Eunjung Song

NdgR, an IclR-like regulator involved in amino-acid-dependent growth, quorum sensing, and antibiotic production in Streptomyces coelicolor

NdgR (regulator for nitrogen source-dependent growth and antibiotic production), an IclR-like regulator, has been initially identified as a binding protein to the promoters of doxorubicin biosynthetic genes in Streptomcyes peucetius by DNA affinity capture assay method. NdgR is well conserved throughout the Streptomcyes species and many other bacteria such as Mycobacteria and Corynebacteria. In Streptomcyes coelicolor, ndgR deletion mutant showed slow cell growth and defects in differentiation and enhances the production of actinorhodin (ACT) in minimal media containing certain amino acids where wild-type strain could not produce ACT. Although deletion mutant of ndgR showed different antibiotic production in minimal media containing Leu or Gln, it only showed reduced mRNA expression levels of the genes involved in leucine metabolism. Neither NdgR-dependent expression of glnA nor direct binding of NdgR protein to glnA, glnII, and glnR promoters was observed. However, ScbR, which is governed by NdgR shown by gel mobility shift assay, binds to promoter of glnR, suggesting indirect regulation of glutamine metabolism by NdgR. NdgR protein binds to intergenic region of ndgR–leuC, and scbR–scbA involved in γ-butyrolactone. Two-dimensional gel analysis has shown a global effect of ndgR deletion in protein expression, including up-regulated proteins involved in ACT synthesis and down-regulation of chaperones such as GroEL, GroES, and DnaK. These results suggest a global regulatory role for NdgR in amino acid metabolisms, quorum sensing, morphological changes, antibiotic production, and expression of chaperonines in S. coelicolor.

Mass spectrometric screening of transcriptional regulators involved in antibiotic biosynthesis in Streptomyces coelicolor A3(2)

DNA-affinity capture assay (DACA) coupled with liquid chromatography–tandem mass spectrometry analysis was applied to identify the transcriptional regulators involved in the biosynthesis of actinorhodin (Act) and undecylprodigiosin (Red) in Streptomyces coelicolor. The aim of this analysis was to determine the specific transcriptional regulators binding to the promoter region of actII-ORF4 or redD. The results of the DACA, as the first screening tool, identified eight proteins, including AdpA, as candidate regulators binding to those promoter regions. To show the direct physical relationship between the regulators and promoters, we purified four regulators over-expressed in soluble form in Escherichia coli and subjected these to an electrophoretic mobility shift assay (EMSA). The results of the EMSA appeared to be compatible with the DACA results for those regulators. A null mutant was also constructed for one of these regulators, SCO6008, which showed early Red production and quite delayed Act production in R5− medium. These observations suggest that DACA can be widely used to find new regulators and that the regulator SCO6008 may be involved in antibiotic production through its binding to the redD promoter.

The ROK Family Regulator Rok7B7 Pleiotropically Affects Xylose Utilization, Carbon Catabolite Repression, and Antibiotic Production in Streptomyces coelicolor

Members of the ROK family of proteins are mostly transcriptional regulators and kinases that generally relate to the control of primary metabolism, whereby its member glucose kinase acts as the central control protein in carbon control in Streptomyces. Here, we show that deletion of SCO6008 (rok7B7) strongly affects carbon catabolite repression (CCR), growth, and antibiotic production in Streptomyces coelicolor. Deletion of SCO7543 also affected antibiotic production, while no major changes were observed after deletion of the rok family genes SCO0794, SCO1060, SCO2846, SCO6566, or SCO6600. Global expression profiling of the rok7B7 mutant by proteomics and microarray analysis revealed strong upregulation of the xylose transporter operon xylFGH, which lies immediately downstream of rok7B7, consistent with the improved growth and delayed development of the mutant on xylose. The enhanced CCR, which was especially obvious on rich or xylose-containing media, correlated with elevated expression of glucose kinase and of the glucose transporter GlcP. In liquid-grown cultures, expression of the biosynthetic enzymes for production of prodigionines, siderophores, and calcium-dependent antibiotic (CDA) was enhanced in the mutant, and overproduction of prodigionines was corroborated by matrix-assisted laser desorption ionization-time-of-flight analysis. These data present Rok7B7 as a pleiotropic regulator of growth, CCR, and antibiotic production in Streptomyces.

