Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.     

Effects of photoperiodicity and light irradiance on phosphate-limited Oscillatoria agardhii in chemostat cultures

Oscillatoria sp. strain 23 is a filamentous, non-heterocystous cyanobacterium that fixes nitrogen aerobically. Although, in this organism nitrogenase is inactivated by oxygen a high tolerance is observed. Up to a pO₂ of 0.15 atm, oxygen does not have any measurable effects on acetylene reduction. Higher concentrations of oxygen inhibited the activity to a relatively high degree. Evidence for two mechanisms of oxygen protection of nitrogenase in this cyanobacterium was obtained. A high rate of synthesis of nitrogenase may allow the organism to maintain a certain amount of active enzyme under aerobic conditions. Secondly, a switch off/on mechanism may reversibly convert the active enzyme into a non-active form which is insensitive to oxygen inactivation after a sudden and short-term exposure to high oxygen concentrations. It is conceived that these mechanisms in addition to a temporal separation of nitrogen fixation from oxygenic photosynthesis sufficiently explain the regulation process of aerobic nitrogen fixation in this organism.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - CAP chloramphenicol     

Effects of photoperiodicity and light irradiance on phosphate-limited Oscillatoria agardhii in chemostat cultures

The cyanobacterium Oscillatoria agardhii was grown in continuous culture under various light conditions in order to study the interactions of light on phosphorus-limited growth. Under severe P-limiting (light-saturating) conditions, a low chlorophyll a and C-phycocyanin content was found. In addition, the light-harvesting capacity, reflected in the values of P _max (maximum light-saturated oxygen production rate) and (photosynthetic affinity), were low compared to light-limited cultures.Reduction of the light climate, either by reduction of the length of the photoperiod or light-intensity, resulted in an increase in light-harvesting capacity (higher pigment content, P _m and ) during growth under P-limiting conditions. Light-induced changes in P _max and could be related to the relative growth rate, being the actual growth rate as a fraction of the growth rate which would be observed under light-limiting conditions.Under P-limiting conditions, reduction of the light-climate caused a reduction in dry weight of the culture. This decrease was mainly due to a decrease in carbohydrate content of the cells. Under all conditions tested, carbohydrates were found to accumulate during the light-period and to be consumed during the dark-period.Evaluation of carbohydrate consumption in the dark yielded a specific maintenance rate constant of 0.001 h^-1. This observation leads to the conclusion that the specific maintenance rate constant is independent on the character of the growth rate limiting nutrient for O. agardhii.     

Semi-automatic identification of punching areas for tissue microarray building: the tubular breast cancer pilot study

Background  

Tissue MicroArray technology aims to perform immunohistochemical staining on hundreds of different tissue samples simultaneously. It allows faster analysis, considerably reducing costs incurred in staining. A time consuming phase of the methodology is the selection of tissue areas within paraffin blocks: no utilities have been developed for the identification of areas to be punched from the donor block and assembled in the recipient block.     

Growth of harmful marine algae in multispecies cultures

Mixtures of harmful and harmless algae were grown in discontinuouslydiluted batch cultures under ammonium, nitrate and phosphatelimitation, and at different irradiances (20–500 µjnolquanta m^–2 s^–1). The species used were Chrysochromulinapolylepis, Emiliania huxleyi type B, Rhodomonas sp., the dinoflagellalesFibrocapsa japonica, Gymnodinium simplex, Gyrodinium aure-olum,Heterocapsa triquetra, Heterosigma carterae, Prorocentrum micansand Alexandrium tamarense, the diatoms Chaetoceros socialis,Cymatosira belgica, Ditylum brightwellii, Laudcria borealis,Odon-telta aunla, Pseudonitzschia pungens, Streptotheca tamesis,and the cyanobacterium Synechococcus. Their growth responsein the mixed algal cultures is discussed in relation to theirabundance in different natural habitats. In comparison withthe other non-diatoms, the mixotrophic C.polylepis grew fastunder all tested nutrient and light limitations.Emiliania huxleyigrew well under nitrogen (N) limitation (with nitrate as N source)and at irradiance levels from 15 up to 500 µmol quantam^–2 s^–1. No growth of calcifying cells could bedetected under N limitation when ammonium was used as N source.Rhodomonas grew reasonably well under ammonium-N limitationand grew fast at the highest irradi-ance. The dinoflagellateswere poor competitors compared to the Prymnesiophyceae. Theenvironmental fitness of the Prymnesiophyceae appears to beclosely related to the reproductive capacity of the vegetativestage, whereas the natural distribution of dinoflagellates seemsmore closely dependent on the generative reproduction-relatedspecific life cycle characteristics of the individual species.The marine diatoms include a mixture of both types of species.Some marine diatom species clearly have the capability to outcompetenon-diatoms under different types of nutrient and light limitationswhen silicate is in excess. Other diatoms seem to be poor competitors.     

