Assimilation mechanisms of Bacillus subtilis

Abstract

Transport systems in bacteria enable uptake of nutrients and release of useless or even toxic material out of the cell but currently these cellular processes remain poorly characterised. Despite establishing the whole genome sequence of Bacillus subtilis, the use of bioinformatics analyses to identify transport systems has not been effective as expected as in vivo experiments are necessary to demonstrate the expression and the specificity of transporters. This thesis set out to develop methods to identify the substrates of transporters and to characterise their regulation and role in cellular metabolism. In this pilot work amino acids were chosen as the nutrients to study and develop the methods to look at uptake of other molecules. The initial focus was to understand how B. subtilis utilises available nutrients and for this work it was necessary to develop a defined medium that was compatible with both analytical methods and permitted good growth of the bacterial strains. Through this work an understanding of which amino acids are preferentially taken up by B. subtilis and how specific auxotroph’s altered assimilation of amino acids were obtained. Combining with other work, alanine metabolism was indicated poorly defined, and consequently this became a focus of both genetic and phenotypic analyses. The work also indicated that a conventional genetic approach would not permit the identification of transporters for other amino acids and so transcriptional approach was used to try and define genes that were up-regulated in strains starved for specific amino acids. It was expected that some would be involved in the uptake of the limited amino acid. But the results indicated a more complex picture of transcriptional regulation that implies that uptake systems are potentially constitutive and starvation for one specific amino acid tends to result in a global stress response rather than the up-regulation of discrete set of genes to resolve the problem. The work presented details of methods to look at the nutritional priority of bacterial strain using B. subtilis as a model system. The work also clarifies the metabolism of alanine at the level of synthesis, extracellular assimilation and degradation I also showed that specific amino acid starvation did not directly result in the stringent response as might be expected. In summary, this work provides a new perspective on bacterial growth in rich media and the potential factors that limit growth as well as provides an insight into the uptake repertoire of B. subtilis with respect to amino acids. An understanding of which has application in synthetic biology with respect to harnessing of bacterial metabolism for the production of specific bio-molecules.