Enhancing the antibacterial function of probiotic bacteria: when less is more

Antibiotic resistant bacteria undoubtedly represent a major public health threat and is a global problem compounded by dwindling treatment options. The mammalian gut is a major reservoir for the carriage of antibiotic resistant bacteria and thus represents a target for prophylactic interventions. Probiotic bacteria confer multiple health benefits, including preventing the growth, colonisation, or carriage of harmful bacteria in the gut. Bacteriocins are antibacterial peptides produced by diverse bacteria and their production is tightly regulated and coordinated at the transcriptional level. A popular strategy for enhancing the antibacterial properties of probiotic bacteria is to retrofit them with the ability to overproduce heterologous bacteriocins constitutively or in response to host or pathogen signal from synthetic promoters. But how the dysregulated overproduction of heterologous bacteriocins affects physiology and antibacterial efficacy of the retrofitted probiotic bacteria is often overlooked. We have conferred the prototypical probiotic Escherichia coli strain Nissle (EcN) the ability to produce wild-type, elevated and reduced amounts of the bacteriocin microcin C (McC) from its naive promoter and in response to native regulatory signals. We show that EcN bacteria that produce the lowest amount of McC display the highest antibacterial efficacy with little to none undesired collateral physiological impact. The results highlight considerations researchers may take into account when retrofitting probiotic bacteria with heterogenous gene products for therapeutic, prophylactic or diagnostic purposes.