Identification of Streptomyces coelicolor genes involved in regulation of antibiotic synthesis.

To define genetic elements that regulate antibiotic synthesis, we screened for mutations that visibly blocked synthesis of Streptomyces coelicolor's two pigmented antibiotics and found mutant strains in which all four antibiotics were blocked. The responsible mutations defined two loci, absA and absB. Two additional approaches to defining genes have been taken: isolation of cloned genes with a dominant negative effect on antibiotic synthesis and isolation of genes which, in multicopy, can compensate for specific mutational blocks. These genes apparently function in a global regulatory pathway (or network) for control of antibiotic synthesis.     

Asymmetric cross‐regulation between the nitrate‐responsive NarX–NarL and NarQ–NarP two‐component regulatory systems from Escherichia coli K‐12

The NarX–NarL and NarQ–NarP sensor–response regulator pairs control Escherichia coli gene expression in response to nitrate and nitrite. Previous analysis suggests that the Nar two‐component systems form a cross‐regulation network in vivo. Here we report on the kinetics of phosphoryl transfer between different sensor–regulator combinations in vitro. NarX exhibited a noticeable kinetic preference for NarL over NarP, whereas NarQ exhibited a relatively slight kinetic preference for NarL. These findings were substantiated in reactions containing one sensor and both response regulators, or with two sensors and a single response regulator. We isolated 21 NarX mutants with missense substitutions in the cytoplasmic central and transmitter modules. These confer phenotypes that reflect defects in phospho‐NarL dephosphorylation. Five of these mutants, all with substitutions in the transmitter DHp domain, also exhibited NarP‐blind phenotypes. Phosphoryl transfer assays in vitro confirmed that these NarX mutants have defects in catalysing NarP phosphorylation. By contrast, the corresponding NarQ mutants conferred phenotypes indicating comparable interactions with both NarP and NarL. Our overall results reveal asymmetry in the Nar cross‐regulation network, such that NarQ interacts similarly with both response regulators, whereas NarX interacts preferentially with NarL.     

Analysis and validation of the pho regulon in the tacrolimus-producer strain Streptomyces tsukubaensis: differences with the model organism Streptomyces coelicolor

Applied Microbiology and Biotechnology - Inorganic and organic phosphate controls both primary and secondary metabolism in Streptomyces genus. Metabolism regulation by phosphate in Streptomyces...     

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.     

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).     

Interaction of SCO2127 with BldKB and its possible connection to carbon catabolite regulation of morphological differentiation in Streptomyces coelicolor

In Streptomyces coelicolor, the sco2127 gene is located upstream of the gene encoding for glucose kinase. This region restores sensitivity to carbon catabolite repression (CCR) of Streptomyces peucetius var. caesius mutants, resistant to 2-deoxyglucose (Dog(R)). In order to search for the possible mechanisms behind this effect, sco2127 was overexpressed and purified for protein-protein interaction studies. SCO2127 was detected during the late growth phase of S. coelicolor grown in a complex media supplemented with 100 mM glucose. Pull-down assays using crude extracts from S. coelicolor grown in the same media, followed by far-western blotting, allowed detection of two proteins bound to SCO2127. The proteins were identified by MALDI-TOF mass spectrometry as SCO5113 and SCO2582. SCO5113 (BldKB) is a lipoprotein ABC-type permease (～66 kDa) involved in mycelium differentiation by allowing the transport of the morphogenic oligopeptide Bld261. SCO2582, is a putative membrane metalloendopeptidase (～44 kDa) of unknown function. In agreement with the possible role of SCO2127 in mycelium differentiation, delayed aerial mycelium septation and sporulation was observed when S. coelicolor A3(2) was grown in the presence of elevated glucose concentrations (100 mM), an effect not seen in a Δ-sco2127 mutant derived from it. We speculate that SCO2127 might represent a key factor in CCR of mycelium differentiation by interacting with BldKB.     

Identification of a DNA sequence associated with plasmid integration in Streptomyces coelicolor A3(2)

Summary We identified a DNA element of length about 1 kb that is present in two copies in the chromosome of Streptomyces coelicolor A3(2) and is also present on the plasmid SCP1 which has been carefully defined genetically, but never isolated as extrachromosomal DNA.A copy of the element is close (within 5 kb) of a gene coding for an extracellular agarase in the chromosome of S. coelicolor A3(2) and in an NF strain, in which SCP1 has integrated into the chromosome, the agarase gene has been deleted. The element has properties reminiscent of Insertion Sequences in Escherichia coli, but it is not yet know if it can transpose.     

Identification of genes involved in siderophore transport in Streptomyces coelicolor A3(2)

The potential iron siderophore transporter genes have been determined from the genome sequence of Streptomyces coelicolor A3(2). One of these gene clusters, cdtABC, was disrupted and characterized to determine its role in the uptake of the siderophores produced by S. coelicolor. Resistance to the siderophore-like antibiotics, salmycin and albomycin, was tested in the parent and cdtABC mutant, showing that the parent, but not the mutant, was sensitive to salmycin, while both were resistant to albomycin. Ferrioxamine competition assays against salmycin suggest that the uptake of salmycin is via a ferrioxamine transport system. However, Fe-55 ferrioxamine B uptake experiments did not reveal any difference between the parent and mutant. This suggests that CdtABC specifically transports salmycin, while ferrioxamine uptake maybe substituted by another transport system.     

A two‐component regulatory system VfmIH modulates multiple virulence traits in Dickeya zeae

Bacterial pathogen Dickeya zeae strain EC1 produces antibiotics‐like phytotoxins called zeamines, which are major virulence determinants encoded by the zms gene cluster. In this study, we identified a zeamine‐deficient mutant with a Tn5 insertion in a gene designated as vfmI encoding a two‐component system (TCS) sensor histidine kinase (HK), which is accompanied by vfmH encoding a response regulator (RR) at the same genetic locus. Domain analysis shows this TCS is analogous to the VfmIH of D. dadantii, with typical characteristics of sensor HK and RR, respectively, and sharing the same operon. Deletion of either vfmI or vfmH resulted in decreased production of zeamines and cell wall degrading enzymes (CWDEs), and alleviated virulence on rice seeds and potato tubers. In D. dadantii 3937, VfmH was shown to bind to the promoters of vfmA and vfmE, while in D. zeae EC1, VfmH could bind to the promoters of vfmA, vfmE and vfmF. RNA‐seq analysis of strain EC1 and its vfmH mutant also showed that the TCS positively regulated a range of virulence genes, including zms, T1SS, T2SS, T3SS, T6SS, flagellar and CWDE genes.     

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.