bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of sewage effluent for human immunodeficiency virus (HIV) using infectivity assay and reverse transcriptase polymerase chain reaction
Authors: Palmer Carol J. , Bonilla G. Fred , Tsai Yu-Li , Lee Moon H. , Javier Brenda J. , Siwak Edward B.
Published: October 2009

Environmental survival of human immunodeficiency virus type 1 (HIV-1) is an important public health concern. Survival of HIV in waste water is of particular interest to those who work at treatment facilities and to the general public who have contact with rivers or ocean water receiving treated sewage effluent. Other researchers have reported that HIV can be detected in waste water. Their studies, however, detected homologous nucleic acid sequences but did not attempt to determine infectivity. The current study tested primary and secondary effluent from a major metropolitan sewage agency for the presence of HIV-1 using reverse transcriptase polymerase chain reaction (RT-PCR), HIV-1 p24 antigen enzyme-linked immunosorbent assay, and infectivity testing. For RT-PCR, primers SK38/SK39 and M667/AA55 were used to identify HIV-1 RNA sequences from concentrated and extracted sewage samples. Infectivity assays employed donor peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin. Coxsackievirus B4, echovirus 7, and poliovirus 1, enteroviruses normally present in sewage, were tested for replication in PBMCs. Poliovirus 1 was found to infect the PBMCs. To eliminate other enteroviruses that may also infect the PBMCs and interfere with HIV-1 testing, concentrated sewage was treated with human immunoglobulin (free of HIV antibodies) and poliovirus antisera before infectivity assays were performed. All treated sewage samples tested negative for HIV-1 by all methods used. HIV-1 seeded into sewage, however, remained infectious in the assay, indicating that the sewage water sample did not interfere with HIV infectivity nor was it toxic to the PBMCs.Key words: HIV, sewage, RT-PCR, infectivity.
Characterization of diacetin B, a bacteriocin fromLactococcus lactissubsp.lactisbv.diacetylactisUL720
Authors: Ali D. , Lacroix C. , Simard R. E. , Thuault D. , Bourgeois C. M.
Published: October 2009

Fourteen Lactococcus lactis strains showing inhibitory activity against Listeria innocua SICC 4202 were isolated from different French raw milks and raw milk cheeses and screened for bacteriocin production by the triple layer method under conditions that eliminate the effects of lactic acid and hydrogen peroxide. Three bacteriocinogenic strains (two Lactococcus lactis subsp. lactis bv. diacetylactis UL719 and UL720 and one Lactococcus lactis subsp. lactis UL730) were selected for their high capacity to inhibit the growth of various food pathogens, including Listeria monocytogenes, Staphylococcus aureus, and clostridial strains. The inhibitory compounds from these three strains are inactivated by selected proteases, indicating their protein nature. They retained their antibacterial activity after heat treatments of 100 °C for 60 min and 121 °C for 20 min, and in the pH range from 2 to 11. The bacteriocin diacetin B produced by strain UL720 has been purified by a pH-dependent adsorption–desorption procedure, followed by reverse-phase high performance liquid chromatography, with a yield of 1.25% of the original activity. Mass spectrometry analysis indicates that the pure peptide has a molecular mass of 4292.32 or 4490.28 Da, while amino acid sequencing allowed the identification of the primary structure of the bacteriocin composed of 37 amino acid residues. The structure of the peptide did not show similarity with other known bacteriocins from lactic acid bacteria.Key words: isolation, Lactococcus lactis subsp. lactis bv. diacetylactis, bacteriocin, diacetin B, purification.
Vibriophage N4 DNA is nonpermuted and terminally redundant
Authors: Ghosh Amar N. , Chakrabarti Bimal K. , Chattopadhyay Dhruba J. , Sil Susmita
Published: October 2009

Phage N4 is one of the five newly isolated phages used in a new phage typing scheme for Vibrio cholerae O1 biovar EITor strains and belongs to the Podoviridae family. Electron microscopic studies showed that the phage N4 has a DNA of 40.4 ± 0.1 kb. A partial denaturation map of the N4 DNA has been constructed. It has been shown with the help of this partial denaturation map that phage N4 genome is nonpermuted. Circularization of the phage genome on treatment with exonuclease III, followed by incubation in 50% formamide, indicates a terminal redundancy.Key words: vibriophage N4, DNA, Vibrio cholerae, denaturation mapping.
Study of the reductive dechlorination of pentachlorophenol by a methanogenic consortium
Authors: Juteau P. , Beaudet R. , McSween G. , Lépine F. , Bisaillon J.-G.
Published: October 2009

Pentachlorophenol (PCP) dechlorination by a methanogenic consortium was observed when glucose, formate, lactate, or yeast extract was present in the mineral medium as a secondary carbon source. Acetate was not a good substrate to sustain dechlorination. The consortium was able to dechlorinate the different monochlorophenols, although the chlorine in position ortho and meta was removed more readily than in para position. Dechlorination was most efficient at 37 °C. At 45 °C, the first PCP dechlorination steps were very rapid, but 3,5-dichlorophenol (3,5-DCP) was not further dechlorinated. At 15 and 4 °C, dechlorination was very slow. The dechlorination of PCP to 3-chlorophenol (3-CP) was still observed after the consortium had been subjected to heat treatment (80 °C, 60 min), suggesting that spore-forming bacteria were responsible. The dechlorinating activity of the consortium was significantly reduced by the presence of hydrogen, 2-bromoethanosulfonic acid (BESA), or sulfate but not of nitrate. The dechlorination of 3-CP was completely inhibited by heat treatment or the presence of BESA, suggesting that a syntrophic microorganism would be involved. Vigorous agitation of the consortium stopped the dechlorination, but the presence of DEAE-Sephacel acting as a support was very efficient in restoring the activity, suggesting that association between certain members of the consortium was important.Key words: pentachlorophenol, dechlorination, anaerobic, methanogenesis.
Population dynamics of endophytic bacteria in field-grown sweet corn and cotton
Authors: McInroy John A. , Kloepper Joseph W.
Published: October 2009

