Use of Direct-Infusion Electrospray Mass Spectrometry To Guide Empirical Development of Improved Conditions for Expression of Secondary Metabolites from Actinomycetes

Zahn, James A.; Higgs, Richard E.; Hilton, Matthew D.

doi:10.1128/aem.67.1.377-386.2001

Cited by 36 publications

(20 citation statements)

References 38 publications

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“…US7 MTCC8723, the absorbance values of metabolites increased for up to 30 days of incubation and then decreased thereafter. This may be due to feedback inhibition by secondary metabolites (Zahn et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Antibiotic-Producing Actinomycetes Isolates

Kumari

Myagmarjav

Prasad

et al. 2013

American Journal of Microbiology

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Actinomycetes are a gram-positive, filamentous subgroup of bacteria most known for antibiotic production. In fact, most of the antibiotics available today have originated from actinomycetes, namely from the genus Streptomyces. Novel bacteria with antimicrobial activities have been discovered from bacterial screen studies for decades and there is still much more yet to be unearthed. One hundred seventy five strains of actinomycetes were isolated from 38 different soil samples from different locations in Patna, India. Potential antibiotic producers were screened against four test microorganisms (Escherichia coli MTCC 739, Staphylococcus aureus MTCC 96, Streptomyces lividans TK23 MTCC 4 and Candida albicans MTCC 227). The bioassay revealed that about 26% of actinomycetes isolates were active against at least one of the test microorganism. Characterization of a selected isolate has led to identification of a unique strain of actinomycetes (MP 525) showing broad-spectrum antibacterial and antifungal properties. The strain MP 525 has been morphologically characterized as Streptomyces sp. and deposited at MTCC, Chandigarh, India with accession number 8723. The 16S rRNA gene of the strain Streptomyces sp. US7 MTCC 8723 was sequenced and the DNA sequence was deposited at NCBI, Bethesda (GenBank accession. No. HQ659005). On the basis of λmax values of culture filtrates, it has been suggested that the strain might be producing LL-E19085-like antibacterial and a flavone glycoside-like antifungal antibiotics, which can be further exploited for industrial and biological applications.

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Section: Discussionmentioning

confidence: 99%

Identification and Characterization of Antibiotic-Producing Actinomycetes Isolates

Kumari

Myagmarjav

Prasad

et al. 2013

American Journal of Microbiology

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“…Fermentation-An aliquot (100 l) of Streptomyces culture (ATCC PTA-3546) was inoculated into 20 ml of a vegetative medium (31) and incubated at 30°C at 165 rpm. After 72 h, 2 ml of the vegetative culture was transferred to 800 ml of fermentation medium (31), incubated at 30°C at 250 rpm, and harvested after 96 h.…”

Section: Methodsmentioning

confidence: 99%

Novel Lipoglycopeptides as Inhibitors of Bacterial Signal Peptidase I

Kulanthaivel

Kreuzman

Strege

et al. 2004

Journal of Biological Chemistry

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Signal peptidase (SPase) I is responsible for the cleavage of signal peptides of many secreted proteins in bacteria. Because of its unique physiological and biochemical properties, it serves as a potential target for development of novel antibacterial agents. In this study, we report the production, isolation, and structure determination of a family of structurally related novel lipoglycopeptides from a Streptomyces sp. as inhibitors of SPase I. Detailed spectroscopic analyses, including MS and NMR, revealed that these lipoglycopeptides share a common 14-membered cyclic peptide core, an acyclic tripeptide chain, and a deoxy-␣-mannose sugar, but differ in the degree of oxidation of the N-methylphenylglycine residue and the length and branching of the fatty acyl chain. Biochemical analysis demonstrated that these peptides are potent and competitive inhibitors of SPase I with K i 50 to 158 nM. In addition, they showed modest antibacterial activity against a panel of pathogenic Gram-positive and Gram-negative bacteria with minimal inhibitory concentration of 8 -64 M against Streptococcus pneumonniae and 4 -8 M against Escherichia coli. Notably, they mechanistically blocked the protein secretion in whole cells as demonstrated by inhibiting ␤-lactamase release from Staphylococcus aureus. Taken together, the present discovery of a family of novel lipoglycopeptides as potent inhibitors of bacterial SPase I may lead to the development of a novel class of broad-spectrum antibiotics.Proteins destined for secretion in both prokaryotic and eukaryotic organisms are initially synthesized as precursors with an amino-terminal extension known as signal (or leader) peptide. The signal sequence is removed by a signal peptidase (SPase) 1 that is localized in the cytoplasmic membrane in bacteria. Cleavage of precursors by SPase leads to the release of secreted proteins from the outer surface of cytoplasmic membrane. In bacteria, two major SPases, SPase I and SPase II with different cleavage specificities, have been identified. SPase I is responsible for processing the majority of secreted proteins (1-3), and SPase II is exclusively involved in processing glyceride-modified lipoproteins (4).SPase I is an attractive target for development of antibacterial agents because of its unique biochemical and physiological properties. It is essential for bacterial viability and growth as demonstrated by gene knockout and other genetic experiments (5-8). It is widely distributed in both Gram-positive and Gramnegative bacteria, as well as in Chlamydia. Genes encoding SPase I have been cloned and sequenced from different bacterial species, including many of clinically relevant bacteria (8, 9). The active domain of bacterial SPase I is exposed to the surface of cytoplasmic membrane as revealed by sequence and topological analysis (10 -12), and thus is accessible to potential inhibitors. In addition, SPases from bacteria and eukaryotic cells are different in composition, location, and possibly catalytic mechanism (13-17). These differences make it ...

