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The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent Pseudomonas fluorescens F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and l -tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent Pseudomonas community. These results suggest that the root-associated Pseudomonas community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant.

372 natural isolates of Rhizobium leguminosarum bv. viciae, rescued from nodules of pea plants grown in an agricultural field in northern Italy, were analyzed by different methods. Three DNA-based fingerprinting techniques were lined up to compare their relative degree of resolution and possible advantages of each approach. The methods included (i) Eckhardt gel plasmid profiles, (ii) pulsed-field gel electrophoresis (PFGE) of genomic large fragment digests, and (iii) random amplified polymorphic DNA (RAPD) profiles, generated with arbitrary primers. The scheme also involved the isolation of a number of different isolates per nodule to estimate the level of intra-nodular variability. It was therefore possible to evaluate the frequency of double and multiple occupancies, and the proportion of the alternative profiles sharing the same nodule, generally resulting in a numerically dominant, main representative accompanied by a secondary one with a slightly different fingerprint. This finding revealed that the different profiles within a nodule are normally due to bacteria derived from the same single invader following genetic alterations possibly occurred during infection, e.g., by plasmid loss. The analysis of 31 nodules revealed 16 different patterns, representing the most frequently occurring nodulation-proficient isolates of the natural soil examined, five of which were found with frequencies around 15%. The sensitivity of the methods in differentiating isolates was compared. The relatedness of the different natural rhizobial isolates was investigated by densitometrical gel analysis of the fingerprints, allowing a comparison of the results. One of the most interesting conclusions was that the degree of information yielded by the plasmid gel profiling alone, carried out by simple visual inspection without software-aided analyses, was surprisingly high, as it enabled a placement of the isolates, whose accuracy, in terms of relatedness, was subsequently confirmed by each of the two genomic methods.Key words: Rhizobium, fingerprinting, DAF, PFGE, plasmid, Eckhardt gel.

In a comparative study, the PCR-based RAPD and ERIC fingerprint methods were evaluated for their resolving power to discriminate among 21 isolates of a natural Rhizobium meliloti population. PCR fingerprint patterns were analysed by using an automated laser fluorescent (ALF) DNA sequencer, thus allowing the automated on-line storage of data. Results obtained were compared to a classification system using insertion sequence (IS) fingerprinting. Both PCR fingerprint methods were comparable in their ability to resolve differences amongst Rh. meliloti isolates. Grouping of strains on the basis of their RAPD as well as their ERIC fingerprints correlated with grouping of strains according to their IS fingerprints. Moreover, strains displaying identical PCR patterns could be further differentiated according to their IS fingerprints, thus allowing a detailed insight into phylogenetic relationship among strains. The automated evaluation of strain-specific fingerprint patterns has the potential to become a valuable tool for studies of bacterial population genetics. Moreover, the rapid identification of single strains, e.g. pathogens in epidemiological studies seems feasible.

Conserved ortholog set (COS) markers are an important functional genomics resource that has greatly improved orthology detection in Asterid species. A comprehensive list of these markers is available at Sol Genomics Network ( http://solgenomics.net/ ) and many of these have been placed on the genetic maps of a number of solanaceous species. We amplified over 300 COS markers from eight potato accessions involving two diploid landraces of Solanum tuberosum Andigenum group (formerly classified as S. goniocalyx, S. phureja), and a dihaploid clone derived from a modern tetraploid cultivar of S. tuberosum and the wild species S. berthaultii, S. chomatophilum, and S. paucissectum. By BLASTn (Basic Local Alignment Search Tool of the NCBI, National Center for Biotechnology Information) algorithm we mapped the DNA sequences of these markers into the potato genome sequence. Additionally, we mapped a subset of these markers genetically in potato and present a comparison between the physical and genetic locations of these markers in potato and in comparison with the genetic location in tomato. We found that most of the COS markers are single-copy in the reference genome of potato and that the genetic location in tomato and physical location in potato sequence are mostly in agreement. However, we did find some COS markers that are present in multiple copies and those that map in unexpected locations. Sequence comparisons between species show that some of these markers may be paralogs. The sequence-based physical map becomes helpful in identification of markers for traits of interest thereby reducing the number of markers to be tested for applications like marker assisted selection, diversity, and phylogenetic studies.

