The Grambacterium Sinor hizobium melilotiis common to many soils worldwide. It is of high agricultural importance due to its ability to fix atmospheric nitrogen in symbiosis with the pasture crop alfalfa (Medieago sativa). In a joint research project about biosafety we focused on the analysis of persistence, spread, and environmental effects of genetically engineered soil bacteria. Therefore, two isogenic S. meliloti strains (GMOs) were marked by chromosomal insertion of a constitutively expressed Jue gene of the firefly Photinus pyralis (Selbitschka et al., 1992, 1995). One strain, regarded as wild type, has the Jue gene inserted downstream the recA gene and is therefore RecA+. The other strain is a RecA· derivative with the Jue gene integrated into the reeA gene. The RecA· strain is tested for its use as a safety strain in the environment since the recA gene is essential for recombination and repair of DNA. Under laboratory conditions using liquid culture and micocosms an increased sensitivity to DNA-damaging agents, a reduced survival, and a reduced nodulation competitivity of the RecA· strain compared to the wild type strain were detected (Selbitschka et al., 1992, 1995; Dammann-Kalinowski et al., 1996). In 1994 and 1995, field release of the two GMOs was carried out at a research site near Braunschweig (Germany). The GMOs were released with a titer of approx. lQG CFU/g soil for the top 25 cm of two model ecosystems, soil columns filled with luvisol (release: September 1994) and small scale field plots (release: April 1995). The luminescence of the GMOs allows to follow their persistence and spread with a detection limit of less than 100 CFU/g soil. For both systems, a decline of the GMO titer for at least one order of magnitude to approx. 10' CFU/g soil was observed within the first four weeks after release. In the soil columns, in the following five months during winter time the titer of both strains dropped to approx. 5 x 104 CFU/g soil. During the next six months, the titer of the RecAstrain remained nearly stable whereas the titer of the RecA + strain showed a

G protein-coupled receptors (GPCRs) are important therapeutic targets for a broad spectrum of diseases and disorders. Obtaining milligram quantities of functional receptors through the development of robust production methods are highly demanded to probe GPCR structure and functions. In this study, we analyzed synergies of the bacterial chaperonin GroEL-GroES and cell-free expression for the production of functionally folded C-X-C chemokine GPCR type 4 (CXCR4). The yield of soluble CXCR4 in the presence of detergent Brij-35 reached ∼1.1 mg/ml. The chaperonin complex added was found to significantly enhance the productive folding of newly synthesized CXCR4, by increasing both the rate (∼30-fold) and the yield (∼1.3-fold) of folding over its spontaneous behavior. Meanwhile, the structural stability of CXCR4 was also improved with supplied GroEL-GroES, as was the soluble expression of biologically active CXCR4 with a ∼1.4-fold increase. The improved stability together with the higher ligand binding affinity suggests more efficient folding. The essential chaperonin GroEL was shown to be partially effective on its own, but for maximum efficiency both GroEL and its co-chaperonin GroES were necessary. The method reported here should prove generally useful for cell-free production of large amounts of natively folded GPCRs, and even other classes of membrane proteins.

Nothing is sterile. Insects, plants, and fungi, highly speciose groups of organisms, conceal a vast fungal biodiversity. An approximation of the total number of fungal species on Earth remains an elusive goal, but estimates should include fungal species hidden in associations with other organisms. Some specific roles have been discovered for the fungi hidden within other life forms, including contributions to nutrition, detoxification of foodstuffs, and production of volatile organic compounds. Fungi rely on associates for dispersal to fresh habitats and, under some conditions, provide them with competitive advantages. New methods are available to discover microscopic fungi that previously have been overlooked. In fungal conservation efforts, it is essential not only to discover hidden fungi but also to determine if they are rare or actually endangered.

Genomic variation between the Sinorhizobium meliloti model strain Rm1021 and the field isolate SM11 was assessed by using the genome-wide S. meliloti Rm1021 Sm6k-oligonucleotide microarray in a comparative genomic hybridisation experiment. Several gene clusters present in the Rm1021 genome are missing in the SM11 genome. In detail, three missing gene clusters were identified for the chromosome, five for megaplasmid pSymA and two for megaplasmid pSymB. To confirm these hybridisation results, the draft genome sequence of the S. meliloti field isolate SM11 was established by 454-pyrosequencing. Three sequencing runs on the ultrafast Genome Sequencer 20 System yielded 112.5 million bases. These could be assembled into 905 larger contigs resulting in a nearly 15-fold coverage of the 7.1 Mb SM11 genome. The missing gene regions identified by comparative genomic hybridisation could be confirmed by the results of the 454-sequencing project. An in-depth analysis of these gene regions resulted in the following findings: (i) a complete type I restriction/modification system encoded by a composite transposon is absent in the chromosome of strain SM11. (ii) Most of the Rm1021 denitrification genes and the complete siderophore biosynthesis operon were found to be missing on SM11 megaplasmid pSymA. (iii) S. meliloti SM11 megaplasmid pSymB lacks a complete cell surface carbohydrate synthesis gene cluster. (iv) Several genes that are absent in the SM11 genome could be assigned to insertion sequences and transposons.

