Socio-Behavioural Barriers to Viral Suppression in the Older Adult Population in Rural South Africa.

South Africa has the largest share of people living with HIV in the world and this population is ageing. The social context in which people seek HIV care is often ignored. Apart from clinical interventions, socio-behavioural factors impact successful HIV care outcomes for older adults living with HIV. We use cross-sectional data linked with demographic household surveillance data, consisting of HIV positive adults aged above 40, to identify socio-behavioural predictors of a detectable viral load. Older adults were more likely to have a detectable viral load if they did not disclose their HIV positive status to close family members (aOR 2.56, 95% CI 1.89-3.46), resided in the poorest households (aOR 1.98, 95% CI 1.23-3.18), or were not taking medications other than ART (aOR 1.83, 95% CI 1.02-1.99) likely to have a detectable. Clinical interventions in HIV care must be supported by understanding the socio-behavioural barriers that occur outside the health facility. The importance of community health care workers in bridging this gap may offer more optimum outcomes for older adults ageing with HIV.

Broadening the ecology of fear: non-lethal effects arise from diverse responses to predation and parasitism.

Research on the 'ecology of fear' posits that defensive prey responses to avoid predation can cause non-lethal effects across ecological scales. Parasites also elicit defensive responses in hosts with associated non-lethal effects, which raises the longstanding, yet unresolved question of how non-lethal effects of parasites compare with those of predators. We developed a framework for systematically answering this question for all types of predator-prey and host-parasite systems. Our framework reveals likely differences in non-lethal effects not only between predators and parasites, but also between different types of predators and parasites. Trait responses should be strongest towards predators, parasitoids and parasitic castrators, but more numerous and perhaps more frequent for parasites than for predators. In a case study of larval amphibians, whose trait responses to both predators and parasites have been relatively well studied, existing data indicate that individuals generally respond more strongly and proactively to short-term predation risks than to parasitism. Apart from studies using amphibians, there have been few direct comparisons of responses to predation and parasitism, and none have incorporated responses to micropredators, parasitoids or parasitic castrators, or examined their long-term consequences. Addressing these and other data gaps highlighted by our framework can advance the field towards understanding how non-lethal effects impact prey/host population dynamics and shape food webs that contain multiple predator and parasite species.

Comparative squamation of the lateral line canal pores in sharks.

The current study collected the first quantitative data on lateral line pore squamation patterns in sharks and assessed whether divergent squamation patterns are similar to experimental models that cause reduction in boundary layer turbulence. In addition, the hypothesis that divergent orientation angles are exclusively found in fast-swimming shark species was tested. The posterior lateral line and supraorbital lateral line pore squamation of the fast-swimming pelagic shortfin mako shark Isurus oxyrinchus and the slow-swimming epi-benthic spiny dogfish shark Squalus acanthias was examined. Pore scale morphology and pore coverage were qualitatively analysed and compared. In addition, pore squamation orientation patterns were quantified for four regions along the posterior lateral line and compared for both species. Isurus oxyrinchus possessed consistent pore scale coverage among sampled regions and had a divergent squamation pattern with multiple scale rows directed dorsally and ventrally away from the anterior margin of the pore with an average divergent angle of 13° for the first row of scales. Squalus acanthias possessed variable amounts of scale coverage among the sampled regions and had a divergent squamation pattern with multiple scale rows directed ventrally away from the anterior margin of the pore with an average angle of 19° for the first row of scales. Overall, the squamation pattern measured in I. oxyrinchus fell within the parameters used in the fluid flow analysis, which suggests that this pattern may reduce boundary layer turbulence and affect lateral line sensitivity. The exclusively ventral oriented scale pattern seen in S. acanthias possessed a high degree of divergence but the pattern did not match that of the fluid flow models. Given current knowledge, it is unclear how this would affect boundary layer flow. By studying the relationship between squamation patterns and the lateral line, new insights are provided into sensory biology that warrant future investigation due to the implications for the ecology, morphology and sensory evolution of sharks.

Infection deflection: hosts control parasite location with behaviour to improve tolerance.

