Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters.Bacterial natural products are excellent lead compounds for drug discovery and have played major roles in the development of pharmaceutical agents in nearly all therapeutic areas (1, 7, 9). Unfortunately, the rate of discovery of new bacterial natural products has decreased, due in part to frequent rediscovery of known compounds (7). An enormous and currently inaccessible reservoir of new natural products is located in the biosynthetic pathways found in the genomes of uncultivated bacteria (18). Heterologous expression of these biosynthetic gene clusters represents a powerful tool for discovering new natural products (20, 21). Herein, we demonstrate that the deltaproteobacterium Myxococcus xanthus is an effective host for heterologous expression of aromatic polyketide biosynthetic pathways. This work expands the scope of polyketide biosynthetic pathways which can be heterologously expressed in M. xanthus and suggests that M. xanthus may be a suitable general host for heterologous expression.Molecular phylogenetic studies have shown that bacterial diversity is enormous, and the vast majority of the diversity is found in uncultivated bacterial species (18). Estimates suggest that 99% of bacteria from the environment are uncultivatable using standard techniques (2, 15, 16). Culture-independent analyses of metagenomic DNA libraries from soil and marine environments indicate that there is a wealth of natural product diversity in these uncultivated strains. For example, analysis of a soil metagenome for a highly conserved region of polyketide synthase genes showed that none of the sequences found were present in the known public databases (5). Polyketide synthases are key enzymes responsible for the production of the polyketide family of natural products in proteobacteria, actinobacteria, and “low-G+C Gram-positive bacteria” (4, 12, 19). Polyketide natural products have been developed into antibiotic, anticancer, and immunosuppressant clinical agents (1, 6, 8). Based on these observations, metagenomic DNA libraries are expected to possess a large number of new polyketide biosynthetic pathways, representing substantial new chemical diversity for drug discovery.Heterologous expression of biosynthetic pathways can play a major role in interrogating metagenomic DNA libraries for new polyketide biosynthetic pathways. Heterologous production of polyketides in hosts such as Streptomyces coelicolor and Streptomyces lividans is an important tool in the identification and characterization of these pathways (6, 8, 17). Results from these studies have shown that Streptomyces strains are good hosts for heterologous production of many polyketides, particularly those from actinomycetes. However, Streptomyces strains have proved to be poor hosts for expression of deltaproteobacterial polyketide biosynthetic pathways, such as those in myxobacteria (10, 17). As polyketide biosynthetic pathways in metagenomic DNA libraries contain both actinomycete- and deltaproteobacterium-derived pathways, a heterologous expression host competent to express pathways of both origins is needed.We examined the ability of the deltaproteobacterium M. xanthus to act as a general heterologous expression host. M. xanthus is a predatory bacterium that undergoes multicellular development in response to nutrient starvation. During development, M. xanthus is known to be an effective host for the heterologous expression of the deltaproteobacterium-derived epothilone D biosynthetic pathway and has been used for the production of epothilone D for clinical trials (17). M. xanthus has also been shown to be an excellent host for the heterologous expression of several other myxobacterial metabolites, including myxothiazol and myxochromide S (3, 11, 22). We demonstrate that M. xanthus can also heterologously express the Streptomyces rimosus oxytetracycline biosynthetic pathway, producing oxytetracycline. This is the first example of a polyketide from a nonmyxobacterial species heterologously expressed in a myxobacterium.To generate an M. xanthus strain capable of heterologously expressing oxytetracycline, the Streptomyces rimosus oxytetracycline biosynthetic pathway (Fig. ​(Fig.1)1) was inserted via homologous recombination into the asgE locus of M. xanthus. The asgE locus of M. xanthus was amplified and inserted into the BglII site of pET28b (Novagen) to produce pMRH02. The oligonucleotides used for the amplification of the asgE locus were 5′-GACGAGATCTGTTGGAAGGTCGGCAACTGG-3′ and 5′-CTTAAGATCTTCCGTGAAGTACTGGCGCAC-3′. The asgE locus provides a chromosomal region for single-crossover homologous recombination into the M. xanthus chromosome. The 32-kb oxytetracycline pathway in S. rimosus was excised from pYT264 (24) and cloned into the EcoRI site of pMRH02 to produce pMRH08. M. xanthus DK1622 was electroporated under standard conditions (13) with pMRH08 to provide an M. xanthus ΔasgE Kan^r mutant. Positive selection for the chromosomal insertion was maintained throughout all experiments by use of kanamycin supplementation (40 μg/ml). This large genomic insertion significantly increased the doubling time for the strain (doubling time, ≈10 h).Open in a separate windowFIG. 1.Oxytetracycline biosynthetic pathway. (A) Enzymatic pathway responsible for formation of oxytetracycline. (B) Oxytetracycline biosynthesis gene cluster from S. rimosus.Oxytetracycline was heterologously produced in M. xanthus under standard rich medium culture conditions and detected in culture broth by liquid chromatography-mass spectrometry (LC-MS). A liquid culture of the mutant strain containing the oxytetracycline gene cluster was cultured for 10 days at 33°C in CTTYE (1.0% Casitone, 0.5% yeast extract, 10.0 mM Tris-HCl, 1.0 mM KH₂PO₄, and 8.0 mM MgSO₄; 100 ml). Acetone (10%, vol/vol) was added to the culture and vigorously mixed. The resulting mixture was extracted with 3 volumes of ethyl acetate to remove the organic soluble materials, including oxytetracycline. The organic extracts were concentrated in vacuo and resuspended in methanol (100 μl). LC-MS analyses were carried out using an Altima Hypersil C₁₈ column (3-μm particle size; 150 mm by 2.1 mm) with a linear gradient of water-acetonitrile (5 to 95%) with 0.05% formic acid over 90 min (0.20 ml/min), followed by positive-ion electrospray ionization (5,500 V) and analysis with a Shimadzu 2010A single quadrupole mass spectrometer. LC-MS analysis indicated that oxytetracycline was present in the fermentation broth (Fig. ​(Fig.2).2). The titer of oxytetracycline was determined to be approximately 10 mg per liter of fermentation broth. Quantification was performed in triplicate by LC-MS analysis using a standard curve generated from commercial oxytetracycline. Negative controls of M. xanthus DK1622 cultures processed under identical conditions did not contain detectable levels of oxytetracycline.Open in a separate windowFIG. 2.LC-MS ion extraction analysis of the molecular ion [M+H]⁺ of standard and culture extracts. (A) Oxytetracycline standard. (B) M. xanthus ΔasgE Kan^r mutant containing the oxytetracycline biosynthetic pathway. (C) Wild-type M. xanthus DK1622.These data indicate that M. xanthus can heterologously express the oxytetracycline polyketide synthase biosynthetic pathway in S. rimosus. Several factors affect the successful heterologous production of polyketide synthase pathways, including codon usage, mRNA stability, functionality of regulatory elements, and the presence of all necessary starter and extender units (14). As codon usages between M. xanthus and the genus Streptomyces are very similar and myxobacteria are known to produce polyketide products requiring a wide diversity of starter and extender units, neither codon usage nor starter and extender unit availability was considered likely to affect the ability of M. xanthus to heterologously express streptomycete biosynthetic pathways. As Streptomyces strains do not appear to be effective at heterologous expression of myxobacterial biosynthetic pathways, we were concerned that Myxococcus and Streptomyces strains may possess substantially different regulatory elements. Our data indicate that the regulatory elements present in streptomycete-derived biosynthetic pathways are sufficient to enable expression of the biosynthetic genes in M. xanthus. Further work exploring the regulatory elements present in myxobacterial polyketide biosynthetic gene clusters is needed to evaluate this hypothesis.This study demonstrates that M. xanthus can heterologously express streptomycete-derived polyketide biosynthetic pathways in addition to myxobacterial polyketide biosynthetic pathways. The observed titer of 10 mg/liter of culture broth is comparable to titers reported for the heterologous expression of myxobacterial polyketide biosynthetic pathways in myxobacteria (11) and streptomycete-derived polyketide biosynthetic pathways in Streptomyces (14, 23) and is sufficient for characterization of the polyketide product. Pseudomonas putida, which has a more favorable growth profile, has been shown to be a good host for heterologous expression of myxobacterial polyketide biosynthetic pathways, with product titers in the range of 0.6 to 40 mg/liter of culture broth (14, 21, 23). The observed breadth of polyketide pathways accessible and the titers of the polyketide products produced make M. xanthus an attractive potential candidate for a “universal” host for facilitating heterologous expression of polyketide biosynthetic pathways derived from environmental samples of metagenomic DNA.     

