Involvement of both cellulose fibrils and a Ca2+-dependent adhesin in the attachment of Rhizobium leguminosarum to pea root hair tips.

We have previously described an assay for the attachment of Rhizobium bacteria to pea root hair tips (cap formation) which was used as a model to study the attachment step in the nodulation process. Under all conditions tested, a positive correlation was observed between the percentage of fibrillated cells and the ability of these bacteria to form caps and to adhere to glass, suggesting that fibrils play a role in the attachment of Rhizobium leguminosarum to pea root hair tips and to glass (G. Smit, J. W. Kijne, and B. J. J. Lugtenberg, J. Bacteriol. 168:821-827, 1986). In the present paper the chemical and functional characterization of the fibrils of R. leguminosarum is described. Characterization of purified fibrils by infrared spectroscopy and cellulase treatment followed by thin-layer chromatography showed that the fibrils are composed of cellulose. Purified cellulose fibrils, as well as commercial cellulose, inhibited cap formation when present during the attachment assay. Incubation of the bacteria with purified cellulase just before the attachment assay strongly inhibited cap formation, indicating that the fibrils are directly involved in the attachment process. Tn5-induced fibril-overproducing mutants showed a greatly increased ability to form caps, whereas Tn5-induced fibril-negative mutants lost this ability. None of these Tn5 insertions appeared to be located on the Sym plasmid. Both types of mutants showed normal nodulation properties, indicating that cellulose fibrils are not a prerequisite for successful nodulation under the conditions used. The ability of the fibril-negative mutants to attach to glass was not affected by the mutations, indicating that attachment to pea root hair tips and attachment to glass are (partly) based on different mechanisms. However, growth of the rhizobia under low Ca2+ conditions strongly reduced attachment to glass and also prevented cap formation, although it had no negative effect on fibril synthesis. This phenomenon was found for several Rhizobium spp. It was concluded that both cellulose fibrils and a Ca2+ -dependent adhesin(s) are involved in the attachment of R. leguminosarum to pea root hair tips. A model cap formation as a two-step process is discussed.     

Structural and electronic properties of the liver fluke hemoglobin heme cavity by nuclear magnetic resonance: hemin isotope labeling

Reconstitution of liver fluke (Dicrocoelium dendriticum) apo-hemoglobin with hemins selectively deuterated at specific positions has permitted the assignment of several heme resonances in the proton nuclear magnetic resonance spectrum of the Met-aquo and Met-cyano forms of the holoprotein. It was established that in the Met-aquo form the meso protons resonate at positions characteristic of a six-co-ordinated in-plane iron. From this, we deduced that the Met-aquo species retains a bound water molecule at pH values as low as 4.5. The orientation of the proximal histidine imidazole ring with respect to the heme group in the cavity was determined through the identification of the heme methyl signals and the analysis of the hyperfine shift pattern in the Met-cyano hemoglobin proton nuclear magnetic resonance spectrum. Compared to sperm whale myoglobin, the heme appears to be rotated by 180 degrees about the alpha, gamma meso-axis. Protein isomers with the heme group in a reversed orientation were not detected, even shortly after reconstitution. In the Met-cyano form, the resonances most affected by the Bohr transition were shown to arise from the heme propionates.     

Specificity of chimpanzee antibodies binding a strain-specific HIV-1 neutralization epitope of the external envelope

Sera from three chimpanzees infected with a primary lymphadenopathy-associated virus (LAV-1) or human T-lymphotropic virus type III (HTLV-IIIB) passage, from two chimpanzees infected with blood from the primary infected chimpanzees, and from one chimpanzee infected with blood from a secondary passage animal all bound the peptides 3B and 3B/RF, sharing the sequence IQRGPGR, with equally high titers. Pepscan analysis confirmed the amino acids Q, R, G, P, and G as irreplaceable in order to retain antigenicity.     

Transformation of Claviceps purpurea using a bleomycin resistance gene

Summary To develop a DNA-mediated transformation system for Claviceps purpurea a vector was constructed using a bleomycin-resistance gene (bleo ^R) fused in frame to the Aspergillus nidulans trp C promoter as a dominant selection marker. The construct was shown to be functional in Aspergillus nidulans and Aspergillus niger and used to transform a wild strain of Claviceps purpurea. Transformats were obtained at low frequencies; they were shown to contain transforming DNA integrated into the chromosomal DNA, probably in multimeric copies and at multiple sites. Combined Southern, Northern and resistance level analysis indicate that the A. nidulans promoter is functional in C. purpurea.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65^th birthday     

