IDENTIFICATION OF BACTERIA IN A BIODEGRADED WALL PAINTING BY DENATURING GRADIENT GEL-ELECTROPHORESIS OF PCR-AMPLIFIED GENE FRAGMENTS CODINGFOR 16S RIBOSOMAL-RNA

Medieval wall paintings are often affected by biodecay, An inventory o f the existing microorganisms associated with the damage to the painti ngs is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking, Nevertheless , fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their meta bolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We us ed a molecular approach based on the detection and identification of D NA sequences encoding rRNA (rDNA) to identify bacteria present on an a ncient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated un der laboratory conditions, To trace the noncultivated fraction of bact eria, total DNA from a biodegraded wall painting sample from a 13th ce ntury fresco was extracted and 194-bp fragments of the 16S rDNA were a mplified with eubacterial primers, The 16S rDNA fragments of uniform l ength obtained from the different bacterial species were separated acc ording to their sequence differences by denaturing gradient gel electr ophoresis (DGGE), By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the correspon ding bacteria, Using this approach, we identified members or close rel atives of the genera Halomonas, Clostridium, and Frankia. To our knowl edge, these groups of bacteria have not yet been isolated and implicat ed by conventional microbiological techniques as contributing to the b iodegradation of wall paintings.