bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequential enrichment of microbial populations exhibiting enhanced biodegradation of crude oil
Authors: Venkateswaran Kasthuri , Harayama Shigeaki
Published: October 2009

The distribution of oil-degrading bacteria in the coastal water and sediments of Hokkaido, Japan, was surveyed. The potential of mixed microbial populations to degrade weathered crude oil was not confined to any ecological components (water or sediment) nor to the sampling stations. One microbial culture that was stable during repeated subculturing degraded 45% of the saturates and 20% of the aromatics present in crude oil in 10 days during the initial screening. The residual hydrocarbons in this culture were extracted by chloroform and dispersed in a fresh seawater-based medium and subsequently inoculated with microorganisms from the first culture. After full growth of the second culture, the residual hydrocarbons were again extracted and dispersed in a fresh medium in which microorganisms from the second culture had been inoculated. This sequential process was carried out six times to enrich those microorganisms that grew on the recalcitrant components of crude oil. After repeated exposure of the residual crude oil to the enriched microorganisms, about 80% of the initially added crude oil was degraded. The cultures obtained after each enrichment cycle were kept, and the degradation of fresh crude oil by the enriched microorganisms was examined. The degradative activity of the enriched cultures increased as the number of enrichment cycles increased. A microbial population that had been selected six times on the residual crude oil could degrade 70% of the saturates and 30% of the aromatics of crude oil. Thus, growth of a microbial population on residual crude oil improved its ability to biodegrade crude oil.Key words: crude oil, biodegradation, sequential enrichment, saturated hydrocarbon, aromatic hydrocarbon.
Low temperature growth, freezing survival, and production of antifreeze protein by the plant growth promoting rhizobacteriumPseudomonas putidaGR12-2
Authors: Sun Xiuying , Griffith Marilyn , Pasternak J. J. , Glick Bernard R.
Published: October 2009

The plant growth promoting rhizobacterium Pseudomonas putida GR12-2 was originally isolated from the rhizosphere of plants growing in the Canadian High Arctic. Here we report that this bacterium was able to grow and promote root elongation of both spring and winter canola at 5 °C, a temperature at which only a relatively small number of bacteria are able to proliferate and function. In addition, the bacterium survived exposure to freezing temperatures, i.e., −20 and −50 °C. In an effort to determine the mechanistic basis for this behaviour, it was discovered that following growth at 5 °C, P. putida GR12-2 synthesized and secreted to the growth medium a protein with antifreeze activity. Analysis of the spent growth medium, following concentration by ultrafiltration, by SDS-polyacrylamide gel electrophoresis revealed the presence of one major protein with a molecular mass of approximately 32–34 kDa and a number of minor proteins. However, at this point it is not known which of these proteins contains the antifreeze activity.Key words: plant growth promoting rhizobacteria, PGPR, bacterial fertilizer, soil bacteria, antifreeze protein.
Preliminary grouping of mutacins
Authors: Morency H. , Trahan L. , Lavoie M. C.
Published: October 2009

All the mutacins described so far have been shown to be different substances. To classify 86 mutacinogenic strains, we determined their activity spectra towards 12 standard oral streptococci and towards themselves, using a deferred antagonism test. The mutacinogenic strains could be grouped into 15 mutacinotypes according to their activity spectra against the 12 standard oral streptococci. By combining the results of their activity spectra and their sensitivity patterns amongst themselves, we defined 24 mutacin groups. Using the 24 type strains corresponding to these groups as producers and as indicators, it will be easier to classify new mutacin-producing isolates.Key words: bacteriocin, mutacin, Streptococcus mutans, inhibitory substance.
Subtyping ofLegionella pneumophilaisolates by arbitrarily primed polymerase chain reaction
Authors: Ledesma E. , Llorca J. , Dasí M. A. , Camaró M. L. , Carbonell E. , Sacristán T. , Martí A. , Pellicer S. , Herrero P.
Published: October 2009

Arbitrarily primed polymerase chain reaction (AP-PCR) was used to differentiate strains of Legionella pneumophila isolated from different water sources in a resort hotel in Benidorm, Alicante, Spain, where an outbreak of Legionnaires' disease occurred among a group of tourists between 65 and 80 years of age. All isolates were L. pneumophila serogroup 1, subtype Pontiac (Knoxville 1). Five different patterns (P1 to P5) were obtained by AP-PCR. The number of bands per pattern varied between 4 and 11. Patterns P1 and P2 represented 60 and 20% of L. pneumophila isolates, respectively. Since different subpopulations of L. pneumophila coexisted (up to three different AP-PCR patterns were identified in a single room), it was not possible to link an individual L. pneumophila strain to the occurrence of this outbreak.Key words: Legionella pneumophila, AP-PCR, subtyping, outbreak.
The two overlapping Azospirillum brasilense upstream activator sequences have differential effects on nifH promoter activity
Authors: Passaglia L. M. P. , Schrank A. , Schrank I. S.
Published: October 2009

The Azospirillum brasilense nifH promoter is positively controlled by the NifA protein bound to the upstream activator sequences (UASs). Two overlapping UASs located at −191 and −182 were identified with the consensus TGT-N10-ACA motif. The role of the two UASs of Azospirillum brasilense nifH promoter was examined by introducing base substitutions in the NifA binding sites. Both the promoter down phenotype of a mutation in UAS2 and increased activation when UAS1 was mutated reveal that the integrity of the UAS2 is required for the efficient activation of nifH promoter. This atypical NifA-binding site may represent a region interacting with two NifA dimers.Key words: Azospirillum brasilense, nif promoters, upstream activator sequence mutagenesis, NifA binding.
Coenzyme A ligases involved in anaerobic biodegradation of aromatic compounds
Authors: Villemur Richard
Published: October 2009

