Influence of material and tube size on DUWLs contamination in a pilot plant

Numerous studies have shown that the water discharged from dental unit waterlines (DUWLs) contains high densities of bacteria, especially non-fermenting Gram negative bacteria. The aim of the present study was to investigate how the material (polyethylene-PE and polytetrafluorethylene-PTFE) and size (1.6 and 4.0 mm) of 4 waterlines in a pilot plant influence the level of contamination in the output water. The water contamination was assessed by analyzing the trend of the heterotrophic plate counts at 22 degrees C as a function of time and by testing for non-fermenting Gram negative bacteria. In all waterlines, the bacterial density increased exponentially during the first months and thereafter remained between 10(4) and 10(6) cfu/ml. However, the plate count at 22 degrees C was lower in the water from PTFE tubes and from larger size tubes. Comamonas acidovorans, Pseudomonas aeruginosa and Pseudomonas fluorescens were isolated. Pseudomonas aeruginosa, responsible for infections associated with dental practice, was never isolated in the output water from PTFE tubes. In order to control bacterial contamination the results suggest the use of waterlines made of PTFE on account of their ability to inhibit the colonization and growth of Pseudomonas aeruginosa.     

Effects of plant growth promoting rhizobacteria (PGPR) on rooting and root growth of kiwifruit (Actinidia deliciosa) stem cuttings

The effects of plant growth promoting rhizobacteria (PGPR) on the rooting and root growth of semi-hardwood and hardwood kiwifruit stem cuttings were investigated. The PGPR used were Bacillus RC23, Paenibacillus polymyxa RC05, Bacillus subtilis OSU142, Bacillus RC03, Comamonas acidovorans RC41, Bacillus megaterium RC01 and Bacillus simplex RC19. All the bacteria showed indole-3-acetic acid (IAA) producing capacity. Among the PGPR used, the highest rooting ratios were obtained at 47.50% for semi-hardwood stem cuttings from Bacillus RC03 and Bacillus simplex RC19 treatments and 42.50% for hardwood stem cuttings from Bacillus RC03. As well, Comamonas acidovorans RC41 inoculations indicated higher value than control treatments. The results suggest that these PGPR can be used in organic nursery material production and point to the feasibility of synthetic auxin (IBA) replacement by organic management based on PGPR.     

Development and application of monoclonal antibodies for in situ detection of indigenous bacterial strains in aquatic ecosystems.

Strain-specific monoclonal antibodies (MAbs) were developed for three different bacterial isolates obtained from a freshwater environment (Lake Plusssee) in the spring of 1990. The three isolates, which were identified by molecular methods, were as follows: Cytophaga johnsonae PX62, Comamonas acidovorans PX54, and Aeromonas hydrophila PU7718. These strains represented three species that were detected in high abundance during a set of mesocosm experiments in Lake Plusssee by the direct analysis of low-molecular-weight RNAs from bacterioplankton. We developed one MAb each for the bacterial isolates PX54 and PU7718 that did not show any cross-reactivity with other bacterial strains by immunofluorescence microscopy. Each MAb recognized the general lipopolysaccharide fraction of the homologous strain. These MAbs were tested successfully for their ability to be used for the in situ detection and counting of bacteria in lake water by immunofluorescence microscopy. During the spring of 1993, A. hydrophila PU7718 showed a depth distribution in Lake Plusssee with a pronounced maximum abundance at 6 m, whereas Comamonas acidovorans PX54 showed a depth distribution with a maximum abundance at the surface. The application of these MAbs to the freshwater samples enabled us to determine the cell morphologies and microhabitats of these strains within their natural environment. The presence of as many as 8,000 cells of these strains per ml in their original habitats 3 years after their initial isolation demonstrated the persistence of individual strains of heterotrophic bacteria over long time spans in pelagic habitats.     

Isolation and characterization of a bacterium which utilizes polyester polyurethane as a sole carbon and nitrogen source

Abstract Various soil samples were screened for the presence of microorganisms which have the ability to degrade polyurethane compounds. Two strains with good polyurethane degrading activity were isolated. The more active strain was tentatively identified as Comamonas acidovorans . This strain could utilize polyester-type polyurethanes but not the polyether-type polyurethanes as sole carbon and nitrogen sources. Adipic acid and diethylene glycol were probably the main degradation products when polyurethane was supplied as a sole carbon and nitrogen source. When ammonium nitrate was used as nitrogen source, only diethylene glycol was detected after growth on polyurethane.     

