Sulfide ameliorates metal toxicity for deep-sea hydrothermal vent archaea

The chemical stress factors for microbial life at deep-sea hydrothermal vents include high concentrations of heavy metals and sulfide. Three hyperthermophilic vent archaea, the sulfur-reducing heterotrophs Thermococcus fumicolans and Pyrococcus strain GB-D and the chemolithoautotrophic methanogen Methanocaldococcus jannaschii, were tested for survival tolerance to heavy metals (Zn, Co, and Cu) and sulfide. The sulfide addition consistently ameliorated the high toxicity of free metal cations by the formation of dissolved metal-sulfide complexes as well as solid precipitates. Thus, chemical speciation of heavy metals with sulfide allows hydrothermal vent archaea to tolerate otherwise toxic metal concentrations in their natural environment.     

Specific glycanforms of type IX collagen accumulate in embryonic chick sterna after 17 days of development

Type IX collagen is a key component of the extracellular matrix of cartilage where it occurs at the surfaces of type II collagen fibrils as a glycanated molecule. The function of the glycosaminoglycan (GAG) side chain of the molecule is, however, unknown. We have shown that type IX collagen in chicken sternal cartilage is synthesized with a unimodal distribution of GAG chain size, but at post 17 days of development three predominant glycanforms of type IX collagen accumulate. Such accumulation did not occur in sterna from day 15 embryos. In day 17 embryos predominant glycanforms were found in the caudal region of the sternum. By day 19 of development the three predominant glycanforms are widespread throughout the caudal and cephalic regions. The results indicate that developmental and anatomical changes occur to type IX collagen that depend on the size of the GAG chain attached to the alpha2(IX) chain of the molecule.      

Estimation of Bacterial Cell Numbers in Humic Acid-Rich Salt Marsh Sediments with Probes Directed to 16S Ribosomal DNA

The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 × 10⁷ to 2.5 × 10⁹ (average, 1.1 × 10⁹ ± 5.2 × 10⁸) cells g of sediment⁻¹. In September, numbers of SRB detected ranged from 5.4 × 10⁸ to 7.3 × 10⁹ (average, 2.5 × 10⁹ ± 1.5 × 10⁹) cells g of sediment⁻¹. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments.     

Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes

Listeria monocytogenes is a foodborne psychrotrophic pathogen that grows at refrigeration temperatures. Previous studies of fatty acid profiles of wild-type and cold-sensitive, branched-chain fatty acid deficient mutants of L. monocytogenes suggest that the fatty acid 12-methyltetradecanoic (anteiso-C(15:0)) plays a critical role in low-temperature growth of L. monocytogenes, presumably by maintaining membrane fluidity. The fluidity of isolated cytoplasmic membranes of wild-type (SLCC53 and 10403S), and a cold-sensitive mutant (cld-1) of L. monocytogenes, grown with and without the supplementation of 2-methylbutyric acid, has been studied using a panel of hydrocarbon-based nitroxides (2N10, 3N10, 4N10, and 5N10) and spectral deconvolution and simulation methods to obtain directly the Lorentzian line widths and hence rotational correlation times (tau(c)) and motional anisotropies of the nitroxides in the fast motional region. tau(c) values over the temperature range of -7 degrees C to 50 degrees C were similar for the membranes of strains SLCC53 and 10403S grown at 10 degrees C and 30 degrees C, and for strain cld-1 grown with 2-methylbutyric acid supplementation (which restores branched-chain fatty acids) at 30 degrees C. However, strain cld-1 exhibited a threefold higher tau(c) when grown without 2-methylbutyric acid supplementation (deficient in branched-chain fatty acids) compared to strains SLCC53, 10403S, and supplemented cld-1. No evidence was seen for a clear lipid phase transition in any sample. We conclude that the fatty acid anteiso-C(15:0) imparts an essential fluidity to the L. monocytogenes membrane that permits growth at refrigeration temperatures.     

Variability in the pKa of histidine side-chains correlates with burial within proteins

Acidic pKas of histidines buried within the protein interior are frequently rationalized on the contradictory basis of either polar interactions within the protein or the effects of a hydrophobic environment. To examine these relationships, we surveyed the buried surface area, depth of burial, polar interactions, and crystallographic temperature factors of histidines of known pKa. It has been found that buried environments of histidines do not always result in acidic pKas. Instead, the variability of histidine pKas increases for residues where the majority of the side-chain is buried. Because buried histidines are always found in mixed polar/apolar environments, multiple environmental contributions to pKa values must be considered. However, the quantitative relationships between heterogeneous environments and pKa values are not immediately apparent from the available data.     

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.     

Diversity and ecology of algae from the Nahal Qishon river,northern Israel

In 59 samples of periphyton and phytoplankton collected in 2002 - 2003 from the Nahal Qishon (Qishon River), northern Israel, we found 178 species from seven divisions of algae and cyanoprocaryotes. Diatoms, clorophytes, and cyanoprocaryotes prevail. Nitzschia and Navicula (Bacillariophyta) are the most abundant. Most of the species are cosmopolitan or widespread, except Lagynion janei (Chrysophyta), which is endemic for the Mediterranean Realm. About 17% of species (26) are new for Israel and five of them represent the first recorded genera: Crinalium endophyticum Crow, Actinocyclus normanii (Gregory) Hustedt, Rhizoclonium hieroglyphicum (Agardh) Kütz (Chlorophyta), Lagynion janei Bourelly, and Stylococcus aureus Chodat. Most of them come from a rare riverine assemblage with red alga Audouinella pygmea, as well as from the estuarine assemblage. Alkaliphiles predominate among the indicators of acidity, with few acidophiles confined to the communities under the impact of industrial wastes. Among the indicators of salinity, most numerous are the oligohalobien-indifferents and species adapted to a moderate salinity level. The relative species richness of ecological groups and the indices of saprobity are correlated with changes in conductivity, pH, and N-nitrate concentration. Indicators of organic pollution fall in the range of betameso- to alfamesosaprobic self-purification grades. Our studies show ecological significance of the Nahal Qishon as a model for a strongly disturbed aquatic ecosystem in the coastal zone of eastern Mediterranean.