Argonaute system of Kordia jejudonensis is a heterodimeric nucleic acid-guided nuclease.

We purified and characterized a prokaryotic argonaute (pAgo) (KjMP) and its associated protein (KjAA) from a bacterium Kordia jejudonensis. The two proteins present as a complex were revealed by the copurification of KjAA with His-tagged KjMP by Ni-NTA affinity column. The KjAA/KjMP complex was a heterodimer evaluated from the molecular weight estimated using size exclusion chromatography. The pAgo complex presented a guide-dependent target DNA cleavage. RNA was the preferred guide; however, DNA also functioned, albeit weakly. Additionally, 5'-phosphorylate or non-phosphorylated guide was equally effective. The purified complex exhibited nonspecific nuclease activity on dsDNA and ssDNA. This is the first study to report that short pAgo and its associated protein form a complex, which has a nucleic acid-guided target recognition and cleavage.

Compiling Multicopy Single-Stranded DNA Sequences from Bacterial Genome Sequences.

A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.

Characterization of cell death in Escherichia coli mediated by XseA, a large subunit of exonuclease VII.

Exonuclease VII (ExoVII) of Escherichia coli is a single strandspecific DNA nuclease composed of two different subunits: the large subunit, XseA, and the small subunit, XseB. In this study, we found that multicopy single-stranded DNAs (msDNAs), Ec83 and Ec78, are the in vivo substrates of ExoVII; the enzyme cuts the phosphodiester bond between the fourth and fifth nucleotides from the 5'end. We used this msDNA cleavage to assess ExoVII activity in vivo. Both subunits were required for enzyme activity. Expression of XseA without XseB caused cell death, even though no ExoVII activity was detected. The lethality caused by XseA was rescued by surplus XseB. In XseA-induced death, cells were elongated and multinucleated, and their chromosomes were fragmented and condensed; these are the morphological hallmarks of apoptotic cell death in bacteria. A putative caspase recognition sequence (FVAD) was found in XseA, and its hypothetical caspase product with 257 amino acids was as active as the intact protein in inducing cell death. We propose that under ordinary conditions, XseA protects chromosome as a component of the ExoVII enzyme, but in some conditions, the protein causes cell death; the destruction of cell is probably carried out by the amino terminal fragment derived from the cleavage of XseA by caspase-like enzyme.

Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli.

Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes.

Human serotonin receptor 3A (5-HT3A) is a ligand-gated ion channel regulated by serotonin. A fusion protein (P9-5-HT3A) of 5-HT3A with the P9 protein, a major envelope protein of bacteriophage phi6, was highly expressed in the membrane fraction of Escherichia coli, and the expressed protein was purified to homogeneity using an affinity chromatography. P9-5-HT3A was observed as mixed oligomers in detergents. The purified P9-5-HT3A was efficiently reconstituted into proteoliposomes, and the serotonin-dependent ion-channel activity of P9-5-HT3A was observed by measuring the increased fluorescence of Fluo-3 attributed to the formation of a complex with the Ca(2+) ions released from the proteoliposomes. Alanine substitution for Trp178 of 5-HT3A abolished the serotonin-dependent ion-channel activity, confirming the importance of Trp178 as a ligand-binding site. Furthermore, the ion-channel activity of the reconstituted P9-5-HT3A was effectively blocked by treatment with ondansetron, an antagonist of 5-HT3A. The bacterial expression system of human 5-HT3A and the proteoliposomes reconstituted with 5-HT3A would provide biophysical and structural analyses of 5-HT3A.

Purification and characterization of recombinant human endothelin receptor type A.

