ELSEVIER FEMS Microbiology Ecology 17 (1995) 247-256 MICROBIOLOGY ECOLOGY Evaluation of prokaryotic diversity by restrictase digestion of 16s rDNA directly amplified from hypersaline environments A.J. Martinez-Murcia, S.G. Acinas, F. Rodriguez-Valera * zyxwvutsrqponmlkjihgfedcb Departamento de GentZtica y Microbiologia, Unioersidad de Alicante, Campus de San Juan, Apartado 374, E-03080 Alicante, Spain Received 9 March 1995; revised 10 April 1995; accepted 10 May 1995 zyxwvutsrqponmlkjihgfedcbaZYXW Abstract A novel molecular strategy to study microbiological diversity is described. This method is based on the restriction digestion of a population of 16s rDNA sequences directly amplified from an environmental sample. Digested fragments separated by polyacrylamide electrophoresis generate characteristic profile data for estimation of diversity and overall similarities between the organisms of different environments. The methodology has been applied to a set of five ponds in a multi-pond solar saltem covering the salinity gradient from about twice that of seawater (6.4%) to NaCl precipitation (30.8%). Bacterial (eubacterial) diversity estimated from the complexity of the banding pattern obtained by restriction of the amplicons from the different ponds decreased with increasing salinity, while for Archaea (archaebacteria) the reverse was true, i.e. the higher the salinity the higher the number of bands. The similarities in taxonomic composition of the prokaryotic populations present in those ponds were evaluated from the number of restriction bands shared by the different samples. The relationships found among the different environments were independent of the enzyme used for digestion and were consistent with previous descriptions obtained by the study of isolates from the different environments. The technique appears to be promising as a rapid method for microbial biodiversity fingerprinting, useful to compare several environments and detect major shifts in species composition of the microbial population. Keywords: Biodiversity; 16s rRNA, Halophilic bacteria; Halophilic archaea; Hypersaline environments 1. Introduction The study of microbial diversity can now be approached by molecular methods using cloning and/or PCR amplification techniques. Highly reli- able diagnostic genes such as 16s rRNA genes can be retrieved directly from total DNA extracted from a natural environment. The amplified genes can be * Corresponding author. Tel: + 34 (6) 5658554; Fax: + 34 (6) 5941787; E-mail: frvalera@vm.Cpd.Ua.Es sequenced and the information used to describe the microbial diversity present in the sample [l-12]. The data include information about non-cultivated (or non-cultivable) microorganisms, which is a signifi- cant improvement over classical pure culture isola- tion techniques for studying microbial diversity. However, the number of samples and sequences per sample is limited by the sequencing capabilities of the laboratory and therefore most studies that have been carried out investigate only about 100 se- quences at the most. To be able to evaluate the biodiversity present in a sample rapidly, and also to 0168-6496/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SSDI 0168-6496(95)00029-l Downloaded from https://academic.oup.com/femsec/article-abstract/17/4/247/505197 by guest on 14 June 2020