Differentiation in Quinolone Resistance by Virulence Genotype in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a leading pathogen that has become increasingly resistant to the fluoroquinolone antibiotics due to widespread prescribing. Adverse outcomes have been shown for patients infected with fluoroquinolone-resistant strains. The type III secretion system (TTSS) is a major virulence determinant during acute infections through the injection of effector toxins into host cells. Most strains exhibit a unique TTSS virulence genotype defined by the presence of either exoS or exoU gene encoding two of the effector toxins, ExoS and ExoU, respectively. Specific TTSS effector genotype has been shown previously to differentially impact virulence in pneumonia. In this study, we examined the relationship between TTSS effector genotype and fluoroquinolone resistance mechanisms in a collection of 270 respiratory isolates. We found that a higher proportion of exoU+ strains were fluoroquinolone-resistant compared to exoS+ strains (63% vs 49%, p = 0.03) despite its lower overall prevalence (38% exoU+ vs 56% exoS+). Results from sequencing the quinolone resistance determining regions (QRDRs) of the 4 target genes (gyrA, gyrB, parC, parE) indicated that strains containing the exoU gene were more likely to acquire ≥2 mutations than exoS+ strains at MICs ≤8 µg/ml (13% vs none) and twice as likely to have mutations in both gyrA and parC than exoS+ strains (48% vs 24% p = 0.0439). Our findings indicate that P. aeruginosa strains differentially develop resistance-conferring mutations that correlate with TTSS effector genotype and the more virulent exoU+ subpopulation. Differences in mutational processes by virulence genotype that were observed suggest co-evolution of resistance and virulence traits favoring a more virulent genotype in the quinolone-rich clinical environment.     

Pseudomonas aeruginosa Population Structure Revisited

At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P. aeruginosa “core lineage” and typically exhibited the exoS ⁺/exoU ⁻ genotype and group B oprL and oprD alleles. This is to our knowledge the first report of an MST analysis conducted on a polyphasic data set.     

Dehalogenation of 4 — Chlorobenzoic Acid by <Emphasis Type="Italic">Pseudomonas</Emphasis> isolates

Twenty three bacterial isolates either pure or consortium were initially screened on the basis of their ability to degrade as well as dechlorinate 4 — chlorobenzoic acid (4-CBA). Based on comparative growth response, three pure isolates Pseudomonas putida GVS-4, Pseudomonas aeruginosa GVS-18 and Pseudomonas aeruginosa GWS-19 and a consortium SW-2 was finally selected for further studies. The enzyme studies performed with cell free extracts revealed that dehalogenase activity was substrate specific with maximum activity at 300 μgml⁻¹ substrate concentration. Catechol 1,2 dioxygenase activity was found to be present in cell free extracts suggesting that 4 — chlorobenzoic acid (4-CBA) is catabolized by ortho-ring cleavage pathway. The dehalogenase enzyme profile showed single enzyme band in case of GVS-4 (Rm 0.76), GVS-18 (Rm 0.84), GWS −19 (Rm 0.85) and two bands in SW-2 (Rm 0.71 & 0.10).     

Population structure of Pseudomonas aeruginosa from five Mediterranean countries: evidence for frequent recombination and epidemic occurrence of CC235

