Proteolytic Activity of Pseudomonas aeruginosa Isolates with TTSS-Mediated Cytotoxicity and Invasiveness to Host Cells

Over 100 of Pseudomonas aeruginosa isolates representing the two TTSS genotypes (exoU ⁻/exoS ⁺ or exoU ⁺/exoS ⁻) were cultured in different media in order to evaluate their proteolytic activities and find a relationship between proteolytic activity and the cytotoxic and/or invasive phenotypes displayed by the strains upon infection of RAW 264.7 murine macrophage-like cells and pulmonary microvascular endothelial cells (PME). The elastolytic activity, protein concentration, and total proteolytic activity (TPA) were measured in culture supernatants. No significant differences were observed in the median elastolytic activities among cytotoxic/noninvasive, noncytotoxic/invasive, and cytotoxic/invasive phenotypes displayed by P. aeruginosa strains. The only significant difference was noted when isolates of the two different TTSS genotypes were grown in a calcium-depleted minimal medium for induction of TTSS (MI). The exoU ⁻/exoS ⁺ isolates showed significant higher levels of the median elastolytic activity when compared to the exoU ⁺/exoS ⁻ isolates. These two groups of isolates secreted the elastase B (LasB) with distinct molecular masses 158 or 116 kD, respectively. The strains of the two TTSS genotypes secreted similar amount of total proteins; however, the higher values of TPA were observed for the isolates of the exoU ⁺ /exoS ⁻ genotype when grown in MI medium. We concluded that there is no direct relationship between secretion of proteases with elastolytic activity and the cytotoxic and/or invasive phenotypes of the isolates observed upon infection of both RAW 264.7 and PME monolayers. Further studies are needed to find out whether others factors beside proteases could influence the mechanism of host cells intoxication mediated by the P. aeruginosa TTSS-delivered toxins.     

ABA may affect the dormancy in triticale caryopses by inhibition of embryo class I RNases,enzymes involved in P<Subscript>i</Subscript> release

The main objective of this study was to analyze the differences in profiles of RNase activities from triticale embryos (Triticosecale, cv. Ugo) between dormant and non-dormant caryopses and to determine the influence of exogenous abscisic acid (ABA) on the activities of these enzymes. The major RNase from the examined tissue was detected following SDS-PAGE, with substrate-based gel assay, described by Yen and Green (Plant Physiol 97:1487–1493, 1991). The activities of enzymes were characterized according to their pH optima, ion dependence, EDTA sensitivity and DNase activity. In embryos with arrested growth (in a natural way by dormancy or artificially by ABA treatment), the activity of two enzymes—24 and 27 kDa—belonging to class I RNases was completely inhibited, whereas that of two other RNases of this family—23 and 25 kDa—was detectable. However, the activity of the class I ribonucleases (enzymes responsible for cellular P_i release) was very low. Moreover, in contrast with non-dormant caryopses, imbibing embryos of dormant or ABA-treated seeds contained 13- and 14-kDa enzymes. These enzymes have not been classified so far, and their specific properties are different from the generally accepted properties of ribonucleolytic enzymes. In addition to the above results, the P_i content in the analyzed samples was determined by the Ames (Methods Enzymol 8:115–118, 1966) method. The results suggest a very low and constant level of inorganic phosphate in dormant samples as well as an evidently decreasing P_i content in embryos under the influence of ABA treatment. The inhibition of the class I RNases activity induced by abscisic acid implies that one of the roles of ABA in seed dormancy may consist in arresting the catabolic release of P_i, which results in retarding the embryo’s growth.     

Revealing Sulfate-Reducing Microorganisms in Oilfield Waters (Flysch Carpathians,South-eastern Poland)

Sulphidogenous microorganism communities were isolated from selected oilfield waters in the Flysch Carpathians of south-eastern Poland. Organisms were incubated using the microcosms method with application of two media: minimal medium and modified Postgate C medium with yeast extract or trisodium citrate or monocyclic hydrocarbons from the BTEX group (benzene, toluene, ethylbenzene, and xylene) as the sole carbon source. Activity of sulphidogenic, autochthonous microorganism communities was noted only on the Postgate medium. Beside active sulphate reduction – max. 70%, ca. 74% biodegradation of organic compounds was also observed in the cultures. The highest content of sulphate-reducing bacteria (SRB) in the COD (ca. 83%) was noted in cultures, in which trisodium citrate and yeast extract were applied as the sole carbon source. Molecular analysis indicated not only the presence of SRB such as Desulfobacterium autothrophicum, Desulfovibrio desulfuricans, but also other microorganisms, e.g., Geobacter metallireducens. All these taxa are obligatory or facultative anaerobes, with metabolism linked mostly with elemental sulphur and/or its oxidized forms, as well as iron. Analysis of the mineral composition of the residues confirmed the presence of elemental sulphur, testifying for the active reduction of sulphates by incompletely oxidizing sulphate reducers assigned to the SRB group. Based on the obtained results, it is concluded that the physical and chemical properties of the oilfield waters are favorable for the growth and development of sulphidogenic microorganism assemblages and mineral-forming processes conducted by them.     

Stepwise sRNA targeting of structured bacterial mRNAs leads to abortive annealing

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Disruption of Homocitrate Synthase Genes in <Emphasis Type="Italic">Candida albicans</Emphasis> Affects Growth But Not Virulence

Two genes, LYS21 and LYS22, encoding isoforms of homocitrate synthase, an enzyme catalysing the first committed step in the lysine biosynthetic pathway, were disrupted in Candida albicans using the SAT1 flipper strategy. The double null lys21Δ/lys22Δ mutant lacked homocitrate synthase activity and exhibited lysine auxotrophy in minimal media that could be fully rescued by the addition of 0.5–0.6 mM l-lysine. On the other hand, its virulence in vivo in the model of disseminated murine candidiasis appeared identical to that of the mother, wild-type strain. These findings strongly question a possibility of exploitation of homocitrate synthase and possibly also other enzymes of the lysine biosynthetic pathway as targets in chemotherapy of disseminated fungal infections.     

Viola roxolanica n. sp.

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