Synthesis and characterization of novel poly cysteine methacrylate nanoparticles and their morphology and size studies.

Polymer nanoparticles (PNPs) have significantly advanced the field of biomedicine, showcasing the remarkable potential for precise drug delivery, administration of nutraceuticals, diagnostics/imaging applications, and the fabrication of biocompatible materials, among other uses. Despite these promising developments, the invention faces notable challenges related to biodegradability, bioactivity, target-site specificity, particle size, carrier efficiency, and controlled release. Addressing these concerns is essential for optimizing the functionality and impact of PNPs in biomedical applications. Here, new poly cysteine methacrylate nanoparticles (PCMANPs), ca. (200 nm) in size have been synthesized from the cysteine methacrylate (CysMA) monomer using different strategies, including emulsion and inverse emulsion polymerization techniques. The monomer was synthesized using the Michael addition reaction, involving the addition of 3-(acryloyloxy)-2-hydroxypropyl methacrylate to the sulfhydryl group (-SH) of the cysteine (Cys) active site, with the aid of dimethyl phenyl phosphine (DMPP) as a nucleophilic agent as previously reported. To enhance nano-polymerization, a thorough exploration of various initiators, including ammonium persulfate (APS) and 4,4'-azobis (4-cyanovaleric acid) (ACVA), alongside surfactants, such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and sodium dodecyl sulfate (SDS), was conducted. Additionally, critical parameters, such as reaction time, temperature, and solvents, were systematically investigated due to their substantial influence on the shape, size, stability, and morphology of the synthesized polymer nanoparticles. This comprehensive approach aims to optimize the synthesis process, ensuring precise control over the key characteristics of the resulting nanoparticles for enhanced performance in diverse applications. Various characterization techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), Raman spectroscopy, Fourier-transform infrared spectroscopy (FTIR), zeta potential, and zeta sizer dynamic light scattering (DLS) analysis, were utilized to investigate purity, morphology, and particle size of the PNPs. As a result, a spherical, monodispersed (homogenized), and stable PCMANP with defined size and morphology was achieved. This may exhibit a remarkable achievement in the future of drug delivery systems and therapeutic index.

Physiological regulation for enhancing biosynthesis of biofilm-inhibiting secondary metabolites in Streptomyces cellulosae.

Although the production of the secondary metabolite is frequently restricted, methods to regulate and optimize their synthesis are extremely beneficial. The current study proposes to enhance the production of antibiofilm metabolite in Streptomyces cellulosae (S. cellulosae). It was isolated from soil by growing on Gause's media and identified by colony morphology and genomic sequencing of 16S rDNA. Antibacterial and antibiofilm activities of the isolates were screened against a series of pathogenic bacteria by agar plug diffusion and 96 well microtiter plate methods, respectively. Physiological regulation of the bacterial bioactivity against biofilm formation was monitored under different cultural conditions. The isolated Streptomyces sequence analysis of the 16S rDNA was 100% identical to the sequence of S. cellulosae strain NBRC 13027. Physical (temperature and pH) and chemical (carbon, nitrogen, and minerals) culture medium factors have shown variable impacts on the growth and bioactive substances of S. cellulosae. Moreover, results of simple linear regression and correlation suggested that most of the physiological regulations with the highest response (r2= 0.85-0.99; p<0.01) and linearly (r= 0.88-0.99; p<0.01) were correlated between microbial biomass and crude extract. Lastly, under different culture growth conditions, biofilm inhibition was tested against Pseudomonas aeruginosa (P. aeruginosa). The physiological regulation results exhibited that 1 µg/mL of the extract was the most efficient concentration against biofilm formation in P. aeruginosa while 3 µg/mL is an effective bactericidal dose against P. aeruginosa. We concluded that S. cellulosae can produce antibacterial and antibiofilm metabolites. Physiological regulation is considered a powerful tool that can be used for increasing the biosynthesis of the active metabolites and biomass.

