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.Coral reefs are important reservoirs of biodiversity in oceanic environments and thus are used as a sensitive indicator of environmental change (23, 39). The number and incidence of diseases that affect corals around the world have dramatically increased over the last four decades (21). As evidence of this worldwide degradation of coral reef ecosystems mounts, interest in identifying the etiological agents of infectious disease in scleractinian corals continues to intensify (21,39) Black band disease (BBD) is among the most important of the globally distributed coral diseases known today, yet the environmental conditions leading to BBD infection are still not well understood (34). BBD results from the rapid migration of a complex mat of microorganisms across the surface of scleractinian coral colonies; this mat lyses healthy coral tissue and leaves dead coral skeleton behind (2, 34). BBD preferentially affects massive-framework-building coral species that serve as the ecological cornerstone of reefs. Therefore, BBD is an important agent in altering coral reef ecosystems.Although BBD is a polymicrobial disease (34, 35), the population of bacteria growing in the BBD biofilm is composed primarily of large filamentous cyanobacteria previously optically identified as the single species Phormidium corallyticum and recently identified as multiple species by molecular techniques (14, 18). Several previous studies have suggested that these cyanobacteria are the BBD pathogens (40). However, the culturing of these cyanobacteria has proved difficult (23, 33), thus preventing tests to fulfill Koch's postulates to determine BBD pathogenicity (34).The aim of the present study was to better understand BBD pathogenesis by analyzing bacterial gene expression in the BBD mat consortia by RNA-arbitrarily primed PCR (RAP-PCR) (5, 9, 16, 43...

A single cyanobacterial ribotype is associated with both red and black bands on diseased corals from Palau

“…Cloning of extracted DNA from field samples using universal primers targeted at a larger region of the 16S rDNA gene would provide a more comprehensive analysis of cyanobacterial populations associated with both red band-and BBD-infected corals. If pathogenicity and virulence are to be studied, an attempt should be made to explore the genes linking identity and function, as has been recently demonstrated for BBD by Frias-Lopez et al (2004a). In the case of Vibrio cholera it has already been shown that the 16S rDNA based identity of strains does not necessarily relate to virulence (Colwell & Huq 1994, Singh et al 2001.…”

Microbial Communities in the Surface Mucopolysaccharide Layer and the Black Band Microbial Mat of Black Band-Diseased Siderastrea siderea

“…There may also be a regional difference in the BBD microbial community. Our study is the first to use molecular techniques to characterize the BBD community on reefs of the northern Caribbean; the other studies have been conducted in the eastern (14) and southern (19)(20)(21)(22) regions. Finally, our novel finding of bacteria associated with toxin-producing dinoflagellates is an important area of BBD etiology that has thus far not been explored.…”

“…In recent years, different molecular techniques have been used to characterize the microbes associated with BBD (14,(19)(20)(21)(22)61). Frias-Lopez and colleagues used terminal restriction fragment length polymorphism and molecular cloning based on 16S rRNA genes to characterize the bacteria and cyanobacteria associated with the BBD community.…”

RNA Arbitrarily Primed PCR and Fourier Transform Infrared Spectroscopy Reveal Plasticity in the Acid Tolerance Response ofStreptococcus macedonicus

“…RAP-PCR was originally adapted from the differential-display PCR method, and it is a powerful technique for the analysis of transcriptional changes, especially those of organisms for which very little or no genetic information is available (6,22). Even though more robust or modern techniques have been developed for gene expression profiling, RAP-PCR has in many cases significant advantages (5,10,36), and it is still being used as a valid method for many studies performed with different organisms (4,17,23,34,37). In our case, we employed this technique to determine transcriptional changes of S. macedonicus due to acid adaptation.…”