Bacterial strains and consortia of bacteria have been isolated for their ability to degrade, under anaerobic conditions, homocyclic monoaromatic compounds, such as phenolic compounds, methylbenzenes, and aminobenzenes. As opposed to aerobic conditions where these compounds are degraded via dihydroxyl intermediates introduced by oxygenases, most of aromatic compounds under anaerobic conditions are metabolized via aromatic acid intermediates, such as nitrobenzoates, hydroxybenzoates, or phenylacetate. These aromatic acids are then transformed to benzoate before the reduction and the cleavage of the benzene ring to aliphatic acid products. One step of these catabolic pathways is the addition of a coenzyme A (CoA) residue to the carboxylic group of the aromatic acids by CoA ligases. This addition would facilitate the enzymatic transformation of the aromatic acids to benzoyl-CoA and the subsequent degradation steps of this latter molecule. Aromatic acid – CoA ligases have been characterized or detected from several bacterial strains that were grown under anaerobic conditions and from an anaerobic syntrophic consortium. They are also involved in the degradation of some aromatic compounds under aerobic conditions. They have molecular masses varying between 48 and 61 kDa, require ATP, Mg2+, and CoASH as cofactors, and have an optimum pH of 8.2–9.3. Amino acid sequence analyses of four aromatic acid–CoA ligases have revealed that they are related to an AMP-binding protein family. Aromatic acid – CoA ligases expressed in anaerobically grown bacterial cells are strictly regulated by the anaerobic conditions and the presence of aromatic acids.Key words: aromatic compounds, coenzyme A ligase, anaerobic microorganisms.
DNA sequence and transcriptional characterization of a β-glucanase gene (celB) fromRuminococcus flavefaciensFD-1
Authors: Vercoe Philip E. , Finks Jennie L. , White Bryan A.
Published: October 2009

The recombinant clone pBAW101 (in pBluescript SK–) contains the celB endoglucanase gene from Ruminococcus flavefaciens FD-1. Subcloning indicated that the endoglucanase activity expressed was present within a 2.4-kb insert (pBAW104). The nucleotide sequence of the celB gene was determined, and upon analysis, revealed an open reading frame of 1943 nucleotides that encodes a polypeptide of 632 amino acids with a molecular weight of 69 414. A putative Shine–Dalgarno sequence was identified 6 bp upstream from the translation start site. The N-terminal 32 amino acid residues were typical of prokaryotic signal sequences. Hydrophobic cluster analysis (HCA) and DNA alignment of CelB to other published β-glucanase polypeptide sequences in GenBank indicate that CelB belongs in HCA cellulase family 44. Primer extension analyses were performed using RNA isolated from R. flavefaciens grown on cellulose and cellobiose, and from Escherichia coli containing the plasmid clone pBAW104. Transcription is initiated at different sites in E. coli and R. flavefaciens. In the case of R. flavefaciens transcription is initiated at a C residue (nucleotides 329), 221 bp upstream from the translation start site. There were no regions resembling E. coli σ70-like promoter sequences present upstream from this putative transcription initiation site. In contrast, numerous transcription initiation sites were identified when RNA from E. coli was used in the primer extension analyses.Key words: Ruminococcus flavefaciens, endoglucanase, transcription, family 44 endoglucanase.
DNA hybridization analysis of thePseudomonas aeruginosaelastase gene [lasB) from different clinical isolates
Authors: Hamood Abdul N. , Griswold John
Published: October 2009

Pseudomonas aeruginosa produces several extracellular virulence factors including elastase (which is encoded by lasB). Recently, we examined several clinical isolates of P. aeruginosa for the production of toxin A, elastase, exoenzyme S, and phospholipase C. Although the majority of the isolates produced a high level of elastase, a few isolates produced either very low or no detectable elastase. In this study, we tried to determine the presence of restriction site heterogeneity within lasB from these isolates and the possible correlation between such heterogeneity and the observed variation in elastase production. Chromosomal DNA from the isolates was digested with different restriction enzymes and examined by Southern blot hybridization experiments using two lasB probes. One lasB probe covers 636 bp of lasB structural gene while the other covers 240 bp of the lasB upstream region. Chromosomal DNA from P. aeruginosa PAO1 and PA103 was used as controls. Results indicate that chromosomal DNA from all isolates hybridized to both lasB probes. Depending on the restriction enzyme used for DNA digestion, lasB from 3 to 12% of the isolates showed different patterns of hybridization with the lasB structural gene probe. However, no difference in the hybridization pattern was seen with the lasB upstream probe. With the exception of one isolate, hybridization of genomic DNA from different isolates (with both probes) produced a single hybridization band. In that isolate, an additional hybridization band was detected. Immunoblotting experiments confirmed that elastase protein is not produced by 6 out of 67 isolates. However, lasB from four of these elastase-deficient strains showed no difference in the hybridization pattern with either lasB probe. These results suggest that (i) lasB is present as a single copy in all but one isolate; (ii) with the exception of one, the lasB upstream region from different P. aeruginosa isolates contains no restriction site polymorphism; (iii) the observed heterogeneity within lasB structural genes is limited; and (iv) variations in the hybridization patterns of lasB from different isolates do not correlate with the differences between these isolates in elastase production.Key words: Pseudomonas aeruginosa, clinical isolates, DNA hybridization, elastase, lasB.
Metabolic characterization ofAcetobacter diazotrophicus
Authors: Alvarez B. , Martínez-Drets G.
Published: October 2009