Expression Level of the mip, pmp18D, and ompA Genes in Chlamydia abortus Isolated from Aborted Ewes.

In this manuscript, we report the proteins macrophage infectivity potentiator (mip, CAB080), major outer membrane protein (momp, CAB048), and polymorphic outer membrane protein (pmp18D, CAB776) that are expressed in different times of pregnancy in mice infected with Chlamydia abortus. Enzootic abortion of ewes (EAE) by C. abortus, an obligate intracellular pathogen, is a critical zoonotic disease-causing significant economic loss to livestock farming globally. This study was carried out for the detection and characterization of macrophage infectivity potentiator (mip, CAB080), major outer membrane protein (momp, CAB048), and polymorphic outer membrane protein (pmp18D, CAB776) using RT-qPCR. These proteins are believed to be expressed as virulence factors in C. abortus isolated from aborted ewes. BALB/c mice (pregnant and nonpregnant) were used as an animal model to be injected intraperitoneally with C. abortus culture in Vero cells since the endometrial lymphoid tissues of these animals resembles that of ewes. Also, the short duration of pregnancy in mice makes them a suitable animal model for obstetric studies. Tissue samples were taken from the mice after 10, 15, and 20 days of pregnancy to compare the expression of the genes mip, pmp18D, and ompA. Transcription level was quantified using RT-qPCR, the GAPDH transcription quantification, as a normalization signal. Abortion occurred in pregnant mice, and apparent differences between the transcriptional levels of the mip, pmp18D, and ompA genes in the samples taken during different time intervals of pregnancy were not observed (p > 0.05). The result indicated that the three bacterial genes, mip, pmp18D, and ompA, play a role as virulence factors in abortion and are differentially expressed in pregnant and nonpregnant animals. Inactivation of the genes is suggested to confirm the hypothesis.

Mastering noninvasive predictive biomarkers to follow up renal transplant patients.

Renal transplantation is the treatment of choice for end-stage renal disease (ESRD) patients. Several cellular processes are regulated via non-coding RNAs by silencing target gene expression. Previous investigations have established a linkage between a number of human microRNAs and kidney failure. This study aims to identify the expression of urinary miR-199a-3p and miR-155-5p as non-invasive biomarkers during post and pre-transplantation over a six-month follow-up period. In addition to the classic chronic renal disease markers (estimated glomerular filtration rate eGFR, Serum creatinine, serum electrolytes, and Antinuclear antibodies ANA test). Urinary miR-199a-3p and miR-155-5p expression levels in 72 adults with diabetic nephropathy and, 42 adults with lupus nephropathy renal transplant recipients. Both were compared with 32 healthy controls prior and post-transplantation. miRNAs were evaluated by quantitative reverse transcription-polymerase chain reaction. Urinary miR-199a-3p significantly (p<0.0001) downregulated in diabetic and lupus nephropathy prior to transplantation and significantly upregulated post-transplantation compared to the control. While urinary miR-155-5p quantities were significantly higher in prior renal transplant patients in comparison with the same patients' post-renal transplantation(P<0.0001). In conclusion, Urinary miR-199a-3p and miR-155-5p can be used as a non-invasive biomarker with high specificity and sensitivity to follow up the renal transplant patients before and post-transplantation instead of biopsy which is complicated by a non-negligible factor.

On Column Binding a Real-Time Biosensor for ß-lactam Antibiotics Quantification.

