Transcriptome analysis of the lactic acid and NaCl-stress response of Lactobacillus plantarum

Many cellular processes are regulated at the transcriptional level. For this reason the transcriptome of a cell, the total set of RNAs under a specific condition, contains information about the biological state of the cell and the genes that play a role under specific circumstances.Transcriptomics is the research method that studies the effect of specific conditions on alterations in the expression levels of complete sets of genes. This thesis describes the application and optimization of transcriptomics and transcriptomics-related techniques in studies on lactic acid and sodium chloride stress-related gene expression in Lactobacillus plantarum WCFS1. The complete genomic sequence of this strain is available, and the organism is used as a model strain for lactic acid bacteria in industrial applications and host-microbe interactions.

In order to maximize specificity and reliability of transcriptome data, a quenching protocol for the isolation of microbial cells from submerged cultures was implemented and validated. The results from this study revealed a significant positive effect of the quenching protocol on the conservation of the transcriptome, and consequently on the reliability and representativity of the gene expression data.

Transcriptome analyses were performed with clone-based microarrays. In order to optimise the potential of clone-based arrays for transcriptomics studies of strains from which the complete genomic sequence is not available, two complementary equations were developed that allow for mathematical design of such an array.The first equation predicts the fraction of genes that is represented on the array in a detectable way and cover at least a set part of the genomic fragment on the array. The second equation predicts the fraction of genes for which information can be interpreted in a quantitative way.

The effect of lactic acid on gene expression in L. plantarum was studied by means of an experimental design that allows for the distinction between the long term effects of lactate, lactic acid, pH, water activity and growth rate. A group of genes was identified that specifically responded to the undissociated form of the lactic acid molecule. This group contained, among others, several genes that have been associated with other stress responses, as well as a highly coherent group of cell surface protein-encoding genes. From these genes, two putative regulators, a peroxide-resistance gene, and one of the cell surface protein encoding clusters were selected for validation of their role under lactic acid-stress. Experiments with mutant strains in which these genes were inactivated or overexpressed gave no specific indication on their relevance under the tested conditions.

In a study on the long term effects of NaCl stress on L. plantarum no increased expression was observed for genes that are typically related to acute osmotic stress. The results indicate a different nature of NaCl-imposed stress on the cells on the long term for which adaptations over multiple functional classes are required. A remarkable observation was the detection of a high number of differentially expressed genes under continous NaCl stress with the consensus sequence for the catabolite responsive element cre in their upstream region.

The results presented contribute to the reliability and applicability of transcriptomics research in microorganisms. Moreover, the results from the studies on the response of L. plantarum towards lactic acid- and NaCl-stress provided new and global insights in the nature of these stresses and may serve a role in strain- and fermentation-optimizations.