Metabolic interlock between the acetolactate synthase isoenzymes and lysine biosynthesis in Escherichia coli K-12

Summary Some of the strains containing mutations in the genes for the acetolactate synthase isoenzymes are temperature sensitive (ts). Suppression of the acetolactate synthase defect due to one of these mutations suppresses also the ts phenotype; moreover, a genetic cross shows that the two phenotypes cannot be dissociated.The ts phenotype is accompanied by a decreased efficiency of transduction with Pl phage. Observations at the light microscope show formation of abnormal cells. Under specific conditions diaminopimelate stimulates growth and restores normal transduction efficiency. The rate of diaminopimelate formed and excreted by non-growing cells decreases when an acetolactate synthase mutation is present.We give evidence that the ts phenotype is due to an increased formation of lysine from diaminopimelate; this causes a starvation for the latter and therefore cell wall abnormalities. In fact, even at the permissive temperature, the lysine pool is 8x increased in a strain with an acetolactate synthase defect, while a slight decrease in the diaminopimelate pool is observed. Moreover, introduction into a ts strain of a mutation in lysA (the gene coding for diaminopimelate decarboxylase) cures the ts phenotype. Finally among the temperature resistant revertants we found some lysine auxotrophs.     

Isolation and visualisation of alkali stable protein/DNA complexes

Distinct polypeptides, 54,000–68,000 daltons in size, are alkali-stably bound to eukaryotic DNA. DNA fragments several hundred base pairs in length associated with these polypeptides are preferentially retained on glass fibre filters from solutions containing 1 M sodium chloride. About 50 percent of the protein/DNA complexes present in total DNA are retained on filters together with about 2 percent of the DNA. This preferential binding is demonstrated (a) by the ratio of ³H and ³⁵S radioactivity retained on filters after filtration of DNA from [³H]thymidine and L-[³⁵S]methionine labelled cells, (b) radioiodination of the material retained on filters and passing filters respectively and (c) by electron microscopical visualisation of the polypeptide component in the complexes after chemical modification with dinitrofluorobenzene (DNFB) followed by incubation with dinitrophenyl (DNP) specific antibodies.     

Utilization of limiting concentrations of ortho-phosphate and production of extracellular organic phosphates in cultures of marine diatoms

The utilization of ortho-phosphate by two coastal marine diatomspecies, Nitzschia closterium and Cyclotella cryptica, was studiedin batch cultures. The hypothesis was tested that thresholdconcentrations in the phosphate uptake determine the lower limitof environmental phosphate, permitting the existence of species.The turn-over time of residual medium phosphate in culturesis {small tilde}10 min, indicating a rapid equilibration ofconcentration dependent on uptake with leakage of ortho-phosphate.Increasing phosphate starvation in cultures diminished the residualortho-phosphate in the range of {small tilde}60–<2nmol l^–1, as measured radiochemically after elution onSephadex® G-10 gel. These concentrations encompass the rangeof limiting phosphate concentration in continuous cultures ofthe few microalgae, for which these concentrations are actuallymeasured. The diatoms excreted {small tilde}20–100 nmolI^–1 of organic phosphate. One dominating compound, probablyan unusual nucleotide, was incompletely or not resorbed underphosphate starvation. In contrast, Nitzschia closterium excretedunder ample phosphate supply a series of three related compounds,probably phospholipids, that were resorbed under depletion.The association of the organic phosphates with macromolecularexudates is interpreted, along with the other observations,as an indication for a hardly explored periplasmatic phosphatemetabolism in these algae. ³Dedicated to Prof. Dr. H.-A. von Stosch in honour of his 75thbirthday. ⁴This study was conducted at the University of Marburg undersupport of the Humboldt Foundation Publication no. 64 of theproject "Biological Research of the Eems-Dollard Estuary".     

The organization of the gene for the human cerebroside sulfate activator protein

The organization of 14 exons covering 97% of the cDNA sequence of human cerebroside sulfate activator protein precursor has been determined from two overlapping EMBL-4 human genomic clones extending over 17kb. All exons and exon/intron splice junctions and five introns were sequenced. Exon 8 consists of only 9 bp and is involved in alternative splicing which generates three different mRNAs of cerebroside sulfate activator precursor.     

Stimulation of human nitric oxide synthase by tetrahydrobiopterin and selective binding of the cofactor.

To check the stimulatory potency of the tetrahydro forms of the two major pteridines occurring in human tissues, neopterin and biopterin, NO synthase was purified 6000-fold from human cerebellum. Tetrahydrobiopterin stimulated the activity up to 4.5-fold in a concentration dependent manner with a maximum above 1 microM, whereas tetrahydroneopterin was completely inactive in concentrations up to 100 microM. Tetrahydrobiopterin, but not neopterin derivatives, were copurified with the NO synthase activity. Our results demonstrate that human cerebellum contains a tetrahydrobiopterin dependent NO synthase activity.     

Effects of permethrin on aquatic organisms in a freshwater stream in south-central Alaska.

Permethrin (0.5%) was applied to individual Lutz spruce, Picea x lutzii Little, to protect them from attack by spruce beetles, Dendroctonus rufipennis (Kirby). Residue levels were monitored in a freshwater stream above, adjacent to, and below the treatment site at intervals before, during, and after treatment. Maximum residue levels in the stream within the treatment site ranged from 0.05 +/- 0.01 ppb 5 h after treatment to 0.14 +/- 0.03 ppb 8-11 h after treatment, with a decrease to 0.02 +/- 0.01 ppb 14 h after treatment. Levels of permethrin in standing pools near the stream within the treatment site were 0.01 +/- 0.01 ppb. Numbers of drifting aquatic invertebrates increased 2-fold during treatment and 4-fold 3 h after treatment and declined to before spray numbers within 9 h. Terrestrial insects did not appear to respond to treatments because none was found in stream drift samples. Trout fry (Dolly Varden), aquatic insect larvae, and periphyton (attached algae) within and below the treatment site during and after treatment did not show signs of mortality compared with an upstream untreated control site.     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.