The interaction in multisubunit non‐ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit‐to‐subunit interaction. We introduced natural docking domains ...into the three‐module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC‐ESI‐MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS‐PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split‐module cases compared to the full‐length wild‐type enzyme.
A breed apart: The non‐ribosomal peptide synthetase (NRPS) XfpS, which produces the peptides xefoampeptides A and B, can be artificially split without loss of biosynthetic activity by introducing docking domains into XfpS. In fact, in this case, the production of both peptides from the split system increased compared to product levels from the full‐length XfpS. Splitting XfpS into three subunits also led to a functional NRPS system.
Although sharing a certain degree of structural uniformity, natural product classes exhibit variable functionalities such as different amino acid or acyl residues. During collision induced ...dissociation, some natural products exhibit a conserved fragmentation pattern close to the precursor ion. The observed fragments result from a shared set of neutral losses, creating a unique fragmentation pattern, which can be used as a fingerprint for members of these natural product classes. The culture supernatants of 69 strains of the entomopathogenic bacteria Photorhabdus and Xenorhabdus were analyzed by MALDI-MS(2), and a database comprising MS(2) data from each strain was established. This database was scanned for concordant fragmentation patterns of different compounds using a customized software, focusing on relative mass differences of the fragment ions to their precursor ion. A novel group of related natural products comprising 25 different arginine-rich peptides from 16 different strains was identified due to its characteristic neutral loss fragmentation pattern, and the structures of eight compounds were elucidated. Two biosynthesis gene clusters encoding nonribosomal peptide synthetases were identified, emphasizing the possibility to identify a group of structurally and biosynthetically related natural products based on their neutral loss fragmentation pattern.
The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of ...the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.
► Promoter exchange as simple way to induce the production of the cryptic blue pigment indigoidine from Photorhabdus. ► Heterologous expression in E. coli as another way to produce indigoidine. ► ...Different regulatory mechanisms for indigoidine biosynthesis in Photorhabdus and E. coli. ► Identification of genes acting as repressors in the indigoidine biosynthesis in Photorhabdus. ► Phylogenetic analysis of indC and comparative cluster analysis indicates horizontal transfer of the indigoidine biosynthesis gene cluster.
The production of the blue pigment indigoidine has been achieved in the entomopathogenic bacterium Photorhabdus luminescens by a promoter exchange and in Escherichia coli following heterologous expression of the biosynthesis gene indC. Moreover, genes involved in the regulation of this previously “silent” biosynthesis gene cluster have been identified in P. luminescens.
In the attempt to establish a reliable real-time PCR protocol for transcriptional analysis of secondary metabolism in
Sorangium cellulosum strain So ce56, a RNA extraction method and a reverse ...transcription protocol was developed. In order to validate chivosazol or etnangien gene cluster transcripts as good candidates to develop the real-time PCR protocol, stability measurements of the transcripts were performed proving both transcripts to be very stable. The chivosazol biosynthetic gene cluster was taken as the test case to evaluate the special problems arising from the large size of the transcripts and the high G/C-content of the encoding DNA. A set of primer pairs targeting the presumed 90
kbp chivosazol transcript at different positions was employed. The production rate of chivosazol was compared to the transcription of the operon in time course experiments revealing that during the logarithmic growth phase transcription is maximally induced and levels out during the stationary phase. Some deviations in transcript numbers could be measured depending on the primer pair used, but cross-evaluation strengthened the notion that the measured numbers reflect the whole transcript quantities and the in vivo level. Finally, a putative promoter located between
chiA and
chiB was examined by using the developed real-time PCR protocol.
PoyD is a radical S‐adenosyl methionine epimerase that introduces multiple D‐configured amino acids at alternating positions into the highly complex marine peptides polytheonamide A and B. This novel ...post‐translational modification contributes to the ability of the polytheonamides to form unimolecular minimalistic ion channels and its cytotoxic activity at picomolar levels. Using a genome mining approach we have identified additional PoyD homologues in various bacteria. Three enzymes were expressed in E. coli with their cognate as well as engineered peptide precursors and shown to introduce diverse D‐amino acid patterns into all‐L peptides. The data reveal a family of architecturally and functionally distinct enzymes that exhibit high regioselectivity, substrate promiscuity, and irreversible action and thus provide attractive opportunities for peptide engineering.