Several studies in recent years have provided evidence that Pseudomonas aeruginosa has a non-clonal population structure punctuated by highly successful epidemic clones or clonal complexes. The role of recombination in the diversification of P. aeruginosa clones has been suggested, but not yet demonstrated using multi-locus sequence typing (MLST). Isolates of P. aeruginosa from five Mediterranean countries (n = 141) were subjected to pulsed-field gel electrophoresis (PFGE), serotyping and PCR targeting the virulence genes exoS and exoU. The occurrence of multi-resistance (≥3 antipseudomonal drugs) was analyzed with disk diffusion according to EUCAST. MLST was performed on a subset of strains (n = 110) most of them had a distinct PFGE variant. MLST data were analyzed with Bionumerics 6.0, using minimal spanning tree (MST) as well as eBURST. Measurement of clonality was assessed by the standardized index of association (I_A ^S). Evidence of recombination was estimated by ClonalFrame as well as SplitsTree4.0. The MST analysis connected 70 sequence types, among which ST235 was by far the most common. ST235 was very frequently associated with the O11 serotype, and frequently displayed multi-resistance and the virulence genotype exoS ⁻/exoU ⁺. ClonalFrame linked several groups previously identified by eBURST and MST, and provided insight to the evolutionary events occurring in the population; the recombination/mutation ratio was found to be 8.4. A Neighbor-Net analysis based on the concatenated sequences revealed a complex network, providing evidence of frequent recombination. The index of association when all the strains were considered indicated a freely recombining population. P. aeruginosa isolates from the Mediterranean countries display an epidemic population structure, particularly dominated by ST235-O11, which has earlier also been coupled to the spread of ß-lactamases in many countries.     

Complementation of Methionine Auxotrophs of Pseudomonas aeruginosa from Cystic Fibrosis

Auxotrophic Pseudomonas aeruginosa are exclusive to respiratory infections in cystic fibrosis (CF) and bronchiectatic patients, and isolates require specific amino acids for growth on minimal media, particularly methionine. Since auxotrophic and prototrophic P. aeruginosa from CF are identical by genotyping, we investigated the genetic events leading to methionine auxotrophy (Met⁻). Most (10/13) Met⁻ strains had the same pattern of growth on methionine precursors and required methionine exclusively for growth. Back mutation to prototrophy was very low (frequencies 10⁻⁸ to <10⁻¹⁰). Complementation of the mutations leading to auxotrophy was achieved for five strains with a genomic library of P. aeruginosa PAO1. Strains with different patterns of growth on methionine precursors were complemented by clones with different restriction patterns, while identical clones complemented strains with the same pattern of growth on methionine precursors. Methionine auxotrophy in P. aeruginosa from CF results from stable chromosomal mutations, and the commonest defect is probably in gene(s) encoding enzymes that convert homocysteine to methionine. Received: 2 August 1997 / Accepted: 23 September 1997     

Nitrogen nutrition influences some biochemical responses to iron deficiency in tolerant and sensitive genotypes of Vitis

The effects of nitrogen source on iron deficiency responses were investigated in two Vitis genotypes, one tolerant to limestone chlorosis Cabernet Sauvignon (Vitis vinifera cv.) and the other susceptible Gloire de Montpellier (Vitis riparia cv.). Plants were grown with or without Fe(III)-EDTA, and with NO₃⁻ alone or a mixture of NO₃⁻ and NH₄⁺. Changes in pH of the nutrient solution and root ferric chelate reductase (FC-R) activity were monitored over one week. We carried out quantitative metabolic profiling (¹H-NMR) and determined the activity of enzymes involved in organic acid metabolism in root tips. In iron free-solutions, with NO₃⁻ as the sole nitrogen source, the typical Fe-deficiency response reactions as acidification of the growth medium and enhanced FC-R activity in the roots were observed only in the tolerant genotype. Under the same nutritional conditions, organic acid accumulation (mainly citrate and malate) was found for both genotypes. In the presence of NH4⁺, the sensitive genotype displayed some decrease in pH of the growth medium and an increase in FC-R activity. For both genotypes, the presence of NH₄⁺ ions decreased significantly the organic acid content of roots. Both Vitis genotypes were able to take up NH₄⁺ from the nutrient solution, regardless of their sensitivity to iron deficiency. The presence of N-NH₄⁺ modified typical Fe stress responses in tolerant and sensitive Vitis genotypes.     

Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1

An alkaline protease produced by Pseudomonas aeruginosa MN1, isolated from an alkaline tannery waste water, was purified and characterized. The enzyme was purified 25-fold by gel filtration and ion exchange chromatography to a specific activity of 82350 U mg⁻¹. The molecular weight of the enzyme was estimated to be 32000 daltons. The optimum pH and temperature for the proteolytic activity were pH 8.00 and 60°C, respectively. Enzyme activity was inhibited by EDTA suggesting that the preparation contains a metalloprotease. Enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺(5 mM), while Ca²⁺ and Mn²⁺ resulted in partial inhibition. The enzyme is different from other Pseudomonas aeruginosa alkaline proteases in its stability at high temperature; it retained more than 90% and 66% of the initial activity after 15 and 120 min incubation at 60°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 291–295. Received 09 June 1999/ Accepted in revised form 24 January 2000     

Intrachromosomal recombination between direct repeats in Penicillium chrysogenum : gene conversion and deletion events

Recombination between direct repeats has been studied in Penicillium chrysogenum using strain TD7-88 (lys⁻ pyr⁺), which contains two inactive copies of the lys2 gene separated by 4.5 kb of DNA (including the pyrG gene) in its genome. Gene conversion leading to products with the lys⁺ pyr⁺ phenotype was observed at a frequency of 1 in 3.2 × 10³ viable spores. Two types of deletion events giving rise to lys⁺ pyr⁻ and lys⁻ pyr⁻ phenotypes were obtained with different frequencies. Southern analysis revealed that gene conversion occurs mainly as a result of crossing over events that remove the BamHI frameshift mutation present in one of the repeats. In lys⁻ pyr⁻ recombinants, the deletion events do not affect the frameshift mutation in the BamHI site, while lys⁺ pyr⁻ recombinants showed repair of the BamHI frameshift mutation and the genotype of the parental non-disrupted strain was restored. In summary, deletion events in P. chrysogenum tend to favor the restoration of the phenotype and genotype characteristic of the parental non-disrupted strain. Received: 9 November 1998 / Accepted: 14 April 1999     

Zinc induced apoptotic death of mouse dendritic cells

Zinc ions (Zn²⁺) are food components with favourable effects in infectious disease. Zn²⁺ is taken up into dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. In other cell types, Zn²⁺ has been shown to stimulate the formation of ceramide, which is in turn known to trigger suicidal cell death. The present study explored whether Zn²⁺ modifies ceramide formation and survival of bone marrow derived DCs. To this end, DCs were isolated from acid sphingomyelinase knockout (asm ^−/−) and corresponding wild type (asm ^+/+) mice and treated with different concentrations of Zn²⁺. Ceramide formation was assessed with anti-ceramide antibodies in FACS and immunohistochemical analysis, sub-G1 cell population by FACS analysis, break down of phosphatidylserine asymmetry by annexin V binding, cell death by propidium iodide incorporation, metabolic cell activity by MTT assay, ROS production from dichlorofluorescein fluorescence and activation of MAPKs by Western blotting. The treatment of asm ^+/+ DCs with low Zn²⁺ concentrations (up to 100 μM) was followed by ceramide formation, increase in sub-G1 cell population and phosphatidylserine exposure, effects blunted in asm ^−/− DCs. The treatment of DCs with C2-ceramide increased the percentage of sub-G1 and apoptotic DCs from both genotypes. Zn²⁺ led to similar activation of MAPKs in asm ^+/+ and asm ^−/− DCs and did not affect ROS production. Higher concentrations of Zn²⁺ led to a marked increase of propidium iodide incorporation in DCs of both genotypes. The present study reveals that in DCs Zn²⁺ triggers ceramide formation, which in turn compromises cell survival.     