This work aimed to develop accurate, quick, and practical tools for the detection of residues of penicillin G antibiotic in biological and non-biological samples. The assays were developed based on the binding mechanism of ß-lactam to penicillin-binding proteins; samples of different concentrations of penicillin G were incubated with in vitro expressed 6X-Histidine-tagged soluble penicillin-binding protein (PBP2x*) of Streptococcus pneumoniae (S. pneumoniae), whereby penicillin G in samples specifically binds to PBP2x*. The fluorescent-labeled ß-lactam analogue Bocillin FL was used as a competent substrate, and two different routes estimated the amounts of the penicillin G. The first route was established based on the differences in the concentration of non-bounded Bocillin FL molecules within the reactions while using a real-time polymerase chain reaction (PCR)-based method for fluorescence detection. The second route depended on the amount of the relative intensity of Bocillin FL bounded to Soluble PBP-2x*, being run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-page), visualized by a ChemiDoc-It®2 Imager, and quantified based on the fluorescence affinity of the competent substrate. While both of the methods gave a broad range of linearity and high sensitivity, the on column based real-time method is fast, non-time consuming, and highly sensitive. The method identified traces of antibiotic in the range 0.01-0.2 nM in addition to higher accuracy in comparison to the SDS-based detection method, while the sensitivity of the SDS-based method ranged between 0.015 and 2 µM). Thus, the on column based real time assay is a fast novel method, which was developed for the first time based on the binding inhibition of a fluorescence competitor material and it can be adapted to screen traces of penicillin G in any biological and environmental samples.

The ROK like protein of Myxococcus xanthus DK1622 acts as a pleiotropic transcriptional regulator for secondary metabolism.

Myxococcus xanthus DK1622 is known as a proficient producer of different kinds of secondary metabolites (SM) with various biological activities, including myxovirescin A, myxalamide A, myxochromide A and DKxanthene. Low production of SM in the wild type bacteria makes searching for production optimization methods highly desirable. Identification and induction of endogenous key molecular feature(s) regulating the production level of the metabolites remain promising, while heterologous expression of the biosynthetic genes is not always efficient because of various complicating factors including codon usage bias. This study established proteomic and molecular approaches to elucidate the regulatory roles of the ROK regulatory protein in the modification of secondary metabolite biosynthesis. Interestingly, the results revealed that rok inactivation significantly reduced the production of the SM and also changed the motility in the bacteria. Electrophoretic mobility shift assay using purified ROK protein indicated a direct enhancement of the promoters encoding transcription of the DKxanthene, myxochelin A, and myxalamide A biosynthesis machinery. Comparative proteomic analysis by two-dimensional fluorescence difference in-gel electrophoresis (2D-DIGE) was employed to identify the protein profiles of the wild type and rok mutant strains during early and late logarithmic growth phases of the bacterial culture. Resulting data demonstrated overall 130 differently altered proteins by the effect of the rok gene mutation, including putative proteins suspected to be involved in transcriptional regulation, carbohydrate metabolism, development, spore formation, and motility. Except for a slight induction seen in the production of myxovirescin A in a rok over-expression background, no changes were found in the formation of the other SM. From the outcome of our investigation, it is possible to conclude that ROK acts as a pleiotropic regulator of secondary metabolite formation and development in M. xanthus, while its direct effects still remain speculative. More experiments are required to elucidate in detail the variable regulation effects of the protein and to explore applicable approaches for generating valuable SM in this bacterium.

Isolation and Identification of Chlamydia abortus from Aborted Ewes in Sulaimani Province, Northern Iraq.

