Biochemical characterization and mutational analysis of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Archaeal NurA protein plays a key role in producing 3'-single stranded DNA used for homologous recombination repair, together with HerA, Mre11, and Rad50. Herein, we describe biochemical characteristics and roles of key amino acid residues of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-NurA). Tba-NurA possesses 5'-3' exonuclease activity for degrading DNA, displaying maximum efficiency at 45 °C-65 °C and at pH 8.0 in the presence of Mn2+. The thermostable Tba-NurA also possesses endonuclease activity capable of nicking plasmid DNA and circular ssDNA. Mutational data demonstrate that residue D49 of Tba-NurA is essential for exonuclease activity and is involved in binding ssDNA since the D49A mutant lacked exonuclease activity and reduced ssDNA binding. The R96A and R129A mutants had no detectable dsDNA binding, suggesting that residues R96 and R129 are important for binding dsDNA. The abolished degradation activity and reduced dsDNA binding of the D120A mutant suggest that residue D120 is essential for degradation activity and dsDNA binding. Additionally, residues Y392 and H400 are important for exonuclease activity since these mutations resulted in exonuclease activity loss. To our knowledge, it is the first report on biochemical characterization and mutational analysis of the NurA protein from Thermococcus.

Biochemical characterization and mechanistic insight of the family IV uracil DNA glycosylase from Sulfolobus islandicus REY15A.

Uracil DNA glycosylase (UDG) can remove uracil from DNA, thus playing an essential role in maintaining genomic stability. Family IV UDG members are mostly widespread in hyperthermophilic Archaea and bacteria. In this work, we characterized the family IV UDG from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A (Sis-UDGIV) biochemically, and dissected the roles of nine conserved residues in uracil excision by mutational analyses. Biochemical data demonstrate that Sis-UDGIV displays maximum efficiency for uracil excision at 50 °C ~ 70 °C and at pH 7.0-9.0. Additionally, the enzyme has displays a weak activity without a divalent metal ion, but maximum activity with Mg2+. Our mutational analyses show that residues E48 and F55 in Sis-UDGIV are essential for uracil removal, and residues E48, F55, R87, R92 and K146 are responsible for binding DNA. Importantly, we systemically revealed the roles of four conserved cysteine residues C14, C17, C86 and C102 in Sis-UDGIV that are required for being ligands of FeS cluster in maintaining the overall protein conformation and stability by circular dichroism analyses. Overall, our work has provided insights into biochemical function and DNA-binding specificity of archaeal family IV UDGs.

A novel Family V uracil DNA glycosylase from Sulfolobus islandicus REY15A.

Uracil DNA glycosylase (UDG) can excise uracil from DNA, thus playing an essential role in counteracting mutations. The genome of the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A encodes one putative Family V UDG (Sis-UDGV). Herein, we provide evidence that Sis-UDGV is a bi-functional glycosylase that can not only excise uracil from DNA, but cleave the generated apurinic/apyrimidinic (AP) site, which differs from other reported mono-functional Family V UDG homologs. Intriguingly, the enzyme can cleave DNA containing an AP site, thus suggesting that it might be involved in AP site repair. Biochemical data demonstrate that Sis-UDGV displays maximum activity for uracil removal at 45 °C ∼ 65 oC and at pH 8.0 ∼ 9.0. Furthermore, Sis-UDGV displays a substrate preference for uracil-containing ssDNA over uracil-containing dsDNA, but has no activity and weak activity for excising hypoxanthine from ssDNA and dsDNA, respectively. Importantly, we dissected the roles of seven conserved residues in Sis-UDGV by mutational analyses, demonstrating that residues D91, E117, E128, H167 and R192 are essential for catalysis. To our knowledge, it is the first report on the novel Family V UDG from Archaea with bi-functionality that harbors glycosylase/AP lyase activity.

Microcystis blooms aggravate the diurnal alternation of nitrification and nitrate reduction in the water column in Lake Taihu.

To explore the effects of Microcystis blooms on nitrogen (N) cycling in the water column, the community structures of the Microcystis-attached and free-living bacteria in Lake Taihu were assessed and a mesocosm experiment was further conducted on the shore of Lake Taihu. The bacterial communities of Microcystis-attached and free-living bacteria were dominated by heterotrophic bacteria, such as Pseudomonas and Massilia, while the relative abundances of the genera related to traditional autotrophic nitrification were surprisingly low. However, the dramatic increase in nitrate (NO3-) levels at the daytime suggested that in the mesocosms nitrification did occur, during which the heterotrophic nitrifiers played a predominant role as revealed by the acetylene inhibition experiment. The ammonium (NH4+) concentrations were always maintained at a low level, indicating that most of the substrates for daytime nitrification originated from organic N. The total N being removed during the experiment was much less than the sum of daily NO3- reduction, while the decrease in NO3- concentration was much higher than the increase in NH4+ concentration during the night, indicating that assimilation was the main explanation for nocturnal NO3- reduction. Thus, the cycling of organic N (remineralization) - heterotrophic nitrification - NO3- assimilation (reduction) promoted by Microcystis blooms aggravates the diurnal variation of NO3- in the water column.

