Divergence Involving Global Regulatory Gene Mutations in an Escherichia coli Population Evolving under Phosphate Limitation

Many of the important changes in evolution are regulatory in nature. Sequenced bacterial genomes point to flexibility in regulatory circuits but we do not know how regulation is remodeled in evolving bacteria. Here, we study the regulatory changes that emerge in populations evolving under controlled conditions during experimental evolution of Escherichia coli in a phosphate-limited chemostat culture. Genomes were sequenced from five clones with different combinations of phenotypic properties that coexisted in a population after 37 days. Each of the distinct isolates contained a different mutation in 1 of 3 highly pleiotropic regulatory genes (hfq, spoT, or rpoS). The mutations resulted in dissimilar proteomic changes, consistent with the documented effects of hfq, spoT, and rpoS mutations. The different mutations do share a common benefit, however, in that the mutations each redirect cellular resources away from stress responses that are redundant in a constant selection environment. The hfq mutation lowers several individual stress responses as well the small RNA-dependent activation of rpoS translation and hence general stress resistance. The spoT mutation reduces ppGpp levels, decreasing the stringent response as well as rpoS expression. The mutations in and upstream of rpoS resulted in partial or complete loss of general stress resistance. Our observations suggest that the degeneracy at the core of bacterial stress regulation provides alternative solutions to a common evolutionary challenge. These results can explain phenotypic divergence in a constant environment and also how evolutionary jumps and adaptive radiations involve altered gene regulation.

A Survey of Fungal Contamination on Books in Public Libraries with Mechanical and Natural Ventilation

Libraries are very propitious environments for the growth of fungi. The great concentration of organic material available for these microorganisms, and often with the lack of adequate ventilation or climate control, would favour this situation. This study was conducted in 2003 to determine the predominant genera of fungi in public libraries by a survey of fungi contaminating the upper surface of books, with and without air conditioning in the city of Sao Paulo, Brazil, in the winter and summer, during the respective periods with high and low levels of airborne fungi in that city. Six libraries were chosen, located on the campus of the University of Sao Paulo, three of them with air conditioning and the other three with natural ventilation. In these six libraries, 31 genera of fungi were identified in total. The genera and frequency of contaminant fungi recovered differed significantly between the libraries with and without air conditioning and in the samples collected in the summer as opposed to the winter. Cladosporium was the most frequent in the libraries with and without air conditioning, and in the winter. Aspergillus was isolated more often in the summer.

Biochemical characterization of potential virulence markers in the human fungal pathogen Pseudallescheria boydii

The ubiquitous Pseudallescheria boydii (anamorph Scedosporium apiospermum) is a saprophytic filamentous fungus recognized as a potent etiologic agent of a wide variety of infections in immunocompromised as well as in immunocompetent patients. Very little is known about the virulence factors expressed by this fungal pathogen. The present review provides an overview of recent discoveries related to the identification and biochemical characterization of potential virulence attributes produced by P. boydii, with special emphasis on surface and released molecules. These structures include polysaccharides (glucans), glycopeptides (peptidorhamnomannans), glycolipids (glucosylceramides) and hydrolytic enzymes (proteases, phosphatases and superoxide dismutase), which have been implicated in some fundamental cellular processes in P. boydii including growth, differentiation and interaction with host molecules. Elucidation of the structure of cell surface components as well as the secreted molecules, especially those that function as virulence determinants, is of great relevance to understand the pathogenic mechanisms of P. boydii.

The opportunistic fungal pathogen Scedosporium prolificans: Carbohydrate epitopes of its glycoproteins

Isolated from the mycelium, of Scedosporium prolificans were complex glycoproteins (RMP-Sp), with three structurally related components (HPSEC). RMP-Sp contained 35% protein and 62% carbohydrate with Rha, Ara, Man, Gal, Glc, and GlcNH(2) in a 18:1:24:8:6:5 molar ratio. Methylation analysis showed mainly nonreducing end- of Galp (13%), nonreducing end- (9%),2-O-(13%), and 3-O-subst. Rhap (7%), nonreducing end-(11%), 2-O-(10%), 3-O-(14%), and 2,6-di-O-subst. Manp units (13%). Mild reductive P-elimination of RMP-Sp gave alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-Manp-(1-->2)-D-Man-ol, with Man-ol substituted at O-6 with beta-D-Galp units, a related pentasaccharide lacking beta-D-Galp units, and beta-D-Galp-(1-->6)-[alpha-D-Manp-(1-->2)]-D-Man-ol in a 16:3:1 w/w ratio. Traces of Man-ol and Rha-ol were detected. ESI-MS showed HexHex-o1 and HCX(3-6)Hex-ol components. Three rhamnosyl units were peeled off successively from the penta- and hexasaccharide by ESI-MS-MS. The carbohydrate epitopes of RMP-Sp differ from those of the glycoprotein of Pseudallescheria boydii, a related opportunistic pathogen. (C) 2007 Elsevier B.V. All rights reserved.