Role of MMP-2 in PKCδ-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: Involvement of a pertussis toxin sensitive protein

Treatment of bovine pulmonary artery smooth muscle with the O₂^•− generating system hypoxanthine plus xanthine oxidase stimulated MMP-2 activity and PKC activity; and inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment of the smooth muscle with SOD (the O₂^•− scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 activity and PKC activity, and reversed the inhibition of Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment with calphostin C (a general PKC inhibitor) and rottlerin (a PKCδ inhibitor) prevented the increase in PKC activity and reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without causing any change in MMP-2 activity in the microsomes of the smooth muscle. Treatment of the smooth muscle with the O₂^•− generating system revealed, respectively, 36 kDa RACK-1 and 78 kDa PKCδ immunoreactive protein profile along with an additional 38 kDa immunoreactive fragment in the microsomes. The 38 kDa band appeared to be the proteolytic fragment of the 78 kDa PKCδ since pretreatment with TIMP-2 abolished the increase in the 38 kDa immunoreactive fragment. Co-immunoprecipitation of PKCδ and RACK-1 demonstrated O₂^•− dependent increase in PKCδ-RACK-1 interaction in the microsomes. Immunoblot assay elicited an immunoreactive band of 41 kDa G_iα in the microsomes. Treatment of the smooth muscle tissue with the O₂^•− generating system causes phosphorylation of G_iα in the microsomes and pretreatment with TIMP-2 and rottlerin prevented the phosphorylation. Pretreatment of the smooth muscle tissue with pertussis toxin reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without affecting the protease activity and PKC activity in the microsomes. We suggest the existence of a pertussis toxin sensitive G protein mediated mechanism for inhibition of Na⁺ dependent Ca²⁺ uptake in microsomes of bovine pulmonary artery smooth muscle under O₂^•− triggered condition, which is regulated by PKCδ dependent phosphorylation and sensitive to TIMP-2 for its inhibition. (Mol Cell Biochem xxx: 107–117, 2005)     

Pyrene-degradation potentials of Pseudomonas species isolated from polluted tropical soils

Three Pseudomonas species isolated from oil polluted soils in Lagos, Nigeria were studied for their pyrene degradation potentials. These isolates exhibited broad substrate specificities for hydrocarbon substrates including polycyclic aromatic hydrocarbons, petroleum fractions and chlorobenzoates. All three isolates tolerated salt concentrations of more than 3%. They resisted ampicillin, cenfuroxime, but susceptible to ofloxacin and ciprofloxacin. Pseudomonas sp. strain LP1 exhibited growth rates and pyrene degradation rates of 0.018 h⁻¹ and 0.111 mg l⁻¹ h⁻¹ respectively, while P. aeruginosa strains LP5 and LP6 had corresponding values of 0.024, 0.082 and 0.017, 0.067 respectively. The overall respective percentage removal of pyrene obtained for strains LP1, LP5 and LP6 after a 30-day incubation period were 67.79, 66.61 and 47.09. Resting cell assay revealed that strain LP1 had the highest uptake rate. Strains LP1, LP5, and LP6 also used the ortho-cleavage pathway. Enzyme study confirmed activity of catechol 1,2-dioxygenase in all with values 0.6823, 0.9199, and 0.8344 μmol min⁻¹ mg⁻¹ respectively for LP1, LP3 and LP6. To the best of our knowledge, ours is the first report of pyrene-degraders from the sub-Saharan African environment.     

Effects of cortisol and fluoride on ion-transporting ATPase activities in cultured osteoblastlike cells

