N-165. Evaluating the Diversity of Anoxyphototrophic Bacteria in Young and Mature Tropical Hypersaline Microbial Mats using Culture-Independent Approaches

F. J. Sanchez-Rivera, C. Rios-Velazquez;
Univ. of Puerto Rico, Mayaguez, PR.

Microbial mats are stratified organosedimentary microbial communities that are vertically distributed in terms of many physical, chemical and biological factors. These microecosystems are typically organized in three layers: a green layer; a pink layer; and a black layer. Each of these layers is characterized by the presence of specific microbial groups. The Tropical Hypersaline Microbial Mats (THMMs) from Puerto Rico exhibit this characteristic layered organization. Our main interest lies in the pink layer, which is mostly populated by Anoxyphototrophic Bacteria (APBs). It is our purpose to assess the diversity of APBs by means of both culture-dependent and culture-independent methods. Here, we present a culture-independent strategy based on the development of pufM gene libraries to evaluate the diversity of APBs in two differentially developed THMMs (mature and young), exposed to seasonal variations (dry and rainy seasons). Due to the fact that mature THMMs exhibit a highly organized and defined structure during both seasons, we expected to obtain a higher degree of diversity in mature mats. Total DNA was extracted from each THMM (rainy season samples) using a chemical-mechanical direct method. In order to detect APBs, pufM (~230bp) was amplified from each sample by PCR. Then, amplicons were cloned onto a vector, which was further transformed into a bacterial host. After obtaining a pufM library, nine clones were randomly selected and tested for the presence of the amplicon. To test whether these clones exhibited differences in their pufM gene composition, these amplicons were subjected to a RFLP analysis using HaeIII restriction enzyme. Based on HaeIII RFLP patterns, six molecular morphotypes were found in the mature mat, whereas only three where found in the young mat. Of the six molecular morphotypes from the mature mat, four of them are unique to that THMM, while only one molecular morphotype from the young mat is unique to that THMM. Both THMMs share two molecular morphotypes, which may suggest that they belong to a common group of microorganisms. In silico analysis is being carried out to assign APBs genus to each molecular morphotype and confirm the common morphotypes in both THMMs.