Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

Background

Brucella abortus is the etiological agent of a worldwide zoonosis called brucellosis. This alpha-proteobacterium is dividing asymmetrically, and PdhS, an essential histidine kinase, was reported to be an old pole marker.

Results

We were interested to identify functions that could be recruited to bacterial poles. The Brucella ORFeome, a collection of cloned predicted coding sequences, was placed in fusion with yellow fluorescent protein (YFP) coding sequence and screened for polar localizations in B. abortus. We report that AidB-YFP was systematically localized to the new poles and at constrictions sites in B. abortus, either in culture or inside infected HeLa cells or RAW264.7 macrophages. AidB is an acyl-CoA dehydrogenase (ACAD) homolog, similar to E. coli AidB, an enzyme putatively involved in destroying alkylating agents. Accordingly, a B. abortus aidB mutant is more sensitive than the wild-type strain to the lethality induced by methanesulphonic acid ethyl ester (EMS). The exposure to EMS led to a very low frequency of constriction events, suggesting that cell cycle is blocked during alkylation damage. The localization of AidB-YFP at the new poles and at constriction sites seems to be specific for this ACAD homolog since two other ACAD homologs fused to YFP did not show specific localization. The overexpression of aidB, but not the two other ACAD coding sequences, leads to multiple morphological defects.

Conclusions

Data reported here suggest that AidB is a marker of new poles and constriction sites, that could be considered as sites of preparation of new poles in the sibling cells originating from cell division. The possible role of AidB in the generation or the function of new poles needs further investigation.     

Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi

Background

Lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production.

Results

The study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termite's digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan- hydrolytic enzymes, pectinases and starch-degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis.

Conclusions

Here we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole body termite extracts evidenced their ability to cleave all types of glycosidic bonds present in plant polysaccharides. The comprehensive proteomic analysis, revealed a complete collection of hydrolytic enzymes including cellulases (GH1, GH3, GH5, GH7, GH9 and CBM 6), hemicellulases (GH2, GH10, GH11, GH16, GH43 and CBM 27) and pectinases (GH28 and GH29).     

Seed germination temperatures of eight Mexican Agave species with economic importance

The genetic diversity of Agave plants is threatened by clonal commercial reproduction and climatic change. Sexual reproduction is uncommon and research on seed germination is scarce. The present study evaluated the seed germination of Agave lechuguilla, Agave striata, Agave americana var. marginata, Agave asperrima, Agave cupreata, Agave duranguesis, Agave angustifolia ssp. tequilana and Agave salmiana at constant temperatures (10, 15, 20, 25, 30, 35 and 40°C). Initial imbibition (after the first 12 h) was significantly variable among species, positively correlated with seed weight (r = 0.6560, P < 0.001) and increased with temperature (from 35% at 10°C to 66% at 40°C). Temperature affected maximum imbibition (83–150%) for A. asperrima, A. lechuguilla, A. salmiana and A. striata; other species averaged 110%. Most germination kinetics best fitted a logistic model, whereas only a few treatments fit a Weibull model. The time to germination onset diminished (P < 0.05) from 125–173 h at 15°C to 68–84 h at 25°C, and then ascended to 84–196 h at 35°C. The mean germination rate and seed germination percentage after 312 h peaked at 25°C (0.50–0.95% seeds/h and 85–99%, respectively) and fell (P < 0.05) to near zero at 10 and 40°C. Temperatures of 10, 35 and 40°C were partially lethal to A. asperrima, A. duranguensis and A. salmiana seeds. The time to germination onset, seed germination percentage after 312 h and mean germination rate are best described by a Gaussian distribution, with its optimum at approximately 25°C. Thus, optimum temperatures are related to the ecological characteristics of each species area.     

Chorioallantoic placentation in Galea spixii (Rodentia,Caviomorpha, Caviidae)

Background  

Placentas of guinea pig-related rodents are appropriate animal models for human placentation because of their striking similarities to those of humans. To optimize the pool of potential models in this context, it is essential to identify the occurrence of characters in close relatives.     

Moss and liverwort xyloglucans contain galacturonic acid and are structurally distinct from the xyloglucans synthesized by hornworts and vascular plants

Xyloglucan is a well-characterized hemicellulosic polysaccharide that is present in the cell walls of all seed-bearing plants. The cell walls of avascular and seedless vascular plants are also believed to contain xyloglucan. However, these xyloglucans have not been structurally characterized. This lack of information is an impediment to understanding changes in xyloglucan structure that occurred during land plant evolution. In this study, xyloglucans were isolated from the walls of avascular (liverworts, mosses, and hornworts) and seedless vascular plants (club and spike mosses and ferns and fern allies). Each xyloglucan was fragmented with a xyloglucan-specific endo-glucanase and the resulting oligosaccharides then structurally characterized using NMR spectroscopy, MALDI-TOF and electrospray mass spectrometry, and glycosyl-linkage and glycosyl residue composition analyses. Our data show that xyloglucan is present in the cell walls of all major divisions of land plants and that these xyloglucans have several common structural motifs. However, these polysaccharides are not identical because specific plant groups synthesize xyloglucans with unique structural motifs. For example, the moss Physcomitrella patens and the liverwort Marchantia polymorpha synthesize XXGGG- and XXGG-type xyloglucans, respectively, with sidechains that contain a beta-D-galactosyluronic acid and a branched xylosyl residue. By contrast, hornworts synthesize XXXG-type xyloglucans that are structurally homologous to the xyloglucans synthesized by many seed-bearing and seedless vascular plants. Our results increase our understanding of the evolution, diversity, and function of structural motifs in land-plant xyloglucans and provide support to the proposal that hornworts are sisters to the vascular plants.     

