A Phylogenomic Study of the OCTase Genes in Pseudomonas syringae Pathovars: The Horizontal Transfer of the argK–tox Cluster and the Evolutionary History of OCTase Genes on Their Genomes

Phytopathogenic Pseudomonas syringae is subdivided into about 50 pathovars due to their conspicuous differentiation with regard to pathogenicity. Based on the results of a phylogenetic analysis of four genes (gyrB, rpoD, hrpL, and hrpS), Sawada et al. (1999) showed that the ancestor of P. syringae had diverged into at least three monophyletic groups during its evolution. Physical maps of the genomes of representative strains of these three groups were constructed, which revealed that each strain had five rrn operons which existed on one circular genome. The fact that the structure and size of genomes vary greatly depending on the pathovar shows that P. syringae genomes are quite rich in plasticity and that they have undergone large-scale genomic rearrangements. Analyses of the codon usage and the GC content at the codon third position, in conjunction with phylogenomic analyses, showed that the gene cluster involved in phaseolotoxin synthesis (argK–tox cluster) expanded its distribution by conducting horizontal transfer onto the genomes of two P. syringae pathovars (pv. actinidiae and pv. phaseolicola) from bacterial species distantly related to P. syringae and that its acquisition was quite recent (i.e., after the ancestor of P. syringae diverged into the respective pathovars). Furthermore, the results of a detailed analysis of argK [an anabolic ornithine carbamoyltransferase (anabolic OCTase) gene], which is present within the argK–tox cluster, revealed the plausible process of generation of an unusual composition of the OCTase genes on the genomes of these two phaseolotoxin-producing pathovars: a catabolic OCTase gene (equivalent to the orthologue of arcB of P. aeruginosa) and an anabolic OCTase gene (argF), which must have been formed by gene duplication, have first been present on the genome of the ancestor of P. syringae; the catabolic OCTase gene has been deleted; the ancestor has diverged into the respective pathovars; the foreign-originated argK–tox cluster has horizontally transferred onto the genomes of pv. actinidiae and pv. phaseolicola; and hence two copies of only the anabolic OCTase genes (argK and argF) came to exist on the genomes of these two pathovars. Thus, the horizontal gene transfer and the genomic rearrangement were proven to have played an important role in the pathogenic differentiation and diversification of P. syringae. Received: 22 May 2001 / Accepted: 26 September 2001     

Protein crystallization under high pressure

Pressure is expected to be an important parameter to control protein crystallization, since hydrostatic pressure affects the whole system uniformly and can be changed very rapidly. So far, a lot of studies on protein crystallization have been done. Solubility of protein depends on pressure. For instance, the solubility of tetragonal lysozyme crystal increased with increasing pressure, while that of orthorhombic crystal decreased. The solubility of subtilisin increased with increasing pressure. Crystal growth rates of protein also depend on pressure. The growth rate of glucose isomerase was significantly enhanced with increasing pressure. The growth rate of tetragonal lysozyme crystal and subtilisin decreased with increasing pressure. To study the effects of pressure on the crystallization more precisely and systematically, hen egg white lysozyme is the most suitable protein at this stage, since a lot of data can be used. We focused on growth kinetics under high pressure, since extensive studies on growth kinetics have already been done at atmospheric pressure, and almost all of them have explained the growth mechanisms well. The growth rates of tetragonal lysozyme decreased with pressure under the same supersaturation. This means that the surface growth kinetics significantly depends on pressure. By analyzing the dependence of supersaturation on growth rate, it was found that the increase in average ledge surface energy of the two-dimensional nuclei with pressure explained the decrease in growth rate. At this stage, it is not clear whether the increase in surface energy with increasing pressure is the main reason or not. Fundamental studies on protein crystallization under high pressure will be useful for high pressure crystallography and high pressure protein science.     

Activation of maturation promoting factor and 26S proteasome assembly accelerated by a high concentration of 1-methyladenine in starfish oocytes

In the oocyte maturation process of the starfish Asterina pectinifera, the extent of inhibition of germinal vesicle breakdown (GVBD) by the proteasome inhibitor MG115 (benzyloxycarbonyl-leucyl-leucyl-norvalinal), as well as the timing of activation of pre-MPF (inactive maturation promoting factor) and 26S proteasome assembly, were found to be dependent on the concentration of the maturation-inducing hormone 1-methyladenine (1-MeAde). Activation of pre-MPF was accelerated by increasing the concentration of 1-MeAde, while there was little effect on the time required for GVBD. Assembly of the 26S proteasome was also accelerated by increasing the concentration of 1-MeAde. These results indicate that a higher concentration of 1-MeAde triggers acceleration of the assembly and increase in the activity of the 26S proteasome, which results in activation of pre-MPF, although there is little effect on the timing of GVBD. It was also clarified that the timing of GVBD is controlled by a rate-liming step after MPF-activation.     

