Transcriptional analysis of biofilm formation and stress response in hyperthermophilic microorganisms

by 1974- Pysz, Marybeth Anne

PYSZ, MARYBETH ANNE. Transcriptional analysis of biofilm formation and stress response in hyperthermophilic microorganisms. (Under the direction of Robert M. Kelly.) The significance of surface colonization and changing thermal conditions in hydrothermal environments motivated examination of biofilm formation and thermal stress response in the model heterotrophic hyperthermophilic microorganisms, Thermotoga maritima and Pyrococcus furiosus. Continuous culture, using maltose-based media and anaerobic conditions at 80°C for T. maritima and 95°C for P. furiosus, was used to generate dense biofilms on nylon mesh and polycarbonate filters; significant amounts of wall growth were observed in the chemostats for both organisms. Transcriptional analysis of biofilmbound cells showed that genetic mechanisms observed for biofilm formation in less thermophilic bacteria applied to T. maritima. L-lactate dehydrogenase (TM1867), NADH oxidase (TM0379), sensor histidine kinase (TM0187), and TetR family transcriptional regulator (TM0823) were among the genes induced in T. maritima biofilms with mesophilic counterparts. Also consistent with cells in mesophilic biofilms was the differential expression of stress-related genes. Thermal stress genes, hrcA (TM0850), grpE (TM0851), and dnaK (TM0373) were up-regulated, indicating that elements of stress response are operational in hyperthermophilic biofilm environments. Expression of stress-related genes in the T. maritima biofilm prompted a study of stress response during heat shock at 90°C. A 407-gene targeted cDNA microarray was used to study the genetic differences between cells at 80°C and cells at 90°C after 0, 5, 10, 20, 30, 60, and 90 minutes. The two major heat shock operons dnaJ-grpE-hrcA (TM0849-TM0850- TM0851) and groEL-groES (TM0505-TM0506), as well as the genes encoding DnaK (TM0373) and heat shock protein class I (TM0374), exhibited maximal induction at early times (~5 minutes), subsequently decreasing to a steady-state level. This expression pattern has also been observed during heat shock of the mesophilic bacteria Escherichia coli and Bacillus subtilis. Also observed was the stress-related response of the SOS regulon involving usrB (TM1761) and recA (TM1859), and the down-regulation of this operon’s repressor lexA (TM1082). Atypical of heat shock response, the majority of genes encoding ATP-dependent proteases, including ClpP (TM0695), ClpQ (TM0521), ClpY (TM0522), LonA (TM1633), and LonB (TM1869), were down-regulated. ATPase Clp C subunits 1 (TM0198) and 2 (TM0873) were both up-regulated, along with ClpX (TM0146) and FtsH (TM0580). The ATP-independent heat shock serine protease HtrA (TM0571) was also induced. A number of other genes not related to stress response also showed significant changes in expression levels. These include transcriptional regulators, genes within the gluconate metabolic pathway, sugar transporters and glycosidases, and sigma factors. Homologs to ?E and ?A were induced during heat shock at 90°C, and suggesting that they are implicated in stress response regulation in T. maritima, although they have not been characterized to date. This work led to the development of chemostat-based methods for generating RNA from hyperthermophiles embedded in anaerobic biofilms that could be used for transcriptional analysis. Such analysis indicated possible connections between the genetic response of biofilm-bound cells and thermal stress response. The results here point to the significance of surface colonization and modification of cellular function arising from thermal changes in the microbial ecology of hydrothermal environments. Transcriptional Analysis of Biofilm Formation and Stress Response in Hyperthermophilic Microorganisms By