Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase.

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.     

Isolation and characterization of fluorescent Pseudomonas associated with the roots of rice and banana grown in Sri Lanka

Bacterial populations in different parts of the rhizosphere of rice and banana in Sri lanka were examined. On rice, the number of aerobic bacteria and the population of fluorescent bacteria were higher in the rhizoplane as compared to the exorhizosphere. However, the opposite was observed with banana. Percentage of fluorescent bacteria was significantly higher on banana (10.8%) than on rice from the wet and dry zones of Sri Lanka (4.3% and 2.7%, respectively). In the endorhizosphere fraction of rice, bacterial populations were very low. Fluorescent bacteria were absent.Based on 33 phenotypical tests, 89 fluorescent isolates were grouped into 5 clusters. The three major clusters covered the isolates belonging to the Pseudomonas fluorescens-putida group, whereas the remaining small clusters contained other UV-fluorescent bacteria. SDS-PAGE of total cell proteins enabled classification of the isolates into one of 12 different protein-polymorphic types. Only a partial correlation was found between the latter classification and the phenotypical one. Cyanogenesis was observed with strains of P. fluorescens only. Isolates P. fluorescens RW9S1 and P. cepacia RW5P1 displayed a potent antagonism against several fungi.     

Production and localization of beta-fructosidase in asynchronous and synchronous chemostat cultures of yeasts.

In synchronized continuous cultures of Saccharomyces cerevisiae CBS 8066, the production of the extracellular invertase (EC 3.2.1.26) showed a cyclic behavior that coincided with the budding cycle. The invertase activity increased during bud development and ceased at bud maturation and cell scission. The cyclic changes in invertase production resulted in cyclic changes in amounts of invertase localized in the cell wall. However, the amount of enzyme invertase present in the culture liquid remained constant throughout the budding cycle. Also, in asynchronous continuous cultures of S. cerevisiae, the production and localization of invertase showed significant fluctuation. The overall invertase production in an asynchronous culture was two to three times higher than in synchronous cultures. This could be due to more-severe invertase-repressive conditions in a synchronous chemostat culture. Both the intracellular glucose-6-phosphate concentration and residual glucose concentration were significantly higher in synchronous chemostat cultures than in asynchronous chemostat cultures. In the asynchronous and synchronous continuous cultures of S. cerevisiae, about 40% of the invertase was released into the culture liquid; it has generally been believed that S. cerevisiae releases only about 5% of its invertase. In contrast to invertase production and localization in the chemostat cultures of S. cerevisiae, no significant changes in inulinase (EC 3.2.1.7) production and localization were observed in chemostat cultures of Kluyveromyces maxianus CBS 6556. In cultures of K. marxianus about 50% of the inulinase was present in the culture liquid.     

Evolution of Volatile Sulfur Compounds during Laboratory-Scale Incubations and Indoor Preparation of Compost Used as a Substrate in Mushroom Cultivation

Volatile sulfur compounds are known to be produced during the preparation of compost used as a substrate in mushroom cultivation. Because they cause odor problems, attempts have been made to reduce the production of these compounds. The influences of temperature and various additions on the production of volatile sulfur compounds from composting material were tested on laboratory-scale preparations. The production of H₂S, COS, CH₃SH, and (CH₃)₂S was proven to be a biological process with an optimal temperature that coincides with the optimal temperature for biological activity. The formation of CS₂ and (CH₃)₂S₂ was shown to be a nonbiological process. The emission of volatile sulfur compounds during the indoor preparation of mushroom compost appeared to be remarkably reduced (about 90%) as compared with the emission during the conventional outdoor process. Introduction of this indoor composting process would result in a significant reduction in environmental pollution.     

