Mixed Continuous Cultures of Polyvinyl Alcohol-Utilizing Symbionts Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C

Stable mixed continuous cultures of Pseudomonas sp. strain VM15C and Pseudomonas putida VM15A, the former of which produced a polyvinyl alcohol (PVA)-degrading enzyme and the latter of which produced an essential growth factor for PVA utilization by strain VM15C, were established with PVA as the sole source of carbon and energy with chemostat cultivation. A high extent of PVA degradation was achieved at dilution rates of less than 0.030/h. The predominant strain in the cultures was the primary metabolizer of PVA, strain VM15C. The growth supporter, strain VM15A, existed as a minor population, although its population was maintained at an almost constant level throughout a dilution region in which the VM15C population decreased markedly as the dilution rate was raised. A crude growth factor which was prepared from a culture supernatant of strain VM15A and increased the specific growth rate of strain VM15C with PVA in an axenic batch culture was also effective for enhancing the VM15C population and PVA degradation in the mixed continuous culture at a high dilution rate (0.064/h). This indicated that the growth-limiting substrate for strain VM15C in the mixed continuous culture is the growth factor produced by strain VM15A.     

Pyrroloquinoline Quinone-Dependent Cytochrome Reduction in Polyvinyl Alcohol-Degrading Pseudomonas sp. Strain VM15C

A polyvinyl alcohol (PVA) oxidase-deficient mutant of Pseudomonas sp. strain VM15C, strain ND1, was shown to possess PVA dehydrogenase, in which pyrroloquinoline quinone (PQQ) functions as a coenzyme. The mutant grew on PVA and required PQQ for utilization of PVA as an essential growth factor. Incubation of the membrane fraction of the mutant with PVA caused cytochrome reduction of the fraction. Furthermore, it was found that in spite of the presence of PVA oxidase, the membrane fraction of strain VM15C grown on glucose without PQQ required PQQ for cytochrome reduction during incubation with PVA. The results provide evidence that PVA dehydrogenase couples with the electron transport chain of PVA-degrading bacteria but that PVA oxidase does not.     

Enhancement of Pyrroloquinoline Quinone Production and Polyvinyl Alcohol Degradation in Mixed Continuous Cultures of Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C with Mixed Carbon Sources

In a mixed continuous culture of Pseudomonas putida VM15A and Pseudomonas sp. strain VM15C with polyvinyl alcohol (PVA) as the sole source of carbon, growth of the PVA-degrading bacterium VM15C and, hence, PVA degradation were limited by the growth factor, pyrroloquinoline quinone, produced by VM15A. Feeding of a carbon source for VM15A, ethanol, with PVA enhanced pyrroloquinoline quinone production and caused increases in the VM15C population and PVA degradation in a mixed continuous culture. There was an optimum range for PVA degradation of the ethanol concentration, although pyrroloquinoline quinone concentrations in continuous mixed cultures increased with increasing ethanol concentration.     

Production of Polyvinyl Alcohol Oxidase by a Symbiotic Mixed Culture

Production of polyvinly alcohol (PVA) oxidase by Pseudomonas sp. strain VM15C, a PVA degrader of a symbiotic PVA-utilizing mixed culture, was examined in various cultures. Despite the absence of PVA in the culture in nutrient broth, VM15C showed approximately the same productivity of PVA oxidase activity as that in the culture with PVA as the sole carbon source, whereas the productivity in the culture with glucose was lower than that of either the nutrient broth or the PVA culture. PVA oxidase activity produced in the nutrient broth culture was predominantly present in the cells, and most of the activity appeared to be in the cytoplasm. In contrast, in the culture with PVA as the sole carbon source, the activity was present in the culture fluid in a larger ratio than in the nutrient broth culture. Thus, production of PVA oxidase activity by this strain was constitutive and repressible, although localization of the produced activity was changed by growth conditions.     

