Specific residues in the N-terminal domain of FimH stimulate type 1 fimbriae assembly in Escherichia coli following the initial binding of the adhesin to FimD usher

Type 1 fimbriae are assembled by the chaperone–usher pathway where periplasmic protein complexes formed between fimbrial subunits and the FimC chaperone are recruited by the outer membrane protein FimD (the usher) for their ordered polymerization and export. FimH adhesin initiates and stimulates type 1 fimbriae polymerization by interacting with FimD. Previously we showed that the N-terminal lectin domain of FimH (N-FimH) is necessary for binding of the adhesin to FimD. In this work, we have selected mutants in N-FimH that reduce the levels of adhesin and type 1 fimbriae displayed in Escherichia coli without altering the levels of FimH in the periplasm. The selected mutations are mostly concentrated in residues G15, N46 and D47. In contrast to other mutations isolated that simply affect binding of FimH to FimD (e.g. C3Y), these variants associate to FimD and alter its susceptibility to trypsin digestion similarly to wild-type FimH. Importantly, their mutant phenotype is rescued when FimD is activated in vivo by the coexpression of wild-type FimH. Altogether, these data indicate that residues G15, N46 and D47 play an important role following initial binding of FimH to FimD for efficient type 1 fimbriae polymerization by this outer membrane usher.     

Metabolic engineering for bioproduction of sugar alcohols

Sugar alcohols find applications in pharmaceuticals, oral and personal care products, and as intermediates in chemical synthesis. While industrial-scale production of these compounds has generally involved catalytic hydrogenation of sugars, microbial-based processes receive increasing attention. The past few years have seen a variety of interesting metabolic engineering efforts to improve the capabilities of bacteria and yeasts to overproduce xylitol, mannitol, and sorbitol. Examples include heterologous expression of yeast xylose reductase in Escherichia coli for the production of xylitol, coexpression of formate dehydrogenase, mannitol dehydrogenase, and a glucose facilitator protein in Corynebacterium glutamicum for mannitol production from fructose and formate, and overexpression of sorbitol-6-phosphate dehydrogenase in lactate dehydrogenase-deficient Lactobacillus plantarum to achieve nearly maximum theoretical yields of sorbitol from glucose.     

Partial purification and characterization of a non-specific acid phosphatase in leaves and root nodules of Phaseolus vulgaris.

Acid phosphatase (ACP) activity in common bean grown with or without 1.5 mM of phosphate has been examined. Leaves and root nodules responded to the absence of an exogenous phosphate source with an increase in ACP activity. Increases in enzyme activity were not associated with the synthesis of new isoforms of the enzyme. We partially purified and characterized the ACPs, which consisted of three proteins, one of leaf and two of nodule. Proteins of leaf migrated at 72 and 51 kDa in SDS-PAGE, whereas that of nodule migrated at 72, 49, 41 and 34 kDa. Enzymes of both organs had a pH optimum of 5.6, and were relatively heat stable. The enzymes exhibit a broad substrate selectivity, with maximal activity obtained with alpha-naphthyl-phosphate, ribulose 1,5-bisphosphate and p-nitrophenyl-phosphate (p-NPP). Potent inhibition by Zn2+, Hg2+, Cu2+, Pb2+, Al3+ and (MoO4)2- was observed.     

Responses of massive and branching coral species to the combined effects of water temperature and nitrate enrichment

The branching coral species Pocillopora damicornis (Linnaeus) and the massive coral species Porites lobata Dana were exposed for 30 days to different temperatures and nitrate concentrations to study the response of the coral-zooxanthella symbiosis. Results suggest that the effect of nitrate enrichment on the polyp-zooxanthella symbiosis varies according to the coral morphology. After the experimental period only 30% of P. damicornis colonies remained healthy, in contrast to 90% of P. lobata. The branching P. damicornis was significantly affected by the addition of nitrate, whereas P. lobata was significantly influenced by water temperature. The two species showed enhanced zooxanthella volume, and chlorophyll contents per cell under high nitrate concentrations. The reduced zooxanthellae density in both species indicated a detrimental influence of the interaction of high nitrate and high temperature. Tissue soluble proteins in P. lobata were significantly reduced by elevated temperature. Results showed that tissue soluble proteins and chlorophylls in P. lobata were from two- to three-fold higher than in P. damicornis. The number of zooxanthellae in P. lobata was double that of P. damicornis. Therefore, we suggest that the slow-growing species P. lobata is better able to cope with changing environmental conditions than the fast-growing coral P. damicornis.     

A new role for the p85-phosphatidylinositol 3-kinase regulatory subunit linking FRAP to p70 S6 kinase activation.

