Molecular and genomic analyses in Clostridium acetobutylicum

[摘要] This dissertation presents molecular and genomic analyses inClostridium acetobutylicum, beginning with an evaluation of Spo0A, a transcriptional regulator required for activation of solvent production genes. While the transcriptional response of Spo0A-dependent genes has been well characterized, this work examines the level of proteins influenced by Spo0A. Proteomic changes are compared when Spo0A levels vary. Six proteins require Spo0A for maximal accumulation. Thirteen proteins have altered levels at the transition to solventogenesis, when Spo0A is active, thus implicating Spo0A in modulating their accumulation. Five proteins were located at more than one position on the two-dimensional gel, suggesting that post-translational modification may be more prevalent in prokaryotes than previously recognized.Because two proteins that require Spo0A are clostridial hydrophobic with a conserved tryptophan (ChW) proteins, this dissertation, secondly, characterizes this family. These proteins are novel and essentially limited toC. acetobutylicum; thus, suggesting roles specific to its unique physiology. Based on sequence features, they are thought to be surface associated proteins. Reporter assays and primer extension experiments establish thatchw14 and chw 16/17 are primarily transcribed by GA during exponential phase. Reporter analysis of chw14 andchw16/17 in the spo0A null strain indicates that Spo0A influences the expression of these genes, while the proteome data suggest that Spo0A is necessary for maximal accumulation of the encoded proteins. Phylogenetic reconstruction suggests that ChW domains function in triplets.Lastly, this dissertation analyzes the enzymatic properties of several butyrate kinase II mutants and examines the genomic arrangement of their encoded genes. At several positions the native amino acid was required for activity while at two positions a conservative change was tolerated. Additionally, the local structural arrangement of a specific trio of amino acids (DFK in BKI and NAL in BKII) may explain the substrate activity differences between these enzymes. A leucine residue in BKII is not expected to make the same cation-pi interactions with the ATP ring structure as the native lysine in its analogous position in BKI. The genomes of related and relevant prokaryotes contain multiple genes coding for carboxylate kinases, thus the presence of multiple isozymes may be common in prokaryotes.

[发布机构] Rice University

[效力级别] biology