Characterization of the human NTAK gene structure and distribution of the isoforms for rat NTAK mRNA

NTAK (neural- and thymus-derived activator for the ErbB kinase, neuregulin-2) is a novel member of the epidermal growth factor (EGF) family. We have isolated and characterized the human NTAK gene, comprising 12 exons spanning in excess of 55 kilobases (kb). The 7. 0kb long mRNA of the human NTAK gene was expressed in the human neuroblastoma SK-N-SH cell line with two alternative isoforms detected. Furthermore, six isoforms have been identified from rat brain and PC-12 cells. Although the alpha isoform of the NTAK gene was found to be expressed in all tissues including brain, the beta isoform was expressed only in rat brain tissues. Potential regulatory regions included consensus binding sites for AP-2, TF-IIIA, Sp-1, and YY-1 located in the 5'-flanking region of the NTAK gene.     

Characterization of iap gene in Listeria monocytogenes strains isolated in Japan

Variation of the iap gene region (407bp) encoding an invasion-associated protein p60 was studied on 12 strains of Listeria monocytogenes of different origin in Japan. These 12 strains are known to have 2 types of serotype (1/2a and 4b) and have a diversity among the strains (Saito et al., 1998). The dye-primer cycle sequencing method was employed to determine the genomic structure, and the nucleotide sequences obtained were compared with those of reference strain SV 1/2a EGD. Differences found in the nucleotides were as follows; point mutations of 33 variations in 32 places; an insertion and 3 deletions of 3 bases; AAT position (po.) 1282-1283, and GCA po. 1307-1309, ACA po. 1412-1414, AAT po. 1439-1444, respectively. Different repeating numbers by 6 base unit, ACA AAT, were also found in the tandem repeat region (po. 1394-1423). Classification of 12 strains was attempted, then 8, 4 and 5 types were obtained from the point mutations, the insertions and deletions, and the repeating numbers, respectively. Consequently, 8 patterns were profiled regardless of each serotype. From these results, genomic structures were partially clarified in the iap gene 407bp of L. monocytogenes isolated in Japan. Then, the possibility of detailed epidemiology for L. monocytogenes infection using a combination of serotype and genome structure was suggested because of the previous polymorphism thought to be due to the nucleotide differences in the region.     

Ascidian sperm lysin system

Fertilization is a precisely controlled process involving many gamete molecules in sperm binding to and penetration through the extracellular matrix of the egg. After sperm bind to the extracellular matrix (vitelline coat), they undergo the acrosome reaction which exposes and partially releases a lytic agent called "lysin" to digest the vitelline coat for the sperm penetration. The vitelline coat sperm lysin is generally a protease in deuterostomes. The molecular mechanism of the actual degradation of the vitelline coat, however, remains poorly understood. In order to understand the lysin system, we have been studying the fertilization mechanism in ascidians (Urochordata) because we can obtain large quantities of gametes which are readily fertilized in the laboratory. Whereas ascidians are hermaphrodites, which release sperm and eggs simultaneously, many ascidians, including Halocynthia roretzi, are strictly self-sterile. Therefore, after sperm recognize the vitelline coat as nonself, the sperm lysin system is thought to be activated. We revealed that two sperm trypsin-like proteases, acrosin and spermosin, the latter of which is a novel sperm protease with thrombin-like substrate specificity, are essential for fertilization in H. roretzi. These molecules contain motifs involved in binding to the vitelline coat. We found that the proteasome rather than trypsin-like proteases has a direct lytic activity toward the vitelline coat. The target for the ascidian lysin was found to be a 70-kDa vitelline coat component called HrVC70, which is made up of 12 EGF-like repeats. In addition to the proteasome system, the ubiquitination system toward the HrVC70 was found to be necessary for ascidian fertilization. In this review, I describe recent progress on the structures and roles in fertilization of the two trypsin-like proteases, acrosin and spermosin, and also on the novel extracellular ubiquitin-proteasome system, which plays an essential role in the degradation of the ascidian vitelline coat.     

Photosynthesis with single-rooted Amaranthus leaves. II. Regulation of ribuelose-1,5-bisphosphate carboxylase,phosphoenolpyruvate carboxylase,NAD-malic enzyme and NAD-malate dehydrogenase and coordination between PCR and C4 photosynthetic metabolism in response to changes in the source-sink balance

We have studied source-sink relationships with a model consisting of single-rooted leaves without petioles. We previously reported that the rate of photosynthesis decreased when C4 model plants prepared from Amaranthus cruentus leaves were subjected to sink-limited conditions by exposure to continuous light for a few days. It was suggested that the inhibition is due to a coordinated decrease in the activity of ribulose-1,5-bisphosphate carboxylase (RuBPcase) and phosphoenol-pyruvate carboxylase (PEPcase), both essential enzymes for photosynthesis in C4 plants. We further investigated the mechanisms behind the decreased activity of RuBPcase, PEPcase, NAD-malic enzyme and NAD-malate dehydrogenase. The results suggested that (1) the initial activity of RuBPcase is suppressed by a lowering of the P(i) level in chloroplasts, (2) the inhibition of PEPcase is due to dephosphorylation of the enzyme via the inhibition of PEPcase kinase and PEPcase phosphatase, (3) the inhibition of NAD-malic enzyme and NAD-malate dehydrogenase is derived from the oxidation of these enzymes, and (4) some proteinous factor(s) may be involved in the inhibition of the activity of these latter three enzymes. The significance of a coordinated decrease in these enzymes in response to a change in the source-sink balance is discussed.