The genome of Syntrophorhabdus aromaticivorans strain UI provides new insights for syntrophic aromatic compound metabolism and electron flow

How aromatic compounds are degraded in various anaerobic ecosystems (e.g. groundwater, sediments, soils and wastewater) is currently poorly understood. Under methanogenic conditions (i.e. groundwater and wastewater treatment), syntrophic metabolizers are known to play an important role. This study explored the draft genome of Syntrophorhabdus aromaticivorans strain UI and identified the first syntrophic phenol‐degrading phenylphosphate synthase (PpsAB) and phenylphosphate carboxylase (PpcABCD) and syntrophic terephthalate‐degrading decarboxylase complexes. The strain UI genome also encodes benzoate degradation through hydration of the dienoyl‐coenzyme A intermediate as observed in Geobacter metallireducens and Syntrophus aciditrophicus. Strain UI possesses electron transfer flavoproteins, hydrogenases and formate dehydrogenases essential for syntrophic metabolism. However, the biochemical mechanisms for electron transport between these H₂/formate‐generating proteins and syntrophic substrate degradation remain unknown for many syntrophic metabolizers, including strain UI. Analysis of the strain UI genome revealed that heterodisulfide reductases (HdrABC), which are poorly understood electron transfer genes, may contribute to syntrophic H₂ and formate generation. The genome analysis further identified a putative ion‐translocating ferredoxin : NADH oxidoreductase (IfoAB) that may interact with HdrABC and dissimilatory sulfite reductase gamma subunit (DsrC) to perform novel electron transfer mechanisms associated with syntrophic metabolism.     

Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-3H]Thymidine in a Hypertrophic Lake

The incorporation of [methyl-³H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-³H]thymidine studies of aquatic bacterial production.     

Redesign of a protein–peptide interaction: Characterization and applications

The design of protein–peptide interactions has a wide array of practical applications and also reveals insight into the basis for molecular recognition. Here, we present the redesign of a tetratricopeptide repeat (TPR) protein scaffold, along with its corresponding peptide ligand. We show that the binding properties of these protein–peptide pairs can be understood, quantitatively, using straightforward chemical considerations. The recognition pairs we have developed are also practically useful for the specific identification of tagged proteins. We demonstrate the facile replacement of these proteins, which we have termed T‐Mods (TPR‐based recognition module), for antibodies in both detection and purification applications. The new protein–peptide pair has a dissociation constant that is weaker than typical antibody–antigen interactions, yet the recognition pair is highly specific and we have shown that this affinity is sufficient for both Western blotting and affinity purification. Moreover, we demonstrate that this more moderate affinity is actually advantageous for purification applications, because extremely harsh conditions are not required to dissociate the T‐Mod‐peptide interaction. The results we present are important, not only because they represent a successful application of protein design but also because they help define the properties that should be sought in other scaffolds that are being developed as antibody replacements.     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain TA1

Rhizobium leguminosarum bv. trifolii strain TA1 is an aerobic, motile, Gram-negative, non-spore-forming rod that is an effective nitrogen fixing microsymbiont on the perennial clovers originating from Europe and the Mediterranean basin. TA1 however is ineffective with many annual and perennial clovers originating from Africa and America. Here we describe the features of R. leguminosarum bv. trifolii strain TA1, together with genome sequence information and annotation. The 8,618,824 bp high-quality-draft genome is arranged in a 6 scaffold of 32 contigs, contains 8,493 protein-coding genes and 83 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

Efficient immortalization and morphological transformation of human fibroblasts by transfection with SV40 DNA linked to a dominant marker

Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno-deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40-transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line.     

Ultrastructure and metabolism of skeletal muscle fibres in the tench: Effects of long-term acclimation to hypoxia

Summary Tench (Tinca tinca) were acclimated to either aerated (P _{O 2} 17.6 KPa) or hypoxic (P _{O 2} 1.5 KPa) water for 6 weeks.Acclimation to hypoxia resulted in a decrease in mitochondrial volume fraction in both slow (22.9 to 15.0 %) and fast glycolytic (4.5 to 1.8 %) myotomal muscles fibres (P<0.01).Intermyofibrillar mitochondrial populations (4.4 to 1.2% slow; 0.6 to 0.04% fast fibres) were affected to a greater extent than those in the subsarcolemmal zone (18.5 to 13.8% slow; 3.9 to 1.8% fast fibres). After acclimation to hypoxia, cytochrome-oxidase activities decreased by 31 and 33 % in slow and fast fibres, respectively, but were maintained in the liver.Fibre size remained unchanged and actively differentiating fibres were observed in muscles from both groups of fish. Hypoxia resulted in a significant increase in myofibrillar volume fraction in both slow (43.1 to 56.1 %) and fast glycolytic fibres (73.1 to 82.7%) (P<0.05).Glycogen concentrations (mg/100g tissue) for liver (6616) slow muscle (1892) and fast muscle (334) were similar for fish acclimated to aerated or hypoxic water. Acclimation to hypoxia increased carnitine palmitoyl transferase activity (moles substrate utilised g·dry wt_-1 min^-1) in slow (0.42 to 1.1), fast glycolytic muscle (<0.01 to 0.15) and liver (1.1 to 3.7) indicating an enhanced capacity for fatty acid oxidation.Phosphofructokinase activities of fast glycolytic fibres were similar in fish acclimated to either aerated or hypoxic water, consistent with an unaltered capacity for anaerobic glycogenolysis. Hexokinase activities (moles substate utilised, g·dry wt^-1 min^-1) decreased in fast fibres (1.2 to 0.4) but were maintained in the slow muslce (2.1 to 2.5) and liver (4.5 to 4.8) of hypoxic fish. The activities of phosphofructokinase in slow muscle and phosphofructokinase, pyruvate kinase and lactate dehydrogenase in liver were two times higher in fish acclimated to hypoxia. An enhanced capacity for glycolysis in these tissues may reflect a reduced threshold for anaerobic metabolism during activity and/or an adaptation for acute exposure to anoxia in fish acclimated to hypoxia.Abbreviations/Glossary CO cytochrome oxidase activity - CPT carnitine palmitoyltransferase activity - HK hexokinase activity - LDH lactate dehydrogenase activity - PFK phosphofructokinase activity - PK pyruvate kinase activity - Vv volume fractions of cell components - normoxic fish acclimated to aerated water - hypoxic fish acclimated to reduced oxygen tensions - P _{O 2} partial pressure of oxygen tension A preliminary account of part of this work was presented at theXth European Meeting on Muscle and Cell Motility held at Galway, Ireland, in September 1981