Radikaler Wechsel: Radikalische S‐Adenosylmethionin(SAM)‐Epimerasen bilden eine neue Familie bakterieller Enzyme mit individueller Architektur und Funktion. Drei Vertreter aus Cyanobakterien führen verschiedene D‐Aminosäure‐Muster in All‐L‐Peptide ein. Eigenschaften wie eine hohe Regioselektivität, Substratpromiskuität und ein irreversibler Wirkmechanismus eröffnen Möglichkeiten für das Peptid‐Engineering.
Activation sequence-1 (as-1)-like regulatory cis elements mediate transcriptional activation in response to increased levels of plant signalling molecules auxin and salicylic acid (SA). Our earlier ...work has shown that tobacco cellular as-1-binding complex SARP (salicylic acid responsive protein) is primarily comprised of bZIP protein TGA2.2 and of minor amounts of a protein that cross-reacts with an antibody directed against related bZIP factor TGA2.1. As this protein was significantly smaller than recombinant TGA2.1, the origin of this protein had remained unresolved. Here we demonstrate that it corresponds to a distinct cleavage product of TGA2.1 generated during extract preparation. Overexpression of TGA2.1 led to increased levels of the TGA2.1/TGA2.2 heterodimer which was as effective with regard to enhancing the SA-inducibility of as-1 containing target gene Nt103 as corresponding amounts of the TGA2.2 homodimer. Thus, the TGA2.1 specific N-terminal domain, which had revealed transcriptional activation potential in yeast, did not show enhanced transcriptional activation in planta. TGA2.1 even had a negative effect on the SA-induced expression of the truncated CaMV 35S (-90) promoter that contains an isolated as-1-element upstream of the TATA-box. Plants expressing a TGA mutant deficient in DNA binding (TGA2.1trd) showed reduced levels of SA-inducible Nt103 expression, thus resembling plants expressing the analogous TGA2.2 derivative TGA2.2trd. In contrast to TGA2.2trd, TGA2.1trd did not reduce auxin-induced expression of Nt103 and SA-induced expression of pathogenesis related protein PR-1a, indicating that TGA2.1trd and TGA2.2trd differ in their capacity to outcompete regulatory factors involved in these regulatory pathways.
Motile predatory Myxobacteria are producers of multiple secondary metabolites and, on starvation, undergo concerted cellular differentiation to form multicellular fruiting bodies. These abilities ...demand myxobacterial genomes to encode sophisticated regulatory networks that are not satisfactorily understood. Here, we present two bacterial enhancer binding proteins (bEBPs) encoded in Myxococcus xanthus acting as direct regulators of secondary metabolites intriguingly exhibiting activating and inhibitory effects. Elucidation of a regulon for each bEBP enabled us to unravel their role in myxococcal development, predation, and motility. Interestingly, both bEBPs are able to interact by forming a hetero-oligomeric complex. Our findings represent an alternative mode of operation of bEBPs, which are currently thought to enhance promoter activity by acting as homo-oligomers. Furthermore, a direct link between secondary metabolite gene expression and predation, motility, and cellular development could be shown for the first time.
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► The bEBPs HsfA and MXAN4899 are direct regulators exhibiting activating and inhibiting effects on transcription ► HsfA and MXAN4899 exhibit a mode of operation: hetero-oligomerization ► The bEBPs link secondary metabolite production to development, motility, and predation ► The results open ways of interpretation in elucidation of complex regulatory networks
Volz et al. introduce two bacterial enhancer binding proteins (bEBP) as transcriptional regulators of secondary metabolites in M. xanthus. The approach increases yield of metabolites and shows the link between development, motility, predation, and secondary metabolite production at the transcriptional level.
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