Reversible Signal Binding by the Pseudomonas aeruginosa Quorum-Sensing Signal Receptor LasR

Many members of the LuxR family of acyl-homoserine lactone (acyl-HSL)-dependent quorum-sensing transcriptional activators are thought to have the unusual characteristics of requiring the signal ligand during polypeptide synthesis to fold into an active conformation and of binding signal extraordinarily tightly. This is the case for the N-3-oxo-dodecanoyl-HSL-dependent Pseudomonas aeruginosa virulence regulator LasR. We present evidence that LasR can fold into an active conformation in vivo in the absence of the acyl-HSL ligand. We also present evidence indicating that in the cellular environment, LasR and N-3-oxo-dodecanoyl-HSL readily dissociate. After dissociation, LasR can remain in a properly folded conformation capable of reassociating with signal. We present a new model for the folding and signal binding of LasR and other members of the family of transcription factors to which LasR belongs. Our findings have important implications concerning the cellular responses to decreased environmental concentrations of signals and have implications about potential quorum-sensing inhibition strategies

Quorum Sensing and Expression of Virulence in Pectobacteria

Quorum sensing (QS) is a population density-dependent regulatory mechanism in which gene expression is coupled to the accumulation of a chemical signaling molecule. QS systems are widespread among the plant soft-rotting bacteria. In Pectobacterium carotovorum, at least two QS systems exist being specified by the nature of chemical signals involved. QS in Pectobacterium carotovorum uses N-acylhomoserine lactone (AHL) based, as well as autoinducer-2 (AI-2) dependent signaling systems. This review will address the importance of the QS in production of virulence factors and interaction of QS with other regulatory systems in Pectobacterium carotovorum

The DNA-recognition fold of Sso7c4 suggests a new member of SpoVT-AbrB superfamily from archaea

Organisms growing at elevated temperatures face the challenge of maintaining the integrity of their genetic materials. Archaea possess unique chromatin proteins for gene organization and information processing. We present the solution structure of Sso7c4 from Sulfolobus solfataricus, which has a homodimeric DNA-binding fold forming a swapped β-loop-β ‘Tai-Chi’ topology. The fold is reminiscent of the N-terminal DNA-binding domain of AbrB and MazE. In addition, several amide resonances in the heteronuclear single quantum coherence spectra of Sso7c4 are shifted and broadened with the addition of small amounts of duplex DNA oligomers. The locations of the corresponding amides in the Sso7c4 structure define its DNA-interacting surface. NMR spectra of DNA titrated with the protein further indicated that Sso7c4 interacts with DNA in the major groove. Taken together, a plausible model for the Sso7c4–DNA complex is presented, in which the DNA double helix is curved around the protein dimer

Interaction of triosephosphate isomerase from the cell surface of Staphylococcus aureus and α-(1→3)-mannooligosaccharides derived from glucuronoxylomannan of Cryptococcus neoformans

The glycolytic enzyme triosephosphate isomerase (TPI; EC 5.3.1.1) of Staphylococcus aureus is a candidate adhesion molecule for the interaction between the bacterium and the fungal pathogen Cryptococcus neoformans. TPI may recognize the mannan backbone of glucuronoxylomannan (GXM) of C. neoformans. We purified TPI from extracts of S. aureus surface proteins to investigate its binding by surface plasmon resonance analysis. The immobilized TPI reacted with GXM in a dose-dependent manner. Furthermore, the interactions between staphylococcal TPI and α-(1→3)-mannooligosaccharides derived from GXM were examined. The oligosaccharides exhibited binding with TPI; however, monomeric mannose did not. Differences in the slopes of the sensorgrams were observed between oligosaccharides with an even number of residues versus those with an odd number. A heterogeneous ligand-parallel reaction model revealed the existence of at least two binding sites on TPI. The enzymic activities of TPI were inhibited in a dose-dependent manner by α-(1→3)-mannooligosaccharides larger than triose. The binding of TPI and α-(1→3)-mannotriose near the substrate-binding site was predicted in silico (AutoDock 3.05). An oligosaccharide of size equal to or greater than triose could bind to the site, affecting enzymic activities. Moreover, affinities were indicated, especially for biose and tetraose, to another binding pocket, which would not affect enzymic activity. These data suggest a novel role for TPI, in addition to glycolysis, on the surface of S. aureus

