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

Abstract

The increasing prevalence of antibiotic resistance in bacterial pathogens has renewed focus on natural products with antimicrobial properties. Lantibiotics are ribosomally synthesized peptide antibiotics that are posttranslationally modified to introduce (methyl)lanthionine bridges. Actinomycetes are renowned for their ability to produce a large variety of antibiotics, many with clinical applications, but are known to make only a few lantibiotics. One such compound is planosporicin produced by Planomonospora alba, which inhibits cell wall biosynthesis in Gram-positive pathogens. Planosporicin is a type AI lantibiotic structurally similar to those which bind lipid II, the immediate precursor for cell wall biosynthesis. The gene cluster responsible for planosporicin biosynthesis was identified by genome mining and subsequently isolated from a P. alba cosmid library. A minimal cluster of 15 genes sufficient for planosporicin production was defined by heterologous expression in Nonomuraea sp. strain ATCC 39727, while deletion of the gene encoding the precursor peptide from P. alba, which abolished planosporicin production, was also used to confirm the identity of the gene cluster. Deletion of genes encoding likely biosynthetic enzymes identified through bioinformatic analysis revealed that they, too, are essential for planosporicin production in the native host. Reverse transcription-PCR (RT-PCR) analysis indicated that the planosporicin gene cluster is transcribed in three operons. Expression of one of these, pspEF, which encodes an ABC transporter, in Streptomyces coelicolor A3(2) conferred some degree of planosporicin resistance on the heterologous host. The inability to delete these genes from P. alba suggests that they play an essential role in immunity in the natural producer.

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

“…RNA was extracted from mycelium harvested after 41, 73, 113, and 162 h. Culture supernatants from each time point were assayed for planosporicin production ( Fig. 2), which could be detected in M1304 and the wild-type strain after 41 and 162 h, respectively; M1302, M1303 and M1308 failed to produce the lantibiotic, as observed previously (25). Interestingly, M1304 (ΔpspW::oriT-hyg) exhibited precocious and increased levels of antibiotic activity compared with the wild-type strain (see later for further discussion).…”

“…Previous RT-PCR studies (25) suggested that the psp gene cluster was transcribed in at least three operons, pspABCTUV, pspXWJYZQR, and pspEF. To identify the transcriptional start sites of these putative operons, and to assess whether pspR and pspJ, which are preceded by intergenic regions of 89 and 231 bp, respectively, were also transcribed from additional promoters, high-resolution nuclease S1 protection analyses were carried out using 32 P-5′ end-labeled probes complementary to the predicted 5′ ends of the transcripts of pspA, pspX, pspJ, pspE, and pspR.…”

“…The lantibiotic prevents the growth of other Gram-positive bacteria by inhibiting cell wall biosynthesis (24), and its N-terminal similarity to nisin (26) and microbisporicin (27) suggests an ability to bind to lipid II, the immediate precursor for cell wall biosynthesis (28). Recently, we identified and analyzed the gene cluster from P. alba responsible for the stationary phase production of planosporicin (25). The core biosynthetic genes are pspA (encoding the precursor peptide), pspB [encoding the dehydratase responsible for the dehydration of five serines and two threonines to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively], and pspC (encoding the cyclase responsible for the coupling of five cysteines to four Dha and one Dhb to form four lanthionines and one methyl-lanthionine, respectively) (Fig.…”

“…DSM 14920 (24) and Planomonospora alba (25) and contains one methyl-lanthionine and four lanthionine bridges (Fig. 1).…”

“…anti-σ factor, respectively. Both pspR and pspX are essential for planosporicin biosynthesis (25). ECF σ factors are frequently involved in sensing and responding to cell envelope stress (29) and occur in a range of bacterial genera, but are particularly prevalent in actinomycetes (30).…”