DGGE, to keep the title short, stands for denaturing gradient gel electrophoresis. You may wonder what an electrophoresis technique is doing in our 'of Terms in Biology' section, but it was once a nifty way to explore microbial diversity in environmental samples. And sometimes it still is, which struck me again when I came across one of the most beautiful electrophoresis gels I have ever seen (Figure 2).
DGGE was developed by Leonard Lerman in the late 1970s, and in 1993 adopted for microbial population profiling by Gerard Muyzer. His technique is based on electrophoresis of PCR-amplified 16S rDNA fragments (from the variable V3 region) in polyacrylamide gels containing a linear gradient of denaturants (urea, formaldehyde). For the younger of our readers: in the early 90s, PCR-amplification of rDNA sequences from environmental samples was just about becoming state-of-the-art, while next generation sequencing (NGS) as a routine technique was still some years away.
In DGGE, DNA fragments of the same length but with different base-pair sequences are separated due to differences in mobility of the partially melted DNA molecules, all with their distinct individual melting points, and which is decreased compared with that of their completely double-stranded form. The technique is comparatively simple, theoretically, but requires extremely precisely constructed electrophoresis chambers, and, for reproducible results, meticulous adherence to detailed instructions for gel preparation and gradient mixing (or depends, in the sister technique TGGE, on a carefully controlled temperature gradient for DNA melting). Nothing for the faint‑hearted experimentalist!
With their recent study, Engelen et al. aimed at a better understanding of the complex stratified microbial community composition in the uppermost layer of the hydrothermal sediments of Guaymas Basin (27°00.40'N/111°24.57'W) where oxidative and reductive nitrogen, sulfur, and carbon-cycling populations and processes overlap and coexist. Sediment cores were sampled in 2016 during Alvin (DSV-2) dive 4868 at a depth of 2,003 m and frozen at ─80°C after removal of the surface Beggiatoa mats (Figure 1). Thermal gradient measurements showed that in situ temperatures at the sampling site, starting at bottom water temperature of 3°C right above the sediment surface, reached 60°C at 10 cm, and increased rapidly with depth to over 100°C.
They chose core 4868-7 for DGGE analysis and, after gradually thawing it at room temperature, siphoned‑off the first 3 mm below the surface, which mostly contained porewater that had frozen out of the core and diluted the sediment. They cut the 'trimmed' core into ~2 mm slices down to 4 cm. Measured DNA concentrations in the upper 2 cm were in the range of ~10 µg/g and dropped sharply thereafter to <2 µg/g wet weight. Prior to DGGE, they performed PCR for the individual fractions aiming at ~500 bp products with 16S rDNA specific primers (from the variable V3 region); an important control was that they checked their PCR products by gel electrophoresis to ensure that the primer combination generated a single band of appropriate length!
By DGGE, they found up to 20 clearly distinguishable bands in all gel lanes, whose intensities showed a fine‑graded distribution across the entire depth gradient (Figure 2). They excised the bands, re‑amplified them individually by PCR, and sequenced them. Figure 3 shows the assignment of their samples to bacterial groups, six known for their phylogeny and two unknown ones; all-in-all no big surprises, their samples contained 16S rDNA sequences that were already known from other deep-sea samples. The known ones included, at the genus/family level, free‑living and syntrophic deltaproteobacterial sulfate-reducing bacteria, fermentative Cytophagales, members of the Chloroflexi (Thermoflexia), and Aminicenantes.
Here's why this gel in Figure 2 struck me as "beautiful." First, I prefer to see – when I can assess the signal-to-noise ratio at a glance as here – physical evidence of differences in DNA sequences rather than algorithmically calculated ones. (Call it a whim or "applied aesthetics," but it's certainly not that kind of quantitative biology we microbiologists are educated to do.) Second, all lanes except the penultimate (from the bottom) appear as if they had been electrophoresed – in the lab we say "run" – on one single denaturing gel. That was not the case. You see not only from the white "spacer" between the lanes in the layout of the composite picture that all lanes were run separately (and separately stained and photographed afterwards). You see this also from the ever-so-slight lateral displacement of corresponding bands in the individual lanes, barely perceptible and only visible when zooming in. The degree of reproducibility achieved here is masterful, and when someone masters a tricky experimental technique, the results are occasionally, well... beautiful.
You may wonder why Engelen et al. didn't find Archaea in their survey of prokaryotic diversity by 16S rDNA PCR of core 4868-7. They state:"DGGE analyses of archaeal 16S rDNA gene fragments were attempted repeatedly, but did not result in clearly discernible DGGE patterns or defined DGGE bands, and are therefore not included in this study." But of course they found Archaea! For this, they applied their DGGE technique to conserved genes other than 16S rDNA. They found, also with a clear depth distribution, archaeal and bacterial ammonia monooxygenase sequences (amoA). Moreover, they could confirm the presence of Archaea capable of methanogenesis and methane oxidation by PCR signals for the methyl‑coenzyme M reductase alpha subunit gene (mcrA). Since I wanted to point out DGGE as a technique here, I just mention these results of their study for completeness.
I said above that DGGE "was once a nifty way to explore..." Today, everything that can be sampled can be sequenced in toto, and the microbial diversity in the samples later be assessed by metagenomics. Engelen et al. were well aware that their technique has limitations with respect to resolution. They state:"In contrast to high-throughput sequencing approaches that yield 104 to 105 sequences per sample (see here and here for recent Guaymas Basin examples), the spatial resolution of DGGE gels limits amplicon separation and identification to approximately a few dozen phylotypes, which are visually tracked in different sampling layers and sediment depths."
Another limitation of the resolution capacity of their techniques stems from the sampling procedure, or, more precisely from sample processing (which would equally apply to high‑throughput approaches). Sediments and microbial mats in the Guaymas Basin seafloor show fine-scale topography, small fluid channels and orifice-like openings that collapse during core retrieval, freezing and slicing. The authors do not address that cutting their sample cores into 2 mm slices is a technical feat, but they concede that the complex three-dimensional topography of microbial niche habitats and their inhabitants is likely to be reduced to an "averaged" layering of homogenized sediments (Figure 1). Also, the smooth transitions of the intensities of individual bands between the layers in the DGGE gel may point to such an "averaging" effect (Figure 2).
The closer one gets to the physical dimensions of the "small things" and their habitats, the more elaborate the experimental efforts to elucidate them become. Engelen et al. propose that solutions toward preserving the original microbial community architecture should include the fixation and fluorescence in situ hybridization (FISH) analysis of entire sediment slices containing multiple microbial consortia, as recently demonstrated in Sonora Margin cold seep sediments. To push the resolution even further, perhaps into the sub-micrometer range, there are currently several promising approaches that explore the combination of microscopy with mass spectrometry. It is too early for now, but a future 'Of Terms in Biology' entry in STC may well have the title: CLEM-SIMS (correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry). In principle, this would then be, albeit on a different scale, the combination of Google Maps with Google Street View.