Abortion in small ruminants is a significant problem in Iraq and causes severe economic losses in sheep farms. Chlamydia abortus causes enzootic abortion in ewes and is associated with reproductive problems in sheep in Sulaimani province - Northern Iraq. During a lambing season in 2017, abortion was widespread among several sheep flocks in different regions of Sulaimani (Kalar, Said Sadiq, and Chamchamal), and C. abortus was one of the causes. Accordingly, we carried out this study to isolate and identify C. abortus in aborted ewes in these regions. We collected 30 samples of aborted fetuses from five herds in which abortions had been observed. The pathogen isolation was done by inoculation into embryonated chicken eggs and conventional PCR was used to identify C. abortus in clinical specimens. C. abortus was identified in one of the 30 aborted fetuses (3.33%) from the Kalar district, and all the remaining 29 samples (96.66%) were found positive to Brucella abortus. The gene ompA encoding the outer membrane protein of C. abortus was sequenced and got the accession number MK643153 in NCBI GenBank. The sequence was named C. abortus strain Sul/2017. Our isolate showed 99.79% homology with Sul/014 (accession No. KY399850) and differed from the latter by two amino acid substitutions at E115K and K259N. The topology of the phylogenetic tree based on the ompA gene showed that the isolate belongs to C. abortus and has a common ancestor with isolates of sheep in Iraq and Tunisia with accession numbers KY399850 and HQ62243, respectively.Abortion in small ruminants is a significant problem in Iraq and causes severe economic losses in sheep farms. Chlamydia abortus causes enzootic abortion in ewes and is associated with reproductive problems in sheep in Sulaimani province ­ Northern Iraq. During a lambing season in 2017, abortion was widespread among several sheep flocks in different regions of Sulaimani (Kalar, Said Sadiq, and Chamchamal), and C. abortus was one of the causes. Accordingly, we carried out this study to isolate and identify C. abortus in aborted ewes in these regions. We collected 30 samples of aborted fetuses from five herds in which abortions had been observed. The pathogen isolation was done by inoculation into embryonated chicken eggs and conventional PCR was used to identify C. abortus in clinical specimens. C. abortus was identified in one of the 30 aborted fetuses (3.33%) from the Kalar district, and all the remaining 29 samples (96.66%) were found positive to Brucella abortus. The gene ompA encoding the outer membrane protein of C. abortus was sequenced and got the accession number MK643153 in NCBI GenBank. The sequence was named C. abortus strain Sul/2017. Our isolate showed 99.79% homology with Sul/014 (accession No. KY399850) and differed from the latter by two amino acid substitutions at E115K and K259N. The topology of the phylogenetic tree based on the ompA gene showed that the isolate belongs to C. abortus and has a common ancestor with isolates of sheep in Iraq and Tunisia with accession numbers KY399850 and HQ62243, respectively.

Identification of the Intron 22 and Intron 1 Inversions of the Factor VIII Gene in Iraqi Kurdish Patients With Hemophilia A.

Hemophilia A (HA) is a severe coagulation disorder affecting 1 in 5000 to 10 000 male births. In severe cases, the most deleterious large DNA rearrangements are inversions of intron 22 (Inv22) and intron 1 (Inv1) of the factor VIII (FVIII) gene. These account for 40% to 50% and 1% to 5% of all causative mutations, respectively. Nevertheless, no genetic analysis to identify the actual causative mutation of FVIII, particularly Inv22 and Inv1, among Iraqi Kurdish hemophiliacs has been performed. In this study, we aimed to genotype Inv22 and Inv1 of the FVIII gene in our patients with HA and reveal the genotype/phenotype correlation with the inversion mutations and their role as a risk factor for the development of inhibitors. Analyses of the Inv22 and Inv1 mutations in 80 Iraqi Kurdish patients with HA (60 severe, 18 moderate, and 2 mild) were performed using the inverse shifting-polymerase chain reaction (IS-PCR) method. In severe cases, 46.7% (28/60) had Inv22 and 3.3% (2/60) had Inv1. The genotype/phenotype relation of Inv22 and Inv1 illustrated a statistically significant association (P = .012) between disease severity and inversion mutations. Slightly more patients with Inv22 (39%) developed inhibitors than those without Inv22 (28%; odds ratio = 1.65, 95% confidence interval = 0.56-4.87, P = .361). Inv22 is a major cause of severe HA in Iraqi Kurdish patients, and IS-PCR is a rapid, robust, and effective method that can be applied for carrier detection and prenatal diagnosis of HA in developing countries.

Application of Indirect Linkage Analysis for Carrier Detection of Hemophilia A in Kurdistan Region of Iraq: Usefulness of Intron 18 BclI T>A, Intron 19 HindIII C>T, and IVS7 nt27 G>A Markers.

