Structure at 1.0 Å resolution of a high-potential iron–sulfur protein involved in the aerobic respiratory chain of Rhodothermus marinus

The aerobic respiratory chain of the thermohalophilic bacterium Rhodothermus marinus, a nonphotosynthetic organism from the Bacteroidetes/Chlorobi group, contains a high-potential iron–sulfur protein (HiPIP) that transfers electrons from a bc ₁ analog complex to a caa ₃ oxygen reductase. Here, we describe the crystal structure of the reduced form of R. marinus HiPIP, solved by the single-wavelength anomalous diffraction method, based on the anomalous scattering of the iron atoms from the [4Fe–4S]^3+/2+ cluster and refined to 1.0 Å resolution. This is the first structure of a HiPIP isolated from a nonphotosynthetic bacterium involved in an aerobic respiratory chain. The structure shows a similar environment around the cluster as the other HiPIPs from phototrophic bacteria, but reveals several features distinct from those of the other HiPIPs of phototrophic bacteria, such as a different fold of the N-terminal region of the polypeptide due to a disulfide bridge and a ten-residue-long insertion.     

Interactions between glycolytic enzymes of Mycoplasma pneumoniae

With only 688 protein-coding genes, Mycoplasma pneumoniae is one of the smallest self-replicating organisms. These bacteria use glycolysis as the major pathway for ATP production by substrate-level phosphorylation, suggesting that this pathway must be optimized to high efficiency. In this study, we have investigated the interactions between glycolytic enzymes using the bacterial adenylate cyclase-based two-hybrid system. We demonstrate that most of the glycolytic enzymes perform self-interactions, suggesting that they form dimers or other oligomeric forms. In addition, enolase was identified as the central glycolytic enzyme of M. pneumoniae due to its ability to directly interact with all other glycolytic enzymes. Our results support the idea of the formation of a glycolytic complex in M. pneumoniae and we suggest that the formation of this complex might ensure higher fluxes through the glycolytic pathway than would be possible with isolated non-interacting enzymes.     

BMP12 and BMP13 gene transfer induce ligamentogenic differentiation in mesenchymal progenitor and anterior cruciate ligament cells

Background aimsTo date there are only very few data available on the ligamentogenic differentiation capacity of mesenchymal stromal/progenitor cells (MSC) and anterior cruciate ligament (ACL) fibroblasts.MethodsWe describe the in vitro potential of MSC and ACL cells to undergo ligamentogenic differentiation upon transduction with adenoviral vectors encoding the human cDNA for bone morphogenetic protein (BMP) 12 and BMP13, also known as growth and differentiation factors (GDF) 6 and 7, respectively.ResultsTransgene expression for at least 14 days was confirmed by Western blot analyzes. After 21 days of cell culture within collagen type I hydrogels, histochemical (hematoxylin/eosin (H&E), Azan and van Gieson), immunohistochemical and polymerase chain reaction (PCR) analyzes of the genetically modified constructs of both cell types revealed elongated, viable fibroblast-like cells embedded in a ligament-like matrix rich in collagens, vimentin, fibronectin, decorin, elastin, scleraxis, tenascin, and tenomodulin.ConclusionsIt appears that both MSC and ACL fibroblasts are capable of ligamentogenic differentiation with these factors. This information may aid in the development of biologic approaches to repair and restore ACL after injury.     

Analysis of Lipid Export in Hydrocarbonoclastic Bacteria of the Genus Alcanivorax: Identification of Lipid Export-Negative Mutants of Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9