Effects of Different Sensitization Events on HLA Alloimmunization in Solid Organ Transplantation Patients

OBJECTIVE HLA alloimmunization is caused by various sensitization events, such as transfusion, pregnancy, or organ transplantation. However, the effects of a particular sensitization event on HLA alloimmunization have not been well studied in parallel using an identical test method. We evaluated how different sensitization events affect the panel-reactive antibody (PRA) status in solid organ transplantation candidates. METHODS PRA identification tests were performed on 674 patients (354 males and 320 females) using Luminex assay kits (LIFECODES, Gen-Probe, Stamford, CT, United States). PRA-positive rates (HLA-A, B, or DR antibodies of median fluorescence intensity [MFI] values of ≥1000) and antibody strengths in PRA-positive cases were analyzed according to the different sensitization events and gender. RESULTS PRA (class I and/or II)-positive rates were significantly higher in patients with transfusion (33.0%; P = .001), pregnancy (71.4%; P < .001), or transplantation events (76.9%; P < .001) than in controls without any identifiable sensitization events (5.6%). Transplantation had the strongest immunization effect, especially for class II HLA antigens. Female compared with male patients (60.3% vs 34.2%; P < .001) and retransplantation compared with first transplantation candidates of kidney transplantation (80.2% vs 41.1%; P < .001) showed a significantly higher PRA-positive rate. Retransplantation candidates (MFI 14,164) showed significantly stronger antibody strength than first transplantation candidates (MFI 5456) and those with single sensitization events of transfusion (MFI 4185) or pregnancy (MFI 5548; P < .001 for each). CONCLUSION Solid organ transplantation appears to have the strongest HLA alloimmunization effect followed by pregnancy and transfusion, especially for class II HLA antigens.

Bioconjugation of L-3,4-Dihydroxyphenylalanine Containing Protein with a Polysaccharide

We describe the simple bioconjugation strategy in combination of periodate chemistry and unnatural amino acid incorporation. The residue specific incorporation of 3,4-dihydroxy-l-phenylalanine can alter the properties of protein to conjugate into the polymers. The homogeneously modified protein will yield quinone residues that are covalently conjugated to nucleophilic groups of the amino polysaccharide. This novel approach holds great promise for widespread use to prepare protein conjugates and synthetic biology applications.

Three-dimensional midcourse guidance using neural networks for interception of ballistic targets

A suboptimal midcourse guidance law is obtained for interception of free-fall targets in the three-dimensional (3D) space. Neural networks are used to approximate the optimal feedback strategy suitable for real-time implementation. The fact that the optimal trajectory in the 3D space does not deviate much from a vertical plane justifies the use of the two-dimensional (2D) neural network method previously studied. To regulate the lateral errors in the missile motion produced by the prediction error of the intercept point, the method of feedback linearization is employed. Computer simulations confirm the superiority of the proposed scheme over linear quadratic regulator guidance and proportional navigation guidance as well as its approximating capability of the optimal trajectory in the 3D space.

A novel function of Streptomyces integration host factor (sIHF) in the control of antibiotic production and sporulation in Streptomyces coelicolor

