New Frontiers in Microbial Genome Research

Pühler A, Selbitschka W. New Frontiers in Microbial Genome Research. Journal of Biotechnology. 2011;155(1):1-2

Genome research in the light of ultrafast sequencing technologies

Pühler A, Selbitschka W. Genome research in the light of ultrafast sequencing technologies. JOURNAL OF BIOTECHNOLOGY. 2008;136(1-2):1-2

Genome research on bacteria relevant for agriculture, environment and biotechnology

Pühler A, Selbitschka W. Genome research on bacteria relevant for agriculture, environment and biotechnology. JOURNAL OF BIOTECHNOLOGY. 2003;106(2-3):119-120

Functional genome research on bacteria relevant for agriculture, environment and biotechnology

Pühler A, Selbitschka W. Functional genome research on bacteria relevant for agriculture, environment and biotechnology. JOURNAL OF BIOTECHNOLOGY. 2009;140(1-2):1-2

Horizontal gene transfer among bacteria in terrestrial and aquatic habitats as assessed by microcosm and field studies

Dröge M, Pühler A, Selbitschka W. Horizontal gene transfer among bacteria in terrestrial and aquatic habitats as assessed by microcosm and field studies. BIOLOGY AND FERTILITY OF SOILS. 1999;29(3):221-245.Genetic interactions among bacteria are mediated by one of the three distinct gene-exchange mechanisms: conjugation, transformation or transduction. Conjugative gene exchange relies on mobile elements, such as plasmids, which transfer between donor and recipient cells. In natural transformation, competent cells take up DNA and incorporate it into their genome. Gene transfer via transduction is mediated by bacteriophages which accidentally package donor DNA in their phage head and transfer it to recipient cells. Driven mainly by biosafety research and research into the rapid dissemination of antibiotic resistance, the evaluation of gene flux among bacteria in their natural habitats has become a focus of scientific interest in recent years. Accordingly, gene transfer has been assessed in laboratory-based studies employing model ecosystems, as well as in field experiments. Conjugative gene exchange has been shown to occur under a wide range of environmental conditions. Factors identified as conducive for conjugation include the presence of nutrients provided by the rhizosphere of plants. Studies addressing gene transfer via transformation have demonstrated that naturally transformable bacteria develop competence and take up DNA under in situ conditions. Moreover, DNA has been shown to persist to some extent in the environment, and thus be available for uptake by naturally competent cells. Gene exchange via transduction has been demonstrated under conditions of nutrient depletion and low densities of host cells. Whereas gene transfer is readily observed in the laboratory, more importantly, field studies have provided direct evidence that all three gene transfer mechanisms also occur in nature. DNA transfer frequencies observed in the environment in some cases differed considerably from those obtained under laboratory conditions. Transfers of low frequency observed in laboratory-based experiments have been readily detected in the environment in the presence of selective forces

Phylogenetic analysis of microbial diversity in the rhizoplane of oilseed rape (Brassica napus cv. Westar) employing cultivation-dependent and cultivation-independent approaches

Kaiser O, Pühler A, Selbitschka W. Phylogenetic analysis of microbial diversity in the rhizoplane of oilseed rape (Brassica napus cv. Westar) employing cultivation-dependent and cultivation-independent approaches. MICROBIAL ECOLOGY. 2001;42(2):136-149.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

CONSTRUCTION OF GENE REPLACEMENT VECTORS FOR GRAM- BACTERIA USING A GENETICALLY MODIFIED SACRB GENE AS A POSITIVE SELECTION MARKER

