Anti-microfouling Activity of Glycomyces sediminimaris UTMC 2460 on Dominant Fouling Bacteria of Iran Marine Habitats

Discovery of environmentally safe anti-fouling agent is currently of considerable interest, due to the continuous impact of biofoulers on the marine habitats and the adverse effects of biocides on the environment. This study reports the anti-adhesion effect of marine living Actinobacteria against fouling strains isolated from submerged panels in marine environments of Iran. The extract of Glycomyces sediminimaris UTMC 2460 affected the biofilm formation of Kocuria sp. and Mesorhizobium sp., as the dominant fouling agents in this ecosystem, up to 93.2% and 71.4%, respectively. The metabolic activity of the fouler bacteria was reduced by the extract up to 17 and 9%, respectively. This indicated the bactericidal potency of the extract on cells in the biofilm state that enables the compound to be effective even once the biofilms are established in addition to the inhibition of biofilm initiation. Moreover, extra polymeric substance (EPS) production by fouling bacteria was reduced by 60–70%. The absence of activities against fouling bacteria in suspension and also the absence of toxic effect on Artemia salina showed the harmless ecological effect of the anti-microfouling extract on the prokaryotic and eukaryotic microflora of the studied Iran marine ecosystem. Metabolic profiling of G. sediminimaris UTMC 2460 revealed the presence of compounds with molecular formulae matching those of known anti-fouling diketopiperazines as major components of the extract. These results suggest that the extract of Glycomyces sediminimaris UTMC 2460 could be used as a potentially eco-friendly viable candidate in comparison to the synthetic common commercial anti-microfouling material to prevent the fouling process in marine habitats of Iran

The insect pathogen Serratia marcescens Db10 uses a hybrid non-ribosomal peptide synthetase-polyketide synthase to produce the antibiotic althiomycin

There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1-alb6. Bioinformatic analysis of the proteins encoded by alb1-6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2-Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism

Thioester reduction and aldehyde transamination are universal steps in actinobacterial polyketide alkaloid biosynthesis

Actinobacteria produce a variety of polyketide alkaloids with unusual structures. Recently, it was shown that a type I modular polyketide synthase (PKS) is involved in the assembly of coelimycin P1, a polyketide alkaloid produced by Streptomyces coelicolor M145. However, the mechanisms for converting the product of the PKS to coelimycin P1 remain to be elucidated. Here we show that the C-terminal thioester reductase (TR) domain of the PKS and an ω-transaminase are responsible for release of the polyketide chain as an aldehyde and its subsequent reductive amination. Bioinformatics analyses identified numerous gene clusters in actinobacterial genomes that encode modular PKSs with a C-terminal TR domain and a homolog of the ω-transaminase. These are predicted to direct the biosynthesis of both known and novel polyketide alkaloids, suggesting that reductive chain release and transamination constitutes a conserved mechanism for the biosynthesis of such metabolites

Desferrioxamine biosynthesis : diverse hydroxamate assembly by substrate-tolerant acyl transferase DesC

Hydroxamate groups play key roles in the biological function of diverse natural products. Important examples include trichostatin A, which inhibits histone deacetylases via coordination of the active site zinc(II) ion with a hydroxamate group, and the desferrioxamines, which use three hydroxamate groups to chelate ferric iron. Desferrioxamine biosynthesis in Streptomyces species involves the DesD-catalysed condensation of various N-acylated derivatives of N-hydroxycadaverine with two molecules of N-succinyl-N-hydroxycadaverine to form a range of linear and macrocyclic tris-hydroxamates. However, the mechanism for assembly of the various N-acyl-N-hydroxycadaverine substrates of DesD from N-hydroxycadaverine has until now been unclear. Here we show that the desC gene of Streptomyces coelicolor encodes the acyl transferase responsible for this process. DesC catalyses the N-acylation of N-hydroxycadaverine with acetyl, succinyl and myristoyl-CoA, accounting for the diverse array of desferrioxamines produced by S. coelicolor. The X-ray crystal structure of DesE, the ferrioxamine lipoprotein receptor, in complex with ferrioxamine B (which is derived from two units of N-succinyl-N-hydroxycadaverine and one of N-acetyl-N-hydroxycadaverine) was also determined. This shows that the acetyl group of ferrioxamine B is solvent exposed, suggesting that the corresponding acyl group in longer chain congeners can protrude from the binding pocket, providing insights into their likely functio

