Studying of Gene Expression Involved in Hyaluronic Acid Synthesis in Streptococcus Equi Subsp. Zooepidemicus Using DNA Microarrays and Real-Time PCR

Hyaluronic acid (HA) is an important substance, which is mostly used in pharmaceutical and cosmetic industry. This substance is commonly found in the human body. HA is one of the factors contributing to virulence of microorganisms. Some bacterial strains produce hyaluronic acid in the form of a mucoid capsule that encapsulates the cell to protect bacteria against the immune system of the host organism. One of the main producers is the bacterial strain Streptococcus equi subsp. zooepidemicus. Contipro a.s. uses the strain CO4A to produce hyaluronic acid in large scale. The production strain was obtained by random mutagenesis by UV light. The aim of the work was to study changes in the genome, which led to a significant increase in hyaluronic acid production, using DNA microarray and real-time PCR (qPCR). The genome of the strain CO4A was sequenced and compared to reference ATCC35246 [1]. The size of the genome is 2,167,251 bp and 83 relevant variants (59 SNV and 34 indels) have been identified. Variants in coding regions were annotated and amino acid sequence changes were determined. In SNV mutations there was a change in the amino acid sequence in 45 cases. The change was identified in every case of indel mutations. The expression level of selected groups of genes was monitored in both strains by the method of DNA microarrays. A cascade of increased expression level of amino sugar metabolism genes leading to the synthesis of UDP-N-acetyl glucosamine was observed in strain CO4A (the increase in expression level of these genes compared to ATCC35246 was on average 28 %). Subsequently, the expression of selected genes was verified by qPCR. There was no significant difference in the expression level of the has operon genes of both strains. The effect of supplementation of the culture medium with N-acetylglucosamine (GlcNAc), which is one of the precursors of HA synthesis, was also studied by qPCR. A positive effect of the supplementation of the culture medium with external GlcNAc in the CO4A strain has been recorded. Also, the supplementation has positive effect on the yield of HA from the medium (increase in yield was on average by 17 %). GlcNAc has been shown to have a positive effect on the yield of HA in ATCC35246 strain as well (increase in yield was 9 % on average), but no significant changes in the expression levels were found in selected groups of genes in ATCC35246