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The excretion of nitrogenous waste products in the form of ammonia (NH3) and ammonium (NH4+) is a fundamental process in aquatic organisms. For mytilid bivalves, little is known about the mechanisms and sites of excretion. This study investigated the localization and the mechanisms of ammonia excretion in mytilid mussels. An Rh protein was found to be abundantly expressed in the apical cell membrane of the plicate organ, which was previously described as a solely respiratory organ. The Rh protein was also expressed in the gill, although at significantly lower concentrations, but was not detectable in mussel kidney. Furthermore, NH3/NH4+ was not enriched in the urine, suggesting that kidneys are not involved in active NH3/NH4+ excretion. Exposure to elevated seawater pH of 8.5 transiently reduced NH3/NH4+ excretion rates, but they returned to control values following 24 h acclimation. These mussels had increased abundance of V-type H+-ATPase in the apical membranes of plicate organ cells; however, NH3/NH4+ excretion rates were not affected by the V-type H+-ATPase specific inhibitor concanamycin A (100 nmol l-1). In contrast, inhibition of ciliary beating with dopamine and increased seawater viscosity significantly reduced NH3 excretion rates under control pH (8.0). These results suggest that NH3/NH4+ excretion in mytilid mussels takes place by passive NH3 diffusion across respiratory epithelia via the Rh protein, facilitated by the water current produced for filter feeding, which prevents accumulation of NH3 in the boundary layer. This mechanism would be energy efficient for sessile organisms, as they already generate water currents for filter feeding. [ABSTRACT FROM AUTHOR]

Protein amyloid aggregates epigenetically determine either advantageous or proteinopathic phenotypes. Prions are infectious amyloidogenic proteins, whereas prionoids lack infectivity but spread from mother to daughter cells. While prion amyloidosis has been studied in yeast and mammalian cells models, the dynamics of transmission of an amyloid proteinopathy has not been addressed yet in bacteria. Using time-lapse microscopy and a microfluidic set-up, we have assessed in E scherichia coli the vertical transmission of the amyloidosis caused by the synthetic bacterial model prionoid RepA-WH1 at single cell resolution within their lineage context. We identify in vivo the coexistence of two strain-like types of amyloid aggregates within a genetically identical population and a controlled homogeneous environment. The amyloids are either toxic globular particles or single comet-shaped aggregates that split during cytokinesis and exhibit milder toxicity. Both segregate and propagate in sublineages, yet show interconversion. ClpB ( Hsp104) chaperone, key for spreading of yeast prions, has no effect on the dynamics of the two RepA-WH1 aggregates. However, the propagation of the comet-like species is DnaK ( Hsp70)-dependent. The bacterial RepA-WH1 prionoid thus provides key qualitative and quantitative clues on the biology of intracellular amyloid proteinopathies. [ABSTRACT FROM AUTHOR]

Protein amyloids arise from the conformational conversion and assembly of a soluble protein into fibrilar aggregates with a crossed β-sheet backbone. Amyloid aggregates are able to replicate by acting as a template for the structural transformation and accretion of further protein molecules. In physicochemical terms, amyloids arguably constitute the simplest self-replicative macromolecular assemblies. Similarly to the mammalian proteins PrP and α-synuclein, the winged-helix dimerization (WH1) domain of the bacterial, plasmid-encoded protein RepA can assemble into amyloid fibres upon binding to DNA in vitro. Here we report that a hyper-amyloidogenic functional variant (A31V) of RepA, fused to a red fluorescent protein, causes an amyloid proteinopathy in Escherichia coli with the following features: (i) in the presence of multiple copies of the specific DNA sequence opsp, WH1(A31V) accumulates as cytoplasmatic inclusions segregated from the nucleoid; (ii) such aggregates are amyloid in nature; (iii) bacteria carrying the amyloid inclusions age, exhibiting a fivefold expanded generation time; (iv) before cytokinesis, small inclusions are assembled de novo and transferred to the daughter cells, in which transmission failures cure amyloidosis; and (v) in the absence of inducer DNA, purified cellular WH1(A31V) inclusions seed amyloid fibre growth in vitro from the soluble protein. RepA-WH1 is a suitable bacterial model system for amyloid proteinopathies. [ABSTRACT FROM AUTHOR]

In many plasmid replicons of Gram-negative bacteria, Rep protein dimers are transcriptional self-repressors of their genes, whereas monomers are initiators of DNA replication. Switching between both functions implies conformational remodelling of Rep, and is promoted by Rep binding to the origin DNA repeats (iterons) or chaperones. Rep proteins play another key role: they bridge together two iteron DNA stretches, found either on the same or on different plasmid molecules. These so-called, respectively, ‘ looped’ and ‘ handcuffed’ complexes are thought to be negative regulators of plasmid replication. Although evidence for Rep-dependent plasmid handcuffing has been found in a number of replicons, the structure of these Rep–DNA assemblies is still unknown. Here, by a combination of proteomics, electron microscopy, genetic analysis and modelling, we provide insight on a possible three-dimensional structure for two handcuffed arrays of the iterons found at the origin of pPS10 replicon. These are brought together in parallel register by zipping-up DNA-bound RepA monomers. We also present evidence for a distinct role of RepA dimers in DNA looping. This work defines a new regulatory interface in Rep proteins. [ABSTRACT FROM AUTHOR]