Carbon and nitrogen metabolism were investigated in Acetobacter diazotrophicus Pal 3, a N2-fixing bacterium able to grow at low pH and at high sugar concentration. Enzymatic, respiratory, and uptake studies were performed. The main active pathway for the catabolism of phosphorylated glucose was the pentose phosphate pathway. In addition, A. diazotrophicus directly oxidized glucose, gluconate, and ketogluconates through respiratory chain-linked enzymes. Soluble enzymes for the oxidation of glucose and gluconate were also found. Acetobacter diazotrophicus had a complete tricarboxylic acid cycle with a respiratory chain-linked malate dehydrogenase. The ability to grow on two- and three-carbon substrates would be explained by the presence of gluconeogenesis. Lack of bacterial growth on dicarboxylates was explained by the absence of a transport system. Ammonium assimilation proceeded mainly through glutamate dehydrogenase under ammonium excess but also through energy-demanding glutamine synthetase and glutamate synthase under N2-fixing conditions. Acetobacter diazotrophicus was not able to transport sucrose and its ability to grow on this disaccharide was explained by the presence of an extracellular enzyme with saccharolytic activity.Key words: Acetobacter diazotrophicus, carbon–nitrogen metabolism, extracellular saccharolytic activity, sucrose–succinate uptake.
Tet(O), a protein that mediates ribosomal protection to tetracycline, binds, and hydrolyses GTP
Authors: Taylor Diane E. , Jerome Lori J. , Grewal Jaswinder , Chang Nicholas
Published: October 2009

The tet(O) tetracycline resistance gene, originally cloned from Campylobacter jejuni, mediates resistance by ribosomal protection. Using partially purified Tet(O) protein of 68 000 Da whose identity was verified by ribosomal protection assays, amino terminal sequencing, and immunoblotting using an antibody raised against the deduced 15 amino acids at the carboxyl terminus of the Tet(O) protein, the Tet(O) protein was found to bind to [α-32P]GTP and [3H]GDP using a filter binding assay. [γ-32P]GTP hydrolysis by Tet(O) was also demonstrated and was found to be time dependent with more than 50% of the hydrolysis activity occurring within the first 5 min. The GTPase activity of Tet(O) appears to be ribosome dependent, suggesting that ribosomes act as an effector similar to other G proteins involved in signal transduction.Key words: ribosomes, tetracycline resistance, GTPase, protein synthesis.
Cyanobacteria and black mangroves in Northwestern Mexico: colonization, and diurnal and seasonal nitrogen fixation on aerial roots
Authors: Toledo Gerardo , Bashan Yoav , Soeldner Al
Published: October 2009

Nitrogen fixation and colonization by associative cyanobacteria in the aerial roots (pneumatophores) of black mangrove trees was evaluated in situ at Balandra lagoon, Baja California Sur, Mexico, for 18 consecutive months. Year-round vertical zonation of cyanobacterial colonization was determined along the pneumatophores. The bottom part close to the sediment was colonized mainly by nonheterocystous, filamentous cyanobacteria resembling Lyngbya sp. and Oscillatoria sp. The central zone was colonized mainly by filaments resembling Microcoleus sp. and the upper part was colonized by coccoidal cyanobacteria within defined colonies resembling Aphanothece sp. mixed with undefined filamentous cyanobacteria. Two of the cyanobacteria (Microcoleus sp. and Anabaena sp.) isolated from the pneumatophore were diazotrophs. Massive biofilm production along the pneumatophores was evident throughout the observation period. The surrounding sediment was seasonally dominated by heterocystous Anabaena sp. Glass and dead-wood surfaces incubated for 18 months in the pneumatophore vicinity showed no zonation in the colonization pattern, although they were heavily colonized. In situ N2fixation showed seasonal and diurnal fluctuations. N2fixation was low during winter, increased in early summer, and reached its peak in midsummer. N2fixation in the summer showed diurnal peaks: one in the morning until midday and the second in the late afternoon. N2fixation was at its lowest levels near midnight. Light and water temperature are probably primary environmental factors governing N2fixation on the pneumatophores.Key words: Avicennia germinans, black mangrove, diazotrophic cyanobacteria, nitrogen fixation, pneumatophore.
Peanut rhizobia under salt stress: role of trehalose accumulation in strain ATCC 51466
Authors: Ghittoni Nora E. , Bueno Miguel A.
Published: October 2009

Strain ATCC 51466, a motile peanut Rhizobium sp., showed patterns of utilization of diverse carbon sources characteristic of fast growers. Bacteria had periplasmic neutral glucans with molecular weight close to 3000. When the extracellular concentration of NaCl was raised to 400 mM, the lag phase of the culture was prolonged about threefold and the generation time was increased almost twice. The changes in growth behavior of salt-stressed bacteria were accompanied by the full suppression of periplasmic oligoglucans and the accumulation of cellular trehalose. Almost identical changes in cell-associated oligoglucans were observed after exposing peanut Rhizobium sp. strain ATCC 10317 to hypersalinity. When the osmotic pressure of the medium was augmented by the addition of either 200 mM mannitol or 16% (w/v) polyethylene glycol, cells of strain ATCC 51466 contained decreased levels of oligoglucans and accumulated trehalose. On the other hand, the content of cellular trehalose increased throughout logarithmic and stationary phases of growth of strain ATCC 51466 in a medium supplemented with 400 mM NaCl. When bacterial cultures were shifted from hypersaline to basal media, oligoglucans were the only oligosaccharides detected. The addition of 10 mM proline to bacteria grown under hypersalinity led to a 50% decrease in the level of trehalose and to the accumulation of oligoglucans. The addition of 10 mM glycine betaine to bacteria grown under hypersalinity also produced accumulation of oligoglucans, but the level of trehalose did not decrease. The results presented here are consistent with a role for trehalose as a compatible solute in peanut Rhizobium ATCC 51466, and they suggest that exogenously added proline may act as a compatible solute in preference to trehalose.Key words: periplasmic glucans, trehalose, peanut Rhizobium, osmotic stress.
Calcium ion influx during sporulation in the yeastSaccharomyces cerevisiae
Authors: Suizu Tetsuyoshi , Tsutsumi Hiroko , Kawado Akitsugu , Suginami Koji , Imayasu Satoshi , Murata Kousaku
Published: October 2009

