BAmSA: Visualising transmembrane regions in protein complexes using biotinylated amphipols and electron microscopy

Membrane protein (MP) complexes play key roles in all living cells. Their structural characterisation is hampered by difficulties in purifying and crystallising them. Recent progress in electron microscopy (EM) have revolutionised the field, not only by providing higher-resolution structures for previously characterised MPs but also by yielding first glimpses into the structure of larger and more challenging complexes, such as bacterial secretion systems. However, the resolution of pioneering EM structures may be difficult and their interpretation requires clues regarding the overall organisation of the complexes. In this context, we present BAmSA, a new method for localising transmembrane (TM) regions in MP complexes, using a general procedure that allows tagging them without resorting to neither genetic nor chemical modification. Labels bound to TM regions can be visualised directly on raw negative-stain EM images, on class averages, or on three-dimensional reconstructions, providing a novel strategy to explore the organisation of MP complexes.     

Abundance,diversity and geographic distribution of cassava mosaic disease pandemic‐associated Bemisia tabaci in Tanzania

Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae), one of the most economically important agricultural pests worldwide, is the vector of cassava mosaic geminiviruses that cause cassava mosaic disease (CMD). In East and Central Africa, a severe CMD pandemic that spread from Uganda in the late 1980s still continues to devastate cassava crops. To assess the association of distinct B. tabaci genetic groups with the CMD pandemic, mitochondrial cytochrome oxidase I gene sequences were analysed from whiteflies collected during surveys conducted from 2010 to 2013 in Tanzania. Four genetic groups – Sub‐Saharan Africa 1 (SSA1), Mediterranean, Indian Ocean and East Africa 1, and a group of unknown whitefly species were identified. SSA1 comprised four subgroups: SSA1‐SG1, SSA1‐SG2, SSA1‐SG1/2 and SSA1‐SG3. SSA1‐SG1 was confined to the pandemic‐affected north‐western parts of Tanzania whilst SSA1‐SG2 and SSA1‐SG3 were found in the central and eastern parts not yet affected by the pandemic. The CMD pandemic front was estimated to lie in Geita Region, north‐western Tanzania, and to be spreading south‐east at a rate of ca 26 km/year. The pandemic‐associated B. tabaci SSA1‐SG1 predominated up to 180 km ahead of the CMD front indicating that changes in whitefly population characteristics precede changes in disease characteristics.     

Diversity of symbiotic bacteria associated with Bemisia tabaci (Homoptera: Aleyrodidae) in cassava mosaic disease pandemic areas of Tanzania

All Bemisia tabaci individuals harbour an obligate bacterial symbiont (Portiera aleyrodidarum), and many also harbour non‐essential facultative symbionts. The association of symbiotic bacteria with the various genetic groups of B. tabaci remains unknown for East Africa. This study aimed to assess any association between the various whitefly genetic groups and the endosymbionts they harbour; to investigate if a unique endosymbiont is associated with super‐abundant whiteflies, and to provide baseline information on endosymbionts of whiteflies for a part of East Africa. Whiteflies collected during surveys in Tanzania were genotyped and screened for the presence of the obligate and six secondary symbionts (SS): Rickettsia (R), Hamiltonella (H), Arsenophonus (A), Wolbachia (W), Cardinium (C) and Fritschea (F). The results revealed the presence of Mediterranean (MED), East Africa 1 (EA1), Indian Ocean (IO) and Sub‐Saharan Africa 1 (SSA1) genetic groups of Bemisia tabaci, with SSA1 further clustered into four sub‐groups: SSA1‐SG1, SSA1‐SG2, SSA1‐SG1/2 and SSA1‐SG3. F was completely absent from all of the whiteflies tested while R was always found in double or multiple infections. In general, no particular symbiont appeared to be associated with the super‐abundant SSA1‐SG1 B. tabaci, although A or AC infections were common among infected individuals. The most striking feature of these super‐abundant whiteflies, dominating cassava mosaic disease pandemic areas, was the high prevalence of individuals uninfected by any of the six SS tested. This study of the endosymbionts of B. tabaci in East Africa showed contrasting patterns of infection in crop and weed hosts.     

Polyphosphate Degradation in Stationary Phase Triggers Biofilm Formation via LuxS Quorum Sensing System in Escherichia coli

In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk⁻ppx⁻ or ppk⁻ppx⁻/ppk⁺ strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.     

Identification of an iron-hepcidin complex

Following its identification as a liver-expressed antimicrobial peptide, the hepcidin peptide was later shown to be a key player in iron homoeostasis. It is now proposed to be the 'iron hormone' which, by interacting with the iron transporter ferroportin, prevents further iron import into the circulatory system. This conclusion was reached using the corresponding synthetic peptide, emphasizing the functional importance of the mature 25-mer peptide, but omitting the possible functionality of its maturation. From urine-purified native hepcidin, we recently demonstrated that a proportion of the purified hepcidin had formed iron-hepcidin complexes. This interaction was investigated further by computer modelling and, based on the sequence similarity of hepcidin with metallothionein, a three-dimensional model of hepcidin, containing one atom of iron, was constructed. To characterize these complexes further, the interaction with iron was analysed using different spectroscopic methods. Monoferric hepcidin was identified by MS, as were possibly other complexes containing two and three atoms of iron respectively, although these were present only in minor amounts. UV/visible absorbance and CD studies identified the iron-binding events which were facilitated at a physiological pH. EPR spectroscopy identified the ferric state of the bound metal, and indicated that the iron-hepcidin complex shares some similarities with the rubredoxin iron-sulfur complex, suggesting the presence of Fe(3+) in a tetrahedral sulfur co-ordination. The potential roles of iron binding for hepcidin are discussed, and we propose either a regulatory function in the maturation of pro-hepcidin into active hepcidin or as the necessary link in the interaction between hepcidin and ferroportin.     

Nature of non-transferrin-bound iron: studies on iron citrate complexes and thalassemic sera

Despite its importance in iron-overload diseases, little is known about the composition of plasma non-transferrin-bound iron (NTBI). Using 30-kDa ultrafiltration, plasma from thalassemic patients consisted of both filterable and non-filterable NTBI, the filterable fraction representing less than 10% NTBI. Low filterability could result from protein binding or NTBI species exceeding 30 kDa. The properties of iron citrate and its interaction with albumin were therefore investigated, as these represent likely NTBI species. Iron permeated 5- or 12-kDa ultrafiltration units completely when complexes were freshly prepared and citrate exceeded iron by tenfold, whereas with 30-kDa ultrafiltration units, permeation approached 100% at all molar ratios. A g = 4.3 electron paramagnetic resonance signal, characteristic of mononuclear iron, was detectable only with iron-to-citrate ratios above 1:100. The ability of both desferrioxamine and 1,2-dimethyl-3-hydroxypyridin-4-one to chelate iron in iron citrate complexes also increased with increasing ratios of citrate to iron. Incremental molar excesses of citrate thus favour the progressive appearance of chelatable lower molecular weight iron oligomers, dimers and ultimately monomers. Filtration of iron citrate in the presence of albumin showed substantial binding to albumin across a wide range of iron-to-citrate ratios and also increased accessibility of iron to chelators, reflecting a shift towards smaller oligomeric species. However, in vitro experiments using immunodepletion or absorption of albumin to Cibacron blue–Sepharose indicate that iron is only loosely bound in iron citrate–albumin complexes and that NTBI is unlikely to be albumin-bound to any significant extent in thalassemic sera.