Investigations were designed to gain fundamental information on the microbial ecology of endophytic bacteria in model dicotyledonous and monocotyledonous hosts. Population dynamics of indigenous endophytic bacteria in cotton (Gossypium hirsutum L. 'DES119') and sweet corn (Zea mays L. 'Silver Queen') stems and roots were studied in a 2-year field trial by quantifying culturable bacteria at intervals during the season on three media: R2A, medium SC, and tryptic soy agar. Population dynamics of endophytic bacteria inside cotton petioles and bolls were also determined in 1 year. Endophytes were recovered from sweet corn roots and stems at seedling emergence at mean population densities of 4 log (colony-forming units per gram fresh weight (cfu/g-fw)) for both seasons, and were present throughout most of the growing season at populations ranging from 4 to 6 log(cfu/g-fw) in 1990 and 4 to 7 log(cfu/g-fw) in 1991. Endophytic bacteria were also present at emergence in cotton roots and stems in 1991 but were not detected until 2 days after emergence in 1990. Endophytic populations in cotton roots ranged from 4 to 6 log(cfu/g-fw) for most of the growing season in 1990 and 1991, while populations in cotton stems fluctuated between 3 and 7 log(cfu/g-fw) during both seasons. In cotton petioles, mean populations generally ranged from 1 to 4 log(cfu/g-fw), while no endophytic bacteria were recovered from bolls (minimum detectable limit = 1.30 log(cfu/g-fw)). The relative contribution of seeds and soil as sources of endophytic bacteria recovered from inside plants was assessed using surface-disinfested seed in a potting mix or on water–agar. With sweet corn, the mean endophytic bacterial population in seedlings grown on water agar was below 2 log(cfu/g-fw), while with cotton the mean was 5 log(cfu/g-fw) 6 days after germination. Internal populations resulting from surface-disinfested seed planted in nonsterile potting mix were 6 log(cfu/g-fw) at 6 days after planting with corn but only 2 log(cfu/g-fw) with cotton. These results indicate that endophytic bacteria are natural inhabitants of internal regions of roots and stems and that the endophytes may arise from both seeds and soils.Key words: cotton, sweet corn, endophytes, colonization.
Increased thermal stability of the enzyme content in permeabilized whole cells from the fission yeastSchizosaccharomyces pombeby exogenous trehalose and other compounds
Authors: Fernández J. , Soto T. , Vicente-Soler J. , Cansado J. , Gacto M.
Published: October 2009

Cells of the fission yeast Schizosaccharomyces pombe were permeabilized by treatment with toluene–ethanol. The permeabilized cells lost the bulk of the internal trehalose pool while most of the alkaline phosphatase, invertase, α-glucosidase, or neutral trehalase activities located inside the cells remained unaffected. This system was used as an in situ assay to determine the involvement of trehalose in enzyme protection during thermal treatments. The addition of trehalose to suspensions of permeabilized cells resulted in a sugar-dependent thermoprotection of the internal marker enzymes. This approach demonstrates that in whole cells of the fission yeast trehalose plays a physiological role as a protective molecule against thermal denaturation of cellular enzymes.Key words: permeabilization, fission yeast, trehalose, thermal stability.
Capsulated cells ofAeromonas salmonicidagrown in vitro have different functional properties than capsulated cells grown in vivo
Authors: Garduño Rafael A. , Kay William W.
Published: October 2009

When grown in vivo in the peritoneal cavity of rainbow trout, Aeromonas salmonicida produces a clearly defined capsule with virulence-related functions. Aeromonas salmonicida grown in vitro in a glucose-rich medium (GRM) has also been reported to produce capsular material. Because in vitro mimicry of in vivo induced traits is highly desirable in vaccine design, the extent to which growth in GRM mimicked in vivo growth was examined. Antibodies specific to in vivo grown cells partially labeled the surface of GRM-grown cells, as well as two distinct proteins (81 700 and 41 000 Mr) in immunoblots of mutants with S-layer or lipopolysaccharide defects. GRM-grown strains showed an increased sensitivity to trout serum in contradistinction to the complete serum resistance of in vivo grown cells; as well, GRM-grown cells were more adherent to trout macrophages. Thus in spite of possessing some surface antigens normally expressed in vivo, cells grown on solid GRM did not possess all functional properties of in vivo grown cells.Key words: Aeromonas salmonicida, bacterial capsules, mimicry of in vivo conditions, furunculosis vaccines.
Fetal bovine serum induces changes in fatty acid composition ofTrypanosoma cruziphosphoinositides
Authors: Racagni G. , Lema M. G. de , Hernández G. , Machado-Domenech E. E.
Published: October 2009