Summary Na⁺,K⁺-ATPase, HCO ₃ ⁻ -ATPase, Ca²⁺,Mg²⁺,-ATPase, Ca²⁺-ATPase, and alkaline phosphatase activities were measured in cultures of osteoblastlike cells treated with fluoride and cortisol separately and in combinations. Low concentrations of cortisol increased HCO ₃ ⁻ -ATPase (10⁻¹¹ to 10⁻¹⁸ M cortisol) and alkaline phosphatase (10⁻¹¹ to 10⁻⁹ M cortisol) activities, but higher cortisol concentrations reduced these activities. Na⁺,K⁺-ATPase, Ca²⁺,Mg²⁺-ATPase, and Ca²⁺-ATPase activities tended only to be reduced by cortisol. Fluoride (10⁻⁶ and 5×10⁻⁶ M) increased HCO ₃ ⁻ -ATPase and alkaline phosphatase activities, but these activities were similar to controls in the presence of 10⁻⁵ M fluoride. Ca²⁺,Mg²⁺-ATPase activity was decreased and Na⁺,K⁺-ATPase activity was increased as the concentration of fluoride increased (10⁻⁶ to 10⁻⁵ M). Preliminary experiments with fluoride indicated that lower concentrations (10⁻⁷ M) were without effect. Cortisol concentrations of 10⁻⁹ and 10⁻⁸ M were chosen for studies with combinations of cortisol and fluoride because the effects of these concentrations on alkaline phosphatase activity were opposite, i.e. 10⁻⁹ M increased whereas 10⁻⁸ M decreased activity. Fluoride concentrations of 10⁻⁶, 5×10⁻⁶, and 10⁻⁵ M were chosen because a peak of alkaline phosphatase activity occurred at 5×10⁻⁶ M fluoride. Higher (10⁻⁴ M) and lower (10⁻⁷ M) fluoride concentrations were without effect. The effects of combinations of cortisol and fluoride depend on the enzyme activity measured. Fluoride (10⁻⁶ M) combined with cortisol (10⁻⁹ M) produced a peak of Na⁺,K⁺-ATPase activity. The increased activity obtained with all concentrations of fluoride alone was preserved when fluoride was combined with 10⁻⁸ M cortisol, although the activity tended to be reduced at 5×10⁻⁶ and 10⁻⁵ M fluoride. HCO ₃ ⁻ -ATPase activity was increased by fluoride combined with 10⁻⁸ M cortisol and decreased by fluoride combined with 10⁻⁹ M cortisol compared to the activities obtained with fluoride alone. The decrease in Ca²⁺,Mg²⁺-ATPase activity caused by fluoride alone was prevented by 10⁻⁹ and enhanced by 10⁻⁸ M cortisol, although all treatments produced the same activity at 10⁻⁵ M fluoride. Ca²⁺-ATPase activity tended to be increased by combinations of fluoride and cortisol, but significantly so only at 10⁻⁵ M fluoride in combinations with 10⁻⁸ and 10⁻⁹ M cortisol. Alkaline phosphatase activity was increased by fluoride combined with 10⁻⁹ M cortisol and decreased by fluoride combined with 10⁻⁸ M cortisol compared to the activities obtained with fluoride alone. These results suggest that the abilities of bone cells to regulate ion transport (as reflected in their ion-transporting ATPase activities) are modulated by glucocorticoids and fluoride. Inasmuch as these cells may regulate the ionic composition and concentrations of the bone extracellular fluid (ECF) in vivo, the modulation of their activities by cortisol and fluoride may result in altered bone ECF composition. This work was supported by Grant NAG-2-108 from the National Aeronautics and Space Administration, D.C., and Grant PO1 NS15767 from the National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD.     

Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.     

Bacillus thuringiensis crystal protein (δ-endotoxin) gene expression is independent of early sporulation specific functions

Bacillus thuringiensis produces a parasporal insecticidal crystal protein. The correlation between sporulation and crystal protein production inBacillus thuringiensis var.israelensis was studied. The strain was made resistant’against streptomycin (St^R)-Acrystalliferous (Cry^-) cured derivatives and asporogenous acrystalliferous (Spo⁻ Cry⁻) mutants blocked at an early stage of sporulation were isolated. Plasmid transfer experiments were performed between St^R Spo⁺ Cry⁺ (streptomycin sensitive sporogeneous crystalliferous) and St^RR Spo⁺ Cry⁻ and also between St^s Spo⁺ Cry⁺ and St^R Spo⁻ Cry⁻ strains. StR colonies were selected. Insect toxicity was exhibited by the St^R isolates in both the cases. The process of crystal formation is, therefore, independent of early sporulative events.     