Hemophilia A (HA) is the most common congenital X-linked coagulopathy caused by mutations in the factor VIII gene. One in 5000 to 10 000 male persons worldwide suffer from HA. It is the archetype of high-cost, low-volume disease. Therefore, identification of carriers is crucial to avoid the birth of affected males. Tracking of the defective X chromosome through indirect linkage analysis represents the most practical method for screening for carriers in developing countries. In this study, 227 individuals from 41 families with HA and 100 normal participants were recruited from the Kurdistan region of Iraq and evaluated for intron 18 BclI, intron 19 HindIII, and IVS7 nt 27 markers by polymerase chain reaction restriction fragment length polymorphism and direct sequencing. Among the studied women, 49%, 42%, and 14% were discovered to be heterozygous for BclI, HindIII, and IVS7 markers, respectively. Using BclI, HindIII, and IVS7 markers, 56%, 46%, and 17% of the families were informative, respectively. The combined informativity of these polymorphic sites reaches 66%. The current study illustrates the effectiveness of the BclI and HindIII markers for the diagnosis of HA carriers among the Iraqi Kurdish population.

A Single-Center Study of Viral Respiratory Tract Infections in Hospitalized Children From the Kurdistan Region of Iraq.

Viral respiratory infections are among the most common causes of disease in humans, particularly in young children, and remain a major public health problem worldwide. For many geographic regions, there is limited epidemiological information on the main causative agents of these diseases. In this article, we investigated, in a prospective study, the viral agents leading to acute respiratory disease in children younger than 15 years of age who were admitted to the pediatric emergency unit of a major teaching hospital in Erbil City, capital of the Kurdistan region, Iraq. Nasopharyngeal samples obtained from 269 hospitalized children were analyzed for viral respiratory pathogens using the xTAG Respiratory Virus Panel Fast assay, and the data were correlated with the clinical and demographic information available for these patients. One or more respiratory virus(es) were detected in 203 out of 269 (75.5%) samples. The most frequent viruses were enterovirus/rhinovirus (n = 88; 32.7%), respiratory syncytial virus (n = 55; 20.4%), and human metapneumovirus (n = 36; 13.4%). In 42 samples (15.6%), coinfections with 2 or more respiratory viruses were detected, with enterovirus/rhinovirus, respiratory syncytial virus, human metapneumovirus, and adenovirus being identified as the most common agents in viral coinfections in these patients.

Crocadepsins-Depsipeptides from the Myxobacterium Chondromyces crocatus Found by a Genome Mining Approach.

Analysis of the genome sequence of the myxobacterium Chondromyces crocatus Cm c5 revealed the presence of numerous cryptic megasynthetase gene clusters, one of which we here assign to two previously unknown chlorinated metabolites by a comparative gene inactivation and secondary metabolomics approach. Structure elucidation of these compounds revealed a unique cyclic depsipeptide skeleton featuring ß- and δ-amide bonds of aspartic acid and 3-methyl ornithine moieties, respectively. Insights into their biosynthesis were obtained by targeted gene inactivation and feeding experiments employing isotope-labeled precursors. The compounds were produced ubiquitously by the species Chondromyces crocatus and were found to inhibit the carbon storage regulator-RNA interaction.

Biosynthesis of crocacin involves an unusual hydrolytic release domain showing similarity to condensation domains.

The crocacins are potent antifungal and cytotoxic natural compounds from myxobacteria of the genus Chondromyces. Although total synthesis approaches have been reported, the molecular and biochemical basis guiding the formation of the linear crocacin scaffold has remained unknown. Along with the identification and functional analysis of the crocacin biosynthetic gene cluster from Chondromyces crocatus Cm c5, we here present the identification and biochemical characterization of an unusual chain termination domain homologous to condensation domains responsible for hydrolytic release of the product from the assembly line. In particular, gene inactivation studies and in vitro experiments using the heterologously produced domain CroK-C2 confirm this surprising role giving rise to the linear carboxylic acid. Additionally, we determined the kinetic parameters of CroK-C2 by monitoring hydrolytic cleavage of the substrate mimic N-acetylcysteaminyl-crocacin B using an innovative high-performance liquid chromatography mass spectrometry-based assay.

