Cloning and characterization of the Pseudomonas sp. 61-3 phaG gene involved in polyhydroxyalkanoate biosynthesis.

Pseudomonas sp. 61-3 produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] random copolymer consisting of monomeric units of 4-12 carbon atoms from sugars. The phaG(Ps) gene encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase was cloned from this strain, and homologous expression of this gene under the control of the lac or the native promoter was investigated. Additional copies of the phaG(Ps) gene in Pseudomonas sp. 61-3 led to an increase in both the polyhydroxyalkanoate (PHA) content in the cells and the fraction of medium-chain-length 3HA units in PHA. Disruption of the chromosomal phaG(Ps) gene resulted in an increase in the fraction of the 3HB unit in PHA. The site-directed mutagenesis of the phaG(Ps) gene was carried out to investigate the role of a HX(4)D motif which has been proposed to be related to PhaG activity.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) copolymer from sugars by recombinant Ralstonia eutropha harboring the phaC1Ps and the phaGPs genes of Pseudomonas sp. 61-3.

Heterologous expression of the phaGPs and the phaClPs genes encoding 3-hydroxyacyl acyl carrier protein-coenzyme A transacylase and polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp. 61-3, respectively, was performed in the phbCRe negative mutant, Ralstonia eutropha PHB-4. The recombinant strain of the R. eutropha PHB-4 produced PHA copolymers consisting of 3-hydroxybutyrate (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-3HA) units of 6-12 carbon atoms from sugars. The 3HB fraction in copolymers was very high (95-97 mol%). The PHA content in the recombinant strain could further be increased by the additional introduction of the phbABRe genes from R. eutropha encoding beta-ketothiolase and NADPH-depedent acetoacetyl-coenzyme A reductase. Differential scanning calorimetry analysis of the PHA copolymers produced by the recombinant R. eutropha PHB-4 have indicated that the PHA is a random copolymer of 3HB and mcl-3HA units.

The effect of osmotic stress on the production of nukacin ISK-1 from Staphylococcus warneri ISK-1.

The effects of several additives on the production of a lantibiotic, nukacin ISK-1, from Staphylococcus warneri ISK-1, in batch fermentation were studied. NaCl, KCl and sorbitol stimulated nukacin ISK-1 production. The addition of 1.4 M NaCl increased nukacin ISK-1 activity 1.5-fold over the control, while cell growth and glucose consumption were inhibited. Nukacin ISK-1 production increased with increasing osmolarity of the medium up to about 3 osmol/kg; however, further increases in osmolarity diminished productivity, irrespective of the kind of additive. Northern blot analysis showed that transcription of the nukacin ISK-1 structural gene (nukA) was activated in the presence of 1.4 M NaC1. These data indicate that the stimulation effect was due to osmotic stress, which acted, at least in part, at the transcriptional level on the nukA gene.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3.

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4-12 carbon atoms. The genes encoding beta-ketothiolase (PhbA(Re)) and NADPH-dependent acetoacetyl-CoA reductase (PhbB(Re)) from Ralstonia eutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1(Ps) gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB(-4) to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6-12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1Ps gene only. In addition, recombinant strains of R. eutropha PHB(-4) produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB(-4)/ pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units.

Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis.

Two Pseudomonas aeruginosa genes, termed phaJ1(Pa) and phaJ2(Pa), homologous to the Aeromonas caviae (R)-specific enoyl-CoA hydratase gene (phaJ(Ac)) were cloned using a PCR technique to investigate the monomer-supplying ability for polyhydroxyalkanoate (PHA) synthesis from beta-oxidation cycle. Two expression plasmids for phaJ1(Pa) and phaJ2(Pa) were constructed and introduced into Escherichia coli DH5alpha strain. The recombinants harboring phaJ1(Pa) or phaJ2(Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities, that is, specific for short chain-length enoyl-CoA or medium chain-length enoyl-CoA, respectively. In addition, co-expression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14-29 wt% of cell dry weight from dodecanoate as a sole carbon source. It has been suggested that phaJ1(Pa) and phaJ2(Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle.

A novel lantibiotic, nukacin ISK-1, of Staphylococcus warneri ISK-1: cloning of the structural gene and identification of the structure.