Inhibitory effect of extracts from Brazilian medicinal plants on the adhesion of <Emphasis Type="Italic">Candida albicans</Emphasis> to buccal epithelial cells

Plants are known to produce a plethora of secondary metabolites which are recognized as a useful source of new drugs or drug leads. Extracts and fractions of Schinus terebinthifolius Raddi (Anacardiaceae), Piper regnellii C.D.C. (Piperaceae), Rumex acetosa L. (Polygonaceae), and Punica granatum L. (Punicaceae) were assessed for their antifungal activity against eight clinical isolates of C. albicans. They were also evaluated for their effect on the adhesion of these C. albicans isolates to buccal epithelial cells (BECs). The ethyl acetate fraction from the leaves of S. terebinthifolius showed promising activity, inhibiting the growth of three C. albicans isolates at 7.8 μg ml⁻¹ and significantly inhibiting their adhesion to BEC at 15 μg ml⁻¹ . In addition, this fraction did not show cytotoxic activity against murine macrophages. The results show the potential of the plant extracts studied as a source of new antifungal compounds. Further studies are necessary for isolation and characterization of the active compounds of these plants.     

Na+-K+-Cl− cotransport system in brain capillary endothelial cells: Response to endothelin and hypoxia

Effect of endothelin-1 and chemically induced hypoxia on Na⁺−K⁺−Cl⁻ cotransport activity in cultured rat brain capillary endothelial cells was examined by using⁸⁶Rb⁺ as a tracer for K⁺; bumetanide-sensitive K⁺ uptake was defined as Na⁺−K⁺−Cl⁻ cotransport activity. Endothelin-1, phorbol 12-myristate 13-acetate (PMA), or thapsigargin increased Na⁺−K⁺−Cl⁻ cotransport activity. A protein kinase C inhibitor, bisindolylmaleimide, inhibited PMA- and endothelin-1- (but not thapsigargin-) induced Na⁺−K⁺−Cl⁻ cotransport activity, indicating the presence of both protein kinase C-dependent regulatory mechanisms and protein kinase C-independent mechanisms which involve intracellular Ca²⁺. Oligomycin, sodium azide, or antimycin A increased Na⁺−K⁺−Cl⁻ cotransport activity by 80–200%. Oligomycin-induced Na⁺−K⁺−Cl⁻ cotransport activity was reduced by an intracellular Ca²⁺ chelator (BAPTA/AM) but not affected by bisindolylmaleimide, suggesting the involvement of intracellular Ca²⁺, and not protein kinase C, in hypoxia-induced Na⁺−K⁺−Cl⁻ cotransport activity. Portions were presented at “27th Annual Meeting, The American Society for Neurochemistry” Philadelphia, Pennsylvania, March 2–6, 1996.     

The effect of complex carbon and nitrogen, salt, Tween-80 and acetate on delta-endotoxin production by a Bacillus thuringiensis subsp kurstaki

Delta-endotoxin production by a strain of Bacillus thuringiensis subsp kurstakion complex media based on crude gruel and fish meal was investigated. High proteolytic activities were concomitantly produced with the bioinsecticide. In such complex media, the repressive regulation due to readily consumed carbon sources was partially overcome. In order to improve substrate assimilation, 0.5 g L⁻¹ sodium chloride and 0.1% Tween-80 were supplemented to the production medium, increasing delta-endotoxin yields when using gruel concentrations below 59 g L⁻¹. At and beyond 75 g L⁻¹ gruel, delta-endotoxin yields were not affected in the presence of 0.5 g L⁻¹ NaCl and 0.1% Tween-80, but proteolytic activity yields were remarkably reduced. Thus, the use of sodium chloride and Tween-80 allowed reduction of the initial gruel concentration to 42 g L⁻¹ for the production of 3350 mg L⁻¹ delta-endotoxin, while it was only 3800 mg L⁻¹ with 92 g L⁻¹ gruel. Moreover, similar to 0.5 g L⁻¹ NaCl and 0.1% Tween-80, the use of 10 g L⁻¹ sodium acetate significantly improved delta-endotoxin production and also reduced the proteolytic activity to 250 U ml⁻¹. Received 05 November 1998/ Accepted in revised form 19 August 1999     

H+ and Cl− secretion in the stomach of the teleost fish, Anguilla anguilla : stimulation by histamine and carbachol

