Identification of a Novel DNA Methyltransferase Activity from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A DNA methyltransferase activity was identified in a strain of Bacillus thuringiensis that was found to protect DNA from cleavage by the restriction endonuclease HaeIII at overlapping sites. Site-directed mutagenesis was used to confirm the recognition sequence of the methyltransferase as ACGGC. RID= ID= <E5>Correspondence to:</E5> N. Crickmore; <E5>email:</E5> n.crickmore&commat;sussex.ac.uk Received: 13 September 2002 / Accepted: 7 October 2002     

Visualization of the <Emphasis Type="Italic">cbbL</Emphasis> Gene in Healthy and Starved Chemoautotrophic Nitrifying Bacteria

Three in situ methods of visualizing the cbbL gene in intact cells of nitrifying bacteria at different physiological states (dormant and metabolically active) were compared after epifluorescence microscopy and image analysis. FISH alone showed the weakest signal intensity. Direct in situ PCR, incorporating labeled nucleotides, showed the greatest sensitivity but also the greatest background. The combination of unlabeled in situ PCR followed by FISH showed relatively high sensitivity, along with the lowest background and highest specificity. Although functional gene expression was not examined in this study, visualization of the potential for carbon fixation in heterogeneous cultures of nitrifying bacteria was demonstrated. RID= ID= <E5>Correspondence to:</E5> C.D. Sinigalliano; <E5>email</E5>: sinigall&commat;fiu.edu Received: 9 August 2002 / Accepted: 4 September 2002     

An Antifungal Chitinase Produced by <Emphasis Type="Italic">Bacillus cereus</Emphasis> with Shrimp and Crab Shell Powder as a Carbon Source

The production of inexpensive chitinolytic enzymes is an element in the utilization of shellfish processing wastes. In this study, shrimp and crab shell powder prepared by treating shrimp and crab processing wastes with boiling and crushing was used as a substrate for the isolation of an antifungal chitinase-producing microorganism. Bacillus cereus YQ 308, a strain isolated from the soil samples, excreted one chitinase when cultured in a medium containing 2% (wt/vol) shrimp and crab shell powder as major carbon source. The chitinase, purified by sequential chromatography, had an Mr of 48 kDa and pI of 5.2. The purified chitinase (2 mg/ml) inhibited the hyphal extension of the fungi Fusarium oxysporum and Pythium ultimum. RID= ID= <E5>Correspondence to: </E5>S.-L. Wang; <E5>email:</E5> sabulo&commat;mail.dyu.edu.tw Received: 27 August 2002 / Accepted: 25 September 2002     

2-Ketogluconic Acid Production and Phosphate Solubilization by <Emphasis Type="Italic">Enterobacter intermedium</Emphasis>

Enterobacter intermedium, isolated from grass rhizosphere, exhibited a strong ability to solubilize insoluble phosphate. This bacterium oxidized glucose to gluconic acid and sequentially to 2-ketogluconic acid (2-KGA), which was identified using HPLC and GC-MS. The ability of E. intermedium to solubilize phosphate and produce 2-KGA produce in broth medium containing different components was monitored with air and without air supply. With an air supply, the production of 2-KGA markedly increased to about 110 g/l at day 10 in media containing 0.2 M gluconic acid, while it was about 65 g/l without gluconic acid addition. With an air supply, the concentration of soluble phosphate significantly decreased to 200–250 mg/l in media containing 1% CaCO₃, whereas it was about 1000 mg/l without CaCO₃ addition. Without an air supply, the concentration of 2-KGA and phosphate were negligible throughout the culture period. RID= ID= <E5>Correspondence to: </E5>K.Y. Kim; <E5>email:</E5> kimkil&commat;chonnam.ac.kr Received: 21 August 2002 / Accepted: 25 September 2002     

Humoral Immune Response in Aspergillosis: An Immunodominant Glycoprotein of 35 kDa from Aspergillus flavus