ABC: software for interactive browsing of genomic multiple sequence alignment data

Background  

Alignment and comparison of related genome sequences is a powerful method to identify regions likely to contain functional elements. Such analyses are data intensive, requiring the inclusion of genomic multiple sequence alignments, sequence annotations, and scores describing regional attributes of columns in the alignment. Visualization and browsing of results can be difficult, and there are currently limited software options for performing this task.     

Procathepsin and acid phosphatase are stored in Musca domestica yolk spheres

Yolk spheres present in mature invertebrate oocytes are composed of yolk proteins and proteolytic enzymes. In the fly Musca domestica, yolk proteins are degraded during embryogenesis by a cathepsin-like proteinase that is stored as a zymogen. An acid phosphatase is also active in the yolk spheres during Musca embryogenesis. In this paper we show that procathepsin and acid phosphatase are initially stored by a different pathway from the one followed by yolk protein precursors. Both enzymes are taken up by the oocytes and transitorily stored into small vesicles (lysosomes) surrounding the early yolk spheres. Fusion of both structures, the early yolk spheres and lysosomes, creates the mature yolk spheres.     

Molecular domains of the cellulose/xyloglucan network in the cell walls of higher plants

Cellulose and xyloglucan (XG) assemble to form the cellulose/XG network, which is considered to be the dominant load-bearing structure in the growing cell walls of non-graminaceous land plants. We have extended the most commonly accepted model for the macromolecular organization of XG in this network, based on the structural and quantitative analysis of three distinct XG fractions that can be differentially extracted from the cell walls isolated from etiolated pea stems. Approximately 8% of the dry weight of these cell walls consists of XG that can be solubilized by treatment of the walls with a XG-specific endoglucanase (XEG). This material corresponds to an enzyme-susceptible XG domain, proposed to form the cross-links between cellulose microfibrils. Another 10% of the cell wall consists of XG that can be solubilized by concentrated KOH after XEG treatment. This material constitutes another XG domain, proposed to be closely associated with the surface of the cellulose microfibrils. An additional 3% of the cell wall consists of XG that can be solubilized only when the XEG- and KOH-treated cell walls are treated with cellulase. This material constitutes a third XG domain, proposed to be entrapped within or between cellulose microfibrils. Analysis of the three fractions indicates that metabolism is essentially limited to the enzyme-susceptible domain. These results support the hypothesis that enzyme-catalyzed modification of XG cross-links in the cellulose/XG network is required for the growth and development of the primary plant cell wall, and demonstrate that the structural consequences of these metabolic events can be analyzed in detail.     

Seed size and germination response: a relationship for fire-following plant species exposed to thermal shock

Thermal shock is well known to be an important stimulus for the germination of soil-stored seeds in fire-prone plant communities. Nevertheless, while the overall germination response of different species is known to vary, the interaction between seed size and germination to a range of thermal-shock temperatures is poorly understood. This interaction may be important in regulating post-fire plant community establishment, since larger seeds are able to emerge from deeper within the soil profile than smaller seeds, and are therefore likely to be insulated against high above-ground temperatures by a deeper soil covering. In this experiment we examined how germination of eight co-occurring Western Australian fire-followers was influenced by thermal shock, and whether germination was significantly correlated with seed size. We found that small-seeded species not only showed enhanced germination at higher temperatures, but that their ability to germinate at higher temperatures was also greater than that displayed by larger-seeded species. These findings suggest that while seed size may be a useful general predictor of post-fire recruitment success, under different fire regimes the interaction between seed size, maximum seedling emergence depth, and the ability to withstand different thermal-shock temperatures is complex and may confound recent predictive models.     

Differentiation of naive CD4<Superscript>+</Superscript> T cells towards T helper 2 cells is not impaired in rheumatoid arthritis patients

An impaired differentiation of naive CD4+ T cells towards Th2 cells may contribute to the chronic tissue-destructive T-cell activity in rheumatoid arthritis (RA). The differentiation of naive CD4+ T cells into memory Th2 cells by IL-7 in comparison with that by IL-4 was studied in RA patients and in healthy controls. Naive CD4+ T cells from peripheral blood were differentiated by CD3/CD28 costimulation in the absence of or in the presence of IL-7 and/or IL-4. The production of IFN-gamma and IL-4 was measured by ELISA and by single-cell FACS analysis to indicate Th1 and Th2 cell activity. CD3/CD28 costimulation and IL-7 were early inducers of IL-4 production, but primarily stimulated IFN-gamma production. In contrast, in short-term cultures exogenously added IL-4 did not prime for IL-4 production but suppressed IL-7-induced IFN-gamma production. Upon long-term stimulation of naive CD4+ T cells, IFN-gamma production was differentially regulated by IL-7 and IL-4, but IL-4 production was increased by both IL-7 and IL-4. IL-7 and IL-4 additively induced polarization towards a Th2 phenotype. This susceptibility of naive CD4+ T cells to become Th2 cells upon culture with IL-7 and IL-4 was increased in RA patients compared with that in healthy controls. These findings demonstrate that, in RA patients, differentiation of naive CD4+ T cells towards a Th2 phenotype by CD3/CD28 costimulation, IL-7 and IL-4 is not impaired. The perpetuation of arthritogenic T-cell activity in RA therefore seems not to be the result of intrinsic defects of naive CD4+ T cells to develop towards suppressive memory Th2 cells.