Bacterial integration host factors (IHFs) play important roles in site-specific recombination, DNA replication, transcription, genome organization and bacterial pathogenesis. In Streptomyces coelicolor, there are three putative IHFs: SCO1480, SCO2950 and SCO5556. SCO1480 or Streptomyces IHF (sIHF) was previously identified as a transcription factor that binds to the promoter region of redD, the pathway-specific regulatory gene for the undecylprodigiosin biosynthetic gene cluster. Here we show that production of the pigmented antibiotics actinorhodin and undecylprodigiosin is strongly enhanced in sihf null mutants, while sporulation was strongly inhibited, with an on average 25% increase in spore size. Furthermore, the sihf mutant spores showed strongly reduced viability, with high sensitivity to heat and live/dead staining revealing a high proportion of empty spores, while enhanced expression of sIHF increased viability. This suggests a major role for sIHF in controlling viability, perhaps via the control of DNA replication and/or segregation. Proteomic analysis of the sihf null mutant identified several differentially expressed transcriptional regulators, indicating that sIHF may have an extensive response regulon. These data surprisingly reveal that a basic architectural element conserved in many actinobacteria such as mycobacteria, corynebacteria, streptomycetes and rhodococci may act as a global regulator of secondary metabolism and cell development.

Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor

Abstractγ-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. γ-Butyrolactone receptors are considered important regulatory proteins, and various γ-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters. The SCO0608 protein sequences are not similar to those of any known γ-butyrolactone binding proteins in Streptomyces such as ScbR from S. coelicolor or ArpA from Streptomyces griseus. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of γ-butyrolactone receptor in Streptomyces, and this SCO0608 was named ScbR-like γ-butyrolactone binding regulator (SlbR) in S. coelicolor.

Frequent false-positive reactions in pronase-treated T-cell flow cytometric cross-match tests.

OBJECTIVE Pretransplantation cross-match (XM) is essential in organ transplantation. The flow cytometric XM (FCXM) is the most sensitive cell-based XM technique. Pronase treatment is used to improve the sensitivity and specificity of the B-cell FCXM. Thus, pronase-treated (PT) T cells are tested in a single tube T-cell/B-cell technique. Observing discrepancies between PT and pronase-nontreated (PN) T- FCXM results, we investigated their incidence, clinical significance, and possible causes. METHODS We tested 226 serum samples from 167 kidney transplantation candidates or posttransplantation follow-up patients using PT and PN T-FCXM in parallel using 3-color and 2-color immunofluorescence staining, respectively. We reviewed panel-reactive antibody (PRA) and donor-specific antibody (DSA) status as well as HLA data and clinical outcomes. RESULTS The T-FCXM positive rate was significantly higher among PT versus PN tests (24.3% vs 11.1%; P < .001). Less than half of the PT-positive cases were positive in the PN test (45.5%; 25/55). Discrepancies were observed in 30 cases (13.3%), all of which gave PT(+)/PN(-) results. Our findings suggested that PT(+)/PN(-) results might arise from non-HLA antibodies. Class I DSA-positive rate (6.3% vs 2.2%; P = .45) and antibody-mediated rejection rate (0% vs 16.3%; P = .32) were not different between PT(+)/PN(-) and PT(-)/PN(-) groups. Moreover, 2 cases of PT(+)/PN(-) were observed among HLA-A, B, DR-identical donor-recipient pairs. CONCLUSION Pronase treatment is prone to give false-positive reactions in T-FCXM test probably due to the participation of non-HLA antibodies including autoantibodies. Patients might be inappropriately excluded from receiving organs. In laboratories using PT single tube T/B FCXM, caution is needed to avoid false-positive reporting of results.

Finding new pathway-specific regulators by clustering method using threshold standard deviation based on DNA chip data of Streptomyces coelicolor

In order to identify the regulators involved in antibiotic production or time-specific cellular events, the messenger ribonucleic acid (mRNA) expression data of the two gene clusters, actinorhodin (ACT) and undecylprodigiosin (RED) biosynthetic genes, were clustered with known mRNA expression data of regulators from S. coelicolor using a filtering method based on standard deviation and clustering analysis. The result identified five regulators including two well-known regulators namely, SCO3579 (WlbA) and SCO6722 (SsgD). Using overexpression and deletion of the regulator genes, we were able to identify two regulators, i.e., SCO0608 and SCO6808, playing roles as repressors in antibiotics production and sporulation. This approach can be easily applied to mapping out new regulators related to any interesting target gene clusters showing characteristic expression patterns. The result can also be used to provide insightful information on the selection rules among a large number of regulators.