ALBERT

Notes: Because the ferric uptake regulator (fur ) appears to be an essential gene in Pseudomonas aeruginosa, resistance to manganese was used as an enrichment to isolate strains carrying point mutations in the fur gene in order to assess its role in the co-ordinate expression of siderophores and exotoxin A (ETA). This report describes a detailed molecular and phenotypic characterization of four mutants and one revertant, which carry point mutations in the fur gene. Two parental strains were used in this study. Three mutants were isolated from the widely used strain, PAO1. One of these, CS (cold sensitive), has a mutation in the 5′ non-coding region of the fur gene while the two other mutants derived from this parent have mutations resulting in the following deduced changes in Fur: mutant A2, H86 → R; mutant A4, H86 → Y. The other mutant (C6) and its revertant (C6Rv) were derived from PAO6261, a mutant of PAO1 with a deletion in the anr gene (anaerobic regulation of arginine deiminase and nitrate reduction) that controls anaerobic respiration in P. aeruginosa. Fur from the C6 mutant has an A10 → G mutation while in the C6Rv spontaneous revertant the mutant Gly residue has been changed to Ser at this position. All mutants were examined for alterations in the iron-regulated expression of siderophores and ETA. The A2 and A4 mutants expressed higher levels of siderophores in iron-deficient media and in iron-replete media. The CS mutant constitutively expressed siderophores at 25°C. At 42°C siderophore biosynthesis was iron repressed as in the parental strain PAO1. The deletion of anr in PAO6261 had no apparent effect on the iron-mediated regulation of siderophore synthesis, but the C6 mutant derived from this strain produces siderophores constitutively. The iron-regulated production of siderophores by C6Rv was similar to the parental strain PAO6261 and PAO1. Because one of the parental strains used in this study is an Anr mutant, regulation of ETA production was assessed under aerobic and microaerobic conditions. Iron-dependent repression of ETA synthesis in both parental strains and A2 and A4 mutants was found to be 50–100-fold under aerobic and microaerobic conditions, as assayed by quantitative Western dot-blot assays. By contrast in the CS and C6 mutants, while iron-dependent repression of ETA synthesis was similar to both parental strains under aerobic conditions, ETA production in these mutants was constitutive in a microaerobic environment. RNase protection analysis of toxA and regAB transcription in PAO1, PAO6261 and the C6 mutant corroborated the results of quantitative dot-blot assays of ETA. The mutant Fur proteins were purified and examined for their ability to bind to the promoter of a gene (pvdS ) that positively regulates the expression of siderophores and ETA. Fur from the A2 and A4 mutants and from the C6Rv revertant was able to bind to the target DNA, but with reduced affinity by comparison to wild-type Fur. Fur from the C6 mutant in DNase I footprint experiments failed to protect the promoter region of the pvdS gene, but it retained some weak binding activity in gel mobility shift assays. The data presented in this study not only furnish some additional insights into the structure–function relationships of Fur, but also afford novel perspectives with regard to Fur and the iron-dependent regulation of virulence factors in P. aeruginosa under environmental conditions that have not previously been considered.

