Cloning, purification, and properties of a phosphotyrosine protein phosphatase from Streptomyces coelicolor A3(2)

We have investigated by restriction fragment analysis genomic abnormalities involving the c-myc gene in DNA isolated from adenomas and hepatocellular carcinomas (HCCs). Adenomas and HCCs were induced by the "resistant hepatocyte" protocol in diethylnitrosamine-initiated male F344 rats. Southern-blot analysis of EcoRI-restricted DNA from normal liver, early and late adenomas, 12 weeks (EAs) and 30 weeks (LAs) after initiation, and HCCs, showed 2 bands of 18 and 3.2 kb hybridizing with c-myc, in all tissues. c-myc amplification occurred in almost all HCCs, and in the majority of EAs and LAs. These results were confirmed by dilution analysis. c-myc amplification was also seen in adenomas and HCCs by Southern analysis with HindIII-restricted DNA, and in HCCs by differential PCR. c-myc mRNA increase occurred in all adenomas and HCCs, but it was higher in the lesions showing gene amplification. Moreover, a 13-kb DNA extraband, hybridizing with c-myc, was found in the HindIII-restricted DNA from HCCs, but not in normal liver and adenomas, and a 7.1-kb extra band was present in EcoRI-digested DNA from one LA. EcoRI-restricted DNA from some adenomas exhibited a decrease in intensity of the 18-kb fragment, and an increase in intensity of the 3.2-kb fragment. No alteration in banding pattern occurred in the beta-actin gene in adenomas. These results provide evidence of amplification and some other rearrangements involving the c-myc gene, in pre-malignant and malignant liver lesions, induced by the RH protocol, and suggest a role of c-myc rearrangement in the progression of adenomas to malignancy.     

Analysis of a Streptomyces coelicolor A3(2) locus containing the nucleoside diphosphate kinase (ndk) and folylpolyglutamate synthetase (folC) genes

Conjugates of oligodeoxyribonucleotide phosphorothioate (ODN-PS) with folic acid, retinoic acid, arachidonic acid, and methoxypoly(ethylene glycol)propionic acid have been synthesized. The procedure involved the initial solid-phase preparation of 5'-amino-functionalized ODN-PS using N-pent-4-enoyl-derived (PNT) nucleoside phosphoramidites followed by conjugation of the oligonucleotide either to the ligand acids, using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide as a coupling reagent, or to their corresponding succinimidyl derivatives. Subsequent exposure of the support to aqueous ammonium hydroxide (28%, 2 h, 55 degrees C) resulted in the release of the fully deprotected ODN conjugates, which were purified by reversed-phase HPLC or by preparative polyacrylamide gel electrophoresis. The identity of the oligonucleotide conjugates was confirmed by MALDI-TOF mass spectral analysis.     

Actinorhodin and undecylprodigiosin production in wild-type and relA mutant strains of Streptomyces coelicolor A3(2) grown in continuous culture

In vitro fertilization and embryo transfer, embryo cryo-preservation, multiple pregnancies, embryo reduction, embryo and gamete donation: each of these questions deserves an articulate ethical and medical discussion. In Italy until this moment we do not have a legislation for the regulation of medically assisted reproduction. One of the most suitable means to monitoring large scale initiatives for medically assisted procreation is through registries that collect standardized information on all treatment cycles performed. In 1992, the Italian national registry on medically assisted procreation was established at the Istituto Superiore di Sanità (ISS, the Italian National Health Institute). The Registry collects data sent voluntarily by centers to the ISS for each cycle of treatment started and for each outcome. The data collected can be used to conduct an adequate statistical-epidemiological analysis and to better evaluate results of specific techniques.     

Cell division gene ftsQ is required for efficient sporulation but not growth and viability in Streptomyces coelicolor A3(2)

We show that the cell division gene ftsQ of Streptomyces coelicolor A3(2) is dispensable for growth and viability but is needed during development for the efficient conversion of aerial filaments into spores. Combined with our previous demonstration that ftsZ of S. coelicolor is not needed for viability, these findings suggest that cell division has been largely co-opted for development in this filamentous bacterium. This makes S. coelicolor an advantageous system for the study of cell division genes.     

Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2)

The sigE gene of Streptomyces coelicolor A3(2) encodes an RNA polymerase sigma factor belonging to the extracytoplasmic function (ECF) subfamily. Constructed sigE deletion and disruption mutants were more sensitive than the parent to muramidases such as hen egg white lysozyme and to the CwlA amidase from Bacillus subtilis. This correlated with an altered muropeptide profile, as determined by reverse-phase high-performance liquid chromatography analysis of lytic digests of purified peptidoglycan. The sigE mutants required high levels of magnesium for normal growth and sporulation, overproducing the antibiotic actinorhodin and forming crenellated colonies in its absence. Together, these data suggest that sigE is required for normal cell wall structure. The role of sigmaE was further investigated by analyzing the expression of hrdD, which is partially sigE dependent. The hrdD gene, which encodes the sigmaHrdD subunit of RNA polymerase, is transcribed from two promoters, hrdDp1 and hrdDp2, both similar to promoters recognized by other ECF sigma factors. The activities of hrdDp1 and hrdDp2 were reduced 20- and 3-fold, respectively, in sigE mutants, although only hrdDp1 was recognized by EsigmaE in vitro. Growth on media deficient in magnesium caused the induction of both hrdD promoters in a sigE-dependent manner.     

Actinorhodin is a chromosomally-determined antibiotic in Streptomyces coelicolar A3(2)

Streptomyces coelicolar A3(2) synthesizes a second antibiotic, in addition to the plasmid-determined methylenomycin A. It was identified, primarily on the evidence of mass spectroscopy of its diethyl ester, as actinorhodin, which has been described previously in other strains. It inhibited most Gram-positive bacteria tested, but only at a comparatively high concentration. Five independent mutations leading to lack of actinorhodin synthesis were located between cysD and strA on the chromosome.     

Genetic mapping and characterization of novel mutations which suppress the effect of a relC mutation on antibiotic production in Streptomyces coelicolor A3(2)

Fifteen patients at the University of Mainz were diagnosed as having T3 or T4 carcinomas of the paranasal sinuses that invaded the orbit. Treatment involved surgical resections of tumor with adjacent periorbita but without enucleation in an attempt to preserve vision and/or cosmesis. Survival rates and especially the rate, time and locations of recurrences were investigated. Analysis of the data revealed that preservation of the eye in these cases with advanced sinus carcinomas with orbital tumor involvement did not seem to worsen prognosis. The individual gain in terms of quality of life through the preservation of vision and cosmesis seems to justify the surgical approach used.     

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or alpha-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2'deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo.     

The relA/spoT-homologous gene in Streptomyces coelicolor encodes both ribosome-dependent (p)ppGpp-synthesizing and -degrading activities

Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.     

Developmental control of the heat-shock stress regulon in Streptomyces coelicolor

In the differentiating eubacterium Streptomyces coelicolor, nutritional imbalances activate a developmental programme which involves the heat-shock stress regulon. In liquid batch cultures, the growth curve could be separated into four components: rapid growth 1 (RG1), transition (T), rapid growth 2 (RG2) and stationary (S). Patterns of gene expression in cultures subjected to heat shock in various phases were recorded on two-dimensional gels and analysed using advanced statistical methods. The responses of all heat-shock proteins (HSPs) were highly dependent upon growth phase, thus demonstrating that the four phases of growth were physiologically distinct. For many HSPs, the level of thermal induction attained were closely related to growth stage-determined levels of synthesis before heat shock, thus supporting the idea that developmental and thermal induction of this stress regulon have common control elements. Cluster analysis identified five groups of HSPs displaying similar kinetics of heat- and developmentally induced synthesis, probably reflecting the influence of major regulatory systems. Methods introduced here to analyse the response of groups of genes to multiple simultaneous stimuli should find broad applications to studies of other prokaryotic and eukaryotic regulons.     

redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD

redD and actII-ORF4, regulatory genes required for synthesis of the antibiotics undecylprodigiosin and actinorhodin by Streptomyces coelicolor A3(2), were transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD. Disruption of hrdD had no effect on antibiotic production, indicating that redD and actII-ORF4 are transcribed in vivo by at least one other RNA polymerase holoenzyme. These data provide the first experimental evidence that HrdD can function as a sigma factor.     