Selbitschka W, NIEMANN S, Pühler A. CONSTRUCTION OF GENE REPLACEMENT VECTORS FOR GRAM- BACTERIA USING A GENETICALLY MODIFIED SACRB GENE AS A POSITIVE SELECTION MARKER. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 1993;38(5):615-618.Based on the wide-host-range suicide vector pSUP102 (Simon et al. 1983), gene replacement vectors (pWS232/pWS233) have been developed that facilitate the identification by a positive selection procedure of double recombination events in Gram- bacteria. The vectors contain the tetracycline and gentamicin resistance gene as selectable markers, as well as a modified sacRB gene mediating sucrose sensitivity. In order to increase the versatility of the sacRB gene as a positive selection marker and, hence, of vectors that carry this gene, an EcoRI as well as a HindIII site located within the coding region of the sacRB gene were removed by in-vitro mutagenesis. To test the suitability of the vectors, a Tn5-carrying EcoRI fragment of Rhizobium leguminosarum biovar. viciae VF39 was homogenotized into the wild-type strain, resulting in double recombinants with a Lac- phenotype. Although the Tn5 insertion was flanked on one side by only approximately 100 bp of VF39 homologous DNA, this was sufficient for homologous recombination to occur, and double recombinants could readily be isolated

The construction of recA-deficient Rhizobium meliloti and R. leguminosarum strains marked with gusA or luc cassettes for use in risk-assessment studies

Selbitschka W, Pühler A, Simon R. The construction of recA-deficient Rhizobium meliloti and R. leguminosarum strains marked with gusA or luc cassettes for use in risk-assessment studies. MOLECULAR ECOLOGY. 1992;1(1):9-19.A vector system was developed employing the recA genes of Rhizobium meliloti and Rhizobiurn leguminosarum biovar. viciae as target sequences for the stable genomic integration of foreign DNA. The plasmid vectors can be used either as integration vectors (single cross-over), or as gene replacement vectors (double cross-over). Gene replacement results in the antibiotic-marker-free integration of cloned DNA into the recA genes of R. meliloti and R. leguminosarum bv. viciae. Consequently, the recombinant strains become recombination deficient (RecA(-)). The expression of integrated genes is under the control of the neomycin phosphotransferase II (nptII) promoter of transposon Tn5. The system was used to construct recA mutant strains of R. meliloti and R. leguminosarum bv. viciae, carrying the Escherichia coli gusA gene encoding beta-glucuronidase as well as the firefly (Photinus pyralis) luc gene encoding luciferase as marker genes. The GUS activity in the constructed strains was found to be absolutely stable over more than 100 generations of non-selective growth in liquid culture. The stability was also confirmed in root-nodule passages. In addition, the potential use of the luc gene as a stable genetic marker in the unequivocal identification of tagged strains among indigenous microbes in non-sterile soil was demonstrated. It is proposed to use bioluminescent recA mutants as model organisms in risk assessment studies with genetically engineered Rhizobium strains

Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge

Droge M, Pühler A, Selbitschka W. Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge. Mol Gen Genet. 2000;263(3):471-482.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 IncP beta subgroup, whereas two plasmids were identified as IncP alpha plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncP beta 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

Growth and nodulation competitiveness of Sinorhizobium meliloti L1 (RecA(-)) is less than that of its isogenic strain L33 (RecA(+)) but comparable to that of two S-meliloti wild-type isolates

Niemann S, Pühler A, Selbitschka W. Growth and nodulation competitiveness of Sinorhizobium meliloti L1 (RecA(-)) is less than that of its isogenic strain L33 (RecA(+)) but comparable to that of two S-meliloti wild-type isolates. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 1997;47(5):525-529.Gnotobiotic systems were used to assess the competitive abilities of bioluminescent Sinorhizobium meliloti strains L1 (RecA(-)) and L33 (RecA(+)) for growth and host plant nodulation in the presence of a reconstructed S. meliloti population. Three wild-type strains belonging to infective subgroups of a natural S. meliloti population were chosen as competitors in microcosm studies. Whereas the RecA(+) strain L33 dominated the reconstructed population with respect to growth and alfalfa nodulation, the competitiveness of the RecA(-) strain L1 was reduced compared to that of one of the field strains, but comparable to that of the other field isolates. This result indicates that strain L1, despite its recA mutation, has the potential to compete successfully with a resident S. meliloti population after environmental release