Human endothelin receptor type A (ET(A)) is a G-protein coupled receptor that mediates vasoconstriction of blood vessels. To determine the structural characteristics and signaling mechanism of ET(A), we have expressed recombinant ET(A) as a fusion protein with p9 envelope protein from phi6 bacteriophage. The His-tag-labeled p9-ET(A) fusion protein was highly expressed in the membrane fraction of Escherichia coli and purified to homogeneity by single affinity chromatography after solubilization with detergents. Purified p9-ET(A) appeared as an oligomer and presented mainly as an α-helical structure. The protein also showed specific binding to endothelin-1 (ET-1) and the alpha subunit of G(q) protein with apparent K(D) values of 17 and 20 nM, respectively. An antagonist of ET(A), bosentan, prevented the interaction between p9-ET(A) and ET-1 in a concentration-dependent manner. These results indicate that recombinant p9-ET(A) has a competent conformation for interactions with ET-1 and the alpha subunit of G(q) protein.

Overexpression of outer membrane protein OprT and increase of membrane permeability in phoU mutant of toluene-tolerant bacterium Pseudomonas putida GM730.

Eight toluene-sensitive mutants were previously isolated from the toluene-tolerant bacterium Pseudomonas putida GM730. One of these mutants was TOS6, in which Tn5 had been inserted into phoU. Susceptibility to multiple antibiotics, as well as toluene sensitivity, was increased in the phoU mutant of P. putida GM730. We compared the outer membrane proteins from the phoU mutant and wild-type via two-dimensional gel electrophoresis. A 45 kDa protein was dramatically overexpressed as the result of phoU inactivation, and this protein was identified by peptide mass fingerprinting and microsequencing as a conserved hypothetical protein consisting of 414 amino acids. The protein, designated as OprT, harbors a signal sequence and extended beta-sheets, both of which are features common to the bacterial porins. The rate of ethidium bromide accumulation in TOS6 was higher than in GM730, which indicates that the TOS6 membranes may be more permeable to ethidium bromide than are the membranes of GM730. We propose that the toluene sensitivity and increased antibiotic susceptibility observed in the phoU mutant may be attributable to increased membrane permeability.

Comparisons of recombinant protein expression in diverse natural isolates of Escherichia coli.

We assessed heterologous protein expression in 64 strains obtained from the Escherichia coli Reference (ECOR) collection, a collection representing diverse natural E. coli populations. A plasmid generating a glutathione S-transferase and plant carbonic anhydrase fusion protein (GST-CA) under the control of the tac promoter was introduced into the ECOR strains, and the quantity of the fusion protein was determined by SDS-PAGE. The foreign protein was generated at various levels, from very high (40 strains, high producers) to very low (six strains, low producers). Immunoblotting showed that the high producers expressed approximately 250-500 times more GST-CA protein than the low producers. The results of semi-quantitative RT-PCR showed that the low producers generated mRNA levels comparable to those of the high producers, thereby suggesting that, at least in this case, inefficient translation is a major cause of the low production. We introduced a different plasmid, which expressed a maltose binding protein and plant guanylate kinase fusion protein (MBP-GK) into the six low producers. Interestingly, five of these expressed MBP-GK at very high levels. Thus, we conclude that the production of a particular protein from an expression vector can vary considerably, depending on the host strain. Strains in the ECOR collection could function as useful alternative hosts when a desired level of protein expression is not obtained from commonly used strains, such as E. coli K12 or B derivatives.

Proteomic analysis of heat-stable proteins in Escherichia coli.

Some proteins of E. coli are stable at temperatures significantly higher than 49 degrees C, the maximum temperature at which the organism can grow. The heat stability of such proteins would be a property which is inherent to their structures, or it might be acquired by evolution for their specialized functions. In this study, we describe the identification of 17 heat-stable proteins from E. coli. Approximately one-third of these proteins were recognized as having functions in the protection of other proteins against denaturation. These included chaperonin (GroEL and GroES), molecular chaperones (DnaK and FkpA) and peptidyl prolyl isomerases (trigger factor and FkpA). Another common feature was that five of these proteins (GroEL, GroES, Ahpc, RibH and ferritin) have been shown to form a macromolecular structure. These results indicated that the heat stability of certain proteins may have evolved for their specialized functions, allowing them to cope with harsh environments, including high temperatures.

