Integron gene cassettes: a repository of novel protein folds with distinct interaction sites.

Mobile gene cassettes captured within integron arrays encompass a vast and diverse pool of genetic novelty. In most cases, functional annotation of gene cassettes directly recovered by cassette-PCR is obscured by their characteristically high sequence novelty. This inhibits identification of those specific functions or biological features that might constitute preferential factors for lateral gene transfer via the integron system. A structural genomics approach incorporating x-ray crystallography has been utilised on a selection of cassettes to investigate evolutionary relationships hidden at the sequence level. Gene cassettes were accessed from marine sediments (pristine and contaminated sites), as well as a range of Vibrio spp. We present six crystal structures, a remarkably high proportion of our survey of soluble proteins, which were found to possess novel folds. These entirely new structures are diverse, encompassing all-α, α+ß and α/ß fold classes, and many contain clear binding pocket features for small molecule substrates. The new structures emphasise the large repertoire of protein families encoded within the integron cassette metagenome and which remain to be characterised. Oligomeric association is a notable recurring property common to these new integron-derived proteins. In some cases, the protein-protein contact sites utilised in homomeric assembly could instead form suitable contact points for heterogeneous regulator/activator proteins or domains. Such functional features are ideal for a flexible molecular componentry needed to ensure responsive and adaptive bacterial functions.

Structural genomics of the bacterial mobile metagenome: an overview.

Mobile gene cassettes collectively carry a highly diverse pool of novel genes, ostensibly for purposes of microbial adaptation. At the sequence level, putative functions can only be assigned to a minority of carried ORFs due to their inherent novelty. Having established these mobilized genes code for folded and functional proteins, the authors have recently adopted the procedures of structural genomics to efficiently sample their structures, thereby scoping their functional range. This chapter outlines protocols used to produce cassette-associated genes as recombinant proteins in Escherichia coli and crystallization procedures based on the dual screen/pH optimization approach of the SECSG (SouthEast Collaboratory for Structural Genomics). Crystal structures solved to date have defined unique members of enzyme fold classes associated with transport and nucleotide metabolism.