A glycoprotein preparation containing 70% carbohydrates and 30% proteins was isolated from the mycelium of two strains of Aspergillus flavus and fractionated by ConA-Sepharose affinity chromatography. An immunodominant 35-kDa antigen was detected in a ConA-bound fraction (B fraction). It contained mannose and galactose in a 1.4:1.0 ratio. This antigen seems to be able to elicit an antibody response in patients with aspergillosis and in rabbits immunized with A. flavus whole cells. The carbohydrate units of the BF fraction appeared to be responsible for the antigenicity, since treatment with periodate removed most of the antibody binding capacity. RID= ID= <E5>Correspondence to: </E5>E. Barreto-Bergter; <E5>email:</E5> eliana.bergter&commat;micro.ufrj.br Received: 16 August 2002 / Accepted: 24 October 2002     

Random Amplified Polymorphic DNA Profiles as a Tool for the Identification of Acanthamoeba divionensis

In the present study, we demonstrated the Random Amplified Polymorphism DNA (RAPD) diagnostic validity. In our study, we have analyzed RAPD profiles searching for characteristic and useful bands for Acanthamoeba diagnosis at the species level. We found a distinctive 370-bp band in A. divionensis RAPD patterns, using the OPC14 primer (TGCGTGCTTG) (Operon Technologies, Inc., Alameda, CA). The band specificity was confirmed by hybridization, using it as a probe, against all OPC14 amplifications from 10 different Acanthamoeba species. Once we sequenced this band, we used it to design a specific primer pair which showed positive amplification only in A. divionensis isolates. RID= ID= <E5>Correspondence to:</E5> A. Ortega-Rivas; <E5>email</E5>: aortega&commat;ull.es Received: 5 August 2002 / Accepted: 23 September 2002     

Heterospecific Expression of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Cell Shape Determination Genes <Emphasis Type="Italic">mreBCD</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>*

The divIVB operon of Bacillus subtilis includes the cell shape-associated mre genes, including the membrane-associated proteins MreC and MreD. TnphoA mutagenesis was utilized to analyze a topological model for MreC. MreC has a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large carboxy terminal domain which lies externally to the outer leaflet of the cell membrane. Expression of the B. subtilis MreB protein, or the Mre C and D proteins, results in a morphological conversion of the Escherichia coli host cells from a rod to a roughly spherical cell, morphologically similar to mre-negative mutants of E. coli. Immunolocalization of the MreC protein in B. subtilis revealed that this protein is found at the midcell division site of the bacterial cells, consistent with the postulated role of the Mre proteins in the regulation of septum-specific peptidoglycan synthesis. RID= ID= <E5>Correspondence to: </E5>G.C. Stewart; <E5>email:</E5> stewart&commat;vet.ksu.edu Received: 5 August 2002 / Accepted: 7 October 2002     

Direct Detection and Quantification of Horizontal Gene Transfer by Using Flow Cytometry and <Emphasis Type="Italic">gfp</Emphasis> as a Reporter Gene

A new cultivation-independent method for studying conjugal gene transfer between bacteria was evaluated. The method was based on direct detection and enumeration of donor and transconjugant bacterial cells by flow cytometry. Specific detection of transconjugants was obtained by using a conjugative plasmid tagged with a reporter gene (gfp) encoding green fluorescent protein. A chromosomal encoded repressor (lacI ^q1 ) repressed expression of GFP in the donor bacteria. Enumeration of the donor cells was performed after induction of GFP expression by the addition of inducer isopropyl-thio--D-galactoside (IPTG). The method presented here provided simple and precise quantification of horizontal gene transfer between both Escherichia coli and Pseudomonas putida strains. RID= ID= <E5>Correspondence to: </E5>S. J. S&oslash;rensen; <E5>email:</E5> SJS&commat;MERMAID.MOLBIO.KU.DK Received: 23 September 2002 / Accepted: 2 October 2002     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

nodD Alleles of Sinorhizobium fredii USDA191 Differentially Influence Soybean Nodulation,nodC Expression,and Production of Exopolysaccharides

All Rhizobium strains examined to date have one or multiple alleles of nodD. At least one copy of nodD and the presence of flavonoid exudates are required for nod gene induction and nodulation. Sinorhizobium fredii USDA191 has two copies of nodD. In this study, we demonstrate that inactivation of either copy of nodD caused a reduction in basal levels of expression of nodC. Extra copies of nodD1 had no effect on the expression of nodC when compared with the wild type, but extra copies of nodD2 abolished the inducer requirement, thereby rendering nodC constitutive. A nodD1 mutant was unable to nodulate soybean cultivars Peking and McCall. Inactivation of nodD2 or addition of extra copies of nodD1 or nodD2 caused delayed nodulation on Peking, and reduced the number of nodules on McCall. Both nodD alleles of S. fredii USDA191 appear to be involved in regulation of exopolysaccharide production; however, nodD2 appears to be more important in this respect than nodD1. RID= ID= <E5>Correspondence to: </E5>H.B. Krishnan; <E5>email:</E5> KrishnanH&commat;missouri.edu Received: 13 September 2002 / Accepted: 7 October 2002     

Indole-3-Acetic Acid Production in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> HP72 and Its Association with Suppression of Creeping Bentgrass Brown Patch

Pseudomonas fluorescens HP72, which suppresses the brown patch disease on bentgrass, produces several secondary metabolites, 2,4-diacetylphloroglucinol (2,4-DAPG), HCN, siderophore, and indole-3-acetic acid (IAA). In this study, IAA biosynthesis in strain HP72 was investigated. After several repeated subcultures, the spontaneous IAA low-producing mutant HP72LI was isolated. The IAA low production of the strain HP72LI was due to the low tryptophan side chain oxidase (TSO) activity. Colonization of strain HP72 on the bentgrass root induced root growth reduction, while strain HP72LI did not induce such growth reduction. The colonization ability of strain HP72 on the bentgrass root is higher than that of strain HP72LI. However, as for biocontrol ability, a significant difference in both strains was not detected. IAA production by strain HP72 may play a role in the construction of short root systems and take advantage of root colonization, but does not contribute to the biocontrol properties of P. fluorescens HP72. RID= ID= <E5>Correspondence to: </E5>S. Suzuki. Received: 9 September 2002 / Accepted: 7 October 2002     

Die Hypokotylfarbe als Markierungsfaktor von Genomstufen der Zuckerrübe

Zusammenfassung In zunehmendem Maße werden anisoploideBeta-Rübensorten angebaut, deren zytologische Kontrolle zwecks Feststellung der Genomstufenprozentanteile recht arbeitszeitaufwendig ist. Übereinstimmend mit polnischen Autoren wurde festgestellt, daß die Hypokotylfarbe ein geeigneter Markierungsfaktor für die einzelnen Genomstufen darstellt. Kreuzt man tetraploide Pflanzen, die ein grünes Hypokotyl besitzen, mit diploiden Pflanzen, die ein rosa Hypokotyl aufweisen, so erhält man von dem tetraploiden Partner tetraploide grüne und triploide hellbraune, von dem diploiden Partner diploide rosa und triploide hellbraune Nachkommenschaften. Die in bezug auf die Hypokotylfarbe heterozygoten Pflanzen kann man demnach von den homozygot grünen und homozygot rosa Individuen unterscheiden. Die Kreuzung diploid grünxtetraploid rosa ist für diese Zwecke nicht brauchbar, da sich die triploiden Heterozygoten mit einem grünen und zwei rosa Allelen in der Hypokotylfarbe nicht deutlich von den homozygoten rosa Pflanzen abheben. Auf die Bedeutung dieser Markierungsmöglichkeit für bestimmte Forschungsprobleme, die Züchtung und die Saatgutkontrolle wird hingewiesen.     

Enzymatic Browning and Biochemical Alterations in Black Spots of Pineapple [Ananas comosus (L.) Merr.]