Elucidation of gephyronic acid biosynthetic pathway revealed unexpected SAM-dependent methylations.

Gephyronic acid, a cytostatic polyketide produced by the myxobacterium Cystobacter violaceus Cb vi76, exhibits potent and selective eukaryotic protein synthesis inhibition. Next-generation sequencing of the C. violaceus genome revealed five type I polyketide synthases and post-PKS tailoring enzymes including an O-methyltransferase and a cytochrome P450 monooxygenase. Seven methyltransferase (MT) domains embedded within the PKS subunits were found to install the methyl branches throughout the gephyronic acid skeleton. A rare loading domain from the GNAT superfamily also contains an embedded MT domain that catalyzes the in situ production of an isobutyryl starter unit. Phylogenetic analysis identified new motifs that distinguish MT domains located in PKS pathways with in cis acyltransferase (AT) domains from MT domains located in PKS pathways with trans AT enzymes. The identification of the gene cluster sets the stage for the generation of a heterologous expression system, which will allow further investigation of selective eukaryotic protein synthesis inhibitors through the generation of gephyronic acid analogues.

Synthetic biotechnology to study and engineer ribosomal bottromycin biosynthesis.

Bottromycins represent a promising class of antibiotics binding to the therapeutically unexploited A-site of the bacterial ribosome. By inhibiting translation they are active against clinically important pathogens, such as vancomycin-resistant Enterococci. Structurally, bottromycins are heavily modified peptides exhibiting various unusual biosynthetic features. To set the stage for compound modification and yield optimization, we identified the biosynthetic gene cluster, used synthetic biotechnology approaches to establish and improve heterologous production, and generated analogs by pathway genetic engineering. We unambiguously identified three radical SAM methyltransferase-encoding genes required for various methylations at unactivated carbons yielding tert-butyl valine, methyl-proline, and ß-methyl-phenylalanine residues, plus a gene involved in aspartate methyl-ester formation. Evidence for the formation of the exo-thiazole unit and for a macrocyclodehydration mechanism leading to amidine ring formation is provided.

Unusual carbon fixation gives rise to diverse polyketide extender units.

Polyketides are structurally diverse and medically important natural products that have various biological activities. During biosynthesis, chain elongation uses activated dicarboxylic acid building blocks, and their availability therefore limits side chain variation in polyketides. Recently, the crotonyl-CoA carboxylase-reductase (CCR) class of enzymes was identified in primary metabolism and was found to be involved in extender-unit biosynthesis of polyketides. These enzymes are, in theory, capable of forming dicarboxylic acids that show any side chain from the respective unsaturated fatty acid precursor. To our knowledge, we here report the first crystal structure of a CCR, the hexylmalonyl-CoA synthase from Streptomyces sp. JS360, in complex with its substrate. Structural analysis and biochemical characterization of the enzyme, including active site mutations, are reported. Our analysis reveals how primary metabolic CCRs can evolve to produce various dicarboxylic acid building blocks, setting the stage to use CCRs for the production of unique extender units and, consequently, altered polyketides.

Mining the cinnabaramide biosynthetic pathway to generate novel proteasome inhibitors.

The cinnabaramides and salinosporamides are mixed PKS/NRPS natural products isolated from a terrestrial streptomycete and a marine actinomycete, respectively. They interfere with the proteasome and thus potentially inhibit the growth of cancer cells. The compounds exhibit a γ-lactam-ß-lactone bicyclic ring structure attached to a cyclohexenyl unit and a PKS side chain. As a first step towards improving anticancer activity and permitting genetic approaches to novel analogues, we have cloned and characterized the cinnabaramide biosynthetic genes from Streptomyces sp. JS360. In addition to the expected PKS and NRPS genes, the cluster encodes functionalities for the assembly of the hexyl side chain precursor. The corresponding enzymes exhibit relaxed substrate specificities towards a number of synthesized precursors, enabling production of novel chlorinated cinnabaramides. These were isolated and analyzed for activity, revealing that derivatives bearing a chlorine atom in the PKS side chain show higher inhibitory potentials towards the proteasome's proteolytic subunits (especially the trypsin and chymotrypsin units) and higher cytotoxicities towards human tumor cell lines than the parent cinnabaramide A. Although their activities towards the proteasome were weaker than that of salinosporamide A, the cinnabaramides were found to inhibit the growth of various fungi with greater potency.

