14 NSFBIO:Identifying Mechanisms for Environmental Adaptation in CMNR Bacteria: A Systems Analysis of GeneRegulation of AlternativeSubstrateMetabolism

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Members of the Corynebacteria-Mycobacteria-Nocardia-Rhodococcus (CMNR) group of bacteria are best recognized for their impact on human health and they include the causative agent of tuberculosis (TB; Mycobacterium tuberculosis), leprosy (Mycobacterium leprae) and diphtheria (Corynebacterium diptheriae). However, many members of this group are also of great significance to agriculture, biotechnology, and environmental issues. CMNR bacteria also include animal (Mycobacterium bovis, Rhodococcus equi, and species of Corynebacteria and Nocardia) and plant (Rhodococcus) pathogens with a broad host range. They also are widely used in industry. Examples include Corynebacterium glutamicum which is used for large-scale manufacturing of amino acids such as L-glutamate and bacteria that are known to accumulate triacyglycerols (TAGs), oils that are the direct building blocks for biodiesel. Furthermore, some CMNR bacteria are also used for a process termed bioremediation due to their ability to break down harmful and persistent pollutants in the environment. A common theme among the CMNR bacteria is a lipid-rich cell envelope that contains fatty acids and lipids that are distinct and not found in other bacteria. This complexity is reflected in the relatively large proportion of lipid metabolism related genes in the genomes of these bacteria. The diversity of habitats colonized by CMNR bacteria is rooted in their ability to adapt to new environments by dynamically altering the composition of complex fatty acids in their unique cell envelope. This proposal will test the hypothesis that depending on the environment and available substrates, the CMNR bacteria selectively alter the composition of their cell envelope. We propose that the bacteria achieve by using expanded families of genes involved in the uptake and biosynthesis of cell envelope 'building blocks'. To elucidate how CMNR bacteria achieve this we will first test the ability of a model CMNR bacterium, Mycobacterium smegmatis, to grow on different substrates that can potentially act as 'building blocks'. We will select growth conditions that lead to cell envelope alterations and determine the transciptome (a global picture of the levels to which genes are expressed) of M. smegmatis growing under the select conditions. The information obtained from these studies will then be analysed using in-house computational tools (a program called EGRIN 2.0) to generate models of gene regulatory networks. In other words, we will 'reverse engineer' these networks in the model bacterium. This model will subsequently allow us, by comparing published genomes, to characterize common and unique regulatory mechanisms across CMNR bacteria. Model predictions will be tested by analyzing consequences of specific gene deletions on cell envelope composition of representative CMNR bacteria under relevant conditions. This inter-institutional proposal combines the complementary expertise of the US and UK based research groups. The systems biology expertise of the US-based PI with the UK-based PI's expertise in characterizing CMNR bacteria lipid metabolism and cell envelope biochemistry will allow us to address the fundamental and biotechnologically important question of how CMNR bacteria adapt to new environments.