Fetal bovine serum (FBS) is a necessary constituent of the culture media employed to foster the growth of Trypanosoma cruzi epimastigote forms. In different laboratories, the serum is used at final concentrations of 5 or 10%. We have normally supplemented the complex medium with 10% FBS. Under this condition we have described the fatty acid composition of the total lipids and of the phosphoinositide fractions. Additionally, we have reported the increase of polyphosphoinositides and phosphatidic acid after cholinergic stimulation. Since further attempts to reproduce these results with 5% FBS in the culture medium were not successful, the effect of the FBS concentration on the fatty acid composition of phospholipids from the T. cruzi epimastigote forms was thoroughly examined. This work showed that when the FBS concentration supplementing the culture medium was reduced from 10 to 5%, the fatty acid composition of the phosphoinositides was altered while the other major phospholipids were not significantly affected. The most relevant result was the decrease in the content of linoleic acid (18:2) and the increase of palmitoleic acid (16:1) in phosphatidylinositol 4,5-bisphosphate. Phosphatidylinositol (PI) and phosphatidylinositol phosphate also exhibited similar changes in the same fatty acids. The C2fatty acid composition of the phosphoinositides, under the same conditions, is also reported here for the first time.Key words: Trypanosoma cruzi, fatty acids, phosphoinositides, fetal bovine serum, phospholipids.
Effect of glucose on the superoxide dismutase production in fungal strainHumicola lutea
Authors: Angelova Maria B. , Genova Lubka K. , Slokoska Ludmila S. , Pashova Svetlana B.
Published: October 2009

The effect of glucose on superoxide dismutase (SOD) activity produced in Humicola lutea 110 was determined. The relatively high glucose concentration in the medium did not repress SOD levels in the cells. Glycerol, a nonfermentable carbon source, caused a slight stimulation of SOD synthesis. Furthermore, the specific rates of enzyme production in the medium with different glucose concentration showed an insignificant difference. Cyclic AMP had no effect on SOD levels. The shift in metabolism as glucose was depleted resulted in an increase in the rate of synthesis of both isocitric dehydrogenase and SOD. Pentachlorophenol and paraquat, which cause the production of superoxide radicals, caused an increase in SOD activity. These results led us to conclude that it is superoxide ion rather than glucose that is controlling SOD levels.Key words: Humicola lutea, fungi, superoxide dismutase, intracellular enzyme, glucose effect.
Pseudomonas aeruginosastrain MA01 aerobically metabolizes the aminodinitrotoluenes produced by 2,4,6-trinitrotoluene nitro group reduction
Authors: Alvarez Marc A. , Kitts Christopher L. , Unkefer Pat J. , Botsford James L.
Published: October 2009

Many microbes reduce the nitro substituents of 2,4,6-trinitrotoluene (TNT), producing aminodinitrotoluenes (ADNTs). These compounds are recalcitrant to further breakdown and are acutely toxic. In a search for organisms capable of metabolizing ADNTs, a bacterial strain was isolated for the ability to use 2-aminobenzoate (anthranilate) as sole C-source. This isolate, Pseudomonas aeruginosa MA01, metabolized TNT by first reducing one nitro group to form either 2-amino-4,6-dinitrotoluene (2ADNT) or 4-amino-2,6-dinitrotoluene (4ADNT). However, strain MA01 was distinct from other TNT-reducing organisms in that it transformed these compounds into highly polar metabolites through an O2-dependent process. Strain MA01 was able to cometabolize TNT, 2ADNT, and 4ADNT in the presence of a variety of carbon and energy sources. During aerobic cometabolism with succinate, 45% of uniformly ring-labeled [14C]TNT was transformed to highly polar compounds. Aerobic cometabolism of purified [14C]2ADNT and [14C]4ADNT with succinate as C-source produced similar amounts of these polar metabolites. During O2-limited cometabolism with succinate as C-source and nitrate as electron acceptor, less than 8% of the [14C]TNT was transformed to polar metabolites. Purified 2,6-diamino-4-nitrotoluene was not metabolized, and while 2,4-diamino-6-nitrotoluene was acetylated, the product (N-acetyl-2,4-diamino-6-nitrotoluene) was not further metabolized. Therefore, strain MA01 metabolized TNT by oxidation of the ADNTs and not by reduction the remaining nitro groups on the ADNTs.Key words: 2,4,6-trinitrotoluene, aminodinitrotoluene, Pseudomonas aeruginosa, cometabolism.
Preference in the nodulation of Phaseolus vulgaris cultivar RAB39
Authors: Montealegre C. , Graham P. H. , Kipe-Nolt J. A.
Published: October 2009

The low nodule occupancy achieved by superior nitrogen-fixing inoculant strains is a problem in the production of many traditional legume species, including Phaseolus vulgaris. Cultivars that select for inoculant strains, rather than nodulate with ineffective indigenous rhizobia, offer one approach to the resolution of this problem. In this study we identify a bean cultivar, RAB39, that nodulates preferentially with Rhizobium tropici, including the inoculant-quality strain UMR1899. This preference in nodulation was not affected by temperature or pH, though strain preference by a second, alkaline-producing Rhizobium etli strain did vary markedly with temperature. When RAB39 was inoculated with 1:1 mixtures of UMR1899 and seven serologically distinct strains of Rhizobium etli, more than 75% of the nodules formed by each strain pair contained UMR1899. A number of studies have reported close correlations between the percent nodule occupancy and speed of nodulation, measured using the root-tip-marking procedure. Exceptions to this correlation were evident in the present study, suggesting that host preference in nodulation was not related to differences in the parameters normally used to estimate speed of nodulation. The preference of RAB39 for R. tropici, a species of Rhizobium that occurs at low frequency in most bean soils, and especially for the inoculant-quality strain UMR1899, provides a tool to overcome the lack of response to inoculation in common bean.Key words: competition, nodulation efficiency, strain preference, common bean.
In vitro colonization and increase in nitrogen fixation of seedling roots of black mangrove inoculated by a filamentous cyanobacteria
Authors: Toledo Gerardo , Bashan Yoav , Soeldner Al
Published: October 2009

