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Cell wall biosynthesis represents a valid target for antibacterial action but only a limited number of chemical structure classes selectively interact with specific enzymes or protein structures like transporters of the cell envelope. The integral membrane protein MraY translocase is essential for peptidoglycan biosynthesis catalysing the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate, thereby generating the cell wall intermediate lipid I. Not present in eukaryotic cells, MraY is a member of the superfamily of yet not well-understood integral membrane enzymes which involve proteins for bacterial lipopolysaccharide and teichoic acid or eukaryotic N-linked saccharides biosynthesis. Different natural nucleoside antibiotics as inhibitors of MraY translocase have been discovered comprising a glycosylated heterocyclic pyrimidin base among other potential lipid-, peptidic- or sugar moieties. Caprazamycins are liponucleoside antibiotics isolated from Streptomyces sp. MK730-62F2. They possess activity in vitro against Gram-positive bacteria, in particular against the genus Mycobacterium including M. intracellulare, M. avium and M. tuberculosis. Structural elucidation revealed the (+)-caprazol core skeleton as a unique moiety, the caprazamycins share with other MraY inhibitors such as the liposidomycins, A-90289 and the muraminomicins. They also share structural features such as uridyl-, aminoribosyl- and fatty acyl-moieties with other MraY translocase inhibitors like FR-900493 and the muraymycins. Intensive studies on their biosynthesis during the last decade identified not only common initial biosynthetic steps, but also revealed possible branching points towards individual biosynthesis of the respective compound. Structural diversity of caprazamycins was generated by feeding experiments, genetic engineering of the biosynthetic gene clusters and chemical synthesis for structure activity relationship studies with its target, MraY translocase., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The shoot endophytic biocontrol strain Pseudomonas chlororaphis subsp. aurantiaca PB-St2 produces a wide range of exoproducts, including enzymes and antibiotics. The production of exoproducts is commonly tightly regulated. In order to get a deeper insight into the regulatory network of PB-St2, the strain was systematically investigated regarding its quorum sensing systems, both on the genetic and metabolic level. The genome analysis of PB-St2 revealed the presence of four putative acyl homoserine lactone (AHL) biosynthesis genes: phzI, csaI, aurI, and hdtS. LC-MS/MS analyses of the crude supernatant extracts demonstrated that PB-St2 produces eight AHLs. In addition, the concentration of all AHL derivatives was quantified time-resolved in parallel over a period of 42 h during the growth of P. aurantiaca PB-St2, resulting in production curves, which showed differences regarding the maximum levels of the AHLs (14.6 nM- 1.75 μM) and the production period. Cloning and heterologous overexpression of all identified AHL synthase genes in Escherichia coli proved the functionality of the resulting synthases PhzI, CsaI, and AurI. A clear AHL production pattern was assigned to each of these three AHL synthases, while the HdtS synthase did not lead to any AHL production. Furthermore, the heterologous expression study demonstrated unequivocally and for the first time that AurI directs the synthesis of two 3-oxo-AHLs., Competing Interests: The authors have declared that no competing interests exist.

Polarized growth in eukaryotes requires polar multiprotein complexes. Here, we establish that selection and maintenance of cell polarity for growth also requires a dedicated multiprotein assembly in the filamentous bacterium, Streptomyces coelicolor. We present evidence for a tip organizing center and confirm two of its main components: Scy (Streptomyces cytoskeletal element), a unique bacterial coiled-coil protein with an unusual repeat periodicity, and the known polarity determinant DivIVA. We also establish a link between the tip organizing center and the filament-forming protein FilP. Interestingly, both deletion and overproduction of Scy generated multiple polarity centers, suggesting a mechanism wherein Scy can both promote and limit the number of emerging polarity centers via the organization of the Scy-DivIVA assemblies. We propose that Scy is a molecular "assembler," which, by sequestering DivIVA, promotes the establishment of new polarity centers for de novo tip formation during branching, as well as supporting polarized growth at existing hyphal tips.

The Streptomyces coelicolor A3(2) sporulation gene whiB is the paradigm of a family of genes (wbl, whiB-like) that are confined to actinobacteria. The chromosome of S. coelicolor contains 11 wbl genes, among which five are conserved in many actinobacteria: whiB itself; whiD, a sporulation gene; wblC, which is required for multi-drug resistance; and wblA and wblE, whose roles had previously been little studied. We succeeded in disrupting wblA and the six non-conserved genes, but could not disrupt wblE. Although mutations in the six non-conserved wbl genes (including some multiple wbl mutants) produced no readily detectable phenotype, mutations in wblA had novel and complex effects. The aerial mycelium of wblA mutants was coloured red, because of the ectopic presence of pigmented antibiotics (actinorhodin and undecylprodigiosin) normally confined to lower parts of wild-type colonies, and consisted almost entirely of non-sporulating, thin, straight filaments, often bundled together in a fibrillar matrix. Rare spore chains were also formed, which exhibited wild-type properties but were genetically still wblA mutants. A wblA mutant achieved higher biomass than the wild-type. Microarray analysis indicated major transcriptional changes in a wblA mutant: using a relatively stringent cut-off, 183 genes were overexpressed, including genes for assimilative primary metabolism and actinorhodin biosynthesis, and 103 were underexpressed, including genes associated with stages of aerial hyphal growth. We suggest that WblA is important in both the slow-down of biomass accumulation and the change from aerial hyphal initial cells to the subapical stem and apical compartments that precede sporulation; and that the mutant aerial mycelium consists of recapitulated defective aerial hyphal initial cells.