Rhizobium meliloti strains isolated from alfalfa plants grown in a mining recultivation field, in a model ecosystem (microcosm) and in soil core containers were characterized by two new taxonomic methods, fingerprinting and handprinting, using insertion sequence elements (IS) as hybridization probes. The diversity of strains within the field population could first be detected with IS-fingerprinting, whereby nearly three times more groups of Rhizobium meliloti strains could be identified in comparison to the groups according to plasmid profiles. This complexity and diversity of the rhizobial population was also detected in microcosm studies. Strains identified among the field population were also detected in the microcosm studies. The persistence of rhizobia in soil was demonstrated in soil core samples held in a cold room for 2 years. A decrease in the genomic diversity of the R. meliloti population upon soil storage was observed. A novel monitoring method, IS-handprinting, in which the presence of certain endogenous insertion elements within a strain is registered, was successfully employed to characterize genetically the field R. meliloti strains with simplicity and speed. In contrast to IS-fingerprinting, IS-handprinting is based on a simple plus-or-minus detection, which is sufficient for a taxonomic characterization. Both methods, using a non-radioactive detection system, are sensitive enough to detect one copy of an insertion element in a strain's genome. IS-fingerprinting, with its fine resolution, would be suitable for ecological studies of individual strains in any complex ecosystem, whereas IS-handprinting would be suitable for monitoring strains and characterizing large numbers of strains.

The nucleotide sequence of a 3.4-kb EcoRI-PstI DNA fragment of Xanthomonas campestris pv. campestris revealed two open reading frames, which were designated xanA and xanB. The genes xanA and xanB encode proteins of 448 amino acids (molecular weight of 48,919) and 466 amino acids (molecular weight of 50,873), respectively. These genes were identified by analyzing insertion mutants which were known to be involved in xanthan production. Specific tests for the activities of enzymes involved in the biosynthesis of UDP-glucose and GDP-mannose indicated that the xanA gene product was involved in the biosynthesis of both glucose 1-phosphate and mannose 1-phosphate. The deduced amino acid sequence of xanB showed a significant degree of homology (59%) to the phosphomannose isomerase of Pseudomonas aeruginosa, a key enzyme in the biosynthesis of alginate. Moreover, biochemical analysis and complementation experiments with the Escherichia coli manA fragment revealed that xanB encoded a bifunctional enzyme, phosphomannose isomerase-GDP-mannose pyrophosphorylase.

On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a clear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliloti strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.

Cosmid clones able to restore exopolysaccharide production in possibly insertion sequence element-induced surface mutants of Xanthomonas campestris pv. campestris were isolated. By fragment-specific Tn5-lac mutagenesis of one of the cosmids, pXCB1002, a new DNA region which is involved in exopolysaccharide biosynthesis and which is organized into at least 12 complementation groups was identified.

A series of broad-host-range expression and lac fusion vectors, based on RSF1010 derivatives, was constructed. The expression vectors contain various promoters (pNm, plac, ptac and pS1) for expression of foreign genes. The efficiency of the promoters was determined in Escherichia coli, Rhizobium meliloti, Rhizobium leguminosarum and Pseudomonas putida by beta-galactosidase activity measurements. Of the promoters assayed in E. coli, the most effective is the tac promoter, whereas in soil bacteria the appropriate promoter for overexpression of foreign genes is the NmR promoter. The GmR gene, serving as a selectable marker for the plasmids, was efficiently expressed in R. meliloti as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thus, pGm was also used to construct an expression vector. The translational fusion vectors allow the identification and characterization of promoter-carrying cloned fragments on the translational level, whereas the transcriptional fusion vectors can be used to identify and to study promoters on cloned fragments. All lac fusion vectors contain the E. coli lacZ gene or the complete lac operon facilitating quantification of expression.

Three types of new variants of the broad-host-range transposen tn5 are described. (i) Tn5-mob derivatives with the new selective resistance (R) markers Gm R, Sp R and TcR facilitate the efficient mobilization of replicons within a wide range of Gram-negative bacteria, (ii) Promoter probe transposons carry the promoterless reporter genes lacZ, npt II, or luc, and NmR GmR or TcR as selective markers. These transposons can be used to generate transcriptional fusions upon insertion, thus facilitating accurate determinations of gene expression, (iii) Tn5-P-out derivatives carry the npt- or tac -promoter reading out from the transposen, and TcR, NmR or GmR genes. These variants allow the constitutive expression of downstream genes. The new Tn5 variants are available on mobilizable Escherichia coli vectors suitable as suicidal carriers for transposen mutagenesis of non-E. coli recipients and some on a phage λ mutant to be used for transposen mutagenesis in E. coli.