Overproduction and high-yield purification of phospholipase A from the outer membrane of Escherichia coli K-12

The cloned gene for the outer-membrane-bound phospholipase A from Escherichia coli was placed under control of the strong PL promoter of phage lambda. Induction of PL resulted in a 250-fold overexpression up to about 2% total cellular protein. This overproduced enzyme was indistinguishable from the wild-type enzyme. A homogeneous phospholiphase A preparation was obtained in high yield from overproducing bacteria, using the zwitterionic detergent C12-Sulfobetaine and anion-exchange chromatography. Detergent gradients were found to exert great influence on the elution characteristics. Considerations for the choice of optimal detergent gradients are discussed. The purified enzyme migrated as a single 29-kDa band in SDS/polyacrylamide gels, and required Ca(II) for activity. Maximum activity was displayed by enzyme samples taken from solutions with detergent concentrations near the critical micelle concentration. However, upon switching from high to optimal detergent concentration, maximum activity was restored in several hours, probably reflecting a slow conformational transition of the protein. Because the final pure protein was found to hydrolyze phospholipids in the intact erythrocyte membrane, a densely packed bilayer, we assume that this protein is in its biological native state.     

Regulation of phosphate uptake kinetics inOscillatoria agardhii

In order to study phosphate uptake kinetics the cyanobacteriumOscillatoria agardhii was grown in continuous culture under a phosphorus limitation. The affinity of the uptake system reflected in the initial slope of the uptake rate versus external substrate concentration curve (dV/ds) was found to be unaffected by the growth wate.The maximum phosphate uptake rate (V _m ) decreased as the growth rate was increased. Attempts were made to relate the decrease ofV _m to the increase in phosphorus content of the cells that occurred a higher growth rates. Accumulation of phosphate during pulse experiments indeed resulted in a decrease ofV _m . However feedback regulation ofV _m by accumulated phosphorus was found to occur only to a small extent in steady state growing cells. The main part of the regulation of the activity of the phosphate uptake system seemingly is determined by a long term process that is, at least longer than 2 h. The presence of short term feedback inhibition by accumulated phosphorus on the activity of the uptake system provides an explanation of the phenomenon thatOscillatoria agardhii is not able to grow at near _max growth rates under a phosphorus limitation.     

Theoretical considerations on growth kinetics and physiological adaptation of nutrient-limited phytoplankton

In many experimental ecological studies on phytoplankton species the growth response of an organism to the prevailing environmental conditions have been studied. Curves relating specific growth rate () to the external nutrient concentration (S) have been constructed to compare nutrient-limited growth of different species under steady state conditions. Microorganisms adapt their physiology to a certain limitation in order to optimize growth. Therefore the shape of the /S curve is closely related to the way a micoorganism adapts its physiology. To see how physiological adaptation and growth rate are interconnected to each other, both can be related to the internal concentration of the growth-limiting nutrient. How the growth rate is related to the internal nutrient concentration is presented in a mathematical equation.Many phytoplankton species during growth under different nutrient limitations show a linear relationship between and the reciprocal value of the internal nutrient content (= Yield). This was originally observed by Droop. The model presented here gives a theoretical explanation of this phenomenon.     

Immunocytochemical analysis of P-fimbrial structure: localization of minor subunits and the influence of the minor subunit FsoE on the biogenesis of the adhesin

Antibodies recognizing the non-adhesive minor P-fimbral subunit protein E and the P-fimbrial adhesin were used in an immunocytochemical analysis of P-fimbrial structure. It was demonstrated that P-fimbriae of the serotypes F71, F72 and F11 carry their adhesin in a complex with protein E. These complexes are commonly found at the tip of the fimbrial structure. In P-fimbriae of serotype F9, expressed by the uropathogenic Escherichia coli strain 21086, adhesin-protein E complexes are localized at the tips as well as along the shafts of the fimbriae. Protein E of F71 fimbriae (FsoE) plays a catalysing role in the biogenesis of the adhesin, but has no effect on the eventual localization of the adhesin.