Aggregate-formation by Clostridium butyricum

Summary Clostridium butyricum was grown in a glucose-limited chemostat culture at a dilution rate of 0.1 h^–1 at pH 6.0. With 0.9% w/v input glucose in the medium the cells were found to grow in suspension and glucose was fermented completely to acetate and butyrate. An increase in the input concentration of glucose resulted in increased concentrations of end-products, but not all extra glucose was consumed. It could be demonstrated that this was due to a lowering of the maximal growth rate by elevated levels of butyric acid. However, prolonged growth in the presence of high glucose concentrations led to an increase in biomass. This was caused by the selection of a variant that was less sensitive to butyrate. This variant was able to form aggregates in an anaerobic gas-lift reactor at high dilution rates. Inoculation of these aggregates in a conventional chemostat culture with high glucose input resulted in an aggregated culture that remained stable for at least 6 months, and in which all glucose was consumed. Whether the organisms grew in suspension or in aggregates was found to be determined by the concentration of butyrate. The isolation of aggregate-forming variants from chemostat cultures leads to a very simple and new type of immobilization technique.Offprint requests to: G. R. Zoutberg     

Rapid detection of chromosome 16 inversion in acute nonlymphocytic leukemia, subtype M4: regional localization of the breakpoint in 16p

The pericentric inversion of chromosome 16 characteristic for acute nonlymphocytic leukemia, subtype M4, was detected in five patients by means of nonradioactive in situ hybridization of complete cosmids. First, five cosmids situated along the short arm of chromosome 16 were used to map the breakpoint of the inversion distal to the rare folate-sensitive fragile site FRA16A. Then, the use of two cosmids on either side of the breakpoint, combined with a probe specific for the centromeric region of chromosome 16, readily detected the inversion, even in poor metaphase spreads.     

Influence of soil type on the transfer of plasmid RP4p from Pseudomonas fluorescens to introduced recipient and to indigenous bacteria

Abstract Transfer of plasmid RP4p from introduced Pseudomonas fluorescens to a co-introduced recipient strain or to members of the indigenous bacterial population was studied in four different soils of varying texture planted with wheat. Donor and recipient strains showed good survival in the four soils throughout the experiment. The numbers of transconjugants found in donor and recipient experiments in two soils, Ede loamy sand and Löss silt loam were significantly higher in the rhizosphere than in corresponding bulk soil. In the remaining two soils, Montrond and Flevo silt loam, transconjugant numbers were not significantly higher in the rhizosphere than in the bulk soil.
The combined utilization of a specific bacteriophage eliminate the donor strain and the pat sequence as a specific marker to detect RP4p was found to be very efficient in detecting indigenous transconjugants under various environmental conditions. The numbers of indigenous transconjugants were consistently higher in rhizosphere than bull soil. A significant rhizosphere effect on transconjugant numbers of transconjugants were recovered from Flevo and Montrond silt loam; these soils possess characteristics such as clay or organic matter contents which may be favorable to conjugation.     

Localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to chromosomes Xq24-q25 and 5q23-q31.

The chromosomal localizations of two closely related human DNA repair genes, HHR6A and HHR6B, were determined by in situ hybridization with biotinylated probes. HHR6A and HHR6B (human homolog of yeast RAD6) encode ubiquitin-conjugating enzymes (E2 enzymes), likely to be involved in postreplication repair and induced mutagenesis. The HHR6B gene was assigned to human chromosome 5q23-q31, whereas the HHR6A gene was localized on the human X chromosome (Xq24-q25). This latter assignment was confirmed with an X-specific human-mouse/hamster somatic cell hybrid panel. Southern blot analysis points to an X and an autosomal localization of HHR6A and HHR6B, respectively, in the mouse. The potential involvement of these genes in human genetic disorders is discussed.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage phiR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10 transconjugants per g of soil when 10 donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx RpP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Characterization of caulobacters isolated from wastewater treatment systems.

Caulobacters are generally assumed to be found only in environments of low organic content; however, we readily isolated strains from a variety of sewage treatment system designs and locations, and 33 distinct strains were characterized. Most were morphologically similar, having the crescent-shaped cell body, short stalk, and hexagonally packed, paracrystalline surface (S) layer characteristic of several Caulobacter crescentus laboratory strains. Upon closer examination, they were distinguishable on the basis of protein band profiles on polyacrylamide gel electrophoresis, gross colony characteristics, or holdfast composition or by DNA restriction fragment length polymorphism analysis with flagellin and S-layer gene probes. Most of the isolates contained one or more high-molecular-weight plasmids and were resistant to a number of antibiotics, characteristics generally not shared with caulobacters isolated from other sources. Six of the 33 strains were retained because they did not fit the typical isolate profile; these strains are overrepresented in our collection compared with their relative proportion in wastewater treatment systems. By colony hybridization and restriction fragment length polymorphism analysis, all of these and one typical isolate showed less homology than the others to the surface array gene of a laboratory strain (C. crescentus CB15), and three hybridized less strongly with the flagellin gene from the same strain. In sum, although the strains were distinguishable, caulobacters from the wastewater treatment systems we examined were relatively homogenous, were similar to characterized laboratory strains, and, with exceptions, could probably be reliably detected as a group by gene probes derived from C. crescentus strains.