Detecting Bacterial Cell Viability in Few µL Solutions from Impedance Measurements on Silicon-Based Biochips.

Using two different types of impedance biochips (PS5 and BS5) with ring top electrodes, a distinct change of measured impedance has been detected after adding 1-5 µL (with dead or live Gram-positive Lysinibacillus sphaericus JG-A12 cells to 20 µL DI water inside the ring top electrode. We relate observed change of measured impedance to change of membrane potential of L. sphaericus JG-A12 cells. In contrast to impedance measurements, optical density (OD) measurements cannot be used to distinguish between dead and live cells. Dead L. sphaericus JG-A12 cells have been obtained by adding 0.02 mg/mL of the antibiotics tetracycline and 0.1 mg/mL chloramphenicol to a batch with OD0.5 and by incubation for 24 h, 30 °C, 120 rpm in the dark. For impedance measurements, we have used batches with a cell density of 25.5 × 108 cells/mL (OD8.5) and 270.0 × 108 cells/mL (OD90.0). The impedance biochip PS5 can be used to detect the more resistive and less capacitive live L. sphaericus JG-A12 cells. Also, the impedance biochip BS5 can be used to detect the less resistive and more capacitive dead L. sphaericus JG-A12 cells. An outlook on the application of the impedance biochips for high-throughput drug screening, e.g., against multi-drug-resistant Gram-positive bacteria, is given.

Microbial Diversity in an Arid, Naturally Saline Environment.

The Arava Valley in is a rock desert within the Great African Rift valley. Soil from this area is covered with a salt crust. Here, we report microbial diversity from arid, naturally saline samples collected near Ein Yahav from the Arava Valley by culture-independent as well as culture-dependent analysis. High-throughput sequencing of the hypervariable region V4 of the 16S rRNA gene revealed that the microbial community consists of halophiles from the domain Bacteria as well as Archaea. Bacterial diversity was mainly represented by the genus Salinimicrobium of the order Flavobacteriales within the phylum Bacteroidetes, from the gammaproteobacterial orders Alteromonadales and Oceanospirillales as well as representatives from the order Bacillales of the phylum Firmicutes. Archaeal diversity was dominated by euryarchaeal Halobacteria from the orders Halobacteriales, Haloferacales, and Natrialbales. But more than 40% of the sequences affiliated with Archaea were assigned to unknown or unclassified archaea. Even if taxonomic resolution of the 16S rRNA gene V4 region for Archaea is limited, this study indicates the need of further and more detailed studies of Archaea. By using culture-dependent analysis, bacteria of the order Bacillales as well as archaea from all three halobacterial orders Halobacteriales, Haloferacales, and Natrialbales including potentially novel species from the genera Halorubrum and Haloparvum were isolated.

The Year-Long Development of Microorganisms in Uncompacted Bavarian Bentonite Slurries at 30 and 60 °C.

In the multibarrier concept for the deep geological disposal of high-level radioactive waste (HLW), bentonite is proposed as a potential barrier and buffer material for sealing the space between the steel canister containing the HLW and the surrounding host rock. In order to broaden the spectra of appropriate bentonites, we investigated the metabolic activity and diversity of naturally occurring microorganisms as well as their time-dependent evolution within the industrial B25 Bavarian bentonite under repository-relevant conditions. We conducted anaerobic microcosm experiments containing the B25 bentonite and a synthetic Opalinus Clay pore water solution, which were incubated for one year at 30 and 60 °C. Metabolic activity was only stimulated by the addition of lactate, acetate, or H2. The majority of lactate- and H2-containing microcosms at 30 °C were dominated by strictly anaerobic, sulfate-reducing, and spore-forming microorganisms. The subsequent generation of hydrogen sulfide led to the formation of iron-sulfur precipitations. Independent from the availability of substrates, thermophilic bacteria dominated microcosms that were incubated at 60 °C. However, in the respective microcosms, no significant metabolic activity occurred, and there was no change in the analyzed biogeochemical parameters. Our findings show that indigenous microorganisms of B25 bentonite evolve in a temperature- and substrate-dependent manner.

Peptidoglycan as major binding motif for Uranium bioassociation on Magnetospirillum magneticum AMB-1 in contaminated waters.

The U(VI) bioassociation on Magnetospirillum magneticum AMB-1 cells was investigated using a multidisciplinary approach combining wet chemistry, microscopy, and spectroscopy methods to provide deeper insight into the interaction of U(VI) with bioligands of Gram-negative bacteria for a better molecular understanding. Our findings suggest that the cell wall plays a prominent role in the bioassociation of U(VI). In time-dependent bioassociation studies, up to 95 % of the initial U(VI) was removed from the suspension and probably bound on the cell wall within the first hours due to the high removal capacity of predominantly alive Magnetospirillum magneticum AMB-1 cells. PARAFAC analysis of TRLFS data highlights that peptidoglycan is the most important ligand involved, showing a stable immobilization of U(VI) over a wide pH range with the formation of three characteristic species. In addition, in-situ ATR FT-IR reveals the predominant strong binding to carboxylic functionalities. At higher pH polynuclear species seem to play an important role. This comprehensive molecular study may initiate in future new remediation strategies on effective immobilization of U(VI). In combination with the magnetic properties of the bacteria, a simple technical water purification process could be realized not only for U(VI), but probably also for other heavy metals.

The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease.

Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.

Strong conservation of the human NF2 locus based on sequence comparison in five species.

We analyzed 137 kb covering human neurofibromatosis 2 ( NF2) tumor suppressor locus and orthologous loci from baboon, mouse, rat, and pufferfish Takifugu rubripes. A predominant feature of human-rodent conservation is a very similar distribution of conserved islands, regarding length, position, and degree of identity. By use of a threshold of 75% identity over > or =100 bp of gap-free alignment, comparisons of human-mouse sequences resulted in 3.58% for extra-exonic conservation, which can be compared to 4.5% of exonic sequence content within the human locus. We identified a duplication of neurofibromin 2 in pufferfish, which resulted in two putative proteins with 74% and 76% identity to the human protein. One distinct island (called inter 1), conserved between all analyzed species, was located between promoters of the NIPSNAP1 and NF2 genes. Inter 1 might represent a novel regulatory element, important for the function of this locus. The high level of intronic conservation in the NF2 locus suggests that a number of unknown regulatory elements might exist within this gene. These elements could be affected by disease-causing mutations in NF2 patients and NF2-associated tumors. Alternatively, this conservation might be explained by presence of not yet characterized transcriptional unit(s) within this locus.