3-Ketosteroid 9α-hydroxylase is an essential factor in the pathogenesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis H37Rv contains the kshA ( Rv3526 ) and kshB ( Rv3571 ) genes, encoding 3-ketosteroid 9α-hydroxylase (KSH). Consistent with their predicted roles, the Δ kshA and Δ kshB deletion mutants of M. tuberculosis H37Rv were unable to use cholesterol and 4-androstene-3,17-dione as primary carbon and energy sources. Interestingly, Δ kshA and Δ kshB mutants were also unable to metabolize the steroid substrate 5α-androstane-3,17-dione, whereas wild-type M. tuberculosis H37Rv could. The deletion of either of these genes lead to rapid death of the microorganism in murine infection models and in macrophages, showing that kshA and kshB are essential factors for M. tuberculosis pathogenesis. Penta-acylated trehalose (PAT) biosynthesis was altered in the Δ kshB mutant, but not the Δ kshA mutant. The Δ kshB mutant synthesizes all other types of lipids. The Δ kshB mutant had a thickened outer layer in its cell wall. KshB thus appears to be involved in multiple processes, probably as a reductase of different oxygenases. We conclude that an impaired 3-ketosteroid 9α-hydroxylase activity is the cause of the highly attenuated phenotype of our M. tuberculosis H37Rv mutants.     

Cytosolic pyruvate,orthophosphate dikinase functions in nitrogen remobilization during leaf senescence and limits individual seed growth and nitrogen content

The protein content of seeds determines their nutritive value, downstream processing properties and market value. Up to 95% of seed protein is derived from amino acids that are exported to the seed after degradation of existing protein in leaves, but the pathways responsible for this nitrogen metabolism are poorly defined. The enzyme pyruvate,orthophosphate dikinase (PPDK) interconverts pyruvate and phosphoenolpyruvate, and is found in both plastids and the cytosol in plants. PPDK plays a cardinal role in C₄ photosynthesis, but its role in the leaves of C₃ species has remained unclear. We demonstrate that both the cytosolic and chloroplastic isoforms of PPDK are up‐regulated in naturally senescing leaves. Cytosolic PPDK accumulates preferentially in the veins, while chloroplastic PPDK also accumulates in mesophyll cells. Analysis of microarrays and labelling patterns after feeding ¹³C‐labelled pyruvate indicated that PPDK functions in a pathway that generates the transport amino acid glutamine, which is then loaded into the phloem. In Arabidopsis thaliana, over‐expression of PPDK during senescence can significantly accelerate nitrogen remobilization from leaves, and thereby increase rosette growth rate and the weight and nitrogen content of seeds. This indicates an important role for cytosolic PPDK in the leaves of C₃ plants, and allows us to propose a metabolic pathway that is responsible for production of transport amino acids during natural leaf senescence. Given that increased seed size and nitrogen content are desirable agronomic traits, and that efficient remobilization of nitrogen within the plant reduces the demand for fertiliser applications, PPDK and the pathway in which it operates are targets for crop improvement.     

Design of a series of bicyclic HIV-1 integrase inhibitors. Part 1: Selection of the scaffold

HIV integrase inhibitors based on a novel bicyclic pyrimidinone core is presented. Nine variations of the core scaffold are evaluated leading to optimization of the 6:6 core giving compound 48 with an EC₅₀ of 3 nM against wild type HIV infected T-cells.     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.     

Completed Genome Sequence of the Anaerobic Iron-Oxidizing Bacterium Acidovorax ebreus Strain TPSY

