Developing robust protein analysis profiles to identify bacterial acid phosphatases in genomes and metagenomic libraries

Abstract

Phylogenetic analysis of more than 4000 annotatedbacterial acid phosphatases was carried out. Ouranalysis enabled us to sort these enzymes into thefollowing three types: (1) class B acid phosphatases,which were distantly related to the other types,(2) class C acid phosphatases and (3) generic acidphosphatases (GAP). Although class B phospha-tases are found in a limited number of bacterial fami-lies, which include known pathogens, class C acidphosphatases and GAP proteins are found in a vari-ety of microbes that inhabit soil, fresh water andmarine environments. As part of our analysis, wedeveloped three profiles, named Pfr-B-Phos, Pfr-C-Phos and Pfr-GAP, to describe the three groups ofacid phosphatases. These sequence-based profileswere then used to scan genomes and metagenomesto identify a large number of formerly unknown acidphosphatases. A number of proteins in databasesannotated as hypothetical proteins were also identi-fied by these profiles as putative acid phosphatases.To validate these in silico results, we cloned genesencoding candidate acid phosphatases from geno-mic DNA or recovered from metagenomic libraries orgenes synthesized in vitro based on proteinsequences recovered from metagenomic data.Expression of a number of these genes, followed byenzymatic analysis of the proteins, further confirmedthat sequence similarity searches using our profilescould successfully identify previously unknown acidphosphatases