Mechanism of melatonin action

Melatonin transduces the effect of photoperiod on the neuroendocrine system. Synthesis of melatonin in the pineal gland is well described, but the location of its target(s) and the mechanism of its action are little known. In attempt to localize melatonin target(s), the presence of high affinity binding sites in rat brain was determined. Such sites were detected in discrete brain areas, including the hypothalamus and anterior pituitary. Subcellular analysis indicated these binding sites were on plasma membranes, which suggests that melatonin modulates cell functions through intracellular second messengers. The effects of melatonin on second messengers were studied using the neonatal anterior pituitary, in which melatonin is known to inhibit the LHRH-induced release of LH. Studies on the effects of melatonin on second messenger indicated [corrected] that melatonin inhibits accumulation of cAMP and cGMP as well as synthesis of diacylglycerol and release of arachidonic acid. Time-course analysis indicates that inhibition by melatonin of the LHRH-induced release of LH increases following long preincubation. Since the effect of melatonin on LHRH-induced release of LH is prevented by dibutyryl cAMP, we conclude that melatonin might act by inhibiting production of cAMP.     

New markers for the neurofibromatosis-2 region generated by microdissection of chromosome 22

To identify new DNA markers around the neurofibromatosis-2 gene on human chromosome 22, the critical region (22q12-q13.1) was microdissected and microcloned from GTG-banded metaphase chromosomes. Eighteen thousand recombinant clones were obtained. Twenty-seven of 55 clones tested (50%) detected single-copy DNA sequences. Nine of nine clones analyzed in detail were found to map to chromosome 22. Interestingly one clone (EAN04) is part of the leukemia inhibitory factor gene which has previously been mapped to 22q11.2-q13.1. Four clones (EAN01, EAN47, EAN57, and EAN68) detect DNA polymorphisms. These probes were used to compare constitutional and tumor genotypes of 41 patients with acoustic neurinoma. Loss of constitutional heterozygosity was identified in 17 of 31 informative cases (55%). From our data we conclude that the microdissection library is a valuable resource for physical and genetic mapping studies in neurofibromatosis-2.     

A host factor absent from Lactococcus lactis subspecies lactis MG1363 is required for conjugative transposition

In matings between Lactococcus lactis strains, the conjugative transposons Tn916 and Tn919 are found in the chromosome of the transconjugants in the same place as in the chromosome of the donor, indicating that no transposition has occurred. In agreement with this, the frequency of L. lactis transconjugants from intraspecies matings is the same whether the donor contains the wild-type form of the transposon or the mutant Tn916-int1, which has an insertion in the transposon's integrase gene. However, in intergeneric crosses with Bacillus subtilis or Enterococcus faecalis donors, Tn916 and Tn919 transpose to different locations on the chromosome of the L. lactis transconjugants. Moreover, Tn916 and Tn919 could not be transferred by conjugation from L. lactis and B. subtilis, E. faecalis or Streptococcus pyogenes. This suggests that excision of these elements does not occur in L. lactis. When cloned into E. coli with adjacent chromosomal DNA from L. lactis, the conjugative transposons were able to excise, transpose and promote conjugation. Therefore, the inability of these elements to excise in L. lactis is not caused by a permanent structural alteration in the transposon. We conclude that L. lactis lacks a factor required for excision of conjugative transposons.     

Chironomid pupal exuviae and larvae of two storage reservoirs in The Netherlands

For three years chironomid larvae were collected bimonthly with a grab sampler in two deep storage reservoirs in The Netherlands. Chironomid pupal exuviae were collected monthly, from April to November, with a handnet. Net samples yielded more taxa than grab samples. The taxa found in net samples originated from more habitats, than taxa found in grab samples. The relative contribution of Orthocladiinae was much larger in net samples. Based on the results obtained in the two reservoirs, some advantages and disadvantages of both sampling methods are discussed.     

Predicting the distribution of plant communities using annual rainfall and elevation: an example from southern Africa

Abstract. Using the results of a total floristic survey of two veld types (Arid Sweet Bushveld and Mixed Bushveld) in the northeastern Transvaal, South Africa, we linked median annual rainfall from a surface response model to each of 139 samples. The samples had been classified floristically into 15 plant communities. These communities represent two broad divisions, corresponding with the concepts embodied in the two veld types. Using contingency tables, we defined the conditions of median annual rainfall and elevation for each of the veld types. Using a geographic analysis system we predicted the distribution of the veld types in an area of 120 000 km² outside the study area. The predicted distribution was validated by comparison with a digitized version of the Acocks map. We conclude that the defined conditions of median annual rainfall and elevation provide confident criteria for the definition of these veld types.