Penicillium funiculosum Thom. was consistently isolated from pineapple-infected fruitlet (black spots). Polyphenol oxidase, peroxidase, and laccase activities were determined in extracts from contiguous and infected fruitlets. Healthy fruitlets showed a rather high level of polyphenol oxidase (optimum pH 7.0), and this activity was tremendously increased (×10) in contiguous infected fruitlets. Furthermore, infected fruitlets also exhibited laccase activity (optimum pH 4.0), while peroxidase was rather constant in both fruitlets. Browning reactions were attributed to qualitative and quantitative modifications of the enzymatic equipment (polyphenol oxidase and laccase) (p<0.0001). In infected fruiltets, sucrose and L-malic acid were present at significantly lower amounts than in healthy ones, likely owing to fungal metabolism (p<0.0001), whereas cell wall material was three times higher, which could be viewed as a defense mechanism to limit expansion of the mycelium. RID= ID= <E5>Correspondence to: </E5>S. Avallone; <E5>email:</E5> avallone&commat;siarc.cnearc.fr Received: 3 September 2002 / Accepted: 25 September 2002     

Amino Acid Substitution of the Largest Subunit of Yeast RNA Polymerase II: Effect of a Temperature-Sensitive Mutation Related to G1 Cell Cycle Arrest

A mammalian temperature-sensitive mutant tsAF8 shows cell cycle arrest at nonpermissive temperatures in mid-G₁ phase. DNA sequence comparison of the largest subunit of RNA polymerase II (Rpb1) from the wild-type and the mutant shows that the mutant phenotype results from a (hemizygous) C-to-A variation at nucleotide 944 in one rpb1 allele, giving rise to an Ala-to-Asp substitution at residue 315 in the protein. This amino acid substitution was introduced into the Schizosaccharomyces pombe rpb1 gene. Whereas tsAF8 cells showed growth defects and altered Rpb1 distribution at nonpermissive temperatures, yeast cells harboring this amino acid substitution did not show apparent temperature sensitivity. The effect of another temperature-sensitive Rpb1 mutation was also small. These results suggest that mutation of the rpb1 gene, which is critical in mammalian cells, may not be deleterious in yeast cells. RID= ID= <E5>Correspondence to: </E5>K. Sugaya; <E5>email:</E5> k_sugaya&commat;nirs.go.jp Received: 2 September 2002 / Accepted: 7 October 2002     

Evolution alpiner Populationen vonEuphrasia (Scrophulariaceae): Die mittel- bis kleinblütigen,drüsenhaarigen Arten

A more precise taxonomic concept ofE. hirtella and its infraspecific synonymy is presented. Its diploid nature (2n = 22) is confirmed. Within the European area ofE. hirtella five different races may be recognised: typical, brandisii, capitulata, Rofan and Bretagne. Taxonomic rank is not yet attributed to these races. The heterogeneous taxonomic assembly E. drosocalyx is disentangled. The type refers to products of hybrid introgression ofE. rostkoviana-characters (long glandular hairs) intoE. minima.

Former contributions of this series areEhrendorfer & Vitek (1984) andGreilhuber & al. (1984).     

Protoplasmic streaming in the giant unicellular green algaAcetabularia mediterranea

Summary Protoplasmic Streaming inAcetabularia mediteranea has been studied by microcinematography in 1. germinating zygotes, 2. germlings before the differentiation of rhizoids and apices, 3. young cells with rhizoids and apices, 4. vegetative cells-several centimeters in length, 5. cells with a maximum sized cap, containing secondary nuclei, and 6. cells after cyst formation. Intracellular transport is found to occur at a network-system of thin filaments and at a different system of headed streaming bands. At the network of filaments chloroplasts are found to move at a velocity of 1–2 m/sec. Headed streaming bands move along the filaments and may lead without interruption from the rhizoid to the apex of the cell andvice versa. The front zone of the streaming bands is occupied by a leading cytoplasmic head-structure. Small vesicles, polyphosphate granula and secondary nuclei are the predominant moving structures in headed streaming bands. The velocity of these particles is found to be 3–11 m/sec. The filament system is found during all developmental stages. Headed streaming bands are undetectable in germinating zygotes and develop from small cytoplasmic droplets in germlings to broad heavily loaded bands in the huge vegetative cell.Transport of secondary nuclei by headed streaming bands is not observed during mitotic divisions and after cyst formation, though moving bands are still present for several weeks after cyst formation.     

