<Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>. The most important bacterial warfare agents — <Emphasis Type="Italic">review</Emphasis>

There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.     

Effects of sugars and growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> growth of <Emphasis Type="Italic">Dactylorhiza</Emphasis> species

The influence of sugars and growth regulators on shoot and root growth of Dactylorhiza species was studied under in vitro conditions. The seedling development was stimulated with the application of glucose and sucrose at concentration of 10 g dm⁻³ each. The improvement of shoot growth rate and shoot length was enhanced by cytokinins N ⁶-(2-isopentenyl)adenine or N ⁶-benzyladenine and their combination with auxin indolebutyric acid (IBA). The root growth rate and root length of seedlings increased in the presence of IBA and α-naphthaleneacetic acid. Individual Dactylorhiza species showed statistically significant differences in shoot and root development depending on sugar and growth regulator combinations.     

Frequencies of the <Emphasis Type="Italic">DRB1</Emphasis>, <Emphasis Type="Italic">DQA1</Emphasis>, <Emphasis Type="Italic">DQB1</Emphasis> and <Emphasis Type="Italic">TNFA</Emphasis> alleles in immigrant population of west Siberia

Based on population analysis of the DRB1, DQA1, DQB1 and TNFA allele frequency distribution patterns, regional features of immunogenetic structure of the population of West Siberia were investigated. Statistically significant linkage disequilibrium within the HLA class II region, as well as between the TNFA and DRB1, DQA1, and DQB1 was demonstrated. Population frequency distribution patterns of two- and multilocus haplotypes were examined.     

Structural characteristics of the chloroplast <Emphasis Type="Italic">rpS16</Emphasis> intron in <Emphasis Type="Italic">Allium sativum</Emphasis> and related <Emphasis Type="Italic">Allium</Emphasis> species

The intron sequence of chloroplast rpS16 and the secondary structure of its pre-mRNA were characterized for the first time in 26 Allium sativum accessions of different ecologo-geographical origins and seven related Allium species. The boundaries and main stem-loop consensus sequences were identified for all six domains of the intron. Polymorphism was estimated for the total intron and its regions. The structural regions of the rpS16 intron proved to be heterogeneous for mutation rate and spectrum. Mutations were most abundant in domains II and IV, and transition predominated in domains I, III, V, and VI. In addition to structural elements and motifs typical for group IIB introns, several Allium-specific micro- and macrostructural mutations were revealed. A 290-bp deletion involving domains III and IV and part of domain V was observed in A. altaicum, A. fistulosum, and A. schoenoprasum. Several indels and nucleotide substitutions were found to cause a deviation of the pre-mRNA secondary structure from the consensus model of group II introns.     

Isolation of <Emphasis Type="Italic">madA</Emphasis> homologs in <Emphasis Type="Italic">Pilobolus crystallinus</Emphasis>

Pilobolus crystallinus shows unique photoresponses at various growing stages. cDNAs for putative photoreceptors were cloned from this fungus. Three genes named pcmada1, pcmada2, and pcmada3 were identified from the PCR fragments, and amplified with degenerated primers for the LOV domain, which is conserved in many blue-light receptors. Deduced amino acid sequences for PCMADA1, PCMADA2, and PCMADA3 had one light-oxygen-voltage (LOV)-sensing and two PER-ARNT-SIM (PAS) domains. A zinc finger DNA-binding motif was conserved in the C-terminals of PCMADA1 and PCMADA3. However, PCMADA2 lacked the zinc finger motif. Expression of pcmada1 was suppressed by blue light whereas that of pcmada3 was promoted by blue-light irradiation.     

Overexpression of the Chitosanase Gene in <Emphasis Type="Italic">Fusarium solani</Emphasis> via <Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-Mediated Transformation

To overexpress the chitosanase gene (csn) in F. solani, a vector based on pCAMBIA 1300 was constructed. The csn gene, which is under control of the Aspergillus nidulans gpdA promoter and A. nidulans trpC terminator, was introduced back into the F. solani genome by Agrobacterium tumefaciens-mediated transformation, and the herbicide-resistance gene bar from Streptomyces hygroscopicus was used as the selection marker. Transformants which showed a significant increase in chitosanase production (~2.1-fold than control) were obtained. Southern blot analysis indicated that most transformants had a single-copy T-DNA integration.     

Influence of medium components on growth kinetics of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Starting with the standard complex medium HL-5C, the influence of different medium components on the growth behavior of Dictyostelium discoideum was investigated. For this purpose, each component was individually deleted from the complex medium HL-5C, and the overall concentration of all components as well as the kinds and concentrations of carbon source were varied. The effects of the supplementation with major components of synthetic medium like vitamins, trace metals, inorganic salts were also investigated. When glucose being the carbon source was deleted from the standard HL-5C medium, the maximal cell density then had a drastic drop. Deletion of yeast extract also caused lower maximal cell density. Of all the carbohydrates investigated only maltose and glucose led to higher final cell concentration. Adding inorganic salts such as CaCl₂ and MgCl₂ to HL-5C medium significantly improved the cell growth, whereas addition of vitamins or trace metals had little effect on cell growth. A semi-empirical model is employed to simulate the cell growth.     