Anti-parasite behaviour can reduce parasitic infections, but little is known about how such behaviours affect infection location within the host's body and whether parasite distribution ultimately affects tolerance of infection. To assess these questions, we exposed both anaesthetized (no behaviour) and non-anaesthetized Hyla femoralis tadpoles to plagiorchiid cercariae (larval trematodes), and quantified resistance, tolerance (relationship between mass change and infection intensity) and encystment location. Non-anaesthetized tadpoles had significantly more infections in their tail region than anaesthetized tadpoles, which had the majority of their infections in the head. This pattern indicates that parasites preferred to infect the head, but that hosts shunted infections to the tail when possible. Furthermore, there was a significant effect of encystment location on tolerance, with head-infected tadpoles having poorer tolerance to infection than tail-infected tadpoles. Variance partitioning suggests that, among infected tadpoles, behaviour contributed more to tolerance than resistance. These results suggest that, in addition to using behaviour to resist parasites, H. femoralis tadpoles also use behaviour to enhance infection tolerance by deflecting infections posteriorly, away from their vital sensory organs. These findings highlight the need to assess how widespread and important behaviour is to the tolerance of infections.

[Radiological comparison between two procedures for ventral spondylodesis: autologous iliac crest bone graft vs bovine bone graft]. / Radiologischer Vergleich zweier Verfahren zur ventralen Spondylodese: Autologer trikortikaler Beckenkammspan vs. boviner Spongiosablock.

BACKGROUND: The objective of this study was the radiological evaluation of osseous integration of autologous iliac crest graft and bovine bone graft after spondylodesis based on a standardized score. MATERIAL AND METHODS: Spondylodesis was performed on 18 sheep, divided into 2 groups, 1 with an autologous iliac crest graft and the other with a bovine bone graft. Computed tomography was performed 12 and 24 weeks postoperatively. The osseous integration was assessed by the Tübinger Score. RESULTS: The evaluation of the CT scans demonstrated a significantly better osseous integration of the autologous iliac crest graft compared to the bovine bone graft. CONCLUSIONS: Based on our results, the bovine bone graft as a transplant for spondylodesis is inadvisable.

Purification and structure elucidation of the by-product of new regulator of antibiotic production and differentiation of Streptomyces.

Streptomyces globisporus 1912, a producer of the antitumor antibiotic landomycin E, forms the new low-molecular signaling molecule N-methylphenylalanyl-dehydrobutyrine diketopiperazine (BDD) and its complex and unstable by-product which restore, like the A-factor in Streptomyces griseus 773, landomycin E and streptomycin biosynthesis, and sporulation of the defective mutants S. globisporus 1912-B2 and S. griseus 1439, respectively. Here, we report the purification and structure elucidation of two compounds with R(f)0.8 by HPLC, LC/MS and 1HMR analysis. These compounds have m/z 338 and 384, accordingly, and each of them is presented by two stereoisomers containing BDD in their structure. A hypothesis explaining the composition and regulatory properties of these unstable compounds is presented.

Co-occurring diagnoses among FMR1 premutation allele carriers.

Following the discovery of two disorders associated with premutation alleles of the fragile X mental retardation gene (FMR1), primary ovarian insufficiency [fragile X-associated primary ovarian insufficiency (FXPOI)] and a tremor/ataxia syndrome [fragile X-associated tremor/ataxia disorder (FXTAS)], numerous studies have examined other potential co-morbid conditions, including neuropsychological deficits. Here, the frequency of self-reported diagnoses obtained through medical history interviews from FMR1 premutation carriers and non-carriers aged 18-50 were analyzed. Study subjects included 537 women, 334 of whom carry the premutation and 151 men, 37 of whom carry the premutation. Men with the premutation did not report any medical conditions at higher rates compared with non-carriers, controlling for age, ethnicity/race, and household income. Women with the premutation reported mental health disorders [i.e. attention deficit hyperactivity disorder (ADHD), anxiety, depression] significantly more often than non-carriers. However, after adjusting for covariates, these increased rates were not statistically significant. Additional follow-up analyses examined the consequence of ovarian dysfunction as a cause of co-occurring conditions. Women with an indication of ovarian insufficiency (i.e. irregular cycles) reported higher rates of thyroid problems and depression/anxiety. Because only women, not men, reported these conditions more often, the relationship between FXPOI and hormone irregularities in women should be explored for a potential link with the increase in the reported medical conditions.

Properties of lanK-based regulatory circuit involved in landomycin biosynthesis in Streptomyces cyanogenus S136.

