Werner Selbitschka

Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis

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.

Horizontal gene transfer as a biosafety issue: A natural phenomenon of public concern

The transfer of genetic information between distantly or even unrelated organisms during evolution had been inferred from nucleotide sequence comparisons. These studies provided circumstantial evidence that in rare cases genes had been laterally transmitted amongst organisms of the domains bacteria, archaea and eukarya. Laboratory-based studies confirmed that the gene pools of the various domains of organisms are linked. Amongst the bacterial gene exchange mechanisms transduction, transformation and conjugation, the latter was identified as the mechanism with potentially the broadest host range of transfer. Previously, the issue of horizontal gene transfer has become important in the context of biosafety. Gene transfer studies carried out under more natural conditions such as in model ecosystems or in the environment established that all gene transfer mechanisms worked under these conditions. Moreover, environmental hot-spots were identified where favourable conditions such as nutrient enrichment increased the probability of genetic exchange among bacteria. In particular, the phytosphere was shown to provide conducive conditions for conjugative gene exchange. Concern has been expressed that transfer of recombinant DNA (e.g. antibiotic resistance genes) from genetically modified organisms (GMOs) such as transgenic plants to phytosphere bacteria may occur and thus contribute to the undesirable spread of antibiotic resistance determinants. Studies which were performed to address this issue clearly showed that such a transfer occurs, if at all, at extremely low frequency.

Evaluation of the resolving power of three different DNA fingerprinting methods to discriminate among isolates of a natural Rhizobium meliloti population

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.

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

Abstract 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 IncPβ subgroup, whereas two plasmids were identified as IncPα plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncPβ 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.

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

Abstract 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

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 a-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 b- and g-subclasses of the Proteobacteria, which were present in the clone library at about 14%. Most of the clones of the a-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 a-Proteobacteria were recovered on complex medium, most of the b-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.

The complete nucleotide sequence and environmental distribution of the cryptic, conjugative, broad-host-range plasmid pIPO2 isolated from bacteria of the wheat rhizosphere

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.

Construction of gene replacement vectors for Gram− bacteria using a genetically modified sacRB gene as a positve selection marker

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.

Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae

SummaryDNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA−Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 by coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA− mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.

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

A vector system was developed employing the recA genes of Rhizobium meliloti and Rhizobium 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 (nptll) promoter of transposon Tn5. The system was used to construct recA mutant strains of R. meliloti and R. leguminosarum by. viciae, carrying the Escherichia coli gusA gene encoding β–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.