Differential expression of superoxide dismutases containing Ni and Fe/Zn in Streptomyces coelicolor

Streptomyces coelicolor contains two distinct superoxide dismutase (SOD) activities detected on native PAGE. The level of each changed differently depending on growth media and scarcely responded to paraquat, a superoxide-generating agent. The total SOD activity doubled in late exponential phase compared with that in mid-exponential phase and less than double upon treatment with plumbagin, another superoxide-generating agent. The two SODs from S. coelicolor ATCC 10147 (Müller) strain were purified to near homogeneity. SOD1, a tetramer of 13.4-kDa subunits, was found to be a novel type of SOD containing 0.74 mol nickel/mol subunit as determined by atomic absorption spectroscopy. SOD2, a tetramer of 22.2-kDa subunits, was found to contain 0.36 mol iron and 0.26 mol zinc/mol subunit. The N-terminal amino acid sequences of both SODs were determined. SOD2 is similar to manganese-containing superoxide dismutases (MnSODs) and iron-containing superoxide dismutases (FeSODs) from other organisms, whereas SOD1 is less similar to known SODs but still contains a few conserved amino acids. The effects of metals and chelating agents on the expression of these two SODs were examined. The presence of nickel at micromolar concentrations in growth media induced the expression of SOD1 (nickel-containing superoxide dismutase; NiSOD), whereas the expression of SOD2 (iron/zinc-containing superoxide dismutase; FeZnSOD) was repressed. The changes in SOD activities were positively correlated with the amount of each enzyme as determined by immunoblotting, suggesting that metals do not modulate the activity per se but the amount of each protein.     

Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor

This report reviews published information on the clinical pharmacokinetics of antitumour agents in patients with liver dysfunction, associated with primary liver disease or liver metastases. Information was available for anthracyclines and their related compounds, antimetabolites, cyclophosphamide, vinca alkaloids, taxanes and epipodophyllotoxins. Changes in the pharmacokinetic profile or metabolism in patients with mild or severe hepatobiliary dysfunction are described and the relationships between serum levels, parameters employed for measuring hepatic function and toxic or therapeutic effects are examined. Current knowledge of the pharmacokinetics of antineoplastic agents in liver disease is far from complete, mostly obtained in small numbers of non-homogeneous patients often presenting only moderate liver dysfunction, and empirical guidelines for dose assessment are still largely applied in clinical practice. Because of the complex pathophysiological mechanisms of liver insufficiency in cancer patients, there is still doubt whether endogenous markers are useful. Although caution in treating cancer patients with liver insufficiency is compulsory, for most compounds there seems no need to recommend dose reductions for moderate impairment. However, for the tubulin acting agents, vincristine, vinblastine and possibly for paclitaxel and docetaxel, there is strong evidence that dose adjustment is mandatory in order to avoid excessive neutropenia and neurotoxicity.     

Extracellular complementation and the identification of additional genes involved in aerial mycelium formation in Streptomyces coelicolor

Morphogenesis in the bacterium Streptomyces coelicolor involves the formation of a lawn of hair-like aerial hyphae on the colony surface that stands up in the air and differentiates into chains of spores. bld mutants are defective in the formation of this aerial mycelium and grow as smooth, hairless colonies. When certain pairs of bld mutants are grown close to one another on rich sporulation medium, they exhibit extracellular complementation such that one mutant restores aerial mycelium formation to the other. The extracellular complementation relationships of most of the previously isolated bld mutants placed them in a hierarchy of extracellular complementation groups. We have screened for further bld mutants with precautions intended to maximize the discovery of additional genes. Most of the 50 newly isolated mutant strains occupy one of three of the previously described positions in the hierarchy, behaving like bldK, bldC, or bldD mutants. We show that the mutations in some of the strains that behave like bldK are bldK alleles but that others fall in a cluster at a position on the chromosome distinct from that of any known bld gene. We name this locus bldL. By introducing cloned genes into the strains that exhibit bldC or bldD-like extracellular complementation phenotypes, we show that most of these strains are likely to contain mutations in genes other than bldC or bldD. These results indicate that the genetic control of aerial mycelium formation is more complex than previously recognized and support the idea that a high proportion of bld genes are directly or indirectly involved in the production of substances that are exchanged between cells during morphological differentiation.     

