An alkaliphilic cyclodextrin glycosyltransferase from a new Bacillus agaradhaerens WN-I strain isolated from an Egyptian soda lake: Purification and properties

Alkaliphilic bacteria were isolated from soil and water samples obtained from Egyptian soda lakes in the Wadi Natrun area. Screening for cyclodextrin glycosyltransferase- producing alkaliphilic bacteria resulted in the isolation of 15 positive strains. Strain WN-I was selected as the best producer of CGTase. 16S rDNA sequence analysis and DNA-DNA hybridization identified the isolate as Bacillus agaradhaerens. The enzyme was purified to homogeneity up to 21 fold by starch adsorption and anion exchange chromatography with a yield of 26.40%. The pure enzyme was a monomer with an estimated molecular weight of 85 kDa. The enzyme was stable, at 25°C, over a pH range of 5.0 to 11, with a maximum activity at pH 9.0. The enzyme activity exhibited an optimum temperature of 55°C and was stable at 40°C for at least 1 h. Thermal stability was improved in the presence of maltodextrin, starch or CaCl2. The enzyme was slightly stimulated by CaCl2, KC1 and BaCl2 but was completely inhibited in the presence of FeCl2 and strongly inhibited by ZnCl2 and CoCl2 and to a lower extent by CuCl2, EDTA, 2-mercaptoethanol, and dithiothritol. The enzyme produced mainly β-CD (71.20% of the total cyclodextrin amount). The enzyme had higher cyclyzation activity (1.9 fold higher) toward Paselli starch than soluble starch.Key words: Alkaliphiles, soda lakes, cyclodextrin glycosyltransferase, Bacillus agaradhaeren, purification, 16S rDNA

Bioreduction of Cr (VI) by potent novel chromate resistant alkaliphilic Bacillus sp. strain KSUCr5 isolated from hypersaline Soda lakes

Isolation of Cr (VI) resistant alkaliphilic bacteria from sediment and water samples collected from Wadi Natrun hypersaline Soda lakes (located in northern Egypt), resulted in isolation of several alkaliphilic bacterial strains that can tolerate up to 2.94 g/l of Cr (VI) in alkaline medium. However, with increasing Cr (VI) concentration up to 29.4 g/l, only one strain, KSUCr5, was able to tolerate up to 22 g/l (75 mM) and with MIC value of 23.5 g/l (80 mM) in alkaline medium (pH 10.5) containing 10% NaCl. Based on the 16S rRNA gene analysis, strain KSUCr5 was identified as Bacillus sp. with 99% similarity and was referred to as Bacillus sp. KSUCr5. In addition, Bacillus sp. strain KSUCr5 showed high tolerance to several other heavy metals including Cd2+ (50 mM), Mo2+ (75 mM), Mn2+ (100 mM), Cu2+ (2 mM), Ni2+ (100 mM), Pb (75 mM), Co2+ (5 mM) and Zn2+ (2 mM). Strain KSUCr5 was shown to be of a high efficiency in detoxifying chromate, as it could rapidly reduce up to 40 mg/l of Cr(VI) to a non detectable level over 24 h. In addition, at initial Cr(VI) concentration of 60 to 80 and 100 mg/l, 100% of the chromate reduction was achieved within 48 and 72 h, respectively. Strain KSUCr5 could reduce Cr(VI) efficiently  over a wide range of initial Cr(VI) concentrations (10 to 300 mg/l) in alkaline medium under aerobic conditions without significant effect on the bacterial growth. It was able to reduce Cr(VI) in a wide range of NaCl (0 to 20%) with a maximum reduction yield at concentration of 0 to 1.5%, indicating the halo tolerance nature of the bacterium. It was found that addition of glucose and Na2CO3 to the culture medium caused a dramatic increase in Cr(VI)-reduction by Bacillus sp. strain KSUCr5. The maximum chromate removal was exhibited in alkaline medium (pH 10) containing 1.2% Na2CO3, 1.5% glucose and 1% NaCl and at incubation temperature of 35°C and culture shaking of 150 rpm. Under optimum Cr (VI) reduction conditions, Cr(VI) concentration of 80 mg/l was completely reduced within 24 h, with reduction rate of 3.3 mg h-1 which is one of the highest Cr(VI) reduction rate under high alkaline conditions, compared with other microorganisms that has been reported so far. Furthermore, the presence of other metals such as Ni2+, Mo2+, Cu2+ and Mn2+ at concentration of 100 mg/l together with Cr(VI) in the culture medium slightly increased Cr(VI)-reduction by the strain KSUCr5. Moreover, the isolate, Bacillus sp.  strain KSUCr5, exhibited an ability to repeatedly reduce hexavalent chromium without any amendment of nutrients, suggesting its potential application in continuous bioremediation of Cr(VI). The results reveal the possible isolation of potent heavy metals resistant bacteria from extreme environment such as hypersaline Soda lakes and their application in bioremediation of heavy metals.Key words: Chromate reduction, bioremediation, heavy metals, Bacillus sp., Soda lakes

In vitro growth inhibition of bloodstream forms of Trypanosoma brucei and Trypanosoma congolense by iron chelators

African trypanosomes exert significant morbidity and mortality in man and livestock. Only a few drugs are available for the treatment of trypanosome infections and therefore, the development of new anti-trypanosomal agents is required. Previously it has been shown that bloodstream-form trypanosomes are sensitive to the iron chelator deferoxamine. In this study the effect of 13 iron chelators on the growth of Trypanosoma brucei, T. congolense and human HL-60 cells was tested in vitro. With the exception of 2 compounds, all chelators exhibited anti-trypanosomal activities, with 50% inhibitory concentration (IC(50)) values ranging between 2.1 – 220 μM. However, the iron chelators also displayed cytotoxicity towards human HL-60 cells and therefore, only less favourable selectivity indices compared to commercially available drugs. Interfering with iron metabolism may be a new strategy in the treatment of trypanosome infections. More specifically, lipophilic iron-chelating agents may serve as lead compounds for novel anti-trypanosomal drug development

Liver cell therapy: is this the end of the beginning?

The prevalence of liver diseases is increasing globally. Orthotopic liver transplantation is widely used to treat liver disease upon organ failure. The complexity of this procedure and finite numbers of healthy organ donors have prompted research into alternative therapeutic options to treat liver disease. This includes the transplantation of liver cells to promote regeneration. While successful, the routine supply of good quality human liver cells is limited. Therefore, renewable and scalable sources of these cells are sought. Liver progenitor and pluripotent stem cells offer potential cell sources that could be used clinically. This review discusses recent approaches in liver cell transplantation and requirements to improve the process, with the ultimate goal being efficient organ regeneration. We also discuss the potential off-target effects of cell-based therapies, and the advantages and drawbacks of current pre-clinical animal models used to study organ senescence, repopulation and regeneration

In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where (together with fibrin and platelets) it initiates the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin detected S. aureus in endocarditic vegetations via noninvasive fluorescence or PET imaging. These prothrombin derivatives bound to staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor