DNA Persistence in a Sink Drain Environment.

Biofilms are organized structures composed mainly of cells and extracellular polymeric substances produced by the constituent microorganisms. Ubiquitous in nature, biofilms have an innate ability to capture and retain passing material and may therefore act as natural collectors of contaminants or signatures of upstream activities. To determine the persistence and detectability of DNA passing through a sink drain environment, Bacillus anthracis strain Ames35 was cultured (6.35 x 107 CFU/mL), sterilized, and disposed of by addition to a sink drain apparatus with an established biofilm. The sink drain apparatus was sampled before and for several days after the addition of the sterilized B. anthracis culture to detect the presence of B. anthracis DNA. Multiple PCR primer pairs were used to screen for chromosomal and plasmid DNA with primers targeting shorter sequences showing greater amplification efficiency and success. PCR amplification and detection of target sequences indicate persistence of chromosomal DNA and plasmid DNA in the biofilm for 5 or more and 14 or more days, respectively

Agarose gel images for <i>Bacillus anthracis</i> PCR samples.

<p>Depicted in A, from left to right, is 100 bp ladder, no-template control (NTC) and 3 reactions utilizing BA-5449 primer set, NTC and 3 reactions utilizing <i>cya</i>-1 primer set, NTC and 3 reactions utilizing <i>PA</i>7/6 primer set and 100 bp ladder. Depicted in B, from left to right, are duplicate reactions with bleached <i>Bacillus anthracis</i> template material and duplicate reactions with autoclaved <i>Bacillus anthracis</i> template material, with all 4 reactions utilizing the ITSeub primer pair.</p

Primers utilized for <i>Bacillus anthracis</i> & eubacteria DNA amplification and the expected amplicons.

<p>Primers utilized for <i>Bacillus anthracis</i> & eubacteria DNA amplification and the expected amplicons.</p

Identification of Differential Gene Expression in Bacteria Associated with Coral Black Band Disease by Using RNA-Arbitrarily Primed PCR

RNA-arbitrarily primed PCR techniques have been applied for the first time to identify differential gene expression in black band disease (BBD), a virulent coral infection that affects reef ecosystems worldwide. The gene activity for the BBD mat on infected surfaces of the brain coral Diploria strigosa was compared with that for portions of the BBD mat that were removed from the coral and suspended nearby in the seawater column. The results obtained indicate that three genes (DD 95-2, DD 95-4, and DD 99-9) were up-regulated in the BBD bacterial mat on the coral surface compared to the transcript base levels observed in the BBD mat suspended in seawater. Clone DD 95-4 has homology with known amino acid ABC transporter systems in bacteria, while clone DD 99-9 exhibits homology with chlorophyll A apoprotein A1 in cyanobacteria. This protein is essential in the final conformation of photosystem I P700. DD 95-2, the only gene that was fully repressed in the BBD mat samples suspended in seawater, exhibited homology with the AraC-type DNA binding domain-containing proteins. These transcriptional activators coordinate the expression of genes essential for virulence in many species of gram-negative bacteria

Digital capillary electrophoresis gel image from LabChip GX acquired using HT DNA Hi Sens(itivity) Reagent Kit.

<p>Sample wells contain, from left to right: LabChip GX Ladder, No-Template Control, Control Isolated <i>Bacillus anthracis</i> strain Ames35 DNA, Day 1 Autoclaved Sample prior to disposal, Day 1 Bleached Sample prior to disposal, 6-round 1 testing samples (Day 2, Day 3, Day 5, Day 6, Day 10 & Day 14 Samples.), and 4-round 2 testing samples (Day 3, Day 11, Day 21 & Day 22 Samples). Duplicate PCR amplification sample data for each time point obtained, but not shown.</p

Sampling tips with female tap ends to attach to the tip of the sampling device.

<p>Various sample materials shown, from left to right: copper wire, nylon fibers, and polyether foam.</p

Cyanobacteria Associated with Coral Black Band Disease in Caribbean and Indo-Pacific Reefs

For 30 years it has been assumed that a single species of cyanobacteria, Phormidium corallyticum, is the volumetrically dominant component of all cases of black band disease (BBD) in coral. Cyanobacterium-specific 16S rRNA gene primers and terminal restriction fragment length polymorphism analyses were used to determine the phylogenetic diversity of these BBD cyanobacteria on coral reefs in the Caribbean and Indo-Pacific Seas. These analyses indicate that the cyanobacteria that inhabit BBD bacterial mats collected from the Caribbean and Indo-Pacific Seas belong to at least three different taxa, despite the fact that the corals in each case exhibit similar signs and patterns of BBD mat development