Staphylococcus warneri ISK-1, which we had previously reported as Pediococcus sp. ISK-1, produces a novel bacteriocin, nukacin ISK-1. Edman degradation of the chemically reduced nukacin ISK-1 produced a sequence of 27 amino acids, 7 of which were unidentified. Using single-specific-primer-PCR product as a probe, a 3.6-kb HindIII fragment containing the nukacin ISK-1 structural gene (nukA) was cloned and sequenced. The deduced amino acid sequence of nukacin ISK-1 had 57 amino acids, including a 30-amino acid leader region. The propeptide sequence showed significant similarity to those of lacticin-481 type lantibiotics. In the region upstream of nukA, a part of a long open reading frame (ORF), designated as nukM, encoding a putative modification enzyme was oriented in the opposite direction. In the region downstream of nukA, ORF1 was found in which the sequence of the putative translational product was similar to various response regulatory proteins.

Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant strains of Pseudomonas sp. 61-3.

Pseudomonas sp. 61-3 (phbC::tet) strain, which is a phbCPs-disrupted mutant, accumulated a random copolymer consisting of (R)-3-hydroxybutyrate (3HB) and (R)-medium-chain-length 3-hydroxyalkanoate (3HA) units of 6-12 carbon atoms, but the 3HB fraction in the copolymer was less than 50 mol %, resulting in the formation of an amorphus polymer. Therefore, the genes encoding beta-ketothiolase (PhbARe) and NADPH-dependent acetoacetyl-CoA reductase (PhbBRe) from Ralstonia eutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with the phaC1Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in the phbCPs-disrupted mutant. The recombinant strains synthesized P(3HB-co-3HA) copolymers with very high 3HB compositions (up to 94 mol %) from glucose. The number-average molecular weights of P(3HB-co-3HA) were in the range of 349 x 10(3) to 605 x 10(3). The structure and physical properties of P(3HB-co-3HA) copolymers were characterized by 1H- and 13C-NMR spectrometry, differential scanning calorimetry, and mechanical tensile measurement. P(94% 3HB-co-3HA) copolymer was demonstrated to have good physical properties and to be a flexible material with moderate toughness, similar to low-density polyethylene.

Co-expression of 3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHA production in Escherichia coli HB101 strain.

The Escherichia coli 3-ketoacyl-ACP reductase gene (fabGEc) was cloned using a PCR technique to investigate the metabolic link between fatty acid metabolism and polyhydroxyalkanoate (PHA) production. Three plasmids respectively harboring fabGEc and the poly-3-hydroxyalkanoate synthesis genes phaCAc and phaC1Ps from Aeromonas caviae and Pseudomonas sp. 61-3 respectively were constructed and introduced into E. coli HB101 strain. On a two-stage cultivation using dodecanoate as the sole carbon source, recombinant E. coli HB101 strains harboring fabGEc and phaC genes accumulated PHA copolymers (about 8 wt% of dry cell weight) consisting of several (R)-3-hydroxyalkanoate units of C4, C6, C8, and C10. It has been suggested that overexpression of the fabGEc gene leads to the supply of (R)-3-hydroxyacyl-CoA for PHA synthesis via fatty acid degradation.

Intravenous bolus infusion of heparin for circulatory insufficiency after finger replantation.

When heparin 2,500 to 5,000 U was administered by intravenous bolus infusion to 13 fingers that had developed arterial thrombosis after replantation, complete survival was achieved in seven of 13 fingers. If fingers demonstrating partial necrosis were included, survival was obtained in 11 of 13 fingers. In nine fingers in which survival was obtained and follow-up observation was possible, sensory recovery was favorable, and there was no limitation in the use of the affected fingers, although mild atrophy of the pulp and nail deformity were noted in fingers with partial necrosis. Using a model of arterial thrombosis created in the carotid arteries of Sprague-Dawley rats, the authors investigated how the intravenous bolus infusion of heparin affected thrombolysis. During the early phase of arterial thrombosis when the thrombus is fragile, the intravenous bolus of heparin affected the balance between coagulation and fibrinolysis, facilitating the thrombolytic process, and increasing the rate of recanalization of the occluded arteries. Since an intravenous bolus injection of heparin is an easy procedure, without the risk of any severe side effects, this method should be considered in such cases of arterial thrombosis, with attention paid to the general condition of the patient.

Cloning and molecular analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3.

Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer (poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]) consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In the phb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), beta-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed by phbBPs-phbAPs-phbCPs. Upstream from phbBPs was found the phbRPs gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbRPs gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbRPs in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of beta-galactosidase activity from a transcriptional phb promoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPs is a positive regulatory protein controlling the transcription of phbBACPs in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) were flanked by a PHA depolymerase gene (phaZPs), and two adjacent open reading frames (ORF1 and phaDPs), and the gene order was ORF1, phaC1Ps, phaZPs, phaC2Ps, and phaDPs. Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida and Ralstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.

Purification and Partial Identification of Bacteriocin ISK-1, a New Lantibiotic Produced by Pediococcus sp. ISK-1.

Bacteriocin ISK-1 is a proteinaceous inhibitory substance produced by Pediococcus sp. ISK-1 isolated from well-aged Nukadoko. Bacteriocin ISK-1 was purified by acid treatment, ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase HPLC from the culture supernatant of Pediococcus sp. ISK-1. Purification of bacteriocin ISK-1 resulted in a 30-fold increase in the specific activity and the recovery was 17%. Molecular mass of bacteriocin ISK-1 measured by fast atom bombardment-mass spectrometry was 2,960. The amino acid composition analysis of bacteriocin ISK-1 showed that it contained unusual amino acids such as lanthionine and/or 3-methyllanthionine, which is a characteristic of lantibiotics. The N-terminal amino acid sequence analysis indicated the first seven N-terminal amino acid residues as NH2-K-K-K-S-G-V-I. The primary sequence showed significant similarity to the lantibiotics lacticin 481 from Lactococcus lactis and variacin from Micrococcus varians, which suggests that bacteriocin ISK-1 is a novel lantibiotic belonging to a lacticin-481 type.

Nisin Z Production by Lactococcus lactis IO-1 Using Xylose as a Carbon Source.

Lactococcus lactis IO-1 was able to use xylose as a carbon source for nisin Z production; the yield based on sugar consumption was about 20% superior to that made with glucose under the same fermentation conditions. The optimal conditions for nisin Z production were with 4% xylose at pH 6.0 and 37°C. Addition of 0.1 M CaCl2 increased nisin Z production specifically, but not cell growth, acid production, or xylose consumption, and resulted in the maximum nisin Z activity of about 1.5 times that without CaCl2.

A bacteriocin of strain Pediococcus sp. ISK-1 isolated from Nukadoko, bed of fermented rice bran.

Pediococcus sp. ISK-1 isolated in our laboratory from well-aged Nukadoko, produces a bacteriocin which has a unique antimicrobial spectrum among pediocins. The bacteriocin was stable at acidic pH, and more than 60% of antimicrobial activity still remained even after being autoclaved at 121 degrees C for 20 min in the pH range of 3 to 8. This is the first report dealing with a bacteriocin produced by lactic acid bacteria isolated from Nukadoko.

Lantibiotic nisin Z fermentative production by Lactococcus lactis IO-1: relationship between production of the lantibiotic and lactate and cell growth.

The influence of several parameters on the fermentative production of nisin Z by Lactococcus lactis IO-1 was studied. Considerable attention has been focused on the relationship between the primary metabolite production of bacteriocin and lactate and cell growth, which has so far not been clarified in detail. Production of nisin Z was optimal at 30 degrees C and in the pH range 5.0-5.5. The addition of Ca2+ to the medium showed a stimulating effect on the production of nisin Z. A maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl2. It was about three times higher than that obtained under the optimal conditions for cell growth and lactic acid production.

Anterior stabilization, instrumentation, and decompression for post-traumatic kyphosis.

Thirty-seven patients underwent surgery for late post-traumatic kyphosis in the lumbar, thoracolumbar, or thoracic spine. Indications for surgery included: increasing deformity, pain, and persistent neurologic deficit with paraparesis in eight, and development of late spinal stenosis in a further nine patients. All patients underwent anterior correction with Kostuik-Harrington instrumentation. Seventeen patients with neurologic deficit underwent decompression over appropriate levels as well. No posterior fusions or instrumentation were carried out. Stable arthrodesis with correction of the deformity occurred in 36 of 37 patients with only one nonunion. Pain was reduced significantly in 78% of patients. Late neurologic improvement of a significant functional degree occurred in three of eight paraparetics. All patients with spinal stenosis had relief of their symptoms and signs.