Metallopeptidase inhibitors arrest vital biological processes in the fungal pathogen Scedosporium apiospermum

P>Scedosporium apiospermum is an emerging agent of opportunistic mycoses in humans. Previously, we showed that mycelia of S. apiospermum secreted metallopeptidases which were directly linked to the destruction of key host proteins. In this study, we analysed the effect of metallopeptidase inhibitors on S. apiospermum development. As germination of inhaled conidia is a crucial event in the infectious process of S. apiospermum, we studied the morphological transformation induced by the incubation of conidia in Sabouraud-dextrose medium at 37 degrees C. After 6 h, some conidia presented a small projection resembling a germ-tube. A significant increase, around sixfold, in the germ-tube length was found after 12 h, and hyphae were exclusively observed after 24 h. Three distinct metallopeptidase inhibitors were able to arrest the transformation of conidia into hyphae in different ways; for instance, 1,10-phenanthroline (PHEN) completely blocked this process at 10 mu mol l-1, while ethylenediamine tetraacetic acid (EDTA) and ethylene glycol-bis (beta-aminoethyl ether; EGTA) only partially inhibited the differentiation at up to 10 mmol l-1. EGTA did not promote any significant reduction in the conidial growth, while PHEN and EDTA, both at 10 mmol l-1, inhibited the proliferation around 100% and 65%, respectively. The secretion of polypeptides into the extracellular environment and the metallopeptidase activity secreted by mycelia were completely inhibited by PHEN. These findings suggest that metallo-type enzymes could be potential targets for future therapeutic interventions against S. apiospermum.

The CATH Hierarchy Revisited-Structural Divergence in Domain Superfamilies and the Continuity of Fold Space

This paper explores the structural continuum in CATH and the extent to which superfamilies adopt distinct folds. Although most superfamilies are structurally conserved, in some of the most highly populated superfamilies (4% of all superfamilies) there is considerable structural divergence. While relatives share a similar fold in the evolutionary conserved core, diverse elaborations to this core can result in significant differences in the global structures. Applying similar protocols to examine the extent to which structural overlaps occur between different fold groups, it appears this effect is confined to just a few architectures and is largely due to small, recurring super-secondary motifs (e.g., alpha beta-motifs, alpha-hairpins). Although 24% of superfamilies overlap with superfamilies having different folds, only 14% of nonredundant structures in CATH are involved in overlaps. Nevertheless, the existence of these overlaps suggests that, in some regions of structure space, the fold universe should be seen as more continuous.

The CATH classification revisited-architectures reviewed and new ways to characterize structural divergence in superfamilies

The latest version of CATH (class, architecture, topology, homology) (version 3.2), released in July 2008 (http://www.cathdb.info), contains 1 14215 domains, 2178 Homologous superfamilies and 1110 fold groups. We have assigned 20 330 new domains, 87 new homologous superfamilies and 26 new folds since CATH release version 3.1. A total of 28 064 new domains have been assigned since our NAR 2007 database publication (CATH version 3.0). The CATH website has been completely redesigned and includes more comprehensive documentation. We have revisited the CATH architecture level as part of the development of a `Protein Chart` and present information on the population of each architecture. The CATHEDRAL structure comparison algorithm has been improved and used to characterize structural diversity in CATH superfamilies and structural overlaps between superfamilies. Although the majority of superfamilies in CATH are not structurally diverse and do not overlap significantly with other superfamilies, similar to 4% of superfamilies are very diverse and these are the superfamilies that are most highly populated in both the PDB and in the genomes. Information on the degree of structural diversity in each superfamily and structural overlaps between superfamilies can now be downloaded from the CATH website.

Metabolomic assessment with CE-MS of the nutraceutical effect of Cystoseira spp extracts in an animal model

There is a need of scientific evidence of claimed nutraceutical effects, but also there is a social movement towards the use of natural products and among them algae are seen as rich resources. Within this scenario, the development of methodology for rapid and reliable assessment of markers of efficiency and security of these extracts is necessary. The rat treated with streptozotocin has been proposed as the most appropriate model of systemic oxidative stress for studying antioxidant therapies. Cystoseira is a brown alga containing fucoxanthin and other carothenes whose pressure-assisted extracts were assayed to discover a possible beneficial effect on complications related to diabetes evolution in an acute but short-term model. Urine was selected as the sample and CE-TOF-MS as the analytical technique to obtain the fingerprints in a non-target metabolomic approach. Multivariate data analysis revealed a good clustering of the groups and permitted the putative assignment of compounds statistically significant in the classification. Interestingly a group of compounds associated to lysine glycation and cleavage from proteins was found to be increased in diabetic animals receiving vehicle as compared to control animals receiving vehicle (N6, N6, N6-trimethyl-L-lysine, N-methylnicotinamide, galactosylhydroxylysine, L-carnitine, N6-acetyl-N6-hydroxylysine, fructose-lysine, pipecolic acid, urocanic acid, amino-isobutanoate, formylisoglutamine. Fructoselysine significantly decreased after the treatment changing from a 24% increase to a 19% decrease. CE-MS fingerprinting of urine has provided a group of compounds different to those detected with other techniques and therefore proves the necessity of a cross-platform analysis to obtain a broad view of biological samples.