Systematic biochemical analysis of somatic missense mutations in DNA polymerase ß found in prostate cancer reveal alteration of enzymatic function.

DNA polymerase ß is essential for short-patch base excision repair. We have previously identified 20 somatic pol ß mutations in prostate tumors, many of them missense. In the current article we describe the effect of all of these somatic missense pol ß mutations (p.K27N, p.E123K, p.E232K, p.P242R, p.E216K, p.M236L, and the triple mutant p.P261L/T292A/I298T) on the biochemical properties of the polymerase in vitro, following bacterial expression and purification of the respective enzymatic variants. We report that all missense somatic pol ß mutations significantly affect enzyme function. Two of the pol ß variants reduce catalytic efficiency, while the remaining five missense mutations alter the fidelity of DNA synthesis. Thus, we conclude that a significant proportion (9 out of 26; 35%) of prostate cancer patients have functionally important somatic mutations of pol ß. Many of these missense mutations are clonal in the tumors, and/or are associated with loss of heterozygosity and microsatellite instability. These results suggest that interfering with normal polymerase ß function may be a frequent mechanism of prostate tumor progression. Furthermore, the availability of detailed structural information for pol ß allows understanding of the potential mechanistic effects of these mutants on polymerase function.

Structural and biochemical analysis of the asc operon encoding 6-phospho-beta-glucosidase in Pectobacterium carotovorum subsp. carotovorum LY34.

An asc operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated from a genomic library in a screen for beta-glucosidase activities. Sequence analysis of the 5618-bp cloned DNA fragment (accession number AY622309) showed three open reading frames (ascG, ascF, and ascB) that are predicted to encode 375, 486, and 476 amino acid proteins, respectively. The AscG ORF shared a high similarity with the Escherichia coli AscG repressor. The AscF ORF shared 81% identity with the E. coli AscF PTS enzyme II(asc), while the AscB ORF was highly similar to 6-phospho-beta-glucosidases and is a member of the glycosyl hydrolase family 1. The purified AscB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. It exhibited maximal activity at pH 7.0 and 40 degrees C, and its activity was enhanced in the presence of Mg(2+) and Ca(2+). The molecular weight of the enzyme was estimated to be 53 000 Da by SDS-PAGE. Two conserved glutamate residues (Glu(182) and Glu(374)) were shown to be important for AscB activity.

Cloning and characterization of a thermostable intracellular alpha-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8.

The gene encoding an intracellular alpha-amylase, AmyB (TM1650), from Thermotoga maritima MSB8, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli. The AmyB enzyme hydrolyzed alpha-1,4 starch linkage. The amyB gene is 1269 bp in length, encoding a protein of 422 amino acids (calculated molecular mass of 50187 Da). The molecular weight of the enzyme was estimated to be 50000 Da by SDS-PAGE after starch-nondenaturing-PAGE. The amino acid sequence of AmyB showed less than 12% identity to other amylases, but contained four regions that are highly conserved among alpha-amylases. The AmyB alpha-amylase exhibited maximal enzymatic activity at pH 7.0 and its optimum temperature for activity was 70 degrees C. Like the alpha-amylases of many other organisms, the thermostability of T. maritima MSB8 alpha-amylase, AmyB expressed in E. coli was enhanced in the presence of Ca(2+) (10 mM).

Cloning of the cel8Y gene from Pectobacterium chrysanthemi PY35 and its comparison to cel genes of soft-rot Pectobacterium.

The phytopathogenic Pectobacterium chrysanthemi (Pch) PY35 secretes multiple isozymes of plant cell wall degrading enzyme cellulases. We cloned a second cel gene that encodes cellulase in Pch PY35. The inserted 2 kb fragment was subcloned in order to geneate pPY710 (cel8Y). The structural organization of the cel8Y gene consists of an open reading frame (ORF) of 999 bp that encodes 332 amino acid residues with a signal peptide of 23 amino acids. The predicted amino acid sequence of Cel8Y was very similar to that of Cellulomonas uda, but completely different from that of the Cel5Z of Pch PY35. It belonged to the glycoside hydrolase family 8, based on amino acid sequence similarities in contrast to Cel5Z of Pch PY35, which was confirmed as family 5. Cel8Y was not closely related to the known cellulases of Pectobacterium. It had the conserved region of the glycoside hydrolase family 8, ASDGDVLIAWALLKAGNKW. The apparent molecular mass of the Cel8Y protein was calculated to be approximately 34 kDa by a carboxymethylcellulosesodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). The Cel8Y had a calculated pl of 6.49. It was optimally active at pH 7 with an approximate optimal temperature around 40 degrees C. The cellulase activity of Cel8Y was lower than that of Cel5Z.

