Phenotypic degeneration occurs during sector formation in Metarhizium anisopliae

AIMS: The formation of sectors was observed during subculturing of an isolate of the entomopathogenic fungus Metarhizium anisopliae, a fungus used for biological control of insect pests. The aim of the investigation was to establish whether sector formation was accompanied by changes in physiological characters. METHODS AND RESULTS: Four degenerative morphological states, with reduced sporulation capacity, were characterized. Subcultures were taken from each sector and four new culture lines established. The new lines were further subcultured every 21 d. A physiological assessment of each line was undertaken after 42 d using TLC of secondary metabolites and fluorogenic enzyme tests. Full sporulation capacity was not regained on subculture, although some cultures recovered partially. Changes in secondary metabolite profiles and the loss in detection of activity of specific enzymes were observed. CONCLUSIONS: Sector formation was frequently accompanied by changes in the ability to produce secondary metabolites and enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results illustrate the importance of maintaining the stability of important cultures during routine subculture. The consequences could have significant implications if degenerate cultures are used as inocula for liquid fermentation cultures or industrial scale production.     

Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites

The paired helical filaments (PHF) found in Alzheimer's disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain.     

Mammalian twisted gastrulation is essential for skeleto-lymphogenesis

Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4). However, TSG function in vivo is still unclear. We isolated a TSG cDNA as a secreted molecule from the mouse aorta-gonad-mesonephros region. Here we show that TSG-deficient mice were born healthy, but more than half of the neonatal pups showed severe growth retardation shortly after birth and displayed dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus was atrophic, and phosphorylation of SMAD1 was augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, our findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, our findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency.     

Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein

AcrAB of Escherichia coli, an archetype among bacterial multidrug efflux pumps, exports an extremely wide range of substrates including solvents, dyes, detergents and antimicrobial agents. Its expression is regulated by three XylS/AraC family regulators, MarA, SoxS and Rob. Although MarA and SoxS regulation works by the alteration of their own expression levels, it was not known how Rob, which is constitutively expressed, exerts its regulatory action. We show here that the induction of the AcrAB efflux pump by decanoate and the more lipophilic unconjugated bile salts is mediated by Rob, and that the low-molecular-weight inducers specifically bind to the C-terminal, non-DNA-binding domain of Rob. Induction of Rob is not needed for induction of AcrAB, and we suggest that the inducers act by producing conformational alterations in pre-existing Rob, as was suggested recently (Rosner, Dangi, Gronenborn and Martin, J Bacteriol 184: 1407-1416, 2002). Decanoate and unconjugated bile salts, which are present in the normal habitat of E. coli, were further shown to make the bacteria more resistant to lipophilic antibiotics, at least in part because of the induction of the AcrAB efflux pump. Thus, it is likely that E. coli is protecting itself by the Rob-mediated upregulation of AcrAB against the harmful effects of bile salts and fatty acids in the intestinal tract.     

In Vivo Synergy Between Green Tea Extract and Levofloxacin Against Enterohemorrhagic Escherichia coli O157 Infection

We studied the synergistic effects of Japanese green tea extract (JGTE) and levofloxacin (LVFX) against enterohemorrhagic Escherichia coli (EHEC) infection in a gnotobiotic mouse model. Mice fed on JGTE conferred a significant degree of protection against an oral challenge with EHEC. Complete elimination of the bacteria from the mice, was however, difficult. The combination of JGTE and LVFX increased the survival rate and reduced damage to target organs. Thus, dietary supplementation with JGTE improved the therapeutic effects of antibiotic treatment. Received: 28 July 2000 / Accepted: 19 September 2000     

Cloning and disruption of the b-isopropylmalate dehydrogenase gene (LEU2 ) of Pichia stipitis with URA3 and recovery of the double auxotroph

Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (PsURA3) to disrupt P. stipitis LEU2 in a P. stipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX26) was selected for resistance to 5′-fluoroorotic acid to obtain P. stipitis FPL-UC7 (ura3-3). A URA3:lacZ“pop-out” cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu⁻ Ura⁺ phenotype. The ura3 marker was recovered from this strain by plating cells onto 5′-fluoroorotate and screening for spontaneous URA3 deletion mutants. Excision of the flanked PsURA3 gene resulted in the Leu⁻Ura⁻ phenotype. The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replication-sequence-based plasmids. Received: 17 June 1997 / Received revision: 10 September 1997 / Accepted: 14 October 1997     

Xylitol formation by Candida boidinii in oxygen limited chemostat culture

Summary Production of xylitol by Candida boidinii NRRL Y-17213 occurs under conditions of an oxygen limitation. The extent to which substrate is converted to xylitol and its coproducts (ethanol, other polyols, acetic acid), and the relative flow rates of substrate to energetic and biosynthetic pathways is controlled by the degree of oxygen limitation.With decrease in oxygen concentration in the inlet gas, for a constant dilution rate of 0.05 1/h. the specific oxygen uptake rate decreased from 1.30 to 0.36 mmol/gh Xylitol was not produced at specific oxygen uptake rates above 0.91 mmol/gh. Upon shift to lower oxygen rates, specific xylitol production rate increased more rapidly than specific ethanol production rate:Nomenclature D dilution rate (1/h) - DOT dissolved oxygen tension (%) - m_o2 maintenance coefficient (mmol O₂/g cell mass h) - q_o2 specific oxygen uptake rate (mmol O₂/g cell mass h) - q_s specific xylose uptake rate (g xylose/g cell mass h) or (mmol xylose/g cell mass h) - q_x specific xylitol production rate (g xylitol/ g cell mass h) or (mmol xylitol/ g cell mass h) - q_e specific ethanol production rate (g ethanol/ g cell mass h) or (mmol ethanol/ g cell mass h) - q_CO2 specific carbon dioxide production rate (mmol CO₂/g cell mass h) - S xylose concentration (g/1) - Y_cm/s cell mass yield coefficient, (g cell mass/mmol xylose) or (g cell mass/ g xylose consumed) - Y_cm/O2 cell mass yield coefficient, (g cell mass/mmol O₂) - Y_X/S xylitol yield coefficient (g xylitol/g xylose consumed) - Y_x/O2 xylitol yield coefficient (g xylitol/mmol O₂) - Y_e/s ethanol yield coefficient (g ethanol/g xylose consumed) - OUR oxygen uptake rate (mmol O₂/1h) - specific growth rate (1/h)     

Light and electron microscopic studies on experimental nocardia-toxicosis in mice

An exotoxin (HS-6) produced by Nocardia otitidiscaviarum isolated from certain lesions of cutaneous nocardiosis of a male 82-year-old patient induced severe injuries in the pancreas, liver, stomach, small intestine, heart, thymus and kidney of male ICR mice. Mice given Nocardia-free preparation of HS-6 at a dose of 1 mg/kg of body weight developed several autophagic vacuoles in the pancreas and liver within 20 min after the i.p. injection. Thereafter, the autophagic vacuoles increased in number and size with time. About 24 hr after the administration of HS-6, the liver showed marked accumulation of fat droplets in the cytoplasm of the hepatocytes. Although they contained abundant autophagic vacuoles in the regions of RER, there were no lipomatoses in the acinar cells of the pancreas, those of the chief cells and smooth muscle cells of the stomach, Paneth cells, goblet cells, smooth muscle cells of the small intestine, and plasma cells in the digestive tract. Biochemical examinations revealed that HS-6 had no significant effect on the protein synthesis of reticulocytes. Inoculation of the Nocardia into the mouse peritoneal cavities caused marked granulomatoses in the pancreas, liver and regional lymph nodes, but did not develop autophagic vacuoles in RER regions of these organs.