Symbiotic Utilization of Polyvinyl Alcohol by Mixed Cultures

Polyvinyl alcohol (PVA)-utilizing cultures were obtained from various sources. They were mixed cultures even after cyclical transfer to liquid and plate media with PVA as a sole source of carbon. Component bacteria were isolated from the several mixed cultures, and it was shown that PVA was utilized symbiotically by two bacterial members which could not utilize PVA in each respective pure culture. From a mixed culture, strains VM15, VM15A (Pseudomonas putida) and VM15C (Pseudomonas sp.) were isolated as members essential for PVA utilization. VM15C was the predominant strain in the mixed-culture population and produced PVA-degrading enzyme. The culture supernatant of VM15A enabled VM15C to grow on PVA. VM15A was presumed to supply VM15C with a unique growth stimulant which was distinct from usual growth factors.     

Purification of membrane-bound polyvinyl alcohol oxidase in Pseudomonas sp. VM15C

Abstract Polyvinyl alcohol (PVA) was utilized by a symbiotic mixed culture which was composed of Pseudomonas putida VM15A and Pseudomonas sp. VM14C. The PVA oxidase was found in the culture fluid, membrane, and cytosol fractions of VM15C. The membrane-bound PVA oxidase was purified by several steps of chromatography. The enzyme (p I = 9.6) exhibited the maximum activity at pH 8.0 to 8.4 and 45°C, and utilized secondary alcohol as well as PVA. The enzyme showed the PVA dehydrogenating activity linking with phenazine ethosulfate, indicating the possibility that PVA oxidation is coupled with an electron transport chain on the bacterial membrane.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

Isolation and characterization of a novel poly(vinyl alcohol)-degrading bacterium, Sphingopyxis sp. PVA3

We have isolated a poly(vinyl alcohol) (PVA)-degrading bacterium from an activated sludge sample obtained from the drainage of a dyeing factory. Enrichment cultures were performed in media containing PVA as the sole or major carbon source. After several rounds of cultivation on liquid and solid media, we were able to isolate a single colony with PVA-degrading ability (strain PVA3). The bacterium could degrade PVA in the absence of symbionts or cofactors such as pyrroloquinoline quinone (PQQ). Over 90% of PVA, at an initial concentration of 0.1%, was degraded within a 6-day cultivation. Degradation was confirmed by both iodometric methods and gel permeation chromatography. Examination of the PVA attached to the cells revealed a large increase in carbonyl groups, suggesting the oxidation of hydroxyl groups of the polymer on the surfaces of cells. Addition of PQQ to the culture medium did not enhance the growth and the PVA-degrading rates of strain PVA3. Furthermore, we found that cells grown on PVA generated hydrogen peroxide upon the addition of PVA. The results strongly suggest that the initial oxidation of PVA is mediated via a PVA oxidase, and not a PQQ-dependent dehydrogenase. A biochemical and phylogenetic characterization of the bacterium was performed. The sequence of the 16S ribosomal RNA gene of the bacterium indicated a phylogenetic position of the strain within the genus Sphingopyxis, and the strain was therefore designated Sphingopyxis sp. PVA3.     

Cell surface structure enhancing uptake of polyvinyl alcohol (PVA) is induced by PVA in the PVA-utilizing <Emphasis Type="Italic">Sphingopyxis</Emphasis> sp. strain 113P3