Heterotrophic plate counts and the isolation of bacteria from mineral waters on selective and enrichment media

The heterotrophic plate counts of 15 brands of bottled non-carbonated mineral waters were examined and found to be generally high and variable. Four selective or enrichment media for the enumeration of coliforms (m-Endo LES and m-lauryl sulphate agar) and Pseudomonas aeruginosa (cetrimide-nalidixic acid agar and malachite green broth) were used to isolate several species of Gram-negative bacteria. Strains identified as CDC gr IVc-2 and Comamonas (Ps.) acidovorans were the two most commonly isolated. Considerable variation in populations was seen between the brands, as well as between two batches of the same mineral water.     

Production of R-(-)-Ketoprofen from an Amide Compound by Comamonas acidovorans KPO-2771-4

R-(-)-2-(3(prm1)-Benzoylphenyl)propionic acid [R-(-)-ketoprofen] was produced from racemic 2-(3(prm1)-benzoylphenyl)propionamide (keto-amide) by the isolated bacterial strain Comamonas acidovorans KPO-2771-4. Sodium fumarate as the carbon source and 2-azacyclononanone or isobutyronitrile as the enhancer in the culture medium were effective for bacterial growth and the enhancement of R-(-)-ketoprofen-producing activity. R-(-)-Ketoprofen produced from the keto-amide by resting cells was present in 99% enantiomeric exess. C. acidovorans KPO-2771-4 has an R-enantioselective amidase for keto-amide because the purified amidase from the bacterium hydrolyzed keto-amide, producing optically pure R-ketoprofen and ammonia.     

Factors affecting the freeze-fracture morphology of in vivo polyhydroxyalkanoate granules

Interesting morphologies were observed when Comamonas acidovorans containing polyhydroxyalkanoates (PHA) of various compositions was freeze-fractured at temperatures far below the glass transition temperatures of PHA. In vivo granules of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comparatively showed the most ductility, and could be stretched extensively. Contrary to the uniform needle-type deformation shown by the poly(3-hydroxybutyrate) homopolymer when fractured at -110 degrees C, copolymers containing 3-hydroxyvalerate units showed various deformation structures. Similar observations were made when in vivo granules of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) were freeze-fractured, although the ductility of the latter was much reduced. In addition, it was found that fracturing at -160 degrees C resulted in decreased ductility of the PHA granules with the concomitant increase in the number of mushroom-type deformation structures. Our results suggest that PHA granules with higher resistance to freeze-fracture deformation show less ductility, and therefore produce the mushroom-type morphology. This is the first report on the freeze-fracture morphology of PHA copolymers containing short-chain-length monomers.     

Use of a simplified cell blot technique and 16S rRNA-directed probes for identification of common environmental isolates.

A simple technique in which rRNA-targeted oligodeoxynucleotide probes are used to identify bacteria immobilized on membranes is described. By using colony lifts, bacteria are directly transferred from plates to untreated nitrocellulose membranes. Alternatively, cells resuspended from colonies can be applied to membranes by using a vacuum manifold under high-salt conditions. Blotted cells are baked and hybridized under stringent conditions by using standard protocols. Treatment of blotted cells with sodium dodecyl sulfate, urea, formaldehyde, or protease had no apparent effect on hybridization signals. Hybridization to rRNA from cells that had been stored refrigerated for 6 days was readily detected; however, fivefold more cells (approximately 10(7) cells) were required to obtain hybridization signals comparable to those generated by cells not subjected to storage. The sequences of oligonucleotide probes specific for Pseudomonas cepacia, Comamonas testosteroni, and Acinetobacter calcoaceticus and a group probe identifying Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas fluorescens, Comamonas acidovorans, and "Flavobacterium" lutescens are presented. In conjunction with the colony lift hybridization procedure, bacteria isolated from river water were identified by using these probes.     

