for each probe were calculated by averaging the conservation values stated in the ARB database (27) for those positions (more ...)
Helix 18 showed quite dramatic changes in accessibility. Since we assume that what was measured for E. coli
in terms of in situ accessibility would to a large degree also apply to other species, it is interesting to compare our results to other findings. Probe Nsv443, specific for the genera Nitrosolobus
, and Nitrosovibrio
of the chemolithotrophic, ammonia-oxidizing bacteria of the beta subclass of the class Proteobacteria
, gives bright in situ signals (19
), whereas a probe with similar specificity, Nsp452, could be used only as a PCR primer (21
) and did not work for fluorescent in situ hybridization (21a
). The results of our detailed study of helix 18 in E. coli
are in line with this observation. Eco440 is in class I, whereas Eco455 is in class VI, showing not much more than background fluorescence.
As for helix 18, the accessibility of helix 6 is quite high in the proximal stem region between probes Eco60 and Eco70 in the 5′ half and probes Eco89 and Eco91 in the 3′ half. Accessibility of the distal part of this helix, including the loop, is much lower, with relative intensities of Eco79, Eco84, and Eco87 of only 3, 6, and 10%, respectively. Here, only probe Eco79 has some self-complementarity (the four 3′ nucleotides could fold back), whereas Eco84 and Eco87 target only the loop and the 3′ half of the helix, thereby excluding self-complementarity as the major reason for the observed inaccessibility. The probe-to-probe changes in signal intensity (Table ) for the 10 probes between Eco60 and Eco91 are gradual and allow the cause of inaccessibility in E. coli
to be mapped quite accurately to about positions 87 to 90. This takes into account that a lack of binding near the end of a short duplex is generally less destabilizing than an internal hindrance (26
). Obviously, the interactions that almost completely block binding of Eco79, Eco84, and Eco87 are restricted to a very narrow region. Interestingly, several probes that successfully target helix 6 in other bacteria exclude the region that is blocked in E. coli
and target either the 5′ half (MPA60 [Microthrix parvicella
, positions 60 to 77] [10
], AER66 [Aeromonas
sp., positions 66 to 83] [14
], Pst67 [Pseudomonas stutzeri
, positions 67 to 84] [1
], and Hau66 [Herpetosiphon aurantiacus
, positions 66 to 84] [1a
]) or the proximal part of the 3′ half (ARC94, positions 94 to 111 [25
]). These bacteria include both members of the gamma subclass of the class Proteobacteria
sp. and P. stutzeri
), of which E. coli
is also a member, and members of only distantly related phyla of the Bacteria
branch] and M. parvicella
[a gram-positive bacterium with a high G+C content of the DNA]). Again, this is a good indication that the in situ accessibilities that we determined for E. coli
apply to other microorganisms as well.
The same is true for probes targeting helix 22. Empirical data acquired by the testing of numerous probes for various bacteria (1a
) had suggested that the second bulge on helix 22 (positions 640 to 643) could be a strong hindrance for probe binding. Our fine mapping with probes complementary to positions 621 to 656 confirms this. Eco621, Eco627, Eco632, and Eco639 show only background fluorescence, whereas Eco645, targeting the adjacent sequence, already has an increased relative fluorescence intensity of 22%. In reviews, Ehresmann et al. (9
) as well as Malhorta and Harvey (18
) have summarized numerous results from both cross-linking and nuclease protection assays that indicate several interactions of helix 22 with small subunit proteins S8, S16, and S17. Of special interest for our study is the interaction between protein S8 and positions 642 and 643.
Another recent report on the conformation of the 16S rRNA is also in line with our data. Lodmell and Dahlberg (16
) have postulated a conformational switch in the proximal stem region of helix 30, including positions 885 to 890 and 910 to 912, that should occur during mRNA translation. This switch requires an open conformation in the vicinity, and we indeed found good accessibility of helix 30 and other core regions. With the large amount of data presented in our study, this type of speculation could be continued. However, we have to state here again that our primary goal was mapping of 16S rRNA in situ probe accessibility and not an analysis of the higher-order structure of the small subunit of the ribosome. One should always keep in mind that we worked on formaldehyde-fixed E. coli
cells with 18-mers that necessarily integrate accessibility over larger regions. Nevertheless, we hope that our data will be of interest to experts in the field of ribosome conformation.
The only major unexpected finding in our study was the low accessibility of the 5′ half of helix 23. About one-quarter of the >200 probes developed in our laboratory over the last 8 years target this region (see, e.g., reference 4
). There are two reasons for this: first, the variability of the nucleotides in this region makes it easy to find signature sequences on about the genus level, and second, the accessibility was in most cases very high, yielding bright fluorescence signals. However, the quantification of probes targeting the 5′ half of helix 23 of E. coli
yielded values of between only 12 and 23% of the signal of probe Eco1482. Flow cytometric quantification of the signals conferred by probes targeting this helix in three other species, A. calcoaceticus
, Z. ramigera
, and C. testosteroni
, clearly demonstrated that the in situ accessibility of helix 23 is indeed generally very good. Obviously, for this particular region E. coli
is a bad model for other organisms. Even though the 16S rRNA is a highly conserved molecule, there are differences in the primary and higher-order structures that will be more pronounced the more distantly related two organisms are. Certain probe target sites that are considered to be open in most species can be deleted in certain species, and obviously, from our E. coli
map nothing can be deduced about the accessibility of insertions that are present in other phyla.
With these limitations for the transferability of our E. coli data to other species, there is still a clear need to test every newly developed probe on reference organisms before it is used with natural samples for the quantification and in situ localization of individual cells. However, we hope that this publication will contribute to a higher probability of successful design of oligonucleotide probes for in situ hybridization.