A field study was conducted with genetically modified sinorhizobium meliloti strains L1 (RecA-) and L33 (RecA+), both tagged with the firefly luciferase luc gene as an identification marker. The strains' fate was studied over a time period of five years. Both strains were rapidly outcompeted for alfalfa nodulation by an indigenous population. In summary, this study demonstrates the usefulness of tagging bacteria designed for environmental releases by the firefly luciferase gene and the high resilience of soil bacteria to allow the establishment of foreign bacterial populations. This work was supported by grants of the German Federal Ministry of Education and Research (BMBF). We thank Penny Hirsch for valuable discussion concerning the preparation of the peat-based inoculants. The contribution of the Bayer-ische Staatsministerium fur Ernahrung, Landwirtschaft und Forsten for making available the field release site in Stras-smoos is gratefully acknowledged. We thank Rudolf Beck and the team of the Strassmoos experimental plant station for their excellent support during the field study.

A field release experiment was carried out to study the fate of the isogenic, firefly luciferase (luc) gene-tagged Sinorhizobium meliloti strains L1 (RecA-) and L33 (RecA+) in the environment. Both strains were released at concentrations of approximately 10(6) cfu g(-1) soil in replicate and randomized field plots, which had been sown with alfalfa (Medicago sativa). The survival of both strains during the following 7 years could be subdivided into three phases: a sharp decline for more than two orders of magnitude within the first 4 months (phase I), followed by fluctuations around an average number of 10(4) cfu g(-1) soil for nearly 4 years (phase II), and a further decline to approximately 60 cfu g(-1) (phase III). At most sampling dates, no significant differences in the survival of both strains were detected, indicating that the recA gene function was dispensable under these environmental conditions. During the field inoculation, both strains were dispersed accidentally by wind in small numbers to noninoculated field plots. Strain L33 established at a concentration of more than 10(3) cfu g(-1) soil with subsequent seasonal fluctuations. Although strain L1 must have been disseminated to a similar extent, it could never be recovered from noninoculated field plots, indicating that the recA mutation interfered with the strain's capability to establish there. At the beginning of the field experiment, an indigenous alfalfa-nodulating population was below the limit of detection. In the following years, however, an indigenous population arose, which finally outcompeted both strains for saprophytic growth and alfalfa nodulation. RecA- strain L1 was outcompeted for alfalfa nodulation slightly faster than its RecA+ counterpart L33. The diversity of the indigenous population was characterized by employing the Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction fingerprint method. Typing of 2731 root nodule isolates revealed a total of 38 fingerprint groups. More than 80% of the isolates could be grouped into six dominant fingerprint groups, indicating that a few dominant bacterial strain types had outcompeted the released strains.

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation–related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.

Im Rahmen des bislang einzigen Freilandexperiments mit gentechnisch veränderten Bakterien in Deutschland wurde die Entwicklung leuchtmarkierter Rhizobienstämme an zwei Freisetzungsstandorten in Niedersachsen und Bayern über einen Zeitraum von fünf beziehungsweise drei Jahren verfolgt. Als Modellorganismen für die gezielte Freisetzung in die Umwelt wurden Knöllchenbakterien eingesetzt, die in Symbiose mit der Nutzpflanze Luzerne zur biologischen Stickstofffixierung befähigt sind. Damit das Verhalten der Stämme in der Umwelt untersucht werden kann, wurden diese mit dem Leuchtgen des Glühwürmchens versehen, so dass die Stämme bei Anwesenheit des Substrats Luziferin Licht aussenden und damit zweifelsfrei identifiziert werden können. Gleichzeitig diente das Experiment zur Prüfung eines biologischen Eingrenzungskonzepts, das auf der Verwendung eines rekombinationsdefekten „Sicherheitsstammes”︁ basiert. Für beide Standorte wurde gezeigt, dass die freigesetzten Rhizobien nach längerer Zeit von der endogenen Population verdrängt werde. Die biologische Eingrenzung freigesetzter Bakterien durch Verwendung eines rekombinationsdefekten Stammes konnte unter Freilandbedingungen nicht demonstriert werden.