Electron microscopy and image analysis reveal common principles of organization in two large protein complexes: groEL-type proteins and proteasomes

In an attempt to settle the question of whether the multicatalytic proteinase or proteasome exist in all three kingdoms of life--eukaryotes, archaebacteria, and eubacteria--we have undertaken a search for them in the eubacterium Comamonas acidovorans. We have, in fact, isolated and purified a cylinder-shaped particle. However, according to various structural and biochemical criteria this turned out to be more reminiscent of the groEL protein from Escherichia coli and its homologs than to proteasomes of eukaryotic or archaebacterial origin. N-terminal sequencing provided definite proof for its belonging to this family of molecular chaperonins. Image analysis of electron micrographs revealed that the C. acidovorans groEL-like protein and proteasomes in spite of their significantly different dimensions have certain principles of organization in common.     

Analysis of the catalytic domain of the lysin of the lactococcal bacteriophage Tuc2009 by chimeric gene assembling

Abstract Dye-linked alcohol dehydrogenase from Rhodopseudomonas acidophila strain M402, able to oxidize polyethylene glycols, was purified to homogeneity. The monomeric enzyme, having a molecular mass of 72 kDa, contains one PQQ and one haem c per enzyme molecule. In other respects also, the enzyme is very similar to the type I quinohaemoprotein alcohol dehydrogenases known to occur in Comamonas testosteroni, Comamonas acidovorans , and Pseudomonas putida species. However, dissimilarities exist with respect to the isoelectric points and the substrate specificities. On reinvestigating the substrate specificity of the C. testosteroni enzyme, it also appeared to exhibit good activity towards polyethylene glycols. Based on what has been reported for the polyethylene glycol-oxidizing alcohol dehydrogenase of Sphingomonas macrogoltabidus , this enzyme is quite different from that of R. acidophila . Keywords: Polyethylene glycol dehydrogenase activity; Alcohol dehydrogenase; PQQ; Haem c ; Rhodopseudomonas acidophila     

Multiparametric analysis of waterline contamination in dental units.

Microbial contamination of dental unit waterlines is thought to be the result of biofilm formation within the small-bore tubing used for these conduits. Systematic sampling of 121 dental units located at the dental school of Université de Montréal showed that none of the waterlines was spared from bacterial contamination. Multilevel statistical analyses showed significant differences between samples taken at the beginning of the day and samples taken after a 2-min purge. Differences were also found between water from the turbine and the air/water syringe. Random variation occurred mainly between measurements (80%) and to a lesser extent between dental units (20%). In other analyses, it was observed to take less than 5 days before initial bacterial counts reached a plateau of 2 x 10(5) CFU/ml in newly installed waterlines. Sphyngomonas paucimobilis, Acinetobacter calcoaceticus, Methylobacterium mesophilicum, and Pseudomonas aeruginosa were the predominant isolates. P. aeruginosa showed a nonrandom distribution in dental unit waterlines, since 89.5% of the all the isolates were located in only three of the nine clinics tested. Dental units contaminated by P. aeruginosa showed significantly higher total bacterial counts than the others. By comparison, P. aeruginosa was never isolated in tap water remote from or near the contaminated dental unit waterlines. In conclusion, dental unit waterlines should be considered an aquatic ecosystem in which opportunistic pathogens successfully colonize synthetic surfaces, increasing the concentration of the pathogens in water to potentially dangerous levels. The clinical significance of these findings in relation to routine dental procedures is discussed.     

Identification of xenobiotic-degrading isolates from the beta subclass of the Proteobacteria by a polyphasic approach including 16S rRNA partial sequencing.

Nineteen gram-negative, aerobic, biodegradative isolates were identified by using a polyphasic taxonomic approach. The presence of the specific polyamine 2-hydroxyputrescine and the presence of a ubiquinone with eight isoprenoid units in the side chain (ubiquinone Q-8) allowed allocation of these organisms to the beta subclass of the Proteobacteria. On the basis of the results of additional characterization experiments (i.e., API 20NE tests, determinations of soluble protein patterns, and DNA-DNA hybridization experiments), we classified six isolates as either Comamonas testosteroni, Comamonas acidovorans, or Alcaligenes xylosoxidans subsp. denitrificans. By using the same criteria we allocated two additional isolates to the genus Alcaligenes. A comparison of a 16S rRNA fragment (positions 1220 to 1377; Escherichia coli nomenclature) indicated that the remaining isolates should be allocated as follows: one is a member of C. testosteroni and one is a member of Acidovorax facilis, as confirmed by the results of additional DNA-DNA hybridizations; two others probably belong to the family Alcaligenaceae; six are related to "Alcaligenes eutrophus"; and one, strain NRRL 12228, occupies an isolated position.     

