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Bacterial diversity and structure of biofilms: Investigating using molecular methods.

Over the years, the stream biofilm project team has developed and used a wide range of methods to analyse stream biofilms, from the point of view of their bacterial and protozoan diversity as in terms of their structure. Below we describe some of the main methods we currently use in our research.

Sampling Methodology:


Figure 1: Biofilm sampled with a Speci-sponge

Replicate samples are taken from stream rocks using Speci-sponges (Figure 1). The sponges are then transported to the laboratory on ice where the biofilm biomass is removed from the sponge, and stored at -80 ºC.

DNA Extraction

DNA is extracted from each biofilm sample using the method of Miller et al. (1999) for all samples where molecular methods are employed. This method combines a bead-beating methodology with chloroform-isoamyl alcohol extraction, followed by precipitation of the extracted DNA with isopropanol.

(Reference - Miller, DN., Bryant, JE., Madsen, EL and Ghiorse, WC (1999) Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Applied and Environmental Microbiology 65: 4715-4724).

Analysis of Genetic Diversity by Polymerase Chain Reaction

Once the DNA has been extracted from the biofilm biomass, two main approaches are used in our laboratory to characterise the bacterial communities from their DNA (i) characterising the community structure using DNA fingerprinting techniques and (ii) identifying specific organisms within biofilm samples using DNA sequence analysis.

DNA Fingerprinting Techniques

The DNA fingerprinting technique most commonly used within our laboratory is Automated Ribosomal Intergenic Spacer Analysis (ARISA). This technique has only recently been used for the evaluation of aquatic bacterial communities. This PCR-based method creates a fingerprint of the microbial community structure from profiles of the 16S-23S intergenic spacer (IGS) region of bacteria, based upon the length of the amplified nucleotide sequence, which displays significant heterogeneity between species. The primers used in this approach (LDBact and SDBact) flank each side of the IGS region within bacterial rRNA genes (Figure 2). This method generates approximately 800 variables for each sample, representing the length (in bp.) of the intergenic spacer region of the constituent bacteria, and creating an accurate profile of the bacterial community structure (Figure 3).


Figure 2: The areas of the bacterial genome that we target for molecular analysis of community structure and composition. The primers employed to enable the phylogenetic identity of bacteria from the sequence of their 16S rRNA genes are PB36 and PB38. The primers SDBact and LDBact flank either side of the highly variable intergenic region between bacterial 16S and 23S rRNA genes and are used for ARISA.


Figure 3. Example of an ARISA profile generated from a stream biofilm sample. Data are peak height (fluorescence; y axes) and fragment length (nucleotide base pairs; x axes). Sample data (green); size ladder (orange).

DNA Sequence Analysis

To identify the bacteria present within a biofilm sample, the primers PB36 and PB38 (Figure 2) are used to target the bacterial 16S ribosomal gene which is highly conserved within the bacterial genome. Analysis of the sequence of the amplified PCR fragment enables the identity of bacterial strains to be elucidated (via comparison with DNA sequences on the NCBI BLAST database). Once the sequences of sufficient PCR fragments have been identified, the composition of bacterial communities between sites and treatments may be compared (Figure 4).


Figure 4. Composition of the bacterial community within a stream biofilm identified by sequence analysis of 16S rRNA genes

 

 

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Stream Biofilm Research Group
School of Biological Sciences
The University of Auckland
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