Triacylglycerols (TAGs), wax esters (WEs), and polyhydroxyalkanoates (PHAs) are the major hydrophobic compounds synthesized in bacteria and deposited as cytoplasmic inclusion bodies when cells are cultivated under imbalanced growth conditions. The intracellular occurrence of these compounds causes high costs for downstream processing. Alcanivorax species are able to produce extracellular lipids when the cells are cultivated on hexadecane or pyruvate as the sole carbon source. In this study, we developed a screening procedure to isolate lipid export-negative transposon-induced mutants of bacteria of the genus Alcanivorax for identification of genes required for lipid export by employing the dyes Nile red and Solvent Blue 38. Three transposon-induced mutants of A. jadensis and seven of A. borkumensis impaired in lipid secretion were isolated. All isolated mutants were still capable of synthesizing and accumulating these lipids intracellularly and exhibited no growth defect. In the A. jadensis mutants, the transposon insertions were mapped in genes annotated as encoding a putative DNA repair system specific for alkylated DNA (Aj17), a magnesium transporter (Aj7), and a transposase (Aj5). In the A. borkumensis mutants, the insertions were mapped in genes encoding different proteins involved in various transport processes, like genes encoding (i) a heavy metal resistance (CZCA2) in mutant ABO_6/39, (ii) a multidrug efflux (MATE efflux) protein in mutant ABO_25/21, (iii) an alginate lyase (AlgL) in mutants ABO_10/30 and ABO_19/48, (iv) a sodium-dicarboxylate symporter family protein (GltP) in mutant ABO_27/29, (v) an alginate transporter (AlgE) in mutant ABO_26/1, or (vi) a two-component system protein in mutant ABO_27/56. Site-directed MATE, algE, and algL gene disruption mutants, which were constructed in addition, were also unable to export neutral lipids and confirmed the phenotype of the transposon-induced mutants. The putative localization of the different gene products and their possible roles in lipid excretion are discussed. Beside this, the composition of the intra- and extracellular lipids in the wild types and mutants were analyzed in detail.Almost all prokaryotes synthesize lipophilic storage substances as an integral part of their metabolism under limited nitrogen or phosphorus conditions if there is an excess of a suitable carbon source at the same time. The accumulated storage lipids serve as energy and carbon sources during starvation periods, and they are mobilized again under conditions of carbon and energy deficiency. The majority of the members of many genera synthesize hydrophobic polymers, such as poly(3-hydroxybutyrate) (PHB) or other types of polyhydroxyalkanoates (PHAs), whereas the accumulation of triacylglycerols (TAGs; trioxoesters of glycerol and long-chain fatty acids [FAs]) or wax esters (WEs; oxoesters of primary long-chain fatty acids and primary long-chain fatty alcohols) occurs in fewer prokaryotes (66). TAG accumulation has been reported for species of the genera Streptomyces, Mycobacterium, Nocardia, Rhodococcus (4, 6, 65), and recently also Alcanivorax and other hydrocarbonoclastic marine bacteria (32). Accumulation of WEs has been frequently reported for species of the genus Acinetobacter (66) but also for marine bacteria, such as Marinobacter (50) and Alcanivorax (11, 32).In general, the accumulation of at least one type of these compounds occurs intracellularly under imbalanced growth conditions in almost all prokaryotes. The localization of neutral lipids in marine organisms is not restricted to the cell cytoplasm, as extracellular lipid deposition has been shown in studies with Alcaligenes sp. PHY9 and Pseudomonas nautica (24). The production of extracellular wax esters by Alcanivorax jadensis T9 growing on hexadecane was described a few years ago (11). Species of the genus Alcanivorax belong to an unusual group of marine hydrocarbon-degrading bacteria, which have been recognized and described over the past few years and were shown to play an important role in the biological removal of petroleum hydrocarbons from contaminated sites (69). Species of the genus Alcanivorax are, like some species of the genera Neptunomonas (27) and Marinobacter (23), marine hydrocarbon-degrading bacteria. Moreover, Alcanivorax and related bacteria constitute the group of obligate hydrocarbonoclastic marine bacteria (OHCB), which exhibit a narrow range of utilizable carbon sources (obligate hydrocarbon utilization), with only a few species being able to metabolize substrates other than hydrocarbons (69). Alcanivorax borkumensis SK2 became a model strain of OHCB, and its importance and pivotal role in hydrocarbon biodegradation have recently been emphasized (33). The predominance of A. borkumensis in early stages of petroleum degradation has also been reported in microcosm studies as well as for a field-scale experiment (26).From a biotechnological point of view, the production of extracellular lipids is important. Secretion of lipophilic products into the culture medium rather than its intracellular accumulation can significantly reduce the costs of product recovery. Another advantage is that the production of WEs and TAGs would not be directly limited by cell density or cell volume. Until now, the mechanism responsible for the export of lipids in bacteria of the genus Alcanivorax or other bacteria had not been known. In this study, we report on a screening procedure to select mutants defective in lipid export for identification of the gene(s) involved in the export mechanism. After transposon-induced mutagenesis we found different mutants which were not able to export TAGs (mutants of A. borkumensis) when the cells were cultivated in the presence of pyruvate as the sole carbon source. Mutants of A. jadensis defective in export of WEs and/or wax diesters (DE) were also identified. The possible influences of the gene products on the export mechanism in Alcanivorax species were analyzed and are discussed.     

A new species of <Emphasis Type="Italic">Laboulbenia</Emphasis> and new records from Panama

Species of Laboulbenia on ground beetles (Coleoptera, Carabidae) collected in a mountain rainforest in Western Panama are described and illustrated. A new species of Laboulbenia on carabids of the genus Platynus (Platynini) in Panama is proposed. It differs from the other species of Laboulbenia mainly by curved thalli and longitudinally twisted wall cells of the perithecia with lips oriented towards strongly branched appendages. L. decipiens, L. pseudomasei, L. subpunctata, and L. tenera are newly recorded for Panama. Only one species collected during the survey is already known for Panama, L. flagellata.     