An isolate of the filamentous cyanobacterium Microcoleus sp. was obtained from black mangrove aerial root (pneumatophore) and inoculated onto young mangrove seedlings to evaluate N2-fixation and root-colonization capacities of the bacterium under in vitro conditions in closed-system experiments. N2fixation (acetylene reduction) gradually increased with time and reached its peak 5 days after inoculation. Later, it decreased sharply. The level of N2fixation in the presence of the plant was significantly higher than the amount of nitrogen fixed by a similar quantity of cyanobacteria on a N-free growth medium. The main feature of this root colonization was the gradual production of a biofilm in which the cyanobacterial filaments were embedded. Visible biofilm production increased with time until it completely covered the entire root system of the plant. The in-and-out movement of cyanobacterial filaments from the biofilm probably allows colonization of uncolonized portions of the root several days after the initial inoculation. This is, to the best of our knowledge, the first report of the artificial inoculation of cyanobacteria on marine mangroves.Key words: Avicennia germinans, beneficial bacteria, biofilm, black mangrove, diazotrophic cyanobacteria, Microcoleus sp., nitrogen fixation, plant growth-promoting bacteria, root colonization.
Growth phase dependent resistance to oxidative stress in a phytopathogen Xanthomonas oryzae pv. oryzae
Authors: Vattanaviboon Paiboon , Praituan Wipa , Mongkolsuk Skorn
Published: October 2009

Xanthomonas oryzae pv. oryzae, a rice bacterial pathogen, showed growth phase dependent resistance to oxidative stress killing. Stationary phase cells were much more resistant to killing concentrations of H2O2, organic peroxides, and a superoxide generator (menadione) than cells from early log and mid-log phases. The stationary phase stress resistance phenotype did not require de novo protein synthesis. Also, nutrient starvation or media metabolites were not inducing signals for the phenotype. The stationary phase stress resistance did not apply to all types of stress. For example, X. oryzae pv. oryzae was equally sensitive to heat and pH 5.5 stress at all growth phases tested. This pattern of stationary phase resistance to stress differs from observations in other bacteria and could be important in plant–microbe interactions.Key words: phytopathogen, stationary growth phase, resistance to oxidative stress killing.
Oxidation–reduction of methanol, formaldehyde, serine, and formate inMethanosphaera stadtmanaeusing14C short- and long-term labelling
Authors: Lin Z. , Sparling R.
Published: October 2009

Methanosphaera stadtmanae derives its energy from the reduction by H2of CH3OH, but not CO2, indicating there is a block in the CO2methanogenesis pathway. Both14CH4and14CO2production were detected in whole cells using [14C]formaldehyde or [14C]serine as substrate.14CO2was also observed from [14C]formate in both whole cells and cofactor-depleted cell-free extracts, and NADP-dependent formate dehydrogenase activity was detected. Both formate and serine blocked the formation of14CO2from formaldehyde in whole cells. The results confirmed that enzymes involved in the reduction of carbon from the level of methylene-tetrahydromethanopterin in a common methanogenic pathway and a tetrahydromethanopterin-dependent serine hydroxymethyltransferase were present in this organism. However, the production of14CH4could not be observed from [14C]formate or14CO2plus H2. [14C]Formate was incorporated specifically into histidine and RNA. [14C]Methanol was also found to label rRNA and cytoplasmic proteins, especially corrinoid proteins.Key words: methanogenesis, formate dehydrogenase, formaldehyde oxidation, C1intermediates.
New mini-Tn5 derivatives for insertion mutagenesis and genetic engineering in Gram-negative bacteria
Authors: Alexeyev M. F. , Shokolenko I. N. , Croughan T. P.
Published: October 2009

Five mini-Tn5 derivatives encoding resistance to Km, Cm, Gm, Tc, and Sm, coupled with the polylinker of the pBluescriptII plasmid, were constructed. These derivatives are carried by an ampicillin-resistant plasmid that has a conditional origin of replication from plasmid R6K and origin of conjugal transfer from the broad host range plasmid RP4. The new vectors are smaller than those previously described and possess numerous unique restriction sites inside the minitransposons for gene cloning in addition to SfiI and NotI sites found in their predecessors.Key words: R6K γ-origin, suicide vectors, RP4 oriT, chromosomal insertion.
Oligonucleotide probes based on 16S rRNA sequences for the identification of four Azospirillum species
Authors: Kabir Md Mahbubul , Faure Denis , Haurat Jacqueline , Normand Philippe , Bally René , Jacoud Colette , Wadoux Patrick
Published: October 2009

Partial sequences of the 16S rRNA molecules of nine strains belonging to four Azospirillum species were used to design species-specific oligonucleotide probes. Azospirillum strains sequences were analyzed and three homologous fragments containing 16 nucleotides were determined. These three probes were found to be characteristic of A. lipoferum (Al), A. irakense (Ai), and A. brasilense/amazonense species (Aba) and of few nontarget organisms. The specificity of these three probes was tested both against sequences in the GenBank data base and in numerous colony hybridization experiments. As a few non-target organisms hyridized with the different Azospirillum probes, the use of these probes in bulk soil hybridization is not permitted. However, their use together with specific isolation techniques is validated.Key words: Azospirillum, bacterial isolation, hybridization, oligonucleotide probe, sequence analysis, 16S rRNA.
Occurrence of heterotrophic bacteria and fungi in cold and hot water distribution systems using water of different quality
Authors: Zacheus Outi M. , Martikainen Pertti J.
Published: October 2009