LanK is TetR-like regulatory protein recently shown to regulate the export and glycosylation of landomycins in Streptomyces cyanogenus S136. Here, several properties of the lanK-mediated regulation were deciphered. LanK seems to function as oligomer as evident from experiments in vitro. In vivo, it is able to recognize various landomycins with altered aglycon structure and the minimal concentration of landomycin A sensed by LanK lies in low nanomolar range. Coexpression studies showed that the positive regulatory gene lanI upregulates lanK-dependent lan genes once the negative LanK-regulation is cancelled. Gene lanK can be useful for the construction of biosensor strains for sensitive and specific identification of producers of landomycin-like molecules with long glycosidic chains.

Effects of temperature and viscosity on miracidial and cercarial movement of Schistosoma mansoni: ramifications for disease transmission.

Parasites with complex life cycles can be susceptible to temperature shifts associated with seasonal changes, especially as free-living larvae that depend on a fixed energy reserve to survive outside the host. The life cycle of Schistosoma, a trematode genus containing some species that cause human schistosomiasis, has free-living, aquatic miracidial and cercarial larval stages that swim using cilia or a forked tail, respectively. The small size of these swimmers (150-350 µm) dictates that their propulsion is dominated by viscous forces. Given that viscosity inhibits the swimming ability of small organisms and is inversely correlated with temperature, changes in temperature should affect the ability of free-living larval stages to swim and locate a host. By recording miracidial and cercarial movement of Schistosoma mansoni using a high-speed camera and manipulating temperature and viscosity independently, we assessed the role each factor plays in the swimming mechanics of the parasite. We found a positive effect of temperature and a negative effect of viscosity on miracidial and cercarial speed. Reynolds numbers, which describe the ratio of inertial to viscous forces exerted on an aquatic organism, were <1 across treatments. Q10 values were <2 when comparing viscosity treatments at 20 °C and 30 °C, further supporting the influence of viscosity on miracidial and cercarial speed. Given that both larval stages have limited energy reserves and infection takes considerable energy, successful transmission depends on both speed and lifespan. We coupled our speed data with mortality measurements across temperatures and discovered that the theoretical maximum distance travelled increased with temperature and decreased with viscosity for both larval stages. Thus, our results suggest that S. mansoni transmission is high during warm times of the year, partly due to improved swimming performance of the free-living larval stages, and that increases in temperature variation associated with climate change might further increase transmission.

A Novel Nanoconjugate of Landomycin A with C60 Fullerene for Cancer Targeted Therapy: In Vitro Studies.

INTRODUCTION: Landomycins are a subgroup of angucycline antibiotics that are produced by Streptomyces bacteria and possess strong antineoplastic potential. Literature data suggest that enhancement of the therapeutic activity of this drug may be achieved by means of creating specific drug delivery systems. Here we propose to adopt C60 fullerene as flexible and stable nanocarrier for landomycin delivery into tumor cells. METHODS: The methods of molecular modelling, dynamic light scattering and Fourier transform infrared spectroscopy were used to study the assembly of C60 fullerene and the anticancer drug Landomycin A (LA) in aqueous solution. Cytotoxic activity of this nanocomplex was studied in vitro towards two cancer cell lines in comparison to human mesenchymal stem cells (hMSCs) using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test and a live/dead assay. The morphology of the cells incubated with fullerene-drug nanoparticles and their uptake into target cells were studied by scanning electron microscopy and fluorescence light microscopy. RESULTS: The viability of primary cells (hMSCs, as a model for healthy cells) and cancer cell lines (human osteosarcoma cells, MG-63, and mouse mammary cells, 4T1, as models for cancer cells) was studied after incubation with water-soluble C60 fullerenes, LA and the mixture C60 + LA. The C60 + LA nanocomplex in contrast to LA alone showed higher toxicity towards cancer cells and lower toxicity towards normal cells, whereas the water-soluble C60 fullerenes at the same concentration were not toxic for the cells. CONCLUSIONS: The obtained physico-chemical data indicate a complexation between the two compounds, leading to the formation of a C60 + LA nanocomposite. It was concluded that immobilization of LA on C60 fullerene enhances selectivity of action of this anticancer drug in vitro, indicating on possibility of further preclinical studies of novel C60 + LA nanocomposites on animal tumor models.

Reduced memory B cells in patients with hyper IgE syndrome.

