Complete genome sequence of Chlamydia psittaci NRM_5 strain isolated from the fecal samples of a wild Indian ring-necked parakeet (Psittacula krameri manillensis) in Japan.

We report here the whole-genome sequence of the Chlamydia psittaci NRM_5 strain isolated from the fecal samples of wild Indian ring-necked parakeet (Psittacula krameri manillensis) in Japan. The sequence type is ST35, which is known to be associated with pigeons and doves, indicating the potential for transmission among bird species.

Cooperative roles of introns 1 and 2 of tobacco resistance gene N in enhanced N transcript expression and antiviral defense responses.

The tobacco virus resistance gene N contains four introns. Transient expression of transcripts from an N transgene containing these introns and driven by the native promoter in the presence of the elicitor of tobacco mosaic virus resulted in its increased expression. The requirement of the native promoter, the elicitor, or the individual introns for enhanced expression of N has not been fully studied. Here, we determined that 35S promoter-driven N transcript expression could be enhanced in the presence of the four introns regardless of the co-expression of the virus elicitor in tobacco. Function analyses using a series of N transgenes with different combination of introns revealed that the presence of intron 1 more so than intron 2 allowed higher accumulation of premature and mature N transcripts; however, both introns were important for not only enhanced gene expression but also for induction of cell death in tobacco and induced local resistance to spread of virus in Nicotiana benthamiana. Our findings indicate that introns 1 and 2 cooperatively contribute to N expression and virus resistance.

Fungal Carbonyl Sulfide Hydrolase of Trichoderma harzianum Strain THIF08 and Its Relationship with Clade D ß-Carbonic Anhydrases.

Carbonyl sulfide (COS) is the most abundant and long-lived sulfur-containing gas in the atmosphere. Soil is the main sink of COS in the atmosphere and uptake is dominated by soil microorganisms; however, biochemical research has not yet been conducted on fungal COS degradation. COS hydrolase (COSase) was purified from Trichoderma harzianum strain THIF08, which degrades COS at concentrations higher than 10,000 parts per million by volume from atmospheric concentrations, and its gene cos (492 bp) was cloned. The recombinant protein purified from Escherichia coli expressing the cos gene converted COS to H2S. The deduced amino acid sequence of COSase (163 amino acids) was assigned to clade D in the phylogenetic tree of the ß-carbonic anhydrase (ß-CA) family, to which prokaryotic COSase and its structurally related enzymes belong. However, the COSase of strain THIF08 differed from the previously known prokaryotic COSase and its related enzymes due to its low reactivity to CO2 and inability to hydrolyze CS2. Sequence comparisons of the active site amino acids of clade D ß-CA family enzymes suggested that various Ascomycota, particularly Sordariomycetes and Eurotiomycetes, possess similar enzymes to the COSase of strain THIF08 with >80% identity. These fungal COSase were phylogenetically distant to prokaryotic clade D ß-CA family enzymes. These results suggest that various ascomycetes containing COSase contribute to the uptake of COS by soil.

Plant Protein-Mediated Inhibition of Virus Cell-to-Cell Movement: Far-Western Screening and Biological Analysis of a Plant Protein Interacting with a Viral Movement Protein.

Cell-to-cell movement via plasmodesmata is a crucial step for plant RNA viruses to determine their host ranges. Many viruses including Tomato mosaic virus (ToMV) encode one or more movement proteins (MPs) that are indispensable for cell-to-cell movement. During movement processes, MPs are thought to interact directly with many plant proteins that may be involved in supporting or inhibiting cell-to-cell movement of viruses. In order to understand the molecular mechanisms that regulate viral spread positively or negatively, it is important to discover such MP-interacting plant proteins and analyze their functions in viral cell-to-cell movement in efficient ways. In this chapter, we provide protocols of a radioisotope-based far-western screening strategy to construct a λ phage cDNA library from a nonhost Brassica campestris (syn. rapa) for ToMV and identify plant proteins that bind directly to the 32P-labeled probe of ToMV MP, and subsequently a biolistic bombardment method to examine whether a plant protein selected have a function as an inhibitory factor that can interfere with virus cell-to-cell movement.

Cloning and expression of a gene encoding a novel thermostable thiocyanate-degrading enzyme from a mesophilic alphaproteobacteria strain THI201.