Bioremediation of soils contaminated with the herbicide 2-sec-butyl-4,6-dinitrophenol (dinoseb).

A novel soil treatment method for achieving the removal of dinoseb (2-sec-butyl-4,6-dinitrophenol) from contaminated soils was investigated. One soil contained dinoseb as the major contaminant, although several other hazardous compounds were also present. A second soil was highly contaminated with dinoseb. Dinoseb was not degraded in these soils under the aerobic conditions at each site. Pretreatment of the soils by the addition of a starchy potato-processing by-product and flooding with phosphate buffer stimulated the consumption of oxygen and nitrate from the soils, thereby lowering the redox potential and creating anaerobic conditions. Anaerobiosis (Eh less than -200 mV) promoted the establishment of an anaerobic microbial consortium that degraded dinoseb completely, without the formation of the polymerization products seen under aerobic or microaerophilic conditions. When dinoseb was present at low concentrations in a chronically contaminated soil, the natural microflora was capable of establishing anaerobic conditions and degrading dinoseb as a result of starch degradation. Inoculation of this soil with an aerobic starch-degrading microorganism and then an acclimated, anaerobic, dinoseb-degrading consortium did not improve dinoseb degradation. In a second acutely contaminated soil, these inoculations improved dinoseb degradation rates over those of uninoculated controls.     

