Novel regulators of antibiotic biosynthesis in Serratia sp. ATCC 39006

Abstract

Antibiotic resistance is an increasing global concern. Moreover, discovery of new antibiotics has dramatically declined over previous decades. Thus, there is a growing need for these compounds. Understanding of the regulatory processes that control antibiotic biosynthesis may prove exploitable in their controlled hyperproduction. The enterobacterium Serratia sp. ATCC 39006 (S39006) is a useful Gram-negative model for studies on the biosynthesis and regulation of bioactive secondary metabolites, particularly two antibiotics – prodigiosin and a carbapenem. Both compounds are tightly regulated in response to various physiological and environmental signals, including quorum sensing. This project aimed to identify and characterise new regulatory genes that modulate antibiotic biosynthesis in S39006. Novel regulators were sought through random transposon mutagenesis, using the wild type (WT) as parental strain and the prodigiosin production phenotype (red pigment) for screening of mutants. Colonies displaying altered pigmentation were thus selected. The transposon insertion effects on pigmentation were verified by transducing the mutations out into the WT genetic background using a generalised transducing phage. From the confirmed mutants, two strains were chosen for deeper characterisation: one carrying an insertion in wzt and another in an intergenic region. Disruption of wzt, encoding an ABC-transporter of the O-antigen, caused elevated production of both antibiotics. It was also pleiotropic, affecting features such as swimming motility and virulence. Pleiotropy was also found in the intergenic mutant: exhibiting enhanced antibiotic synthesis, and modified motility and virulence capacity. This intergenic insertion then led to the study of VfmE, an AraC-family transcription regulator, which activates antibiotic synthesis. This regulator was previously associated with virulence in phytopathogenic Dickeya strains, operating via a secondary quorum sensing system encoded in the vfm (virulence factor modulating) cluster, which uses an unknown signalling molecule. The members of this Vfm system were also found in S39006 and, by using transposon mutagenesis, their effect on antibiotic production was disclosed. To pinpoint the mechanisms by which wzt, the intergenic, and vfmE mutations act, double mutants with known S39006 regulators were generated to establish connections to the wider regulatory network. Finally, cutting-edge proteomics analysis was conducted for the intergenic mutant. In summary, functional characterisation of the regulatory mutations and their physiological impacts on the modulation of antibiotic production and associated wider pleiotropy were dissected here. This work thus provides insights into the mechanisms modulating antibiotic biosynthesis in S39006 via new regulators.