Abstract
Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 104 clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.
Highlights
The immune system plays an important role in the prevention of bacterial infections
Detection of chemotactic factors by their receptors triggers the activation of cellular movement and results in quickly congregating at an infection site by attraction by cytokines expressed by activated endothelium, mast cells, and macrophages or by bacterial by-products [2]
A well-known example of a formyl peptide is N-formyl-Met-Leu-Pro, which was synthesized by mimicking bacteria
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The discovery of new amino acid ligases has been performed by homology search using the sequences of known enzymes or by selecting candidate sequences that have unique ATP-binding domains in the ligase, and succeeded in expressing them in host cells such as Escherichia coli for functional analysis [6]. As this approach requires the full-length sequence information of the genes or genomic sequence, it is mainly performed on established strains. Domain, an ATP-binding motif that is a conserved domain involved in peptide synthethase, and the gene cluster activating neutrophils was identified
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