New species and records of cercosporoid hyphomycetes from Panama

Cercosporoid hyphomycetes on living leaves of plants were collected in Panama, identified, described, and illustrated. Newly described species are Passalora guraniae from Gurania sp. (Cucurbitaceae), Pseudocercospora arrabidaeae from Arrabidaea cf. candicans (Bignoniaceae), Pseudocercospora hymenaeae from Hymenaea courbaril (Caesalpinioideae/Fabaceae), Pseudocercospora solandrae from Solandra sp. (Solanaceae), and Verrucisporota struthanthicola from Struthanthus sp. (Loranthaceae). New reports for Panama are Cercospora glauciana from a new host plant genus (Rhynchospora, Cyperaceae), Pseudocercospora acalyphicola from a new host plant species (Acalypha macrostachya, Euphorbiaceae), Pseudocercospora cecropiae, Pseudocercospora cecropiicola, Pseudocercospora cecropiigena, Pseudocercospora mirandensis, and Ramularia rubella.     

Effects of betaine and condensed molasses solubles on nitrogen balance and nutrient digestibility in piglets fed diets deficient in methionine and low in compatible osmolytes

A balance experiment was carried out to investigate the effects of betaine monohydrate (BET) or betaine derived from condensed molasses solubles (CMS) as a substitute for methionine and choline on nitrogen (N) balance and total tract nutrient digestibility in weaned piglets. The experiment included four treatments with 32 barrows with an average initial body weight (BW) of 13.5 kg. The supplementation of DL-methionine and choline (positive control = PC) to the basal diet, which was deficient in methionine and low in compatible osmolytes in the form of betaine or its precursor choline (negative control = NC) resulted in a significant increase in N retention of 0.8 g/d. The substitution of DL-methionine and choline with BET or CMS did not affect N retention compared to the PC and the NC treatment either. Feeding the PC diet increased the digestibilities of organic matter, NDF, ADF, NFE, crude ash, Ca, P, methionine, tryptophan and cystine by 1.9%, 7.3%, 9.7%, 1.1%, 6.3%, 13.9%, 7.7%, 15.9%, 4.3% and 2.8%, respectively, and tended (p < 0.20) to increase the digestibilities of most other amino acids by 1.6-3.4%. Digestibility of CP, EE (HCl), Mg and Na was 3.1% (p=0.09), 5.1% (p=0.09), 5.1% (p= 0.06) and 3.3% (p= 0.17) higher, respectively, when compared to the NC treatment. BET and CMS supplementation increased most nutrient digestibilities in the same magnitude as for the PC treatment. In summary, the supplementation of betaine, originating from different sources, to a diet with low contents of compatible osmolytes increased in particular the fermentation of fibre and enhanced mineral absorption. The supplementation of the NC with DL-methionine was more efficient in improving N retention than the replacement of DL-methionine by betaine originating from BET or CMS.     

Euglena gracilis rhodoquinone:ubiquinone ratio and mitochondrial proteome differ under aerobic and anaerobic conditions

Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1beta subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1alpha, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP(+) oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.     

Single eubacterial origin of eukaryotic sulfide:quinone oxidoreductase,a mitochondrial enzyme conserved from the early evolution of eukaryotes during anoxic and sulfidic times

Mitochondria occur as aerobic, facultatively anaerobic, and, in the case of hydrogenosomes, strictly anaerobic forms. This physiological diversity of mitochondrial oxygen requirement is paralleled by that of free-living alpha-proteobacteria, the group of eubacteria from which mitochondria arose, many of which are facultative anaerobes. Although ATP synthesis in mitochondria usually involves the oxidation of reduced carbon compounds, many alpha-proteobacteria and some mitochondria are known to use sulfide (H2S) as an electron donor for the respiratory chain and its associated ATP synthesis. In many eubacteria, the oxidation of sulfide involves the enzyme sulfide:quinone oxidoreductase (SQR). Nuclear-encoded homologs of SQR are found in several eukaryotic genomes. Here we show that eukaryotic SQR genes characterized to date can be traced to a single acquisition from a eubacterial donor in the common ancestor of animals and fungi. Yet, SQR is not a well-conserved protein, and our analyses suggest that the SQR gene has furthermore undergone some lateral transfer among prokaryotes during evolution, leaving the precise eubacterial lineage from which eukaryotes obtained their SQR difficult to discern with phylogenetic methods. Newer geochemical data and microfossil evidence indicate that major phases of early eukaryotic diversification occurred during a period of the Earth's history from 1 to 2 billion years before present in which the subsurface ocean waters contained almost no oxygen but contained high concentrations of sulfide, suggesting that the ability to deal with sulfide was essential for prokaryotes and eukaryotes during that time. Notwithstanding poor resolution in deep SQR phylogeny and lack of a specifically alpha-protebacterial branch for the eukaryotic enzyme on the basis of current lineage sampling, a single eubacterial origin of eukaryotic SQR and the evident need of ancient eukaryotes to deal with sulfide, a process today germane to mitochondrial quinone reduction, are compatible with the view that eukaryotic SQR was an acquisition from the mitochondrial endosymbiont.