Activation of a casB gene encoding ß-glucosidase of Pectobacterium carotovorum subsp. carotovorum LY34.

Two cas genes were isolated from Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34). Sequence analysis of the 4873 bp cloned DNA fragment (accession number AY866383) revealed two open reading frames (casF and casB) that are predicted to encode 658 and 467 amino acid proteins, respectively. The CasF protein is similar to other PTS enzyme II components. casB encodes ß-glucosidase, a member of the glycosyl hydrolase family 1. An inverted repeat sequence was identified in the casB promoter region, and was hypothesized to have a negative effect on casB transcription. Replacement of the casB promoter of Pcc LY34 with the bglB promoter activated the casB gene, consistent with the repeats inhibiting expression of casB. Purified CasB enzyme was estimated to be 53,000 Da by SDS-PAGE, and hydrolyzed salicin, arbutin, pNPG, and MUG. CasB exhibited maximal activity toward pNPG at pH 7.0 and 40 °C, and Mg(2+) is essential for its activity. Two conserved glutamate residues (Glu(177) and Glu(366)) were shown to be important for CasB activity.

Cloning and biochemical analysis of ß-glucoside utilization (bgl) operon without phosphotransferase system in Pectobacterium carotovorum subsp. carotovorum LY34.

ß-Glucosidases are widespread in bacteria and involved in the metabolism of various carbohydrate substrates. Studying of ß-glucoside utilization (bgl) operons on operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) will help us understanding how ß-glucoside utilization (bgl) operon can cooperate with other systems in bacterium caused soft-rot disease. Pcc LY34 causes soft-rot disease in plants and expresses multiple enzymatic forms of ß-glucosidases. To fully explore the ß-glucoside utilization system in Pcc LY34, we have isolated a bgl operon from a genomic library for screening of ß-glucosidase activities. Sequence analysis of a 3050bp cloned DNA fragment (accession number AY870655) shows two open reading frames (bglY and bglK) that are predicted to encode proteins of 474 and 278 amino acid residues, respectively. Pair wise similarity analysis suggests BglY is a beta-glucosidase (a member of glycosyl hydrolase family 1) and BglK is a transcriptional antiterminator protein. bglY promoter region contains an inverted repeat sequence similar to transcriptional terminator. Different from other four ß-glucoside utilization operons of Pcc LY34 strain, BglY contains signal peptide sequences as extracellular ß-glucosidase. Comparisons of five ß-glucoside utilization operons of Pcc LY34 strain showed that bglYK operon does not have phosphotransferase system domain which are responsible for sugar transportation. BglY shares 33-44% identity with other four ß-glucosidases of Pcc LY34 strain. Enzyme assay showed that purified BglY enzyme hydrolyzed salicin, arbutin, pNPG, and MUG, and exhibited maximal activity at pH 7.0 and 40°C. This activity was enhanced Mg(2+). Site-directed mutagenesis revealed E166 and E371 are critical of BglY's ß-glucosidase activity.

Cloning and comparison of third beta-glucoside utilization (bglEFIA) operon with two operons of Pectobacterium carotovorum subsp. carotovorum LY34.

A third bgl operon containing bglE, bglF, bglI, and bglA was isolated from Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34). The sequences of BglE, BglF, and Bgll were similar to those of the phosphotransferase system (PTS) components IIB, IIC, and IIA respectively. BglF contains important residues for the phosphotransferase system. The amino acid sequence of BglA showed high similarity to various 6-phospho-beta-glucosidases and to a member of glycosyl hydrolase family 1. Sequence and structural analysis also revealed that these four genes were organized in a putative operon that differed from two operons previously isolated from Pcc LY34, bglTPB (accession no. AY542524) and ascGFB (accession no. AY622309). The transcription regulator for this operon was not found, and the EII complexes for PTS were encoded separately by three genes (bglE, bglF, and bglI). The BglA enzyme had a molecular weight estimated to be 57,350 Da by SDS-PAGE. The purified beta-glucosidase hydrolyzed salicin, arbutin, rhoNPG, rhoNPbetaG6P, and MUG, exhibited maximal activity at pH 7.0 and 40 degrees C, and displayed enhanced activity in the presence of Mg2+ and Ca2+. Two glutamate residues (Glu178 and Glu378) were found to be essential for enzyme activity.