The serine/threonine kinase p70 S6 kinase (p70S6K) phosphorylates the 40 S ribosomal protein S6, modulating the translation of an mRNA subset that encodes ribosomal proteins and translation elongation factors. p70S6K is activated in response to mitogenic stimuli and is required for progression through the G(1) phase of the cell cycle and for cell growth. Activation of p70S6K is regulated by phosphorylation of seven different residues distributed throughout the protein, a subset of which depends on the activity of p85/p110 phosphatidylinositol 3-kinase (PI3K); in fact, the phosphorylation status of Thr(229) and Thr(389) is intimately linked to PI3K activity. In the full-length enzyme, however, these sites are also acutely sensitive to the action of FKBP 12-rapamycin-associated protein (FRAP). The mechanism by which PI3K and FRAP cooperate to induce p70S6K activation remains unclear. Here we show that the p85 regulatory subunit of PI3K also controls p70S6K activation by mediating formation of a ternary complex with p70S6K and FRAP. The p85 C-terminal SH2 domain is responsible for p85 coupling to p70S6K and FRAP, because deletion of the C-terminal SH2 domain inhibits complex formation and impairs p70S6K activation by PI3K. Formation of this complex is not required for activation of a FRAP-independent form of p70S6K, however, underscoring the role of p85 in regulating FRAP-dependent p70S6K activation. These studies thus show that, in addition to the contribution of PI3K activity, the p85 regulatory subunit plays a critical role in p70S6K activation.     

A Kinetic Examination of Penicillin Acylase Stability in Water-organic Solvent Systems at Different Temperatures

We have studied the thermal stability of penicillin acylase from Streptomyces lavendulae in water-organic solvent monophasic systems at the range of temperatures between 40 and 60°C. We found a linear correlation between the log λP value of the solvent and the activation free energy for denaturation ( ΔG d ) at all temperatures tested. Thermodynamic analysis of the results indicates that solvents with log λP ≤-2.3 have protective effects, whereas solvents with log λP ≥-1.8 are deleterious for penicillin acylase.     

Estimation of coancestry in Iberian pigs using molecular markers

Genetic markers provide a useful tool toestimate pairwise coancestry betweenindividuals in the absence of a known pedigree. Inthe present work 62 pigs from two relatedstrains of Iberian breed, Guadyerbas andTorbiscal, belonging to a conservationprogramme with completely known pedigrees since1945, have been genotyped for 49microsatellites. Four coefficients thatsummarise molecular resemblance betweenindividuals together with eightestimators of coancestry have been calculatedfrom this information. Their values werecompared with the genealogical coancestry,calculated from the complete or partialpedigree. The eight estimations obtained usingmolecular information substantiallyunderestimate the coancestry calculated usingthe genealogical analysis. The correlationbetween the estimates and the genealogicalvalues was also calculated. This correlationwas high, between 0.78 and 0.93 for differentestimators, when all pairwise comparisons amongthe 62 animals were considered. However, thecorrelation decreases remarkably to 0.49–0.69and 0.37–0.47 for the Guadyerbas and Torbiscalpopulations respectively, when they wereanalysed separately. All the correlations weresimilar to those obtained when using simplecoefficients of molecular resemblance such asmolecular coancestry or similarity indexes.Finally, simulations were carried out tofurther explore the results obtained. It isconcluded that lack of information on theallele frequencies in the base population mayexplain the bias of these estimators inpopulations with complex pedigrees.     

Test for positional candidate genes for body composition on pig chromosome 6

One QTL affecting backfat thickness (BF), intramuscular fat content (IMF) and eye muscle area (MA) was previously localized on porcine chromosome 6 in an F₂ cross between Iberian and Landrace pigs. This work was done to study the effect of two positional candidate genes on these traits: H-FABP and LEPR genes. The QTL mapping analysis was repeated with a regression method using genotypes for seven microsatellites and two PCR-RFLPs in the H-FABP and LEPR genes. H-FABP and LEPR genes were located at 85.4 and 107 cM respectively, by linkage analysis. The effects of the candidate gene polymorphisms were analyzed in two ways. When an animal model was fitted, both genes showed significant effects on fatness traits, the H-FABP polymorphism showed significant effects on IMF and MA, and the LEPR polymorphism on BF and IMF. But when the candidate gene effect was included in a QTL regression analysis these associations were not observed, suggesting that they must not be the causal mutations responsible for the effects found. Differences in the results of both analyses showed the inadequacy of the animal model approach for the evaluation of positional candidate genes in populations with linkage disequilibrium, when the probabilities of the parental origin of the QTL alleles are not included in the model.