Effect of alkali treatment time and extraction time on agar from <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis>

The effects of alkali treatment time and extraction time of native agar and alkali treated agar obtained from Gracilaria vermiculophylla were studied. The response characteristics were mainly agar yield and gel strength. Alkali treatment was carried out at 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h. Agar yield and gel strength decreased with the increase in the time of the alkali treatment. The highest yield (15.3%) and highest gel strength (1,064 g cm⁻²) were obtained at 0.5 h, and therefore this time was used for the next experiment. The extraction of both native and alkali treated agars was carried out at 1.5, 2.0, 2.5, and 3.0 h. The best extraction time for alkali treated agar was 1.5 h, and for native agar 2.5 h. The alkali treated agar obtained with the different alkali treatment and extraction times showed higher melting (92.4–99.7°C) and gelling (35.7–39.6°C) temperatures. Native agar was lower in melting (60.2–64.1°C) and gelling (20.4–23.4°C) temperatures. The 3,6-anhydrogalactose content decreased with increasing alkali treatment time, with the opposite effect during the extraction of native and alkali treated agars.     

Effect of abscisic acid and proline on <Emphasis Type="Italic">in vitro</Emphasis> flowering in <Emphasis Type="Italic">Vigna aconitifolia</Emphasis>

An experiment was taken up to find out possibilities of manipulating the in vitro flowering in moth bean. Abscisic acid (ABA) and proline both alone and in combination influenced days to flower induction, number of flowers per plant, number of pods per plant and seeds per pod. Frequency of flowering plants approached 100 % at 1 and 3 μM ABA and 800 μM proline. The range of flowering period (3 to 23.6 d) has also been influenced by various treatments.     

Decolourisation and dephenolisation potential of selected <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Nigri</Emphasis> strains – <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> in olive mill wastewater

Aspergillus section Nigri strains Aspergillus aculeatus Ege-K 258, A. foeditus var. pallidus Ege-K156, A. niger Ege-K 4 and A. tubingensis Ege-K 265 were used to treat olive mill wastewater (OMW) in an investigation aimed at exploring their dephenolisation and decolourisation ability and, consequently, the economic feasibility of using any or all of these strains in a pre-treatment step in the processing of OMW. Of these strains A. tubingensis Ege-K 265 resulted in an 80% decolourisation of twofold-diluted OMW and a 30% decolourisation of undiluted OMW; in addition, it was able to remove approximately 30% of all phenolic compounds in both twofold-diluted and undiluted OMW. We conclude that A. tubingensis Ege-K 265 could be effectively used in the pre-treatment step of a combined aerobic-anaerobic process to solve the environmental problems caused by OMW in Mediterranean countries.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Life cycle of <Emphasis Type="Italic">Aylax hypecoi</Emphasis> (<Emphasis Type="Italic">Insecta: Hymenoptera: Cynipidae</Emphasis>), a gall inducer on <Emphasis Type="Italic">Hypecoum</Emphasis> ssp. (<Emphasis Type="Italic">Papaveraceae</Emphasis>)

The life cycle and developmental stages of Aylax hypecoi (Trotter, 1913, Hymenoptera: Cynipidae: Aylacini) were studied in detail. Aylax hypecoi is known to induce galls in fruits of two Hypecoum species — H. imberbe and H. geslini (Papaveraceae) and the larva develops in host plant fruits. The morphology and development of egg, larva and pupa were investigated, which has previously not been done. The shape and size of terminal-instar larvae and associated galls are sex-specific. Overwintering stage, adult emergence and flying periods, and egg productivity were studied also.     

Effect of the fungicide Prochloraz-Mn on the cell wall structure of <Emphasis Type="Italic">Verticillium fungicola</Emphasis>

The chemical structure of the cell wall of two isolates of Verticillium fungicola collected from diseased fruit bodies of the commercial mushroom Agaricus bisporus treated with the fungicide Prochloraz-Mn was analyzed. The isolates were obtained during different periods of time and grown in the absence and presence of the LD₅₀ values of the fungicide for V. fungicola. In addition, another V. fungicola isolate collected previous to the routine utilization of Prochloraz-Mn but grown under the same conditions was also analyzed. The overall chemical composition of the cell wall from the three isolates showed detectable differences in their basic components, with a significant decrease in the protein content in fungicide-treated cells. This inhibitory effect was partially compensated by an increase in neutral and/or aminated carbohydrates and was accompanied by appreciable modifications of polysaccharide structure, as deduced after methylation analysis and gas-liquid chromatography-mass spectrometry (GLC-MS). Moreover, differences in hyphal morphology caused by the fungicide were observed by transmission electron microscopy (TEM). Accepted 2 May 2002 Electronic Publication     

