Thermotolerance and molecular chaperone function of an SGT1-like protein from the psychrophilic yeast, <Emphasis Type="Italic">Glaciozyma antarctica</Emphasis>

The ability of eukaryotes to adapt to an extreme range of temperatures is critically important for survival. Although adaptation to extreme high temperatures is well understood, reflecting the action of molecular chaperones, it is unclear whether these molecules play a role in survival at extremely low temperatures. The recent genome sequencing of the yeast Glaciozyma antarctica, isolated from Antarctic sea ice near Casey Station, provides an opportunity to investigate the role of molecular chaperones in adaptation to cold temperatures. We isolated a G. antarctica homologue of small heat shock protein 20 (HSP20), GaSGT1, and observed that the GaSGT1 mRNA expression in G. antarctica was markedly increased following culture exposure at low temperatures. Additionally, we demonstrated that GaSGT1 overexpression in Escherichia coli protected these bacteria from exposure to both high and low temperatures, which are lethal for growth. The recombinant GaSGT1 retained up to 60 % of its native luciferase activity after exposure to luciferase-denaturing temperatures. These results suggest that GaSGT1 promotes cell thermotolerance and employs molecular chaperone-like activity toward temperature assaults.     

Differential accumulation of Xanthomonas campestris pv. campestris proteins during the interaction with the host plant: Contributions of an in vivo system

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot, a highly destructive disease that affects all brassicas. This work aimed to study the interaction Xcc–Brassica oleracea using an in vivo system in an attempt to identify proteins involved in pathogenicity. We used label‐free shotgun 2D‐nanoUPLC/MS^E to analyze Xcc proteins in three conditions: in the interaction with susceptible (REK) and resistant (REU) plants and in culture medium (control condition). A model of Xcc–susceptible host interaction is proposed and shows that Xcc increases the abundance of several crucial proteins for infection and cell protection. In this study, we also confirmed the differential expression by qPCR analysis of selected genes. This is the first report showing a large‐scale identification of proteins in an in vivo host plant condition. Considering that most studies involving phytopathogens are in vitro (growth in culture medium or in plant extract), this work contributes with relevant information related to the plant–pathogen interaction in planta.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Optimization of medium composition for the production of antimicrobial activity by Bacillus subtilis B38

An antimicrobial activity produced by Bacillus subtilis B38 was found to be effective against several bacteria, including pathogenic and spoilage microorganisms such as, Listeria monocytogenes, Salmonella enteridis, and clinical isolates of methicillin‐resistant Staphylococcus species. Nutrients such as carbon, nitrogen sources, and inorganic salts enhanced the production level of the antibacterial activity by B. subtilis B38. A first screening step showed that lactose, ammonium succinate, and manganese most influenced both cell growth and antibacterial activity production. These three factors varied at two levels in eight experiments using full factorial design. Results indicated that maximum cell growth (OD = 10.2) and maximum production of antibacterial activity (360 AU/mL) were obtained in a modified medium containing 1.5% (w/v) lactose, 0.15% (w/v) ammonium succinate, and 0.3 mg/L manganese. Depending on the indicator strain used, the antibacterial activity was 2‐ to 4‐fold higher in the modified culture medium than in TSB medium under the same conditions. Thin layer chromatography‐bioautography assay showed the presence of three active spots with R_f values of 0.47, 0.7, and 0.82 in TSB medium. However, the inhibition zone of two spots (R_f values of 0.7 and 0.82) was slightly larger in the modified medium. Moreover, a large zone of inhibition with an R_f value of 0.3, was observed in this modified medium, instead of the spot having an R_f value of 0.47. These results suggest that the nutrients act as environmental factors, quantitatively and qualitatively affecting the production of antibacterial compounds by B. subtilis B38. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A generic model of electron transport in mitochondria

In this paper, a simplified, generic model of mitochondrial metabolism is explored. In particular the following question is addressed: To what extent are phenomena observed in experiments and simulations of mitochondrial metabolism generic, in the sense that they must occur in all models with this basic structure? Of particular interest are the electron transport chain and oxidative phosphorylation, and how flux through the system and the redox states of intermediates respond to physiologically important stimuli. These stimuli include changes in substrate supply (NADH/FADH(2)), in oxygenation, and in membrane proton gradient/ATP demand. Analytical techniques are used to show that certain experimentally observed effects must occur in the generic model. These include the responses of both flux and redox states to changed substrate and oxygen concentrations. At the same time other effects, such as the responses of redox states to changes in proton gradient, are dependent on the details of the model, and are not common to every model with the same basic structure. The phenomenon of saturation in response to large inputs is also discussed.     

Effect of salinity on plant growth and biological activities of Carthamus tinctorius L. extracts at two flowering stages

In the present study, we were interested in the effect of salt stress on phenolic and carotenoid contents, antioxidant and antimicrobial activity of two varieties of Carthamus tinctorius (Jawhara and 104) flowers. For this purpose, C. tinctorius flowers from plants grown under four saline treatments (0, 5, 10 and 15 g/L NaCl) were collected at two development stages. As salinity increased up to 10 g/L, results showed that total phenols, flavonoids, condensed tannins and carotenoid contents increased with salinity. Such variability might be of great importance in terms of valorizing this plant as a source of naturally secondary metabolites. Furthermore, our results showed an enhancement of antioxidant activity which was evaluated by four different test systems (DPPH, β-carotene–linoleic acid, chelating and reducing power assays) with increasing stress severity. Obtained results showed that, for the two varieties, salt effect was more pronounced at post flowering stage than full flowering one. The sensitivity test of the methanolic extracts of the harvested flowers was applied against seven human pathogenic bacteria and three yeast strains. Salinity reduced significantly the antimicrobial activity of flower extracts.