A novel mechanism of immunity controls the onset of cinnamycin biosynthesis in Streptomyces cinnamoneus DSM 40646
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Streptomyces cinnamoneus DSM 40646 produces the Class II lantibiotic cinnamycin which possesses an unusual mechanism of action, binding to the membrane lipid phosphatidylethanolamine (PE) to elicit its antimicrobial activity. A comprehensive analysis of the cinnamycin biosynthetic gene cluster has unveiled a novel mechanism of immunity in which the producing organism methylates its entire complement of PE prior to the onset of cinnamycin production. Deletion of the PE methyl transferase gene cinorf10, or the two-component regulatory system (cinKR) that controls its expression, leads not only to sensitivity to the closely related lantibiotic duramycin, but also abolishes cinnamycin production, presumably reflecting a fail-safe mechanism that serves to ensure that biosynthesis does not occur until immunity has been established.
KeywordsLantibiotic Phosphatidylethanolamine Regulation Resistance Induction
Mutational analysis of the cin gene cluster
Transcriptional organisation of the cin gene cluster and a key role for cinR1 in activating cinnamycin production
To determine the transcriptional organisation of the cin gene cluster and to ultimately gain insights into the mechanisms underlying the regulation of cinnamycin production, nuclease S1 protection studies were carried out revealing at least nine transcriptional start sites (Fig. 2a), five of which are involved in expressing genes required for biosynthesis of the lantibiotic (organised in the likely the transcription units cinorf7AMX, cinTH, cinKR, cinorf10, and cinR1). cinR1, at the right end of the cin cluster, encodes a member of the SARP family of regulatory proteins ; the binding sites for these transcriptional activators are often characterised by a series of hexameric nucleotide repeats that are separated from each other by one turn (ca. 11 nt) of the DNA helix with the gene proximal repeat situated one and a half helical turns from the −10 promoter element recognised by RNA polymerase. Interestingly, three such repeats are located upstream of the cinorf7 transcriptional start site (Fig. 2b). Together with the 1800-fold reduction (determined by qRTPCR; data not shown) in the level of transcription of cinorf7 upon deletion of cinR1, this suggests that CinR1 is the direct transcriptional activator of the likely cinorf7AMX biosynthetic operon. Previously, we had noted the presence of an inverted repeat sequence located between cinA and cinM ; nuclease S1 protection studies revealed that this sequence, as we had speculated, functions as a transcriptional attenuator, presumably ensuring an appropriate stoichiometry of the precursor peptide CinA and the biosynthetic enzymes CinM (lanthionine synthetase) and CinX. While Cinorf7 has been shown to be required for formation of the lysino-alanine bridge , no enzymatic function for the protein has been identified; given that it is likely to be produced in similar quantities to the precursor peptide, it is conceivable that it performs a chaperone-like role in the formation of this unusual post-translational modification.
cinorf10 encodes a PE monomethyltransferase
Expression of cinorf10 in S. lividans results in MMPE accumulation and confers resistance to duramycin
The cinorf10 coding sequence was next cloned into the integrative Streptomyces vector pIJ10257  under the control of the constitutive ermE* promoter and introduced into S. lividans 1326 by conjugation. TLC analysis of lipid extracts of the cinorf10-containing strain again revealed an additional lipid spot, the mobility of which was again consistent with MMPE (Fig. 3c). This additional lipid spot was not detected in a strain containing only the pIJ10257 vector, but a spot of similar mobility was observed in extracts from the strain expressing the bradyrhizobial pmtA.
Deletion of cinL, cinR, or cinK in S. cinnamoneus results in sensitivity to duramycin as well as abolishing cinnamycin production
The mutational analysis described earlier had shown that deletion of cinorf10, cinR, or cinK in S. cinnamoneus resulted in loss of cinnamycin production. To assess the effect of these deletions on immunity to cinnamycin, bioassays were carried out revealing that each of the mutants had become sensitive to duramycin (Fig. 4b), consistent with a role for cinKR in regulating cinorf10 expression and potentially that establishment of immunity is a pre-requisite for biosynthesis. Both resistance to duramycin and notably cinnamycin production was restored to each of the deletion strains following the introduction of pIJ10257 with cinorf10 expressed from the ermE* promoter (data not shown); the ability of constitutively expressed cinorf10 to suppress the non-producing phenotype of the mutants indicates that the only essential role for CinKR in cinnamycin biosynthesis is to regulate cinorf10 expression.
Expression of PEMT activity is induced by sub-inhibitory concentrations of duramycin
In an attempt to address whether induction might reflect a direct interaction of duramycin with CinK, rather than membrane stress sensed by the sensor kinase, cultures were also treated with a variant of cinnamycin (P9Q) that lacks antimicrobial activity and that is thought not to bind to PE. In cinnamycin, P9 contributes to the PE-binding pocket and may interact directly with the ethanolamine head group of the phospholipid [10, 19]. Replacement of P9 with a Q (glutamine) residue containing a bulky side group would likely interfere with the insertion of the glycerophosphoethanolamine head group into its binding pocket. The ability of the variant to induce PEMT activity (Fig. 6a) suggests that induction is not a consequence of membrane stress. Moreover, since immunity to cinnamycin appears to be a pre-requisite for production, the ability of the strain to produce deoxycinnamycin, which lacks antibiotic activity, after deletion of cinX is again consistent with specific induction by the lantibiotic, rather than a response to membrane perturbation.
When cultures were treated with daptomycin, a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus and whose lipophilic tail is believed to insert into the bacterial cell membrane causing rapid membrane depolarization and potassium ion efflux, and thus membrane stress , at concentrations that caused a fourfold reduction in growth rate and biomass accumulation, there was no induction of PEMT activity (Fig. 6b), again consistent with a direct role for the lantibiotic independent of its antimicrobial activity in inducing immunity.
Expression of PEMT activity occurs in a growth phase-dependent manner and precedes cinnamycin production
Deletion of cinorf10 abolishes cinnamycin production but has no effect on cinorf7 and cinM transcription
The lysino-alanine bridge of cinnamycin is required for antibacterial activity
Our earlier deletion analysis had shown that cinorf7 is essential for cinnamycin biosynthesis in S. cinnamoneus. More recently, co-expression studies in E. coli have shown that the same gene was required for lysino-alanine bridge formation and that the latter was required for antibacterial activity . In a complementary approach, the substrate flexibility of CinM and CinX was exploited using an in vivo expression system developed by Jesus Cortes (Novacta Biosystems) to generate cinnamycin variants in S. cinnamoneus in which S6 or K19 were replaced with A. Production of both variants (S6A, 2042 Da; K19A, 1983 Da) was readily confirmed by MALDI-ToF mass spectrometry, but both failed to inhibit B. subtilis in bioassays, confirming the previous E. coli expression studies.
We thank Anthony Appleyard (Novacta Biosystems) for the gift of the cinnamycin P9Q variant, Jesus Cortes (Novacta Biosystems) for the plasmid expression system used to generate the S6A and K19A derivatives, Gerhard Saalbach (John Innes Centre) for assistance with mass spectrometry, and Kim Findlay (John Innes Centre) for scanning electron microscopy. This work was supported financially by the Biotechnological and Biological Sciences Research Council (BBSRC, UK) Institute Strategic Programme Grant “Understanding and Exploiting Plant and Microbial Secondary Metabolism” (BB/J004561/1), BBSRC Grant 208/P08242 and DTI/BBSRC Grant APG20593. This article is dedicated to Professor Arnold L. Demain on his 90th birthday for the immense contribution that he has made to industrial microbiology and biotechnology.
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