Biochemical and functional characterization of a eukaryotic-type protein kinase,SpkB, in the cyanobacterium,Synechocystis sp. PCC 6803

On the basis of the genome sequence, the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for eukaryotic-type protein kinase belonging to Pkn2 subfamily ( spkA approximately spkG). Previously, SpkA was shown to have protein kinase activity and to be required for cell motility. Here, the role of the spkB was examined. The spkB gene was expressed in Escherichia coli as a fusion protein with His-tag, and the protein was purified by Ni(2+) affinity chromatography. The eukaryotic-type protein kinase activity of the expressed SpkB was demonstrated as autophosphorylation to itself and phosphorylation of the general substrate proteins. SpkB showed autophosphorylation activity in the presence of both Mg(2+) and Mn(2+), but not in Ca(2+). Phenotype analysis of spkB disruptant of Synechocystis revealed that spkB is required for cell motility, but not for phototaxis. These results suggest that SpkB is the eukaryotic-type protein kinase, which regulates cellular motility via protein phosphorylation like SpkA.     

Cloning and nucleotide sequence analysis of the chloramphenicol resistance gene on conjugative R plasmids from the fish pathogen Photobacterium damselae subsp. piscicida

Transferable resistance to various drugs was investigated in Photobacterium damselae subsp. piscicida from Japan. Drug resistances were transferred via plasmids of 100, 50, and 40 kb. Resistance to chloramphenicol (Cmr) was transferred on plasmids of all 3 sizes. The Cmr gene (cat) was cloned from the 50 kb plasmids pPDP8511 and pPDP9106 transferred from P. damselae subsp. piscicida strains isolated in different years and places in Japan. Subcloning localized the cat to within 1.5 kb HindIII-HincII (or PstI) fragments. Nucleotide sequences of the coding and flanking region of the cat were determined as 1607 bp (HindIII-HincII fragment) in pPDP8511 and 1568 bp (HindIII-PstI fragment) in pPDP9106, which corresponded with the sequence from nucleotides 40 to 1607 in pPDP8511. The nucleotide sequences identified an open reading frame (ORF) encoding 213 amino acid residues with a calculated molecular mass of about 24.8 kDa, a size consistent with the molecular mass of known cat gene products, and the ORF had maximum homology (99.5%) with a Type II CAT variant from Haemophilus influenzae.     

Molecular cloning,sequencing and expression of the gene encoding a novel chitinase A from a marine bacterium, <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. PE2, and its domain structure

The pchA gene encoding chitinase A (PchA) from a Pythium porphyrae cell-wall-degrading marine bacterium, Pseudomonas sp. PE2, was cloned and characterized. The deduced PchA was a modular enzyme composed of an N-terminal signal peptide, a glycoside hydrolase family 18 catalytic domain that was responsible for the chitinase activity, the chitin-binding domains (ChBDs), and the carbohydrate-binding modules (CBM). The amino acid sequence of ChBD(PchA) was highly conserved in the CBM family 12 that also accommodates ChBDs without an AKWWTQG motif, a domain commonly found in bacterial chitinase and Streptomyces griseus protease C. Interestingly, CBM(PchA) showed significant sequence homology to the C-terminal region of endoglucanase B from Cellvibrio mixtus, which is a member of CBM family 6. This is the first report of a chitinase possessing a domain with high similarity to CBM family 6. Deletion analysis indicated clearly that ChBD(PchA) might play an important role in the binding of native chitin and chitosan, but not processed chitin. CBM(PchA) also appeared to play such a role in the binding of xylan and Avicel. These results suggest that the C-terminal region of PchA might be a key component in the binding of chitin in the cell walls of P. porphyrae or other structural components of marine organisms.     

Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80-93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.     

