Pronova Biopharma - Vurdering av utslipp til Sandefjordsfjorden

Det er gjort en vurdering av hvordan avløpsvann fra Pronova Biopharma kan påvirke oksygenforholdene i Sandefjordsfjorden. Arbeidet har bl.a. omfattet strømmålinger, oksygenprøver og bruk av modeller. Hovedkonklusjonen er at til vanlig vil utslippet bare i meget liten grad påvirke oksygenforholdene. De viktigste elementene i fjordens oksygenbudsjett varierer med tiden, og høsten er den mest sårbare perioden. Man kan ikke se helt bort fra at et sammentreff av flere ugunstige faktorer da kan skape en periode med en merkbar redusert konsentrasjon (til vannkvalitetsklasse Mindre God) i en lokal vannmasse omkring utslippet. Ingen undersøkelser har imidlertid påvist at dette har skjedd.Pronova Biopharma AS, Sandefjord v/ A.L. Steneru

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32?36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component

Type B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins

The Type IX Secretion System: Advances in Structure, Function and Organisation

The type IX secretion system (T9SS) is specific to the Bacteroidetes phylum. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilises the T9SS to transport many proteins—including its gingipain virulence factors—across the outer membrane and attach them to the cell surface. Additionally, the T9SS is also required for gliding motility in motile organisms, such as Flavobacterium johnsoniae. At least nineteen proteins have been identified as components of the T9SS, including the three transcription regulators, PorX, PorY and SigP. Although the components are known, the overall organisation and the molecular mechanism of how the T9SS operates is largely unknown. This review focusses on the recent advances made in the structure, function, and organisation of the T9SS machinery to provide further insight into this highly novel secretion system

Protein substrates of a novel secretion system are numerous in the bacteroidetes phylum and have in common a cleavable C-Terminal secretion signal, extensive post-translational modification, and cell-surface attachment

The research outputs in this collection have been funded in whole or in part by the National Health and Medical Research Council (NHMRC).Published VersionCopyright © 2012 American Chemical SocietyThe secretion of certain proteins in Porphyromonas gingivalis is dependent on a C-terminal domain (CTD). After secretion, the CTD is cleaved prior to extensive modification of the mature protein, probably with lipopolysaccharide, therefore enabling attachment to the cell surface. In this study, bioinformatic analyses of the CTD demonstrated the presence of three conserved sequence motifs. These motifs were used to construct Hidden Markov Models (HMMs) that predicted 663 CTD-containing proteins in 21 fully sequenced species of the Bacteroidetes phylum, while no CTD-containing proteins were predicted in species outside this phylum. Further HMM searching of Cytophaga hutchinsonii led to a total of 171 predicted CTD proteins in that organism alone. Proteomic analyses of membrane fractions and culture fluid derived from P. gingivalis and four other species containing predicted CTDs (Parabacteroides distasonis, Prevotella intermedia, Tannerella forsythia, and C. hutchinsonii) demonstrated that membrane localization, extensive post-translational modification, and CTD-cleavage were conserved features of the secretion system. The CTD cleavage site of 10 different proteins from 3 different species was determined and found to be similar to the cleavage site previously determined in P. gingivalis, suggesting that homologues of the C-terminal signal peptidase (PG0026) are responsible for the cleavage in these species.10.1021/pr400487

Type B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins

A proposed model for the roles and interactions of PorK, PorL, PorM, PorN and PG0189 in the T9SS.

<p>PorK and PorN interact to form a ring-shaped structure that is localised in the periplasm and tethered to the outer membrane via the PorK lipid moieties (lipid shown as black line). This structure may be further stabilised by its association with the PG0189 outer membrane protein. It is proposed that the PorK and PorN rings assemble around the periplasmic extensions of the unknown OM secretion pore. Both PorL and PorM have transmembrane spanning domains and are proposed to transduce energy from the inner membrane (PMF) or cytosol (ATP) and power secretion of the T9SS substrates through the transient interactions with the PorK/N complex. The topology of the PorL and PorM inner membrane proteins is not known.</p

Cryo transmission electron micrographs of the purified PorK/N complex.

<p>(A) First column -top views of the complex, second column- side views, third column—tilted views. The side views show two major rings and within each major ring two distinct sub-rings were observed, shown by the black arrow heads. Note the thin electron dense band between the two major rings, shown by the white arrow head, which may represent the lipid tails overlap of the PorK lipoprotein. (B) Purified native PorK/N complex from the wild type has the same structure as from the mutant (W50ABK*WbaP) at that resolution. (C) LDAO treated complex, the association between the two major rings has been ablated and single complexes are now noticeable. Scale bars 20 nm. (D) A 3-D reconstruction of the PorK/N ring. (E) Schematic representations of the PorK/N rings. The double major ring form (<i>in-vitro</i>) is proposed to be an artifact of the two major rings interacting via the PorK lipid shown in purple. Whereas the <i>in-vivo</i> form is proposed to be a single major ring composed of PorN (dark blue) and PorK (light blue) with the PorK lipid (purple) involved in anchorage to the OM which is represented by two white parallel lines.</p

Absence of EDSL on <i>porK</i>, <i>porN</i>, <i>porL</i> and <i>porM</i> mutants.

<p>Cryo-EM micrographs of whole cells (A) Wild type (B) <i>porK</i> mutant (C) <i>porN</i> mutant (D) <i>porL</i> mutant and (E) <i>porM</i> mutant. A magnified section is shown on the bottom of each image. Arrows point to the electron-dense surface layer (EDSL) that is absent in the mutants. Scale bars 100 nm.</p

Electron micrographs of negatively stained PorK/N complex.

<p>Complexes in fraction 6 of CsCl density gradients were stained with uranyl acetate and observed under TEM. (A) Homogenous ring-shaped structures of PorK/N complex. Scale bar 100 nm (B) Top view of the complex forming a ring with 32–36 subunits (white arrow heads). Scale bar 20 nm. (C) The first two columns show side views of the complex where two major rings were observed (black arrowheads). Scale bar 20 nm. The third column shows the tilted view of the complex verifying the presence of two rings. (D) A higher resolution image obtained by virtual section (0.76 nm) through an electron tomogram of a negatively stained complex. At this resolution individual subunits (32) were observed. Scale bar 20 nm.</p