Development of a simple cell lysis method for recombinant DNA using bacteriophage lambda lysis genes.

In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

A proteomic approach to study msDNA function in Escherichia coli.

Retron is a prokaryotic genetic element that produces multicopy single-stranded DNA covalently linked to RNA (msDNA) by a reverse transcriptase. It was found that cells producing a large amount of msDNA, rather than those that did not, showed a higher rate of mutation. In order to understand the molecular mechanism connecting msDNA production to the high mutation rate the protein patterns were compared by two dimensional gel electrophoresis. Ten proteins were found to be differentially expressed at levels more than three fold greater in cells with than without msDNA, nine of which were identified by MALDI TOF MS. Eight of the nine identified proteins were repressed in msDNA-producing cells and, surprisingly, most were proteins functioning in the dissimilation of various carbon sources. One protein was induced four fold greater in the msDNA producing cells and was identified as a 30S ribosomal protein S2 involved in the regulation of translation. The molecular mechanism underlying the elevated mutation in msDNA-producing cell still remains elusive.

Proteomic analysis revealed a strong association of a high level of alpha1-antitrypsin in gastric juice with gastric cancer.

To investigate the pathology of gastric disorders, we compared the proteomic patterns of gastric juice from patients with various gastric disorders. In healthy subjects pepsin A, pepsin B and gastric lipase were the major proteins detected by two-dimensional gel electrophoresis. These digestive enzymes were not detected in 60% of gastric cancer cases (18 out of 30 analyzed cases). Interestingly, an extraordinary amount of alpha(1)-antitrypsin was observed in these cases. In contrast to gastric cancer cases, alpha(1)-antitrypsin was detected in only 5% of patients (three out of 56) with chronic atrophic gastritis, and the detection frequency went up as the disease developed (one of four intestinal metaplasia cases, two of seven tubular adenoma cases, a single examined case of hyperplastic polyp and 60% of gastric cancer). Zymography showed that a 60 kDa protease strongly associated with alpha(1)-antitrypsin and mass spectrometric analysis revealed that the gastric alpha(1)-antitrypsin was a protease-cleaved form. Our data suggest that alpha(1)-antitrypsin and 60 kDa protease may serve as good diagnostic and prognostic markers for conditions associated with gastric cancer.

Defining the plant disulfide proteome.

There is considerable interest in redox regulation and new targets for thioredoxin and glutaredoxin are now being identified. It would be of great benefit to the field to have a list of all possible candidates for redox regulation--that is all disulfide proteins in plant. We developed a simple and very powerful method for identifying proteins with disulfide bonds in vivo. In this method, free thiols in proteins are fully blocked by alkylation, following which disulfide cysteines are converted to sulfhydryl groups by reduction. Finally, proteins with sulfhydryls are isolated by thiol affinity chromatography. Our method is unique in that membrane proteins as well as water-soluble proteins are examined for their disulfide nature. By applying this method to Arabidopsis thaliana we identified 65 putative disulfide proteins, including 20 that had not previously been demonstrated to be regulated by redox state. The newly identified, possibly redox-regulated proteins include: violaxanthin de-epoxidase, two oxygen-evolving enhancer proteins, carbonic anhydrase, photosystem I reaction center subunit N, photosystem I subunit III, S-adenosyl-L-methionine carboxyl methyltransferase, guanylate kinase, and bacterial mutT homolog. Possible functions of disulfide bonding in these proteins are discussed.

Characterization of a novel single-stranded RNA mycovirus in Pleurotus ostreatus.