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region.

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.

Analysis of bgl operon structure and characterization of beta-glucosidase from Pectobacterium carotovorum subsp. carotovorum LY34.

A putative bgl operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated. Sequence analysis of the 5,557 bp cloned DNA fragment (accession no. AY542524) showed three open reading frames (bglT, bglP, and bglB) predicted to encode 287, 633, and 468 amino acid proteins respectively. BglT and BglP ORFs show high similarity to that of the Pectobacterium chrysanthemi ArbG antiterminator and ArbF permease respectively. Also, the latter contains most residues important for phosphotransferase activity. The amino acid sequence of BglB showed high similarity to various beta-glucosidases and is a member of glycosyl hydrolase family 1. The purified BglB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. The molecular weight of the enzyme was estimated to be 53,000 Da by SDS-PAGE. The purified beta-glucosidase exhibited maximal activity at pH 7.0 and 40 degrees C, and its activity was enhanced in the presence of Mg(2+). Two glutamate residues (Glu(173) and Glu(362)) were found to be essential for enzyme activity.

Activity enhancement of Cel5Z from Pectobacterium chrysanthemi PY35 by removing C-terminal region.

The phytopathogenic bacterium Pectobacteium chrysanthemi PY35 secretes Cel5Z endoglucanase belonging to the glycoside hydrolase family 5 of EC 3.2.1.4. The mutation of cel5Z::Omega gene was constructed by cloning the 2.0-kb SmaI fragment containing the streptomycin/spectinomycin-resistance gene of pHP45(Omega) into the BalI site of pPY100. The insertion of Omega fragment generated a new stop codon, removing the Ser/Thr-rich linker region and the cellulose binding domain (CBD) in the C-terminal region of cel5Z gene. By subsequent subcloning from this 4.9-kb fragment (pPY1001), a 1.0-kb (pPY1002) fragment was obtained and designated as cel5Z::Omega. The cel5Z::Omega gene had an open reading frame (ORF) of 1011 bp, encoding 336 amino acids, starting with an ATG codon and ending with a new TGA stop codon. The molecular mass of the Cel5Z::Omega protein in E. coli transformant appeared to be 32 kDa by SDS-PAGE analysis in the presence of carboxymethyl-cellulose (CMC). The Cel5Z::Omega protein hydrolyzed CMC with 1.7-fold higher activity than the intact Cel5Z cellulase.

Endophytic bacillus sp. isolated from the interior of balloon flower root.

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G+C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35.

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.

Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16S rDNA sequence.

Phylogenetic analysis of archaea in the rumen ecosystem was analysed by PCR of 16S rDNA from the bovine rumen using archaea-specific primers. The libraries were constructed from rumen fluid (AF), rumen solid (AS), and rumen epithelium (AE) from a rumen-fistulated Korean cow (Hanwoo). The 45 AF clones could be divided into three groups and the largest group was affiliated with the Methanomicrobiaceae family (96% of clones). The AF clones contained a high proportion of unidentifiable clones (67%). The 39 AE clones could be divided into two groups and the largest group was also affiliated with the Methanomicrobiaceae family (95% of clones). The AE clones contained a low proportion of unidentifiable clones (5%). The 20 AS clones could be divided into two groups that were affiliated with either the Methanobacteriaceae family (55%) or the Methanomicrobiaceae family (45%). The AS clones contained a moderate proportion of unidentifiable clones (40%). The predominant family of whole rumen archaea was found to belong to the Methanomicrobiaceae (85%). Methanomicrobiaceae were predominant in the rumen epithelium and the rumen fluid while Methanobacteriaceae were predominant in the rumen solid. One clone from the rumen fluid and two clones from the rumen epithelium contained rDNA sequences of Non-Thermophilic-Crenarchaeota (NTC) and Thermophilic-Crenarchaeota (TC), respectively, which have not previously been described from the rumen.