Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglossicida, as a candidate for disease control

Two types of bacteriophage specific to Pseudomonas plecoglossicida, the causative agent of bacterial hemorrhagic ascites disease in cultured ayu fish (Plecoglossus altivelis), were isolated from diseased ayu and the rearing pond water. One type of phage, which formed small plaques, was tentatively classified as a member of the family Myoviridae, and the other type, which formed large plaques, was classified as a member of the family Podoviridae. All 27 strains of P. plecoglossicida examined, which were isolated from diseased ayu from geographically different areas in 1991 to 1999, exhibited quite similar sensitivities to either type of phage. One strain of P. plecoglossicida was highly virulent for ayu, and the 50% lethal dose (LD(50)) when intramuscular injection was used was 10(1.2) CFU fish(-1); in contrast, phage-resistant variants of this organism were less virulent (LD(50), >10(4) CFU fish(-1)). Oral administration of phage-impregnated feed to ayu resulted in protection against experimental infection with P. plecoglossicida. After oral administration of P. plecoglossicida cells of this bacterium were always detected in the kidneys of control fish that did not receive the phage treatment, while the cells quickly disappeared from the phage-treated fish. Bacterial growth in freshwater was lower in the presence of phage, and the number of phage PFU increased rapidly. These results suggest that it may be possible to use phage to control the disease caused by P. plecoglossicida.     

Analysis of gyrB and toxR gene sequences of Vibrio hollisae and development of gyrB- and toxR-targeted PCR methods for isolation of V. hollisae from the environment and its identification

Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio (toxR). A portion of the gyrB sequence of V. hollisae was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the corresponding Vibrio parahaemolyticus gyrB sequence. The toxR gene of V. hollisae was cloned utilizing a htpG gene probe derived from the V. parahaemolyticus htpG gene, which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized for differentiation of V. hollisae from other Vibrio species and from microorganisms found in marine fish. PCR methods targeting the two gene sequences were established. Both PCR methods were shown to specifically detect the respective target sequences of V. hollisae but not other organisms. A strain of V. hollisae added at a concentration of 1 to 10(2) CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37 degrees C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the V. hollisae toxR gene. We conclude that screening of seafood samples by this 35-cycle, V. hollisae toxR-specific PCR, followed by isolation on a differential medium and identification by the above htpG- and toxR-targeted PCR methods, can be useful for isolation from the environment and identification of V. hollisae.     

Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.     

Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.     

Phosphorylation of tau is regulated by PKN

For the phosphorylation state of microtubule-associated protein, tau plays a pivotal role in regulating microtubule networks in neurons. Tau promotes the assembly and stabilization of microtubules. The potential for tau to bind to microtubules is down-regulated after local phosphorylation. When we investigated the effects of PKN activation on tau phosphorylation, we found that PKN triggers disruption of the microtubule array both in vitro and in vivo and predominantly phosphorylates tau in microtubule binding domains (MBDs). PKN has a catalytic domain highly homologous to protein kinase C (PKC), a kinase that phosphorylates Ser-313 (= Ser-324, the number used in this study) in MBDs. Thus, we identified the phosphorylation sites of PKN and PKC subtypes (PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -zeta, and -lambda) in MBDs. PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352. There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a novel phosphorylation-dependent antibody recognizing phosphorylated tau at Ser-320, showed immunoreactivity in Chinese hamster ovary cells expressing tau and the active form of PKN, but not in Chinese hamster ovary cells expressing tau and the inactive form of PKN. The immunoreactivity for phosphorylated tau at Ser-320 increased in the presence of a phosphatase inhibitor, FK506 treatment, which means that calcineurin (protein phosphatase 2B) may be involved in dephosphorylating tau at Ser-320 site. We also noted that PKN reduces the phosphorylation recognized by the phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by specific phosphorylation of tau, which leads to disruption of tubulin assembly.     

Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors

A major shortcoming to the use of adeno-associated virus (rAAV) vectors is their limited packaging size. To overcome this hurdle, we split an expression cassette and cloned it into two separate vectors. The vectors contained either a nuclear localizing Escherichia coli lacZ transgene (nlslacZ) with a splice acceptor, or the human elongation factor 1alpha ( EF1alpha) gene enhancer/promoter(s) (EF1alphaEP) with a splice donor. We co-injected a promoter-less nlslacZ vector with a vector containing either a single EF1alphaEP or a double copy of the EF1alphaEP in a head-to-head orientation, into the portal vein of mice. Gene expression, measured by both transduction efficiency and quantitation of the recombinant protein, was as much as 60-70% of that obtained from mice that received a single vector containing a complete EFalphaEP/nlslacZ expression cassette. This two-vector approach may allow development of gene therapy strategies that will carry exogenous DNA sequences with large therapeutic cDNAs and/or regulatory elements.     

External chiral ligand-induced enantioselective versions of the [2,3]-Wittig sigmatropic rearrangement

The external chiral ligand-induced enantioselective [2,3]-Wittig rearrangements of crotyl benzyl ethers and crotyl propargylic ethers are described. The most notable is that treatment of (E)-crotyl propargylic ethers with a t-butyllithium/(S;S)-bis(oxazoline) complex provides a relatively high enantioselectivity (up to 89% ee), together with a high threo-diastereoselectivity. Furthermore, examples of the "asymmetric catalytic version" of the rearrangement of crotyl benzyl ethers are presented.     

Purification and characterization of cyclohexanone 1,2-monooxygenase from Exophiala jeanselmei strain KUFI-6N

Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer-Villiger monooxygenases.