Mixed culture bioconversion of 16-dehydropregnenolone acetate to androsta-1,4-diene-3,17-dione: optimization of parameters

Bioconversion of 16-dehydropregnenolone acetate (16-DPA) to androsta-1,4-diene-3,17-dione (ADD), an intermediate for the production of female sex hormones, by mixed culture of Pseudomonas diminuta MTCC 3361 and Comamonas acidovorans MTCC 3362 is reported. Various physicochemical parameters for the bioconversion of 16-DPA to ADD have been optimized in shake flask cultures. Nutrient broth inoculated with actively growing co-culture proved ideal for bacterial growth and bioconversion. A temperature range of 35-40 degrees C was most suitable; higher or lower temperatures adversely affected the bioconversion. Dimethylformamide below 2% concentration was the most suitable carrier solvent. Maximum conversion was recorded at 0.5 mg mL(-1) 16-DPA. A pH of 5.0 yielded a peak conversion of 62 mol % in 120 h incubation period. Addition of 9alpha-hydroxylase inhibitors failed to prevent further breakdown of ADD to nonsteroidal products. 16-DPA conversion in a 5 L fermenter followed a similar trend.     

Degradation of cocaine by a mixed culture of Pseudomonas fluorescens MBER and Comamonas acidovorans MBLF.

A mixed culture that could utilize cocaine as the sole source of carbon and energy for growth was isolated by selective enrichment. The individual microorganisms within this mixed culture were identified as Pseudomonas fluorescens (termed MBER) and Comamonas acidovorans (termed MBLF). Each microorganism was shown to be unable to grow to any appreciable extent on 10 mM cocaine in the absence of the other. C. acidovorans MBLF was found to possess an inducible cocaine esterase which catalyzed the hydrolysis of cocaine to ecgonine methyl ester and benzoate. C. acidovorans was capable of growth on benzoate at concentrations below 5 mM but was unable to metabolize ecgonine methyl ester. P. fluorescens MBER was capable of growth on either benzoate as the sole source of carbon or ecgonine methyl ester as the sole source of carbon and nitrogen. P. fluorescens MBER was found to initiate the degradation of ecgonine methyl ester via ecgonine, pseudoecgonine, and pseudoecgonyl-coenzyme A. Subcellular studies resulted in the identification of an ecgonine methyl esterase, an ecgonine epimerase, and a pseudoecgonyl-coenzyme A synthetase which were induced by growth on ecgonine methyl ester or ecgonine. Further metabolism of the ecgonine moiety is postulated to involve nitrogen debridging, with the production of carbonyl-containing intermediates.     

Heterotrophic plate counts and the isolation of bacteria from mineral waters on selective and enrichment media

M anaia , C.M., N unes , O.C., M orais , P.V. & da C osta , M.S. 1990. Heterotrophic plate counts and the isolation of bacteria from mineral waters on selective and enrichment media. Journal of Applied Bacteriology 69 , 871–876.
The heterotrophic plate counts of 15 brands of bottled non-carbonated mineral waters were examined and found to be generally high and variable. Four selective or enrichment media for the enumeration of coliforms (m-Endo LES and m-lauryl sulphate agar) and Pseudomonas aeruginosa (cetrimide-nalidixic acid agar and malachite green broth) were used to isolate several species of Gram-negative bacteria. Strains identified as CDC gr IVc-2 and Comamonas (Ps.) acidovorans were the two most commonly isolated. Considerable variation in populations was seen between the brands, as well as between two batches of the same mineral water.     

High-level expression and characterization of a highly functional Comamonas acidovorans xanthine dehydrogenase in Pseudomonas aeruginosa

An improved procedure is described for the high-level expression of Comamonas acidovorans XDH in Pseudomonas aeruginosa PAO1-LAC. The level of functional expression (56 mg protein/L culture) is found to be 7-fold higher than that observed in Escherichia coli and 30-fold higher than that induced in C. acidovorans. Co-expression of the xdhC gene is required for maximal level of functional expression. Comparison of purified preparations of XDH expressed in the absence of xdhC (XDH(AB)) with that expressed in its presence (XDH(ABC)) shows the increased level of activity due to the level of Mo incorporation. The Fe and FAD contents of expressed enzymes are independent of xdhC co-expression. Electron paramagnetic resonance spectroscopy, circular dichroism spectroscopy, metal analysis, and kinetic properties of recombinant purified XDH(ABC) are identical with those exhibited by the native enzyme. This expression system should serve as a valuable tool for further biophysical and mechanistic investigations of xanthine dehydrogenase by site-directed mutagenesis. A method is also described to evaluate the suitability of P. aeruginosa and other organisms as potential expression hosts for five different sources of xdh genes.     