Notes: The effect of bentazone at 10 and 100 ppm in two soils on microbial activities and populations was investigated for 32 weeks. The higher concentration caused reductions in carbon dioxide output and oxygen uptake in both soils, In soil I (pH 6.4, organic C 1.7%) an increase in total mineral nitrogen occurred throughout but in soil H (pH 5.6, organic C 3.7%) a reduction in both mineralization of nitrogen and nitrification was observed from week 22 onwards. In general, populations of fungi and actinomycetes were resistant to or stimulated by bentazone, especially in soil II at 100 ppm. With bacteria, including cellulolytic and spore formers, cellulolytic fungi and actinomycetes, there was some inhibition even at the low concentration, effects being more pronounced in soil II than in soil I. The herbicide at 20 ppm had very little effect on growth and morphology of four fungal species in pure culture.Residue analyses showed that bentazone degraded more rapidly in soil II than in soil I although appreciable effects occurred throughout the experiment in both soils. Evaluation simultanée de diverses réactions de la microflore du sol à la bentazone. L'effet de la bentazone à 10 et 100 ppm sur les activités et les populations microbiennes a étéétudié pendant 32 semaines. La concentration la plus forte a provoqué des réductions du dégagement de dioxyde de carbone et de l'absorption d'oxygéne dans les deux sols. Dans le sol I (pH 6,4, teneur en carbone organique 1,7%) un accroissement de I'azote mineral total s'est constamment manifesté, mais, dans le sol II (pH 5,6, teneur en carbone organique 3,7%) une réduction simultanée de la minéralisation de I'azote et de la nitrification a été observée a partir de la 22ème semaine. D'une façon générale, les populations de champignons et d'actinomycétes se sont montrées résistantes à la bentazone, ou même stimulées par elle, en particulier pour le sol II à la concentration de 100 ppm. Avec les bactéries, ycompris les formes sporultés et cellulolytiques, les champignons cellulolytiques et les aainomycétes, il s'est produit une certaine inhibition même à la concentration faible, ces cffets étant plus prononcés dans le sol II que dam le sol I. L'herbicide à 20 ppm n'a eu qu'une trés faible action sur la croissance et la morphologie de quatre espéces de champignons en culture pureDes analyses de résidus ont montré que la bentazone s'est dégradée plus rapiderment dans le sol II que dans le sol I, bien que des effets appréciables soieni apparus dans toute l'expérience avec les deux sols. Gleichzeitige Erfassung verschiedener Reaktionen der Bodenmikroflora auf Bentazon Es wurde die Wirkung von Bentazon (10 und 100 ppm) über 32 Wochen auf die mikrobielle Aktivität und auf die Mikroorganismenpopulationen zweier Boden untersucht. Durch die höhere Konzentration ging in beiden Boden die Abgabe von Kohlendioxid und die Aufnahme von Sauerstoff zurück. Im Boden I (pH 6,4; org. C l,7%) wurde während der gesamten Versuchszeit ein Anstieg des mineralischen Stickstoffs festgestellt, während im Boden II (pH 5,6; org. C 3,7%) ein Rückgang in der Stickstoffmineralisierung und der Nitrifikation ab der 22. Woche zu beobachten war. Im allgemeinen waren die Pilz- und Actinomycetenpopulationen gegenüber Bentazon resistent oder sie wurden durch das Herbizid gefördert, besonders im Boden II bei 100 ppm. Beiden Bakterien, einschliesslich zellulolytischer und sporenbildender Arten, den zellulolytischen Pilzen und Actinomyceten wurde eine geringe Hemmung, sogar bei der niedrigen Konzentration, festgestellt. Diese Effekte waren im Boden II stärker als im Boden I ausgeprägt. In Reinkultur hatten 20 ppm Bentazon nur eine gennge Wirkung auf das Wachstum und die Morphologie von vier Pilzarten.Rückstandsuntersuchungen ergaben, dass Bentazon im Boden II schneller ais im Boden I abgebaut wurde, es kam aber in beiden Böden zu deutlichen Effekten.

Notes: Effects of glyphosate, paraquat, trifluralin and atrazine on activities of dehydrogenase, phosphatase and urease in one soil were measured. Only glyphosate at 21.6 kg/ha was found to inhibit the enzyme activities and generally the results were not statistically significant. Enzyme activity associated with micro-organisms proliferating in soil supplemented with lucerne meal was similarly not affected by the herbicides. Interpretation of results from enzyme activity measurements in soils treated with herbicides is discussed. It is proposed that effects of natural stress can be used to judge the relative importance of herbicide induced change.

Notes: Examples from the literature and from the authors’own laboratory demonstrate that herbicides can exert adverse effects on the soil micro-flora, depending on concentration, though often at higher rates than the herbicidal dose. Examples refer to changes in microbial growth (asulam, linuron, paraquat) and equilibrium (metoxuron), respiration and nitrification in samples from field experiments (linuron, simazine) and laboratory experiments (bentazone, glyphosate, barban, etc.), enzyme activities (urease, phosphatase; barban, chlorpropham, linuron) and the decay of sprayed vegetation (glyphosate, paraquat).

Notes: Summary Prenatal diagnosis of cystic fibrosis by microvillar enzyme assay on amniotic fluid supernatant has been carried out on 258 sequential pregnancies with a 1 in 4 recurrence risk, all with known outcome. In general the three enzymes evaluated, γ-glutamyltranspeptidase, aminopeptidase M and the intestinal isoenzyme of alkaline phosphatase, showed a high degree of concordance. However, there were two unusual patterns of microvillar enzyme activity; in seven cases a low γ-glutamyltranspeptidase activity was associated with elevated values of intestinal alkaline phosphatase, and in ten cases there were isolated low values of intestinal alkaline phosphatase. The former pattern was found to be associated with cystic fibrosis in five cases, while the latter was associated with a normal outcome in all ten cases. A retrospective analysis of enzyme values suggested that the optimal system for minimizing false positives and false negatives was to define foetal cystic fibrosis as a sample where two of the three microvillar enzymes were below a cut-off of half the median value for the gestational week. If such scoring were applied to the cases where conventional microvillar enzyme patterns were observed, the false positive rate was 2.3% and the false negative rate 4.4% between 17 and 20 weeks of gestation.