Polyvinyl alcohol (PVA)-utilizing Sphingopyxis sp. 113P3 (reidentified from Sphingomonas sp. 113P3) removed almost 0.5% PVA from culture supernatants in 4 days. Faster degradation of 0.5% PVA was performed by the periplasmic fraction. The average molecular size of PVA in the culture supernatant or cell-bound PVA was gradually shifted higher, suggesting that lower molecular size molecules are degraded faster. Depolymerized products were found in neither the culture supernatant nor the cell-bound fraction; however they were recovered from the periplasmic fraction. As extracellular or cell-associated PVA oxidase activity was almost undetectable in strain 113P3, degradation of PVA must be performed by periplasmic PVA dehydrogenase after uptake into the periplasm. Following the consumption of PVA, a dent appeared on the cell surface on day 2 and increased in size and depth for 4 days and was maintained for 8 days. Ultrastructural change on the cell surface was only observed in PVA medium, but not in nutrient broth (NB), suggesting that the change is induced by PVA. Fluorescein-4-isothiocyanate-labeled PVA was bound more to cells grown in PVA than to cells grown in NB. No binding was found with PVA-grown cells treated with formaldehyde. Thus, a dent on the cell surface seems to be related to the uptake of PVA.     

Properties and roles of bacterial symbionts of polyvinyl alcohol-utilizing mixed cultures

From several polyvinyl alcohol (PVA)-utilizing mixed cultures, two component bacterial strains essential for PVA utilization were isolated, and their properties and roles in PVA utilization were studied. Each pair of essential component strains consisted of a type I strain, which produced a PVA-degrading enzyme and constituted the predominant population of the mixed culture in PVA, and a type II strain, which produced a certain growth stimulant for the former strain. All of the type I strains were taxonomically identical and assigned as Pseudomonas sp. In contrast, type II strains were taxonomically different from each other, belonging to Pseudomonas spp. and Alcaligenes sp. PVA utilization occurred in each mixed culture of a type I strain with Pseudomonas putida VM15A as a substitute for the type II strain of the original pair and also in each mixed culture of a type II strain with Pseudomonas sp. VM15C. The growth rates of these substituted, mixed cultures differed from each other.     

Properties and roles of bacterial symbionts of polyvinyl alcohol-utilizing mixed cultures.

From several polyvinyl alcohol (PVA)-utilizing mixed cultures, two component bacterial strains essential for PVA utilization were isolated, and their properties and roles in PVA utilization were studied. Each pair of essential component strains consisted of a type I strain, which produced a PVA-degrading enzyme and constituted the predominant population of the mixed culture in PVA, and a type II strain, which produced a certain growth stimulant for the former strain. All of the type I strains were taxonomically identical and assigned as Pseudomonas sp. In contrast, type II strains were taxonomically different from each other, belonging to Pseudomonas spp. and Alcaligenes sp. PVA utilization occurred in each mixed culture of a type I strain with Pseudomonas putida VM15A as a substitute for the type II strain of the original pair and also in each mixed culture of a type II strain with Pseudomonas sp. VM15C. The growth rates of these substituted, mixed cultures differed from each other.     

Variations in the Alternative Oxidase in Chlamydomonas Grown in Air or High CO(2)

Chlamydomonas in the resting phase of growth has an equal capacity of about 15 micromole O₂ uptake per hour per milligram of chlorophyll for both the cytochrome c, CN-sensitive respiration, and for the alternative, salicylhydroxamic acid-sensitive respiration. Alternative respiration capacity was measured as salicylhydroxamic acid inhibited O₂ uptake in the presence of CN, and cytochrome c respiration capacity as CN inhibition of O₂ uptake in the presence of salicylhydroxamic acid. Measured total respiration was considerably less than the combined capacities for respiration. During the log phase of growth on high (2-5%) CO₂, the alternative respiration capacity decreased about 90% but returned as the culture entered the lag phase. When the alternative oxidase capacity was low, addition of salicylic acid or cyanide induced its reappearance. When cells were grown on low (air-level) CO₂, which induced a CO₂ concentrating mechanism, the alternative oxidase capacity did not decrease during the growth phase. Attempts to measure in vivo distribution of respiration between the two pathways with either CN or salicylhydroxamic acid alone were inconclusive.     

