Environmental Microbiology (2004) 6(7), 678–685 doi:10.1111/j.1462-2920.2004.00619.x © 2004 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 20046 7678685Original ArticleDesign of new probes for Geodermatophilaceae membersC. Urzì, V. La Cono and E. Stackebrandt Received 7 November, 2003; accepted 4 February, 2004. *For correspondence. E-mail Clara.Urzi@unime.it; Tel. (+39) 090 6765196; Fax: (+39) 090 392733. Design and application of two oligonucleotide probes for the identification of Geodermatophilaceae strains using fluorescence in situ hybridization (FISH) Clara Urzì, 1 * Violetta La Cono 1 and Erko Stackebrandt 2 1 Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Salita Sperone, 31, I-98166 Messina, Italy. 2 DSMZ – German Collection of Microorganisms and Cell Cultures GmbH, Manscheroder Weg 1b, D-38124, Braunschweig, Germany. Summary Bacteria of the family of Geodermatophilaceae are actively involved in the decay processes [Urzì, C. and Realini, M. (1998) Int Biodeterior Biodegrad 42: 45–54; Urzì, C., Salamone, P., Schumann, P., and Stacke- brandt, E. (2000) Int J Syst Evol Microbiol 50: 529–536] of stone monuments. Characterization of isolates includes phenotypic, chemotaxonomic and genetic analysis often requiring long-term procedures. The use of specific probes for members of Geodermato- philaceae family could be useful for the easy detec- tion of those strains colonizing rock surfaces and involved in the biodeterioration. Two 16S rRNA- targeted oligonucleotide probes were designed for the specific detection of members of the family Geo- dermatophilaceae using fluorescence in situ hybrid- ization (FISH); one probe specific for members of the two genera Geodermatophilus/Blastococcus and the second for members of the genus Modestobacter. Introduction It has become evident in recent years that a great variety of actinomycetes must be considered major colonizers of stone surfaces exposed to outdoor and indoor environ- ments (Urzì et al., 2000; 2003). Among these, up to 90– 100% of strains isolated from various rock samples (Urzì et al., 2001) belong to three genera of the family Geoder- matophilaceae (Stackebrandt et al., 1997), i.e. Geoder- matophilus (Luedemann, 1968), Blastococcus (Ahrens and Moll, 1970; Urzì et al., 2004) and Modestobacter (Mevs et al., 2000). These bacteria were also often isolated from decaying building stones and it has been suggested that they are actively involved in the decay processes (Urzì and Realini, 1998; Urzì et al., 2000). All these surfaces were in fact characterized by a high level of alteration, including the formation of grey and/or orange diffused patinas and, moreover, by phenomena of alveolization, detachment, biopitting, crumbling and powdering (Urzì et al., 2001). The characterization of isolates resulting in their generic affiliation include time- and cost consuming phenotypic, chemotaxonomic and genomic analysis (Urzì et al., 2001; 2004) and, if restricted to mainly morpholog- ical characterization, were not discriminative. Fluorescence in situ hybridization (FISH), circumvent- ing a lengthy identification process, is a rapid and highly valuable tool for the cultivation-independent identification of individual microbial cells from environmental samples using rRNA-targeted oligonucleotide probes (Amann et al., 1990). Since then, the scientific literature witnesses the description and application of a broad battery of taxon- specific oligonucleotide probes (see Amann et al., 1995; 2001) for rapid determination of organisms which are dif- ficult to either differentiate by traditional criteria or to obtain pure cultures. Design of new probes for specific taxa of microorganisms thus allows the application of a top-to-bottom approach for the characterization of the microbial community structure. In recent years, FISH has also been applied in the field of works of arts to study Eubacteria and Archea involved in the biodeterioration of surfaces (Piñar et al., 2001; Urzì and Albertano, 2001; Urzì et al., 2003). However, as sequences of only a few members of the family of Geo- dermatophilaceae were determined, no specific probes had been designed. In this study we apply two probes to pure cultures isolated recently (Urzì et al., 2000; 2003) one specific for members of the two genera Geodermato- philus and Blastococcus, the second for members of the genus Modestobacter, in order to obtain a rapid tool for the identification that targets all type strains and genomi- cally related isolates. As a result of the fact that most of these microorganisms live on rocks, especially in dry climates, they tend to cluster in more or less thick aggregates with the conse- quent reduction of cell permeability and the accessibility