In the present study, the influence of short-term osmotic variations on some electrophysiological properties related to NaCl absorption across posterior gills of Chasmagnathus granulatus was investigated. The transepithelial potential difference (V[sub te]) of isolated and perfused gills increased significantly when hyposmotic saline (699mosmoll[sup -1]) was used instead of isosmotic solution (1045 mosmol 1[sup -1]). A reduction of the concentration of Na[sup +] or CI[sup -] at constant osmolarity did not produce any change in V[sub te]. Transepithelial short-circuit current (I[sub sc]) and conductance (G[sub te]), measured with split gill lamellae mounted in a modified Ussing chamber, also increased after changing to hyposmotic salines (I[sub sc]: from -89.0±40.8 μA cm[sup -2] to -179.3±37.0 μA cm[sup -2]; G[sub te]: from 40.5±16.9mScm[sup -2] to 47.3±l5.8mS cm[sup - 2]). The observed effects of reduced osmolarity were fast, reversible and gradually dependent on the magnitude of the osmotic variation. The acitivity of the Na[sup +]/K[sup +]-ATPase increased significantly after perfusion with hyposmotic saline, from 18.73±6.35 μmol P[sub i] h[sup -1] mg[sup -1] to 41.84±14.54 μmol P[sub i] h[sup 1-] mg[sup -1]. Theophylline maintained part of the elevated V[sub te] induced by hyposmotic saline, suggesting that an increased cellular cyclic AMP level is involved in the response to reduced osmolarity. In summary, the results indicate that the hemolymph osmolarity regulates active transbranchial NaCl absorption by modulating the activity of the basolateral Na[sup +]/K[sup +]-ATPase and by changing a conductive pathway, probably at the apical membrane. [ABSTRACT FROM AUTHOR]

1. The ability to oxidize quinate is elicited in Acinetobacter calco-aceticus not only by growth on quinate but also by growth on protocatechuate or its precursors such as shikimate and p-hydroxybenzoate. Accordingly the synthesis of a quinate dependent dehydrogenase was found to be induced by protocatechuate. Moreover, this enzyme seems to belong to the same coordinate block of enzymes responsible for the conversion of shikimate to β-ketoadipyl-CoA. 2. Analogies between quinate and shikimate oxidation m A. calco-aceticus have been substantiated by showing that both quinate dehydrogenase and shikimate dehydrogenase activities rely on a single enzyme. 3. Several auxotrophs that have lost the ability to utile both quinate and shikimate as sole source of carbon and energy have been isolated. Particutarly relevant for this study are two of these mutants in which the dehydrogenase is severely impaired and correspondingly their ability to oxidize the mentioned hydroaromatic compounds is substantially reduced. The properties of these auxotrophs and their revertants confirm the key role of the dehydrogenase in the dissimilation of both quinate and shikimate. The rationale of the control of this enzyme by protocatechuate is discussed. [ABSTRACT FROM AUTHOR]

0948 02 [ABSTRACT FROM AUTHOR]

Upon binding to short specific dsDNA sequences in vitro, the N-terminal WH1 domain of the plasmid DNA replication initiator RepA assembles as amyloid fibres. These are bundles of single or double twisted tubular filaments in which distorted RepA-WH1 monomers are the building blocks. When expressed in Escherichia coli, RepA-WH1 triggers the first synthetic amyloid proteinopathy in bacteria, recapitulating some of the features of mammalian prion diseases: it is vertically transmissible, albeit non-infectious, showing up in at least two phenotypically distinct and interconvertible strains. Here we report B3h7, a monoclonal antibody specific for oligomers of RepA-WH1, but which does not recognize the mature amyloid fibres. Unlike a control polyclonal antibody generated against the soluble protein, B3h7 interferes in vitro with DNA-promoted or amyloid-seeded assembly of RepA-WH1 fibres, thus the targeted oligomers are on-pathway amyloidogenic intermediates. Immuno-electron microscopy with B3h7 on thin sections of E. coli cells expressing RepA-WH1 consistently labels the bacterial nucleoid, but not the large cytoplasmic aggregates of the protein. This observation points to the nucleoid as the place where oligomeric amyloid precursors of RepA-WH1 are generated, and suggests that, once nucleated by DNA, further growth must continue in the cytoplasm due to entropic exclusion. [ABSTRACT FROM AUTHOR]