Sonja Selenska-Pobell

Bacterial interactions with uranium: An environmental perspective

The presence of actinides in radioactive wastes is of major concern because of their potential for migration from the waste repositories and long-term contamination of the environment. Studies have been and are being made on inorganic processes affecting the migration of radionuclides from these repositories to the environment but it is becoming increasingly evident that microbial processes are of importance as well. Bacteria interact with uranium through different mechanisms including, biosorption at the cell surface, intracellular accumulation, precipitation, and redox transformations (oxidation/reduction). The present study is intended to give a brief overview of the key processes responsible for the interaction of actinides e.g. uranium with bacterial strains isolated from different extreme environments relevant to radioactive repositories. Fundamental understanding of the interaction of these bacteria with U will be useful for developing appropriate radioactive waste treatments, remediation and long-term management strategies as well as for predicting the microbial impacts on the performance of the radioactive waste repositories.

Bacterial diversity in soil samples from two uranium waste piles as determined by rep-APD, RISA and 16S rDNA retrieval

The bacterial diversity in two uranium waste piles was studied. Total DNA was recovered from a large number of soil samples collected from different sites and depths in the piles using two procedures for direct lysis. Significant differences in the bacterial composition of the samples were revealed by the use of rep-APD, RISA and 16S ARDREA. The 16S rDNA analyses showed that the uranium wastes were dominated by Acidithiobacillus ferrooxidans and by several Pseudomonas species classified in the γ-subdivision of the Proteobacteria. The three kinds of A. ferrooxidans 16S and IGS rDNA specific fragments that were found corresponded to the three phylogenetic groups recognised in this species. This microdiversity probably reflects the genetic adaptation of the uranium waste strains to different concentrations of heavy metals.

Spectroscopic and Microscopic Characterization of Uranium Biomineralization in Myxococcus xanthus

In this work, synchrotron-based X-ray absorption spectroscopy (XAS) and transmission electron microscopy (TEM) studies were carried out to elucidate at molecular scale the interaction mechanisms of Myxococcus xanthus with uranium at different pH values. Extended X-ray absorption fine structure (EXAFS) spectroscopic measurements showed that there are significant differences in the structural parameters of the U complexes formed by this bacterium at pH 2 and 4.5. At very low acidic pH of 2, the cells accumulated U(VI) as organic phosphate-metal complexes. At pH 4.5, however, the cells of this bacterium precipitated U(VI) as meta-autunite-like phase. TEM analyses demonstrated that at pH 2 the uranium accumulates were located mainly at the cell surface, whereas at pH 4.5 a uranium precipitation occurred on the cell wall and within the extracellular polysaccharides (EPS) characteristic of this bacterium. Dead/live staining studies showed that 30% and 50% of the uranium treated cell populations were alive at pH 2 and 4.5, respectively. The precipitation of U(VI) as mineral meta-autunite-like phase is possibly due to the bacterial acidic phosphatase activity. The precipitation of uranium as mineral phases may lead to more stable U(VI) sequestration that may be suitable for remediation purposes. These observations, combined with the very high uranium accumulation capability of the studied bacterial cells indicate that M. xanthus may significantly influence the fate of uranium in soil environments where these bacterial species are mainly found.

Sequences around the fragmentation sites of the large subunit ribosomal RNA in the family Rhizobiaceae

We demonstrated that the representatives of the family Rhizobiaceae possess, instead of one single 23S rRNA molecule, three different sets of 23S-like rRNA fragments with sizes of about: 135 b and 2.6 kb (set 1); 135 b, 400 b, and 2.2 kb (set 2); 135 b and two molecules of about 1.3 kb (set 3). In two of the fragmentations, intervening sequences – IVS I and IVS II – are involved. The IVS I is connected to a cleavage of the 23S rRNA primary transcript into two molecules (135 b and 2.6 kb large). The IVS II is located at position 543 of the gene, and it leads to an additional processing of the 2.6 kb rRNA species into two molecules with sizes of about 400 b and 2.2 kb. In contrast to the IVS I, which is a common feature of all rhizobia, the IVS II is present in a limited number of strains only. The primary and secondary structures of the regions of the unmatured 23S rRNA transcript possessing IVS I (helix 9) and IVS II (helix 25) were analysed. On the basis of our analyses we propose secondary structure models of the parts of the matured 23S rRNA-like molecules of rhizobia corresponding to the helices 9 and 25. The third fragmentation of the rhizobial 23S rRNA represents a break in the central part of the 2.6 kb-large rRNA and it leads to an occurence of two fragments with approximately equal size of about 1.3 kb. We have demonstrated that the central fragmentation is not connected to the presence of IVSs but probably to a minor change in the nucleotide sequence in the central part of the 2.6 rRNA.