Strain THI201, a member of the alphaproteobacteria, is a novel thiocyanate (SCN(-))-degrading bacterium isolated from lake water enriched with potassium thiocyanate (KSCN). This bacterium carries the enzyme thiocyanate hydrolase (SCNase) that hydrolyses thiocyanate to carbonyl sulfide and ammonia. Characterization of both native and recombinant SCNase revealed properties different from known SCNases regarding subunit structure and thermostability: SCNase of strain THI201 was composed of a single protein and thermostable. We cloned and sequenced the corresponding gene and determined a protein of 457 amino acids of molecular mass 50 267 Da. Presence of a twin-arginine (Tat) signal sequence of 32 amino acids was found upstream of SCNase. The deduced amino acid sequence of SCNase showed 83% identity to that of a putative uncharacterized protein of Thiobacillus denitrificans ATCC 25259, but no significant identity to those of three subunits of SCNase from Thiobacillus thioparus strain THI115. The specific activities of native and recombinant enzyme were 0.32 and 4-15 µmol min(-1) (mg protein)(-1), respectively. The maximum activity of SCNase was found in the temperature range 30-70 °C. The thiocyanate-hydrolysing activity in both enzymes was decreased by freeze-thawing, although 25-100% of the activity of recombinant protein could be retrieved by treating the enzyme at 60 °C for 15 min. Furthermore, both native and recombinant enzymes retained the activity after pre-treatment of the protein solution at temperatures up to 70 °C.

Analysis of the cell death-inducing ability of the ethylene response factors in group VIII of the AP2/ERF family.

The ethylene response factor (ERF) family is one of the largest families of plant-specific transcription factors. We have shown previously that the overexpression of the gene for NtERF3, a tobacco transcriptional repressor containing the ERF-associated amphiphilic repression (EAR) motif in the C-terminal region, induces hypersensitive reaction (HR)-like cell death. Many EAR motif-containing ERFs, including NtERF3, are clustered in group VIII of the ERF family. In this study, we aimed at revealing the cell death-inducing ability of group VIII ERFs and the correlation between ERFs and HR. The results showed that many of the EAR motif-containing ERFs classified into subgroup VIII-a of Arabidopsis, rice, and tobacco had cell death-inducing ability in tobacco leaves. Seven AtERFs in subgroup VIII-b did not induce cell death; however, some ERFs in subgroup VIII-b of rice and tobacco showed cell death-inducing ability. An expression analysis of group VIII ERFs in HR-inducing tobacco suggested that the cell death-inducing ability of NtERFs was not necessarily associated with induction of HR. In addition, it was revealed that the EAR motif-containing AtERFs in subgroup II-a also showed cell death-inducing ability. The influence of sequence variation in the EAR motif on the ability to induce cell death is also discussed.

Carbonyl sulfide hydrolase from Thiobacillus thioparus strain THI115 is one of the ß-carbonic anhydrase family enzymes.

Carbonyl sulfide (COS) is an atmospheric trace gas leading to sulfate aerosol formation, thereby participating in the global radiation balance and ozone chemistry, but its biological sinks are not well understood. Thiobacillus thioparus strain THI115 can grow on thiocyanate (SCN(-)) as its sole energy source. Previously, we showed that SCN(-) is first converted to COS by thiocyanate hydrolase in T. thioparus strain THI115. In the present work, we purified, characterized, and determined the crystal structure of carbonyl sulfide hydrolase (COSase), which is responsible for the degradation of COS to H2S and CO2, the second step of SCN(-) assimilation. COSase is a homotetramer composed of a 23.4 kDa subunit containing a zinc ion in its catalytic site. The amino acid sequence of COSase is homologous to the ß-class carbonic anhydrases (ß-CAs). Although the crystal structure including the catalytic site resembles those of the ß-CAs, CO2 hydration activity of COSase is negligible compared to those of the ß-CAs. The α5 helix and the extra loop (Gly150-Pro158) near the N-terminus of the α6 helix narrow the substrate pathway, which could be responsible for the substrate specificity. The k(cat)/K(m) value, 9.6 × 10(5) s(-1) M(-1), is comparable to those of the ß-CAs. COSase hydrolyzes COS over a wide concentration range, including the ambient level, in vitro and in vivo. COSase and its structurally related enzymes are distributed in the clade D in the phylogenetic tree of ß-CAs, suggesting that COSase and its related enzymes are one of the catalysts responsible for the global sink of COS.

Extracellular histone induces plasma hyaluronan-binding protein (factor VII activating protease) activation in vivo.

Plasma hyaluronan-binding protein (PHBP), an activator of factor VII and prourokinase, is a serine protease circulating as a single-chain proenzyme (pro-PHBP). Pro-PHBP converts to the active two-chain form through autoproteolysis, and effectors that modulate autoactivation can regulate PHBP-mediated processes. Here, we show that histone promotes pro-PHBP autoactivation in vivo. Histone bound to pro-PHBP and promoted intermolecular pro-PHBP binding. Histone-mediated pro-PHBP activation in plasma leads to the formations of bradykinin and PHBP-α(2)-antiplasmin complex as well as histone degradation. Pro-PHBP activation was observed in the circulation of mice after injection of histone or lipopolysaccharide, which induced septic response accompanying extracellular histone release. Our results suggest pathophysiological relevance of histone-dependent pro-PHBP activation in hyperinflammatory process.

