Contribution of ExsFA and ExsFB proteins to the localization of BclA on the spore surface and to the stability of the bacillus anthracis exosporium

Spores of Bacillus anthracis, the etiological agent of anthrax, and the closely related species Bacillus cereus and Bacillus thuringiensis, possess an exosporium, which is the outermost structure surrounding the mature spore. It consists of a paracrystalline basal layer and a hair-like outer layer. To date, the structural contribution of only one exosporium component, the collagen-like glycoprotein BclA, has been described. It is the structural component of the hair-like filaments. Here, we describe two other proteins, ExsFA and ExsFB, which are probably organized in multimeric complexes with other exosporium components, including BclA. Single and double exsF deletion mutants were constructed and analyzed. We found that inactivation of exsF genes affects the BclA content of spores. BclA is produced by all mutants. However, it is partially and totally released after mother cell lysis of the DeltaexsFA and DeltaexsFA DeltaexsFB mutant strains, respectively. Electron microscopy revealed that the exsF mutant spores have defective exosporia. The DeltaexsFA and DeltaexsFA DeltaexsFB spore surfaces are partially and totally devoid of filaments, respectively. Moreover, for all mutants, the crystalline basal layer appeared unstable. This instability revealed the presence of two distinct crystalline arrays that are sloughed off from the spore surface. These results indicate that ExsF proteins are required for the proper localization of BclA on the spore surface and for the stability of the exosporium crystalline layers.     

Diversity of the C-terminal portion of the biphenyl dioxygenase large subunit

The biphenyl dioxygenase (BPDO) catalyses a stereospecific dioxygenation of biphenyl and analogs of it. Aside from being involved in the destruction and detoxification of toxic pollutants in soil, in the context of the green chemistry concept, this enzyme is a promising biocatalyst to design new more selective and more environmentally friendly approaches to manufacture fine chemicals. At this time, most of our knowledge about the variability of key residues determining the substrate specificity and regiospecificity of the enzyme oxygenase component (BphAE) toward biphenyl analogs and about the effect of altering these residues on catalytic properties is based on investigations made with BphAEs from cultured organisms and engineered enzymes derived from them. The purpose of this work was to examine the diversity of the amino acid sequence patterns of the alpha subunit (BphA) C-terminal domain deduced from PCR products amplified from DNA extracted from cultured bacteria of various phylogenetic lines and from the soil microflora of PCB-contaminated soils. Of special interest were segments of the C-terminal portion called regions I, III and IV. Altogether, the phylogenetic tree obtained from aligning the deduced amino acid sequences of BphAs C-terminal domain from cultured bacteria belonging to various ecological niches and from uncultured soil bacteria reveals that most of the BphAs were linked to the three clusters of BphAs previously reported. However, few belong to new branches that diverge from the previously known branches showing a high diversity of BphAs in natural environment. Furthermore, data show a wide distribution of BphAs with family linkages that not only crosses bacterial taxonomic frontiers but also ecological niches. Nevertheless, in spite of this divergence, the sequence patterns of regions III and IV amino acids that are known to influence substrate specificity and regiospecificity are rather conserved among BphAs and the pattern was independent of the family cluster to which they belong. In most cases, regions III and IV amino acid patterns are closer to those of Pseudomonas pseudoalcaligenes KF707 BphA1 than to the most versatile Burkholderia xenovorans LB400 BphA. This might suggest that the PCB-degrading potency of soil bacteria is closer to the one observed for KF707 BphAE than from LB400 BphAE. However, the fact that among less than 20 PCR products amplified from soil DNA that we have sequenced, one of them was very homologous to that of LB400 BphA and in addition, residues 335 and 336 of LB400 were replaced by residues that previous enzyme engineering had shown to extend the range of PCB substrate used by the enzyme strongly suggest that PCB-degrading bacteria are evolving in soil to optimize their PCB-degrading capacity.     

Effect of culture medium composition on inhibition of growth ofNeisseria gonorrhoeae by lactobacilli

The role of genital microorganisms in resistance to gonococcal infection is usually based on their in vitro inhibition of gonococcal growth. Three different culture media (GC, DSA, and MRS) were evaluated for their ability to support the growth of 23 lactobacilli strains and the detection of the antigonococcal activity of these bacteria. The MRS medium was the most suitable medium for the growth of lactobacilli since it favored a good growth of all the lactobacilli strains tested, but it was inhibitory toNeisseria gonorrhoeae. Decreasing the concentration of Tween 80, ammonium citrate, and sodium acetate to one-tenth of their original concentrations yielded a modified MRS medium which still supported good growth of the lactobacilli and was no longer inhibitory to the gonococci. While GC medium did not allow any detection of the production of antigonococcal activity by the lactobacilli, both modified MRS and DSA media allowed the detection of this activity by the agar overlay technique. The use of modified MRS medium is recommended since it is less selective than DSA medium for the growth of lactobacilli.     

Can tobacco have a potentially beneficial effect to our health?

With urgent pressure to clean up the contaminated environment, new approaches are needed. Phyto- and rhizoremediation using plants and related bacteria is a promising approach, but has its inborn limitations. To overcome the slow performance of the process, transgenic plants have been prepared specifically tailored for phytoremediation purposes. Our projects addressed a group of widespread synthetic organic xenobiotics, polychlorinated biphenyls (PCBs), and heavy metals as representatives of inorganic contaminants. Beside basic research studies in the field of phyto/rhizoremediation of the mentioned toxicants we focused on genetically modified plants as a highly promising tool for these purposes. We tried to prepare tobacco plants expressing the bacterial enzyme responsible for cleaving PCBs, coded by the gene bphC from the bacterial biphenyl operon. The expression of bphC product in fusion with the green fluorescent protein is described together with evaluation of the twice increased resistance of transgenic seeds towards PCBs. The other model is addressing improvement of cadmium accumulation by preparing plants bearing fused transgenes of metal binding protein (yeast metallothionein) with an introduced additional metal binding domain--polyhistidine anchor with high affinity to metals. The genetically modified plants exhibit 190% Cd accumulation of the control in harvestable parts, higher resistance and lower Cd content in roots. The performance of the plants in real contaminated soil is also evaluated.     

Comparison between Itraconazole and Cotrimoxazole in the Treatment of Paracoccidiodomycosis

Background

There are no published reports on studies comparing itraconazole (ITC), sulfamethoxazole-trimethoprim (cotrimoxazole, CMX), and ITC followed by CMX (ITC/CMX) in the treatment of paracoccidiodomycosis. This study aimed to compare the efficacy, effectiveness, safety and time to clinical and serologic cure in paracoccidioidomycosis patients treated with ITC or CMX, the antifungal agents most widely used.

Methodology

A quasi-experimental study was performed in 177 patients with a confirmed or probable diagnosis of paracoccidioidomycosis. Treatment was divided into two stages: 1) initial, which was continued until clinical cure was achieved and the erythrocyte sedimentation rate decreased to normal values; 2) complementary, which was continued until serologic cure was achieved. Medians were compared via the Mann-Whitney test, and frequencies were compared via the chi-squared test. The assessment of variables as a function of time was performed using Kaplan-Meier curves and Cox regression. The significance level was established as p≤0.05.

Principal Findings

No difference was found in the efficacy and effectiveness of the initial treatment of 47 individuals given ITC and 130 individuals given CMX; however, the time to clinical cure was shorter in the former compared with the latter group (105 vs. 159 days; p = 0.001), specifically in patients with the chronic form. Efficacy and effectiveness of the three regimens were similar in the complementary treatment; however, the time to serologic cure was shorter when ITC (161 days) or CMX (495 days) was used compared with ITC/CMX (881 days) [p = 0.02]. The independent predictors of a shorter time to serologic cure were treatment with ITC [risk ratio = 6.61 (2.01–21.75)] or with CMX [risk ratio = 5.11 (1.91–13.67)]). The prevalence of side effects was lower with ITC (6.4%) than with CMX (20.0%; p = 0.03).

Conclusions

Since ITC induced earlier clinical cure and was better tolerated than CMX, such triazole should be considered the first-choice for PCM treatment.     

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.