Identification of Two Putative Acyltransferase Genes Potentially Implicated in Dithiolopyrrolone Biosyntheses in Saccharothrix algeriensis NRRL B-24137

Abstract

The dithiolopyrrolone class of antibiotics has been known to display bacteriostatic activity against both Gram-positive and Gram-negative bacteria and exert other biological activities. Acyltransferase activities are proposed to be responsible for the structural diversity of dithiolopyrrolones produced by Saccharothrix algeriensis NRRL B-24137. Moreover, two activities, pyrrothine N-acetyltransferase and pyrrothine N-benzoyltransferase, are reported to catalyze the formation, respectively, to thiolutin and benzoyl-pyrrothine (BEP) in this bacterium. In this study, two genes encoding two putative acyltransferases were identified in S. algeriensis. The first one, actA, was identified by bioinformatic analysis and by analogy to an acetyltransferase, hlmA, identified in holomycin biosynthetic gene cluster in Streptomyces clavuligerus. The second was identified by purification of both enzymes from the bacterial biomass which provided a semipurified extract. The microsequencing of tryptic peptides from the final protein preparation yielded sequences of eight different fragments, two of them encoded by one gene, actB, in S. algeriensis genome bank. The alignment of actB against the GenBank database revealed significant homology to acyltransferase family. Differential expression of these genes, actA and actB, was then investigated in three different media: (i) semisynthetic medium (SSM), which promotes the production of thiolutin; (ii) SSM supplemented by 1.25 mM benzoic acid (SSM + BA), which promotes the production of both thiolutin and BEP; and (iii) tryptic soy broth (TSB) in which no dithiolopyrrolone derivatives were detected.

The final acylation step in aromatic dithiolopyrrolone biosyntheses: Identification and characterization of the first bacterium N-benzoyltransferase from Saccharothrix algeriensis NRRL B-24137

“…Another transferase, or several transferases, must be responsible for converting pyrrothine/holothin to their related products. Finally, if we couple these results with our previous results concerning the transcriptional analyses of actA on the two media SSM and SSM supplemented by benzoic acid [15], it is clear now that the role of benzoic acid as well as the similar compounds seemed to be benzoyl-CoA precursor supply than their role as gene inducer.…”

“…Protein expression was induced at different conditions. The conditions were a combination of three different temperatures (15,25 and 37 • C) and three different IPTG concentrations (100, 500 and 1000 M). For small scale, free-cell extract was prepared as follows, the biomass of 25 mL was obtained after centrifugation and resuspended in 1 mL of lysis buffer (Tris-HCl 50 mM pH 8), then it was transferred to a Fast Protein Blue tube (MP Biomedicals, Irvine, CA, USA).…”

“…More recently, we have proposed two ORFs, actA and actB to be involved in dithiolopyrrolones biosynthesis in Sa. algeriensis [15]. Furthermore, we have tried to reveal the role of benzoic acid on the production of BEP.…”

Efficacy of Saccharothrix algeriensis NRRL B-24137 to suppress Fusarium oxysporum f.sp. vasinfectum induced wilt disease in cotton

“…SA NRRL B-24137 is a gram positive bacterium isolated from Sahara soil, which produces numerous bioactive metabolites, belonging to pyrrothine, known as dithiolopyrrolone derivatives ( Lamari et al, 2002 ; Merrouche et al, 2010 ; Merrouche et al, 2011 ). SA NRRL B-24137 has been reported having some antibacterial and antifungal activities ( Merrouche et al, 2011 ; Muzammil et al, 2012 ; Saker, Lebrihi & Mathieu, 2014 ). As a whole, the prime objective of this study is the evaluation of biocontrol potential of well documented SA NRRL B-24137 for the first time against Fusarium wilt in cotton plants in greenhouse conditions.…”

Identification, structure and function of the methyltransferase involved in the biosynthesis of the dithiolopyrrolone antibiotic xenorhabdin

“…To test this hypothesis, we produced holomycin in X. doucetiae FRM16. Since holomycin and XRDs only differ in the length of their acyl side chain, we replaced the XRD specific acyltransferase gene xrdE in strain ∆hfq PBAD_xrdA by either hlmA, the gene encoding the acetyltransferase in holomycin BGC of S. clavuligerus ATCC 27064 2 , or actA, the coding sequence of the putative acetyltransferase involved in thiolutin biosynthesis from S. algeriensis NRRL B-24137 13,14 .…”