Over-expression of putative transcriptional coactivator KELP interferes with Tomato mosaic virus cell-to-cell movement.

Tomato mosaic virus (ToMV) encodes a movement protein (MP) that is necessary for virus cell-to-cell movement. We have demonstrated previously that KELP, a putative transcriptional coactivator of Arabidopsis thaliana, and its orthologue from Brassica campestris can bind to ToMV MP in vitro. In this study, we examined the effects of the transient over-expression of KELP on ToMV infection and the intracellular localization of MP in Nicotiana benthamiana, an experimental host of the virus. In co-bombardment experiments, the over-expression of KELP inhibited virus cell-to-cell movement. The N-terminal half of KELP (KELPdC), which had been shown to bind to MP, was sufficient for inhibition. Furthermore, the over-expression of KELP and KELPdC, both of which were co-localized with ToMV MP, led to a reduction in the plasmodesmal association of MP. In the absence of MP expression, KELP was localized in the nucleus and the cytoplasm by the localization signal in its N-terminal half. It was also shown that ToMV amplified normally in protoplasts prepared from leaf tissue that expressed KELP transiently. These results indicate that over-expressed KELP interacts with MP in vivo and exerts an inhibitory effect on MP function for virus cell-to-cell movement, but not on virus amplification in individual cells.

Activation of prothrombin by two subtilisin-like serine proteases from Acremonium sp.

Two novel subtilisin-like serine proteases (AS-E1 and -E2) that activate prothrombin have been identified in a culture of the fungus Acremonium sp. The enzymes were purified through repeated hydrophobic interaction chromatography. The N-terminal sequences of AS-E1 (34.4 kDa) and AS-E2 (32 kDa) showed high similarity to the internal sequences of two distinct subtilisin-like hypothetical proteins from Chaetomium globosum. Both enzymes proteolytically activated prothrombin to meizothrombin(desF1)-like molecules, while the activation cleavage seemed to occur at a site (Tyr(316)-Ile(317)) that is four residues proximal to the canonical Xa cleavage site (Arg(320)-Ile(321)). Both enzymes inhibited plasma clotting, possibly due to extensive degradation of fibrinogen and production of meizothrombin(desF1)-like molecule.

Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K.

Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, gammaCys131-SO(2)H. When the SCNase alpha, beta and gamma subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (alphabetagamma)(4), like native SCNase but exhibited no catalytic activity. Metal analysis indicated that SCNase was expressed as an apo-form irrespective of the presence of cobalt in the medium. On the contrary, SCNase co-expressed with P15K, encoded just downstream of SCNase genes, in cobalt-enriched medium under the optimized condition (SCNase((+P15K))) possessed 0.86 Co atom/alphabetagamma trimer and exhibited 78% of the activity of native SCNase. SCNase((+P15K)) showed a UV-Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase((+P15K)) had the gammaCys131-SO(2)H modification. These results indicate that SCNase((+P15K)) is the active holo-SCNase. P15K is likely to promote the functional expression of SCNase probably by assisting the incorporation of cobalt ion.

Brassinosteroid deficiency due to truncated steroid 5alpha-reductase causes dwarfism in the lk mutant of pea.

The endogenous brassinosteroids in the dwarf mutant lk of pea (Pisum sativum) were quantified by gas chromatography-selected ion monitoring. The levels of castasterone, 6-deoxocastasterone, and 6-deoxotyphasterol in lk shoots were reduced 4-, 70-, and 6-fold, respectively, compared with those of the wild type. The fact that the application of brassinolide restored the growth of the mutant indicated that the dwarf mutant lk is brassinosteroid deficient. Gas chromatography-selected ion monitoring analysis of the endogenous sterols in lk shoots revealed that the levels of campestanol and sitostanol were reduced 160- and 10-fold, respectively, compared with those of wild-type plants. These data, along with metabolic studies, showed that the lk mutant has a defect in the conversion of campest-4-en-3-one to 5alpha-campestan-3-one, which is a key hydrogenation step in the synthesis of campestanol from campesterol. This defect is the same as that found in the Arabidopsis det2 mutant and the Ipomoea nil kbt mutant. The pea gene homologous to the DET2 gene, PsDET2, was cloned, and it was found that the lk mutation would result in a putative truncated PsDET2 protein. Thus it was concluded that the short stature of the lk mutant is due to a defect in the steroidal 5alpha-reductase gene. This defect was also observed in the callus induced from the lk mutant. Biosynthetic pathways involved in the conversion of campesterol to campestanol are discussed in detail.

Interaction of tomato mosaic virus movement protein with tobacco RIO kinase.

