Aerobic spore-forming bacteria as detrimental infectants in plant tissue cultures

Summary Aerobic spore-forming bacteria were isolated from plant tissue cultures from a commercial plant cultivation station. Bacillus circulans was found to be a detrimental infectant as a serious consequence of the heat-resistance of the endospores of these bacteria. They were extremely motile, utilized several growth-promoting factors, and could be eliminated by early microscopical identification, killing by heat treatment, or by using antibiotics or disinfecants.Dedicated to Professor Dr. Gustav Kortüm on the occasion of his 80th birthday     

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting regeneration of transgenic plants

We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587–593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by -glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.Abbreviations BAP benzylaminopurine - CTAB hexadecyltrimethylammoniumbromide - Na₂EDTA ethylenediamine-tetra-acetate ferric-sodium salt - FeNaEDTA ethylenediamine-tetra-acetate ferric-sodium salt - GA₃ gibberellic acid 3 - GusA -glucuronidase - gusA -glucuronidase gene of Escherichia coli - IAA indole acetic acid - IBA indole butyric acid - 2iP N6-2-isopentenyl adenine - NAA naphthalene acetic acid - nptII neomycinphosphotransferase II gene - bar phosphinothricin acetyl transferase gene - PCR polymerase chain reaction - PPT phosphinothricin - STS silver thiosulphate - T-DNA transferred DNA - TDZ thidiazuron - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide - Zea trans-Zeatin     

Characterization of devA, a gene required for the maturation of proheterocysts in the cyanobacterium Anabaena sp. strain PCC 7120.

Mutant M7, obtained by transposon mutagenesis of the cyanobacterium Anabaena sp. strain PCC 7120, is impaired in the development of mature heterocysts. Under aerobic conditions, the mutant is unable to fix N2 because of a deficiency of at least two components of the oxygen-protective mechanisms: a hemoprotein-coupled oxidative reaction and heterocyst-specific glycolipids. DNA contiguous with the inserted transposon was recovered from the mutant and sequenced. The transposon had inserted itself within a 732-bp open reading frame designated devA. The wild-type form of devA, obtained from a lambda-EMBL3 library of Anabaena sp. DNA, had the identical sequence. Directed mutagenesis of devA in the wild-type strain showed that the phenotype of the mutant was caused by insertion of the transposon. The wild-type form of devA on a shuttle vector complemented the mutation in M7. Expression of devA by whole filaments, monitored following nitrogen stepdown by using luxAB as the reporter, increased ca. eightfold during differentiation; the increase within differentiating cells was much greater. The deduced sequence of the DevA protein shows strong similarity to the ATP-binding subunit of binding protein-dependent transport systems. The product of devA may, therefore, be a component of a periplasmic permease that is required for the transition from a proheterocyst to a mature, nitrogen-fixing heterocyst.     

CHARACTERIZATION OF TWO CHLORELLA VULGARIS (CHLOROPHYCEAE) STRAINS ISOLATED FROM WASTEWATER OXIDATION PONDS1

We report here on the characterization and isolation of two ecotypes of Chlorella vulgaris Beyerinck that coexist in wastewater reservoirs. One ecotype (C1) contains high amounts of chlorophyll b, is capable of autotrophic growth, and can utilize only a few organic solutes for growth. The second ecotype (C2) contains low amounts of chlorophyll b, requires vitamin B₁₂, and can support its growth with a broad range of organic compounds. Of the two ecotypes, the latter showed slower growth rates when light was the sole source of energy. Cells of C2-type Chlorella attained higher photosynthetic activities than C1-type cells at saturating irradiances. However, their low chlorophyll b content and lower light utilization efficiency suggest that C2-type Chlorella contains relatively low amounts of light-harvesting antennae, a disadvantage in severely light-limited ecosystems like wastewater reservoirs. We hypothesize that the two Chlorella types coexist by adopting different lifestyles: C1-type cells rely largely on their photosynthetic potential for energy conservation and growth, whereas C2-type cells may exploit their heterotrophic properties for this purpose.     