Negative regulation of violacein biosynthesis in Chromobacterium violaceum

In Chromobacteium violaceum, the purple pigment violacein is under positive regulation by the N-acylhomoserine lactone CviI/R quorum sensing system and negative regulation by an uncharacterized putative repressor. In this study we report that the biosynthesis of violacein is negatively controlled by a novel repressor protein, VioS. The violacein operon is regulated negatively by VioS and positively by the CviI/R system in both C. violaceum and in a heterologous Escherichia coli genetic background. VioS does not regulate the CviI/R system and apart from violacein, VioS and quorum sensing regulate other phenotypes antagonistically. Quorum sensing regulated phenotypes in C. violaceum are therefore further regulated providing an additional level of control

Pest categorisation of Pantoea stewartii subsp. stewartii

Following a request from the European Commission, the EFSA Plant Health Panel performed a pest categorisation of Pantoea stewartii subsp. stewartii (hereafter P. s. subsp. stewartii). P. s. subsp. stewartii is a Gram-negative bacterium that causes Stewart’s vascular wilt and leaf blight of sweet corn and maize, a disease responsible for serious crop losses throughout the world. The bacterium is endemic to the USA and is now present in Africa, North, Central and South America, Asia and Ukraine. In the EU, it is reported from Italy with a restricted distribution and under eradication. The bacterium is regulated according to Council Directive 2000/29/EC (Annex IIAI) as a harmful organism whose introduction and spread in the EU is banned on seeds of Zea mays. Other reported potential host plants include various species of the family Poaceae, including weeds, rice (Oryza sativa), oat (Avena sativa) and common wheat (Triticum aestivum), as well as jackfruit (Artocarpus heterophyllus), the ornamental Dracaena sanderiana and the palm Bactris gasipaes, but there is uncertainty about whether these are hosts of P. s. subsp. stewartii or of the other subspecies. The pest could enter the EU via host plants for planting (including seed) and via insect vectors from neighbouring countries. Host plants are widely distributed and climatic conditions are conducive in the EU. P. s. subsp. stewartii could spread by movement of host plants for planting (including seeds) and insect vectors. Impacts could occur on maize and rice. Methods to certify pest freedom of maize seeds are available. The main knowledge gaps concern the availability of vectors in the EU, the level of susceptibility of the maize cultivars grown in the EU, the virulence of strains in recent outbreaks, and the host range of the bacterium. The criteria assessed by the Panel for consideration as a potential quarantine pest are met

Elemental biochemical analysis of the polysaccharides in the extracellular matrix of the yeast Saccharomyces cerevisiae

In yeast multicellular aggregates, such as biofilms and colonies, cells are supported by a yeast extracellular matrix (yECM) of glycosidic nature, the composition of which is mostly unknown. Saccharomyces cerevisiae ECM was produced, extracted and partitioned. An analytical-grade pure glycoside fraction was obtained, fractionated by anionic exchange liquid chromatography and analyzed by gas chromatography–mass spectrometry and polyacrylamide gel electrophoresis. Two different molecular weight polysaccharides were found, composed of glucose, mannose and small relative amounts of galactose. One of the polysaccharides had a low molecular weight, compatible with the association with glycoproteins abundantly occurring in yECM. In addition, these polysaccharide species were separated by diaminopropane agarose gel electrophoresis and induced metachromatic shift, suggesting chemical substitution, which was corroborated by anticoagulation activity. This was shown to be associated with the double deletion of the yeast homologues of the mammalian Hedgehog modulators Hhatl and Hhat, respectively yeast Gup1 and Gup2. These results pioneer the study of the molecular biology of the ECM supporting S. cerevisiae multicellular aggregates such as biofilms.The polysaccharide analysis was performed at the Laboratory of Glycoconjugates Biochemistry and Cellular Biology, UFRJ, Brazil. Fábio Faria-Oliveira was supported by a PhD scholarship (SFRH/BD/45368/2008) from FCT (Fundação para a Ciência e a Tecnologia). This work was funded by Marie Curie Initial Training Network GLYCOPHARM (PITN-GA-2012-317297), and FCT/MEC through Portuguese funds (PIDDAC)–PEst-OE/ BIA/UI4050/2014.info:eu-repo/semantics/publishedVersio