Essential control of an endothelial cell ISOC by the spectrin membrane skeleton

Mechanism(s) underlying activation of store-operated Ca2+ entry currents, ISOC, remain incompletely understood. F-actin configuration is an important determinant of channel function, although the nature of interaction between the cytoskeleton and ISOC channels is unknown. We examined whether the spectrin membrane skeleton couples Ca2+ store depletion to Ca2+ entry. Thapsigargin activated an endothelial cell ISOC (-45 pA at -80 mV) that reversed at +40 mV, was inwardly rectifying when Ca2+ was the charge carrier, and was inhibited by La3+ (50 microM). Disruption of the spectrin-protein 4.1 interaction at residues A207-V445 of betaSpIISigma1 decreased the thapsigargin-induced global cytosolic Ca2+ response by 50% and selectively abolished the endothelial cell ISOC, without altering activation of a nonselective current through cyclic nucleotide-gated channels. In contrast, disruption of the spectrin-actin interaction at residues A47-K186 of betaSpIISigma1 did not decrease the thapsigargin-induced global cytosolic Ca2+ response or inhibit ISOC. Results indicate that the spectrin-protein 4.1 interaction selectively controls ISOC, indicating that physical coupling between calcium release and calcium entry is reliant upon the spectrin membrane skeleton.     

Kinetics of the sodium-dependent glutamine transporter in human intestinal cell confluent monolayers.

The intestinal epithelium metabolism of glutamine plays a critical role in inter-organ nitrogen flow. Although it is known that glutamine is the primary oxidative energy source and nucleotide precursor in intestinal cells, the luminal uptake of glutamine by the apical surface of enterocytes is poorly understood. In this study we have uncovered the sodium-dependent transporter system responsible for L-glutamine uptake by the apical membrane of a human intestinal epithelial cell line. The sodium-dependent Michaelis constant (Km) = 247 +/- 45 microM glutamine, and Jmax = 4.44 +/- 0.65 x 10(-9) mole min-1(mg protein)-1 (37 degrees C). Glutamine shares the transporter with alanine, as demonstrated by unlabeled glutamine inhibition of [3H]alanine uptake kinetics with a purely competitive-type inhibition pattern, and glutamine inhibition Ki = 205 +/- 18 microM by Dixon analysis. The inhibition pattern for a series of amino acid analogs indicated that this intestinal apical membrane sodium-dependent transporter for glutamine is distinct from any other transport system found in membranes of non-intestinal cells.     

Frequent beneficial mutations during single-colony serial transfer of Streptococcus pneumoniae

The appearance of new mutations within a population provides the raw material for evolution. The consistent decline in fitness observed in classical mutation accumulation studies has provided support for the long-held view that deleterious mutations are more common than beneficial mutations. Here we present results of a study using a mutation accumulation design with the bacterium Streptococcus pneumoniae in which the fitness of the derived populations increased. This rise in fitness was associated specifically with adaptation to survival during brief stationary phase periods between single-colony population bottlenecks. To understand better the population dynamics behind this unanticipated adaptation, we developed a maximum likelihood model describing the processes of mutation and stationary-phase selection in the context of frequent population bottlenecks. Using this model, we estimate that the rate of beneficial mutations may be as high as 4.8×10(-4) events per genome for each time interval corresponding to the pneumococcal generation time. This rate is several orders of magnitude higher than earlier estimates of beneficial mutation rates in bacteria but supports recent results obtained through the propagation of small populations of Escherichia coli. Our findings indicate that beneficial mutations may be relatively frequent in bacteria and suggest that in S. pneumoniae, which develops natural competence for transformation, a steady supply of such mutations may be available for sampling by recombination.     