The changes in intra- and (or) extra-cellular concentrations of Ca2+, Mg2+, K+, and Na+during sporulation of a MATa/MATα diploid yeast of Saccharomyces cerevisiae were examined in a nutrition-deprived medium with potassium acetate. Among these, Ca2+in external medium was preferentially incorporated into cells, and sporulation was induced when the magnitude of free Ca2+gradient between cytosol [Ca2+]iand external medium [Ca2+]oreached more than 3 × 103([Ca2+]i/[Ca2+]o = 3.5 × 103). The result indicated that the meiosis and (or) sporulation signal of the yeast S. cerevisiae was generated through elevated Ca2+influx rather than release from the internal Ca2+stores.Key words: Saccharomyces cerevisiae, calcium signalling, Ca2+influx, meiosis, sporulation.
Differences among strains of Bradyrhizobium in fatty acid–methyl ester analysis
Authors: Graham P. H. , Sadowsky M. J. , Tighe S. W. , Thompson J. A. , Date R. A. , Howieson J. G. , Thomas R.
Published: October 2009

Fatty acid–methyl ester (FAME) and two-dimensional principal component analysis of 89 strains of Bradyrhizobium, most of which were from soybean, distinguished five groups of bradyrhizobia. These included one cluster containing several isolates previously designated as Bradyrhizobium elkanii, and two related clusters containing strains previously identified as belonging to Bradyrhizobium japonicum groups IA and IB. Not all of the organisms evaluated clustered with the B. japonicum and B. elkanii strains. A number of Bradyrhizobium strains isolated from soybean in Korea and northern Thailand had FAME profiles so different from the B. japonicum and B. elkanii strains as to warrant separation at the species level. A slow-growing isolate from Lupinus also had a FAME profile very different from those of the other bradyrhizobia. Results obtained in this study were generally in agreement with those obtained using other taxonomic approaches, suggesting that FAME analysis provides a relatively simple and reliable procedure for the initial characterization of Bradyrhizobium isolates.Key words: fatty acid analysis, Bradyrhizobium taxonomy, FAME analysis, strain identification.
Alterations in cell pigmentation, protein expression, and photosynthetic capacity of the cyanobacteriumOscillatoria tenuisgrown under low iron conditions
Authors: Trick Charles G. , Wilhelm Steven W. , Brown Christopher M.
Published: October 2009

To better describe the iron-limited nutrient status of aquatic photosynthetic microorganisms, we examined the effects of iron limitation on pigment content, maximum rates of photosynthetic oxygen evolution, and respiratory oxygen consumption in the filamentous cyanobacterium Oscillatoria tenuis Ag. Within the range of iron (4.2 × 10−5–5.1 × 10−9 M FeCl3), growth rates were not limited by photosynthetic capacity but rather by another, as of yet undetermined, iron-requiring cellular function. We have also investigated membrane proteins that are induced when the cells are grown in low iron medium. Using membrane fractionation techniques we were able to recognize specific proteins localized in the outer membrane and periplasmic space of O. tenuis. The recovery of growth rates at low iron levels occurred in parallel with the induction of these proteins and the production of extracellular siderophores. The additional iron acquired by this high affinity transport system did not reestablish photosynthesis in O. tenuis to the iron-satiated level but did reestablish growth to iron-replete levels. Oscillatoria tenuis appears to invoke an alternate physiology to compensate for iron deficiency.Key words: cyanobacteria, iron, Oscillatoria tenuis, periplasmic proteins, photosynthesis.
Poly(β-hydroxybutyrate) extrusion from pleomorphic cells ofAzotobacter vinelandiiUWD
Authors: Page William J. , D'elia Luis , Sherburne Richard , Graham Lori L.
Published: October 2009

Azotobacter vinelandii UWD cells fill with up to 80% (per dry mass) poly(β-hydroxybutyrate) (PHB) after 24 h growth in medium containing sugars and fish peptone. However, peptones were not usually added to Azotobacter culture as they induced pleomorphism and compromised cell wall strength. This study examines the morphology of these PHB-producing pleomorphic cells in the transmission electron microscope. PHB-producing cells incubated for 18–24 h were most frequently 2–3 μm diameter spheres containing up to 20 PHB inclusions/cross section, or a calculated ≈ 100 inclusions/cell volume. These inclusions tended to be of small size (≈ 0.5 μm diameter) and became fewer and larger in older cells. The most striking feature of these pleomorphic cells was the apparent extrusion of polymer from the cells. It is unlikely that PHB extrusion is an active process from a viable cell as there was considerable cell wall damage at the point of polymer extrusion. The results suggest that the extrusion of PHB may be the result of polymer expansion, caused by the dehydration of the specimen for transmission electron microscopy, coupled with the inability of the pleomorphic cell wall to retain the expanding polymer. Thus, freeze-substituted sections of similar cells that were prepared without chemical dehydration did not extrude PHB. However, lysed cells prepared for transmission electron microscopy by chemical dehydration also did not extrude PHB, which suggests differences in the fluidity of the PHB in intact cell inclusions and lysed cell granules.Key words: poly(β-hydroxybutyrate), inclusions, polymer expansion, dehydration artifact.
Bioengineering of poly(β-hydroxyalkanoates) for advanced material applications: incorporation of cyano and nitrophenoxy side chain substituents
Authors: Kim Ohyoung , Gross Richard A. , Rutherford Denise R.
Published: October 2009