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“…Broth cultures of 43 actinomycetes (17) were started from cryogenic stocks in a vegetative medium that contained (per liter) 30 g of tryptic soy broth (Difco Laboratories, Detroit, Mich.), 3 g of yeast extract (Sigma Chemical Co., St. Louis, Mo. ), 2 g of MgSO 4 , 5 g of glucose, and 4 g of maltose.…”

Section: Methodsmentioning

confidence: 99%

“…Researchers interested in identifying secondary metabolite-eliciting growth conditions for a large, uncharacterized collection of microorganisms may find this rapid tool useful for evaluating multiple growth conditions with statistically large numbers of microorganisms (17). Given a defined set of growth conditions, this tool may also prove to be useful for identifying particularly productive microorganisms for the construction of a screening library.…”

Section: Vol 67 2001 Rapid Characterization Of Extracts By Es-msmentioning

confidence: 99%

Rapid Method To Estimate the Presence of Secondary Metabolites in Microbial Extracts

Higgs

Zahn

Gygi

et al. 2001

Appl Environ Microbiol

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Screening microbial secondary metabolites is an established method to identify novel biologically active molecules. Preparation of biological screening samples from microbial fermentation extracts requires growth conditions that promote synthesis of secondary metabolites and extraction procedures that capture the secondary metabolites produced. High-performance liquid chromatography (HPLC) analysis of fermentation extracts can be used to estimate the number of secondary metabolites produced by microorganisms under various growth conditions but is slow. In this study we report on a rapid (approximately 1 min per assay) surrogate measure of secondary metabolite production based on a metabolite productivity index computed from the electrospray mass spectra of samples injected directly into a spectrometer. This surrogate measure of productivity was shown to correlate with an HPLC measure of productivity with a coefficient of 0.78 for a test set of extracts from 43 actinomycetes. This rapid measure of secondary metabolite productivity may be used to identify improved cultivation and extraction conditions by analyzing and ranking large sets of extracts. The same methods may also be used to survey large collections of extracts to identify subsets of highly productive organisms for biological screening or additional study.Microbial extracts have been and continue to be a productive source of new biologically active molecules for drug discovery (2, 13). It is estimated that more than 30% of worldwide human pharmaceutical sales have compounds from natural sources as their origin (12). With advances in genomics and high-throughput screening (HTS) technology, many new therapeutic targets are accessible for identifying pharmaceutical agents. In addition to the historical practice of screening microbial fermentation extracts for antibiotic activity, extracts can now routinely be screened with a variety of new functional, receptor binding, enzyme inhibition, and protein-protein interaction assays. HTS formats used at many large pharmaceutical research organizations are, however, generally incompatible with complex fermentation extracts. The reasons for the incompatibility include nonspecific interference with assay systems, cost in dollars and time to identify and dereplicate active components from a complex mixture, and adverse physical properties for automated liquid-handling equipment. Therefore, additional investment in selection and preparation of fermentation extracts is one strategy to align natural product drug discovery with today's automated HTS assay systems. A typical approach to improve the compatibility of fermentation extracts with HTS was recently reported by Schmid et al., who described a multistage automated solid-phase extraction (SPE) system (12).To make the best use of finite resources in natural product discovery organizations, it is important to identify collections of organisms that produce secondary metabolites, culture conditions that generally support secondary metabolite synthesis, and sample prepar...

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Use of Direct-Infusion Electrospray Mass Spectrometry To Guide Empirical Development of Improved Conditions for Expression of Secondary Metabolites from Actinomycetes

Cited by 36 publications

References 38 publications

Identification and Characterization of Antibiotic-Producing Actinomycetes Isolates

Identification and Characterization of Antibiotic-Producing Actinomycetes Isolates

Novel Lipoglycopeptides as Inhibitors of Bacterial Signal Peptidase I

Rapid Method To Estimate the Presence of Secondary Metabolites in Microbial Extracts

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