The microbiological quality of cold and hot water samples of 67 Finnish buildings was studied. Most of the buildings were apartment buildings receiving their cold water from municipal groundwater or surface water plants. Disinfection with chlorine was applied in all the surface water plants and 33% of the groundwater plants. Water samples in buildings were taken from incoming cold water, from the hot water main just before and after the heat exchanger, and from a tap or shower in an apartment. The viable counts of mesophilic bacteria and fungi and total cell counts were higher in cold than in hot water samples. In hot water, the microbial counts were higher in samples from taps and showers than from the mains. In taps and showers, the decrease in hot water temperature probably increased the microbial numbers. Thermophilic bacteria appeared with high numbers in all the hot water samples, but thermophilic fungi were found in only one sample. Bacterial biomass and mean cell volume were greater in processed surface water than in processed groundwater samples. Disinfection with chlorine reduced the viable plate counts, although the chlorine concentration was extremely low in the water samples studied.Key words: heterotrophic bacteria, fungi, distribution system, groundwater, surface water.
Cytology of irregular growth forms of Ophiostoma ulmi and Ophiostoma novo-ulmi growing through millipore filter membranes and sterilized elm wood sections
Authors: Ouellette G. B. , Côté C. , Méthot N. , Chamberland H. , Lafontaine J. -G.
Published: October 2009

When Ophiostoma ulmi or Ophiostoma novo-ulmi are grown on either 0.22- or 0.45-μm millipore filter membranes placed on impoverished agar medium, the fungus grows through these membranes and takes on various irregular pleomorphic growth forms (P-forms). Links of continuity between these forms and the more regular ones have been shown using light, confocal, and transmission electron microscopy. Tests with labelled probes, such as gold-complexed wheat germ agglutinin for chitin and β-exoglucanase for cellulosic β-1,4-glucans, have indicated that in P-forms deposition of chitin is much altered but is less so in the case of cellulosic glucan. The cytology of these forms compared with the regular fungal ones is also very different, particularly with reference to mitochondria and nuclei. Also, numerous vesiculate structures were noted in the rarely septate P-forms. Similar irregular forms with opaque contents were produced by these fungi when they were grown on sterilized elm wood sections. When these latter samples were fixed by high-pressure freezing, the following main features were noted: fungal cells with a very thin wall, slightly labelled for chitin but more intensely for cellulosic glucans; well-preserved structures, such as plasmalemma and endoplasmic reticulum; and a slightly opaque, fibril-containing extracellular sheath. Differences in labelling for galactose, whether of wall layers or cell contents, were also observed in regular and P-forms. Electron opaque bodies that labelled strongly for galactose were also numerous in P-forms in some samples.Key words: transmission electron microscopy, high-pressure freezing, gold labelling, extracellular sheaths, wall constituents.
Description of two new species ofHalomonas:Halomonas israelensissp.nov. andHalomonas canadensissp.nov.
Authors: Huval J. Hansel , Latta Roger , Wallace Rebecca , Kushner Donn J. , Vreeland Russell H.
Published: October 2009

Six well-known strains of halotolerant bacteria, including two strains previously identified only as NRCC 41227 and Ba1have been compared using 125 phenotypic characters and DNA–DNA hybridization. Although these strains represent some of the most heavily studied salt-tolerant bacteria, they have never been taxonomically compared. The data presented show that these bacteria form a relatively homogeneous group related at the genus level. The taxonomic comparison showed that these six organisms represented four distinct species all related above the 65% Jaccard coefficient level. In addition to two previously identified bacterial species, Halomonas elongata (ATCC 33173T) and Halomonas halodurans (ATCC 29686T), the strains included in this study represent two previously unnamed Halomonas species. These two new taxa have been assigned the names Halomonas israelensis (ATCC 43985T) and Halomonas canadensis (NRCC 41227T = ATCC 43984). DNA–DNA hybridizations show that these two species are related to the type species H. elongata at 54.9 and 48.9%, respectively.Key words: Halomonas, taxonomy, israelensis, canadensis, halotolerant.
Genetic recombinants inTrichoderma pseudokoningii(Rifai) without typical parasexuality
Authors: Bagagli Eduardo , Furlaneto Márcia Cristina , Pizzirani-Kleiner Aline , Azevedo João Lúcio
Published: October 2009

Heterokaryons were obtained by hyphal anastomosis in Trichoderma pseudokoningii using strains with morphologic and double auxotrophic genetic markers. Stable haploid and unstable hyperhaploid recombinants were detected in monosporic colonies derived from heterokaryons and selected in appropriate media or by an enrichment filtration technique. Diploids were not observed and the recombinant frequencies were higher than those found in fungi with a typical parasexual cycle. The mechanism of recombinant emergence may involve a transient diploid formation followed by chromosomal losses at the hyphal stages, resulting in haploid and hyperhaploid recombinants. The possibility of intrachromosomal rearrangement with the involvement of cytoplasmatic DNA as a heterokaryotic transformation system cannot be excluded.Key words: Trichoderma pseudokoningii, parasexual process, transformation, evolution.
The association of bdellovibrios with surfaces in the aquatic environment
Authors: Williams Henry N. , Kelley Jacqueline I. , Baer Marcie L. , Turng Been-Foo
Published: October 2009

In the aquatic environment, the association of microorganisms with surfaces represents an important phase in the life cycle and ecology of many microbial species. However, previous studies to understand the ecology of the bdellovibrios have ignored examination of the role of surfaces and surface association. In this report, the association of bdellovibrios with sterile surfaces and the stability of the organisms on abiotic and biotic surfaces were investigated. Sterilized oyster shells, glass slides, and steel and titanium surfaces were submerged in the Patuxent River. Following submersion for periods ranging from 2 h to 1 week, test surfaces were scraped or brushed to remove adherent biofilm surface material. The resulting suspension of surface biofilm was cultured for total numbers of bacterial colony-forming units and bdellovibrio plaque-forming units. In other experiments on the persistence of bdellovibrios on surfaces, mussel shells and tunicates with accumulated biofilm were exposed to repeated treatments of mechanical agitation. Following each treatment, the suspension supernatant was cultured for the numbers of predators released from the surface. The results of the two investigations revealed that bdellovibrios associate with surfaces following brief periods of submersion in natural waters, the type of surface influences the association, the number of the predators recovered increased with the time of submersion to a certain peak, and the predators on surfaces are relatively resistant to removal by physical agitation of the surrounding water.Key words: bdellovibrio, surface association.
Production and rheological properties of a succinoglycan from Pseudomonas sp. 31260 grown on wood hydrolysates
Authors: Meade Maura J. , Tanenbaum S. W. , Nakas J. P.
Published: October 2009

