Novel method for detection of butanolides in Streptomyces coelicolor culture broth, using a His-tagged receptor (ScbR) and mass spectrometry

Gamma-butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using Streptomyces coelicolor as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.     

Isolation and Identification of Novel ADP-Ribosylated Proteins from Streptomyces coelicolor A3(2)

An important role of protein ADP-ribosylation in bacterial morphogenesis has been proposed (J. Bacteriol. 178, 3785-3790; 178, 4935-4941). To clarify the detail of ADP-ribosylation, we identified a new kind of target protein for ADP-ribosylation in Streptomyces coelicolor A3(2) grown to the late growth phase. All four proteins (MalE, BldKB, a periplasmic protein for binding branched-chain amino-acids, and a periplasmic solute binding protein) were functionally similar and participated in the regulation of transport of metabolites or nutrients through the membrane. ADP-ribosylation was likely to occur on a cysteine residue, because the modification group was removed by mercuric chloride treatment. The modification site may be the site of lipoprotein modification necessary for protein export. This report is the first suggesting that certain proteins involved in membrane transport can be ADP-ribosylated.     

Imaging of Streptomyces coelicolor A3(2) with Reduced Autofluorescence Reveals a Novel Stage of FtsZ Localization

Imaging of low abundance proteins in time and space by fluorescence microscopy is typically hampered by host-cell autofluorescence. Streptomycetes are an important model system for the study of bacterial development, and undergo multiple synchronous cell division during the sporulation stage. To analyse this phenomenon in detail, fluorescence microscopy, and in particular also the recently published novel live imaging techniques, require optimal signal to noise ratios. Here we describe the development of a novel derivative of Streptomyces coelicolor A3(2) with strongly reduced autofluorescence, allowing the imaging of fluorescently labelled proteins at significantly higher resolution. The enhanced image detail provided novel localization information for the cell division protein FtsZ, demonstrating a new developmental stage where multiple FtsZ foci accumulate at the septal plane. This suggests that multiple foci are sequentially produced, ultimately connecting to form the complete Z ring. The enhanced imaging properties are an important step forward for the confocal and live imaging of less abundant proteins and for the use of lower intensity fluorophores in streptomycetes.     

Sensitive and Specific Peak Detection for SELDI-TOF Mass Spectrometry Using a Wavelet/Neural-Network Based Approach

SELDI-TOF mass spectrometer''s compact size and automated, high throughput design have been attractive to clinical researchers, and the platform has seen steady-use in biomarker studies. Despite new algorithms and preprocessing pipelines that have been developed to address reproducibility issues, visual inspection of the results of SELDI spectra preprocessing by the best algorithms still shows miscalled peaks and systematic sources of error. This suggests that there continues to be problems with SELDI preprocessing. In this work, we study the preprocessing of SELDI in detail and introduce improvements. While many algorithms, including the vendor supplied software, can identify peak clusters of specific mass (or m/z) in groups of spectra with high specificity and low false discover rate (FDR), the algorithms tend to underperform estimating the exact prevalence and intensity of peaks in those clusters. Thus group differences that at first appear very strong are shown, after careful and laborious hand inspection of the spectra, to be less than significant. Here we introduce a wavelet/neural network based algorithm which mimics what a team of expert, human users would call for peaks in each of several hundred spectra in a typical SELDI clinical study. The wavelet denoising part of the algorithm optimally smoothes the signal in each spectrum according to an improved suite of signal processing algorithms previously reported (the LibSELDI toolbox under development). The neural network part of the algorithm combines those results with the raw signal and a training dataset of expertly called peaks, to call peaks in a test set of spectra with approximately 95% accuracy. The new method was applied to data collected from a study of cervical mucus for the early detection of cervical cancer in HPV infected women. The method shows promise in addressing the ongoing SELDI reproducibility issues.     

Metabolic engineering of Streptomyces coelicolor for enhanced prodigiosins(RED) production

Bacterial prodigiosins are red-colored secondary metabolites with multiple activities,such as anticancer,antimalarial and immunosuppressive,which hold great potential for medical applications.In this study,dramatically enhanced prodigiosins(RED) production in Streptomyces coelicolor was achieved by combinatorial metabolic engineering,including inactivation of the repressor gene ohkA,deletion of the actinorhodin(ACT) and calcium-dependent antibiotic(CDA) biosynthetic gene clusters(BGCs) and multi-copy chromosomal integration of the RED BGC.The results showed that ohkA deletion led to a 1-fold increase of RED production over the wild-type strain M145.Then,the ACT and CDA BGCs were deleted successively based on the AohkA mutant(SBJ101).To achieve multi-copy RED BGC integration,artificial ΦC31 attB site(s) were inserted simultaneously at the position where the ACT and CDA BGCs were deleted.The resulting strains SBJ102(with a single deletion of the ACT BGC and insertion of one artificial attB site) and SBJ103(with the deletion of both BGCs and insertion of two artificial attB sites) produced 1.9-and 6-fold higher RED titers than M145,respectively.Finally,the entire RED BGC was introduced into mutants from SBJ101 to SBJ103,generating three mutants(from SBJ104 to SBJ106) with chromosomal integration of one to three copies of the RED BGC.The highest RED yield was from SBJ106,which produced a maximum level of 96.8 mg g~(-1) cell dry weight,showing a 12-fold increase relative to M145.Collectively,the metabolic engineering strategies employed in this study are very efficient for the construction of high prodigiosin-producing strains.     

