Regulation and physiological roles of the microbial secondary metabolite indole in its producing and non-producing bacterial species

Abstract

Microorganisms produce a diverse range of secondary metabolites which are often characteristic of their producing organisms. They have broad applications in health care and society (e.g., natural antibiotics, pigments, toxins)and biosynthetic genes of novel secondary metabolites are broadly distributed in newly sequenced microbial genomes. Yet, why microorganisms synthesize these substances in natural ecological niches and how their production and presence correlates with microbial physiology remains largely unknown.

Indole is a common microbial secondary metabolite produced by many bacterial species and is an established marker for the identification of Enterobacteriaceae. However, regulation of indole biosynthesis and its physiological functions in its producing species remain unclear.A previous study from our group found that indole production was induced during the anaerobic growth of Escherichia coli. In the first part of my thesis, I systematically investigated indole biosynthesis under various anaerobic metabolic status. While the expression of the tnaCAB indole biosynthetic operon and consequently indole production was induced in rich medium in the presence of several respiratory electron acceptors under anaerobic condition, indole biosynthesis in E. coli was found to be repressed during nitrate respiration under this condition, especially at the log growth phase. This inspired me to systematically investigate indole biosynthesis during anaerobiosis. The tnaCAB operon was found to be repressed by NarXL and NarPQ in an FNR-dependent manner (FNR is an anaerobic global regulator). Biochemical analysis showed that when indole was present during anaerobic nitrate respiration, it was rapidly converted to indole nitrosative derivatives, which led to significantly increased DNA mutation frequency in E. coli. Repression of indole biosynthesis during nitrate respiration provided the bacteria with competitive advantages over indole-producing species. This indicated that production of the secondary metabolite indole was closely coupled with the metabolic status of its producing bacterium to facilitate physiological adaptation. These results demonstrated a physiological process where production of the secondary metabolite was repressed and this genetic repression contributed to cell physiology.

In the second part of my thesis, I further investigated the effect of physiological indole on non-producing bacteria co-existing with indole-producing species. Co-culture of E. coliK-12 MG1655 (indole-producing) and opportunistic pathogen Pseudomonas aeruginosaATCC27853 (non-indole producing) was used as the model system.Co-culturing the two species led to stimulation of pyocyanin (PYO), a pigmented virulence factor in P. aeruginosa, in an indole-dependent manner. This stimulation was found to be mediated by the PQS (Pseudomonas Quinolone Signal) quorum sensing system in ATCC27853.Further studies revealed that indole indeed impacted various physiological activities in ATCC27853 related to its pathogenicity and virulence, and indole pre-treatment led to enhanced aggregation of ATCC27853 on human bronchial epithelial cells (16HBE). These results suggested that indole could influence the physiology of surrounding non-indole producing P. aeruginosa and this interspecies communication has implications in polybacterial infections.

This research provides insightful advancements on the physiology of microbial secondary metabolites, which will benefit the investigation of the complex world of microbial communities.