Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR

Abstract

A novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bourne et al. 1996 Appl. Environ. Microbiol. 62: 4086-4094). We now report the cloning and molecular characterisation of four genes from this Sphingomonas sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolytic enzymes involved in this pathway together with a putative oligopeptide transporter. The heterologously expressed degradation pathway proteins are enzymatically active. Microcystinase (MlrA), the first enzyme in the degradative pathway, is a 336-residue endopeptidase, which displays only low sequence identity with a hypothetical protein from Methanobacterium thermoautotrophicum. Inhibition of microcystinase by EDTA and 1,10-phenanthroline suggests that it is a metalloenzyme. The most likely residues that could potentially chelate an active-site transition metal ion are in the sequence HXXHXE, which would be unique for a metalloproteinase. Situated immediately downstream of mlrA with the same direction of transcription is a gene mlrD, whose conceptual translation (MlrD, 442 residues) shows significant sequence identity and similar potential transmembrane spanning regions to the PTR2 family of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene encodes the enzyme MlrB (402 residues) which cleaves linear microcystin LR to a tetrapeptide degradation product. This enzyme belongs to the "penicillin-binding enzyme" family of active site serine hydrolases. The final gene in the cluster mlrC, is located upstream of the mlrA gene and is transcribed in the opposite direction. It codes for MlrC (507 residues) which mediates further peptidolytic degradation of the tetrapeptide. This protein shows significant sequence identity to a hypothetical protein from Streptomyces coelicolor. It is suspected to be a metallopeptidase based on inhibition by metal chelators. It is postulated on the basis of comparison with other microorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of microcystin LR.

Secondary metabolite gene expression and interplay of bacterial functions in a tropical freshwater cyanobacterial bloom

“…Microcystin may also function to enhance the aggregation of Microcystis cells as often occurs during bloom formation (Gan et al , 2012) and to provide a fitness advantage to Microcystis under oxidative stress (Zilliges et al , 2011). Despite transcript evidence that the microcystin biosynthetic operon was expressed throughout the day-night cycle, concentrations of dissolved microcystin toxin were measured near the limit of detection in this study (Supplementary Figure S2), potentially reflecting low synthesis activity, biodegradation (Bourne et al , 2001; Mou et al , 2013) and/or conversion of microcystin to insoluble conjugates that are not detected by the enzyme-linked immunosorbent assay-based method used in this study (Meissner et al , 2013). …”

“…Betaproteobacterial genera recently implicated in microcystin biodegradation (Mou et al , 2013) were observed among sequenced transcripts (Supplementary Database 1). No significant sequence similarity was observed for the mrlAB -mediated microcystin biodegradation pathway (Bourne et al , 2001; Mou et al , 2013). …”

Presence or Absence of mlr Genes and Nutrient Concentrations Co-Determine the Microcystin Biodegradation Efficiency of a Natural Bacterial Community

“…However, some naturally occurring bacterial populations are reported to effectively degrade MCs [16,17,18]. A MC-LR biodegradation pathway has been characterized in some bacterial strains that involve the action of three specific peptidases and a putative oligopeptide transporter encoded in the mlr A-D gene cluster [19]. The first enzyme encoded by the mlr A gene cleaves the Adda-Arg peptide bond from the MC cyclic structure, resulting in a linearized molecule that is 160 times less toxic [20].…”

Isolation and Characterization of Microcystin Degrading Bacteria from Holy Ponds in India

“…2). These cluster and genes mlrA, mlrB, mlrC and mlrD encode microcystinase, serine protease, peptidase, tripeptidase respectively and others yet to characterize (Bourne et al, 2001;Kormas and Lymperopoulou, 2013;Saito et al, 2003;Shimizu, et al, 2012). Enzyme encoded by mlrA gene cleaves the Adda-Arg peptide bond of MC-LR and convert it from cyclic structure, which render MCs stable against general protease and other factors, to linear form.…”