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.     

Anti-apoptotic mechanism of silkworm hemolymph in HeLa cell apoptosis

Silkworm hemolymph (SH) was found to exhibit anti-apoptotic activities in mammalian and insect cell systems. An anti-apoptotic mechanism of SH was investigated in a staurosporine-induced HeLa cell using flow cytometry, caspase assay, Immunoblot, and Immunochemistry. The addition of 5% SH to the medium resulted in lower intracellular activities of caspase-3 and caspase-9 after 0.6 μM of staurosporine treatment; however, SH did not directly inhibit the activities of those enzymes. This suggests SH inhibits the event upstream of these caspase activation steps, such as mitochondrial level events. We found from Immunoblot and Immunochemistry that cytochrome c release from the mitochondria was blocked by SH. SH also inhibited Bax translocation to the mitochondria. On the contrary, SH did not block the apoptosis when Bax is not involved in promoting apoptosis. With these results, we propose that SH protects mitochondria from apoptosis signal via blocking Bax translocation, and the subsequent apoptotic events are then inhibited. The inhibition of apoptosis using SH and its components may lead to new approaches for the minimization of cell death during commercial animal cell cultures.     

Classical Raman spectroscopic studies of NADH and NAD+ bound to liver alcohol dehydrogenase by difference techniques

We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a "small" molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitrogen and carbon fixation byAnabaena sp. isolated from a rice paddy and grown under P and light limitations

Anabaena sp., isolated from a rice paddy, was investigated for its nitrogen fixation as measured by acetylene reduction activity (ARA) in P-limited continuous and light-limited semi-continuous cultures. Growth rate (μ) under P limitation was a function of cell P content (q _p). Both the photosynthetic capacity (P_max) and photosynthetic efficiency (α) increased with μ when expressed per cell, but not per unit chla. The ARA of steady-state cells under P limitation increased with μ and was linearly related to C-fixation rate. This was apparently a consequence of the control of C-fixation by P limitation. In light-limited cells, steady state ARA, both at the culture light intensity and in the dark, increased asymptotically with μ, but the activity in the dark was only about 51% of that in the light. When the light level of steady-state cells grown at a high in intensity was switched to a low level, ARA decreased exponentially with time. Dark ARA activity also showed a similar decline, but at much lower levels. Thus, ARA depended not only on light history, but also immediate photosynthesis. Steady-state ARA at the ambient intensity or in the dark showed a strong correlation with¹⁴C-fixation rate. ARA of light-limited cells showed the same light-saturation characteristics as their¹⁴C-fixation, with the same initial saturation intensity,I _k. The ratios of P_max to the maximum ARA (ARA_max), and α to the slope of ARA (α_ara) were identical. A comparison of gross to net photosynthesis and N₂ fixation suggested that there was little leakage or excretion of fixed C or N.     

Nutritional regulation of hepatic heme biosynthesis and porphyria through PGC-1alpha

Inducible hepatic porphyrias are inherited genetic disorders of enzymes of heme biosynthesis. The main clinical manifestations are acute attacks of neuropsychiatric symptoms frequently precipitated by drugs, hormones, or fasting, associated with increased urinary excretion of delta-aminolevulinic acid (ALA). Acute attacks are treated by heme infusion and glucose administration, but the mechanisms underlying the precipitating effects of fasting and the beneficial effects of glucose are unknown. We show that the rate-limiting enzyme in hepatic heme biosynthesis, 5-aminolevulinate synthase (ALAS-1), is regulated by the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). Elevation of PGC-1alpha in mice via adenoviral vectors increases the levels of heme precursors in vivo as observed in acute attacks. The induction of ALAS-1 by fasting is lost in liver-specific PGC-1alpha knockout animals, as is the ability of porphyrogenic drugs to dysregulate heme biosynthesis. These data show that PGC-1alpha links nutritional status to heme biosynthesis and acute hepatic porphyria.     

Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays

To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50 degrees C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was approximately 5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 x 10(7) cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r(2) = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r(2) = 0.74). In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.     

The pleckstrin homology domain of phosphoinositide-specific phospholipase Cdelta4 is not a critical determinant of the membrane localization of the enzyme

The inositol lipid and phosphate binding properties and the cellular localization of phospholipase Cdelta(4) (PLCdelta(4)) and its isolated pleckstrin homology (PH) domain were analyzed in comparison with the similar features of the PLCdelta(1) protein. The isolated PH domains of both proteins showed plasma membrane localization when expressed in the form of a green fluorescent protein fusion construct in various cells, although a significantly lower proportion of the PLCdelta(4) PH domain was membrane-bound than in the case of PLCdelta(1)PH-GFP. Both PH domains selectively recognized phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), but a lower binding of PLCdelta(4)PH to lipid vesicles containing PI(4,5)P(2) was observed. Also, higher concentrations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) were required to displace the PLCdelta(4)PH from the lipid vesicles, and a lower Ins(1,4,5)P(3) affinity of PLCdelta(4)PH was found in direct Ins(1,4,5)P(3) binding assays. In sharp contrast to the localization of its PH domain, the full-length PLCdelta(4) protein localized primarily to intracellular membranes mostly to the endoplasmic reticulum (ER). This ER localization was in striking contrast to the well documented PH domain-dependent plasma membrane localization of PLCdelta(1). A truncated PLCdelta(4) protein lacking the entire PH domain still showed the same ER localization as the full-length protein, indicating that the PH domain is not a critical determinant of the localization of this protein. Most important, the full-length PLCdelta(4) enzyme still showed binding to PI(4,5)P(2)-containing micelles, but Ins(1,4,5)P(3) was significantly less potent in displacing the enzyme from the lipid than with the PLCdelta(1) protein. These data suggest that although structurally related, PLCdelta(1) and PLCdelta(4) are probably differentially regulated in distinct cellular compartments by PI(4,5)P(2) and that the PH domain of PLCdelta(4) does not act as a localization signal.