Genetic engineering-based approach to explore the bioactive potential of Pseudoalteromonas rubra S4059, a prodigiosin-producing marine bacterium

Abstract

Antimicrobial resistance is a huge threat to global public health and we urgently need to discover novel antibiotics to treat infectious diseases. Most antibiotics are derived from natural products of microorganisms and they are typically encoded by biosynthetic gene clusters (BGCs) which are collections of the genes encoding biosynthetic enzymes that are responsible for natural compounds production. Mining of bacterial genomes has indicated that there is a large number of BGCs for which we do not yet know the chemistry; so a potentially untapped source of bioactive compounds in microorganisms. More recently, marine bacteria, for instance from the genus Pseudoalteromonas, have received attention due to their production of bioactive compounds, both enzymes and chemicals with drug activities with potential for pharmaceutical and industrial applications. The purpose of this project is to explore the bioactive potential of a pigmented bacterium Pseudoalteromonas rubra S4059 with genetic manipulation and cultivation-based approaches. Genome mining on pigmented Pseudoalteromonas has shown that these species harbor large amounts of orphan BGCs and have a large potential for novel compounds production, however, these orphan BGCs are considered “silent” under laboratory conditions. Mimicking the natural environment using ecological niche associated nutrients such as chitin, can induce secondary metabolite production in some Vibrios and also activate unknown BGCs. Chitin degradation is a trait for pigmented Pseudoalteromonas species and thus, we hypothesized that there also in Pseudoalteromonas could a link between chitin degradation and bacterial secondary metabolites production. Using proteomic, metabolomic and genetic approaches, we found that chitin does not significantly influence secondary metabolites production in S4059 as compared to other carbon sources, however, its degradation products, chitin monomer can induce the overall high production of secondary metabolites in S4059. Surprisingly, we found that growth on natural chitin surface leads to spontaneous mutation of S4059 that abolishes pigment production. The non-pigmented mutants appeared to have a fitness advantage as compared to the wildtype as the degree of autolysis was lower. Thus, we here for the first time report that growth on chitin surface can provide genetic diversity driving bacterial evolution of pigmented Pseudoalteromonas. Furthermore, we used the developed genetic manipulation approach to verify a prodigiosin BGC and found that a blue pigment, dipyrrolyldipyrromethene prodigiosin, was produced by the pigC mutant of P. rubra S4059. In conclusion, this work has contributed by developing a genetic approach to manipulate S4059 and thus has extended the genetic manipulation tools in this genus. Also, this work provides a novel sight on ecological niche driving pigmented Pseudoalteromonas evolution.