Random and Repetitive Primer Amplified Polymorphic DNA Analysis of Five Soil and Two Clinical Isolates of Rahnella aquatilis

Summary PCR patterns of five nitrogen-fixing soil bacterial strains isolated from the rhizosphere of wheat in the vicinity of Bayreuth were generated by PCR using random and repetitive (BOX, ERIC and REP) primers. They have been compared to the patterns (obtained with the same primers) of several Rahnella aquatilis strains (including the type strain of this species - R. aquatilis ATCC 33071), the strain R. aquatilis ATCC 33989 (to whom one of our isolates has been formerly assumed to be related), and two clinical isolates of the same species. As outgroup strains Enterobacter agglomerans 19-78, Escherichia coli W2438 and Pantoea agglomerans 5D representing different species of the family Enterobacteriaceae were used. By all primers used it was clearly shown that the natural isolates belong to the species R. aquatilis . Even more, they have been clustered into two groups around the two ATCC strains 33071 and 33989, respectively. By these analyses, it was possible also to show that the clinical R. aquatilis strains form another, third group. In addition, both kinds of PCR fingerprinting (using arbitrary or repetitive primers) generated highly reproducible patterns when parallel reactions with total DNA extracted by different methods from independent liquid cultures of one and the same strain were performed. The patterns obtained by PCR fingerprinting of total DNA or of cells from fresh colonies or liquid cultures added to the PCR mixture did not differ significantly. The RAPD and rep-APD characterization of the strains studied here is in full agreement with their taxonomical analysis performed by other molecular methods such as micro- and macro-RFLP fingerprinting, ribotyping and 16S rDNA sequencing. On the basis of these results we recommend to apply these simple, fast and cheap methods for identification and discrimination of new environmental and clinical isolates of the species R. aquatilis.

COMPLEXATION OF U(VI) WITH CELLS OF THIOBACILLUS FERROOXIDANS AND THIOMONAS CUPRINA OF DIFFERENT GEOLOGICAL ORIGIN

Recently, we demonstrated that the strain Thiobacillus ferrooxidans ATCC 33020 which is recovered from a uranium mine is taxonomically neither closely related to the type strain of the species Thiobacillus ferrooxidans nor to several other strains of this species stemming from copper and coal mines. In the present work, the interaction was studied of the above-mentioned strains with U(VI). We found that the uranium mine isolate 33020 accumulates significantly higher amounts of U(VI) in comparison to the other T. ferrooxidans strains studied. Extraction studies with sulfuric acid released none, and with EDTA only a small fraction (10—42%) of the U(VI) that was accumulated by the bacteria. The main part of the uranium was irreversibly bound to the biomass. Time-resolved laser fluorescence spectroscopy demonstrated the formation of strong inner-sphere complexes of U(VI) with the bacteria. Moreover, the binding strength of the Thiobacillus ferrooxidans complexes corresponds to the accumulation capability of the strains, i.e., the strongest was the complex with the uranium mine isolate 33020.

How to monitor released rhizobia

Existing methods for detection and identification of rhizobia are reviewed. Some perspectives for development of new and more effective techniques for monitoring of rhizobia in soil and in inoculants are presented. The advantages of the recently developed approach — PCR-genome fingerprinting, by use of arbitrary and repetitive primers, for precise bacterial identification are described. The possible application of this technique for developing taxon-specific rhizobial probes for direct detection of these bacteria in environmental samples is discussed.

Bacterial communities in uranium mining waste piles and their interaction with heavy metals

High diversity and significant differences were found in the structures of bacterial communities present in several U mill tailings and U mining waste piles. Many bacterial strains were successfully cultured from those uranium wastes, most of which are unusually effective in different biotransformations of U. The molecular basis for the selective and reversible binding of U and some other toxic metals by one of the natural bacterial isolates was found to be a novel kind of S-layer protein. Our analysis indicates that uranium wastes are a valuable reservoir for unusual microorganisms prospective for bacteria-based bioremediation.

Bioaccumulation of U(VI) by Sulfolobus acidocaldarius under moderate acidic conditions

Abstract U(VI) accumulation by the acidothermophilic archaeon Sulfolobus acidocaldarius at a moderate acidic pH of 4.5 was investigated. This pH value is relevant for some heavy metal and uranium polluted environments where populations of S. acidocaldarius were found to persist. We demonstrate that U(VI) is rapidly complexed by the archaeal cells. A combination of X-ray absorption spectroscopy and time-resolved laser-induced fluorescence spectroscopy revealed that at pH 4.5 organic phosphate and carboxylic groups are involved in the U(VI) complexation. These results are in contrast to those published for most bacteria which at this pH precipitate U(VI) mainly in inorganic uranyl phosphate phases. As demonstrated by TEM only a limited part of the added U(VI) was biomineralized extracellularly in the case of the studied archaeon. Most of the U(VI) accumulates were localized in a form of intracellular deposits which were associated with the inner side of the cytoplasma membrane. Observed differences in U(VI) bioaccumulation between the studied archaeon and bacteria can be explained by the significant differences in their cell wall structures as well as by their different physiological characteristics.

Random and repetitive primer amplified polymorphic DNA analysis of Bacillus sphaericus

A comparative study of 15 strains representing the five homology groups of Bacillus sphaericus was performed by two PCR methods: RAPD and rep‐PCR fingerprinting. The PCR analysis performed with primers corresponding to the naturally occurring repetitive sequences REP, ERIC and BOX, as well as with three random primers, showed highly variable patterns and allowed differentiation of the strains studied. This demonstrated the high discriminative power of the methods. The cluster analysis revealed a low level of similarity between the different homology groups, and within groups I and III, which is evidence of the high genetic heterogeneity of the species B. sphaericus. Close genetic relatedness was observed for the representatives of group IIA, pathogenic to mosquitoes, which supports the idea for differentiation of this group as a separate species.