Genetic and Functional Analysis of the Biosynthesis of a Non-Ribosomal Peptide Siderophore in Burkholderia xenovorans LB400

Abstract

B. xenovorans LB400 is a model bacterium for the study of the metabolism of aromatic compounds. The aim of this study was the genomic and functional characterization of a non-ribosomal peptide synthetase containing gene cluster that encodes a siderophore in B. xenovorans LB400. The mba gene cluster from strain LB400 encodes proteins involved in the biosynthesis and transport of a hydroxamate-type siderophore. Strain LB400 has a unique mba gene organization, although mba gene clusters have been observed in diverse Burkholderiales. Bioinformatic analysis revealed the presence of promoters in the mba gene cluster that strongly suggest regulation by the ferric uptake regulator protein (Fur) and by the alternative RNA polymerase extracytoplasmic function sigma factor MbaF. Reverse transcriptase PCR analyses showed the expression of iron-regulated transcriptional units mbaFGHIJKL, mbaN, mbaABCE, mbaO, mbaP and mbaD genes under iron limitation. Chrome azurol S (CAS) assay strongly suggests that strain LB400 synthesized a siderophore under iron limitation. Mass spectrometry ESI-MS and MALDI-TOF-MS analyses revealed that the siderophore is a non-ribosomal peptide, and forms an iron complex with a molecular mass of 676 Da. Based on bioinformatic prediction, CAS assay and MS analyses, we propose that the siderophore is L-Nδ-hydroxy-Nδ-formylOrn-D-β-hydroxyAsp-L-Ser-L-Nδ-hydroxy-Nδ-formylOrn-1,4-diaminobutane that is closely related to malleobactin-type siderophores reported in B. thailandensis.

Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family

“…6A). The stereochemistry of 10 of 18 siderophores with TβH Asp -mediated hydroxylation has been reported in the literature (3,25,26,30,32,34,(54)(55)(56)(57). In contrast to the IβH Asp clade, most of the TβH Asp enzymes were reported to hydroxylate Asp to form the 3R product.…”

“…Based on homology to SyrP and OrbG, the existence of a number of β-OHAsp residues in siderophores has been rationalized (7,11,(24)(25)(26)(27)(28) or predicted (29)(30)(31)(32)(33). Recently, Kurth et al (33) used cucF, a β-hydroxylase domain from the cupriachelin (Fig.…”

Iron Acquisition Mechanisms and Their Role in the Virulence of Burkholderia Species

“…For malleobactin, the predicted specificity of the N-terminal adenylation domain changes to β-hydroxytyrosine although the accepted substrate is N 5 -formyl- N 5 -hydroxyornithine. In some species, such as B. thailandensis , the N-terminal N 5 -formyl- N 5 -hydroxyornithine is formylated on the N 2 -amino group upon formation of the tetrapeptide to generate malleobactin E (Franke et al, 2015 ), whereas in B. xenovorans the N-terminal N 5 -formyl- N 5 -hydroxyornithine does not appear to undergo such a tailoring reaction (Vargas-Straube et al, 2016 ) and so we tentatively refer to this siderophore as “malleobactin X.” The N- and C-terminal adenylation domains of PhmA-PhmB are predicted to accept aspartate and cysteine, respectively, but the structure of the product, phymabactin, is unknown, although it is predicted to have siderophore activity based on its genomic context (Esmaeel et al, 2016 ). In all cases, the second and third adenylation domains are predicted to accept aspartate and serine, respectively, which correspond to β-hydroxy-D-aspartate and L-serine in the final product.…”

p-Cymene Promotes Its Catabolism through the p-Cymene and the p-Cumate Pathways, Activates a Stress Response and Reduces the Biofilm Formation in Burkholderia xenovorans LB400

“…xenovorans LB400 is a model bacterium for the study of the degradation of polychlorobiphenyls (PCBs) and a wide range of aromatic compounds [12–18]. Recently, the biosynthesis of a non-ribosomal peptide hydroxamate-type siderophore in strain LB400 has been reported [19]. LB400 genome has a size of 9.73 Mbp distributed in a major chromosome (C1) (4.90 Mbp), a minor chromosome (C2) (3.36 Mbp) and a megaplasmid (MP) (1.47 Mbp) [14].…”