Patterns of energy metabolism and growth kinetics of Kluyveromyces marxianus in whey chemostat culture

The influence of physiological parameters such as carbon substrate flux and O₂ uptake rates on energy metabolism are reported with reference to biomass productivity in whey chemostat culture. The combined results show that oxidoreductive energy metabolism may be attained independently of the yeast reaching its maximum respiratory capacity. A novel metabolic interpretation is presented proposing that a relative imbalance between glycolysis and subsequent oxidative steps alone is sufficient to account for the observed results. By means of a mathematical model the results could be reproduced under all experimental conditions. The new interpretation provides an insight into the manner in which energy mettbolism is regulated and influences growth-related process Kluyveromyces marxianus, as well as other yeasts with similar physiological characteristics. Correspondence to: J. I. Castrillo     

Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers.

A plasma membrane fraction was obtained by the combined use of differential centrifugation and aqueous polymer two-phase partitioning techniques. Vanadate-inhibited ATPase and glucan synthase activities were highly enriched in this fraction, although the presence of ATPase activity which was not inhibited by vanadate, nitrate, molybdate, anyimycin A or azide was also detected. Other intracellular membrane marker activities were present at very low or undetectable levels. A further separation step using Percoll density gradient centrifugation resulted in the separation of a fraction which exclusively contained vanadate-inhibited ATPase activity, and was enriched with silicotungstic-acid-staining membrane material. Latency tests performed on the plasma membrane markers showed that the membrane vesicles were in the right-side-out orientation.     

Synthesis and Stereochemical Characterization of Hydroxy- and Epoxy-derivatives of Rotenone

One to four routes of synthesis are described for 8′-hydroxyrotenone, 5′-hydroxyrotenone, two epimers of 6′,7′-dihydro-6′,7′-dihydroxyrotenone, two epimers of 6′,7′-epoxyrotenone and the four rotenolones derived from each of these compounds. The stereochemical relationships are determined, in each case, by chemical interconversion, ORD and monochromatic rotation to assess the absolute configuration of the B/C ring juncture and by IR, MS and NMR for the cis- or trans-nature of this juncture. The new compounds described are useful standards for studies on the metabolites and photodecomposition products of rotenone insecticide chemical.     

Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity

In the last decades bacterial glycoengineering emerged as a new field as the result of the ability to transfer the Campylobacter jejuni N- glycosylation machinery into Escherichia coli for the production of recombinant glycoproteins that can be used as antigens for diagnosis, vaccines, and therapeutics. However, the identification of critical parameters implicated in the production process and its optimization to jump to a productive scale is still required. In this study, we developed a dual expression glycosylation vector for the production of the recombinant glycoprotein AcrA-O157, a novel antigen that allows the serodiagnosis of the infection with enterohemorrhagic E. coli O157 in humans. Volumetric productivity was studied in different culture media and found that 2xYP had 6.9-fold higher productivity than the extensively used LB. Subsequently, bioreactor batch and exponential-fed-batch cultures were designed to determine the influence of the specific growth rate (μ) on AcrA-O157 glycosylation efficiency, production kinetics, and specific productivity. At μ_max, AcrA glycosylation with O157-polysaccharide and the specific synthesis rate were maximal, constituting the optimal physiological condition for AcrA-O157 production. Our findings should be considered for the design, optimization, and scaling up of AcrA-O157 production and other recombinant glycoproteins attractive for industrial applications.     

The Balearic Islands: a reservoir of cpDNA genetic variation for evergreen oaks

Aim To analyse the role of the Balearic Islands as a refuge area for evergreen Quercus (cork oak: Quercus suber L., holm oak: Q. ilex L., kermes oak: Q. coccifera L.), by using molecular, historical and palaeobotanical data. Location The Western Mediterranean Basin (Balearic Islands, eastern Iberia, Provence, Sardinia, Corsica, Sicily, Malta, Italy, Northern Africa). Methods We sampled 108 populations and used the PCR‐RFLP technique with five universal cpDNA primers to define haplotypes in the sampled populations. Diversity, differentiation parameters and spatial analysis of the populations, using a spatial version of amova , were linked to the geological history of the Western Mediterranean Basin in order to explain the present spatial pattern of the evergreen Quercus populations in the Balearics. Results Evergreen Quercus cpDNA shows a complex structure, with remnants of ancient diversity in the Balearics. Balearic populations of holm oak are related to Iberian populations, while for cork and kermes oaks, we found both Tyrrhenian and Iberian haplotypes. Main conclusions The complex spatial patterns of cpDNA in Balearic evergreen Quercus appears explicable in terms of a combination of physical (vicariance and long distance dispersal) and biological (introgressive hybridization) factors. The Balearics constitute a glacial refuge area and a reservoir of genetic variation with traces of ancient diversity from Messinian–Pliocene stages.     

A genomic island in Brucella involved in the adhesion to host cells: Identification of a new adhesin and a translocation factor

Adhesion to host cells is the first step in the virulence cycle of any pathogen. In Gram‐negative bacteria, adhesion is mediated, among other virulence factors such as the lipopolysaccharides, by specific outer‐membrane proteins generally termed adhesins that belong to a wide variety of families and have different evolutionary origins. In Brucella, a widespread zoonotic pathogen of animal and human health concern, adhesion is central as it may determine the intracellular fate of the bacterium, an essential stage in its pathogenesis. In the present paper, we further characterised a genomic locus that we have previously reported encodes an adhesin (BigA) with a bacterial immunoglobulin‐like domain (BIg‐like). We found that this region encodes a second adhesin, which we have named BigB; and PalA, a periplasmic protein necessary for the proper display in the outer membrane of BigA and BigB. Deletion of bigB or palA diminishes the adhesion of the bacterium and overexpression of BigB dramatically increases it. Incubation of cells with the recombinant BIg‐like domain of BigB induced important cytoskeletal rearrangements and affected the focal adhesion sites indicating that the adhesin targets cell–cell or cell–matrix proteins. We additionally show that PalA has a periplasmic localisation and is completely necessary for the proper display of BigA and BigB, probably avoiding their aggregation and facilitating their transport to the outer membrane. Our results indicate that this genomic island is entirely devoted to the adhesion of Brucella to host cells.     

Iron-molybdenum cofactor synthesis in Azotobacter vinelandii Nif- mutants.

Nif- mutants of Azotobacter vinelandii defective in dinitrogenase activity synthesized iron-molybdenum cofactor (FeMo-co) and accumulated it in two protein-bound forms: inactive dinitrogenase and a possible intermediate involved in the FeMo-co biosynthetic pathway. FeMo-co from both these proteins could activate apo-dinitrogenase from FeMo-co-deficient mutants.     

Phosphatidylethanolamine synthesis is required for optimal virulence of Brucella abortus

The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell.