Anaerocolumna sedimenticola sp. nov., isolated from fresh water sediment

Strain CBA3638^T was isolated from the Geum River sediment, Republic of Korea. The cells of strain CBA3638^T were Gram-stain-positive, strictly anaerobic, rod-shaped, and 0.5–1.0 μm wide, and 4.0–4.5 μm long. Optimal growth occurred at 37 °C, pH 7.0, and 1.0% (w/v) NaCl. Based on the 16S rRNA gene sequence, the phylogenetic analysis showed that strain CBA3638^T belongs to the genus Anaerocolumna in the family Lachnospiraceae, and is most closely related to Anaerocolumna cellulosilytica (94.6–95.0%). The DDH value with A. cellulosilytica SN021^T showed 15.0% relatedness. The genome of strain CBA3638^T consisted of one circular chromosome that is 5,500,435 bp long with a 36.7 mol% G?+?C content. The genome contained seven 16S-5S-23S rRNA operons and one antibiotic resistance-related transporter gene (mefA). Quinones were not detected. The predominant cellular fatty acids were C_16:0 and C_14:0 and the polar lipids were diphosphatidylglycerol, phosphatidylcholine, and uncharacterised polar lipids. Based on the polyphasic taxonomic analysis, we propose strain CBA3638^T as a novel species in the genus Anaerocolumna, with the name Anaerocolumna sedimenticola sp. nov. The type strain is CBA3638^T (=?KACC 21652^T?=?DSM 110663^T).

     

Application of Biofilm-Forming Bacteria on the Enhancement of Organophosphorus Fungicide Degradation

This study aimed to develop technology enhancing the biodegradation efficacy against organophosphorus fungicide with biofilm-forming bacteria in situ. Using the crystal violet staining method, two bacterial strains having biofilm formation capability were isolated and identified as Pseudomonas sp. C7 and Bacillus sp. E5. Compared with the culture of tolclofos-methyl degrader Sphingomonas sp. 224, biofilm formation was improved by co-inoculation with biofilm-forming bacterium Bacillus sp. E5. Evaluated in liquid culture conditions, this two-species mixed consortium was observed to degrade tolclofos-methyl more effectively than Sphingomonas sp. 224 alone, with an approximately 90% degradation efficiency within 48 h of dosing. The improved effectiveness of the consortium biofilm was reflected using soil in situ with an approximately 7% increased degradation ratio over Sphingomonas sp. 224 alone. This is the first report demonstrating improved bioremediation degradation efficacy against tolclofos-methyl exhibited by a consortium biofilm. This work presents a possible effective bioremediation strategy using a specific biofilm composition against pollutants containing organophosphorus compounds in situ.     

Sunxiuqinia dokdonensis sp. nov., isolated from deep sub-seafloor sediment

A novel facultatively anaerobic strain DH1^T was isolated from deep sub-seafloor sediment at a depth of 900 m below the seafloor off Seo-do (the west part of Dokdo Island) in the East Sea of the Republic of Korea. The new strain was characterized using polyphasic approaches. The isolate was Gram-stain-negative, motile by gliding, non-spore-forming rods, oxidase-negative, and catalase-positive; and formed colonies of orange-red color. The NaCl range for growth was 0.5–7.0% (w/v) and no growth was observed in the absence of NaCl. The isolate grew optimally at 30°C, with 2% (w/v) NaCl and at pH 7. The cell-wall hydrolysates contained ribose as a major sugar. The DNA G+C content was 40.8 mol%. The closest related strains are Sunxiuqinia faeciviva JAM-BA0302^T and Sunxiuqinia elliptica DQHS-4^T (97.9 and 96.3% sequence similarity, respectively). The level of DNA-DNA relatedness between strain DH1^T and S. faeciviva JAM-BA0302^T was around 41% (but only 6% between DH1T and S. elliptica DQHS-4^T). The major cellular fatty acids of the isolate were contained iso-C_15:0 (25.9%), anteiso-C_15:0 (16.7%), and summed feature 9 (comprising C_16:0 3-OH and/or unknown fatty acid of dimethylacetal ECL 17.157; 13.2%). The predominant menaquinone was MK-7. On the basis of polyphasic evidence from this study, the isolate was considered to represent a novel species of the genus Sunxiuqinia, for which the name Sunxiuqinia dokdonensis sp. nov. is proposed; the type strain is DH1^T (=KCTC 32503^T =CGMCC 1.12676^T =JCM 19380^T).     