Mining microbial genomes for new natural products and biosynthetic pathways

Analyses of microbial genome sequences have revealed numerous examples of ‘cryptic’ or ‘orphan’ biosynthetic gene clusters, with the potential to direct the production of novel, structurally complex natural products. This article summarizes the various methods that have been developed for discovering the products of cryptic biosynthetic gene clusters in microbes and gives an account of my group's discovery of the products of two such gene clusters in the model actinomycete Streptomyces coelicolor M145. These discoveries hint at new mechanisms, roles and specificities for natural product biosynthetic enzymes. Our efforts to elucidate these are described. The identification of new secondary metabolites of S. coelicolor raises the question: what is their biological function? Progress towards answering this question is also summarized

In vitro biosynthetic studies of bottromycin expand the enzymatic capabilities of the YcaO superfamily

The bottromycins belong to the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Bottromycins exhibit unique structural features, including a hallmark macrolactamidine ring and thiazole heterocycle for which divergent members of the YcaO superfamily have been biosynthetically implicated. Here we report the in vitro reconstitution of two YcaO proteins, BmbD and BmbE, responsible for the ATP-dependent cyclodehydration reactions that yield thiazoline- and macrolactamidine-functionalized products, respectively. We also establish the substrate tolerance for BmbD and BmbE and systematically dissect the role of the follower peptide, which we show serves a purpose similar to canonical leader peptides in directing the biosynthetic enzymes to the substrate. Lastly, we leverage the expanded capabilities of YcaO proteins to conduct an extensive bioinformatic survey to classify known YcaO chemistry. This analysis predicts new functions remain to be uncovered within the superfamily

The Luminosity of SN 1999by in NGC 2841 and the Nature of `Peculiar' Type Ia Supernovae

We present UBVRIJHK photometry and optical spectroscopy of the so-called 'peculiar' Type Ia supernova 1999by in NGC 2841. The observations began one week before visual maximum light which is well-defined by daily observations. The light curves and spectra are similar to those of the prototypical subluminous event SN 1991bg. We find that maximum light in B occurred on 1999 May 10.3 UT (JD 2,451,308.8 +/- 0.3) with B=13.66 +/- 0.02 mag and a color of B_max-V_max=0.51 +/- 0.03 mag. The late-time color implies minimal dust extinction from the host galaxy. Our photometry, when combined with the recent Cepheid distance to NGC 2841 (Macri et al. 2001), gives a peak absolute magnitude of M_B=-17.15 +/- 0.23 mag, making SN 1999by one of the least luminous Type Ia events ever observed. We estimate a decline rate parameter of dm15(B)=1.90 mag, versus 1.93 for SN 1991bg, where 1.10 is typical for so-called 'normal' events. We compare SN 1999by with other subluminous events and find that the B_max-V_max color correlates strongly with the decline rate and may be a more sensitive indicator of luminosity than the fading rate for these objects. We find a good correlation between luminosity and the depth of the spectral feature at 580 nm, which had been attributed solely to Si II. We show that in cooler photospheres the 580 nm feature is dominated by Ti II, which provides a simple physical explanation for the correlation. Using only subluminous Type Ia supernovae we derive a Hubble parameter of H_0=75 +12 -11 km/s Mpc, consistent with values found from brighter events.Comment: 36 preprint pages including 18 figures. Near-IR photometry of the SN has been added to the paper. Scheduled to appear in ApJ vol. 613 (September 2004). High-resolution version available from http://www.nd.edu/~pgarnavi/sn99by/sn99by.p