Dominant-negative mutations in STAT-3 have recently been found in the majority of patients with sporadic or autosomal-dominant hyper IgE syndrome (HIES). Since STAT-3 plays a role in B cell development and differentiation, we analyzed memory B cells in 20 patients with HIES, 17 of which had STAT-3 mutations. All but four patients had reduced non-switched and/or class-switched memory B cells. No reduction in these B cell populations was found in 16 atopic dermatitis patients with IgE levels above 1000 KU/L. There was no correlation between the reduction of memory B cells and the ability to produce specific antibodies. Moreover, there was no correlation between the percentage of memory B cells and the infection history. Analysis of memory B cells can be useful in distinguishing patients with suspected HIES from patients with atopic disease, but probably fails to identify patients who are at high risk of infection.

Anti-Mullerian hormone indicates early ovarian decline in fragile X mental retardation (FMR1) premutation carriers: a preliminary study.

BACKGROUND: Women who carry the fragile X mental retardation (FMR1) premutation are at risk for fragile X-associated primary ovarian insufficiency. Past studies have shown that carriers who are still cycling have increased levels FSH compared with non-carriers. As anti-Mullerian hormone (AMH) has been shown as an excellent marker of ovarian decline, we examined AMH levels among premutation carriers to characterize their ovarian function. METHODS: We determined the level of FSH and AMH in serum samples collected during early follicular phase from women who carried longer FMR1 repeat alleles (defined as >or=70 repeats, n = 40) and those with shorter repeat alleles (<70 repeats, n = 75), identified by DNA analysis. Comparisons were made stratified by age and carrier status. RESULTS: For all age groups, AMH levels were significantly lower among longer repeat allele carriers compared to shorter repeat allele carriers (P = 0.002, 0.006 and 0.020 for women ages 18-30, 31-40 and 41-50 years, respectively). In contrast, increased FSH indicative of early ovarian decline was only evident for longer repeat allele carriers aged 31-40 years (P = 0.089, 0.001 and 0.261 for women ages 18-30, 31-40 and 41-50 years, respectively). CONCLUSIONS: These preliminary data suggest that AMH levels indicate early ovarian decline among women with longer FMR1 repeat alleles; moreover, AMH appears to be a better marker than FSH in identifying this early decline.

Surprising production of a new urdamycin derivative by S. fradiae Delta urdQ/R.

A strain (S. fradiae Delta urdQ/R) with mutations in urdQ and urdR encoding a dTDP-hexose-3,4-dehydratase and a dTDP-hexose-4-ketoreductase, respectively, produces a new urdamycin analogue (urdamycin X) with changes in the polyketide structure. The structure of urdamycin X has been elucidated by NMR spectroscopy. Urdamycin X was not detectable, even in small amounts, in either S. fradiae Delta urdQ, in S. fradiae DeltaurdR or in S. fradiae A0, a mutant lacking all glycosyltransferase genes. Complementation of S. fradiae Delta urdQ/R restored urdamycin A production indicating that the mutations did not cause any polar effect.

Transgenes sustain epigeal insect biodiversity in diversified vegetable farm systems.

Many ecological studies have focused on the effects of transgenes in field crops, but few have considered multiple transgenes in diversified vegetable systems. We compared the epigeal, or soil surface-dwelling, communities of Coleoptera and Formicidae between transgenic and isoline vegetable systems consisting of sweet corn, potato, and acorn squash, with transgenic cultivars expressing Cry1(A)b, Cry3, or viral coat proteins. Vegetables were grown in replicated split plots over 2 yr with integrated pest management (IPM) standards defining insecticide use patterns. More than 77.6% of 11,925 insects from 1,512 pitfall traps were identified to species, and activity density was used to compare dominance distribution, species richness, and community composition. Measures of epigeal biodiversity were always equal in transgenic vegetables, which required fewer insecticide applications than their near isolines. There were no differences in species richness between transgenic and isoline treatments at the farm system and individual crop level. Dominance distributions were also similar between transgenic and isoline farming systems. Crop type, and not genotype, had a significant influence on Carabidae and Staphylinidae community composition in the first year, but there were no treatment effects in the second year, possibly because of homogenizing effects of crop rotations. Communities were more influenced by crop type, and possibly crop rotation, than by genotype. The heterogeneity of crops and rotations in diversified vegetable farms seems to aid in preserving epigeal biodiversity, which may be supplemented by reductions in insecticide use associated with transgenic cultivars.