Minding the gaps: metabolomics mends functional genomics

Two recent studies in PNAS and Nat Chem Biol highlight the power of modern mass-spectrometry techniques for enzyme discovery applied to microbiology. In so doing, they have uncovered new potential targets for the treatment of tuberculosis.Proc Natl Acad Sci USA (2013) 110 28, 11320–11325 doi: 10.1073/pnas.1221597110Nat Chem Biol (2013). doi:10.1038/nchembio.1355. Advance online publication 29 September 2013Many have come to regard metabolism as a well-understood housekeeping activity of all cells, functionally compartmentalized away from other biological processes. However, growing reports of unexpected links between a diverse range of disease states and specific metabolic enzymes or pathways have begun to challenge this view. In doing so, such discoveries have exposed more glaring, and neglected, deficiencies in our understanding of cellular metabolism, triggering a broad resurgence of interest in metabolism.“Metabolomics […] offers a global window into the biochemical state of a cell or organism…”Metabolomics is the newest of the systems-level disciplines and seeks to reveal the physiological state of a given cell or organism through the global and unbiased study of its small-molecule metabolites [1]. Metabolites are the final products of enzymes and enzyme networks, the substrates and products of which often cannot be deduced from genetic information and the levels of which reflect the integrated product of the genome, proteome and environment [2]. Metabolomics thus offers a global window into the biochemical state of a cell or organism, made experimentally possible by the unprecedented discriminatory power and sensitivity of modern mass-spectrometry-based technologies (Fig 1). Two recent reports from the Carvalho and Neyrolles groups, published recently in Proceedings of the National Academy of Science USA and Nature Chemical Biology [3,4], exemplify the rapidly growing impact of metabolomics-based approaches on tuberculosis research.Open in a separate windowFigure 1Modern mass spectrometry illuminates bacterial metabolism. A comparison of activity-based metabolomic profiling with classic metabolic tracing. See the text for details.Within the field of infectious diseases, the deficiencies in our understanding of microbial metabolism have emerged most prominently in the area of tuberculosis research. Despite the development of the first chemotherapies more than 50 years ago, tuberculosis remains the leading bacterial cause of death worldwide, due in part to a failure to keep pace with the emergence of drug resistance [5]. The causes of this shortfall are multifactorial. However, a key contributing factor is our incomplete understanding of the metabolic properties of Mycobacterium tuberculosis (Mtb), its aetiological agent. Unlike most bacterial pathogens, Mtb infects humans as its only known host and reservoir, within whom it resides largely isolated from other microbes. Mtb has thus evolved its metabolism to serve interdependent physiological and pathogenic roles. Yet, more than a century after Koch''s initial discovery of Mtb and 15 years after the first publication of its genome sequence, knowledge of Mtb''s metabolic network remains surprisingly incomplete [6,7,8].“…tuberculosis remains the leading bacterial cause of death worldwide…”As for almost all sequenced microbial genomes, homology-based in silico approaches have failed to suggest a function for nearly 40% of Mtb genes that, presumably, include a significant number of orphan enzyme activities for which no gene has been ascribed [8]. Such approaches have further neglected the impact of evolutionary selection and its ability to dissociate sequence conservation from biochemical activity and physiological function, in order to help optimize the fitness of a given organism within its specific niche. For Mtb, such genes and enzymes represent an especially promising and biologically selective, but untapped, source of potential drug targets.In the study from the Carvalho group, successful application of a recently developed metabolomics assay—known as activity-based metabolomic profiling (ABMP)—allowed the authors to reassign a putatively annotated nucleotide phosphatase (Rv1692) as a D,L-glycerol 3-phosphate phosphatase [3,9]. ABMP was specifically developed to identify enzymatic activities for genes of unknown function by leveraging the analytical discriminatory power of liquid-chromatography-coupled high-resolution mass spectrometry (LC-MS) to analyse the impact of a recombinant enzyme and potential co-factors on a highly concentrated, small-molecule extract derived from the homologous organism (Fig 1). By monitoring for the matched time and enzyme-dependent depletion and accumulation of putative substrates and products, this assay enables the discovery of catalytic activities—rather than simple binding—by using the cellular metabolome as arguably the most physiological chemical library of potential substrates that can be tested, in a label and synthesis-free manner. Moreover, candidate activities assigned by this method can be confirmed by using independent biochemical approaches—such as reconstitution with purified components—and genetic techniques—such as wild-type and genetic knockout, knockdown or overexpression strains. In reassigning Rv1692 as a glycerol phosphate phosphatase, rather than a nucleotide phosphatase, Carvalho and colleagues demonstrate the potential of ABMP to overcome the biochemical challenge of assigning substrate specificity to a member of a large enzyme superfamily—in this case, the haloacid dehydrogenase superfamily. But, perhaps more significantly, they also direct new biological attention to the largely neglected area of Mtb membrane homeostasis, in which Rv1692 might play an important role in glycerophospholipid recycling and catabolism.“…knowledge of Mtb''s metabolic network remains surprisingly incomplete”Neyrolles and colleagues make use of the same metabolomics platform to perform metabolite-tracing studies by using stable-isotope-labelled precursors, which led them to reassign a putatively annotated asparagine transporter (AnsP1) as an aspartate transporter. AnsP1 bears 55% sequence identity and 70% similarity to an orthologue in Salmonella that belongs to the amino acid transporter family 2.A.3.1, whereas aspartate transporters are typically members of the dicarboxylate amino acid:cation symporter family 2.A.23 [4]. This study demonstrates the ability of metabolomic platforms to not only characterize the activity of a given protein within its natural physiological milieu, but also revive classical experimental methods by using modern technologies. The availability of stable (non-radioactive) isotopically labelled precursors has now made it possible to resolve their specific metabolic fates. In this case, such an approach revealed that Mtb can use aspartate as both a carbon and nitrogen source, after its uptake through AnsP1. Looking beyond the specific biochemical assignment of AnsP1 as an aspartate—rather than asparagine—transporter, this study illustrates the potential impact of such discoveries on downstream paths of investigation. In this case, the remarkable application of high-resolution dynamic secondary ion mass spectroscopy to provide the first direct biochemical images of the nutritional environment of the Mtb-infected phagosome.New technologies are often developed in the context of specific needs. However, their impact is usually not realized until extended beyond such contexts, sometimes resulting in major paradigm shifts. The above examples highlight just two emerging possibilities of how metabolomics technologies can be extended beyond the context of global comparisons and provide unique biological insights. To the extent that the analytical power of these platforms can be adapted to other functional approaches, metabolomics promises to pay handsome biochemical and physiological dividends.     