Site-directed mutagenesis of the yeast V-ATPase B subunit (Vma2p)

The B subunit of the vacuolar (H+)-ATPase (V-ATPase) has previously been shown to participate in nucleotide binding and to possess significant sequence homology with the alpha subunit of the mitochondrial F-ATPase, which forms the major portion of the noncatalytic nucleotide binding sites and contributes several residues to the catalytic sites of this complex. Based upon the recent x-ray structure of the mitochondrial F1 ATPase (Abrahams, J.P., Leslie, A.G., Lutter, R., and Walker, J.E. (1994) Nature 370,621-628), site-directed mutagenesis of the yeast VMA2 gene has been carried out in a strain containing a deletion of this gene. VMA2 encodes the yeast V-ATPase B subunit (Vma2p). Mutations at two residues postulated to be contributed by Vma2p to the catalytic site (R381S and Y352S) resulted in a complete loss of ATPase activity and proton transport, with the former having a partial effect on V-ATPase assembly. Interestingly, substitution of Phe for Tyr-352 had only minor effects on activity (15-30% inhibition), suggesting the requirement for an aromatic ring at this position. Alteration of Tyr-370, which is postulated to be near the adenine binding pocket at the noncatalytic sites, to Arg, Phe, or Ser caused a 30-50% inhibition of proton transport and ATPase activity, suggesting that an aromatic ring is not essential at this position. Finally, mutagenesis of residues in the region corresponding to the P-loop of the alpha subunit (H180K, H180G, H180D, N181V) also inhibited proton transport and ATPase activity by approximately 30-50%. None of the mutations in either the putative adenine binding pocket nor the P-loop region had any effect on the ability of Vma2p to correctly fold nor on the V-ATPase to correctly assemble. The significance of these results for the structure and function of the nucleotide binding sites on the B subunit is discussed.     

Assembly of the cell division protein FtsZ into ladder-like structures in the aerial hyphae of Streptomyces coelicolor

In the filamentous bacterium Streptomyces coelicolor, the cell division protein FtsZ is required for the conversion of multinucleoidal aerial hyphae into chains of uninucleoidal spores, although it is not essential for viability. Using immunofluorescence microscopy, we have shown that FtsZ assembles into long, regularly spaced, ladder-like arrays in developing aerial hyphae, with an average spacing of about 1.3 microm. Within individual hyphae, ladder formation was relatively synchronous and extended for distances over 100 microm. These ladders were present only transiently, decreasing in intensity as chromosomes separated into distinct nucleoids and disappearing upon the completion of septum formation. Evidence from the overall intensity of immunofluorescence staining suggested that ladder formation was regulated in part at the level of the accumulation and degradation of FtsZ within individual aerial hyphae. Finally, FtsZ ladder formation was under developmental control in that long arrays of FtsZ rings could not be detected in certain so-called white mutants (whiG, whiH and whiB), which are blocked in spore formation. The assembly of FtsZ into ladders represents the earliest known molecular manifestation of the process of spore formation, and its discovery provides insight into the role of whi genes in the conversion of aerial hyphae into chains of spores. We have also described a novel use of a cell wall-staining technique to visualize apical tip growth in vegetatively growing hyphae.     

MCAT is not required for in vitro polyketide synthesis in a minimal actinorhodin polyketide synthase from Streptomyces coelicolor

BACKGROUND: It has been proposed that Streptomyces malonyl CoA: holo acyl carrier protein transacylases (MCATs) provide a link between fatty acid and polyketide biosynthesis. Two recent studies have provided evidence that the presence of MCAT is essential for polyketide synthesis to proceed in reconstituted minimal polyketide synthases (PKSs). In contrast to this, we previously showed that the holo acyl carrier proteins (ACPs) from type II PKSs are capable of catalytic self-malonylation in the presence of malonyl CoA, which suggests that MCAT might not be necessary for polyketide biosynthesis. RESULTS: We reconstituted a homologous actinorhodin (act) type II minimal PKS in vitro. When act holo-ACP is present in limiting concentrations, MCAT is required by the synthase complex in order for polyketide biosynthesis to proceed. When holo-ACP is present in excess, however, efficient polyketide synthesis proceeds without MCAT. The rate of polyketide production increases with holo-ACP concentration, but at low ACP concentration or equimolar AC:KS:CLF (KS, ketosynthase; CLF, chain length determining factor) concentrations this rate is significantly lower than expected, indicating that free holo-ACP is sequestered by the KS/CLF complex. CONCLUSIONS: The rate of polyketide biosynthesis is dictated by the ratio of holo-ACP to KS and CLF, as well as by the total protein concentration. There is no absolute requirement for MCAT in polyketide biosynthesis in vitro, although the role of MCAT during polyketide synthesis in vivo remains an open question. MCAT might be responsible for the rate enhancement of malonyl transfer at very low free holo-ACP concentrations or it could be required to catalyse the transfer of malonyl groups from malonyl CoA to sequestered holo-ACP.