Identification and analysis of the expression of growth-dependent transcripts detected by RAP-PCR in the haloalkaliphilic archaeon Natrialba magadii

MADRID ENRIQUE; MICAELA CERLETTI; ROBERTO PAGGI; ROSANA DE CASTRO

Carlos Paz, Córdoba

Congreso; VI Congreso Argentino de Microbiología General; 2009

SAMIGE

Extremophiles live in environments with conditions that are lethal for most life forms. Particularly, haloarchaea have developed strategies to survive in the presence of high salt concentrations (>2M NaCl) and high pH values (alkaliphilic haloarchaea). The transition to the stationary phase induces changes in the morphology and physiology of bacteria. Often, such changes are regulated by a cell to cell based-communication mechanism denoted as quorum sensing. The changes induced during the transition from exponential growth to the stationary phase as well as the occurrence of quorum sensing-like mechanisms have been poorly explored in archaea and in organisms thriving in extreme environments. The RNA arbitrarily primed PCR (RAP-PCR) is a powerfull method that can be applied to identify genes that are differentially expressed. Differences in gene expression can be detected using this methodology, as each sample generates its own unique RNA fingerprint for each condition examined. The aim of the present work was to identify those genes that are differentially regulated in response to the transition from the exponential to the stationary phase in the alkaliphilic haloarchaeon Natrialba magadii. The screening was also aimed to those genes whose induction responded to the high cell density and, thus, may be regulated by quorum sensing. By using RAP-PCR, we have previously obtained 9 differentially expressed cDNAs which were cloned, sequenced and analyzed by bioinformatics search. Two of these cDNAs, 500 bp and 350 bp, were further characterized in this work. The 500 bp sequence (L1500) was identified as a HIT protein (histidine triad protein), while the 350 bp sequence (L6350) showed identity to an oligopeptide ABC transporter solute-binding protein. Specific primers were designed based on sequence analysis these cDNAs and used to validate the differential expression pattern by RT-PCR. The relative mRNA level of these transcripts was normalized to the 7S RNA transcript level. The HIT protein mRNA was highly expressed in the exponential phase and decreased 5- fold as the cells entered the stationary phase. On the contrary, the abundance of the ABC transporter mRNA was low in exponentially growing cells and increased 4-fold in the early stationary phase. In addition, the expression of this transcript was also analyzed in response to the presence of stationary phase conditioned medium (CM) showing a 2.5-fold increase in the exponentially growing cells supplemented with CM compared to the control cells. The identification of genes that respond to different growth stages and the characterization of signal molecules involved in this process will help to understand the nature of the molecular mechanism of cellular communication used by the haloarchaea in extreme environmental conditions.