Autoregulatory factors of secondary metabolism and morphogenesis in actinomycetes

The Gram-positive bacterial genus Streptomyces possesses interesting biological aspects, such as the ability to produce a wide variety of secondary metabolites and a mycelial form of growth that culminates in sporulation. A close relationship of secondary metabolism and cell differentiation has been well recognized; secondary metabolism might be a physiological expression of cell differentiation. A variety of diffusible low-molecular-weight chemical substances have been found to function in general as regulatory factors, like "hormones" in eukaryotes, for secondary metabolism and cell differentiation. Among these factors, A-factor has been most extensively studied. This review summarizes recent research on the chemical structures, functions, biosyntheses, and mode of action of these regulatory factors.     

GAP control: regulating the regulators of small GTPases

The small GTPases of the Ras superfamily mediate numerous biological processes through their ability to cycle between an inactive GDP-bound and an active GTP-bound form. Among the key regulators of GTPase cycling are the GTPase-activating proteins (GAPs), which stimulate the weak intrinsic GTP-hydrolysis activity of the GTPases, thereby inactivating them. Despite the abundance of GAPs and the fact that mutations in GAP-encoding genes underlie several human diseases, these proteins have received relatively little attention. Recent studies have addressed the regulatory mechanisms that influence GAP activity. So far, findings suggest that GAP activity is regulated by several mechanisms, including protein-protein interactions, phospholipid interactions, phosphorylation, subcellular translocation and proteolytic degradation.     

Pleiotropic role of VEGF-A in regulating fetal pulmonary mesenchymal cell turnover

Tight regulation of VEGF-A production and signaling is important for the maintenance of lung development and homeostasis. VEGF null mice have provided little insight into the role of VEGF during the later stages of lung morphogenesis. Therefore, we examined the in vitro effects of autocrine and paracrine VEGF-A production and the inhibition of VEGF-A signaling on a Flk-1-negative subset of fetal pulmonary mesenchymal cells (pMC). We hypothesized that VEGF-A receptor signaling regulates turnover of fetal lung mesenchyme in a cell cycle-dependent manner. VEGF receptor blockade with SU-5416 caused cell spreading and decreased proliferation and bcl-2 localization. Nuclear expression of the cell cycle inhibitory protein, p21, was increased with SU-5416 treatment, and p27 was absent. Autocrine VEGF production by pMC resulted in proliferation and p21/p27-dependent contact inhibition. In contrast, exogenous VEGF-A increased cell progression through the cell cycle. Selective activation of Flt by placental growth factor demonstrated the importance of this receptor/kinase in the VEGF-A responsiveness of pMC. The expression and localization of the survival factor bcl-2 was dependent on VEGF. These results provide evidence that VEGF-A plays a critical role in the regulation of fetal pulmonary mesenchymal proliferation, survival, and the subsequent development of normal lung architecture through bcl-2 and p21/p27-dependent cell cycle control.     

Histidine kinases and response regulators in networks

Two-component systems, composed of a histidine kinase (HK) and a response regulator (RR), are the major signal transduction devices in bacteria. Originally it was thought that these two components function as linear, phosphorylation-driven stimulus-response system. Here, we will review how accessory proteins are employed by HKs and RRs to mediate signal integration, scaffolding, interconnection and allosteric regulation, and how these two components are embedded in regulatory networks.     

Coordinated gene networks regulating Arabidopsis plant metabolism in response to various stresses and nutritional cues

The expression pattern of any pair of genes may be negatively correlated, positively correlated, or not correlated at all in response to different stresses and even different progression stages of the stress. This makes it difficult to identify such relationships by classical statistical tools such as the Pearson correlation coefficient. Hence, dedicated bioinformatics approaches that are able to identify groups of cues in which there is a positive or negative expression correlation between pairs or groups of genes are called for. We herein introduce and discuss a bioinformatics approach, termed Gene Coordination, that is devoted to the identification of specific or multiple cues in which there is a positive or negative coordination between pairs of genes and can further incorporate additional coordinated genes to form large coordinated gene networks. We demonstrate the utility of this approach by providing a case study in which we were able to discover distinct expression behavior of the energy-associated gene network in response to distinct biotic and abiotic stresses. This bioinformatics approach is suitable to a broad range of studies that compare treatments versus controls, such as effects of various cues, or expression changes between a mutant and the control wild-type genotype.     

The nitrosophenol system of iron metabolism regulation in actinomycetes

Iron-containing o-nitrosophenol compounds have been isolated from 64 strains of microorganisms of 18 species. The analysis of author's and published data allowed to divide iron-containing microbial compounds into two physiological groups: ferri-ionophores, i. e. compounds participating in active iron transfer, and those immobilizing iron at increase in its content in medium up to 10(-4)-10(-2) M. O-nitrosophenol pigments were found to belong to the latter group. A scheme of participation of o-nitrosophenol compounds in regulation of iron metabolism in actinomycetes has been proposed.     