Human Lung Cancer Cells Grown in an Ex Vivo 3D Lung Model Produce Matrix Metalloproteinases Not Produced in 2D Culture

We compared the growth of human lung cancer cells in an ex vivo three-dimensional (3D) lung model and 2D culture to determine which better mimics lung cancer growth in patients. A549 cells were grown in an ex vivo 3D lung model and in 2D culture for 15 days. We measured the size and formation of tumor nodules and counted the cells after 15 days. We also stained the tissue/cells for Ki-67, and Caspase-3. We measured matrix metalloproteinase (MMP) levels in the conditioned media and in blood plasma from patients with adenocarcinoma of the lung. Organized tumor nodules with intact vascular space formed in the ex vivo 3D lung model but not in 2D culture. Proliferation and apoptosis were greater in the ex vivo 3D lung model compared to the 2D culture. After 15 days, there were significantly more cells in the 2D culture than the 3D model. MMP-1, MMP-9, and MMP-10 production were significantly greater in the ex vivo 3D lung model. There was no production of MMP-9 in the 2D culture. The patient samples contained MMP-1, MMP-2, MMP-9, and MMP-10. The human lung cancer cells grown on ex vivo 3D model form perfusable nodules that grow over time. It also produced MMPs that were not produced in 2D culture but seen in human lung cancer patients. The ex vivo 3D lung model may more closely mimic the biology of human lung cancer development than the 2D culture.     

Efficiency of Root Respiration of a Flood-Tolerant and a Flood-Intolerant Senecio Species as Affected by Low Oxygen Tension

A method was described to determine root growth respiration and root maintenance respiration rate of plants, grown in culture solutions of high and low oxygen concentrations, during linear growth. Root growth respiration of aerobically grown plants was three to four times lower in the flood-intolerant Senecio jacobaea L. than in the flood-tolerant species. Senecio aquaticus Hill. Root growth respiration of Senecio aquaticus Hill decreased by a factor two to three upon transplantation to a culture solution of low oxygen tension. The difference in root growth respiration between aerobically and anaerobically grown Senecio aquaticus Hill was ascribed to the presence of a highly active non-phosphorylating oxidase.     

Denitrification of PVA-immobilized denitrifying photosynthetic bacterium,Rhodobacter sphaeroides

A newly isolated denitrifying strain, Rhodobacter sphaeroides NII2 was immobilized in polyvinyl alcohol (PVA) gel, and the properties of the cells in the gel were examined. The immobilized cells had low or almost no denitrification activity, but the cells were activated by incubation in light with culture medium for denitrification containing 0.5% nitrate and no other nitrogen source. Cells grown in the dark were activated by incubation at an earlier stage and to a higher rate than the light-grown cells. The activation was markedly enhanced in the PVA gel with a low cell concentration. The immobilized cells consumed nitrate with a temporary accumulation of NO₂ and evolved nitrogen gas. The immobilized cells could use various organic compounds as electron donors for denitrification. Thus, the immobilized cells were applied to a continuous treatment of synthetic wastewater using an aparatus devised by this laboratory. The results showed an efficient removal of NO₃-N from the test water.     

Synthesis and localization of L-lactate oxidase in yeasts Yarrowia lipolytica

Conditions for L-lactate oxidase synthesis by the yeast Yarrowia lipolytica were investigated. The enzyme was found to be synthesized during growth on L-lactate in the exponential growth phase. L-lactate oxidase synthesis was also observed on glucose after adaptation to stress conditions (oxidative or thermal stress) during the stationary growth phase after glucose consumption. The cells grown on L-lactate exhibited high levels of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase), which exceeded those of glucose-grown cells. Ultrastructurally, L-lactate-grown cells and the cells grown on glucose and adapted to various stress conditions were also found to be similar, with increased mitochondria, elevated number and size of peroxisomes, and formation of lipid and polyphosphate inclusions. In order to determine the intracellular localization of L-lactate oxidase, the cells were disintegrated by the lytic enzyme complex from Helix pomatia. Centrifugation of the homogenate in Percoll gradient resulted in the isolation of purified fractions of the native mitochondria and peroxisomes. L-lactate oxidase was shown to be localized in peroxisomes.     