Isolation of poly(beta-L-malic acid)-degrading bacteria and purification and characterization of the PMA hydrolase from Comamonas acidovorans strain 7789

Several bacteria were isolated which were able to utilize poly(beta-L-malic acid) as sole carbon source for growth. The poly(beta-L-malic acid) hydrolyzing enzyme of Comamonas acidovorans strain 7789 was detected in the membrane fraction. The enzyme was purified by isolation of crude cell membranes by ultracentrifugation of disrupted cells, solubilization of the membrane fraction with octylglucoside, selective precipitation with 50% saturated ammonium sulfate and preparative isolectric focusing. SDS-PAGE analysis revealed a M(r) of 43,000. The pH optimum was 8.1 and the Km was 0.13 microM (in terms of monomeric units) and 0.0021 microM poly(beta-L-malic acid) at pH 8.1 (100 mM glycylglycine buffer). Addition of NaCl, KCl, CaCl2 or MgCl2 (from 25 to 100 mM) decreased the hydrolase activity, whereas EDTA or polymethane sulfonic acid fluoride had no influence on the enzyme. The depolymerization of poly(beta-L-malic acid) proceeded from the ends of the polyester resulting in the formation of L-malate. Esterase activity was not detectable with p-nitrophenyl acetate or p-nitrophenyl butyrate, which is used to determine for example poly(3-hydroxybutyric acid) depolymerase activity.     

Electrostatic properties of the anion selective porin Omp32 from Delftia acidovorans and of the arginine cluster of bacterial porins

The functional properties of the anion-selective porin Omp32 from the bacterium Delftia acidovorans, formerly Comamonas acidovorans, are determined by the particularly narrow channel constriction and the electrostatic field inside and outside the pore. A cluster of arginines (Arg 38, Arg 75, and Arg 133) determines the electrostatic field close to the constriction zone. Stacked amino acids carrying charges are prone to drastic pK(a) shifts. However, optimized calculations of the titration behavior of charged groups, based on the finite-difference Poisson-Boltzmann technique, suggest that all the arginines are charged at physiological pH. Protonation of the clustered arginines is stabilized by one buried glutamate residue (Glu 58), which is strongly interacting with Arg 75 and Arg 38. This functional arrangement of three charged amino acid residues is of general significance because it is found in the constriction zones of all known 16-stranded porins from the alpha-, beta-, and gamma-proteobacteria.     

An extra large insertion in the polyhydroxyalkanoate synthase from Delftia acidovorans DS-17: its deletion effects and relation to cellular proteolysis

The polyhydroxyalkanoate (PHA) synthase (PhaC(Da)) from Delftia acidovorans DS-17 (formerly Comamonas acidovorans) has a unique large insertion consisting of 40 amino acid residues in the alpha/beta hydrolase fold region. In order to examine whether this insertion is necessary for enzyme function, we generated a mutant gene where the nucleotides encoding the insertion sequence were deleted [phaC(Da)del(342-381)]. The ability of the mutant PhaC(Da) lacking the insertion sequence to produce PHA in recombinant Escherichia coli JM109 was compared with that of wild-type PhaC(Da). The results revealed that the mutant enzyme had approximately one fourth the activity of the wild-type enzyme. However, there was no significant difference in PHA content accumulated in cells harboring either the mutant PhaC(Da) or wild-type PhaC(Da) nor were there any differences in the molecular masses of the produced polymers. Therefore, we have concluded that the characteristic insertion is not indispensable for PHA synthesis. Also, slight cellular proteolysis in E. coli was found specifically for wild-type PhaC(Da) by Western blot analysis. This result prompted us to further examine the proteolytic stability of PhaC(Da) in D. acidovorans. Consequently, it has been suggested that the insertion region of PhaC(Da) is susceptible to cellular proteolysis during accumulation of PHA.