Streptomyces cinnamonensis DSM 1042 produces two types of isoprenoid secondary metabolites: the prenylated naphthalene derivative furanonaphthoquinone I (FNQ I), and isoprenylated phenazines which are termed endophenazines. Previously, a 55 kb gene cluster was identified which contained genes for both FNQ I and endophenazine biosynthesis. However, several genes required for the biosynthesis of these metabolites were not present in this cluster. We now re-screened the cosmid library for genes of the mevalonate pathway and identified a separate genomic locus which contains the previously missing genes. This locus (15 kb) comprised orthologues of four phenazine biosynthesis genes known from Pseudomonas strains. Furthermore, the locus contained a putative operon of six genes of the mevalonate pathway, as well as the gene epzP which showed sequence similarity to a recently discovered class of prenyltransferases. Inactivation and complementation experiments proved the involvement of epzP in the prenylation reaction in endophenazine biosynthesis. This newly identified genomic locus is more than 40 kb distant from the previously identified cluster. The protein EpzP was expressed in Escherichia coli in form of a his-tag fusion protein and purified. The enzyme catalysed the prenylation of 5,10-dihydrophenazine-1-carboxylic acid (dihydro-PCA) using dimethylallyl diphosphate (DMAPP) as isoprenoid substrate. K(m) values were determined as 108 µM for dihydro-PCA and 25 µM for DMAPP., (© 2010 The Authors. Journal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The bacterium Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces prenylated derivatives of phenazine 1-carboxylic acid. From this organism, we have identified the prenyltransferase gene ppzP. ppzP resides in a gene cluster containing orthologs of all genes known to be involved in phenazine 1-carboxylic acid biosynthesis in Pseudomonas strains as well as genes for the six enzymes required to generate dimethylallyl diphosphate via the mevalonate pathway. This is the first complete gene cluster of a phenazine natural compound from streptomycetes. Heterologous expression of this cluster in Streptomyces coelicolor M512 resulted in the formation of prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of ppzP, only nonprenylated phenazine 1-carboxylic acid was formed. Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate dimethylallyltransferase, forming a C-C bond between C-1 of the isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many other prenyltransferases, the reaction of PpzP is independent of the presence of magnesium or other divalent cations. The K(m) value for dimethylallyl diphosphate was determined as 116 microm. For dihydro-PCA, half-maximal velocity was observed at 35 microm. K(cat) was calculated as 0.435 s(-1). PpzP shows obvious sequence similarity to a recently discovered family of prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The present finding extends the substrate range of this family, previously limited to phenolic compounds, to include also phenazine derivatives.

The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e., novE and novG. Functional proof for the role of NovG as a positive regulator of novobiocin biosynthesis had been provided previously, and we now investigated the role of novE. Heterologous expression experiments with the novobiocin biosynthetic gene cluster showed that the entire putative promoter region of novE is required to achieve optimal novobiocin production. Overexpression of novE, using a replicative vector, resulted in an increase of novobiocin formation. In contrast, inactivation of novE by in frame deletion resulted in a strong reduction of novobiocin biosynthesis. Novobiocin production could be restored by an intact copy of novE, but also by the regulatory gene novG. These observations suggest that novE is a positive regulator of novobiocin biosynthesis. NovE was expressed in E. coli and purified. However, in contrast to parallel experiments with NovG, no DNA-binding properties could be shown for NovE. RT-PCR experiments showed that expression of novG was detectable in the absence of NovE, and also that expression of novE occurred in absence of NovG.

In the biosynthetic gene cluster of the aminocoumarin antibiotic clorobiocin, the small ORF cloY encodes a 71 aa protein which shows significant sequence similarity to mbtH from the mycobactin biosynthetic gene cluster of Mycobacterium tuberculosis. mbtH-like genes are frequently found in the biosynthetic gene clusters of peptide antibiotics and siderophores, but their function has remained enigmatic. In a recent publication it has been suggested that these genes may have no function for secondary metabolite biosynthesis. An in-frame deletion of cloY in the clorobiocin cluster has now been carried out. When the modified cluster was expressed in the heterologous host Streptomyces coelicolor M512, clorobiocin was still formed. However, when the two further mbtH-like genes from elsewhere in the host genome were inactivated as well, clorobiocin formation was reduced dramatically. Complementation with cloY or with any of three other mbtH-like genes restored clorobiocin formation. This is the first report proving the requirement of an mbtH-like gene for secondary metabolite formation, and the first proof that different mbtH-like genes can functionally replace each other. Feeding of an mbtH-defective triple mutant strain with an intact 3-amino-4,7-dihydroxy-coumarin moiety restored antibiotic production, showing that cloY is specifically required for the formation of this moiety of the clorobiocin molecule.

