Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast

The Schizosaccharomyces pombe pfh1⁺ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1⁺ gene suppress a temperature-sensitive allele of cdc24⁺, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism. We show that a full-length Pfh1 fusion protein is active as a monomer. The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate. The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1⁺ abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38. Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities. These results indicate that the simultaneous loss-of-function mutations of pfh1⁺ and dna2⁺ (or cdc24⁺) are essential to restore the growth defect. Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol δ-mediated lagging strand displacement DNA synthesis.     

Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

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).     

Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression

Proteins associated with the centrosome play key roles in mitotic progression in mammalian cells. The activity of Cdk1-opposing phosphatases at the centrosome must be inhibited during early mitosis to prevent premature dephosphorylation of Cdh1—an activator of the ubiquitin ligase anaphase-promoting complex/cyclosome—and the consequent premature degradation of mitotic activators. In this paper, we show that reversible oxidative inactivation of centrosome-bound protein phosphatases such as Cdc14B by H₂O₂ is likely responsible for this inhibition. The intracellular concentration of H₂O₂ increases as the cell cycle progresses. Whereas the centrosome is shielded from H₂O₂ through its association with the H₂O₂-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H₂O₂ and facilitating inactivation of centrosome-bound phosphatases. Dephosphorylation of PrxI by okadaic acid–sensitive phosphatases during late mitosis again shields the centrosome from H₂O₂ and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle.     

Genomic analysis of facultatively oligotrophic haloarchaea of the genera Halarchaeum,Halorubrum, and Halolamina,isolated from solar salt

Archives of Microbiology - Extremely halophilic archaea (haloarchaea) belonging to the phylum Euryarchaeota have been found in high-salinity environments. In this study, Halarchaeum sp. CBA1220,...     

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.     

Soluble Guanylate Cyclase α1–Deficient Mice: A Novel Murine Model for Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α₁ subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α₁–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α₁ and β₁ subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.