Watasemycin biosynthesis in Streptomyces venezuelae : thiazoline C-methylation by a type B radical-SAM methylase homologue

2-Hydroxyphenylthiazolines are a family of iron-chelating nonribosomal peptide natural products that function as virulence-conferring siderophores in various Gram-negative bacteria. They have also been reported as metabolites of Gram-positive Streptomyces species. Transcriptional analyses of Streptomyces venezuelae ATCC 10712 revealed that its genome contains a putative 2-hydroxyphenylthiazoline biosynthetic gene cluster. Heterologous expression of the gene cluster in Streptomyces coelicolor M1152 showed that the mono- and dimethylated derivatives, thiazostatin and watasemycin, respectively, of the 2-hydroxyphenylthiazoline enantiopyochelin are two of its metabolic products. In addition, isopyochelin, a novel isomer of pyochelin containing a C-methylated thiazolidine, was identified as a third metabolic product of the cluster. Metabolites with molecular formulae corresponding to aerugine and pulicatins A/B were also detected. The structure and stereochemistry of isopyochelin were confirmed by comparison with synthetic standards. The role of two genes in the cluster encoding homologues of PchK, which is proposed to catalyse thiazoline reduction in the biosynthesis of enantiopyochelin in Pseudomonas protegens, was investigated. One was required for the production of all the metabolic products of the cluster, whereas the other appears not to be involved in the biosynthesis of any of them. Deletion of a gene in the cluster encoding a type B radical-SAM methylase homologue abolished the production of watasemycin, but not thiazostatin or isopyochelin. Feeding of thiazostatin to the mutant lacking the functional PchK homologue resulted in complete conversion to watasemycin, demonstrating that thiazoline C-methylation by the type B radical-SAM methylase homologue is the final step in watasemycin biosynthesis

MmfL catalyses formation of a phosphorylated butenolide intermediate in methylenomycin furan biosynthesis

Using a combination of a synthetic substrate analogue and product standard, MmfL, a homologue of the γ-butyrolactone biosynthetic enzyme AfsA, was shown to catalyse the condensation of dihydroxyacetone phosphate with a β-ketoacyl thioester to form a phosphorylated butenolide intermediate in the biosynthesis of the methylenomycin furans, which induce methlenomycin antibiotic production in Streptomyces coelicolor A3(2). AfsA homologues are also involved in the biosynthesis of 2-akyl-4-hydroxy-3-methyl butenolide inducers of antibiotic production in other Streptomyces species, indicating that diverse signalling molecules are assembled from analogous phosphorylated butenolide intermediates

Unique post-translational oxime formation in the biosynthesis of the azolemycin complex of novel ribosomal peptides from Streptomyces sp. FXJ1.264

Streptomycetes are a rich source of bioactive specialized metabolites, including several examples of the rapidly growing class of ribosomally-biosynthesized and post-translationally-modified peptide (RiPP) natural products. Here we report the discovery from Streptomyces sp. FXJ1.264 of azolemycins A–D, a complex of novel linear azole-containing peptides incorporating a unique oxime functional group. Bioinformatics analysis of the Streptomyces sp. FXJ1.264 draft genome sequence identified a cluster of genes that was hypothesized to be responsible for elaboration of the azolemycins from a ribosomally-biosynthesized precursor. Inactivation of genes within this cluster abolished azolemycin production, consistent with this hypothesis. Moreover, mutants lacking the azmE and azmF genes accumulated azolemycin derivatives lacking the O-methyl groups and an amino group in place of the N-terminal oxime (as well as proteolysed derivatives), respectively. Thus AzmE, a putative S-adenosyl methionine-dependent methyl transferase, is responsible for late-stage O-methylation reactions in azolemycin biosynthesis and AzmF, a putative flavin-dependent monooxygenase, catalyzes oxidation of the N-terminal amino group in an azolemycin precursor to the corresponding oxime. To the best of our knowledge, oxime formation is a hitherto unknown posttranslational modification in RiPP biosynthesis