Novel functional poly(β-hydroxyalkanoates), PHAs, with hyperpolarizable side groups were prepared. Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442 were selected as biocatalysts. These organisms were first grown on citrate (40 mM) before polymer formation was studied in a second stage. The carbon sources in second-stage cultivations were mixtures (total of 15 mM) of octanoate (OA) with either 7.5 or 10 mM para-cyanophenoxyhexanoate (CPH), para-cyanophenoxyvalerate (CPV), para-cyanophenoxybutyrate (CPB), or para-nitrophenoxyhexanoate (NPH). For both organisms, only small deviations in the number of colony-forming units per millilitre were observed when second-stage cultivations containing only 15 mM octanoate were compared with those using mixtures of OA with either CPB, CPV, CPH, or NPH. PHA volumetric yields as a function of organism, carbon source mixture, and culture time are reported. The percent incorporations of substituted phenoxy side groups in the polymer products were determined by1H NMR spectroscopy. Pseudomonas putida formed PHAs with up to 24.2% 3-hydroxy-6-(para-cyanophenoxy)hexanoate side groups when 5 mM OA and 10 mM CPH were used. A dramatic decrease in the percent incorporation of para-cyanophenoxy (CP) by P. putida was observed when the relatively shorter CP-substituted n-alkanoate chain CPV and CPB cosubstrates were used. Use of NPH in place of CPH had deleterious effects on both polymer formation and percent incorporation of substituted phenoxy side groups in P. putida. Pseudomonas oleovorans formed PHAs with only up to ~2% CP side chains when a combination of OA and CPH was used and little to no CP side groups when a combination of OA and CPV or CPB was used. Substitution of NPH in place of CPH led to a modest increase (up to ~5%) in substituted phenoxy side groups. Thus, a new route to first-generation chiral polymer structures for nonlinear optical applications was demonstrated.Key words: Pseudomonas oleovorans, Pseudomonas putida, poly(β-hydroxyalkanoate), poly(3-hydroxy-6-para-cyanophenoxyhexanoate), poly(3-hydroxy-6-para-nitrophenoxyhexanoate), poly(β-hydroxy-para-cyanophenoxyvalerate).
Low molecular weight complexed poly(3-hydroxybutyrate): a dynamic and versatile molecule in vivo
Authors: Reusch Rosetta N.
Published: October 2009

It is increasingly clear that poly(3-hydroxybutyrate) (PHB) is not just an inert storage polymer, confined to certain bacteria, but a ubiquitous, interactive, solvating biopolymer involved in important physiological functions. Low molecular weight PHB, complexed to other macromolecules (c-PHB), is widely distributed in biological cells, being found in representative organisms of nearly all phyla. Complexation modifies the physical and chemical properties of c-PHB, allowing it to pervade aqueous as well as hydrophobic regions of the cell, and as a result c-PHB can be found in cytoplasm and intracellular fluids as well as in membranes and lipoproteins. The lipidic homopolymer associates with other macromolecules primarily via its ester carbonyl oxygens, which can act as hydrogen-bond acceptors or as ligands for coordinate bonds to cations. The spacing of the electron-donating groups along the flexible backbone allows for multiple bonding interactions, and forms the basis for the ability of c-PHB to bind to proteins, or to form ion-conducting complexes with salts. The singular ability of c-PHB to dissolve salts and facilitate their transfer across hydrophobic barriers defines a potential physiological niche for c-PHB in cell metabolism.Key words: polyhydroxybutyrate, polyphosphate, polymer electrolyte, ion transport.
Accumulation of poly(3-hydroxybutyric acid) and overproduction of exopolysaccharides in a mutant of a methylotrophic bacterium
Authors: Breuer Uta , Ackermann Jörg-Uwe , Babel Wolfgang
Published: October 2009

The pink-pigmented facultatively methylotrophic bacterium Methylobacterium rhodesianum MB 126 is able to grow on methanol as the sole source of carbon and energy. Under certain conditions, e.g., limitation of ammonium, phosphate, or oxygen, carbon from methanol is channeled into poly(3-hydroxybutyric acid) (PHB) whereas other polymers or metabolites are hardly overproduced. A mutant of this strain, which we isolated after chemical mutagenesis, is impaired in its ability to synthesize PHB. Under the conditions mentioned above, the mutant still accumulated PHB, but in the absence of ammonium it simultaneously synthesized PHB and a considerable amount of an exopolysaccharide. This phenomenon was surprising insofar as the wild type did not produce exopolysaccharide in such amounts. An attempt was made to elucidate and discuss the possible reasons for these findings.Key words: methylotrophy, serine pathway bacteria, PHB, exopolysaccharides.
Biosynthesis of copolyesters by Hydrogenophaga pseudoflava from various lactones
Authors: Choi Mun Hwan , Song Jae Jun , Yoon Sung Chul
Published: October 2009