Tomato mosaic virus (ToMV) has a regulatory gene encoding a movement protein (MP) that is involved in the cell-to-cell movement of viral RNA through plasmodesmata. To identify the host cell factors interacting with ToMV MP, we used a recombinant MP probe to isolate cDNA clones from a phage expression library of Nicotiana tabacum by a far-Western screening method. One of the cDNA clones encoded an MP-interacting protein, MIP-T7, homologous to the yeast novel protein kinase, Rio1p. We isolated a full-length cDNA by RT-PCR. The putative gene product was designated NtRIO, and shared 33 and 73% amino acid identity with yeast and Arabidopsis RIO kinases, respectively. In vitro analyses using recombinant proteins showed that NtRIO also interacted with a different MP derived from Cucumber mosaic virus. NtRIO had autophosphorylation activity and phosphorylated ToMV MP. Addition of recombinant tobacco casein kinase 2 resulted in a marked increase in the phosphorylation of NtRIO. The interaction between NtRIO and ToMV MP was inhibited by phosphorylation of NtRIO.

The catalytic subunit of protein kinase CK2 phosphorylates in vitro the movement protein of Tomato mosaic virus.

The movement protein (MP) of Tomato mosaic virus (ToMV) was reported previously by us to be phosphorylated in vitro by a cellular protein kinase(s) that exhibited several characteristics of casein kinase 2 (CK2). To characterize further this CK2-like cellular kinase, we have cloned cDNAs encoding the CK2 catalytic subunit from tobacco and compared the properties of the recombinant protein with those of the CK2-like cellular kinase. The recombinant CK2 catalytic subunit formed a complex with ToMV MP and phosphorylated it, similar to the CK2-like cellular kinase. Phosphoamino acid analyses of various mutant MPs altered near the C terminus revealed that the recombinant CK2 catalytic subunit phosphorylated serine-261, while the CK2-like cellular kinase phosphorylated both serine-261 and threonine-256. Both kinases were suggested to phosphorylate an additional serine residue(s) in regions other than the C-terminal peptide. The results are consistent with our previous prediction of involvement of CK2 in phosphorylation of ToMV MP.

Phosphorylation of the movement protein of cucumber mosaic virus in transgenic tobacco plants.

The 3a protein of Cucumber mosaic virus is essential for the cell-to-cell movement of the viral RNA through plasmodesmata. We have introduced an epitope peptide before the stop codon of the 3a protein and cloned the tagged ORF into a binary vector for Agrobacterium-mediated transformation. The established transgenic tobacco lines produced the 3a protein, which was specifically detected with anti-3a and anti-epitope antisera. Metabolic labeling and subsequent immunoprecipitation revealed that [32P]-orthophosphate was incorporated into the 3a protein. The phosphoamino acid analysis indicated that the 3a protein contained phosphoserine but not phosphothreonine or phosphotyrosine. This is the first demonstration of the 3a protein phosphorylation in planta.

Cloning of a tobacco cDNA coding for a putative transcriptional coactivator MBF1 that interacts with the tomato mosaic virus movement protein.

Viral movement through plasmodesmata in host plants depends on the interaction between virus-encoded movement protein (MP) and host proteins. To search for MP-interacting protein (MIP), far-western screening of a tobacco cDNA library was carried out using a recombinant MP of tomato mosaic virus (ToMV) as a probe. One of the positive cDNA clones, designated MIP204, was highly homologous to a class of transcriptional coactivators commonly referred to as multiprotein bridging factor 1 (MBF1). ToMV MP could also bind to the Arabidopsis homologues of MBF1. The recombinant MIP204 bound to MPs of ToMV and a crucifer tobamovirus CTMV-W, but not of cucumber mosaic virus. MPs of ToMV and the related virus may interact with MBF1-like proteins to modulate host gene expression.

Isolation of a cDNA for a nucleoside diphosphate kinase capable of phosphorylating the kinase domain of the self-incompatibility factor SRK of Brassica campestris.

SRK is a plant receptor kinase involved in the self-incompatibility system of Brassica species. During a cDNA screening for the phosphoproteins from a stigma expression library, a clone encoding the nucleoside diphosphate kinase III (Bc-NDPK III) was obtained. After in vitro phosphorylation assays with recombinant proteins, Bc-NDPK III contained mostly phosphoserine. By contrast, the kinase domain of SRK contained phosphoserine and phosphothreonine, both of which were significantly increased by the addition of Bc-NDPK III in the presence of an SRK inhibitor KN-62. The result suggested the possible involvement of Bc-NDPK III in the signal transduction pathway through SRK.

Genetic and immunochemical characterization of thiocyanate-degrading bacteria in lake water.

Natural aquatic and soil samples were screened for the presence of thiocyanate-degrading bacteria. Using thiocyanate supplementation, we established an enrichment culture containing such bacteria from lake water. The dominant bacteria had the scnC-LS5 gene encoding thiocyanate hydrolase, which was closely related to the enzyme found previously in Thiobacillus thioparus THI115 isolated from activated sludge.

In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.

The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.