ERK is involved in the reorganization of somatosensory cortical maps in adult rats submitted to hindlimb unloading

Sensorimotor restriction by a 14-day period of hindlimb unloading (HU) in the adult rat induces a reorganization of topographic maps and receptive fields. However, the underlying mechanisms are still unclear. Interest was turned towards a possible implication of intracellular MAPK signaling pathway since Extracellular-signal-Regulated Kinase 1/2 (ERK1/2) is known to play a significant role in the control of synaptic plasticity. In order to better understand the mechanisms underlying cortical plasticity in adult rats submitted to a sensorimotor restriction, we analyzed the time-course of ERK1/2 activation by immunoblot and of cortical reorganization by electrophysiological recordings, on rats submitted to hindlimb unloading over four weeks. Immunohistochemistry analysis provided evidence that ERK1/2 phosphorylation was increased in layer III neurons of the somatosensory cortex. This increase was transient, and parallel to the changes in hindpaw cortical map area (layer IV). By contrast, receptive fields were progressively enlarged from 7 to 28 days of hindlimb unloading. To determine whether ERK1/2 was involved in cortical remapping, we administered a specific ERK1/2 inhibitor (PD-98059) through osmotic mini-pump in rats hindlimb unloaded for 14 days. Results demonstrate that focal inhibition of ERK1/2 pathway prevents cortical reorganization, but had no effect on receptive fields. These results suggest that ERK1/2 plays a role in the induction of cortical plasticity during hindlimb unloading.     

Reduction of laccase type 1 copper by 3,4-dihydroxyphenylalanine and other catechol derivatives

3,4-Dihydroxyphenylalanine (DOPA) is not a preferred substrate of Rhus vernicifera laccase, as rate constants for the anaerobic reduction of the type 1 cupric atom by L-DOPA (6.3 X 10(1) M-1 s-1), D-DOPA (2.6 X 10(1) M-1 s-1), and L-DOPA methyl ester (2.6 X 10(1) M-1 s-1) are considerably smaller than k1 (catechol) (7 X 10(2) M-1 s-1) and rate constants characteristic of numerous other nonphysiological organic substrates (25 degrees C, pH 7.0, I = 0.5 M). The reactions of DOPA derivatives with laccase are unique, however, in that a two-term rate law pertains: kobsd = k0 + k1[phenol]; k0(L-DOPA) = 7 X 10(-2) s-1. The reactivities of other catechol derivatives (pyrogallol, gallic acid, and methyl gallate) with laccase type 1 copper were also examined.     

Body mass effects of a physical activity and healthy food intervention in middle schools

Objective: To evaluate the effects of a 2‐year middle school physical activity and healthy food intervention, including an environmental and computer‐tailored component on BMI and BMI z‐score in boys and girls. Research Methods and Procedures: A random sample of 15 schools with seventh and eighth graders was randomly assigned to three conditions: an intervention with parental support group, an intervention‐alone group, and a control group. Weight and height were measured at the beginning and end of each school year to assess BMI and BMI z‐score. A physical activity and healthy food program was implemented over 2 school years. Results: In girls, BMI and BMI z‐score increased significantly less in the intervention with parental support group compared with the control group (p < 0.05) or the intervention‐alone group (p = 0.05). In boys, no significant positive intervention effects were found. Discussion: This was the first study evaluating the effectiveness of an intervention combining environmental changes with personal computer‐tailored feedback on BMI and BMI z‐score in middle school children. After 2 school years, BMI and BMI z‐score changed in a more positive direction in girls as a result of the intervention with parental support.     

Increased complexity of radiation-induced chromosome aberrations consistent with a mechanism of sequential formation

Complex chromosome aberrations (any exchange involving three or more breaks in two or more chromosomes) are effectively induced in peripheral blood lymphocytes (PBL) after exposure to low doses (mostly single particles) of densely ionising high-linear energy transfer (LET) alpha-particle radiation. The complexity, when observed by multiplex fluorescence in situ hybridisation (m-FISH), shows that commonly four but up to eight different chromosomes can be involved in each rearrangement. Given the territorial organisation of chromosomes in interphase and that only a very small fraction of the nucleus is irradiated by each alpha-particle traversal, the aim of this study is to address how aberrations of such complexity can be formed. To do this, we applied theoretical "cycle" analyses using m-FISH paint detail of PBL in their first cell division after exposure to high-LET alpha-particles. In brief, "cycle" analysis deconstructs the aberration "observed" by m-FISH to make predictions as to how it could have been formed in interphase. We propose from this that individual high-LET alpha-particle-induced complex aberrations may be formed by the misrepair of damaged chromatin in single physical "sites" within the nucleus, where each "site" is consistent with an "area" corresponding to the interface of two to three different chromosome territories. Limited migration of damaged chromatin is "allowed" within this "area". Complex aberrations of increased size, reflecting the path of alpha-particle nuclear intersection, are formed through the sequential linking of these individual sites by the involvement of common chromosomes.     

Antimicrobial action of nisin against Salmonella typhimurium lipopolysaccharide mutants.

The antimicrobial activity of nisin against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT2 was investigated. Nisin sensitivity was associated with the extent of saccharide deletions from the outer membrane core oligosaccharide. The results indicated that the core oligosaccharide in lipopolysaccharide plays a role in nisin sensitivity.