Differential Gene Expression and Colanic Acid Gene Effects on Biofilm Formation

Bates, Christa Lynn
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Biofilm gene expression is an emerging field of study within bacterial genomics. Investigators strive to understand changes in phenotype, morphology, and metabolism in response to selective pressures within various environments. By understanding the genetic switches activated as a result of phenotype change we may more readily control the growth of non-beneficial bacterial communities. This study analyzed general differential expression due to the maturity of the biofilm community through statistical analyses of microarray data from four and six day biofilm cultures. Molecular sequence analyses were performed to demonstrate genomic affects of insertion or deletion mutations. Lastly, characterization of insertion mutations of wzc with respect to biofilm formation was performed to assess the role of colanic acid in newly formed biofilms. A significant difference was found between gene expression and maturity using the ANOVA. The post mortem analyses of biofilm cultures found significant changes in genes with functions in translation, ribosomal structure and biogenesis, transcription, DNA replication, recombination and repair, cell division and chromosome partitioning, cell envelope biogenesis and outer membrane synthesis, energy production and conversion, carbohydrate transport and metabolism, coenzyme metabolism, lipid metabolism, and secondary metabolite biosynthesis and transport catabolism. These finding suggest a metabolic shift as a result of maturation between four and six day biofilms. A major oversight in current research is pre-mutagenesis sequence analyses to determine the potential genomic expression affects. Molecular sequence analyses were performed to demonstrate methods for pre-mutagenesis sequence characterization. Sequences of up-, down-, and neutrally regulated genes were analyzed along with surrounding sequence up and downstream. Sequences were analyzed in six frames for amino acid sequence, potential overlapping open reading frames (ORFs), protein homology, conserved domain presence, RNA secondary structure, and protein locality. Up-regulated bl995 is potentially involved in outer membrane permeability control, is possibly a trans-acting factor, and is most likely located in the periplasmic area between the inner and outer membranes of Gram-negative bacteria. Neutrally transcribed, bl394 is potentially involved in operons controlling the switch between aerobic and anaerobic growth with a protein localization of cytoplasmic nature. Down-regulated gene, b2852 is suspected to be involved in some type of membrane permeability control, however these results were not clear and the protein localization could not be predicted. These predictions will be used in fixture work as a basis for analyzing phenotypical and morphological changes post-mutagenesis. Specific analyses were performed to assess the role of colanic acid, exopolysaccharide, synthesis on early biofilm formation. Escherichia coli JM83 and Escherichia coli JM83wzc::KmR were transformed with plasmids which expressed functional and single amino acid mutations for wzc. This gene is suggested to prevent colanic acid production when mutated. Colanic acid production, namely fucose a non-dialyzable methyl-pentose, was quantified by growth of all strains in nutrient rich and minimal médias followed by centrifuge extraction from broth, dialysis, lyophilization, concentration, and standardized against L-fucose. Biofilm production was quantified via methods described by George O’Toole and Roberto Kolter. Amounts of colanic acid produced did not correlate to the functionality of wzc, which suggests its lack of influence on exopolysaccharide synthesis within the first 48 hours of growth, It was also found that functionality of wzc does not correlate to biofilm formation. Moreover, mutations in phosphorylation and catalytic sites for colanic acid production may promote biofilm formation. The information obtained herein can be further applied to targeting specific genes and gene products for promoting or discouraging biofilm formation.
Biofilms, Gene expression, Phenotype
Bates, C. L. (2004). <i>Differential gene expression and colanic acid gene effects on biofilm formation</i> (Unpublished thesis). Texas State University-San Marcos, San Marcos, Texas.