A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries

Abstract

19In bacterial functionally related genes comprising metabolic pathways and protein complexes are 20 frequently encoded in operons and are widely conserved across phylogenetically diverse species. The 21 evolution of these operon-encoded processes is affected by diverse mechanisms such gene duplication, 22 loss, rearrangement, and horizontal transfer. These mechanisms can result in functional diversification 23 of gene-families, increasing the potential evolution of novel biological pathways, and serves to adapt 24 evolutionary relationships of operon encoded genes and the evolution of operon organization as a 49 whole. To address this challenge, we present a novel method to study operon evolution by integrating 50 phylogenetic tree based clustering and genomic-context networks. We apply this approach to perform 51 the first systematic survey of all known synthase dependent bacterial biofilm machineries, 52 demonstrating the generalizability of our approach for operons of diverse size, protein family 53 composition, and species distribution. Our approach is able to identify distinct biofilm operon clades 54 across phylogenetically diverse bacteria, resulting from gene rearrangement, duplication, loss, fusion, 55 and horizontal gene transfer. We also elucidate different evolutionary trajectories of Gram-negative and 56Gram-positive biofilm production machineries, and in a companion paper (BIORXIV/2019/768473) 57 present the experimental validation of a novel mode of biofilm production in a subset of Gram-positive 58 bacteria. 59 60

Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

“…1), several genera are noticeably absent, including Listeria and Staphylococci. However, 53 Staphylococcal genomes, largely comprising strains of the human pathogen Staphylococcus aureus , were found from our accompanying bioinformatics analysis to possess operons involved in the production of the extracellular polysaccharide PNAG [25], which likely reflects its key role in virulence [36]. Collectively, the data presented here suggests that many Gram-positive bacteria have a previously unrecognized genetic locus that, at its core, is remarkably similar to the pelABCDEFG operon from P. aeruginosa .…”

“…While this work expanded our knowledge of bacteria with pel operons considerably, the use of PelC as a search sequence limits this analysis to Gram-negative bacteria. To overcome this, we developed a computational pipeline that allows for the unbiased identification of homologous bacterial operons [25]. A search for pel loci using the protein coding sequences of the P. aeruginosa PAO1 pel operon as a reference set identified pelF and pelG loci in 43 Gram-positive phyla, including commonly studied species of Bacilli, Clostridia, and Streptococci [25].…”

“…To overcome this, we developed a computational pipeline that allows for the unbiased identification of homologous bacterial operons [25]. A search for pel loci using the protein coding sequences of the P. aeruginosa PAO1 pel operon as a reference set identified pelF and pelG loci in 43 Gram-positive phyla, including commonly studied species of Bacilli, Clostridia, and Streptococci [25]. Manual examination of the genomic context of the pelFG loci revealed the presence of genes whose protein products are consistent with the functions of PelA, PelD, and PelE when analyzed by BLAST [29] and Phyre 2 [30].…”

“…1) would provide the minimally required components for Pel polymerization and transport across the cytoplasmic membrane of Gram-positive bacteria. We used these genes as a basis to perform a second search of fully sequenced Gram-positive genomes that identified new loci that were missed during the initial screen, ultimately identifying 161 Gram-positive organisms harboring pel- like operons (Table S1; [25]).…”

“…In particular, cellulose [23] and poly-β-(1,6)- N -acetyl-D-glucosamine (PNAG; [24]) have been identified as biofilm components across many bacterial genera. In a companion paper [25], we developed a computational pipeline that enables the unbiased identification of functionally related gene clusters from genome sequence data. Applying this pipeline to all bacterial phyla, we performed a systematic search for synthase-dependent polysaccharide biosynthetic operons involved in cellulose, PNAG, alginate and Pel polysaccharide production.…”