Pseudomonas sp. ATCC 31260 produced substantial amounts of anionic extracellular polysaccharide (EPS) from a mineral acid hydrolysate of wood, prepared using the "Tennessee Valley Authority" process. Partially purified EPS production approached 16.5 g/L (as hexadecyltrimethylammonium bromide precipitate) when the pH of the hydrolysate was initially adjusted to 7.5 and amended with 0.05% each of peptone and yeast extract. This EPS, now characterized as a succinoglycan, is composed of glucose, galactose, succinate, pyruvate, and acetate. Solutions of this EPS are pseudoplastic, and under specified conditions, are rheologically comparable with commercially available xanthan.Key words: bioconversion, rheology, viscosity, hardwood hydrolysate.
Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Rhodococcus ruber
Authors: Anderson Alistair J. , Williams D. Roger , Dawes Edwin A. , Ewing David F.
Published: October 2009

The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of the polyhydroxyalkanoate produced from glucose by Rhodococcus ruber was investigated using nuclear magnetic resonance analysis. Spectra obtained for polymer produced from [6-13C]-, [2-13C]-, and [1-13C]glucose were compatible with carboxylation of pyruvate followed by TCA cycle reactions to yield succinate, which is a precursor of propionyl-CoA in the biosynthesis of 3HV in this organism. Carboxylation of pyruvate may occur directly or involve methylmalonyl-CoA transcarboxylase but the latter process is more probable as it provides a route for recycling oxaloacetate, which is essential for the pathway to operate. The observed incorporation of label from NaH13CO3 into C-3 of 3HV monomers cannot occur via forward reactions of the TCA cycle but may be explained by direct carboxylation of pyruvate, followed by reverse reactions of the TCA cycle to yield succinyl-CoA. This minor route accounts for at least 12% of the carbon flux in the synthesis of 3HV monomer units. Inhibitors of key enzymes of possible pathways for polyhydroxyalkanoate biosynthesis in R. ruber were tested. Addition of monofluoroacetate, an inhibitor of aconitase, to cultures decreased production of 3HV, supporting the proposed major route for 3HV synthesis.Key words: Rhodococcus ruber, polyhydroxyalkanoate, PHA, biosynthesis.
Regulation of PHB metabolism inAlcaligenes eutrophus
Authors: Mansfield David A. , Anderson Alistair J. , Naylor Linda A.
Published: October 2009

Metabolites associated with the poly(3-hydroxybutyrate) (PHB) biosynthetic pathway in Alcaligenes eutrophus were measured to gain an insight into the regulation of PHB synthesis in vivo. Alcaligenes eutrophus was grown in carbon-limited chemostat culture to provide bacteria producing negligible PHB, and in nitrogen-limited chemostat culture to yield PHB-synthesizing bacteria. 3-Hydroxybutyryl-CoA (3HBCoA) was detected only in polymer-accumulating bacteria. The level of coenzyme A (CoASH) was approximately three times higher in the absence of PHB synthesis, in accord with the putative role of this metabolite in the regulation of 3-ketothiolase. The level of acetoacetyl-CoA was, however, similar in PHB-accumulating and nonaccumulating bacteria, suggesting that NADPH-acetoacetyl-CoA reductase may regulate PHB synthesis in bacteria grown under carbon limitation. Immediately after nitrogen exhaustion in batch culture of A. eutrophus, there was an initial large decrease in the weight-average molecular weight, which corresponded to the rapid disappearance of CoASH and the maximum level of 3HBCoA. The decrease in the rate of PHB synthesis in batch culture was consistent with regulation involving NADPH-acetoacetyl-CoA reductase. The disappearance of 3HBCoA coincided with the cessation of PHB synthesis and the maximum level of acetyl-CoA.Key words: metabolites, PHB biosynthesis, regulation, Alcaligenes eutrophus, molecular weight.
Biomimetic, amorphous granules of polyhydroxyalkanoates: composition, mobility, and stabilization in vitro by proteins
Authors: Horowitz Daniel M. , Sanders Jeremy K. M.
Published: October 2009

It is a remarkable feature of poly(3-hydroxybutyrate) (PHB) that although the isolated polymer is highly crystalline, native PHB storage granules in cells are only found in an amorphous, mobile state. It has recently been proposed that the failure of PHB granules to crystallize is simply the result of slow nucleation kinetics that are operative for small, isolated particles. In support of this new model, we present here a straightforward procedure by which pure crystalline PHB can be reconstituted into submicron-size, detergent-coated artificial granules. The artificial granules are amorphous and stable in suspension, and they are essentially indistinguishable from their native counterparts in terms of size, morphology, molecular mobility, and density. Furthermore, when the surfactant coating is removed from the artificial granules by dialysis, the granules crystallize, verifying the nucleation hypothesis. In vivo, the PHB granule surface is likely to consist of both polypeptide and lipid; in vitro it is possible to prepare amorphous PHB granules that are stabilized solely by phospholipids or by any of several common proteins (serum albumin, casein, or ovalbumin). Artificial amorphous granules may be prepared from a variety of different bacterial PHAs and from blends of incompatible polyesters.Key words: polyhydroxyalkanoate, poly(3-hydroxybutyrate), granules, nucleation, latex.
Detection of poly(3-hydroxybutyrate) granules by electron microscopy ofVibrio harveyistained with malachite green
Authors: Sun Weiqun , Teng Kathy , Meighen Edward
Published: October 2009