Synthesis of orthogonally protected (<Emphasis Type="Italic">3R</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)- and (<Emphasis Type="Italic">3S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4,5-diamino-3-hydroxypentanoic acids

Summary.  The paper describes two methods of the synthesis of ethyl (3R,4S)- and (3S,4S)-4-[(benzyloxycarbonyl)amino]-5-[(tert-butyloxycarbonyl)amino]-3-hydroxypentanoates, useful for the syntheses of edeine analogs. Differently N-protected (S)-2,3-diaminopropanoic acid was used as a substrate in both procedures. The absolute configuration of newly generated asymmetric carbon atoms C-3 in β-hydroxy-γ,δ-diamino products was assigned by means of ¹H NMR spectroscopy after their transformation into corresponding piperidin-2-ones. Received May 24, 2002 Accepted October 10, 2002 Published online December 18, 2002 Acknowledgment The authors are indebted to the Faculty of Chemistry, Technical University of Gdańsk for financial support. Authors' address: Zbigniew Czajgucki, M. Sc., Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Technical University of Gdańsk, 11/12 Narutowicza St., 80-952 Gdańsk, Poland, Fax +48 58 347 11 44, E-mail: zmczaj@wp.pl     

Effect of the<Emphasis Type="Italic"> recU</Emphasis> suppressors<Emphasis Type="Italic"> sms</Emphasis> and<Emphasis Type="Italic"> subA</Emphasis> on DNA repair and homologous recombination in<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

In Bacillus subtilis, mutant alleles of the genes sms and subA partially suppress the recombination phenotype of recU cells. When present in an otherwise Rec(+) strain, Delta sms and Delta subA alleles render cells slightly sensitive to DNA-damaging agents, and impair chromosomal transformation (3- to 10-fold reduction), but do not affect plasmid transformation (less than 1.5-fold reduction). The Delta sms and Delta subA alleles were introduced into rec-deficient strains representative of the epistatic groups alpha (recF strain), beta (addA addB), gamma (recH), epsilon (recB, Delta recU and recD strains) and zeta (Delta recS). Both the Delta sms and Delta subA mutations were found to increase sensitivity to DNA-damaging agents in recF, Delta recS and addAB cells. In contrast, the Delta sms mutations decreased the sensitivity of recB, Delta recU, recD and recH cells, and the Delta subA mutation decreased the sensitivity of recB and Delta recU cells to DNA-damaging agents. Functions classified within the epistatic groups alpha, epsilon and zeta are required for intramolecular recombination, measured as plasmid transformation. The Delta sms and Delta subA mutations, which partially suppressed the recombinational repair phenotype of mutants for functions within epistatic group epsilon, enhanced plasmid transformation of recU (recB, recD) and recS cells by 10- to 20-fold. In the absence of the proteins Sms and SubA, the recombination machinery is apparently redirected towards (an) alternative pathway(s). Furthermore, the shared ability of the Delta sms and Delta subA mutations to act as indirect suppressors of recB, recU and recD mutations supports the classification of the recBUD genes within epistatic group epsilon.     

Export of functional <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> alditol oxidase to the periplasm or cell surface of <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.     

Mapping species differences for adventitious rooting in a <Emphasis Type="Italic">Corymbia torelliana</Emphasis> × <Emphasis Type="Italic">Corymbia citriodora</Emphasis> subspecies <Emphasis Type="Italic">variegata</Emphasis> hybrid

Quantitative trait loci (QTL) detection was carried out for adventitious rooting and associated propagation traits in a second-generation outbred Corymbia torelliana × Corymbia citriodora subspecies variegata hybrid family (n = 186). The parental species of this cross are divergent in their capacity to develop roots adventitiously on stem cuttings and their propensity to form lignotubers. For the ten traits studied, there was one or two QTL detected, with some QTL explaining large amounts of phenotypic variation (e.g. 66% for one QTL for percentage rooting), suggesting that major effects influence rooting in this cross. Collocation of QTL for many strongly genetically correlated rooting traits to a single region on linkage group 12 suggested pleiotropy. A three locus model was most parsimonious for linkage group 12, however, as differences in QTL position and lower genetic correlations suggested separate loci for each of the traits of shoot production and root initiation. Species differences were thought to be the major source of phenotypic variation for some rooting rate and root quality traits because of the major QTL effects and up to 59-fold larger homospecific deviations (attributed to species differences) relative to heterospecific deviations (attributed to standing variation within species) evident at some QTL for these traits. A large homospecific/heterospecific ratio at major QTL suggested that the gene action evident in one cross may be indicative of gene action more broadly in hybrids between these species for some traits.