Biosynthesis of the myxobacterial antibiotic corallopyronin A.

Corallopyronin A is a myxobacterial compound with potent antibacterial activity. Feeding experiments with labelled precursors resulted in the deduction of all biosynthetic building blocks for corallopyronin A and revealed an unusual feature of this metabolite: its biosynthesis from two chains, one solely PKS-derived and the other NRPS/PKS-derived. The starter molecule is believed to be carbonic acid or its monomethyl ester. The putative corallopyronin A biosynthetic gene cluster is a trans-AT-type mixed PKS/NRPS gene cluster, containing a beta-branching cassette. Striking features of this gene cluster are a NRPS-like adenylation domain that is part of a PKS-type module and is believed to be responsible for glycine incorporation, as well as split modules with individual domains occurring on different genes. It is suggested that CorB is a trans-acting ketosynthase and it is proposed that it catalyses the Claisen condensation responsible for the interconnection of the two chains. Additionally, the stereochemistry of corallopyronin A was deduced by a combination of a modified Mosher's method and ozonolysis with subsequent chiral GC analyses.

Characterization of a novel type of oxidative decarboxylase involved in the biosynthesis of the styryl moiety of chondrochloren from an acylated tyrosine.

Myxobacteria are soil-dwelling bacteria notable for several unique behavioral features, such as cellular movement by gliding and the formation of multicellular fruiting bodies. More recently they have gained recognition as producers of several unique polyketide and nonribosomal polypeptide metabolites with potential therapeutic value. The biosynthesis of these compounds often involves highly unusual mechanisms including the formation of the chloro-hydroxy-styryl moiety of the chondrochloren antibiotic produced by Chondromyces crocatus Cm c5. Here it is shown that the final product of the chondrochloren megasynthetase is the novel natural product pre-chondrochloren, a carboxylated and saturated derivative of chondrochloren. This compound was isolated from strains harboring mutants of a hypothetical oxidative decarboxylase (CndG) identified in the chondrochloren gene cluster. CndG was heterologously expressed in Escherichia coli and shown to be an FAD-dependent oxidative decarboxylase. Biochemical characterization of the protein was achieved using the intermediate described above as the substrate and yielded chondrochloren by oxidative decarboxylation. It was also demonstrated that the CndG post-assembly line modification of pre-chondrochloren is essential for the biological activity of chondrochloren.

Unusual chemistry in the biosynthesis of the antibiotic chondrochlorens.

The antibiotic chondrochlorens A and B from the myxobacterium Chondromyces crocatus Cm c5 incorporate several unusual structural features, notable among them a shared chloro-hydroxy-styryl functionality and the ethoxy group of chondrochloren B. Our analysis of the chondrochloren gene cluster by targeted gene inactivation coupled with assays in vitro has shed significant light on the biosynthesis of these metabolites. Chlorination of tyrosine occurs early in the pathway, likely on a peptidyl carrier protein-bound intermediate, whereas decarboxylation to the styryl moiety appears to be accomplished by an unprecedented oxidative decarboxylase. We also show that the chondrochloren B ethoxy group arises from initial incorporation by the polyketide synthase of hydroxy malonate as an extender unit, methylation in cis by an O-methyltransferase, followed by a second methylation. This report therefore constitutes a direct demonstration of the involvement of a radical S-adenosylmethionine methylase in bacterial secondary metabolism.