Genes involved in the biosynthesis of PQQ fromAcinetobacter calcoaceticus

From a gene bank of theAcinetobacter calcoaceticus genome a plasmid was isolated that complements four different classes of PQQ^- mutants. Subclones of this plasmid revealed that the four corresponding PQQ genes are located on a fragment of 5 kilobases. The nucleotide sequence of this 5 kb fragment was determined and by means of Tn5 insertion mutants the reading frames of the PQQ genes could be identified. Three of the PQQ genes code for proteins of M_r 29700 (gene I), M_r 10800 (gene II) and M_r 43600 (gene III) respectively. In the DNA region where gene IV was mapped however the largest possible reading frame encodes for a polypeptide of only 24 amino acids. A possible role for this small polypeptide will be discussed. Finally we show that expression of the four PQQ genes inAcinetobacter lwoffi andEscherichia coli lead to the synthesis of the coenzyme in these organisms.     

Assessing toxicity of Lake Diefenbaker (Saskatchewan,Canada) sediments using algal and nematode bioassays

Lake Diefenbaker, on the South Saskatchewan River, Saskatchewan, Canada, receives, on average, 90% of its inflow from snowmelt and rainfall in the Rocky Mountains. The inflowing rivers also receive irrigation return flows and municipal and industrial effluents which may result in the contamination of lake sediments. The sediments were assessed by nematode and algal bioassays.The toxicity of five chemical fractions of the sediment was determined using the nematode Panagrellus redivivus as the test organisms. The results suggest that the sediment chemical fractions frequently inhibit growth and maturation, while lethality was observed at 4 of 12 sites.Samples from 3 of these sites were further evaluated using conventional elutriate Algal Fractionation Bioassays (AFB) with both natural Lake Diefenbaker phytoplankton and a mixed laboratory grown algal culture. The natural phytoplankton showed inhibition at sediment: water ratios of 10: 1; whereas the algal cultures showed both enhancement and inhibition. Evidently, the sediments are frequently toxic to the species tested except for the algal culture. The AFB assesses the mitigative and synergistic effects of contaminants and nutrients and being a conventional elutriate, is more realistic and potentially more acceptable than the chemical fractionation/nematode bioassay technique which essentially considers potential trace organic contaminant effects.     

Effect of culture media and growth phase on the morphology of lactobacilli and on their ability to adhere to epithelial cells

Previous studies have demonstrated that the ability of lactobacilli to attach to and colonize uroepithelial surfaces is an important characteristic that enhances interference against uropathogenic bacteria. This adherence capacity was found to vary amongst lactobacillus strains and with the type of growth medium used to culture the organisms. The present study was undertaken to examine further the effect of culture media and growth phase on lactobacillus adherence to uroepithelial cells in vitro. In addition, a freeze substitution technique was developed to examine the morphology of strainsLactobacillus casei ssrhamnosus RC-17,L. casei GR-1, andL. acidophilus T-13 in relation to growth conditions and adhesion. A growth curve was plotted for strain GR-1, and adherence was found to be lowest for bacteria in early log phase (39 bacteria per uroepithelial cell) and highest in stationary phase (59 bacteria per uroepithelial cell). Strains RC-17 and GR-1 attached in high numbers to uroepithelial cells, whereas T-13 was poorly adherent. The latter formed a long, relatively dense, fibrous capsule after growth in brain heart infusion yeast extract agar, unlike strains GR-1 and RC-17, which formed a short, tightly bound, electron-dense capsule which surrounded the cells in a radial fashion. Growth of RC-17 in batch cultures of human urine, with and without addition of carbohydrates, resulted in formation of an irregular, fibrous extracellular matrix. These experiments illustrate that growth phase and culture conditions affect the extracellular structure of lactobacilli and also affect the adherence capacity of these bacteria. Structural changes mediated by availability of nutrients may partly explain why lactobacilli vary between species and between hosts in their colonization of the urogenital tract.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

The respiratory response to heat shock in Neurospora crassa

A sharp decrease in oxygen uptake occurred in Neurospora crassa cells that were transferred from 30 degrees C to 45 degrees C, and the respiration that resumed later at 45 degrees C was cyanide-insensitive. Energization of mitochondria, measured in vivo with fluorescence microscopy and a carbocyanine dye, also declined sharply in cells at 45 degrees C. Electron microscopy showed no changes in mitochondrial complexity; however, the cytoplasm of heat-shocked cells was deficient in glycogen granules.