Plasmid pIPO2 is a cryptic, conjugative, broad-host-range plasmid isolated from the wheat rhizosphere. It efficiently self-transfers between alpha, beta and gamma Proteobacteria and has a mobilizing/retromobilizing capacity for IncQ plasmids. The complete nucleotide sequence of pIPO2 is presented on the basis of its mini-Tn5::luxABtet-tagged derivative, pIPO2T. The pIPO2 sequence is 39815 bp long and contains at least 43 complete ORFs. Apart from a suite of ORFs with unknown function, all of the genes carried on pIPO2 are predicted to be involved in plasmid replication, maintenance and conjugative transfer. The overall organization of these genes is different from previously described plasmids, but is similar to the genetic organization seen in pSB102, a conjugative plasmid recently isolated from the bacterial community of the alfalfa rhizosphere. The putative conjugative transfer region of pIPO2 covers 23 kb and contains the genes required for DNA processing (Dtr) and mating pair formation (Mpf). The organization of these transfer genes in pIPO2 is highly similar to the genetic organization seen in the environmental plasmid pSB102 and in pXF51 from the plant pathogen Xylella fastidiosa. Plasmids pSB102 and pXF51 have recently been proposed to form a new family of environmental broad-host-range plasmids. Here it is suggested that pIPO2 is a new member of this family. The proposed Mpf system of pIPO2 shares high amino acid sequence similarity with equivalent VirB proteins from the type IV secretion system of Brucella spp. Sequence information was used to design primers specific for the detection of pIPO2. Environmental DNA from a range of diverse habitats was screened by PCR with these primers. Consistently positive signals for the presence of pIPO2 were obtained from a range of soil-related habitats, including the rhizospheres of young wheat plants, of field-grown oats and of grass (all gramineous plants), as well as from the rhizosphere of tomato plants. These data add to the growing evidence that plasmids carry advantageous genes with as yet undefined functions in plant-associated communities.

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101-pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1beta) and RK2 (IncP-1alpha), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.

The structure of the microbial rhizoplane community of the important crop plant oilseed rape was studied by using a culture-dependent as well as a culture-independent approach based on 16S rDNA amplification. After isolation of the microbial community from the rhizoplane of oilseed rape (Brassica napus cv. Westar), the collected suspension was divided into two parts. One part was used for cultivation of bacteria onto three different growth media to establish a culture collection. From the other part of the rhizoplane suspension, genomic DNA was isolated and purified. Thereafter, 16S rDNA was amplified by PCR and cloned to obtain a library of 16S rDNA genes representative for the bacterial communities of this habitat. Phylogenetic 16S rDNA sequence analysis of 103 clones of this library revealed considerable differences from the corresponding nucleotide sequences of 111 cultured bacteria. Whereas the 16S rDNA clone library was dominated by alpha -Proteobacteria and bacteria of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum (51% and 30%, respectively), less than 17% of the cultured bacteria belonged to these two groups. More than 64% of the cultivated isolates were allocated to the beta- and gamma -subclasses of the Proteobacteria, which were present in the clone library at about 14%. Most of the clones of the alpha -Proteobacteria of the library showed highest similarity to Bradyrhizobium sp. No such bacteria were found in the culture collection. Similarly, the second dominant group of the clone library comprising members of the CFB phylum was represented in the culture collection by a single isolate. The phylogenetic analysis of isolates of the culture collection clearly emphasized the need to use different growth media for recovery of rhizoplane bacteria. Whereas most of the alpha -Proteobacteria were recovered on complex medium, most of the beta -Proteobacteria were isolated onto minimal media. Our results demonstrate that the combined approach pursued in this paper is necessary to explore the biodiversity of bacterial rhizoplane communities.

In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses. Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline, streptomycin or spectinomycin, and were recovered at frequencies in the range of 10(-5) to 10(-8) per recipient. A total of 12 distinct plasmids, designated pB1-pB12, was identified. Their sizes ranged between 41 to 69 kb and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids, displaying extremely high transfer frequencies in filter crosses, ranging from 10(-1) to 10(-2) per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncPbeta subgroup, whereas two plasmids were identified as IncPalpha plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncPbeta plasmids at the DNA sequence level. In order to characterize the gene "load" of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide dismutase, parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified.