Hydrogenophaga pseudoflava (formerly Pseudomonas pseudoflava) was able to accumulate a large amount of copolyesters when grown on mixed substrates of glucose and lactones in a batch fermentation. Lactones such as γ-butyrolactone, γ-valerolactone, and higher analogues generally did not support cell growth when used as the sole carbon source. Co-feeding of lactones with glucose enhanced the utilization of lactones for both copolyester accumulation and cell growth. The copolyester from the cells grown on the mixed substrates of glucose (10 g/L) and γ-valerolactone (1–3 mL/L) was poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), while cells grown with γ-butyrolactone (1–3 mL/L) as a cosubstrate produced poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)). The values of parameter D, calculated from the NMR dyad-sequence data for polymer samples obtained after 72 h of cultivation, showed lactone concentration dependences differing among lactones. The time-course data obtained from growth on the cosubstrates of γ-valerolactone (2 mL/L) and glucose (10 g/L) revealed that 3HV-rich copolymers were synthesized in the early growth phase, and the 3HB-rich fraction steadily increased in the later accumulation phase and then peaked at 80 h when γ-valerolactone was depleted. These polyhydroxyalkanoate accumulation profiles suggested a high D value of the final product, whose value was determined to be 3.25. γ-Valerolactone was consumed faster than γ-butyrolactone. The difference between the assimilation behavior of the two lactones was discussed in relation to the heterogeneity of the final copolyester products. A correlation between NMR microstructure and the physiology of polyhydroxyalkanoate accumulation was observed.Key words: microstructural heterogeneity, bacterial copolyesters, lactones, Hydrogenophaga pseudoflava.
Isolation and partial characterization ofBacillus megateriummutants deficient in poly(3-hydroxybutyrate) synthesis
Authors: Floccari Mirtha E. , López Nancy I. , Méndez Beatriz S. , Fürst Ursula Pieper , Steinbüchel Alexander
Published: October 2009

Poly(3-hydroxybutyrate) (PHB)-negative mutants of Bacillus megaterium were isolated following mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. Different strategies were used for isolation, including ultracentrifugation through sucrose gradients and selection for resistance to allyl alcohol. The mutants were detected on agar plates by staining the colonies with Sudan Black. Four mutants did not synthesize any PHB as revealed by gas chromatographic analysis. The enzymatic characterization showed no or extremely low synthase activity for all of the mutants. In contrast, no significant alterations were observed in the β-ketothiolase and the NADPH-dependent acetoacetyl-CoA reductase activities. All mutants sporulated in complete and minimal media.Key words: poly(3-hydroxybutyrate), synthase, mutants, Bacillus megaterium.
Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions
Authors: Steinbüchel Alexander , Aerts Kristin , Liebergesell Matthias , Wieczorek Roman , Babel Wolfgang , Föllner Christina , Madkour Mohamed Hussein , Mayer Frank , Pieper-Fürst Ursula , Pries Andreas , Valentin Henry Erich
Published: October 2009

Some mathematical calculations were done that provided information about the structure and biochemistry of polyhydroxyalkanoic acid (PHA) granules and about the amounts of the different constituents that contribute to the PHA granules. The data obtained from these calculations are compared with data from the literature, which show that PHA granules consist not only of the polyester but also of phospholipids and proteins. The latter are referred to as granule-associated proteins, and they are always located at the surface of the PHA granules. A concept is proposed that distinguishes four classes of structurally and functionally different granule-associated proteins: (i) class I comprises the PHA synthases, which catalyze the formation of ester linkages between the constituents; (ii) class II comprises the PHA depolymerases, which are responsible for the intracellular degradation of PHA, (iii) class III comprises a new type of protein, which is referred to as phasins and which has most probably a function analogous to that of oleosins in oilseed plants, and (iv) class IV comprises all other proteins, which have been found to be associated with the granules but do not belong to classes I–III. Particular emphasis is placed on the phasins, which constitute a significant fraction of the total cellular protein. Phasins are assumed to form a close protein layer at the surface of the granules, providing the interface between the hydrophilic cytoplasm and the much more hydrophobic core of the PHA inclusion.Key words: PHA granules, polyhydroxyalkanoic acids, PHA synthase, Alcaligenes eutrophus, Pseudomonas oleovorans.
Degradation and applications of polyhydroxyalkanoates
Authors: Brandi Helmut , Bachofen Reinhard , Mayer Jörg , Wintermantel Erich
Published: October 2009

A series of tests is available to study the biodegradation of plastic materials under either laboratory or field conditions. Most of the standard methods have been published by the American Society for Testing and Materials. All of them describe techniques to investigate the biodegradation of plastics under laboratory conditions. Microbially formed polyhydroxyalkanoates (PHAs) have been marketed recently as biodegradable plastics. However, currently only a few articles made from PHAs (e.g., bottles) are commercially available. A series of microorganisms (prokaryotes as well as eukaryotes) has been characterized as being able to degrade PHAs. With one exception (Ilyobacter delafieldii), all of them were isolated from aerobic environments. So far, over 10 different extracellular PHA depolymerases have been purified and characterized. Depolymerases that preferentially attack PHAs with monomer units other than 3-hydroxybutyrate have been found only in Pseudomonas fluorescens and Pseudomonas lemoignei.Key words: poly(3-hydroxybutyrate), polyhydroxyalkanoates, biodegradation, industrial applications.
In situ biodegradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in natural waters
Authors: Mergaert J. , Wouters A. , Swings J. , Anderson C.
Published: October 2009