Acidovorax ebreus strain TPSY is the first anaerobic nitrate-dependent Fe(II) oxidizer for which there is a completed genome sequence. Preliminary protein annotation revealed an organism optimized for survival in a complex environmental system. Here, we briefly report the completed and annotated genome sequence of strain TPSY.Microorganisms from diverse anoxic environments are capable of nitrate-dependent Fe(II) oxidation at circumneutral pH (4, 11, 17, 18, 20, 21). Despite their geochemical importance (22), little is known of the underlying biochemical and genetic mechanisms. Genome sequencing of several nitrate-dependent Fe(II) oxidizers will provide insight into this process. By comparing Fe(II) oxidation mechanisms in various organisms, we hope to identify both the conserved and disparate aspects of the metabolism. The genome of Acidovorax ebreus strain TPSY is the first of these to be sequenced.Strain TPSY is a motile, Gram-negative facultative anaerobe isolated from groundwater collected from the U.S. Department of Energy Integrated Field Research Challenge site at Oak Ridge, TN. Growth experiments performed as previously described (21) revealed TPSY''s incapacity for lithoautotrophic growth, which was supported by a lack of genes in the genome encoding any known CO₂ fixation pathways. TPSY did grow mixotrophically with Fe(II) as the electron donor and a 0.1 mM acetate carbon source. 16S rRNA gene sequence analysis placed TPSY in the class Betaproteobacteria with 99.8% similarity to Acidovorax sp. strain JS42 in the family Comamonadaceae.The completed genome consisted of a single circular chromosome of 3,796,573 bp with an average 66.8% G+C content. A total of 3,479 protein-encoding genes were predicted, and 34 (0.98%) had no similarity to public database sequences. Sequencing performed at the Department of Energy Joint Genome Institute (JGI) used Sanger sequencing and 454 pyrosequencing to a depth of 20× coverage. All JGI library construction and sequencing techniques can be found at http://www.jgi.doe.gov/. Sequence assembly, quality assessment, and annotation were performed using the software Phred/Phrap/Consed (www.phrap.com) (6-8), Dupfinisher (10), CRITICA (2), GLIMMER, and GENERATION (5) and the JGI Integrated Microbial Genomes site (12). The completed genome sequence contained 33,341 reads and had an average of ninefold coverage per base and an error rate of <1 in 100,000.TPSY was named in part for its meandering motility, and its genome confirmed the twitching phenotype with the presence of pilT, pilU, and a complete set of flagellar and chemotaxis genes. The ability of TPSY to oxidize simple alcohols and acids with oxygen or nitrate respiration was confirmed by the genome. In addition, biosynthetic pathways for all amino acids except tyrosine and phenylalanine were present. No homologues of chorismate mutase (EC 5.4.99.5), an enzyme required for tyrosine and phenylalanine anabolism, were identified. The genome contained both intact Embden-Meyerhof-Parnas and Entner-Doudoroff pathways, in addition to a pentose phosphate pathway and a trichloroacetic acid cycle.In support of its facultative anaerobicity, a complete set of genes for denitrification and three different terminal oxidases (cytochrome aa₃, cbb₃, and cytochrome d quinol oxidase) were present. The cbb₃ and cytochrome d oxidases, with their high oxygen affinity, putatively enable survival in microaerobic environments (14).TPSY had sequences encoding 30 transposases, 11 integrases, and 11 phage/prophage-related genes. A region of particular interest putatively conferred resistance to lead, arsenate, and mercury: pbrRATARTBC, arsRDAB, and merRPCADE. Evidence suggests horizontal transfer and insertion of this region, as it was flanked on the 5′ end by λ prophage-related genes and the 3′ end encoded a putative Tn21 transposase. Phenotypic studies by the method of Wang et al. (19) revealed MICs of 16 μM phenylmercuric acetate and 250 μM MgCl₂. TPSY was also capable of growth in the presence of arsenate (10 mM) but did not use it as an electron acceptor.Related to phage infection, one CRISPR (clustered, regularly interspaced, short palindromic repeats) region (3, 16) was predicted. The core proteins, the cas1 and cas2 genes, and a csn1 gene formed the CRISPR subtype Nmeni, which is associated with vertebrate pathogens and commensals (9). However, the lack of typical pathogenic type I or III secretion systems such as the hec cluster of Dickeya chrysanthemi (15) or the inv/spa system of Salmonella enterica serovar Typhimurium (13) indicated that TPSY would probably not exhibit a pathogenic lifestyle.     

Description of the novel perchlorate-reducing bacteria Dechlorobacter hydrogenophilus gen. nov., sp. nov. and Propionivibrio militaris, sp. nov.

Novel dissimilatory perchlorate-reducing bacteria (DPRB) were isolated from enrichments conducted under conditions different from those of all previously described DPRB. Strain LT-1^T was enriched using medium buffered at pH 6.6 with 2-(N-morpholino)ethanesulfonic acid (MES) and had only 95% 16S rRNA gene identity with its closest relative, Azonexus caeni. Strain MP^T was enriched in the cathodic chamber of a perchlorate-reducing bioelectrical reactor (BER) and together with an additional strain, CR (99% 16S rRNA gene identity), had 97% 16S rRNA gene identity with Propionivibrio limicola. The use of perchlorate and other electron acceptors distinguished strains MP^T and CR from P. limicola physiologically. Strain LT-1^T had differences in electron donor utilization and optimum growth temperatures from A. caeni. Strains LT-1^T and MP^T are the first DPRB to be described in the Betaproteobacteria outside of the Dechloromonas and Azospira genera. On the basis of phylogenetic and physiological features, strain LT-1^T represents a novel genus in the Rhodocyclaceae; strain MP^T represents a novel species within the genus Propionivibrio. The names Dechlorobacter hydrogenophilus gen. nov., sp. nov and Propionivibrio militaris sp. nov. are proposed.     

Differential Cleavage of Provasopressin by the Major Molecular Forms of SPC3

Abstract: We have investigated the roles of full-length and carboxyl-terminus-truncated forms of the subtilisin-like prohormone convertase SPC3 in the processing of the radiolabeled vasopressin and oxytocin precursors, in vitro. We found SPC3 cleaves provasopressin at both the vasopressin-neurophysin and neurophysin-glycopeptide processing sites. Prooxytocin is cleaved by SPC3 at the oxytocin-neurophysin cleavage site. However, our results reveal differences in processing of provasopressin by the different molecular forms of SPC3. In incubations where the rate of autocatalytic carboxyl-terminus truncation of SPC3 was dramatically reduced, 86-kDa SPC3, which has an unprocessed carboxyl terminus, cleaved provasopressin at the neurophysin-glycopeptide junction. Cleavage at the vasopressin-neurophysin junction only occurred with the appearance of carboxyl-terminus-truncated forms of the enzyme. Incubations containing 64-kDa SPC3 or 64-kDa SPC3-T, a recombinant form of SPC3 truncated 14 amino acids beyond the conserved carboxyl-terminal "P-domain," rapidly cleaved provasopressin at both the vasopressin-neurophysin and neurophysin-glycopeptide junctions. Our results also suggest that prooxytocin is unable to be cleaved by the 86-kDa form of SPC3. We propose that SPC3 should be considered as a candidate endoprotease in the biosynthesis of vasopressin. Furthermore, we suggest that the carboxyl terminus of SPC3 alters the cleavage specificity of SPC3.     

The habitat hectares approach to vegetation assessment: An evaluation and suggestions for improvement

Summary The habitat hectares approach is an explicit, quantitative method for assessing the quality of vegetation by adding scores that are assigned to 10 habitat attributes. We believe it will be more repeatable and transparent than other methods that rely on subjective judgement. However, we have four principal criticisms of the method as it is currently proposed: (i) measurement of some of the attributes may be subject to considerable error that varies among assessors; (ii) the comparison of each measure with a single benchmark does not accommodate appropriate disturbance regimes; (iii) the proposed combination of attributes leads to some apparent internal inconsistencies; and (iv) it is not clear how the method will actually be used in practice. We suggest modifications to address these concerns and improve the proposed method. Finally, we make additional suggestions about the method's potential application, including: separate reporting of the extent and quality of different vegetation types to avoid the inappropriate combination of measures of area and quality; valuing appropriate disturbance regimes in natural areas; and considering very carefully the application of compensation or mitigation measures.     

Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.