A hybrid NRPS-PKS gene cluster related to the bleomycin family of antitumor antibiotics in Alteromonas macleodii strains

Abstract

Although numerous marine bacteria are known to produce antibiotics via hybrid NRPS-PKS gene clusters, none have been previously described in an Alteromonas species. In this study, we describe in detail a novel hybrid NRPS-PKS cluster identified in the plasmid of the Alteromonasmacleodii strain AltDE1 and analyze its relatedness to other similar gene clusters in a sequence-based characterization. This is a mobile cluster, flanked by transposase-like genes, that has even been found inserted into the chromosome of some Alteromonasmacleodii strains. The cluster contains separate genes for NRPS and PKS activity. The sole PKS gene appears to carry a novel acyltransferase domain, quite divergent from those currently characterized. The predicted specificities of the adenylation domains of the NRPS genes suggest that the final compound has a backbone very similar to bleomycin related compounds. However, the lack of genes involved in sugar biosynthesis indicates that the final product is not a glycopeptide. Even in the absence of these genes, the presence of the cluster appears to confer complete or partial resistance to phleomycin, which may be attributed to a bleomycin-resistance-like protein identified within the cluster. This also suggests that the compound still shares significant structural similarity to bleomycin. Moreover, transcriptomic evidence indicates that the NRPS-PKS cluster is expressed. Such sequence-based approaches will be crucial to fully explore and analyze the diversity and potential of secondary metabolite production, especially from increasingly important sources like marine microbes.

Figure 1. The hybrid NRPS-PKS cluster in A . macleodii .

Linear representation of the plasmid pAMDE1. Scale shown in the figure is in Kilobases. GC content and GC skew are shown. The high GC region corresponding to the cluster is shown in red. Genes are color coded and a legend is provided below.

Figure 2. Schematic representation of the hybrid NRPS-PKS cluster of pAMDE1.

Genes are colored according to the inferred function. NRPS and PKS domain architecture is shown inside boxes. NRPS and PKS gene numbers are indicated. C: Condensation domain, A: Adenylation domain, T: thiolation domain, Cy: modified condensation domain, KS: ketosynthase domain, CM: C-methyltransferase ACP: Acyl Carrier Protein domain, TE: thioesterase domain. A scale is shown below.

Figure 3. Phylogenetic tree of KS domains.

All known categories of KS domains are shown in the tree. The branch containing pAMDE1 KS domain and closely related KS domains are shown in detail in the inset on the right. Bootstrap values are shown on the nodes. Ks1: KS of first module of assembly lines, Ksa: KS-alpha, Ksb: KS-beta, Fas: fatty acids, PUFA: Polyunsaturated fatty acids.

Figure 4. Comparison of the protein domains of pAMDE1 to bleomycin related clusters.

NRPS genes are represented in red and the PKS gene in blue. Within the arrows the names of the ORF in pAMDE1 and the homologs in the bleomycin or the other clusters are indicated. The protein domains are shown inside the boxes. In addition, functional classification is shown for condensation domains (C) and the substrate amino acid for each adenylation domain (A). The modules (NRPS-0 to NRPS-9) described for the bleomycin compounds are represented by the black lines above the domains. Light blue arrows indicate putative missing domains in pAMDE1 in comparison to bleomycin. AT and KR domains are also shown in light blue. Blm genes: bleomycin, zbm genes: zorbamycin, tlm genes: tallyzomycin. LCL: catalyzes peptide bond formation between 2 L-amino acids, CYC: heterocyclization domain, modAA: modify the incorporated amino acids, unc: unclassified. A non-functional A-domain is marked with a red-cross.

Figure 5. Phleomycin resistance in Alteromonas macleodii strains.

A) Phleomycin assay of AltDE (DE, lacking the NRPS-PKS cluster) and AltDE1 (DE1, harboring the NRPS-PKS cluster in the plasmid pAMDE1) on marine agar plates. B) Phleomycin assay of strains U7 and U8 (both harbor the NRPS-PKS cluster in their chromosome) and UM7 and U4 (both harbor the NRPS-PKS cluster in their plasmids).

Figure 6. Gene expression of the NRPS-PKS cluster.

The graph shows the estimated gene expression using RPKM values associated with the NRPS-PKS cluster of pAMDE1. Genes are colored according to the inferred function. The numbers below some of the box arrows correspond to ORF numbers for that particular gene.