Investigation of Natural Products from Pseudoalteromonas luteoviolacea and The Expression and Purification of a Serine Protease HtrA Protein from Faecalibacterium prausnitzii
To mimic the native environment of P. luteoviolacea 2ta16 and to potentially turn on silent gene clusters, including an alterochromide-like cluster, cotton scaffolds were added to the culture media. Modification of culturing conditions such as time, temperature, and culture size, along with the addition of cotton scaffolds, appears to produce significant changes in culture phenotype, and with UPLC-PDA-MS analysis, differences in metabolite production are also observed. Supplementation of the cultures with coumaric acid, a key molecule involved in the biosynthesis of alterochromides, was also investigated. New metabolites with distinctive UV absorbance have been observed, and efforts are ongoing to isolate, purify, and characterize these peaks using HPLC, high-resolution mass spectrometry and NMR analysis. Emerging data has begun to show the bidirectional interactions between the gut, the microbiome, and the brain. Decreased levels of Faecalibacterium prausnitzii (F. prausnitzii) in the gut have been associated with irritable bowel disease, irritable bowel syndrome, Crohn’s disease, and ulcerative colitis. It appears that secretory products of F. prausnitzii act on protease-activated receptor-4 (PAR-4) on gut neurons; PAR-4 activation is anti-nociceptive. Genomic analysis of F. prausnitzii revealed several possible serine proteases encoded in the genome. Two of the encoded serine proteases appear to be the most plausible proteins responsible for the observed anti-nociceptive effects. One serine protease is hypothesized to be membrane-bound, while the other is a high temperature requirement A (HtrA) serine protease. Since previous studies examined the supernatant of commensal bacteria, it was postulated that the serine protease would not be membrane-bound and the HtrA serine protease was investigated. To determine what role the serine protease HtrA might play in the gut, the protein was cloned and heterologously expressed in Escherichia coli.