Genetic analysis of A-factor synthesis in Streptomyces coelicolor A3(2) and Streptomyces griseus

A-factor is a potent pleiotropic effector produced by Streptomyces griseus and is essential for streptomycin production and spore formation in this organism. Its production is widely distributed among various actinomycetes including Streptomyces coelicolor A3(2). Genetic analysis of A-factor production was carried out with S. coelicolor A3(2), and two closely linked loci for A-factor mutations (afsA and B) were identified between cysD and leuB on the chromosomal linkage map. In contrast, genetic crosses of A-factor-negative mutants of S. griseus, using a protoplast fusion technique, failed to give a fixed locus for A-factor gene(s) and suggested involvement of an extrachromosomal or transposable genetic element in A-factor synthesis in this organism.     

Identification and cloning of a umu locus in Streptomyces coelicolor A3(2)

The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV and many other DNA-damaging agents. E. coli umu mutants are defective in mutagenesis and slightly more sensitive to DNA-damaging agents. The existence of a umuDC analogue in Streptomyces coelicolor was suggested by data of our previous works. We cloned from Streptomyces coelicolor a fragment of DNA homologous to the E. coli umuDC region that is able to complement the E coli umuC122::Tn5 mutation. Therefore our data suggest that S. coelicolor contains a functional umu-like operon.     

Misaminoacylation and tRNA-dependent transamidation in Streptomyces coelicolor A3(2)

Abstract Streptomyces coelicolor was found to be devoid of glutaminyl-tRNA synthetase. In this bacterium, tRNA^Gln is aminoacylated by glutamyl-tRNA synthetase to yield glutamyl-tRNA^Gln, followed by correction to glutaminyl-tRNA^Gln by a tRNA-dependent amidotransferase.     

Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform

Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25⁺⁴⁰ isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25⁺⁴⁰ as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.     

Identification of genes for mycothiol biosynthesis in Streptomyces coelicolor A3(2)

Mycothiol is a low molecular weight thiol compound produced by a number of actinomycetes, and has been suggested to serve both anti-oxidative and detoxifying roles. To investigate the metabolism and the role of mycothiol in Streptomyces coelicolor, the biosynthetic genes (mshA, B, C, and D) were predicted based on sequence homology with the mycobacterial genes and confirmed experimentally. Disruption of the mshA, C, and D genes by PCR targeting mutagenesis resulted in no synthesis of mycothiol, whereas the mshB mutation reduced its level to about 10% of the wild type. The results indicate that the mshA, C, and D genes encode non-redundant biosynthetic enzymes, whereas the enzymatic activity of MshB (acetylase) is shared by at least one other gene product, most likely the mca gene product (amidase).     

Matrix Assisted Ionization Vacuum (MAIV), a New Ionization Method for Biological Materials Analysis Using Mass Spectrometry

The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) for the mass spectrometric analysis of peptides and proteins had a dramatic impact on biological science. We now report that a wide variety of compounds, including peptides, proteins, and protein complexes, are transported directly from a solid-state small molecule matrix to gas-phase ions when placed into the vacuum of a mass spectrometer without the use of high voltage, a laser, or added heat. This ionization process produces ions having charge states similar to ESI, making the method applicable for high performance mass spectrometers designed for atmospheric pressure ionization. We demonstrate highly sensitive ionization using intermediate pressure MALDI and modified ESI sources. This matrix and vacuum assisted soft ionization method is suitable for the direct surface analysis of biological materials, including tissue, via mass spectrometry.The conversion of large and nonvolatile compounds such as proteins into gas-phase ions is of immense fundamental and practical importance. The 2002 Nobel Prize in Chemistry was awarded for the accomplishment of this conversion via electrospray ionization (ESI)¹ (1) and matrix-assisted laser desorption/ionization (MALDI) (2) interfaced with mass spectrometry (MS) to obtain the molecular weights of proteins with high accuracy. These methods employ high voltage or a laser to form gaseous analyte ions from a wide variety of compounds in solution or a solid matrix, respectively.MALDI interfaced with a time-of-flight (TOF) mass spectrometer produces gas-phase analyte ions in vacuum and is the method of choice for the molecular imaging of biological surfaces. Ionization in vacuum provides excellent ion transmission (3), as well as good spatial resolution achieved using a focused laser beam. However, the analysis of protein complexes is very challenging with MALDI, requiring strategies such as first-shot phenomena (4) and chemical crosslinking (5). The necessity of a laser also makes MALDI less soft than ESI and produces background ions, which can hinder the analysis of small molecules (6, 7). MALDI is also of limited utility on high performance mass-to-charge (m/z) analyzers because of mass range issues related to the formation of singly charged ions, which also produce few fragment ions for structural characterization (8).Multiple charged ions produced directly from solution in ESI bring the m/z ratio within the range of high performance mass spectrometers, allowing the analysis of high-mass compounds. These instruments have advanced features for structural characterization, such as ion mobility spectrometry (IMS) for gas-phase separations (9–11), ultra-high mass resolution and mass accuracy (12–14), and advanced fragmentation such as electron transfer dissociation (ETD) (13, 14). However, ESI is limited for surface characterization, requiring approaches such as desorption-ESI (15) and laser ablation ESI (16), ionization methods that produce multiply charged ions but are not compatible with analyses of larger proteins or fragile complexes.A softer ionization approach is needed in order to observe fragile molecules and molecular complexes in living organisms at low levels directly from tissue and cell cultures, without extensive sample preparation, while retaining spatial information. Ideally, this approach would be compatible with mass spectrometers having advanced capabilities to aid structural characterization directly from surfaces. The new ionization method described here, in which molecules are transferred from solid-phase to gas-phase ions through the simple exposure of a material of interest in a suitable matrix to vacuum, is an advance toward this goal and is of fundamental interest.