Molecular analysis of a plasmid-encoded phenol hydroxylase from Pseudomonas CF600

Pseudomonas strain CF600 is able to utilize phenol and 3,4-dimethylphenol as sole carbon and energy source. We demonstrate that growth on these substrates is by virtue of plasmid-encoded phenol hydroxylase and a meta-cleavage pathway. Screening of a genomic bank, with DNA from the previously cloned catechol 2,3-dioxygenase gene of the TOL plasmid pWW0, was used in the identification of a clone which could complement a phenol-hydroxylase-deficient transposon insertion mutant. Deletion mapping and polypeptide production analysis identified a 1.2 kb region of DNA encoding a 39.5 kDa polypeptide which mediated this complementation. Enzyme activities and growth properties of Pseudomonas strains harbouring this fragment on a broad-host-range expression vector indicate that phenol hydroxylase is a multicomponent enzyme containing the 39.5 kDa polypeptide as one component.     

Plant succession in areas of scorched and blown-down forest after the 1980 eruption of Mount St. Helens,Washington

Abstract. Patterns of plant succession were studied in areas of scorched and blown-down forest resulting from the 1980 eruption of Mount St. Helens, Washington. Changes in species abundance were observed for 7 years in permanent sample plots representing four post-disturbance habitats, or site types. Total plant cover and species richness increased with time on all site types. In blown-down forests supporting snowpack at the time of eruption, understory recovery was dominated by the vegetative regeneration of species persisting through disturbance. In forests without snowpacks, plant survival was poorer. Increases in cover and diversity were dominated first by introduced grasses, then by colonizing forbs characteristic of early successional sites. Epilo-bium angustifolium and Anaphalis margaritacea showed widespread recruitment and clonal expansion throughout the devastated area. As a result, species composition on previously forested sites converged toward that on formerly clearcut sites, where early serai forbs resprouted vigorously from beneath the tephra. Total plant cover and species diversity were poorly correlated with post-disturbance habitat and general site characteristics (e.g. distance from the crater, elevation, slope, and aspect). However, distributions of several life-forms (e.g. low sub-shrubs and tall shrubs) were strongly correlated with depth of burial by tephra and with cover of tree rootwads. Thus, early community recovery may reflect microsite variation or chance survival and recruitment rather than broad-scale gradients in environment or disturbance. Recovery of pre-disturbance composition and structure will undoubtedly be much slower than after other types of catastrophic disturbance. The rate and direction of community recovery will largely depend on the degree to which original understory species survived the eruption.     

Identification of algI and algJ in the Pseudomonas aeruginosa alginate biosynthetic gene cluster which are required for alginate O acetylation.

Mucoid strains of Pseudomonas aeruginosa overproduce alginate, a linear exopolysaccharide Of D-mannuronate and variable amounts of L-guluronate. The mannuronate residues undergo modification by C-5 epimerization to form the L-guluronates and by the addition of acetyl groups at the 0-2 and 0-3 positions. Through genetic analysis, we previously identified algF, located upstream of algA in the 18-kb alginate biosynthetic operon, as a gene required for alginate acetylation. Here, we show the sequence of a 3.7-kb fragment containing the open reading frames termed algI, algJ, and algF. An algI::Tn5O1 mutant, which was defective in algIJFA because of the polar nature of the transposon insertion, produced alginate when algA was provided in trans. This indicated that the algIJF gene products were not required for polymer biosynthesis. To examine the potential role of these genes in alginate modification, mutants were constructed by gene replacement in which each gene (algI, algJ, or algF) was replaced by a polar gentamicin resistance cassette. Proton nuclear magnetic resonance spectroscopy showed that polymers produced by strains deficient in algIJF still contained a mixture of D-mannuronate and L-guluronate, indicating that C-5 epimerization was not affected. Alginate acetylation was evaluated by a colorimetric assay and Fourier transform-infrared spectroscopy, and this analysis showed that strains deficient in algIJF produced nonacetylated alginate. Plasmids that supplied the downstream gene products affected by the polar mutations were introduced into each mutant. The strain defective only in algF expression produced an alginate that was not acetylated, confirming previous results. Strains missing only algJ or algI also produced nonacetylated alginates. Providing the respective missing gene (algI, algJ, or algF) in trans restored alginate acetylation. Mutants defective in algI or algJ, obtained by chemical and transposon mutagenesis, were also defective in their ability to acetylate alginate. Therefore, algI and algJ represent newly identified genes that, in addition to algF, are required for alginate acetylation.     

Molecular analysis of two functional homologues of the S 3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations

The S ₃ allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S ₁ allele, preceded by an 18 amino acid signal sequence. Expression of the S ₃ coding region in Escherichia coli produced a form of the protein, denoted S₃e, which specifically inhibited S₃ pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S₁ protein. In contrast to other S proteins identified to date, S₃ protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S₁ and S₃ proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S ₃ allele with antiserum raised to S₃e, revealed a protein (S ₃ s) which was indistinguishable in pI and M _r from that in the Shirley population. A cDNA encoding S ₃ s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level.     

Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K⁺]_o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca²⁺]_i) caused by Ca²⁺ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca²⁺ sequestration. In medium containing a normal concentration of extracellular Ca²⁺ ([Ca²⁺]_o), thapsigargin caused a sustained rise of intracellular Ca²⁺ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca²⁺]_o in the presence of thapsigargin further increased [Ca²⁺]_i, suggesting that the sustained rise of [Ca²⁺]_i was caused by a thapsigargin-induced Ca²⁺ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca²⁺ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca²⁺]_i and enhanced survival caused by depolarization with elevated [K⁺]_o, suggesting that these effects are mediated by Ca²⁺ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca²⁺]_i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca²⁺ through L-type channels. These results provide additional evidence that increased [Ca²⁺]_i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca²⁺]_i for investigation of this and other Ca²⁺-dependent phenomena. © 1995 John Wiley & Sons, Inc.