Recurrent activating mutations of CD28 in peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of mature T-cell neoplasms with a poor prognosis. Recently, mutations in TET2 and other epigenetic modifiers as well as RHOA have been identified in these diseases, particularly in angioimmunoblastic T-cell lymphoma (AITL). CD28 is the major co-stimulatory receptor in T cells which, upon binding ligand, induces sustained T-cell proliferation and cytokine production when combined with T-cell receptor stimulation. We have identified recurrent mutations in CD28 in PTCLs. Two residues-D124 and T195-were recurrently mutated in 11.3% of cases of AITL and in one case of PTCL, not otherwise specified (PTCL-NOS). Surface plasmon resonance analysis of mutations at these residues with predicted differential partner interactions showed increased affinity for ligand CD86 (residue D124) and increased affinity for intracellular adaptor proteins GRB2 and GADS/GRAP2 (residue T195). Molecular modeling studies on each of these mutations suggested how these mutants result in increased affinities. We found increased transcription of the CD28-responsive genes CD226 and TNFA in cells expressing the T195P mutant in response to CD3 and CD86 co-stimulation and increased downstream activation of NF-κB by both D124V and T195P mutants, suggesting a potential therapeutic target in CD28-mutated PTCLs.

Iterative type II polyketide synthases, cyclases and ketoreductases exhibit context-dependent behavior in the biosynthesis of linear and angular decapolyketides.

BACKGROUND: Iterative type II polyketide synthases (PKSs) produce polyketide chains of variable but defined length from a specific starter unit and a number of extender units. They also specify the initial regiospecific folding and cyclization pattern of nascent polyketides either through the action of a cyclase (CYC) subunit or through the combined action of site-specific ketoreductase (KR) and CYC subunits. Additional CYCs and other modifications may be necessary to produce linear aromatic polyketides. The principles of the assembly of the linear aromatic polyketides, several of which are medically important, are well understood, but it is not clear whether the assembly of the angular aromatic (angucyclic) polyketides follows the same rules. RESULTS: We performed an in vivo evaluation of the subunits of the PKS responsible for the production of the angucyclic polyketide jadomycin (jad), in comparison with their counterparts from the daunorubicin (dps) and tetracenomycin (tcm) PKSs which produce linear aromatic polyketides. No matter which minimal PKS was used to produce the initial polyketide chain, the JadD and DpsF CYCs produced the same two polyketides, in the same ratio; neither product was angularly fused. The set of jadABCED PKS plus putative jadl CYC genes behaved similarly. Furthermore, no angular polyketides were isolated when the entire set of jad PKS enzymes and Jadl or the jad minimal PKS, Jadl and the TcmN CYC were present. The DpsE KR was able to reduce decaketides but not octaketides; in contrast, the KRs from the jad PKS (JadE) or the actinorhodin PKS (ActIII) could reduce octaketide chains, giving three distinct products. CONCLUSIONS: It appears that the biosynthesis of angucyclic polyketides cannot be simply accomplished by expressing the known PKS subunits from artificial gene cassettes under the control of a non-native promoter. The characteristic structure of the angucycline ring system may arise from a kinked precursor during later cyclization reactions involving additional, but so far unknown, components of the extended decaketide PKS. Our results also suggest that some KRs have a minimal chain length requirement and that CYC enzymes may act aberrantly as first-ring aromatases that are unable to perform all of the sequential cyclization steps. Both of these characteristics may limit the widespread application of CYC or KR enzymes in the synthesis of novel polyketides.

Elucidation of the function of two glycosyltransferase genes (lanGT1 and lanGT4) involved in landomycin biosynthesis and generation of new oligosaccharide antibiotics.

BACKGROUND: The genetic engineering of antibiotic-producing Streptomyces strains is an approach that became a successful methodology in developing new natural polyketide derivatives. Glycosyltransferases are important biosynthetic enzymes that link sugar moieties to aglycones, which often derive from polyketides. Biological activity is frequently generated along with this process. Here we report the use of glycosyltransferase genes isolated from the landomycin biosynthetic gene cluster to create hybrid landomycin/urdamycin oligosaccharide antibiotics. RESULTS: Production of several novel urdamycin derivatives by a mutant of Streptomyces fradiae Tü2717 has been achieved in a combinatorial biosynthetic approach using glycosyltransferase genes from the landomycin producer Streptomyces cyanogenus S136. For the generation of gene cassettes useful for combinatorial biosynthesis experiments new vectors named pMUNI, pMUNII and pMUNIII were constructed. These vectors facilitate the construction of gene combinations taking advantage of the compatible MunI and EcoRI restriction sites. CONCLUSIONS: The high-yielding production of novel oligosaccharide antibiotics using glycosyltransferase gene cassettes generated in a very convenient way proves that glycosyltransferases can be flexible towards the alcohol substrate. In addition, our results indicate that LanGT1 from S. cyanogenus S136 is a D-olivosyltransferase, whereas LanGT4 is a L-rhodinosyltransferase.