Electron microscopy of potential morphological changes in Vibrio harveyi cells during development of luminescence revealed lipidlike poly(3-hydroxybutyrate) (PHB) granules at high cell density using the standard glutaraldehyde fixation procedure. On staining with malachite green, much more definitive sponge-like granules with defined membranes were observed. Those spongelike granules were not detected in. V. harveyi dark mutants deficient in the production of the lux autoinducer, N-(3-hydroxybutyroyl)homoserine lactone, which controls expression of luminescence. Addition of autoinducer to the mutants during growth restored not only luminescence but also the synthesis of the spongelike granules. In this study, the spongelike granules, produced on staining V. harveyi cells with malachite green, were measured by stereological analyses. A linear relationship was found between the amount of material in the spongelike granules and the amount of PHB over a wide range of levels modulated by the lux autoinducer and (or) growth conditions, providing strong evidence that staining with malachite green is a powerful technique for identification of PHB granules.Key words: PHB granules, malachite green, bioluminescence.
Artificial granule suspensions of long side chain poly(3-hydroxyalkanoate)
Authors: Marchessault R. H. , Morin F. G. , Wong S. , Saracovan I.
Published: October 2009

In vitro preparation of long side chain poly(3-hydroxyalkanoate) artificial granule suspensions that mimic the "as biosynthesized" inclusions in vivo is reported. Elastomeric poly(3-hydroxyalkanoate) can be made into an aqueous suspension of noncrystalline, submicron-sized particles. Light-scattering measurements on these suspensions showed polymer particles in the range of 90–200 nm in diameter. High-resolution 13C-NMR studies demonstrated the noncrystalline character of the in vitro artificial granules. Upon drying at room temperature, the suspensions yielded a continuous film resulting from the coalescence of these polymers with a low glass transition temperature and low Young's modulus. Aqueous suspensions of poly(3-hydroxyoctanoate) are ideal substrates for enzymatic degradation studies because of their stability and purity, since they are self-stabilized. The range of poly(3-hydroxyalkanoates) that can be converted to artificial granules and the methods of preparation are described.Key words: poly(3-hydroxyalkanoate), artificial granules, in vitro bacterial inclusions, enzyme model substrates, poly(3-hydroxyoctanoate).
Substrate specificities of poly(hydroxyalkanoate)-degrading bacteria and active site studies on the extracellular poly(3-hydroxyoctanoic acid) depolymerase ofPseudomonas fluorescensGK13
Authors: Schirmer Andreas , Matz Claudia , Jendrossek Dieter
Published: October 2009

The isolation of poly(3-hydroxyoctanoic acid)- and poly(6-hydroxyhexanoic acid)-degrading bacteria yielded 28 strains with abilities to degrade various polymers. The most versatile strains hydrolyzed five different polyesters comprising short chain length and medium chain length poly(hydroxyalkanoates). The new isolates together with previously isolated poly(hydroxyalkanoate)-degrading bacteria were classified into 11 groups with respect to their polymer-degrading specificities. All PHA depolymerases studied so far have been characterized by the lipase consensus sequence Gly-X-Ser-X-Gly in their amino acid sequence, which is a known sequence for serine hydrolases. When we replaced the central residue, Ser-172, in the corresponding sequence Gly-Ile-Ser-Ser-Gly of the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, with alanine the enzyme lost its activity completely. This result of the mutational experiment indicates that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases.Key words: poly(hydroxyalkanoates), PHA depolymerases, serine hydrolases, substrate specificity, Pseudomonas fluorescens.
Intracellular poly(3-hydroxybutyrate) depolymerase inAlcaligenes eutrophus
Authors: Saito Terumi , Takizawa Kazuaki , Saegusa Haruhisa
Published: October 2009

The intracellular depolymerization of poly(3-hydroxybutyrate) (PHB) in Alcaligenes eutrophus was investigated. PHB granules of A. eutrophus released D-(−)-3-hydroxybutyrate when incubated at 37 °C. The pH profile of autodigestion of the PHB granules revealed two peaks of activity, one at pH 6–7 and the other at a more alkaline pH. Autodigestion of native PHB granules was inhibited in the presence of Triton X-100. The PHB depolymerase activity was detected in the supernatant from centrifugation at 100 000 × g of the cell extract by using the protease-treated native PHB granules as substrate that lost most of their autodigestive activity. The soluble PHB depolymerase activity increased by about 20-fold or more during PHB synthesis compared with that of PHB-deficient cells, and there was a stringent correlation between PHB content and PHB depolymerase activity in A. eutrophus cells. A soluble PHB depolymerase was partially purified and was inhibited strongly in the presence of diisopropyl fluorophosphate. The pH optimum of the PHB depolymerase in the supernatant fraction was about 8–9. These results indicate that two types of PHB depolymerase may be associated with the native granules, and there was an enzyme with an alkaline pH optimum in the supernatant of the cell extract that seemed to be coregulated with PHB synthesis in the cells.Key words: Alcaligenes eutrophus, poly(3-hydroxybutyrate) depolymerase, poly(3-hydroxybutyrate) granules, biodegradation.
Poly(β-L-malate) hydrolase from Plasmodia ofPhysarum polycephalum
Authors: Korherr Christian , Roth Michael , Holler Eggehard
Published: October 2009

