The collection of bacterial, archaeal, eukaryal and viral microbes in surface waters, oceans soils and wastes together constitute the environmental microbiome. This microbiome is vital to humans and the environment in several ways including but not restricted to nutrient recycling, biotechnological application in the food and beverage industry and genetic engineering technologies. Nonetheless, pathogenic microbes can invade and grow within other organisms causing disease (Egli and Koster).
Different strategies have been used to define microbial community structure across microbial ecosystems. Taxonomic classification based on morphologic and physiological traits of cultured microbial communities is no longer attractive since the fraction of cells that may be cultured is not representative of the overall abundance or diversity the environment (Pernthaler and Glöckner). In light of the foregoing, culture independent assessments of microbial communities become necessary. In this regard, the ribosomal RNA molecules (small subunit, 16S, and 18S, in Eukarya; large subunit 23S and 28S, in Eukarya) sequence divergence among taxa provide attractive alternative options as molecular markers of diversity.
Inorder to detect the presence of specific organisms in environmental samples in this study, organisms specific primers specific for the 16S rRNA of the economically important bacteria such as Planctomycetes Acidobacteria, Actinobacteria, Firmicutes Verrucomicrobia, Bacteroidetes, Chloroflexi, Proteobacteria, Gemmatimonadetes were used.
Materials and methods
- Soil surface samples approximately 2 cm deep were taken randomly from the edges of each of adjacent agricultural plots.
Nucleic acids were extracted from soil samples using the technique described by (Zhou, Bruns and Tiedje) with a few modifications. The sample was mixed with DNA extraction buffer and incubated addition of sodium dodecyl sulfate and heating at 65 °C. Purifiocation was done twice by extraction with an equal volume of chloroform–isoamyl alcohol before precipitating of isopropanol. The concentrations of the extracted DNA were determined by Nanodrop spectrophotometry.
PCR amplification of organism specific 16S rRNA
The extracted DNA was subjected to PCR amplification using primers designed to amplify rRNA genes from a particular group of organisms. The PCR mixtures had 10 ng of a template DNA, 10 pmol of each primer, 10 mmol of dNTP mixture and 2.5 U of Taq DNA polymerase. The thermal profile was as follows: an initial denaturing step at 95 °C for 5 min, 33 cycles consisting of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, and a final elongation step at 72 °C for 10 min.
Agarose gel electophoresis of PCR amplicons
Agarose elctrophoresis was performed to visualize the PCR products allowing the determination of the amplicon sizes and amplification success in general. Individual amplicons were resolved on a 1.2% agarose gel at 80 V for one hour to allow for size fractionation. The ethidium bromide stained DNA was then visualized under UV light.
The average DNA yield using the procedure described in the materials and methods section gave approximately 150ng of DNA. Proteobacteria, Acidobacteria, Actinobacteria, Verrucomicrobia samples gave single strong PCR products of the expected sizes using their respective specific primers. For these organisms, there was no amplicon produced using the primers specific for other organisms effectively rendering the alternative primers as suitable negative controls. Bacteroidetes, Chloroflexi, Planctomycetes also gave single but weak bands with their respective primers. No amplicons were recorded for both Gemmatimonadetes, and Firmicutes organisms.
Discussion and conclusion
There is compelling evidence to show that 16S rRNA genes from soil bacteria are affiliated with several phyla each making varying contributions to the different soil bacterial communities. As regards the bacterial community sampled in this study, the Proteobacteria, Acidobacteria, Actinobacteria, Verrucomicrobia seem to be the dominant phyla at least as judged by the intensity of the PCR amplicons. Although there are several other phyla, the soil sampled seems to be less dominated by these with Bacteroidetes, Chloroflexi, and Planctomycetes seeming to be the next abundant phyla.
Although the rRNA approach is the principal tool for these kinds of studies, it often leads to underestimation of bacterial diversity due to polymerase chain reaction (PCR) primers bias coupled with the inefficiency of DNA extraction techniques. In view of these facts, it is difficult to comment on the absolute absence or otherwise of the Gemmatimonadetes, and Firmicutes organisms. Despite these limitations, rRNA approach remains one of the most important tools to assess microbial diversity. A thorough study of its potential bias becomes a priority.
In relation to the environmental microbiome, it is uncertain as to what extent the variations in microbial communities are influenced by different biological, chemical, and physical factors. This will obviously form an interesting topic for further investigation. Nonetheless, a proper understanding of what the microbial populations is in the environment, as well as evaluation of the available technologies for estimating this diversity will be of critical value for informing health-related questions and understanding biogeochemical cycles.
Egli, Thomas and Wolfgang Koster. "Pathogenic microbes in water and food: changes and challenges." FEMS Microbiology Reviews 26.2 (2006).
Pernthaler, Jakob and Frank Oliver Glöckner. Fluorescence in situ hybridization with rRNA-targeted. Bremen: Max-Planck-Institute for Marine Microbiology, 1998.
Zhou, J, M A Bruns and JM Tiedje. "DNA recovery from soils of diverse composition." American Society for Microbiology 62.2 (1996): 316-322.