Heterologous expression of a Photorhabdus luminescens syrbactin-like gene cluster results in production of the potent proteasome inhibitor glidobactin A

Syrbactins are cyclic peptide derivatives which are known to inhibit the eukaryotic proteasome by irreversible covalent binding to its catalytic sites. The only two members of this family characterized to date, syringolin A and glidobactin A, are secreted by certain strains of Pseudomonas syringae pv. syringae and strain K481-B101 from the order Burkholderiales, respectively. Syrbactins are the products of mixed non-ribosomal peptide/polyketide synthases encoded by gene clusters with a characteristic architecture. Similar, but not identical gene clusters are present in several other bacterial genomes, including that of Photorhabdus luminescens subsp. laumondii TT01, which is therefore hypothesized to be able to produce a syrbactin-type proteasome inhibitor. Here we report the cloning of the putative syrbactins synthetase encoding gene cluster of Ph. luminescens into a cosmid vector and its heterologous expression in Pseudomonas putida. Analysis of culture supernatants of transformed Ps. putida by HPLC and mass spectrometry revealed the presence of glidobactin A, indicating that the syrbactins-like gene cluster of Ph. luminescens encodes a glidobactin A synthetase and that this organism has the capacity to synthesize glidobactin A.     

Morphology of the proboscis of Hubrechtella Juliae (nemertea,pilidiophora): Implications for pilidiophoran monophyly

The proboscis of Hubrechtella juliae was examined using transmission electron microscopy, scanning electron microscopy, and confocal laser scanning microscopy to reveal more features of basal pilidiophoran nemerteans for morphological and phylogenetic analysis. The proboscis glandular epithelium consists of sensory cells and four types of gland cells (granular, bacillary, mucoid, and pseudocnidae‐containing cells) that are not associated with any glandular systems; rod‐shaped pseudocnidae are 15–25 μm in length; the central cilium of the sensory cells is enclosed by two rings of microvilli. The nervous plexus lies in the basal part of glandular epithelium and includes 26–33 (11–12 in juvenile) irregularly anastomosing nerve trunks. The proboscis musculature includes four layers: endothelial circular, inner diagonal, longitudinal, and outer diagonal; inner and outer diagonal muscles consist of noncrossing fibers; in juvenile specimen, the proboscis longitudinal musculature is divided into 7–8 bands. The endothelium consists of apically situated support cells with rudimentary cilia and subapical myocytes. Unique features of Hubrechtella's proboscis include: acentric filaments of the pseudocnidae; absence of tonofilament‐containing support cells; two rings of microvilli around the central cilium of sensory cells; the occurrence of subendothelial diagonal muscles and the lack of an outer diagonal musculature (both states were known only in Baseodiscus species). The significance of these characters for nemertean taxonomy and phylogeny is discussed. The proboscis musculature in H. juliae and most heteronemerteans is bilaterally arranged, which can be considered a possible synapomorphy of Hubrechtellidae + Heteronemertea (= Pilidiophora). J. Morphol. 274:1397–1414, 2013. © 2013 Wiley Periodicals, Inc.     

Mechanism of activation of elongation factor Tu by ribosome: Catalytic histidine activates GTP by protonation

Elongation factor Tu (EF-Tu) is central to prokaryotic protein synthesis as it has the role of delivering amino-acylated tRNAs to the ribosome. Release of EF-Tu, after correct binding of the EF-Tu:aa-tRNA complex to the ribosome, is initiated by GTP hydrolysis. This reaction, whose mechanism is uncertain, is catalyzed by EF-Tu, but requires activation by the ribosome. There have been a number of mechanistic proposals, including those spurred by a recent X-ray crystallographic analysis of a ribosome:EF-Tu:aa-tRNA:GTP-analog complex. In this work, we have investigated these and alternative hypotheses, using high-level quantum chemical/molecular mechanical simulations for the wild-type protein and its His85Gln mutant. For both proteins, we find previously unsuggested mechanisms as being preferred, in which residue 85, either His or Gln, directly assists in the reaction. Analysis shows that the RNA has a minor catalytic effect in the wild-type reaction, but plays a significant role in the mutant by greatly stabilizing the reaction’s transition state. Given the similarity between EF-Tu and other members of the translational G-protein family, it is likely that these mechanisms of ribosome-activated GTP hydrolysis are pertinent to all of these proteins.     

Corruption of the Intra-Gene DNA Methylation Architecture Is a Hallmark of Cancer

Epigenetic processes - including DNA methylation - are increasingly seen as having a fundamental role in chronic diseases like cancer. It is well known that methylation levels at particular genes or loci differ between normal and diseased tissue. Here we investigate whether the intra-gene methylation architecture is corrupted in cancer and whether the variability of levels of methylation of individual CpGs within a defined gene is able to discriminate cancerous from normal tissue, and is associated with heterogeneous tumour phenotype, as defined by gene expression. We analysed 270985 CpGs annotated to 18272 genes, in 3284 cancerous and 681 normal samples, corresponding to 14 different cancer types. In doing so, we found novel differences in intra-gene methylation pattern across phenotypes, particularly in those genes which are crucial for stem cell biology; our measures of intra-gene methylation architecture are a better determinant of phenotype than measures based on mean methylation level alone (K-S test in all 14 diseases tested). These per-gene methylation measures also represent a considerable reduction in complexity, compared to conventional per-CpG beta-values. Our findings strongly support the view that intra-gene methylation architecture has great clinical potential for the development of DNA-based cancer biomarkers.     

Previous Motor Experience Enhances Courtship Behavior in Male Cricket <Emphasis Type="Italic">Gryllus bimaculatus</Emphasis>

In crickets Gryllus bimaculatus, flight has been shown to be able to promote aggressive encounters between males and to suppress escape behavior. The aim of this study was to examine the influence of flight on male behavior in male–female interactions. We found that flown males demonstrate enhanced courtship behavior. The latency of calling song was significantly shorter, while the relative total duration of singing as well as the duration of singing episodes longer in flown males than in the control. Mating rate was also significantly higher in the experimental group containing flown males. The results suggest that, in addition to previously reported effects on aggressiveness and escape, flying has a profound accelerating effect on male courtship behavior.     

Protein L5 is crucial for in vivo assembly of the bacterial 50S ribosomal subunit central protuberance

In the present work, ribosomes assembled in bacterial cells in the absence of essential ribosomal protein L5 were obtained. After arresting L5 synthesis, Escherichia coli cells divide a limited number of times. During this time, accumulation of defective large ribosomal subunits occurs. These 45S particles lack most of the central protuberance (CP) components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33 and L35) and are not able to associate with the small ribosomal subunit. At the same time, 5S rRNA is found in the cytoplasm in complex with ribosomal proteins L18 and L25 at quantities equal to the amount of ribosomes. Thus, it is the first demonstration that protein L5 plays a key role in formation of the CP during assembly of the large ribosomal subunit in the bacterial cell. A possible model for the CP assembly in vivo is discussed in view of the data obtained.     

New Zn complexes based on 1,2,4-triazoles: Synthesis, structure and luminescence

The reaction of 3-(pyridin-2-yl)-5-(2-aminophenyl)-1H-1,2,4-triazole and salicylaldehyde or benzaldehyde leads a new type of 1,2,4-triazole’s derivats. Reactions of these ligands with zinc acetate in ethanol lead to the formation of two new complexes with azomethine or dihydrotriazaindolizine form of ligands. The structures of these complexes were investigated by X-ray analysis. The complexes showed strong green-blue luminescence in solid state.