Isolation of lipopolysaccharides and the effect of Polymyxin B on the outer membrane of Corynebacterium autotrophicum

Lipopolysaccharides (LPS) from Corynebacterium autotrophicum were isolated and analyzed. Autotrophically grown cells contained 2–5 mg of partly purified LPS per g dry weight of lyophilized cells. Serological cross reaction with Lipid A antigen of Salmonella minnesota confirmed the presence of LPS in C. autotrophicum. Electron microscopy of negatively stained Polymyxin B-treated cells showed formation of blebs on the Outer Membrane indicating an interaction of Polymyxin B specifically with LPS. Up to now, no Gram-positive organisms are known which contain any LPS. Thus, C. autotrophicum, though giving opposite results when the Gram-staining reaction was applied by several authors, has to be classified into the group of Gram-negative bacteria.Non-Common Abbreviations LPS lipopolysaccharide - KDO 2-keto-3-deoxyoctonate     

Biodiversity of poly-extremophilic Bacteria: Does combining the extremes of high salt, alkaline pH and elevated temperature approach a physico-chemical boundary for life?

Bacterial microorganisms that grow optimally at Na⁺ concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review focuses on the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance of extremely halophilic eubacteria; the focus is on those with alkaline pH optima, above 8.5, and elevated temperature optima, above 50°C. If all three conditions are required for optimal growth, these microorganisms are termed "poly-extremophiles". However, only a very few extreme halophiles able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated so far. Therefore the question is: do the combined extreme growth conditions of the recently isolated poly-extremophiles, i.e., anaerobic halophilic alkalithermophiles, approach a physico-chemical boundary for life? These poly-extremophiles are of interest, as their adaptive mechanisms give insight into organisms' abilities to survive in environments which were previously considered prohibitive to life, as well as to possible properties of early evolutionary and extraterrestrial life forms.     

Comparison of Energy and Growth Yields for Desulfitobacterium dehalogenans during Utilization of Chlorophenol and Various Traditional Electron Acceptors

Desulfitobacterium dehalogenans grew with formate as the electron donor and 3-chloro-4-hydroxyphenylacetate (3-Cl-4-OHPA) as the electron acceptor, yielding Y_X/formate, Y_X/2e⁻, and Y_X/ATP ranging from 3.2 to 11.3 g of biomass (dry weight)/mol, thus indicating that energy was conserved through reductive dechlorination. Pyruvate was utilized as the electron donor and acceptor, yielding stoichiometric amounts of acetate and lactate, respectively, and a Y_{X/reduced acceptor} of 13.0 g of biomass (dry weight)/mol. The supplementation of pyruvate-containing medium with additional electron acceptors, such as 3-Cl-4-OHPA, nitrate, fumarate, or sulfite, caused pyruvate to be replaced as the electron acceptor and nearly doubled the Y_X/ATP (Y_{X/acetate formed}). A comparison of the yields for 3-Cl-4-OHPA with those for other traditional electron acceptors indicates that the dehalogenation reaction led to the formation of similar amounts of energy equivalents. The various electron acceptors were used concomitantly with 3-Cl-4-OHPA in nonacclimated cultures, but the utilization rates and amounts utilized differed.     

Advances in development of a genetic system for Thermoanaerobacterium spp.: expression of genes encoding hydrolytic enzymes, development of a second shuttle vector, and integration of genes into the chromosome

Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system, Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticum JW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of the Thermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.     

Thermosediminibacter oceani gen. nov., sp. nov. and Thermosediminibacter litoriperuensis sp. nov., new anaerobic thermophilic bacteria isolated from Peru Margin