We have developed a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related to Escherichia coli. The system consists of two components: special E. coli donor strains and derivatives of E. coli vector plasmids. The donor strains (called mobilizing strains) carry the transfer genes of the broad host range IncP–type plasmid RP4 integrated in their chromosomes. They can utilize any gram negative bacterium as a recipient for conjugative DNA transfer. The vector plasmids contain the P–type specific recognition site for mobilization (Mob site) and can be mobilized with high frequency from the donor strains. The mobilizable vectors are derived from the commonly used E. coli vectors pACYC184, pACYC177, and pBR325, and are unable to replicate in strains outside the enteric bacterial group. Therefore, they are widely applicable as transposon carrier replicons for random transposon insertion mutagenesis in any strain into which they can be mobilized but not stably maintained. The vectors are especially useful for site–directed transposon mutagenesis and for site–specific gene transfer in a wide variety of gram negative organisms.

We have shown by physical and genetic means that there are two megaplasmids in all strains of Rhizobium meliloti that we have studied. Megaplasmids from several strains of R. meliloti were mobilized to Agrobacterium tumefaciens and to other Rhizobium strains using the Tn5-Mob system. We were also able to resolve these two megaplasmids in agarose gels for most strains, and to show that only one of them hybridized to nif and nod genes. Transfer of this plasmid, the pSym, to Agrobacterium, R. leguminosarum, and R. trifolii strains conferred on these recipients the ability to nodulate alfalfa ineffectively. The second megaplasmid did not appear to have a direct role in nodule initiation. However, we were able to complement extracellular polysaccharide (EPS-) mutants of R. meliloti by transferring this second megaplasmid into them. Furthermore, Tn5-induced EPS- mutants of R. meliloti 2011, which produced ineffective (Fix-) nodules of abnormal morphology, were shown by hybridization and complementation to carry mutations in this second megaplasmid. This demonstrates that both megaplasmids of R. meliloti are necessary for the effective nodulation of alfalfa.

High-spin states in 224,226,228Th were investigated using conversion-electron and γ-ray spectroscopic methods following 226 Ra (α, x n ) reactions. The Kπ = 0+ and 0− rotational bands are observed, which in 224,226Th merge into a single band characteristic for reflection asymmetric shape. The systematics of the level energies and El transition rates of 220–230Th are discussed in terms of a stable octupole deformation.

We report the first identification of novel viruses, and sequence of an entire viral genome, by a single step of high-throughput parallel sequencing of small RNAs from diseased, as well as symptomless plants. Contigs were assembled from sequenced total siRNA from plants using small sequence assembly software and could positively identify RNA, ssDNA and dsDNA reverse transcribing viruses and in one case spanned the entire genome. The results present a novel approach which cannot only identify known viral pathogens, occurring at extremely low titers, but also novel viruses, without the necessity of any prior knowledge.

The broad-host-range vectors pSUP104, pSUP106, pSUP204, pSUP304, and pSUP404 are based on conventional Escherichia coli vectors (such as pBR325 and pACYC184) which have been modified to include the mobilization and broad-host-range replication functions of the IncQ plasmid RSF1010. These vector plasmids now can be maintained in a wide range of bacterial genera including Rhizobium, Agrobacterium, and Pseudomonas. They are efficiently mobilized by RP4 and thus are of particular interest for bacteria refractory to transformation. They offer the selection markers and cloning sites characteristic of the basic E. coli vectors. Therefore, they can be applied and adapted to a variety of cloning strategies. However, the cloning of very large fragments (e.g., in cosmid hybrids of pSUP106) was found to affect the stability of the recombinant molecules in a Rec+ background. This instability was not observed with smaller inserts of about 5 kilobases.

Publisher Summary This chapter describes the experimental application of a vector system for Rhizobium that enables efficient transposon mutagenesis, site-specific insertion of selectable markers, creation of deletions in predetermined DNA regions, complementation studies, and high-frequency transfer of any DNA molecule between different strains. The essential ingredients of the system are the broad host range transfer and mobilization functions of plasmid RP4, transposable elements, mainly Tn5, and usual Escherichia coli specific vector plasmids. The major area of research in Rhizobium genetics is the identification, characterization, and manipulation of symbiotic genes. Modern recombinant DNA techniques have been applied to the analysis of cloned rhizobial genes in their parental environment by the construction of broad host range vector plasmids. The antibiotic resistance plasmid RP4 is known to be self-transmissible to any Gram-negative bacterium. The advantages of the use of antibiotic resistance transposons as mutagenic agents are insertion of a transposon into a gene leads to a complete loss of its function, transposon insertion mutation can be directly selected so that the yield of a transposon mutagenesis experiment is 100%, and the antibiotic resistance marker carried by the transposon acts as a probe for the genetic and physical location of the affected gene.