Characterization of a novel type III alcohol dehydrogenase from Thermococcus barophilus Ch5.

The genome of the hyperthermophilic and piezophilic euryarchaeaon Thermococcus barophilus Ch5 encodes three putative alcohol dehydrogenases (Tba ADHs). Herein, we characterized Tba ADH547 biochemically and probed its catalytic mechanism by mutational studies. Our data demonstrate that Tba ADH547 can oxidize ethanol and reduce acetaldehyde at high temperature with the same optimal temperature (75 °C) and exhibit similar thermostability for oxidization and reduction reactions. However, Tba ADH547 has different optimal pH for oxidation and reduction: 8.5 for oxidation and 7.0 for reduction. Tba ADH547 is dependent on a divalent ion for its oxidation activity, among which Mn2+ is optimal. However, Tba ADH547 displays about 20% reduction activity without a divalent ion, and the maximal activity with Fe2+. Furthermore, Tba ADH547 showcases a strong substrate preference for 1-butanol and 1-hexanol over ethanol and other alcohols. Similarly, Tba ADH547 prefers butylaldehyde to acetaldehyde as its reduction substrate. Mutational studies showed that the mutations of residues D195, H199, H262 and H274 to Ala result in the significant activity loss of Tba ADH547, suggesting that residues D195, H199, H262 and H274 are responsible for catalysis. Overall, Tba ADH547 is a thermoactive ADH with novel biochemical characteristics, thereby allowing this enzyme to be a potential biocatalyst.

Biochemical characterization and mutational studies of a novel 3-methlyadenine DNA glycosylase II from the hyperthermophilic Thermococcus gammatolerans.

The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes a putative 3-methlyadenine DNA glycosylase II (Tg-AlkA). Herein, we report biochemical characterization and catalytic mechanism of Tg-AlkA. The recombinant Tg-AlkA can excise hypoxanthine (Hx) and 1-methlyadenine (1-meA) from dsDNA with varied efficiencies at high temperature. Notably, Tg-AlkA is a bi-functional glycosylase, which is sharply distinct from all the reported AlkAs. Biochemical data show that the optimal temperature and pH of Tg-AlkA for removing Hx from dsDNA are ca.70 °C and ca.7.0-8.0, respectively. Furthermore, the Tg-AlkA activity is independent of a divalent metal ion, and Mg2+ stimulates the Tg-AlkA activity whereas other divalent ions inhibit the enzyme activity with varied degrees. Mutational studies show that the Tg-AlkA W204A and D223A mutants abolish completely the excision activity, thereby suggesting that residues W204 and D223 are involved in catalysis. Surprisingly, the mutations of W204, D223, Y139 and W256 to alanine in Tg-AlkA lead to the increased affinity for binding DNA substrate with varied degrees, suggesting that these residues are flexible for conformational change of the enzyme. Therefore, Tg-AlkA is a novel AlkA that can remove Hx and 1-meA from dsDNA, thus providing insights into repair of deaminated and alkylated bases in DNA from hyperthermophilic Thermococcus.

Optimization of the culture condition for an antitumor bacterium Serratia proteamacula 657 and identification of the active compounds.

Exploration of novel active anti-tumor compounds from marine microbes for pharmaceutical applications has been a continuously hot spot in natural product research. Bacterial growth and metabolites may greatly vary under different culture conditions. In this study, the effects of different culture conditions and medium components on the growth and bioactive metabolites of Serratia proteamacula 657, an anti-tumor bacterium found in our previous study, were investigated. The results showed that lower temperature, weak acidic condition and solid fermentation favored the bacterial growth and the production of active compounds. Four components in the culture medium, NaCl, peptone, yeast extract and MgSO4, were found important to the bacterial growth and active compounds production in medium optimization. Under the optimized condition of solid state fermentation at pH 6.0-7.0, 23-25 °C, with the MgSO4-free medium containing 10.0 g/L peptone, 1.0 g/L yeast extract and 19.45 g/L NaCl, the antitumor activity of S. proteamacula 657 and the yield of crude extracts increased about 15 times and 6 times than the sample obtained in the original liquid fermentation, respectively. The active components in the metabolites of S. proteamacula 657 were identified as a homolog of prodigiosin, a red bacterial pigment, based on the analysis of the NMR and GC-MS. The bacterium S. proteamacula 657, which is adapted to lower temperature, produced prodigiosin-like pigments with highly antitumor activity, suggesting the bacterium is a potential new source for prodigiosin production.