David A. Hodgson

Primary metabolism and its control in streptomycetes: a most unusual group of bacteria.

Streptomycetes are Gram-positive bacteria with a unique capacity for the production of a multitude of varied and complex secondary metabolites. They also have a complex life cycle including differentiation into at least three distinct cell types. Whilst much attention has been paid to the pathways and regulation of secondary metabolism, less has been paid to the pathways and the regulation of primary metabolism, which supplies the precursors. With the imminent completion of the total genome sequence of Streptomyces coelicolor A3(2), we need to understand the pathways of primary metabolism if we are to understand the role of newly discovered genes. This review is written as a contribution to supplying these wants. Streptomycetes inhabit soil, which, because of the high numbers of microbial competitors, is an oligotrophic environment. Soil nutrient levels reflect the fact that plant-derived material is the main nutrient input; i.e. it is carbon-rich and nitrogen- and phosphate-poor. Control of streptomycete primary metabolism reflects the nutrient availability. The variety and multiplicity of carbohydrate catabolic pathways reflects the variety and multiplicity of carbohydrates in the soil. This multiplicity of pathways has led to investment by streptomycetes in pathway-specific and global regulatory networks such as glucose repression. The mechanism of glucose repression is clearly different from that in other bacteria. Streptomycetes feed by secreting complexes of extracellular enzymes that break down plant cell walls to release nutrients. The induction of these enzyme complexes is often coordinated by inducers that bear no structural relation to the substrate or product of any particular enzyme in the complex; e.g. a product of xylan breakdown may induce cellulase production. Control of amino acid catabolism reflects the relative absence of nitrogen catabolites in soil. The cognate amino acid induces about half of the catabolic pathways and half are constitutive. There are reduced instances of global carbon and nitrogen catabolite control of amino acid catabolism, which again presumably reflects the relative rarity of the catabolites. There are few examples of feedback repression of amino acid biosynthesis. Again this is taken as a reflection of the oligotrophic nature of the streptomycete ecological niche. As amino acids are not present in the environment, streptomycetes have rarely invested in feedback repression. Exceptions to this generalization are the arginine and branched-chain amino acid pathways and some parts of the aromatic amino acid pathways which have regulatory systems similar to Escherichia coli and Bacillus subtilis and other copiotrophic bacteria.

The dynamic architecture of the metabolic switch in Streptomyces coelicolor

BackgroundDuring the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.ResultsSurprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.ConclusionsOur study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.

Glucose Repression of Carbon Source Uptake and Metabolism in Streptomyces coelicolor A3(2) and its Perturbation in Mutants Resistant to 2-Deoxyglucose

SUMMARY: A newly devised method to obtain diffuse growth of Streptomyces coelicolor A3(2) in liquid minimal medium was used to study glucose repression. Although diauxic growth was not obtained, glucose repression of uptake of 14C-labelled carbon sources was demonstrated. Active, arabinose-induced, arabinose transport was repressed at the level of transcription by glucose. Of two glycerol-inducible glycerol transport systems, one was glucose-inhibited but not repressed (and operated by facilitated diffusion), whilst the other (an active transport system) was glucose-repressed. Active transport systems for galactose and fructose which did not require induction by their respective sugars were both inhibited by glucose. Galactose- and fructose-metabolizing enzymes were inducible by the respective sugars, but only in the absence of glucose. This was because glucose both inhibited galactose and fructose transport and repressed the metabolic enzymes concerned. Constitutive active glucose uptake was also demonstrated in arabinose-grown cells. Mutants that grew on arabinose or glycerol in the presence of 2-deoxy-glucose were glucose-derepressed for both soluble carbon source utilization and extracellular agarose. Three glucose-derepressed mutants were studied in detail. One of these could not utilize glucose (and probably lacks glucose kinase), whilst the other two could utilize glucose to differing degrees.

Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes.

