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L-Rhamnose is an essential component of the cell wall and plays roles in mediating virulence and adhesion to host tissues in many microorganisms. Glucose-1-phosphate thymidylyltransferase (RmlA, EC 2.7.7.24) catalyzes the first reaction of the four-step pathway of Lrhamnose biosynthesis, producing dTDP-D-glucose from dTTP and glucose-1-phosphate. Three RmlA homologues of varying size have been identified in the genome of a thermophilic archaeon, Sulfolobus tokodaii strain 7. In this study, we report the heterologous expression of the largest homologue (a 401 residue-long ST0452 protein) and characterization of its thermostable activity. RmlA enzymatic activity of this protein was detected from 65 to 100°C, with a half-life of 60 min at 95°C and 180 min at 80°C. Analysis of a deletion mutant lacking the 170-residue C-terminal domain indicated that this region has an important role in the thermostability and activity of the protein. Analyses of substrate specificity indicated that the enzymatic activity of the full-length protein is capable of utilizing ␣-D-glucose-1-phosphate and N-acetyl-D-glucosamine-1-phosphate but not ␣-D-glucosamine-1-phosphate. However, the protein is capable of utilizing all four deoxyribonucleoside triphosphates and UTP. Thus, the ST0452 protein is an enzyme containing both glucose-1-phosphate thymidylyltransferase and N-acetyl-D-glucosamine-1-phosphate uridylyltransferase activities. This is the first report of a thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities.

Human peptidylarginine deiminase type IV (PAD4), a Ca(2+)-dependent enzyme known to convert arginine residues to citrulline residues in histones, has been shown to be associated with the development of rheumatoid arthritis. Recently, it was noted that the human peptidylarginine deiminase type IV gene (PADI4) regulates the expression of estrogen-responsive genes by modifying the methylated arginine sites in histones H3 and H4. In this study, we demonstrated that PADI4 was expressed in MCF-7 cells and was responsive to estrogen at the transcriptional level. Using the luciferase reporter gene fused to wild-type or mutated 5'-flanking region of PADI4, we characterized that as few as 348 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Chromatin immunoprecipitation and small interfering RNA assays revealed that activator protein-1, Sp1/Sp3, and nuclear factor-Y were cis-acting factors bound to the minimal promoter of PADI4 and that they regulated gene expression in a cooperative manner. Moreover, it was indicated that estrogen stimulated PADI4 expression through binding of estrogen receptor (ER)-alpha to the upstream of the PADI4 gene and ERalpha-mediated enhancement of activator protein-1, Sp1, and nuclear factor-Y levels. These findings indicated that estrogen stimulated PADI4 expression through both of the classical and nonclassical ER-mediated pathways.

Most marine bacteria produce exopolysaccharides (EPS), and bacterial EPS represent an important source of dissolved organic carbon in marine ecosystems. It was proposed that bacterial EPS rich in uronic acid is resistant to mineralization by microbes and thus has a long residence time in global oceans. To confirm this hypothesis, bacterial EPS rich in galacturonic acid was isolated from Alteromonas sp. JL2810. The EPS was used to amend natural seawater to investigate the bioavailability of this EPS by native populations, in the presence and absence of ammonium and phosphate amendment. The data indicated that the bacterial EPS could not be completely consumed during the cultivation period and that the bioavailability of EPS was not only determined by its intrinsic properties, but was also determined by other factors such as the availability of inorganic nutrients. During the experiment, the humic-like component of fluorescent dissolved organic matter (FDOM) was freshly produced. Bacterial community structure analysis indicated that the class Flavobacteria of the phylum Bacteroidetes was the major contributor for the utilization of EPS. This report is the first to indicate that Flavobacteria are a major contributor to bacterial EPS degradation. The fraction of EPS that could not be completely utilized and the FDOM (e.g., humic acid-like substances) produced de novo may be refractory and may contribute to the carbon storage in the oceans.

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Most marine bacteria can produce exopolysaccharides (EPS). However, very few structures of EPS produced by marine bacteria have been determined. The characterization of EPS structure is important for the elucidation of their biological functions and ecological roles. In this study, the structure of EPS produced by a marine bacterium, Alteromonas sp. JL2810, was characterized, and the biosorption of the EPS for heavy metals Cu2+, Ni2+, and Cr6+ was also investigated. Nuclear magnetic resonance (NMR) analysis indicated that the JL2810 EPS have a novel structure consisting of the repeating unit of [-3)-α-Rhap-(1→3)-α-Manp-(1→4)-α-3OAc-GalAp-(1→]. The biosorption of the EPS for heavy metals was affected by a medium pH; the maximum biosorption capacities for Cu2+ and Ni2+ were 140.8 ± 8.2 mg/g and 226.3 ± 3.3 mg/g at pH 5.0; however, for Cr6+ it was 215.2 ± 5.1 mg/g at pH 5.5. Infrared spectrometry analysis demonstrated that the groups of O-H, C=O, and C-O-C were the main function groups for the adsorption of JL2810 EPS with the heavy metals. The adsorption equilibrium of JL2810 EPS for Ni2+ was further analyzed, and the equilibrium data could be better represented by the Langmuir isotherm model. The novel EPS could be potentially used in industrial applications as a novel bio-resource for the removal of heavy metals.

The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme catalyzes reversibly the intra-molecular phosphoryl interconverting reaction of mannose-6-phosphate and mannose-1-phosphate or glucose-6-phosphate and glucose-1-phosphate. Glucose-6-phosphate and glucose-1-phosphate are known to be utilized for energy metabolism and cell surface construction, respectively. PMM/PGM has been isolated from many microorganisms. By performing similarity searches using existing PMM/PGM sequences, the homologous ORFs PH0923 and PH1210 were identified from the genomic data of Pyrococcus horikoshii OT3. Since PH0923 appears to be part of an operon consisting of four carbohydrate metabolic enzymes, PH0923 was selected as the first target for the investigation of PMM/PGM activity in P. horikoshii OT3. The coding region of PH0923 was cloned and the purified recombinant protein was utilized for an examination of its biochemical properties. The enzyme retained half its initial activity after treatment at 95 degrees C for 90 min. Detailed analyses of activities showed that this protein is capable of utilizing a variety of metal ions that are not utilized by previously characterized PMM/PGM proteins. A mutated protein with an alanine residue replacing the active site serine residue indicated that this residue plays an important but non-essential role in PMM/PGM activity.