Silvia Berkner

Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus–Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the β-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this β-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.

Expanding and understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus

Although Sulfolobus species are among the best studied archaeal micro-organisms, the development and availability of genetic tools has lagged behind. In the present paper, we discuss the latest progress in understanding recombination events of exogenous DNA into the chromosomes of Sulfolobus solfataricus and Sulfolobus acidocaldarius and their application in the construction of targeted-deletion mutant strains.

Development and validation of a method for determination of trace levels of alkylphenols and bisphenol A in atmospheric samples.

A method has been developed and validated in order to assess the occurrence of the alkylphenols tert-octylphenol and the isomers of technical nonylphenol as well as bisphenol A in gasphase and aerosol samples of a remote area. Gasphase samples were adsorbed to XAD2 resin, aerosol samples were taken on glass fiber filters. After ultrasonic extraction, clean-up by column chromatography and silylation of the analytes, ten nonylphenol peaks were quantified separately using a GC-MSD-SIM method. The absolute limits of detection and determination are in the range of a few pg per compound, which is a prerequisite for the quantification of the analytes in relatively unpolluted air. The precision of the whole analytical method is in the range of 1-17% and the recoveries range from 57% to 80%. Problems were encountered during method development due to the tendency of the analytes to sorb to glass surfaces. Silanisation of glassware was crucial to achieve acceptable recoveries. The widespread use of the analytes in plastic resins resulted in sample contamination. For this reason a careful choice of sampling material was necessary. Measured concentrations in gasphase samples (lower nanogram per m3 range) and aerosol samples (upper picogram per m3 range) are one to three orders of magnitude below already published concentrations.

Genetic tools for Sulfolobus spp : vectors and first applications

Sulfolobus species belong to the best-studied archaeal organisms but have lacked powerful genetic methods. Recently, there has been considerable progress in the field of Sulfolobus genetics. Urgently needed basic genetic tools, such as targeted gene knockout techniques and shuttle vectors are being developed at an increasing pace. For S. solfataricus knockout systems as well as different shuttle vectors are available. For the genetically more stable S. acidocaldarius shuttle vectors have been recently developed. In this review we summarize the currently available genetic tools and methods for the genus Sulfolobus. Different transformation protocols are discussed, as well as all so far developed knockout systems and Sulfolobus–Escherichia coli shuttle vectors are summarized. Special emphasis is put on the important vector components, i.e., selectable markers and Sulfolobus replicons. Additionally, the information gathered on different Sulfolobus strains with respect to their use as recipient strains is reviewed. The advantages and disadvantages of the different systems are discussed and aims for further improvement of genetic systems are identified.

Antibiotic resistance and the environment—there and back again

Today, it is difficult to imagine a time when bacterial infections caused a terrible toll of disability and death. The discovery and use of antibiotics in medicine in the 20th century, together with better hygiene and vaccination programs, has drastically increased human life expectancy. In a way, however, these antibiotic ‘silver bullets’ have become victims of their own success, and most people are no longer aware of the deadly threat of bacterial infections. As a result, we have become lax and even irresponsible: We use antibiotics at every opportunity to treat mild or even viral infections, and we have used them in massive amounts in agriculture as a preventive measure against bacterial diseases, and to promote the growth of poultry, beef and pigs in animal farms. > Since the beginning of the antibiotic era in the first half of the 20th century, antibiotics and antibiotic resistance genes have been introduced to or have spread to almost every ecosystem on earth. This complacent attitude is about to change. The past few decades have seen the rise of antibiotic‐resistant bacterial strains that cause increasingly severe, difficult to treat, and sometimes even fatal infections. Multi‐drug‐resistant strains of Mycobacterium tuberculosis, Staphylococcus aureus and various Enterococci species are now nearly untreatable with standard antibiotics and pose a growing threat to patients in hospitals and the community at large. In addition, contact with farm animals has been identified as another source for bacterial pathogens. However, the problem does not end at the hospital entrance or the farmyard gate; the environment is a huge source of antibiotic resistance. In fact, antibiotics and antibiotic resistance determinants are a natural phenomenon and have been present in the environment long before humans discovered and begun to use antibiotics. Microorganisms produce antibiotics to gain a growth advantage and to defend …

An Active Nonautonomous Mobile Element in Sulfolobus islandicus REN1H1

In the crenarchaeote Sulfolobus islandicus REN1H1, a mobile element of 321 bp length has been shown to be active. It does not contain terminal inverted repeats and transposes by a replicative mechanism. This newly discovered element has been named SMN1 (for Sulfolobus miniature noninverted repeat transposable element).

Characterisation of the novel restriction endonuclease SuiI from Sulfolobus islandicus.

A restriction endonuclease activity from Sulfolobus islandicus REN2H1 was purified by phosphocellulose and cation exchange chromatography. The enzyme cuts DNA at the recognition site GCwGC as could be shown by restriction analysis of plasmids and short synthetic duplex DNA. The cleavage occurs after the first guanosine base and is inhibited by 5-methyl-cytosine methylation. The restriction activity is salt-sensitive and has an optimal activity around 70°C.

Nanopharmaceuticals: Tiny challenges for the environmental risk assessment of pharmaceuticals.

Many new developments and innovations in health care are based on nanotechnology. The field of nanopharmaceuticals is diverse and not as new as one might think; indeed, nanopharmaceuticals have been marketed for many years, and the future is likely to bring more nanosized compounds to the market. Therefore, it is time to examine whether the environmental risk assessment for human pharmaceuticals is prepared to assess the exposure, fate, and effects of nanopharmaceuticals in an adequate way. Challenges include the different definitions for nanomaterials and nanopharmaceuticals, different regulatory frameworks, the diversity of nanopharmaceuticals, the scope of current regulatory guidelines, and the applicability of test protocols. Based on the current environmental risk assessment for human medicinal products in the European Union, necessary adaptations for the assessment procedures and underlying study protocols are discussed and emerging solutions identified.

Identification of the minimal replicon and the origin of replication of the crenarchaeal plasmid pRN1

We have determined the minimal replicon of the crenarchaeal plasmid pRN1. It consists of 3097 base pairs amounting to 58% of the genome of pRN1. The minimal replicon comprises replication operon orf56/orf904 coding for a transcriptional repressor and the replication protein of pRN1. An upstream region of 64 bp that contains the promoter of the replication operon is essential as well as 166 bp of sequence downstream of the orf904 gene. This region contains a putative transcriptional terminator and a 100 nucleotides long stem–loop structure. Only the latter structure was shown to be required for replication. In addition replication was sustained when the stem–loop was displaced to another part of the pRN1 sequence. By mutational analysis we also find that the integrity of the stem–loop structure is required to maintain the replication of pRN1‐derived constructs. As similar stem–loop structures are also present in other members of the pRN family, we suggest that this conserved structural element could be the origin of replication for the pRN plasmids. Further bioinformatic analysis revealed that the domain structure of the replication protein and the presence of a similar stem–loop structure as the putative replication origin are also found in several bacteriophages.