Identification of an acetoacetyl coenzyme A synthetase-dependent pathway for utilization of L-(+)-3-hydroxybutyrate in Sinorhizobium meliloti

D-(-)-3-Hydroxybutyrate (DHB), the immediate depolymerization product of the intracellular carbon store poly-3-hydroxybutyrate (PHB), is oxidized by the enzyme 3-hydroxybutyrate dehydrogenase to acetoacetate (AA) in the PHB degradation pathway. Externally supplied DHB can serve as a sole source of carbon and energy to support the growth of Sinorhizobium meliloti. In contrast, wild-type S. meliloti is not able to utilize the L-(+) isomer of 3-hydroxybutyrate (LHB) as a sole source of carbon and energy. In this study, we show that overexpression of the S. meliloti acsA2 gene, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase, confers LHB utilization ability, and this is accompanied by novel LHB-CoA synthetase activity. Kinetics studies with the purified AcsA2 protein confirmed its ability to utilize both AA and LHB as substrates and showed that the affinity of the enzyme for LHB was clearly lower than that for AA. These results thus provide direct evidence for the LHB-CoA synthetase activity of the AcsA2 protein and demonstrate that the LHB utilization pathway in S. meliloti is AcsA2 dependent.     

Characterization of the Escherichia coli CcmH protein reveals new insights into the redox pathway required for cytochrome c maturation

The CcmH protein of Escherichia coli is encoded by the last gene of the ccm gene cluster required for cytochrome c maturation. A mutant in which the entire ccmH gene was deleted failed to synthesize both indigenous and foreign c-type cytochromes. However, deletion of the C-terminal hydrophilic domain homologous to CycH of other gram-negative bacteria affected neither the biogenesis of indigenous c-type cytochromes nor that of the Bradyrhizobium japonicum cytochrome c ₅₅₀. This confirmed that only the N-terminal domain containing a conserved CXXC motif is required in E. coli. PhoA fusion analysis showed that this domain is periplasmic. Site-directed mutagenesis of the cysteines of the CXXC motif revealed that both cysteines are required for cytochrome c maturation during aerobic growth, whereas only the second cysteine is required for cytochrome c maturation during anaerobic growth. The deficiency of the point mutants was complemented when 2-mercapto-ethanesulfonic acid was added to growing cells; other thiol compounds did not stimulate cytochrome c formation in these strains. We propose a model for the reaction sequence in which CcmH keeps the heme binding site of apocytochrome c in a reduced form for subsequent heme ligation. Received: 7 September 1998 / Accepted: 15 November 1998     

Comparing lactate and glycerol as a single-electron donor for sulfate reduction in fluidized bed reactors

Among the greatest challenges to the full implementation of biological sulfate reduction are the cost and availability of the electron source. With the development of the biofuel industry, new organic substrates have become available. Therefore, this work sought to compare the performance of a sulfidogenic process utilizing either lactate or glycerol as the substrate for sulfate-reducing bacteria (SRB) growth. Although sulfate reduction is energetically more favorable with lactate, glycerol is a less expensive alternative because excess production is forecasted with the worldwide development of the biodiesel industry. Continuous experiments were performed in a fluidized bed (FB) reactor containing activated carbon as a carrier for a mixed bacterial population composed of sulfate-reducing and fermentative bacteria. During the lactate-fed phases, incomplete oxidation of lactate to acetate by SRB was the dominant metabolic pathway resulting in as much as 90 % sulfate reduction and high acetate concentrations (2.7 g L^?1). Conversely, in the glycerol-fed phases, glycerol degradation resulted from syntrophic cooperation between sulfate-reducing and fermentative bacteria that produce butyrate along with acetate (1.0 g L^?1) as oxidation products. To our knowledge, this is the first report of butyrate formation during sulfate reduction in a glycerol-fed continuous-flow reactor. Sulfate concentrations were reduced by about 90 % (from 2,000 to 100–300 mg L^?1) when glycerol was being fed to the reactor. Since the FB reactor was able to stand a change from lactate to glycerol, this reactor is recommended as the preferred option should glycerol be selected as a cost-effective alternative to lactate for continuous sulfate reduction.     

Association of Liposome-Encapsulated Trivalent Antimonial with Ascorbic Acid: An Effective and Safe Strategy in the Treatment of Experimental Visceral Leishmaniasis

Background:

Visceral leishmaniasis (VL) is a chronic debilitating disease endemic in tropical and subtropical areas, caused by protozoan parasites of the genus Leishmania. Annually, it is estimated the occurrence of 0.2 to 0.4 million new cases of the disease worldwide. Considering the lack of an effective vaccine the afflicted population must rely on both, an accurate diagnosis and successful treatment to combat the disease. Here we propose to evaluate the efficacy of trivalent antimonial encapsulated in conventional liposomes, in association with ascorbic acid, by monitoring its toxicity and efficacy in BALB/c mice infected with Leishmania infantum.

Methodology/Principal Findings:

Infected mice were subjected to single-dose treatments consisting in the administration of either free or liposome-encapsulated trivalent antimony (SbIII), in association or not with ascorbic acid. Parasite burden was assessed in the liver, spleen and bone marrow using the serial limiting dilution technique. After treatment, tissue alterations were examined by histopathology of liver, heart and kidney and confirmed by serum levels of classic biomarkers. The phenotypic profile of splenocytes was also investigated by flow cytometry. Treatment with liposome-encapsulated SbIII significantly reduced the parasite burden in the liver, spleen and bone marrow. Co-administration of ascorbic acid, with either free SbIII or its liposomal form, did not interfere with its leishmanicidal activity and promoted reduced toxicity particularly to the kidney and liver tissues.

Conclusions/Significance:

Among the evaluated posological regimens treatment of L. infantum-infected mice with liposomal SbIII, in association with ascorbic acid, represented the best alternative as judged by its high leishmanicidal activity and absence of detectable toxic effects. Of particular importance, reduction of parasite burden in the bone marrow attested to the ability of SbIII-carrying liposomes to efficiently reach this body compartment.     

Growth Factor Stimulation Improves the Structure and Properties of Scaffold-Free Engineered Auricular Cartilage Constructs

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.     

A new and aberrant species of Dugesia (Platyhelminthes,Tricladida, Dugesiidae) from Madagascar

In this paper we report a new species of Dugesia of the family Dugesiidae from Madagascar, representing the fourth species of freshwater planarian known from this global biodiversity hotspot. In some respects the new species is aberrant, when compared with its congeners, being characterized by a head with smoothly rounded auricles, a peculiar course of the oviducts, including the presence of a common posterior extension, and by the asymmetrical openings of the vasa deferentia at about halfway along the seminal vesicle. Further, it is characterized by a ventral course of the ejaculatory duct with a terminal opening, very long spermiducal vesicles and unstalked cocoons. Its diploid chromosome complement with 18 chromosomes represents an uncommon feature among fissiparous species of Dugesia.     

Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget’s disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.