Identify a Microbe:

Using DNA Sequence (16S rRNA gene) for Strain Identification

This primer set amplifies a roughly 1450 bp fragment of a gene encoding the 16S rRNA gene from all members of the domain Bacteria (Lane, 1991; Stackebrandt & Liesack, 1993). All organisms on Earth have one or more 16S/18S rRNA genes, but this primer set was originally shown to be specific for the domain Bacteria. However, recent work has shown that these primers can also amplify the 18S rRNA gene from certain corals (Galkiewicz & Kellogg, 2008). More importantly, the original 27F primer misses some key subgroups in the domain Bacteria, so we use the 27F-YM+3/1492R primer mix to optimize strain coverage (Frank et al., 2008). The annealing temperature for this PCR reaction is 48C. This primer set can be purchased separately or as part of a kit that includes colony DNA isolation and sequencing of PCR amplicons.

Products:

16S rRNA Gene Primer Set. Enough for 10 PCR reactions $15 + S/H
Identify-a-Microbe Kit reagents Enough for 10 DNA isolations, PCR reactions, & DNA sequencing (sent out) $140 + S/H

Bacteria are Everywhere

The famous paleontologist and science writer Stephen J. Gould once wrote that there has never been an "age of insects", "age of dinosaurs", or "age of mammals". There has really only been an "age of bacteria" because these relatively tiny organisms have dominated the earth since its earliest biotic beginnings in so many ways (Gould, 1996). By "little b" bacteria, Gould meant prokaryotes of which there are two kinds. The domain Bacteria includes the bacteria we all know - the gut inhabitant and sometimes pathogen E. coli, Lactobacillus species involved in yogurt and cheese production, and Streptococcus pyogenes that can cause strep throat, just to name a few. The domain Archaea is a very different group of prokaryotes and we didn't even know they existed as a distinct group until the late 1970's, but it turns out they are also ubiquitous (Woese & Fox, 1977). We find bacteria in all kinds of recognizable habitats, oceans, lakes, and soil, but also in strange places like glaciers, hot springs, deep sea thermal vents, and on or inside other organisms. They be found at temperatures from 0oC to over 100oC, at pH's from < 1 to>10, at great pressures, at salt concentrations so high the salt starts to fall out of solution, and across a broad range of nutrient concentrations. In many of these places, we can also find some eukaryotic microbes - algae, fungi, and others. Yet, we are still left with our question of "how do we know who they are?"

For bacteria, the choice for decades has been plating samples out on various culture media and then taking isolated colonies and restreaking them on fresh plates to ensure that each colony is a pure culture of one particular bacterial strain. If it is a pure culture, then every colony on the restreaked plate will look the same as each other. Once we are sure that a particular strain is pure, colonies of that strain can then be used to identify it using a variety of biochemical assays (e.g., can it break down urea), stains (e.g., the very popular Gram stain), and growth-based tests (e.g., can the strain grow in very salty conditions such as the presence of 10% NaCl).

Sometimes, we may be interested in a particular bacterial strain, but we cannot use the standard culture-based lab methods - we cannot figure out how to get that strain to grow up on a plate or in a broth culture. Evidence has accumulated during the last two decades that cultured-based identification methods are not good estimators of microbial diversity because they catch only a small percentage (0.5-5%) of the microbial diversity present in any habitat at any time (Staley & Konopka, 1985). Even after more than a century of efforts, we only can get a minority of bacteria to grow in culture (Kaeberlein, Lewis, & Epstein, 2002). Does that mean we cannot identify unculturable bacteria? No, we just have to either deal with them as part of an overall community (see our Metagenomic DNA Analysis kits) or we have to find a way to obtain a "mostly pure" sample of the bacterial strain of interest where the vast majority of the organisms are the same kind. This sometimes happens in nature or we can enrich for it (e.g., Winogradsky column).