A new group of anaerobic thermophilic bacteria was isolated from enrichment cultures obtained from deep sea sediments of Peru Margin collected during Leg 201 of the Ocean Drilling Program. A total of ten isolates were obtained from cores of 1–2 m below seafloor (mbsf) incubated at 60°C: three isolates came from the sediment 426 m below sea level with a surface temperature of 9°C (Site 1227), one from 252 m below sea level with a temperature of 12°C (Site 1228), and six isolates under sulfate-reducing condition from the lower slope of the Peru Trench (Site 1230). Strain JW/IW-1228P from the Site 1228 and strain JW/YJL-1230-7/2 from the Site 1230 were chosen as representatives of the two identified clades. Based on the 16S rDNA sequence analysis, these isolates represent a novel group with Thermovenabulum and Caldanaerobacter as their closest relatives. The temperature range for growth was 52–76°C with an optimum at around 68°C for JW/IW-1228P and 43–76°C with an optimum at around 64°C for JW/YJL-1230-7/2. The pH^25C range for growth was from 6.3 to 9.3 with an optimum at 7.5 for JW/IW-1228P and from 5 to 9.5 with an optimum at 7.9–8.4 for JW/YJL-1230-7/2. The salinity range for growth was from 0% to 6% (w/v) for JW/IW-1228P and from 0% to 4.5% (w/v) for JW/YJL-1230-7/2. The G+C content of the DNA was 50 mol% for both JW/IW-1228P and JW/YJL-1230-7/2. DNA–DNA hybridization yielded 52% similarity between the two strains. According to 16S rRNA gene sequence analysis, the isolates are located within the family, Thermoanaerobacteriaceae. Based on their morphological and physiological properties and phylogenetic analysis, it is proposed that strain JW/IW-1228P^T is placed into a novel taxa, Thermosediminibacter oceani, gen. nov., sp. nov. (DSM 16646^T=ATCC BAA-1034^T), and JW/YJL-1230-7/2^T into Thermosediminibacter litoriperuensis sp. nov. (DSM 16647^T =ATCC BAA-1035^T).An erratum to this article can be found at     

Distribution of genes encoding the microbial non-oxidative reversible hydroxyarylic acid decarboxylases/phenol carboxylases

Bacterial non-oxidative, reversible multi subunit hydroxyarylic acid decarboxylases/phenol carboxylases are encoded by the three clustered genes, B, C, and D, of approximately 0.6, 1.4, and 0.2 kb, respectively. There are more than 160 homologues in the database with significant similarity to gene B (homology to ubiX) and C (ubiD) distributed in all three microbial domains, however, homologues to gene D, are not numerous ( approximately 15). The occurrence of the entire BCD gene cluster encoding for either identified or presumptive hydroxyarylic acid decarboxylase to date has been revealed in Sedimentibacter hydroxybenzoicus (unique genes arrangement CDB), Streptomyces sp. D7, Bacillus subtilis, B. licheniformis, E. coli O157:H7, Klebsiella pneumoniae, Enterobacter cloacae, Shigella dysenteriae, Salmonella enterica, S. paratyphi, S. typhimurium, S. bongori, and S. diarizonae. The corresponding genes from S. hydroxybenzoicus, B. subtilis, Streptomyces sp. D7, E. coli O157:H7, K. pneumoniae, and S. typhimurium were cloned and expressed in E. coli DH5alpha (void of analogous genes), and shown to code for proteins exhibiting non-oxidative hydroxyarylic acid decarboxylase activity.     

Complete genome sequence of the anaerobic, halophilic alkalithermophile Natranaerobius thermophilus JW/NM-WN-LF

The genome of the anaerobic halophilic alkalithermophile Natranaerobius thermophilus consists of one 3,165,557-bp chromosome and two plasmids (17,207 bp and 8,689 bp). The present study is the first to report the completely sequenced genome of an anaerobic polyextremophile and genes associated with roles in regulation of intracellular osmotic pressure, pH homeostasis, and growth at elevated temperatures.     

A simple theory of peptide interactions on a membrane surface: excluded volume and entropic order

A simple theory of the interactions of peptides bound onto a lipid membrane is developed, modeling the peptides as rods on a surface. At low peptide surface-concentration, excluded volume dominates the peptide-peptide interactions and the orientation of the peptides is random, resulting in an isotropic configuration. However, at high peptide density on the membrane, the peptides become orientationally ordered, resulting in an anisotropic configuration. This effect is entirely entropic in origin, and simply reflects the fact that peptides can be exchanged more easily on the surface if they are equally aligned, resulting in a larger number of possible configurations. In three dimensions, this phenomenon corresponds to the well-known isotropic-nematic phase transition. In two dimensions, the problem is not as well understood. The theoretical treatment presented here yields a simple, manageable expression which can be compared with experimental data. Two-dimensional ordering results in an increase in the apparent binding constant of peptides to membranes at high concentration of peptides relative to what is expected from the effect of excluded volume alone. The possible implications of side-by-side alignment for several biological processes, such as peptide translocation across membranes and plaque formation in Alzheimer's disease, are discussed.