This is the first report of generalized transduction in the Gram‐positive, food‐borne pathogen Listeria monocytogenes. Bacteriophages were isolated from the environment and from lysogens, or were obtained from other laboratories. Of the 59 bacteriophages tested, 34 proved to be capable of transduction. We exploited the ability of L. monocytogenes to grow at room temperature and isolated bacteriophages that were incapable of growth at 37°C. Transductions at this temperature therefore eliminated transductant killing and lysogeny, as did inclusion of citrate and the use of a low multiplicity of infection. Transducing bacteriophages were found for each of the well‐characterized L. monocytogenes strains: EGD, 10403, Mack (serotype1/2a), L028 (serotype 1/2c), Scott A (serotype 4b) and strains from the Jalisco and Halifax, Nova Scotia outbreaks (serotype 4b). P35 (φLMUP35) is a particularly useful generalized transducing bacteriophage with a wide host range (75% of all serotype 1/2 strains tested). Its disadvantages are that it is small and transduction is relatively infrequent. U153(φCU‐SI153/95) is larger than P35 and transduction frequency increased 100‐fold, but it has a very narrow host range. We demonstrated interstrain transduction and used transduction to test linkage between transposon insertions and mutant phenotypes in a variety of strains.

Light‐induced carotenogenesis in Myxococcus xanthus: light‐dependent membrane sequestration of ECF sigma factor CarQ by anti‐sigma factor CarR

Light‐induced carotenogenesis in Myxococcus xanthus is under the control of the carQRS operon. CarQ, a proposed extracytoplasmic (ECF) RNA polymerase sigma factor, is required for expression of the operon and the carC gene that encodes phytoene dehydrogenase. CarR, an inner membrane protein in Escherichia coli, is essential for carQRS promoter inactivation in the dark. CarS is required for the light‐dependent expression of the promoter of the carB gene cluster that encodes the rest of the structural genes for carotenogenesis. Regulation of carQRS is dependent on the stoichiometry of CarQ and CarR. Increasing the copy number of carQ over carR led to constitutive carotenogenesis, as did loss of translational coupling between carQ and carR. The severity of the constitutive phenotype depended on the distance between the uncoupled genes. When expressed in M. xanthus, a CarR:β‐galactosidase fusion protein disappeared in the light. We propose that anti‐sigma factor CarR sequesters CarQ to the membrane in the dark, but, in the light, loss of CarR leads to release of the sigma factor.

A response-regulator-like activator of antibiotic synthesis from Streptomyces coelicolor A3(2) with an amino-terminal domain that lacks a phosphorylation pocket.

In Streptomyces coelicolor A3(2), bldA mutants that lack the tRNA for the rare leucine codon UUA fail to make the red undecylprodigiosin antibiotic complex. To find out why, red-pigmented while bald (Pwb) derivatives of a bldA mutant were isolated. Using a cloning strategy that allowed for (and demonstrated) dominance of the mutations, they were localized to the red gene cluster. By using insert-mediated integration of a phi C31 phage-based vector, one of the Pwb mutations was more precisely located between red structural genes to a segment of approximately 1 kb about 4 kb from the known pathway-specific regulatory gene redD. The segment contained most of an ORF (redZ) encoding a protein (RedZ) with end-to-end similarity to response regulators of diverse function from a variety of bacteria. Remarkably, in RedZ hydrophobic residues replace nearly all of the charged residues that usually make up the phosphorylation pocket present in typical response regulators, including the aspartic acid residue that is normally phosphorylated by a cognate sensory protein kinase. A single TTA codon in redZ provided a potential explanation for the bldA-dependence of undecylprodigiosin synthesis. This codon was unchanged in three Pwb mutants, but further analysis of one of the mutants revealed a potential up-promoter mutation. It seems possible that a combination of low-level natural translation of the UUA codon by a charged non-cognate tRNA, coupled with increased transcription of redZ in the Pwb mutant allows the accumulation of a threshold level of the RedD protein.

Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis

We report the occurrence of an isomerase with a putative (βα)8‐barrel structure involved in both histidine and trypto‐phan biosynthesis in Streptomyces coelicolor A3(2) and Mycobacterium tuberculosis HR37Rv. Deletion of a hisA homologue (SCO2050) putatively encoding N ′‐[(5′‐phosphoribosyl)‐formimino]‐5 amino‐imidazole‐4‐carboxamide ribonucleotide isomerase from the chromosome of S. coelicolor A3(2) generated a double auxotrophic mutant for histidine and tryptophan. The bifunctional gene SCO2050 and its orthologue Rv1603 from M. tuberculosis complemented both hisA and trpF mutants of Escherichia coli. Expression of the E. coli trpF gene in the S. coelicolor mutant only complemented the tryptophan auxo‐trophy, and the hisA gene only complemented the histidine auxotrophy. The discovery of this enzyme, which has a broad‐substrate specificity, has implications for the evolution of metabolic pathways and may prove to be important for understanding the evolution of the (βα)8‐barrels.

Light‐induced carotenogenesis in Myxococcus xanthus: DNA sequence analysis of the carR region

The carR region encodes a light‐inducible promoter, a negative regulator of the promoter and a trans‐acting activator that controls the light‐inducible Myxococcus xanthus carotenoid biosynthesis regulon. DNA sequence analysis revealed, downstream of the promoter, three translationally coupled genes, carQ, carR and carS. Sequencing of mutations demonstrated that carR encoded the negative regulator and was an integral membrane protein. Mutant construction and sequencing revealed that carS was the trans‐acting activator and that carQ was a positive regulator of the promoter. Neither gene encodes proteins with known sequence‐specific DNA‐binding motifs. The sequence of the light‐inducible promoter region, identified by primer extension analysis, showed similarity to the consensus sequence of the Escherichia coli stress response (‘heat‐shock’) promoters.

Light-induced carotenogenesis in Myxococcus xanthus: functional characterization of the ECF sigma factor CarQ and antisigma factor CarR

Illumination of dark‐grown Myxococcus xanthus with blue light leads to the induction of carotenoid synthesis. Central to this response is the activation of the light‐inducible promoter, PcarQRS, and the transcription of three downstream genes, carQ, carR and carS. Sequence analysis predicted that CarQ is a member of the ECF (extracytoplasmic function) subfamily of RNA polymerase sigma factors, and that CarR is an inner membrane protein. Genetic analysis strongly implied that CarR is an antisigma factor that sequesters CarQ in a transcriptionally inactive complex. Using in vitro transcription run‐off assays, we present biochemical evidence that CarQ functions as a bacterial sigma factor and is responsible for transcription initiation at PcarQRS. Similar experiments using the crtI promoter failed to implicate CarQ in direct transcription of the crtI gene. Experiments using the yeast two‐hybrid system demonstrated a protein–protein interaction betweefn CarQ and CarR, providing evidence of a CarQ–CarR complex. The yeast two‐hybrid system data also indicated that CarR is capable of oligomerization. Fractionation of M. xanthus membranes with the detergent sarkosyl showed that CarR was associated with the inner membrane. Furthermore, CarR was found to be unstable in illuminated stationary phase cells, providing a possible mechanism by which the CarR–CarQ complex is disrupted.

Metabolic modeling and analysis of the metabolic switch in Streptomyces coelicolor

BackgroundThe transition from exponential to stationary phase in Streptomyces coelicolor is accompanied by a major metabolic switch and results in a strong activation of secondary metabolism. Here we have explored the underlying reorganization of the metabolome by combining computational predictions based on constraint-based modeling and detailed transcriptomics time course observations.ResultsWe reconstructed the stoichiometric matrix of S. coelicolor, including the major antibiotic biosynthesis pathways, and performed flux balance analysis to predict flux changes that occur when the cell switches from biomass to antibiotic production. We defined the model input based on observed fermenter culture data and used a dynamically varying objective function to represent the metabolic switch. The predicted fluxes of many genes show highly significant correlation to the time series of the corresponding gene expression data. Individual mispredictions identify novel links between antibiotic production and primary metabolism.ConclusionOur results show the usefulness of constraint-based modeling for providing a detailed interpretation of time course gene expression data.