The fundus of an eel stomach was mounted in an Ussing chamber and bathed with control Ringer on the serosal side and with unbuffered solution on the mucosal side. The gastric mucosa exhibited a mucosa negative transepithelial voltage (V _t), a “short circuit” current (I _SC) and a small spontaneous acid secretion rate (J _H). All these parameters were abolished by cimetidine treatment. Bilateral ion substitution experiments in tissues lacking spontaneous acid secretion suggested that a net Cl⁻ transport from serosa to mucosa was responsible for the genesis of the I _SC in the absence of H⁺ secretion. Serosal application of histamine (10⁻⁴ mol · l⁻¹) or carbachol (10⁻⁴ mol · l⁻¹) stimulated both I _SC and J _H. The action of carbachol was independent of histamine. The control as well as the histamine-stimulated I _SC was sensitive to both serosal bumetanide (10⁻⁵ mol · l⁻¹), inhibitor of the Na⁺-K⁺-2Cl⁻ cotransport, and 4,4-diisothiocyano-stilbene-2,2-disulphonic acid (DIDS, 5 · 10⁻⁴ mol · l⁻¹), inhibitor of the Cl⁻-HCO⁻ ₃ exchange, while the I _SC stimulated by carbachol was nullified by serosal DIDS. These data suggested that the non-acidic Cl⁻ uptake across the serosal membrane was linked to the activity of both Na⁺-K⁺-2Cl⁻ cotransport and Cl⁻-HCO⁻ ₃ antiporter; histamine stimulated both transporters while carbachol was limited to the anion exchanger. The finding that the acid secretion was strictly dependent on serosal Cl⁻ and was completely blocked by serosal DIDS suggested that the Cl⁻ accompanying H⁺ secretion entered the cell through the serosal membrane by the Cl⁻-HCO⁻ ₃ exchange. In addition, the acid secretion stimulated by carbachol was also dependent on serosal Na⁺ and sensitive to the application of 5-N-N-dimethyl-amiloride in the serosal bath, suggesting that the increased activity of the Cl⁻-HCO⁻ ₃ during carbachol treatment was linked to the activation of serosal Na⁺-H⁺ exchange. The inhibitory effect of luminal omeprazole (10⁻⁴ mol · l⁻¹) on acid secretion suggested the presence of the H⁺-K⁺ pump on the luminal membrane. Accepted: 18 September 1997     

Nitrogen and phosphorus utilization in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> isolated from Laguna de Bay,Philippines

Phytoplankton supports fisheries and aquaculture production. Its vital role as food for aquatic animals, like mollusks, shrimp, and fish cannot be overemphasized. Because of its contribution as a food source for fish, the growth kinetics of Microcystis aeruginosa, a dominant cyanobacterium in the lake, was studied. The regular occurrence of M. aeruginosa is experienced during the months of May to July or from September to November in Laguna de Bay, the largest freshwater lake in the Philippines. M. aeruginosa was collected from Laguna de Bay, isolated, and established in axenic conditions. Data on the growth kinetic parameters for nitrate-nitrogen and phosphate-phosphorus utilization by M. aeruginosa gave the following values: half-saturation constant (K _s ), 0.530 mg N. L⁻¹ and 0.024 mg P. L⁻¹ respectively; maximum growth rate (μ _max ), 0.671. d⁻¹ and 0.668. d⁻¹ respectively; maximum cell yield, 6.5 and 6.54 log, cells. ml⁻¹ respectively; nutrient level for saturated growth yield, 8.71 mg N. L⁻¹ and 0.22 mg P. L⁻¹ respectively; and minimum cell quota (Q ₀ ), 2.82 pg N. cell⁻¹ and 0.064 pg P. cell⁻¹ respectively. The low K _s value and high maximum growth rate (μ _max ) for phosphorus by M. aeruginosa would suggest a high efficiency of phosphorus utilization. On the other hand, the high K _s value for nitrogen indicated a low rate of uptake for this nutrient.