A mycovirus, named oyster mushroom spherical virus (OMSV), was isolated from cultivated oyster mushrooms with a severe epidemic of oyster mushroom Die-back disease. OMSV was a 27-nm spherical virus encapsidating a single-stranded RNA (ssRNA) of 5.784 kb with a coat protein of approximately 28.5 kDa. The nucleotide sequence of the virus revealed that its genomic RNA was positive strand, containing 5784 bases with seven open reading frames (ORF). ORF1 had the motifs of RNA-dependent RNA polymerases (RdRp) and helicase. ORF2 encoded a coat protein. ORF3 to 7 could encode putative polypeptides of approximately 12, 12.5, 21, 14.5, and 23 kDa, respectively, but none of them showed significant similarity to any other known polypeptides. The 5' end of the viral RNA was uncapped and the 3' end was polyadenylated with 74 bases. Genomic structure and organization and the derived amino acid sequence of RdRp and helicase domain were similar to those of tymoviruses, a plant virus group.

Identifying the major proteome components of Helicobacter pylori strain 26695.

The whole genome sequences of Helicobacter pylori strain 26695 have been reported. Whole cell proteins of H. pylori strain 26695 cells were obtained and analyzed by two-dimensional electrophoresis, using immobilized pH gradient strips. The most abundant proteins were shown in the region of pI 4.0-9.5 with molecular masses from 10 to 100 kDa. Soluble proteins were precipitated by the use of 0-80% saturated solutions of ammonium sulfate. Soluble proteins precipitated by the 0-40% saturations of ammonium sulfate produced similar spot profiles and their abundant protein spots had acidic pI regions. However, a number of soluble proteins precipitated by more than 60% saturation of ammonium sulfate were placed in the alkaline pI regions, compared to those precipitated by 40% saturation. In addition, we have performed an extensive proteome analysis of the strain utilizing peptide MALDI-TOF-MS. Among the 345 protein spots processed, 175 proteins were identified. The identified spots represented 137 genes. One-hundred and fifteen proteins were newly identified in this study, including DNA polymerase III beta-subunit. These results might provide guidance for the enrichment of H. pylori proteins and contribute to construct a master protein map of H. pylori.

Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry.

Protein identification by peptide mass fingerprinting, using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), plays a major role in large proteome projects. In order to develop a simple and reliable method for protein identification by MALDI-TOF MS, we compared and evaluated the major steps in peptide mass fingerprinting. We found that the removal of excess enzyme from the in-gel digestion usually gave a few more peptide peaks, which were important for the identification of some proteins. Internal calibration always gave better results. However, for a large number of samples, two step calibrations (i.e. database search with peptide mass from external calibration, then the use of peptide masses from the search result as internal calibrants) were useful and convenient. From the evaluation and combination of steps that were already developed by others, we established a single overall procedure for peptide identification from a polyacrylamide gel.

A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase.

A cDNA, PHCC-TPx, specifying a protein highly homologous to known phospholipid hydroperoxide glutathione peroxidases was isolated from a Chinese cabbage cDNA library. PHCC-TPx encodes a preprotein of 232 amino acids containing a putative N-terminal chloroplast targeting sequence and three conserved Cys residues (Cys(107), Cys(136), and Cys(155)). The mature form of enzyme without the signal peptide was expressed in Escherichia coli, and the recombinant protein was found to utilize thioredoxin (Trx) but not GSH as an electron donor. In the presence of a Trx system, the protein efficiently reduces H(2)O(2) and organic hydroperoxides. Complementation analysis shows that overexpression of the PHCC-TPx restores resistance to oxidative stress in yeast mutants lacking GSH but fails to complement mutant lacking Trx, suggesting that the reducing agent of PHCC-TPx in vivo is not GSH but is Trx. Mutational analysis of the three Cys residues individually replaced with Ser shows that Cys(107) is the primary attacking site by peroxide, and oxidized Cys(107) reacts with Cys(155)-SH to make an intramolecular disulfide bond, which is reduced eventually by Trx. Tryptic peptide analysis by matrix-assisted laser desorption and ionization time of flight mass spectrometry shows that Cys(155) can form a disulfide bond with either Cys(107) or Cys(136).