Enzymatic synthesis of high-molecular-mass poly-gamma-glutamate and regulation of its stereochemistry

For the first time, we succeeded in synthesizing in vitro poly-gamma-glutamate (PGA) with high molecular masses (>1,000 kDa) by the use of enzyme-associated cell membranes from Bacillus subtilis subsp. chungkookjang. The activity for PGA synthesis, however, was readily lost in the presence of critical concentrations of detergents tested in micelles. The optimum pH for the reaction was found to be approximately 7.0. We examined the effects of some divalent cations on PGA synthesis and found that Mg(2+) was essential in catalysis and that Zn(2+) additionally boosted the activity. In contrast, Fe(2+) and Ca(2+) acted as inhibitors. Mn(2+) did not apparently influence the in vitro formation of PGA. DL-Glutamate (D isomer content, 60 to 80%) apparently served as the best substrate; d-Glutamate was preferable to the L isomer as a substrate. When D- and L-glutamate were used for the reaction, the elongated chains of PGAs were composed of the D- and L-isomers, respectively. Our results suggest that the stereochemical properties of enzymatically synthesized PGAs substantially depend on the stereochemistry (DL ratio) of glutamate as the substrate. Furthermore, genetic analysis indicated that all the pgsB, -C, and -A gene products, which are responsible for PGA production by B. subtilis cells, were also indispensable for enzymatic PGA synthesis.     

Generation of influenza A virus NS2 (NEP) mutants with an altered nuclear export signal sequence

The NS2 (NEP) protein of influenza A virus contains a highly conserved nuclear export signal (NES) motif in its amino-terminal region (12ILMRMSKMQL21, A/WSN/33), which is thought to be required for nuclear export of viral ribonucleoprotein complexes (vRNPs) mediated by a cellular export factor, CRM1. However, simultaneous replacement of three hydrophobic residues in the NES with alanine does not affect NS2 (NEP) binding to CRM1, although the virus with these mutations is not viable. To determine the extent of sequence conservation required by the NS2 (NEP) NES for its export function during viral replication, we randomly introduced mutations by degenerative mutagenesis into the region of NS cDNA encoding the NS2 (NEP) NES and then attempted to generate mutant viruses containing these alterations by reverse genetics. Sequence analysis of the recovered viruses showed that although some of the mutants possessed amino acids other than those conserved in the NES, hydrophobicity within this motif was maintained. Nuclear export of vRNPs representing all of the mutant viruses was completely inhibited in the presence of a CRM1 inhibitor, leptomycin B, as was the transport of wild-type virus, indicating that the CRM1-mediated pathway is responsible for the nuclear export of both wild-type and mutant vRNPs. The vRNPs of some of the mutant viruses were exported in a delayed manner, resulting in limited viral growth in cell culture and in mice. These results suggest that the NES motif may be an attractive target for the introduction of attenuating mutations in the production of live vaccine viruses.     

Development of immune cellular biosensing system for assessing chemicals on inducible nitric oxide synthase signaling activator

An immune cellular biosensing system has been constructed to assess immunomodulating effects of chemicals. Production of nitric oxide in the immune cellular biosensing system was used as readout of an immune cellular response for assessing the immunomodulating effects of chemicals. The macrophage-like cell line RAW264.7, which has signaling pathways of inducible nitric oxide synthase, was employed in the cellular biosensing system. The immune cellular biosensing system consisted of a Pt counter electrode, an Ag/AgCl reference electrode, and a gold electrode onto which a polyion complex layer was coated to allow adherence of the RAW264.7 cells. As the results of evaluating effects of a polyion complex layer on cell viabilities by using WST-8 assay, the polyion complex layer did not affect RAW264.7 cells. The polyion-coated gold electrode could measure NO without the drawback of electrochemical interference that occurs with differential pulse voltammetry. The detection limit of the immune cellular biosensing system was 4.2 nM released NO as measured by double potential step chronoamperometry. The potent immune activating abilities of lipopolysaccharide and interferon-gamma could be assessed by the cellular biosensing system; NO release from cells was detected within 600 ms.