Mechanisms of neutralization of toxSAS from toxin-antitoxin modules.

Toxic small alarmone synthetase (toxSAS) enzymes constitute a family of bacterial effectors present in toxin-antitoxin and secretion systems. toxSASs act through either translation inhibition mediated by pyrophosphorylation of transfer RNA (tRNA) CCA ends or synthesis of the toxic alarmone adenosine pentaphosphate ((pp)pApp) and adenosine triphosphate (ATP) depletion, exemplified by FaRel2 and FaRel, respectively. However, structural bases of toxSAS neutralization are missing. Here we show that the pseudo-Zn2+ finger domain (pZFD) of the ATfaRel2 antitoxin precludes access of ATP to the pyrophosphate donor site of the FaRel2 toxin, without affecting recruitment of the tRNA pyrophosphate acceptor. By contrast, (pp)pApp-producing toxSASs are inhibited by Tis1 antitoxin domains though occlusion of the pyrophosphate acceptor-binding site. Consequently, the auxiliary pZFD of AT2faRel is dispensable for FaRel neutralization. Collectively, our study establishes the general principles of toxSAS inhibition by structured antitoxin domains, with the control strategy directly coupled to toxSAS substrate specificity.

Biochemical and X-ray analyses of the players involved in the faRel2/aTfaRel2 toxin-antitoxin operon.

The aTfaRel2/faRel2 operon from Coprobacillus sp. D7 encodes a bicistronic type II toxin-antitoxin (TA) module. The FaRel2 toxin is a toxic small alarmone synthetase (toxSAS) that inhibits translation through the pyrophosphorylation of uncharged tRNAs at the 3'-CCA end. The toxin is neutralized by the antitoxin ATfaRel2 through the formation of an inactive TA complex. Here, the production, biophysical analysis and crystallization of ATfaRel2 and FaRel2 as well as of the ATfaRel2-FaRel2 complex are reported. ATfaRel2 is monomeric in solution. The antitoxin crystallized in space group P21212 with unit-cell parameters a = 53.3, b = 34.2, c = 37.6 Å, and the best crystal diffracted to a resolution of 1.24 Å. Crystals of FaRel2 in complex with APCPP, a nonhydrolysable ATP analogue, belonged to space group P21, with unit-cell parameters a = 31.5, b = 60.6, c = 177.2 Å, ß = 90.6°, and diffracted to 2.6 Šresolution. The ATfaRel2-FaRel2Y128F complex forms a heterotetramer in solution composed of two toxins and two antitoxins. This complex crystallized in two space groups: F4132, with unit-cell parameters a = b = c = 227.1 Å, and P212121, with unit-cell parameters a = 51.7, b = 106.2, c = 135.1 Å. The crystals diffracted to 1.98 and 2.1 Šresolution, respectively.

Bacterial contamination of boar semen affects the litter size.

One hundred and fifteen semen samples were collected from 115 different boars from two farms in Cuba. The boars belonged to five different breeds. Evaluation of the semen sample characteristics (volume, pH, colour, smell, motility of sperm cells) revealed that they meet international standards. The samples were also tested for the presence of agglutinated sperm cells and for bacterial contamination. Seventy five percent of the ejaculates were contaminated with at least one type of bacteria and E. coli was by far the major contaminant, being present in 79% of the contaminated semen samples (n=68). Other contaminating bacteria belonged to the genera Proteus (n=31), Serratia (n=31), Enterobacter (n=24), Klebsiella (n=12), Staphylococcus (n=10), Streptococcus (n=8) and Pseudomonas (n=7). Only in one sample anaerobic bacteria were detected. Pearson's analysis of the data revealed that there is a positive correlation between the presence of E. coli and sperm agglutination, and a negative correlation between sperm agglutination and litter size. One-way ANOVA and post hoc Tukey analysis of 378 litters showed that the litter size is significantly reduced when semen is used that is contaminated with spermagglutinating E. coli above a threshold value of 3.5x10(3)CFU/ml.

Stability, subunit interactions and carbohydrate-binding of the seed lectin from Pterocarpus angolensis.

From 1 kg of defatted Pterocarpus angolensis (mukwa tree) seed meal, 21.6 grams of an alpha,D-mannose/glucose-specific lectin can be purified on mannose-Sepharose. Relative affinities for several (oligo)saccharides and glycoproteins were studied by haemagglutination-inhibition. Gel filtration shows that the lectin exists as a dimer above pH 5 and as a monomer below pH 3.5. This is confirmed by studies on the release of lectin subunits that were adsorbed from solution to lectin monomers immobilized onto Eupergit-c. From the gel filtration patterns it is calculated that a residue with pK(a) of about 4.4 is involved in dimer dissociation. Titration of glutamic acids (E60, E209) is postulated to be involved. CD spectroscopy shows that the secondary structure of the lectin is unchanged between pH 1 and 12.5, and that the tertiary structure remains unchanged between pH 5 and 12. In the acid pH region, reversible spectral changes occur that may be due to the titration of one or more amino acids with a pK(a) value of 3.9-4.2, probably aspartic acid. These residues are implicated in sugar-binding but not in dimerization of the lectin. Only at pH 12.5, irreversible denaturation occurs. Mukwa lectin displays full carbohydrate-binding capacity between pH 4 and 12, as is concluded from ELLA (Enzyme Linked Lectin Assay) using ovalbumin and fetuin, and from binding of the same glycoproteins to immobilized lectin monomers. The lectin is rapidly and fully reversibly demetallized at pH 2.5 with 5 mM EDTA. The demetallized lectin is completely devoid of sugar-binding activity. Mukwa lectin is a very thermostable molecule (at least till 85 degrees C). However, addition of non-ionic detergents substantially lowers its thermostability.