Feed-Forward Regulation of Microbisporicin Biosynthesis in Microbispora corallina

Abstract

Lantibiotics are ribosomally synthesized, posttranslationally modified peptide antibiotics. Microbisporicin is a potent lantibiotic produced by the actinomycete Microbispora corallina and contains unique chlorinated tryptophan and dihydroxyproline residues. The biosynthetic gene cluster for microbisporicin encodes several putative regulatory proteins, including, uniquely, an extracytoplasmic function (ECF) factor, MibX , a likely cognate anti-factor, MibW, and a potential helix-turn-helix DNA binding protein, MibR. Here we examine the roles of these proteins in regulating microbisporicin biosynthesis. S1 nuclease protection assays were used to determine transcriptional start sites in the microbisporicin gene cluster and confirmed the presence of the likely ECF sigma factor ؊10 and ؊35 sequences in five out of six promoters. In contrast, the promoter of mibA, encoding the microbisporicin prepropeptide, has a typical Streptomyces vegetative sigma factor consensus sequence. The ECF sigma factor MibX was shown to interact with the putative anti-sigma factor MibW in Escherichia coli using bacterial two-hybrid analysis.MibX autoregulates its own expression but does not directly regulate expression of mibA. On the basis of quantitative reverse transcriptase PCR (qRT-PCR) data, we propose a model for the biosynthesis of microbisporicin in which MibR functions as an essential master regulator and the ECF sigma factor/anti-sigma factor pair, MibX /MibW, induces feed-forward biosynthesis of microbisporicin and producer immunity.

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

“…These modifications are unique to NAI-107 within the lantibiotic class of compounds and are rare in other ribosomally synthesized peptides. Genetic analysis previously identified the biosynthetic gene cluster and revealed insights into the pathway-specific regulation by an extracytoplasmic function -factor⅐anti--factor complex (26,27).…”

The antibiotic planosporicin coordinates its own production in the actinomycete Planomonospora alba

“…Thus, there is a feed-forward mechanism of regulation in which the production of a small, apparently subinhibitory concentration of the antibiotic results in activation of planosporicin biosynthesis throughout the substrate mycelium, much of which has not reached the stage of maturity at which normal, stationary phase biosynthesis of the antibiotic would occur and thus ensuring that sufficient quantities of the lantibiotic are made to provide P. alba with a competitive advantage. Feed-forward regulation of antibiotic biosynthesis by an immature form of the antibiotic was proposed (but not demonstrated) for microbisporicin biosynthesis (19), and biosynthetic pathway intermediates, as well as the final product, have been postulated to activate export and potentially immunity mechanisms in streptomycetes (39)(40)(41)(42).…”

“…Although actinomycetes have not generally been recognized as prolific producers of potentially useful lantibiotics, two actinomycete compounds, NVB302 [an actagardine derivative (9)] and NAI-107 [also known as microbisporicin (10)] are currently in clinical development as anti-infectives, whereas a third, Moli1901 (also known as lancovutide or duramycin, a structural analog of cinnamycin), promises to be a useful adjunct for the treatment of cystic fibrosis (11,12). Actinomycete RiPPs that have been analyzed genetically include cinnamycin (13), the spore-associated proteins SapB (14) and SapT (15), michiganin (16), actagardine (17), deoxyactagardine (18), microbisporicin (19,20), cypemycin (21), venezuelin (22), and grisemycin (23).…”

Cloning and Analysis of the Planosporicin Lantibiotic Biosynthetic Gene Cluster of Planomonospora alba

“…The closest homologue of PspW is MibW, which has been shown to interact with MibX to negatively regulate microbisporicin production (49). Thus, PspW is predicted to be a negative regulator of planosporicin production.…”

“…The closest homologue of PspX is MibX, which is an activator of microbisporicin biosynthesis in M. corallina (49). ECF -factors are frequently negatively regulated by membrane-associated anti-sigma factors that are encoded by adjacent and translationally coupled genes (50).…”