The biodegradation of samples of poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxybutyrate-co-10%-3-hydroxyvalerate) (P(3HB-co-10%-3HV)), and poly(3-hydroxybutyrate-co-20%-3-hydroxyvalerate) (P(3HB-co-20%-3HV)) was investigated in situ in natural waters. The degradation was studied by decrease in mass, molecular weight, and tensile strength. In two freshwater ponds the polymers were slowly degraded. After half a year of submersion the mass loss was less than 7%. After 358 days in a freshwater canal, 34% mass loss was recorded for the homopolymer, and 77% for the P(3HB-co-10%-3-HV) samples, while the P(3HB-co-20%-3HV) samples had completely disappeared. In seawater in the harbour of Zeebrugge, P(3HB) samples lost 31% of their initial mass, and the copolymers 49–52%, within 270 days. In all of these environments, the degradation rate was faster during the summer, when the temperature of the water was higher. No relevant changes in molecular weight could be detected, indicating that the degradation took place only at the surface of the samples. The degradation resulted in considerable loss of tensile strength of the copolymer samples. Ninety-two microorganisms, mainly bacteria, able to degrade P(3HB) in polymer overlayer plates, were isolated and identified by fatty acid analysis. The isolates from one freshwater pond belonged mainly to the bacterial genus Acidovorax, while the microorganisms from the other freshwater pond belonged to various bacterial genera, to Streptomyces, and to the mould genus Penicillium. Most of the 31 bacterial isolates from seawater were identified as Alteromonas haloplanktis. The results demonstrate that P(3HB) and P(3HB-co-3HV) samples are effectively biodegradable in natural waters under real-life conditions and reveal the biodiversity of the microflora responsible for this biodegradation.Key words: poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), biodegradation, seawater, freshwater.
Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase ofComamonassp. and of its structural gene
Authors: Jendrossek Dieter , Backhaus Martina , Andermann Meike
Published: October 2009

The poly(3-hydroxybutyrate) (PHB) depolymerase structural gene of Comamonas sp. (phaZCsp) was cloned in Escherichia coli and identified by halo formation on PHB-containing solid medium. The nucleotide sequence of a 1719 base pair MboI fragment was determined and contained one large open reading frame (ORF1, 1542 base pairs). This open reading frame encoded the precursor of the PHB depolymerase (514 amino acids; Mr, 53 095), and the deduced amino acid sequence was in agreement with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 26 onwards. Analysis of the deduced amino acid sequence revealed a domain structure of the protein: a signal peptide that was 25 amino acids long was followed by a catalytic domain of about 300 amino acids, a fibronectin type III (Fn3) modul sequence, and a putative PHB-specific substrate-binding site. By comparison of the primary structure with that of other polyhydroxyalkanoate (PHA) depolymerases, the catalytic domain apparently contained a catalytic triad of serine, histidine, and aspartate. In addition, a conserved region resembling the oxyanion hole of lipases was present. The catalytic domain was linked to a C-terminal putative substrate-binding site by a sequence about 90 amino acids long resembling the Fn3 modul of fibronectin and other eukaryotic extracellular matrix proteins. A threonine-rich region, which was found in four of five PHA depolymerases of Pseudomonas lemoignei, was not present in the Comamonas sp. depolymerase. The similarities with and differences from other PHA depolymerases are discussed.Key words: biodegradable polymer, poly(3-hydroxybutyrate) depolymerase, serine hydrolase, catalytic triad, Comamonas sp., fibronectin type III modul, substrate-binding site.
Biodegradation of cyclic and substituted linear oligomers of poly(3-hydroxybutyrate)
Authors: Brandl Helmut , Aeberli Brigitte , Bachofen Reinhard , Schwegler Ingrid , Müller Hans-Martin , Bürger Michael H. , Hoffmann Torsten , Lengweiler Urs D. , Seebach Dieter
Published: October 2009

Cyclic oligo(3-hydroxybutyrate), oligo(3-HB), was synthesized and purified, resulting in oligolides that contained three to seven (R)-3-hydroxybutyrate units (triolides up to heptolides). In addition, linear 3-HB octamers obtained as either tert-butyl or methyl esters were substituted with different end groups at the hydroxy end. The hydroxy terminus was replaced by either a benzyloxy, trifluoroacetoxy, crotonyloxy, (S)-3-hydroxybutyryloxy, or fluorenylmethylcarbonyloxy (FMOC) group. P(3-HB) hairpin loops occurred on the surface of certain regions of the polymer, especially of lamellar crystallites. Cyclic 3-HB oligomers provide a model system for these loops. It is assumed that they provide attachment points for the depolymerizing enzymes. All of the (R)-oligolides tested were degraded except the (R)-triolide. Triolides were not degraded, suggesting that enzymatic attack was prevented presumably by steric hindrance on the rigid ring system. Unsubstituted linear octamers were degraded. Biodegradation was prevented when the hydroxy terminus was protected by the FMOC group, but was not dependent on a free hydroxy terminal group: all other protecting groups did not prevent degradation. Substitution of the carboxy end by a methyl or tert-butyl ester group did not influence biodegradation.Key words: poly(3-hydroxybutyrate), oligolides, biodegradation, clear zone test.
High-level poly(β-hydroxybutyrate) production in recombinantEscherichia coliin sugar-free, complex medium
Authors: Kalousek S. , Lubitz W.
Published: October 2009