The survival and vertical translocation of two isogenic, luciferase marker gene (luc)-tagged Sinorhizobum meliloti strains, L33 (RecA(+)) and L1 (RecA(-)) was studied under field conditions over a period of 2 years in a soil which was deficient in indigenous S, meliloti. Both strains were inoculated separately at the end of the growing season of 1994 onto replicate field lysimeters (diameter 32 cm) seeded with alfalfa (Medicago sativa). From an initial density of 10(6) cfu g(-1) soil in the A(p)-horizon (0-25 cm depth), populations of both strains declined during winter to 3 x 10(4) cfu g(-1) One year after the field release, a significantly increased titer of the RecA(+) strain was detected (P less than or equal to 0.05). Removal of the green parts of alfalfa from the lysimeters, 79 weeks after inoculation, resulted in a significant decline of the RecA- (2.3 x 10(3) cfu g(-1)) and a slight increase of the RecA(+) strain (9.0 x 10(3) cfu g(-1)). Throughout the whole monitoring period, marker gene-tagged cells were exclusively located in the A(p)-horizon and not below. No inoculated cells were detected in flow-through rain water (threshold of detection 10 cfu ml(-1)) even though each lysimeter was percolated with an average of 42.51 during this study. Single luciferase positive cells could be detected in the A(p)-horizons of non-inoculated lysimeters, which were located between the inoculated lysimeters using nodulation assays. Cultivation methods failed to detect these cells. The bioluminescent nodules were almost exclusively caused by strain L33 and not by L1, indicating that the RecA(-) strain was less competitive in alfalfa nodulation. Soil chemical properties and quantities of microbial populations, culturable on four different growth media, were not affected by the S. meliloti inoculations. This study demonstrates the usefulness of small scale lysimeter field releases to assess the performance and potential ecological effects of genetically modified bacterial inoculants, (C) 2000 Elsevier Science Ltd. All rights reserved.

ISRm14 is 2695 basepairs (bp) in size and bordered by 22 bp imperfect inverted repeats (IRs). A 9-bp target sequence is duplicated upon ISRm14 transposition. The DNA strand that putatively encodes the transposase enzyme carries three open reading frames (ORFs) designated ORFs 1 to 3, which specify putative proteins of 15.9 kDa, 13.1 kDa, and 61.1 kDa, respectively. According to its structural characteristics, ISRm14 belongs to the recently proposed IS66 family of IS elements. The ORFs1 to 3 encoded putative proteins displayed significant similarities to ORFs of the previously unrecognized IS element ISEc8, which is inserted adjacent to the locus of enterocyte effacement (LEE) pathogenicity island of Escherichia coli EDL933. Analyses of the distribution of ISRm14 in a natural S. meliloti population showed its widespread occurrence in 66% of the strains tested with a copy number ranging from 1 to 6.

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1 and ISRm220-13-5 are 1481 and 1550 base pairs (bp) in size, respectively. ISRm102F34-1 is bordered by 15 bp imperfect terminal inverted repeat sequences (two mismatches), whereas the terminal inverted repeat of ISRm220-13-5 has a length of 16 bp (two mismatches). Both insertion sequence elements generate a 6-bp target duplication upon transposition. The putative transposase enzymes of ISRm102F34-1 and ISRm220-13-5 consist of 449 or 448 amino acid residues with predicted molecular weights of 50.7 or 51.3 kDa and theoretical isoelectric points of 10.8 or 11.1, respectively. ISRm102F34-1 is identical in 98.9% of its nucleotide sequence to an apparently inactive copy of an insertion sequence element, designated ISRm7, which flanks the left-end of the nodule formation efficiency (nfe) region of plasmid pRmeGR4b of S. meliloti strain GR4. ISRm102F34-1 and ISRm220-13-5 are closely related since they show an overall identity of 57.0% at the nucleotide sequence level and of 47.3% at the deduced amino acid level of their putative transposases. Both insertion sequence elements displayed significant similarity to the Xanthomonas campestris ISXc6 and its homolog IS1478a. Since none of these insertion sequence elements could be allocated to existing families of insertion sequence elements, a new family is proposed. Analysis of the distribution of ISRm102F34-1/ISRm7 in various local S. meliloti populations sampled from Medicago sativa, Medicago sphaerocarpa and Melilotus alba host plants at different locations in Spain revealed its presence in 35% of the isolates with a copy number ranging from 1 to 5. Furthermore, ISRm102F34-1/ISRm7 homologs were identified in other rhizobial species.

The enterobacterial repetitive intergenic consensus (ERIC)-PCR method was employed to generate genomic amplification products of Sinorhizobium meliloti strain 2011. Eleven distinctive PCR fragments obtained in PCR reactions by using the ERIC2 primer were cloned and their partial or complete nucleotide sequences established. DNA sequences that extended past the ERIC2 primer region were not conserved among the 11 PCR fragments and showed no sequence similarity to the enterobacterial ERIC consensus sequence. Thus, repetitive ERIC or ERIC-like sequences seem not to be an integral part of the S. meliloti genome. An amplification product of S. meliloti 2011 was identified which was present in S. meliloti strains but absent in other rhizobial species. Based on the nucleotide sequence information, a pair of PCR primers was designed and used for PCR amplification of sequences of S. meliloti laboratory strains 2011, L5–30, AK631 and 102F34. Nucleotide sequence analysis of the amplification products revealed a 100% DNA sequence conservation. Database searches showed that the DNA fragment putatively encodes the C-terminal part of a protein displaying similarity to 2-hydroxyacid dehydrogenases of various organisms. The newly designed PCR primers should be useful for the rapid identification of S. meliloti isolates.