‘Candidatus Dichloromethanomonas elyunquensis’ gen. nov., sp. nov., a dichloromethane-degrading anaerobe of the Peptococcaceae family

Taxonomic assignments of anaerobic dichloromethane (DCM)-degrading bacteria remain poorly constrained but are important for understanding the microbial diversity of organisms contributing to DCM turnover in environmental systems. We describe the taxonomic classification of a novel DCM degrader in consortium RM obtained from pristine Rio Mameyes sediment. Phylogenetic analysis of full-length 16S rRNA gene sequences demonstrated that the DCM degrader was most closely related to members of the genera Dehalobacter and Syntrophobotulus, but sequence similarities did not exceed 94% and 93%, respectively. Genome-aggregate average amino acid identities against Peptococcaceae members did not exceed 66%, suggesting that the DCM degrader does not affiliate with any described genus. Phylogenetic analysis of conserved single-copy functional genes supported that the DCM degrader represents a novel clade. Growth strictly depended on the presence of DCM, which was consumed at a rate of 160 ± 3 μmol L^?1 d^?1. The DCM degrader attained 5.25 × 10⁷ ± 1.0 × 10⁷ cells per μmol DCM consumed. Fluorescence in situ hybridization revealed rod-shaped cells 4 ± 0.8 μm long and 0.4 ± 0.1 μm wide. Based on the unique phylogenetic, genomic, and physiological characteristics, we propose that the DCM degrader represents a new genus and species, ‘Candidatus Dichloromethanomonas elyunquensis’.     

The chondroitin polymerase K4CP and the molecular mechanism of selective bindings of donor substrates to two active sites

Bacterial chondroitin polymerase K4CP is a multifunctional enzyme with two active sites. K4CP catalyzes alternative transfers of glucoronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to elongate a chain consisting of the repeated disaccharide sequence GlcAbeta1-3GalNAcbeta1-4. Unlike the polymerization reactions of DNA and RNA and polypeptide synthesis, which depend upon templates, the monosaccharide polymerization by K4CP does not. To investigate the catalytic mechanism of this reaction, we have used isothermal titration calorimetry to determine the binding of the donor substrates UDP-GlcA and UDP-GalNAc to purified K4CP protein and its mutants. Only one donor molecule bound to one molecule of K4CP at a time. UDP-GlcA bound only to the C-terminal active site at a high affinity (K(d)=6.81 microm), thus initiating the polymerization reaction. UDP-GalNAc could bind to either the N-terminal or C-terminal active sites at a low affinity (K(d)=266-283 microm) but not to both sites at the same time. The binding affinity of UDP-GalNAc to a K4CP N-terminal fragment (residues 58-357) was profoundly decreased, yielding the average K(d) value of 23.77 microm, closer to the previously reported K(m) value for the UDP-GalNAc transfer reaction that takes place at the N-terminal active site. Thus, the first step of the reaction appears to be the binding of UDP-GlcA to the C-terminal active site, whereas the second step involves the C-terminal region of the K4CP molecule regulating the binding of UDP-GalNAc to only the N-terminal active site. Alternation of these two specific bindings advances the polymerization reaction by K4CP.     

Characterization of HTT Inclusion Size,Location, and Timing in the zQ175 Mouse Model of Huntington′s Disease: An In Vivo High-Content Imaging Study

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2–12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.     

Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis.     

Properties of phosphoribulokinase from Thiobacillus neapolitanus

Partially purified preparations of ribulose-5-phosphate kinase (specific activity, 50 to 125 mumoles per min per mg of protein) were employed in a series of kinetic experiments in the presence of several concentrations of H(+), Mg(2+), adenosine triphosphate (ATP), and phosphoenolpyruvate (PEP). The pH optimum of the enzyme was found to be 7.9; at this pH and above, response of the enzyme to variations in ATP concentration was hyperbolic, exhibiting a K(m) of 7 x 10(-4)m ATP. At pH values below the optimum the response to ATP was sigmoidal, as it was throughout the entire pH range in the presence of PEP at a concentration greater than 5 x 10(-4)m. In the presence of PEP the pH optimum shifted to pH 8.4. In contrast, phosphoribulokinase from spinach exhibited hyperbolic responses throughout its pH range with no inhibition caused by PEP. Thiobacillus neapolitanus phosphoribulokinase was inhibited by PEP in a sigmoidal manner; however, in the presence of suboptimal concentrations of Mg(2+) the addition of PEP caused significant stimulation of activity. It is postulated that the enzyme consists of interacting subunits with several sites on the enzyme for binding ATP and with several separate sites binding PEP. It is suggested that PEP functions as a regulator of CO(2) fixation when the organism is under conditions of unlimited concentrations of substrate and CO(2).     