Novel Miscanthus hybrids: Modelling productivity on marginal land in Europe using dynamics of canopy development determined by light interception

New biomass crop hybrids for bioeconomic expansion require yield projections to determine their potential for strategic land use planning in the face of global challenges. Our biomass growth simulation incorporates radiation interception and conversion efficiency. Models often use leaf area to predict interception which is demanding to determine accurately, so instead we use low-cost rapid light interception measurements using a simple laboratory-made line ceptometer and relate the dynamics of canopy closure to thermal time, and to measurements of biomass. We apply the model to project the European biomass potentials of new market-ready hybrids for 2020–2030. Field measurements are easier to collect, the calibration is seasonally dynamic and reduces influence of weather variation between field sites. The model obtained is conservative, being calibrated by crops of varying establishment and varying maturity on less productive (marginal) land. This results in conservative projections of miscanthus hybrids for 2020–2030 based on 10% land use conversion of the least (productive) grassland and arable for farm diversification, which show a European potential of 80.7–89.7 Mt year⁻¹ biomass, with potential for 1.2–1.3 EJ year⁻¹ energy and 36.3–40.3 Mt year⁻¹ carbon capture, with seeded Miscanthus sacchariflorus × sinensis displaying highest yield potential. Simulated biomass projections must be viewed in light of the field measurements on less productive land with high soil water deficits. We are attempting to model the results from an ambitious and novel project combining new hybrids across Europe with agronomy which has not been perfected on less productive sites. Nevertheless, at the time of energy sourcing issues, seed-propagated miscanthus hybrids for the upscaled provision of bioenergy offer an alternative source of renewable energy. If European countries provide incentives for growers to invest, seeded hybrids can improve product availability and biomass yields over the current commercial miscanthus variety.     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

Characterization of Herpetosiphon spec. —A gliding filamentous bacterium from bulking sludge

Summary Filamentous bacteria were isolated from bulking activated sludge and identified as Herpetosiphon spec. The Gram-negative filaments are more than 500 m long and they show gliding motility. The bacteria grown in artificial media (J- or EC-medium), in shaken cultures yield about 3 g cells per liter. Optimum growth was observed at 25°C and pH 7.2. The colonies are either uncoloured or bright red depending on the cultivation medium. The isolated bacteria exhibit lytic activity towards cells of Escherichia coli and Klebsiella pneumoniae. The G+C ratio of the five strains from different bulking sludge samples was found to be between 48.7 moles% and 49.0 moles%.     

Recovery of intestinal membrane binding sites for K88 E. coli from pig mucosal organ cultures

Summary Putative receptors for K88+ E. coli from piglet intestinal epithelium were released into the organ culture medium and were demonstrated by direct binding with K88+ E. coli through the utilization of an in vitro binding procedure or by immunoprecipitation with K88 antigen.Incorporation of ¹⁴C-glucosamine by newborn to day old and 3-week to 6-week old piglet jejunal and ileal mucosa, in organ culture, occurred throughout the 24 hr culture period. Uptake in both age groups and both areas of the intestine was similar with a somewhat greater incorporation by the older age group.Secretion of ¹⁴C-glucosamine-labeled components into the culture medium was demonstrated by gel filtration of the concentrated medium. Some large molecular weight components eluted in the void volume in excess of 2 x 10⁶ daltons. A second peak of activity was spread from approximately 690K to 25K daltons. All eluted fractions demonstrated binding to K88+ E. coli.Antibodies to purified brush borders from susceptible pigs produced prominent precipitation bands following double diffusion with concentrated organ culture media which confirmed that the organ culture media contained labeled proteins of brush border origin.Immunoprecipitation of the intestinal mucosal organ culture media with K88+ pili and pilus antisera, followed by electrophoresis with SDS and reduced conditions, demonstrated a subunit of approximately 35K daltons.     

Mean corpuscular hemoglobin is increased in Martin-Bell syndrome

Summary Studying the blood picture of 11 patients with Martin-Bell syndrome, we found the erythrocytes relatively hyperchromic when compared to the data from 171 matched controls living in the same institution. Because mean corpuscular hemoglobin is increased also in patients with folic acid deficiency states, we feel that our data provide further evidence that Martin-Bell syndrome is an inherited disease of folate metabolism.The data were first presented at the 18th Meeting of the Gesellschaft für Anthropologie und Humangenetik, Münster/Westf., October 5–8, 1983