The relationships between the structure of paddy levees and the plant species diversity in cultural landscapes on the west side of Lake Biwa,Shiga, Japan

Paddy levees form networks of narrow linear habitats and play various roles in cultural landscapes. Traditional landscapes on the west side of Lake Biwa consist of paddy field terraces and both stone and soil levees that have been maintained by paddy field management using local resources. Paddy levees in this study site are principally classified into five different types. Our study points out how differences in paddy levee structure as well as in management practices influence the plant species. Seventeen paddy levee transects were split into four habitat types based on their species components by TWINSPAN. Spatial characteristics and physical structures of paddy levees depended on natural conditions and human activities. The species–area curves of each levee type showed a clear distinction: the soil, stone and abandoned curves were steep, while the concrete and consolidated ones were gentle. The vegetation on consolidated levees was utterly different from the vegetation on traditional levee types from the aspect of species richness and species components. Soil type levees contained various woody plant species and included more diverse and indigenous plant species than abandoned type levees.     

Die bursa- und schwanzstrukturen und ihre aberrationen bei den strongylina (Nematoda) morphologische studien zum problem der pluri-und paripotenzerscheinungen

Zusammenfassung In der Einleitung ist das Ziel der Arbeit in den wesentlichsten Punkten herausgestellt.Die Bursastrukturen (Bursavelum und Rippen bzw. Papillen) der parasitischen Strongylina lassen sich von den entsprechenden Bildungen der freilebenden Rhabditina, vor allem der Gattung Rhabditis, ableiten und in ihren Einzelgliedern homologisieren.Die im Laufe der Phylogenie bei den Strongylina auftretenden strukturellen Transformationen lassen sich auf einige wenige, relativ einfache morphogenetische Grundvorgänge zurückführen, die da sind: Wachstumsallometrien, Materialkompensationen, Organverschmelzungen und Spaltungen (Fissationen), Rudimentationen und ähnliche Vorgänge.Innerhalb der Strongylina Bursa ist ein Gefälle der Wachstumsgradienten feststellbar, das sich vom Zentrum der Bursa sowohl nach distal als auch proximalwärts abschwdcht. Zunehmende Förderung der zentral gelegenen Organe (Rippen) führt zu entsprechender Reduktion der peripheren Bursastrukturen, was vor allem im terminalen Schwanzabschnitt auffällt und zur Ausbildung des oft nur noch als Rudiment vorhandenen Dorsalrippenkomplexes führt. Letzterer entspricht in seiner Gesamtheit der Schwanzspitze der peloderen Rhabditiden mit den Papillen 9 und 10.Die bei Rhabditis moist getrennten Papillen 7 und 8 sind bei allen Strongylina zu einer Rippe (Externodorsal-Rippe) verschmolzen, die jedoch in manchen Aberrationen durch Abspaltung eines akzessorischen Astes ihre wahre Natur (als Verschmelzungsprodukt) zu erkennen gibt (Atavismus).Da dieselben Transformationsvorgänge innerhalb der Strongylina mehrfach unabhängig voneinander wirksam geworden sind, treten bestimmte Strukturformen als Parallelbildungen in verschiedenen phylogenetischen Union auf (polytope Entstehung).Zahlreich untersuchte Bildungsabweichungen (Aberrationen), deren Bedeutung für die Morphologie kurz umrissen wird, erschöpfen sich in den gleichen strukturellen Transformationstypen, die auch bei der Evolution der verschiedenen Union der Strongylina nachweisbar sind. Die Aberrationen führen daher häufig zu Atavismen oder zu Parallelvariationen (homologe Variationen").Die Zahl der Umwandlungsmbglichkeiten (Potenzen) der Bursastrukturen innerhalb der Strongylina ist beschränkt (Paripotenz im Sinne Haeckers). Bestimmte Arten (und Entwicklungshnien) haben jeweils nur bestimmte Potenzen realisiert. Andere können jedoch latent (virtuell) im Kryptotypus vorhanden sein, ohne normalerweise in Erscheinung. zu treten. In bestimmten Aberrationen können sie jedoch plötzlich realisiert werden, so ihr latentes Vorhandensein demonstrierend (Pluripotenz).Wie lange bestimmte Potenzen in einer Gruppe erhalten bleiben konnen, verdeutlichen auch die Schwanzhocker weiblicher Nematoden, als zum Bauplan der Nematoden gehbrende Bildungen. Die Potenz zur Ausbildung dieser Strukturen kommt offensichtlich sehr vielen Nematoden-Arten zu, wird jedoch nur in relativ wenigen Fällen, aber innerhalb der verschiedenen Gruppen bald hier, bald dort (disjunkte Verbreitung), realisiert. Es handelt sich bei den Schwanzhöckern um rudimentäre Organe, die bei keiner Nematoden-Art mehr voll ausgebildet erhalten sind. Ihre Rudimentation beruht zum Teil auf Materialentzug, als Folge von Unkonstruktionen der Schwanzregion, wobei die Adultstadien zuerst betroffen werden (Aphanisie nach Sewertzoff).Bei den in Chiropteren parasitierenden Strongylacanthinae haben sich Schwanzhöcker noch bei allen Arten erhalten, was ein offensichtlich archaisches Merkmal darstellt. Bei anderen Nematoden, denen sie nur im Larvalstadium zukommen, treten sie wohl durch Fötalisation in seltenen Fällen auch bei den adulten Stadien wieder auf.Alle speziellen Bursaformen der Strongylina lassen sich durch relativ wenige und einfache Transformationsvorgänge aus einem durch Abstraktion gewonnenen diagrammatischen Typus ableiten (Prinzip der variablen Proportionen" nach Troll).Die typisierten Umwandlungsvorgänge decken sich weitgehend mit den von Remane allgemein gefaßten strukturellen Typen der Realmutationen. Da sie bei den beobachteten Aberrationen, deren Entstehung auf dem Wege über Realmutationen sehr wahrscheinlich ist, in homologer Weise auftreten, kann das innerhalb der Strongylina zu beobachtende Evolutionsphänomen auf Realmutationen zurückgeführt warden.Obwohl sich die untersuchten strukturellen Transformationen in dem systematisch relativ wait gefaßten Rahmen einer Unterordnung abspielen (transspezifische Evolution nach Rensch), handelt es sich bei der von uns bevorzugten Terminologie (nach Woltereck und Remane), unter Berücksichtigung des Charakters der Umwandlungen, doch nur um Vorgänge, die in den Bereich der Mikroevolution fallen.     