Isolation and Characterization of a Catabolite Repression-Insensitive Mutant of a Methanol Yeast, Candida boidinii A5, Producing Alcohol Oxidase in Glucose-Containing Medium

Mutants exhibiting alcohol oxidase (EC 1.1.3.13) activity when grown on glucose in the presence of methanol were found among 2-deoxyglucose-resistant mutants derived from a methanol yeast, Candida boidinii A5. One of these mutants, strain ADU-15, showed the highest alcohol oxidase activity in glucose-containing medium. The growth characteristics and also the induction and degradation of alcohol oxidase were compared with the parent strain and mutant strain ADU-15. In the parent strain, initiation of alcohol oxidase synthesis was delayed by the addition of 0.5% glucose to the methanol medium, whereas it was not delayed in mutant strain ADU-15. This showed that alcohol oxidase underwent repression by glucose. On the other hand, degradation of alcohol oxidase after transfer of the cells from methanol to glucose medium (catabolite inactivation) was observed to proceed at similar rates in parent and mutant strains. The results of immunochemical titration experiments suggest that catabolite inactivation of alcohol oxidase is coupled with a quantitative change in the enzyme. Mutant strain ADU-15 was proved to be a catabolite repression-insensitive mutant and to produce alcohol oxidase in the presence of glucose. However, it was not an overproducer of alcohol oxidase and, in both the parent and mutant strains, alcohol oxidase was completely repressed by ethanol.     

Differential Mitochondrial Electron Transport through the Cyanide-Sensitive and Cyanide-Insensitive Pathways in Isonuclear Lines of Cytoplasmic Male Sterile, Male Fertile, and Restored Petunia

Three pairs of isonuclear lines of cytoplasmic male sterile (CMS) and fertile Petunia cells (Petunia hybrida [Hook] Vilm. and Petunia parodii L.S.M.) grown in suspension culture were examined for sensitivity to inhibitors of respiratory electron transport at time-points after transfer into fresh media. Cells from CMS lines differed from cells of fertile lines in their utilization of the cyanide-insensitive oxidase pathway. Under our culture regime, after approximately 3 days of culture cells from the CMS lines exhibited much lower cyanide-insensitive, salicylhydroxamic acid-sensitive respiration than cells from the fertile lines. This respiratory difference was shown to be specific to the mitochondrial alternative oxidase pathway by using other characteristic inhibitors of mitochondrial electron transport in experiments with isolated mitochondria. Immature anthers from CMS plants also showed lower alternative oxidase activity relative to anthers from male fertile plants, but no such difference was detected in leaf tissue, ovary or perianth tissue, or anthers collected just prior to anthesis. A cell line from a fertile plant carrying a nuclear fertility restorer gene and the CMS cytoplasm exhibited increased activity of the alternative pathway compared with the CMS lines.     

Isolation and Culture Characterization of a New Polyvinyl Alcohol-Degrading Strain: Penicillium sp. WSH02-21

A fungal strain able to grow on polyvinyl alcohol (PVA) as sole carbon source was isolated from activated sludge of a textile factory. Morphological characteristics showed that this strain belonged to Penicillium sp., and, to our knowledge, this is the first report of PVA degradation by a strain of Penicillum sp. When 0.5% PVA was used as the carbon source in culture medium, it could be completely degraded after 12 days. This strain was found to produce and secrete an inducible PVA-degrading enzyme. High PVA concentration and oxygen transfer were favourable for PVA-degrading enzyme synthesis by Penicillium sp. cultured in shake-flasks. Moreover, Penicillum sp. cultured in PVA medium may spontaneously produce more catalase to decompose H₂O₂, a product of PVA oxidation by PVA oxidase, for protection of the cells from H₂O₂ damage. This revised version was published online in July 2006 with corrections to the Cover Date.