Regardless of how we obtain a particular bacterial strain, by culture, straight from nature, or from enrichment, how can we identify that strain without having to assume that it is culturable? A genetics tool comes to the rescue! We typically think of genetics as just a set of concepts (e.g., gene, chromosome) and processes (e.g., meiosis, recombination), but genetics is also an experimental toolbox that can be used to address questions throughout the life sciences. One of the tools in our genetics toolbox is a technique called Polymerase Chain Reaction or PCR (Sakai et al., 1985). In PCR, a stretch of DNA is replicated over and over and over again until you have a workable quantity of it. The specificity in PCR comes from the choice of short stretches of single-stranded DNA called primers onto which DNA Polymerase adds nucleotides during DNA replication. Those primers might be specific to a DNA sequences found only in a species, a genus, a family, or some higher level taxon (e.g., Blackwood, Oaks, & Buyer, 2005). Any gene is a potential PCR target; it just depends on the question you are addressing.

Since we don't know the identity of our unknown bacterial strain, we will use PCR primers targeted to a gene found in all organisms - the 16S/18S rRNA gene. This gene encodes one of the rRNA molecules that are used to construct ribosomes, the essential cell machinery that catalyzes the formation of new proteins. The 16S/18S rRNA gene is highly conserved across all life on Earth, meaning that if you compare the gene sequence from two different organisms you will be easily able to tell that it is pretty much the same along its length. However, it is not 100% identical. Rather, the gene sequence has slowly changed over evolutionary time - in other words, from the moment one group of organisms is split into two and they don't reproduce or exchange DNA with each other anymore, then mutations will slowly accumulate in the 16S/18S rRNA gene such that the two groups are now slightly different in their DNA sequences. The longer the time since the divergence of the group, the more differences between their DNA sequences. This is the basic idea behind using DNA and protein sequences to understand evolutionary relationships and is the basis for the currently accepted tree of life (Woese & Fox, 1977). These differences in gene sequence can also used to identify unknown organisms by comparing their 16S/18S rRNA gene sequence to a database of sequences from known organisms. Certain parts of the 16S/18S rRNA gene mutate faster than others and that dictates what regions are used for PCR amplification. With PCR, you don't have to grow the organisms and you don't have to have the entire genome intact. As long as you can amplify a portion of the 16S/18S rRNA gene and determine its sequence, you have a chance to identify the organism.

Starting Ideas for Projects:

  • Your students can identify some habitat of interest, be it commonplace, rare, or weird, and sample the habitat and try to grow up some of its microbial community members on culture plates. Each isolated colony on a plate is a potential study subject. We would recommend first streaking a colony for pure culture on a new plate, but then your students can isolate genomic DNA from a pure colony and use PCR to amplify a portion of the 16S/18S rRNA gene. Then we will help you get the sequence of that amplified DNA fragment and you can finally compare that sequence to database of known sequences to potentially identify your unknown organism.

References

Blackwood, C.B., A. Oaks, & J.S. Buyer, 2005. Applied Environ. Micro. 71:6193-8. Phylum- and class-specific PCR primers for general microbial community analysis.

Frank, J.A., C.I. Reich, S. Sharma, J.S. Weisbaum, B.A. Wilson, & G.J. Olsen, 2008. Applied Environ. Micro. 74:2461-70. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes.

Galkiewicz, J.P., & C.A. Kellogg, 2008. Applied Environ. Micro. 74:7828-31. Cross-kingdom amplification using Bacteria-specific primers: complications for studies of coral microbial ecology.

Gould, 1996. Full House, pp. 175-192, New York: Harmony Books.

Kaeberlein, Lewis, & Epstein, 2002. Science 296:1127-9. Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment.

Lane, D. J. 1991. p. 115-175. In Nucleic Acid Techniques in Bacterial Systematics.

Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115-147. In E. Stackebrandt & M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons, New York, NY.

Saiki et al., 1985. Science 230:1350-4. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.

Stackebrandt, E., and W. Liesack. 1993. Nucleic acids and classification, p. 152-189. In M. Goodfellow & A. G. O’Donnell (ed.), Handbook of new bacterial systematics. Academic Press, London, England.

Staley & Konopka, 1985. Annual Rev. Micro. 39: 321-46. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats.

Woese & Fox, 1977. Proc. Natl. Acad. Sci. USA 74:5088-90. Phylogenetic structure of the prokaryotic domain: the primary kingdoms.