MATERIALS AND METHODS
By Karen A. Sandell
The isolates used in this research project were deep-water (> 120 feet seawater) invertebrate-associated (primarily sponges) eubacteria maintained in the HBMCC. Samples were collected by Harbor Branch Oceanographic Institution’s underwater submersibles such as the Johnson Sea Link I and II. The isolation methods used involved the sampling of the invertebrate via aseptic technique upon surfacing. The invertebrate tissue was ground in sterile seawater and the subsequent supernatant was diluted in sterile seawater before plating onto a series of media designed to recover a diverse range of heterotrophic microbes. Media ranged from extremely nutrient poor (60% seawater, 40% deionized water, trace metals, phosphate, agar), to nutrient rich (Difco Marine Agar 2216) and included a large variety of carbon sources (e.g. chitin, simple and complex sugars and mucin). Isolation media were also designed to include both antibiotics and/or extract of the host tissue.
Bacterial isolates were maintained on Difco Marine Agar 2216 slants and, in almost all cases, were freshly streaked on Difco Marine Agar 2216 plates before DNA extraction. Difco Marine Agar 2216 contains synthetic seawater ingredients (NaCl, MgCl2, Na2SO4, CaCl2, KCl, etc.) along with peptone and yeast extract as a source of organic carbon.
Bacterial cells for DNA extraction were collected with a sterile 1ul loop either directly from isolates maintained on agar slants or re-grown cultures of these isolates streaked on agar plates. The cells were added to 125 µl of Chelex-100 (Bio-Rad Inc.) made as a 5% solution in sterile distilled water. Total genomic DNA was then extracted using the standard protocol for Chelex-100 (De Lamballerie et al. 1992).
POLYMERASE CHAIN REACTION (PCR)
PCR amplified the number of copies of the 16S small subunit (SSU) rRNA gene region. Universal 16S primers Ecoli9 5’-GATTTTGATCCTGGCTCAG-3’ (equal to Lane 1991 "27F" primer) and Loop27rc 5’-GACTACCAGGGTATCTAATC-3’ (Lopez et al. 1999) amplified around 750 base pairs of the 5’ end of the 16S SSU rRNA gene under standard PCR conditions: initial 94°C denature for 2 min.; 34 cycles of 94°C denature for 1 min., 53°C annealing for 1 min., and 72°C extension for 1 min.; 72°C extension for 30 min; 4°C hold). Each 30.1 ul reaction contained around 1 ng of DNA template, 3ul of 10X PCR reaction buffer, 3ul of 1mM dNTP nucleotide mix, 1 ul of each primer (25 pmol/ul), and 0.1 ul (0.5 U) of Taq DNA polymerase (Promega). A positive control (with previously amplifiable DNA) and negative control (no template added) was run for every PCR performed. All PCR products were visualized by gel electrophoresis using ethidium bromide stained 1% agarose gel (MetaPhor-FMC BioProducts) in 0.5X TBE buffer.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)
RFLP was utilized as a primary screen for genetic variation in SSU PCR products. This method is a PCR-based fingerprinting technique in which the
amplified DNA is digested by a restriction endonuclease. When the digestion product is electrophoresed on an agarose gel (2%), the various sized fragments separate according to size and result in a species-specific banding pattern (DeLong 1998). Three tetrameric (4-base cutting) restriction endonucleases were used in order to increase the chances of detecting unique RFLP patterns: RsaI, HaeIII, and HhaI (Invitrogen). RsaI and HaeIII restriction patterns were obtained for all isolates. HhaI was used for samples that did not cut with either RsaI or HaeIII, or for instances where further distinction was necessary. Moyer et al. (1996) performed computer-simulated RFLP analysis on 100 SSU rRNA gene sequences from the Ribosomal Database Project (RDP) database and found that the use of three restriction enzymes can detect >99% of different bacterial taxa. Gel electrophoresis images were digitally captured on an Eagle Eye scanner (Stratagene). The imager’s accompanying software, RFLPscan (Scanalytics, Billerica, MA) was used to objectively calculate the molecular weight of each RFLP band.
DNA templates were purified using a Qiagen QIAquick™ PCR Purification Kit and Sephadex (Amersham Biosciences) columns before cycle sequencing using an ABI Prism™ Big Dye® Terminator Cycle Sequencing Ready Reaction Kit. Automated sequencing using ABI DNA sequencers was conducted at the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida in Gainesville, FL. Once the sequence results were obtained and edited into contiguous 16S rDNA fragments, a BLAST (Basic Local Alignment Search Tool) search was performed using the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST/). The BLAST search compares the 16S sequences obtained to a database of known sequences in order to find the closest taxonomic match (Altschul et al. 1997). As a comparison, sequence matches were also obtained from the Ribosomal Database Project database (http://rdp.cme.msu.edu/html/). This website is dedicated exclusively to ribosomal RNA structure and sequence information (Maidak et al. 1996, Maidak et al. 1999).
Isolates with RFLP bands of similar molecular weight were grouped accordingly. Because agarose gels have a limited amount of resolution, there will be an inherent amount of error in sizing bands, which increases with size of the band. The most precise RFLP fragment sizes can be derived from the DNA sequence data.
Microsoft Access also allowed comparisons between the RFLP patterns (matched to their taxonomic identity) across different invertebrate hosts, depths, and geographic locations. The University of California San Diego Super Computer Biology Workbench (http://workbench.sdsc.edu/) provided “virtual” RFLP data for all sequences obtained in order to verify that the calculated band lengths for the RFLPs were correct.
Sequence alignments were obtained using the CLUSTAL W program located on University of California San Diego Super Computer Biology Workbench. These alignments were used to reconstruct phylogenies using maximum likelihood, and distance (numerical) procedures using PAUP (phylogenetic analysis using parsimony) v 4.0b3a (Swofford 1999), MEGA, and other phylogenetic analysis programs. All inferred trees were subject to bootstrap evaluations and rooted using the outgroup method (Hillis et al. 1996). The subsequent cladograms assessed the overall diversity between the sequenced SSU rDNA fragments obtained in this project.
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Flow chart of sample processing procedures.