A 68-kDa extracellular glycoprotein from Physarum polycephalum that hydrolyses specifically poly(β-L-malic acid) by removing monomers of L-malic acid in an exolytic manner has been purified and characterized. The enzyme was purified 1740-fold from the culture medium by ammonium sulfate precipitation, hydrophobic interaction chromatography on butyl-Toyopearl, and gel permeation chromatography on Superdex 200 to a specific activity of 9.0 μmol∙min−1∙mg−1. The hydrolase was also purified from the cytosol, which contained 1 mg in 43 g cells in contrast to 1 mg extracellular enzyme in 28 L of culture medium. The pH optimum was pH 3.5 as a result of the effect of an acidic side chain on Vmaxand the preferred binding of poly(β-L-malate) in the ionized form. Intracellular hydrolase was only marginally active on [14C]poly(β-L-malate) that had been injected into plasmodia. Poly(L-aspartate), poly(L-glutamate), poly(vinyl sulfate), and poly(acrylate) were neither bound nor degraded by the hydrolase. Poly(β-hydroxybutyric acid), which was considered the reduced form of poly(β-L-malate), was not a substrate. The enzyme is neither a metallo- nor a serine-esterase, and is distinct from poly(3-hydroxybutyric acid) depolymerases. It is related to a glucosidase with respect to hydrophobic interaction chromatography, the pH-activity dependence, and its inhibition with mercuribenzoate, N-bromosuccinimide, and D-gluconolactone, but not the use of the substrates.Key words: poly(β-L-malate), polymalatase, Physarum polycephalum, biodegradative polymer.
Production of poly(3-hydroxybutyric acid) by recombinantEscherichia colistrains: genetic and fermentation studies
Authors: Lee Sang Yup , Chang Ho Nam
Published: October 2009

A number of Escherichia coli strains including K12, B, W, XL1-Blue, DH5α, HB101, JM109, and C600 were transformed with the stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid biosynthesis genes, and were subsequently compared for their ability to synthesize and accumulate poly(3-hydroxybutyric acid) (PHB). The rate of PHB synthesis, the extent of PHB accumulation, and PHB yield from glucose varied considerably from one strain to another. Strains XL1-Blue and B harboring pSYL105 synthesized PHB at the highest rate to a final concentration of ca. 7 g/L in complex medium containing 20 g glucose/L. With an aim to reduce the cost of the medium, the effect on PHB accumulation of supplementing a defined medium with complex nitrogen sources was examined. A PHB concentration of 81 g/L could be obtained in 41 h from a pH-stat fed-batch culture of XL1-Blue(pSYL105) in a semidefined medium. When the availability of acetyl-CoA was increased by supplementing the medium with complex nitrogen sources, amino acids, or oleic acid, PHB synthesis by recombinant E. coli was enhanced.Key words: polyhydroxyalkanoic acid, poly(3-hydroxybutyric acid), fed batch, Escherichia coli.
Hemicellulose as a potential substrate for production of poly(β-hydroxyalkanoates)
Authors: Ramsay Juliana A. , Hassan Marie-Claire Aly , Ramsay Bruce A.
Published: October 2009

Pseudomonas cepacia was evaluated for its ability to utilize xylose, a major hemicellulosic sugar of hardwoods, for the production of the biodegradable, thermoplastic poly(β-hydroxybutyrate) (PHB). This culture produced 2.6 g∙L−1 of biomass containing 60% (w/w) PHB when grown in shake flasks on an ammonium-limited, mineral salts medium containing 10 g∙L−1 of xylose. Batch fermentation data showed that growth and PHB production kinetics on xylose were similar to previously published results for the same microorganism on fructose. On xylose, the maximum specific growth rate, the maximum specific PHB production rate (based on total biomass minus PHB biomass), the overall yield of biomass produced from substrate consumed, the yield of PHB produced from substrate consumed (YPHB/S), and the percentage of PHB were 0.22 h−1, 0.072 g∙g−1∙h−1, 0.29 g∙g−1, 0.11 g∙g−1 and 45% (w/w), respectively. A high maintenance energy (0.119 g of xylose∙g of biomass−1∙h−1) is probably responsible for the low overall yield. However, the product yield, YPHB/S, was still the highest reported for any microorganism grown on pentosic sugars. Using the YPHB/S of 0.11 g∙g−1, it was estimated that the substrate cost (in terms of hydrolyzed hemicellulose) for PHB production would be similar to that of cane molasses and half that of bulk glucose.Key words: poly(β-hydroxybutyrate), PHB, hemicellulose, xylose, Pseudomonas cepacia.
Chemical synthesis of poly(β-hydroxybutyrate) by the polymerization of (R,S)-β-butyrolactone with aluminoxane catalysts
Authors: Lenz Robert W. , Yang John , Wu Bin , Harlan C. Jeff , Barron Andrew R.
Published: October 2009

Racemic β-butyrolactone can be polymerized to crystalline polymers of reasonably high molecular weight by the use of aluminoxane catalysts. Investigations in this and other laboratories have shown that these polymers can be fairly highly isotactic, and in that form they are closely related in structure and physical properties to a reserve polyester produced by many bacteria: poly(β-hydroxybutyrate). In the present investigation, Ziegler–Natta type aluminoxane catalyst systems and a new aluminoxane derivative, tert-butylaluminoxane, were evaluated in attempts to polymerize racemic β-butyrolactone in higher yields to polymers of higher molecular weight and higher stereoregularity. The addition of group 4 metallocenes as cocatalysts for both methylaluminoxane and iso-butylaluminoxane catalysts gave higher yields of the isotactic polymer than those obtained with these catalysts alone, but surprisingly, the tert-butylaluminoxane catalysts produced predominantly syndiotactic rather than isotactic polymers.Key words: poly(β-hydroxybutyrate), β-butyrolactone, aluminoxane catalysts, isotactic poly(β-hydroxybutyrate), syndiotactic poly(β-hydroxybutyrate).