Oxidative cleavage of premithramycin B is one of the last steps in the biosynthesis of the antitumor drug mithramycin.

BACKGROUND: Mithramycin is a member of the clinically important aureolic acid group of antitumor drugs that interact with GC-rich regions of DNA nonintercalatively. These drugs contain a chromophore aglycon that is derived from condensation of ten acetate units (catalyzed by a type II polyketide synthase). The aglycones are glycosylated at two positions with different chain length deoxyoligosaccharides, which are essential for the antitumor activity. During the early stages of mithramycin biosynthesis, tetracyclic intermediates of the tetracycline-type occur, which must be converted at later stages into the tricyclic glycosylated molecule, presumably through oxidative breakage of the fourth ring. RESULTS: Two intermediates in the mithramycin biosynthetic pathway, 4-demethyl-premithramycinone and premithramycin B, were identified in a mutant lacking the mithramycin glycosyltransferase and methyltransferase genes and in the same mutant complemented with the deleted genes, respectively. Premithramycin B contains five deoxysugars moieties (like mithramycin), but contains a tetracyclic aglycon moiety instead of a tricyclic aglycon. We hypothesized that transcription of mtmOIV (encoding an oxygenase) was impaired in this strain, preventing oxidative breakage of the fourth ring of premithramycin B. Inactivating mtmOIV generated a mithramycin nonproducing mutant that accumulated premithramycin B instead of mithramycin. In vitro assays demonstrated that MtmOIV converted premithramycin B into a tricyclic compound. CONCLUSIONS: In the late stages of mithramycin biosynthesis by Strepyomyces argillaceus, a fully glycosylated tetracyclic tetracycline-like intermediate (premithramycin B) is converted into a tricyclic compound by the oxygenase MtmOIV. This oxygenase inserts an oxygen (Baeyer-Villiger oxidation) and opens the resulting lactone. The following decarboxylation and ketoreduction steps lead to mithramycin. Opening of the fourth ring represents one of the last steps in mithramycin biosynthesis.

The NDP-sugar co-substrate concentration and the enzyme expression level influence the substrate specificity of glycosyltransferases: cloning and characterization of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster.

BACKGROUND: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2x D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. RESULTS: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. CONCLUSIONS: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.

Identification of a sugar flexible glycosyltransferase from Streptomyces olivaceus, the producer of the antitumor polyketide elloramycin.

BACKGROUND: Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which is produced by Streptomyces olivaceus Tü2353. Structurally is a tetracyclic aromatic polyketide derived from the condensation of 10 acetate units. Its chromophoric aglycon is glycosylated with a permethylated L-rhamnose moiety at the C-8 hydroxy group. Only limited information is available about the genes involved in the biosynthesis of elloramycin. From a library of chromosomal DNA from S. olivaceus, a cosmid (16F4) was isolated that contains part of the elloramycin gene cluster and when expressed in Streptomyces lividans resulted in the production of a non-glycosylated intermediate in elloramycin biosynthesis, 8-demethyl-tetracenomycin C (8-DMTC). RESULTS: The expression of cosmid 16F4 in several producers of glycosylated antibiotics has been shown to produce tetracenomycin derivatives containing different 6-deoxysugars. Different experimental approaches showed that the glycosyltransferase gene involved in these glycosylation events was located in 16F4. Using degenerated oligoprimers derived from conserved amino acid sequences in glycosyltransferases, the gene encoding this sugar flexible glycosyltransferase (elmGT) has been identified. After expression of elmGT in Streptomyces albus under the control of the erythromycin resistance promoter, ermEp, it was shown that elmG can transfer different monosaccharides (both L- and D-sugars) and a disaccharide to 8-DMTC. Formation of a diolivosyl derivative in the mithramycin producer Streptomyces argillaceus was found to require the cooperative action of two mithramycin glycosyltransferases (MtmGI and MtmGII) responsible for the formation of the diolivosyl disaccharide, which is then transferred by ElmGT to 8-DMTC. CONCLUSIONS: The ElmGT glycosyltransferase from S. olivaceus Tü2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several L- and D-sugars and also a diolivosyl disaccharide into the aglycon 8-DMTC. ElmGT is an example of sugar flexible glycosyltransferase and can represent an important tool for combinatorial biosynthesis.