Rhodospirillum sodomense,sp. nov., a Dead Sea rhodospirillum species

A new species of halophilic anoxygenic purple bacteria of the genus Rhodospirillum is described. The new organism, isolated from water/sediment of the Dead Sea, was vibrio-shaped and an obligate halophile. Growth was best at 12% NaCl, with only weak growth occurring at 6% or 21% NaCl. Growth occurred at Mg²⁺ concentrations up to 1 M but optimal growth was obtained at 0.05–0.1 M Mg²⁺. Bromide was well tolerated as an alternative anion to chloride. The new organism is an obligate phototroph, growing photoheterotrophically in media containing yeast extract and acetate or a few other organic compounds. Growth of the Dead Sea Rhodospirillum species under optimal culture conditions was slow (minimum t_d 20 h). Cells contained bacteriochlorophyll a and carotenoids of the spirilloxanthin series and mass cultures were pink in color. Absorption spectra revealed the presence of a B875 (light-harvesting I) but no B800/B850 (light-harvesting II) photopigment complex. The new organism shares a number of properties with the previously described halophilic phototrophic bacterium Rhodospirillum salinarum and was shown to be related to this phototroph by 16S rRNA sequencing. However, because of its salinity requirements, photosynthetic properties, and isolation from the Dead Sea, the new phototroph is proposed as a new species of the genus Rhodospirillum, R. sodomense.     

A highly specialized flavin mononucleotide riboswitch responds differently to similar ligands and confers roseoflavin resistance to Streptomyces davawensis

Streptomyces davawensis is the only organism known to synthesize the antibiotic roseoflavin, a riboflavin (vitamin B(2)) analog. Roseoflavin is converted to roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide in the cytoplasm of target cells. (Ribo-)Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for the biosynthesis and transport of riboflavin. Streptomyces davawensis is roseoflavin resistant, and the closely related bacterium Streptomyces coelicolor is roseoflavin sensitive. The two bacteria served as models to investigate roseoflavin resistance of S. davawensis and to analyze the mode of action of roseoflavin in S. coelicolor. Our experiments demonstrate that the ribB FMN riboswitch of S. davawensis (in contrast to the corresponding riboswitch of S. coelicolor) is able to discriminate between the two very similar flavins FMN and RoFMN and shows opposite responses to the latter ligands.     

Plasma glycoprotein profiling for colorectal cancer biomarker identification by lectin glycoarray and lectin blot

Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient; hence, the need for simple blood tests that could be used for the early detection of CRC. In this work, we have developed methods for glycoproteomics analysis to identify plasma markers with utility to assist in the detection of colorectal cancer (CRC). Following immunodepletion of the most abundant plasma proteins, the plasma N -linked glycoproteins were enriched using lectin affinity chromatography and subsequently further separated by nonporous silica reversed-phase (NPS-RP)-HPLC. Individual RP-HPLC fractions were printed on nitrocellulose coated slides which were then probed with lectins to determine glycan patterns in plasma samples from 9 normal, 5 adenoma, and 6 colorectal cancer patients. Statistical tools, including principal component analysis, hierarchical clustering, and Z-statistics analysis, were employed to identify distinctive glycosylation patterns. Patients diagnosed with colorectal cancer or adenomas were shown to have dramatically higher levels of sialylation and fucosylation as compared to normal controls. Plasma glycoproteins with aberrant glycosylation were identified by nano-LC-MS/MS, while a lectin blotting methodology was used to validate proteins with significantly altered glycosylation as a function of cancer progression. The potential markers identified in this study for diagnosis to distinguish colorectal cancer from adenoma and normal include elevated sialylation and fucosylation in complement C3, histidine-rich glycoprotein, and kininogen-1. These potential markers of colorectal cancer were subsequently validated by lectin blotting in an independent set of plasma samples obtained from 10 CRC patients, 10 patients with adenomas, and 10 normal subjects. These results demonstrate the utility of this strategy for the identification of N -linked glycan patterns as potential markers of CRC in human plasma, and may have the utility to distinguish different disease states.     

Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress

Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of the icaADBC gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn917 insertions of two isogenic, icaADBC-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium S. epidermidis 1457. The insertion site was the same in both of the mutants and was located in the first gene, rsbU, of an operon highly homologous to the sigB operons of Staphylococcus aureus and Bacillus subtilis. Supplementation of Trypticase soy broth with NaCl (TSB(NaCl)) or ethanol (TSB(EtOH)), both of which are known activators of sigB, led to increased biofilm formation and PIA synthesis by S. epidermidis 1457. Insertion of Tn917 into rsbU, a positive regulator of alternative sigma factor sigma(B), led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSB(EtOH), the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSB(NaCl), the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn917 insertion of mutant M15 to the independent and biofilm-producing strain S. epidermidis 8400. In 11 clinical S. epidermidis strains, a restriction fragment length polymorphism of the sigB operon was detected which was independent of the presence of the icaADBC locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.     

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.