It is evident that the knowledge about biological nitrogen fixation was enormously increased when genetic systems in free-living and symbiotic nitrogen fixing bacteria were developed and used for the analysis of their nif (nitrogen fixation) genes. Due to its close relationship to Eschericia coli, Klebsiella pneumoniae, a facultatively anaerobic procaryotic microorganism, was initially selected to analyze nif genes (Streicher et al., 1971; Dixon, Postgate, 1971). For other Gram-negative nitrogen fixing microorganisms, genetic systems were recently developed and are now used to compile information in the field of nif genetics. To review all the information available would certainly exceed the scope of this paper. Therefore, we concentrate on three different nitrogen fixing species which are currently being analyzed in our laboratory: the facultative anaerobe Klebsiella pneumoniae, the phototrophic Rhodopseudomonas capsulata and the symbiotic nitrogen fixing Rhizobium meliloti. For these three species, the genetic techniques developed will be outlined. It is our special intention to show that break-throughs are often achieved following the development of new methodology.

Mutants of Rhizobium meliloti strain 2011 were induced by general Tn5 transposition using pBR325-Mob-Tn5 as a mobilising vector and inoculated on sterilized seedlings of Medicago sativa (alfalfa). After 3 or 4 weeks plants were examined for nodulation and also tested by the acetylene reduction assay. Of about 2400 mutants screened by this procedure, 23 showed defects or significant morphological and physiological changes in their symbiotic properties. In total, nine mutants failed to induce nodule formation. Among these there were five auxotrophic mutants (two for leucine, two for valine/isoleucine and one mutant with two Tn_5 insertions but unknown auxotrophic markers). Further auxotrophic mutants (arginine, adenine and cysteine) induced small nodules which sometimes were tumorlike in their appearance. These mutants did not fix nitrogen. Among the ineffective mutants, there were three prototrophic ones with an altered colony type.

Slow (m.soleus) and fast (m.tibialis anterior) muscles of the rabbit were subjected to indirect long-term intermittent stimulation (3 weeks, 8 hrs daily) with a frequency pattern of 10 imp/sec. Whereas no changes were observed in case of the slow muscle, stimulation induced profound changes in the fast tibialis anterior muscle. These consisted in a rearrangement of the enzyme activity pattern of energy-supplying metabolism, e.g. decrease in glycogenolytic and glycolytic enzyme activities and severalfold increase in key enzymes of aerobic endoxidation of substrates in beta-oxidation and the citric acid cycle. Concomitant with the increase in aerobic oxidative capacity, there was an increased resistance to fatigue. Histochemical studies revealed a strong increase in mitochondria of all fibres. The bimodal distribution of fibre cross-sectional area in the normal tibialis anterior muscle was changed by stimulation into a more homogeneous population of fibres with a smaller cross-sectional area. Despite a 50% increase in time to peak of isometric twitch contraction no changes were observed in the fibre population with regard to myofibrillar ATPase reaction in quantitative evaluation of whole cross-sections of the muscles. The percentage of fibres histochemically classified as slow amounted to 2.8% and 3.1% in control and stimulated tibialis anterior muscle. Nevertheless the data suggest a transformation of the fibre population under the influence of long-term intermittent stimulation.

Agrobacterium tumefaciens strains LBA275 and LBA290 were cured of their cryptic plasmid pAtC58 by the introduction of the Rhizobium meliloti plasmid pRme41a, which is incompatible with pAtC58. pRme41a and pTiC58, the resident Ti plasmid of LBA275, were subsequently eliminated by growth at supraoptimal temperature (40 degrees C). The resulting plasmid-free Agrobacterium strains, UBAPF1 and UBAPF2, have proved extremely useful for the study of Rhizobium plasmids. The loss of the cryptic plasmid pAtC58 has no effect on the tumor-forming ability of the Agrobacterium strains; when the Ti plasmid is present, normal tumors are formed on Kalanchoe daigremontiana.

A DNA fragment of the broad host range plasmid RP4 carrying the cis-acting DNA recognition site for conjugative DNA transfer between bacterial cells (Mobsite) was cloned into the kanamycin-neomycin resistance transposon Tn5. Using conventrional transposon mutagenesis techniques the new transposon, called Tn5-Mob, can easily be inserted into the host DNA of gram-negative bacteria. A host replicon carrying Tn5-Mob is then mobilizable into any other gram-negative species if the transfer functions of plasmid RP4 are provided in trans. The potential of Tn5-Mob was demonstrated by mobilizing Rhizobium meliloti plasmids as well as the E. coli chromosome at high frequencies.

Positive selection procedures for the isolation of transposable DNA elements from Gram-negative bacteria are described. The use of endogenous insertion sequences for the identification and characterization of wild-type bacterial populations is discussed.