The Gene Cluster for the Biosynthesis of the Glycopeptide Antibiotic A40926 by Nonomuraea Species

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A Derivative of the Thiopeptide GE2270A Highly Selective against Propionibacterium acnes

A chemical derivative of the thiopeptide GE2270A, designated NAI003, was found to possess a substantially reduced antibacterial spectrum in comparison to the parent compound, being active against just a few Gram-positive bacteria. In particular, NAI003 retained low MICs against all tested isolates of Propionibacterium acnes and, to a lesser extent, against Enterococcus faecalis. Furthermore, NAI003 showed a time- and dose-dependent killing of both a clindamycin-resistant and a clindamycinsensitive P. acnes isolate. Gel shift experiments indicated that, like the parent compound, NAI003 retained the ability to bind to elongation factors Tu (EF-Tus) derived from Escherichia coli, E. faecalis, or P. acnes, albeit with reduced efficiency. In contrast, EF-Tus derived from the NAI003-insensitive Staphylococcus aureus or Streptococcus pyogenes did not bind this compound. These results were confirmed by in vitro studies using a hybrid translation system, which indicated that NAI003 can inhibit most efficiently protein synthesis driven by the P. acnes EF-Tu. P. acnes mutants resistant to NAI003 were isolated by direct plating. With one exception, all analyzed strains carried mutations in the tuf gene, encoding EF-Tu. Because of its selective effect on P. acnes in comparison to resident skin flora, NAI003 represents a promising candidate for the topical treatment of acne, which has already completed a phase 1 clinical study

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

BACKGROUND: PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. RESULTS: Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. CONCLUSIONS: The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria

N-acetyl-cysteinylated streptophenazines from Streptomyces

Here, we describe two N-acetyl-cysteinylated streptophenazines (1 and 2) produced by the soil-derived Streptomyces sp. ID63040 and identified through a metabolomic approach. These metabolites attracted our interest due to their low occurrence frequency in a large library of fermentation broth extracts and their consistent presence in biological replicates of the producer strain. The compounds were found to possess broad-spectrum antibacterial activity while exhibiting low cytotoxicity. The biosynthetic gene cluster from Streptomyces sp. ID63040 was found to be highly similar to the streptophenazine reference cluster in the MIBiG database, which originates from the marine Streptomyces sp. CNB-091. Compounds 1 and 2 were the main streptophenazine products from Streptomyces sp. ID63040 at all cultivation times but were not detected in Streptomyces sp. CNB-091. The lack of obvious candidates for cysteinylation in the Streptomyces sp. ID63040 biosynthetic gene cluster suggests that the N-acetyl-cysteine moiety derives from cellular functions, most likely from mycothiol. Overall, our data represent an interesting example of how to leverage metabolomics for the discovery of new natural products and point out the often-neglected contribution of house-keeping cellular functions to natural product diversification

Elucidating the molecular physiology of lantibiotic NAI-107 production in <i>Microbispora </i>ATCC-PTA-5024

BACKGROUND: The filamentous actinomycete Microbispora ATCC-PTA-5024 produces the lantibiotic NAI-107, which is an antibiotic peptide effective against multidrug-resistant Gram-positive bacteria. In actinomycetes, antibiotic production is often associated with a physiological differentiation program controlled by a complex regulatory and metabolic network that may be elucidated by the integration of genomic, proteomic and bioinformatic tools. Accordingly, an extensive evaluation of the proteomic changes associated with NAI-107 production was performed on Microbispora ATCC-PTA-5024 by combining two-dimensional difference in gel electrophoresis, mass spectrometry and gene ontology approaches. RESULTS: Microbispora ATCC-PTA-5024 cultivations in a complex medium were characterized by stages of biomass accumulation (A) followed by biomass yield decline (D). NAI-107 production started at 90 h (A stage), reached a maximum at 140 h (D stage) and decreased thereafter. To reveal patterns of differentially represented proteins associated with NAI-107 production onset and maintenance, differential proteomic analyses were carried-out on biomass samples collected: i) before (66 h) and during (90 h) NAI-107 production at A stage; ii) during three time-points (117, 140, and 162 h) at D stage characterized by different profiles of NAI-107 yield accumulation (117 and 140 h) and decrement (162 h). Regulatory, metabolic and unknown-function proteins, were identified and functionally clustered, revealing that nutritional signals, regulatory cascades and primary metabolism shift-down trigger the accumulation of protein components involved in nitrogen and phosphate metabolism, cell wall biosynthesis/maturation, lipid metabolism, osmotic stress response, multi-drug resistance, and NAI-107 transport. The stimulating role on physiological differentiation of a TetR-like regulator, originally identified in this study, was confirmed by the construction of an over-expressing strain. Finally, the possible role of cellular response to membrane stability alterations and of multi-drug resistance ABC transporters as additional self-resistance mechanisms toward the lantibiotic was confirmed by proteomic and confocal microscopy experiments on a Microbispora ATCC-PTA-5024 lantibiotic-null producer strain which was exposed to an externally-added amount of NAI-107 during growth. CONCLUSION: This study provides a net contribution to the elucidation of the regulatory, metabolic and molecular patterns controlling physiological differentiation in Microbispora ATCC-PTA-5024, supporting the relevance of proteomics in revealing protein players of antibiotic biosynthesis in actinomycetes

Multi-omics Study of Planobispora rosea, Producer of the Thiopeptide Antibiotic GE2270A

Planobispora rosea is the natural producer of the potent thiopeptide antibiotic GE2270A. Here, we present the results of a metabolomics and transcriptomics analysis of P. rosea during production of GE2270A. The data generated provides useful insights into the biology of this genetically intractable bacterium. We characterize the details of the shutdown of protein biosynthesis and the respiratory chain associated with the end of the exponential growth phase. We also provide the first description of the phosphate regulon in P. rosea. Based on the transcriptomics data, we show that both phosphate and iron are limiting P. rosea growth in our experimental conditions. Additionally, we identified and validated a new biosynthetic gene cluster associated with the production of the siderophores benarthin and dibenarthin in P. rosea. Together, the metabolomics and transcriptomics data are used to inform and refine the very first genome-scale metabolic model for P. rosea, which will be a valuable framework for the interpretation of future studies of the biology of this interesting but poorly characterized species. IMPORTANCE Planobispora rosea is a genetically intractable bacterium used for the production of GE2270A on an industrial scale. GE2270A is a potent thiopeptide antibiotic currently used as a precursor for the synthesis of two compounds under clinical studies for the treatment of Clostridium difficile infection and acne. Here, we present the very first systematic multi-omics investigation of this important bacterium, which provides a much-needed detailed picture of the dynamics of metabolism of P. rosea while producing GE2270A

A community resource for paired genomic and metabolomic data mining

Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.Peer reviewe

Minimum Information about a Biosynthetic Gene cluster

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog