Identification of differential gene expression in bacteria associated with coral black band disease by using RNA-arbitrarily primed PCR

Abstract

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.

FIG. 1.

T-RFLP profiles of the BBD mat bacterial communities. (a and A) HhaI-digested PCR products from BBD mat samples taken from coral and BBD mat samples suspended in seawater, respectively. (b and B) MspI-digested PCR products from BBD mat samples taken from coral and BBD mat samples suspended in seawater, respectively. (c and C) RsaI-digested PCR products from BBD mat samples taken from coral and BBD mat samples suspended in seawater, respectively.

FIG. 2.

Formaldehyde agarose gel showing RNA extractions of BBD microbial mats growing in situ on coral surfaces (lanes 1 and 2) and suspended in seawater (lanes 3 and 4).

FIG. 3.

Example of RAP-PCR results obtained by using a high-resolution agarose gel and the Lnr95 primer. Samples a and a' are duplicates from the same sample of RNA extracted from an in situ BBD mat growing on the coral surface. Samples b and b' are duplicates from the same sample of RNA extracted from a BBD mat suspended in seawater. Arrows indicate the positions of molecular weight standards not visible in the photo.

FIG. 4.

RT-PCR analysis to assess relative differences in gene expression. Clone designations are given above the pictures. (A) BBD mat growing in situ on coral; (B) BBD mat in seawater.