Synthesis and Antiviral Activities of 1,3-Oxathiolanyl Nucleosides with 5-Hydroxymethyl Substituent

Abstract

Novel 1,3-oxathiolanyl pyrimidine nucleosides with 5-hydroxymethyl substituent were synthesized starting from _d-mannose and evaluated for antiviral activities against HIV-1, HSV type 1,2 and HCMV.     

Design and Synthesis of Dually Branched 5′-Norcarbocyclic Adenosine Phosphonodiester Analogue as a New Anti-HIV Prodrug

A novel 3′,4′-dimethyl-5′-norcarbocyclic adenosine phosphonic acid was prepared using acyclic stereoselective route from 4-hydroxybutan-2-one (4). To improve the cellular permeability and enhance the anti-HIV activity of this phosphonic acid, a (bis)SATE phosphonodiester nucleoside prodrug (20) was prepared and its chemical stability was evaluated. The newly synthesized bis(SATE) analogue (20) and its parent nucleoside phosphonic acid (18) were assayed for anti-HIV activity using an in vitro assay system in a CEM cell line.     

Design and Synthesis of Novel Threosyl-5′- Deoxyphosphonic Acid Purine Analogues as Potent Anti-Hiv Agents

The discovery of threosyl phosphonate nucleoside (PMDTA, EC₅₀ = 2.53 μM) as a potent anti-HIV agent has led to the synthesis and biological evaluation of 5 ′-deoxyversions of threosyl phosphonate nucleosides from 1,4-dihydroxy-2-butene. The synthesized nucleoside phosphonic acid analogues 14 and 19 were tested for anti-HIV activity as well as cytotoxicity. The adenine analogue 14 exhibits moderate in vitro anti-HIV-1 activity (EC₅₀ = 12.6 μM).     

Synthesis and Anti-Hiv Activity of 4′-Modified Cyclopentenyl Pyrimidine C-Nucleosides

Novel syntheses of 4′-modified cyclopentenyl pyrimidine C-nucleosides were performed via C-C bond formation using S_N2 alkylation via the key intermediate mesylates 6 and 16, which were prepared from acyclic ketone derivatives. When antiviral evaluation of synthesized compound was performed against various viruses such as HIV-1, HSV-1 and HSV-2, isocytidine analogue 20 showed moderate anti-HIV activity in CEM cell line (EC₅₀ = 13.1 μmol).7