Hybrid isoprenoid secondary metabolite production in terrestrial and marine actinomycetes

Terpenoids are among the most ubiquitous and diverse secondary metabolites observed in nature. Although actinomycete bacteria are one of the primary sources of microbially derived secondary metabolites, they rarely produce compounds in this biosynthetic class. The terpenoid secondary metabolites that have been discovered from actinomycetes are often in the form of biosynthetic hybrids called hybrid isoprenoids (HIs). HIs include significant structural diversity and biological activity and thus are important targets for natural product discovery. Recent screening of marine actinomycetes has led to the discovery of a new lineage that is enriched in the production of biologically active HI secondary metabolites. These strains represent a promising resource for natural product discovery and provide unique opportunities to study the evolutionary history and ecological functions of an unusual group of secondary metabolites.     

Pleiotropic mutants of Aspergillus nidulans altered in carbon metabolism.

Summary Mutants altered in carbon catabolite regulation have been isolated by selecting for mutants of theareA217 strain capable of using acetamide as the sole nitrogen source in the presence of sucrose. In addition tocreA mutants described previously by Arst and Cove, strains with mutations in two new genes,creB andcreC, have been found. ThecreB andcreC mutants grow poorly on some sole carbon sources and have low levels of some enzymes of carbon catabolism e.g. -galactosidase and D-quinate dehydrogenase. ThecreB andcreC mutants are hypersitive to fluoroacetate, fluoroacetamide and allyl alcohol in the presence of glucose or sucrose but not glycerol; and the enzymes, acetamidase, and alcohol dehydrogenase, are less sensitive to carbon catabolite repression than the wild-type strain. Extracellular protease and -glucosidase enzyme activities are elevated increB andcreC mutants, while L-proline and L-glutamate uptake capacities are lower in both the presence and absence of glucose. Interactions betweencreA, B and C mutations have been investigated in double mutants, and the dominance properties ofcreB andcreC mutants determined. The results indicate that thecreB andcreC genes may have a regulatory role in the control of carbon catabolism.     

Gene probes for the detection of 6-deoxyhexose metabolism in secondary metabolite-producing streptomycetes

DNA probes were designed from the streptomycin production genes strDELM of Streptomyces griseus involved in the biosynthesis of the 6-deoxyhexose (6DOH) dihydrostreptose which could detect the genomic fragments coding for 6DOH formation in other actinomycetes strains. In about 70% of the 43 strains tested at least one signal could be detected with strD-, strE- or strLM-specific probes. Evidence is presented that the hybridizing genes are mostly clustered and probably engaged in the formation of secondary metabolites. Because of the wide-spread use of 6DOH constituents in natural products these probes should allow to detect a vast array of different secondary metabolic gene clusters in actinomycetes.     

Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora

Here we report associations between secondary metabolite production and phylogenetically distinct but closely related marine actinomycete species belonging to the genus Salinispora. The pattern emerged in a study that included global collection sites, and it indicates that secondary metabolite production can be a species-specific, phenotypic trait associated with broadly distributed bacterial populations. Associations between actinomycete phylotype and chemotype revealed an effective, diversity-based approach to natural product discovery that contradicts the conventional wisdom that secondary metabolite production is strain specific. The structural diversity of the metabolites observed, coupled with gene probing and phylogenetic analyses, implicates lateral gene transfer as a source of the biosynthetic genes responsible for compound production. These results conform to a model of selection-driven pathway fixation occurring subsequent to gene acquisition and provide a rare example in which demonstrable physiological traits have been correlated to the fine-scale phylogenetic architecture of an environmental bacterial community.     

Genetic methods and strategies for secondary metabolite yield improvement in actinomycetes

The foundation for any strain improvement program is efficient random chemically-induced mutagenesis coupled with highly reproducible fermentation and product assays. The broad spectrum of spontaneous mutations can be leveraged in some cases by direct selection of mutants with desired traits. Transposons containing outward-reading promoter activity might be used to enhance yields by inducing promoter fusions, disrupting negative regulatory elements, or disrupting genes involved in competing pathways. Transposons might also be used to identify and clone positive regulatory genes. As knowledge of the key elements in the fermentation process and secondary metabolite biosynthesis grows, gene cloning and targeted gene duplication becomes an important tool. Duplication of genes involved in rate limiting steps can be achieved to improve product yields by inserting the desired gene(s) into neutral sites in the chromosome by homologous recombination or by site-specific integration. The probabilities and frequencies of success of the molecular genetic approaches should increase with an increasing knowledge of key factors influencing product yields. This knowledge can be broadened dramatically by a combination of structural and functional genomics, gene disruption analysis and metabolic modeling. Protoplast fusion can be used to recombine beneficial traits from any of the other approaches.     

Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering

Production of secondary metabolites is a process influenced by several physico-chemical factors including nutrient supply, oxygenation, temperature and pH. These factors have been traditionally controlled and optimized in industrial fermentations in order to enhance metabolite production. In addition, traditional mutagenesis programs have been used by the pharmaceutical industry for strain and production yield improvement. In the last years, the development of recombinant DNA technology has provided new tools for approaching yields improvement by means of genetic manipulation of biosynthetic pathways. These efforts are usually focused in redirecting precursor metabolic fluxes, deregulation of biosynthetic pathways and overexpression of specific enzymes involved in metabolic bottlenecks. In addition, efforts have been made for the heterologous expression of biosynthetic gene clusters in other organisms, looking not only for an increase of production levels but also to speed the process by using rapidly growing and easy to manipulate organisms compared to the producing organism. In this review, we will focus on these genetic approaches as applied to bioactive secondary metabolites produced by actinomycetes.