In Streptomyces coelicolor ParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci in S. coelicolor vegetative hyphae. In contrast, in aerial hyphae regularly spaced bright foci accompanied sporulation-associated chromosome condensation and septation.

Ribonucleotide reductases (RNRs) catalyse the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs. We show here that the Streptomyces coelicolor M145 nrdAB genes encoding an oxygen-dependent class I RNR are co-transcribed with nrdS, which encodes an AraC-like regulatory protein. Likewise, the class II oxygen-independent RNR nrdJ gene forms an operon with a likely regulatory gene, nrdR, which encodes a protein possessing an ATP-cone domain like those present in the allosteric activity site of many class Ia RNRs. Deletions in nrdB and nrdJ had no discernible effect on growth individually, but abolition of both RNR systems, using hydroxyurea to inactivate the class Ia RNR (NrdAB) in the nrdJ deletion mutant, was lethal, establishing that S. coelicolor possesses just two functional RNR systems. The class II RNR (NrdJ) may function to provide a pool of deoxyribonucleotide precursors for DNA repair during oxygen limitation and/or for immediate growth after restoration of oxygen, as the nrdJ mutant was slower in growth recovery than the nrdB mutant or the parent strain. The class Ia and class II RNR genes show complex regulation. The nrdRJ genes were transcribed some five- to sixfold higher than the nrdABS genes in vegetative growth, but when nrdJ was deleted, nrdABS transcription was upregulated by 13-fold. In a reciprocal experiment, deletion of nrdB had little effect on nrdRJ transcription. Deletion of nrdR caused a dramatic increase in transcription of nrdJ and to a less extent nrdABS, whereas disruption of cobN, a gene required for synthesis of coenzyme B12 a cofactor for the class II RNR, caused similar upregulation of transcription of nrdRJ and nrdABS. In contrast, deletion of nrdS had no detectable effect on transcription of either set of RNR genes. These results establish the existence of control mechanisms that sense and regulate overall RNR gene expression.

The left ends of the biosynthetic gene clusters of novobiocin ( nov), clorobiocin ( clo) and coumermycin A(1) ( cou) from Streptomyces spheroides (syn. S. caeruleus) NCIMB 11891, S. roseochromogenes var. oscitans DS 12.976 and S. rishiriensis DSM 40489 were cloned and sequenced. Sequence comparison suggested that novE, cloE and couE, respectively, represent the borders of these three clusters. Inactivation of novE proved that novE does not have an essential catalytic role in novobiocin biosynthesis, but is likely to have a regulatory function. The gene products of novF and cloF show sequence similarity to prephenate dehydrogenase and may produce 4-hydroxyphenylpyruvate (4HPP) as a precursor of the substituted benzoate moiety of novobiocin and clorobiocin. Coumermycin A(1) does not contain this benzoate moiety, and correspondingly the coumermycin cluster was found not to contain a functional novF homologue. The coumermycin biosynthetic gene cluster apparently evolved from an ancestral cluster similar to those of novobiocin and clorobiocin, and parts of the ancestral novF homologue have been deleted in this process. No homologue to novC was identified in the gene clusters of clorobiocin and coumermycin, questioning the postulated involvement of novC in aminocoumarin biosynthesis. Heterologous expression of novDEFGHIJK in Streptomyces lividans resulted in the formation of 2,4-dihydroxy-alpha-oxy-phenylacetic acid, suggesting that at least one of the proteins encoded by these genes may participate in a hydroxylation reaction.

Glutamine synthetase I (GSI) enzyme activity in Streptomyces coelicolor is controlled post-translationally by the adenylyltransferase (GlnE) as in enteric bacteria. Although other homologues of the Escherichia coli Ntr system (glnK, coding for a PII family protein; and glnD, coding for an uridylyltransferase) are found in the S. coelicolor genome, the regulation of the GSI activity was found to be different. The functions of glnK and glnD were analysed by specific mutants. Surprisingly, biochemical assay and two-dimensional PAGE analysis showed that modification of GSI by GlnE occurs normally in all mutant strains, and neither GlnK nor GlnD are required for the regulation of GlnE in response to nitrogen stimuli. Analysis of the post-translational regulation of GlnK in vivo by two-dimensional PAGE and mass spectrometry indicated that it is subject to both a reversible and a non-reversible modification in a direct response to nitrogen availability. The irreversible modification was identified as removal of the first three N-terminal amino acid residues of the protein, and the reversible modification as adenylylation of the conserved tyro-sine 51 residue that is known to be uridylylated in E. coli. The glnD insertion mutant expressing only the N-terminal half of GlnD was capable of adenylylating GlnK, but was unable to perform the reverse deadenylylation reaction in response to excess ammonium. The glnD null mutant completely lacked the ability to adenylylate GlnK. This work provides the first example of a PII protein that is modified by adenylylation, and demonstrates that this reaction is performed by a homologue of GlnD, previously described only as a uridylyltransferase enzyme.