Alkane inducible proteins in Geobacillus thermoleovorans B23

<p>Abstract</p> <p>Background</p> <p>Initial step of β-oxidation is catalyzed by acyl-CoA dehydrogenase in prokaryotes and mitochondria, while acyl-CoA oxidase primarily functions in the peroxisomes of eukaryotes. Oxidase reaction accompanies emission of toxic by-product reactive oxygen molecules including superoxide anion, and superoxide dismutase and catalase activities are essential to detoxify them in the peroxisomes. Although there is an argument about whether primitive life was born and evolved under high temperature conditions, thermophilic archaea apparently share living systems with both bacteria and eukaryotes. We hypothesized that alkane degradation pathways in thermophilic microorganisms could be premature and useful to understand their evolution.</p> <p>Results</p> <p>An extremely thermophilic and alkane degrading <it>Geobacillus thermoleovorans </it>B23 was previously isolated from a deep subsurface oil reservoir in Japan. In the present study, we identified novel membrane proteins (P16, P21) and superoxide dismutase (P24) whose production levels were significantly increased upon alkane degradation. Unlike other bacteria acyl-CoA oxidase and catalase activities were also increased in strain B23 by addition of alkane.</p> <p>Conclusion</p> <p>We first suggested that peroxisomal β-oxidation system exists in bacteria. This eukaryotic-type alkane degradation pathway in thermophilic bacterial cells might be a vestige of primitive living cell systems that had evolved into eukaryotes.</p

Cleavage of a DNA–RNA–DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA–RNA junction with prokaryotic RNases HII

AbstractWe have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA–RNA–DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5′-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA–RNA junction (5′-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA

A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon

AbstractA RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein

Isolation of TBP-interacting protein (TIP) from a hyperthermophilic archaeon that inhibits the binding of TBP to TATA-DNA

AbstractWe have isolated TBP (TATA-binding protein)-interacting protein (TIP) from cell lysates of a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1, by affinity chromatography with TBP-agarose. Based on the internal amino acid sequence information, PCR primers were synthesized and used to amplify the gene encoding this protein (Pk-TIP). Determination of the nucleotide sequence and characterization of the recombinant protein revealed that Pk-TIP is composed of 224 amino acid residues (molecular weight of 25 558) and exists in a dimeric form. BIAcore analyses for the interaction between recombinant Pk-TIP and recombinant Pk-TBP indicated that they interact with each other with an equilibrium dissociation constant, KD, of 1.24–1.46 μM. A gel mobility shift assay indicated that Pk-TIP inhibited the interaction between Pk-TBP and a TATA-DNA. Pk-TIP may be one of the archaeal factors which negatively regulate transcription

Recent advances in telomerase-based medicine for targeting cancers

[Curriculum Vitae & Summary] International Symposium on Tumor Biology in Kanazawa 2004 / Kanazawa, Japan February 12 and 13, 200

Middle Ear Carcinoid Tumor : Case Report and Literature Review

We herein report a rare case of a carcinoid tumor observed in the middle ear. The 51-year-old female patient presented to our hospital with an 8-year history of mild deafness and sensation of obstruction within the left ear. Otoscopic examination revealed a pinkish mass in the hypotympanum, which was visible through an intact tympanic membrane. Computed tomography (CT) of the temporal bone showed a well-circumscribed mass of soft tissue density filling the left hypotympanum. The provisional clinical diagnosis was cholesteatoma or another type of tumor (e.g. glomus jugulare tumor or facial nerve neurilemmoma). Transcanal tympanotomy was performed under general anesthesia to explore the lesion. At surgery, a well-delineated, encapsulated pinkish mass was found in the hypotympanum. It was not adherent to the tympanic membrane or ossicles. The mass arose, via a narrow pedicle, from the lining epithelium at the promontory. Complete resection of the mass was performed with careful clearance around the ossicles and epithelium at the promontory. Histological examination showed a carcinoid tumor composed of islands, cords, and nests of tumor cells in a fibrous stroma. Immunohistochemistry revealed granular cytoplasmic positivity for chromogranin A. No general symptoms of carcinoid syndrome were observed in this patient. Endoscopic examination revealed no evidence of other carcinoid tumors in the gastrointestinal and respiratory tracts. Eight years postoperatively, no evidence of recurrence or metastatic disease has been observed

Gentisate 1,2-dioxygenase from Xanthobacter polyaromaticivorans 127W.

A putative gentisate 1,2-dioxygenase was encoded in the dibenzothiophene degradation gene cluster (dbd) from Xanthobacter polyaromaticivorans 127W. The deduced amino acid sequence showed high sequence similarity with gentisate dioxygenases from Pseudomonas alcaligenes (AAD49427, 65% identical), Bradyrhizobium japonicum (NP_766750, 64%), and P. aeruginosa (ZP_00135722, 54%), and moderate similarity with 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7 (BAA31235, 33%) and salicylate dioxygenase from Pseudaminobacter salicylatoxidans (AAQ91293, 33%). The enzyme, GDOxp, was heterologously produced in Escherichia coli and purified to homogeneity. GDOxp formed a tetramer and exhibited high dioxygenase activity against 1,4-dihydroxy 2-naphthoate as well as gentisate, suggesting unusually broad substrate specificity. GDOxp easily released ferrous ion under unfavorable temperature and pH conditions to become an inactive monomer protein. An inactive monomer protein can reconstitute a tetramer structure and restore enzyme activity in a cooperative manner upon the addition of ferrous ion. Chymotryptic digestion and protein truncation experiments suggested that the N-terminal region is important for the tetramerization of GDOxp

Biofilm formation by a Bacillus subtilis strain that produces γ-polyglutamate

The extracellular matrix produced by Bacillus subtilis B-1, an environmental strain that forms robust floating biofilms, was purified, and determined to be composed predominantly of -polyglutamate (-PGA), with a molecular mass of over 1000 kDa. Both biofilm formation and -PGA production by B. subtilis B-1 increased with increasing Mn2+ or glycerol concentration. -PGA was produced in a growth-associated manner in standing culture, and floating biofilms were formed. However, -PGA was produced in a non-growth-associated manner in shaking culture conditions. When B. subtilis B-1 was grown in a microaerated culture system, floating biofilm formation and -PGA production were significantly retarded, suggesting that oxygen depletion is involved in the initial steps of floating biofilm formation in standing culture. Proteomic analysis of membrane proteins demonstrated that flagellin, oligopeptide permease and Vpr protease precursor were the major proteins produced by cells in a floating biofilm and a colony