Meiotic analysis ofElymus caucasicus,E. longearistatus,and their interspecific hybrids with twenty-threeElymus species (Triticeae,Poaceae)

Interspecific hybridizations were carried out between the two tetraploidsElymus caucasicus andE. longearistatus, and 23 tetraploids and hexaploids ofElymus containing SH, SY, SYH, and SYW genomes and representing various geographical regions. Meiotic pairing was studied in the two target species and their hybrids. It is concluded from this study that (i) interspecific hybridization is fairly easy to perform although strong reproductive barriers exist between the species; (ii)Elymus caucasicus andE. longearistatus are allotetraploids, and share the diverged SY genomes; (iii) the divergence of SY genomes is correlated with the geographic distance between theElymus spp. studied.     

Further cytogenetic analyses of the Croatian triploid shallot “Ljutika” (Allium cepa var.viviparum, Alliaceae) and its comparison with the Indian triploid “Pran”

Feulgen and silver-stained karyotypes and meiosis of two triploid viviparous onion forms (Allium cepa var.viviparum), the Croatian Ljutika and the Indian Pran, were comparatively analyzed. The results of chromosome measurements show that Ljutika and Pran are karyologically not identical, although significant similarities were found in the morphology of their chromosomes. Five geographically distant clones of Ljutika showed good agreement in the number and gross morphology of the chromosomes and in the number and position of NORs and interphase nucleoli. Heterotrivalents were predominant in meiosis of Ljutika but a relatively high frequency of higher multivalents together with univalents and bivalents were also observed. The relationship between Ljutika and Pran and their possible origin are discussed.