A promoter-probe vector (pHX200) was constructed using the broad-host-range cosmid pLA2917 and a promoterless xylE gene of Pseudomonas as the reporter gene. Insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene moxF (methanol oxidation) in front of the xylE gene in pHX200V-47 resulted in high-level expression of the xylE gene product -catechol 2,3-dioxygenase -in Methylobacterium organophilum XX. The specific activity of the enzyme was four times higher in methanol-grown M. organophilum XX culture than in succinate-grown culture. Interestingly, the insertion of the same fragment in the opposite orientation in front of the xylE gene (pHX200V-74) also led to elevated catechol2,3-dioxygenase activity. This promoter activity was also methanol regulated. A total of 21 methanol-regulated promoter clones were identified that originate from three gene clusters (groups V, VI and VII) on the M. organophilum XX chromosome involved in methanol oxidation. Vector pHX200 and its derivatives were successfully mobilized into cells of three phylogenetically diverse methylotrophic bacteria : Methylophilus methylotrophus AS1, Methylobacterium extorquens AM1 and Methylobacterium sp. DM4. The reporter gene (xylE) was functionally expressed in all three bacteria with the aid of a proper promoter. Transcriptional fusions of methanol-regulated promoters with the xyZE gene were mobilized into Mox-mutants of M. organophilum XX and M. extorquens AM1 to study the roles of methanol oxidation genes, especially regulatory genes. It appeared that vector pHX200 is an efficient promoter probe with wide host-range and an excellent tool for studies of structure and function of promoters/regulators.

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 10 7 CFU per g of roots soon after sowing to less than 10 2 CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.

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To assess effects of plant crop species on rhizosphere ascomycete communities in the field, we compared a wheat monoculture and an alternating crop rotation of wheat and potato. Rhizosphere soil samples were taken at different time points during the growing season in four consecutive years (1999-2002). An ascomycete-specific primer pair (ITS5-ITS4A) was used to amplify internal transcribed spacer (ITS) sequences from total DNA extracts from rhizosphere soil. Amplified DNA was analyzed by denaturing gradient gel electrophoresis (DGGE). Individual bands from DGGE gels were sequenced and compared with known sequences from public databases. DGGE gels representing the ascomycete communities of the continuous wheat and the rotation site were compared and related to ascomycetes identified from the field. The effect of crop rotation exceeded that of the spatial heterogeneity in the field, which was evident after the first year. Significant differences between the ascomycete communities from the rhizospheres of wheat in monoculture and one year after a potato crop were found, indicating a long-term effect of potato. Sequencing of bands excised from the DGGE gels revealed the presence of ascomycetes that are common in agricultural soils.

Nucleotide sequence analysis of the mxcQ and mxcE loci, required for the synthesis of methanol dehydrogenase in Methylobacterium organophilum XX, has revealed two open reading frames that show significant similarity to sequences of prokaryotic two-component systems, especially MxaY and MxaX proteins of another met h y lotro phic bacterium, Paracoccus denitrificans. Cel Ifree extracts and DNA-column-fractionated proteins from wild-type M. organophilum XX cells grown on methanol or succinate contained protein(s) that were able to bind specifically to the upstream region of methanol dehydrogenase large subunit gene (mxaF). In contrast, cell-free extracts from mxcQ and mxcE mutant strains of M. organophilum XX had zero or reduced binding activity towards the promoter fragments of the mxaF gene. This is consistent with the involvement of the mxcQ and mxcE genes in transcriptional regulation of methanol dehydrogenase synthesis. Analyses of sequential deletions of the mxaf upstream region have defined the functional boundary of the promoter/operator region of this gene and identified one nucleotide segment as essential to the activation of mxaf.