The poly(β-hydroxybutyrate) (PHB) biosynthetic genes of Alcaligenes eutrophus that are organized in a single operon (phbCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type phb operon from plasmid p4A leads to the formation of 10 or 50–80% PHB/cell dry mass when the cells are grown in Luria–Bertani medium alone or supplemented with 1% glucose (w/v), respectively. To further stimulate PHB formation independent of additional carbon source in Luria–Bertani medium, molecular methods have been applied to provide efficient E. coli transcription and translation signals for the PHB synthase gene (phbC). The lac promoter present upstream of the phbC sequence allows its expression to be controlled depending on the LacI status of the chosen host strain. The T7 gene 10 ribosome binding site is utilized for translational initiation. PHB production in E. coli was compared in strains either harboring plasmid p4A containing the intact phbCAB operon or harboring two compatible plasmids carrying the β-ketothiolase (phbA) and acetoacetyl-CoA-reductase (phbB) genes under transcriptional control of the lac promoter–operator region and also carrying separately the phbC gene with its natural promoter sequence. In addition, plasmid pSYN allowing the phbC gene to be expressed under new transcription and translation conditions combined with plasmid pUMS gave rise to the same amount of PHB formation (70% PHB cell dry mass) in E. coli when grown in Luria–Bertani medium without glucose supplement.Key words: PHB production, engineered phbCAB operon.
Strategies for the sustainable production of new biodegradable polyesters in plants: a review
Authors: Leij Feike R. van der , Witholt Bernard
Published: October 2009

In this study we review relevant pathways with regard to the production of poly(3-hydroxyalkanoates) (PHA) with medium chain length monomers in higher plants. On the basis of what is known of the genetics and the biochemistry of PHA formation in bacteria, and of fatty acid metabolism in various organisms, a number of possibilities for PHA production in model plants and in economically important crop plants are listed. Along with the molecular biology of PHA synthesis and fatty acid metabolism, we discuss theoretical and environmental considerations, metabolic engineering strategies, and plant transformation systems.Key words: polyhydroxyalkanoate, fatty acid, starch, potato, Arabidopsis.
Multi-inhibition kinetic model for the growth ofAlcaligenes eutrophus
Authors: Belfares Lamia , Perrier Michel , Ramsay Bruce A. , Ramsay Juliana A. , Jolicoeur Mario , Chavarie Claude
Published: October 2009

During the growth phase of Alcaligenes eutrophus, it was observed that the concentrations of glucose and ammonium ions are inhibitory at a certain level, which affects the specific growth rate. Moreover, it has been established from previous work that (an) unidentified substance(s) inhibit(s) the growth at biomass concentrations above 10 g∙L−1. A model taking into account these multiple inhibitory factors is proposed to predict the growth of A. eutrophus. The model parameters were determined by batch experiments except for the parameter associated with inhibition at high biomass concentration. This parameter was estimated by a classical optimization method. The validity of the model was tested by comparing the model predictions with experimental data obtained in batch and transient continuous fermentations and with literature data.Key words: A. eutrophus, inhibitory product, high biomass concentration.
Fermentative production of poly(β-hydroxybutyric acid) from xylose viaL-lactate by a two-stage culture method employingLactococcus lactisIO-1 andAlcaligenes eutrophus
Authors: Tanaka Kenji , Katamune Kouji , Ishizaki Ayaaki
Published: October 2009

A two-stage culture method employing Lactococcus lactis IO-1 and Alcaligenes eutrophus was developed for the production of polyhydroxyalkanoic acid (PHA) from xylose via L-lactate. In this culture method, xylose was converted into L-lactic acid and acetic acid by a culture of L. lactis IO-1, and the organic acids were then converted into PHA by a culture of A. eutrophus. Alcaligenes eutrophus grew at a specific growth rate of 0.61/h; however, the growth rate decreased as the lactate concentration in the medium increased. When the supernatant of the IO-1 culture broth containing 10-g/L L-lactate was used as a medium for A. eutrophus in batch culture, the cell concentration increased to 8.5 g/L in 24 h and 55% (w/w) of the content of the cells was found to be poly(β-hydroxybutyric acid), P(3HB). Furthermore, fed-batch culture of A. eutrophus was carried out with feeding of L-lactic acid to maintain the L-lactate concentration around 3.0 g/L. As a result, 41.0 g/L of cells and 28.7 g/L of P(3HB) were produced after 17 h of cultivation.Key words: polyhydroxybutyric acid, xylose, L-lactic acid, Alcaligenes eutrophus, Lactococcus lactis.
On-line analysis of Alcaligenes eutrophus fermentations
Authors: Meier-Schneiders M. , Grosshans U. , Busch C. , Weikmann W. , Steinbuchel A.
Published: October 2009

Improvements in the poly(β-hydroxybutyrate) (PHB) productivity of Alcaligenes eutrophus can be achieved by using genetically engineered strains. Knowledge of their metabolic regulation can be obtained from fermentation experiments. Certain events may not be noticed owing to the typically low frequency of sampling. To avoid information gaps, a newly developed fermenter-calorimeter was used for batch experiments allowing comprehensive on-line monitoring of fermentations. PHB-producing strain HI 6 and PHB-lacking mutant PHB−4 were selected, since they are well characterized with respect to the metabolism of PHB. For both strains, the profiles of thermograms, oxygen dissolved in the medium, and carbon dioxide formation were in good agreement with the off-line analysis. The on-line measurements showed characteristic differences between strains PHB−4 and H16. During fed-batch cultivations of PHB-accumulating strain H16, rapid autolysis of the cells was observed.Key words: Alcaligenes eutrophus, PHB, fermentation, calorimetry, on-line analysis, growth efficiency.