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PubMed2004

Antimicrobial efficacy of a new antibiotic-loaded poly(hydroxybutyric-co-hydroxyvaleric acid) controlled release system.

OBJECTIVE: Failure of orthopaedic devices, mainly femoral hip replacements, due to infection is of increasing medical importance. There is a need for improved antibiotic delivery systems in the treatment of orthopaedic infections and here we have evaluated polyhydroxyalkanoate formulations for their suitability as a constant delivery system for gentamicin. METHODS: Gentamicin was incorporated in poly(hydroxybutyric-co-hydroxyvalerate) (PHBV) with 8% or 12% hydroxyvalerate (HV) content at 2:1 or 5:1 (weight to weight) ratio. In conjunction with an elution study, a scanning electron microscopy and a porosity study were carried out to explore physical characteristics of the complexes before and after the leaching effect. The antibacterial effectiveness of the complexes was analysed in a bacterial adhesion assay using clinical isolates of Staphylococcus haemolyticus and Staphylococcus aureus. In addition, the polymers were exposed to pooled human blood to test their biocompatibility in both static and dynamic environments. RESULTS: We have shown that increasing the HV content from 8% to 12% leads to a faster release of the integrated antibiotic. An increase in antibiotic content enhanced the homogeneity while decreasing the permeability of the complexes and reducing the release rate. A significant reduction in the number of the adherent S. aureus and gentamicin-resistant S. haemolyticus within a 48 h exposure to our formulations confirmed the effectiveness of the PHBV/gentamicin complexes. Finally, these formulations did not alter the haemodynamics of the pooled blood samples after an extended period of time. CONCLUSION: Taken together, the PHBV/gentamicin formulations may prove to be effective preventive therapeutic modalities in implant-related Staphylococcus infections.

Anti-Bacterial Agents↗
PubMed2003

Enhanced synthesis of poly(3-hydroxybutyrate) in recombinant Escherichia coli by means of error-prone PCR mutagenesis, saturation mutagenesis, and in vitro recombination of the type II polyhydroxyalkanoate synthase gene.

Type II synthase (PhaC1(Ps)) for polyhydroxyalkanoate (PHA) from Pseudomonas sp. 61-3 was subjected to an in vitro evolution system including PCR-mediated mutagenesis in order to improve the function of PhaC1(Ps) in terms of its ability to produce poly(3-hydroxybutyrate) [P(3HB)] in recombinant Escherichia coli. Based on our established in vivo assay system, two positions (Ser325 and Gln481) where mutations provided remarkable increases in P(3HB) synthesis were identified. Saturation mutagenesis at these positions was carried out to explore whether there might be more beneficial sequences for P(3HB) synthesis than those identified in the point mutation library. As a result, five single mutants [S325C (T) and Q481M (K, R)] gave rise to highly enhanced P(3HB) synthesis. Drastically enhanced P(3HB) synthesis (up to 340- to 400-fold the amount of that of the wild type) was further achieved by generation of all five variants of the double mutants combining the codons for residues 325/481. It is feasible that the replacement of Ser (specific for type II synthase) by Thr (specific for type I synthase) at position 325 resulted in acquiring greater P(3HB) synthesis ability as exhibited by type I synthases. The other hot spot, 481, that positively contributes to enhanced P(3HB) synthesis is located adjacent to a His479, a residue that forms a putative catalytic diad that can be inferred by sequence alignment.

Acyltransferases↗
PubMed2006

Thiolysis of poly(3-hydroxybutyrate) with polyhydroxyalkanoate synthase from Ralstonia eutropha.

Poly(3-hydroxybutyrate) (PHB) is synthesized from 3-hydroxybutyryl-CoA by polyhydroxyalkanoate synthase and hydrolyzed by PHB depolymerase. In this study, we focused on the reverse reaction of polyhydroxyalkanoate synthase, and propose the possibility that PHB can be degraded through a novel process, that is thiolysis of PHB with CoASH. Polyhydroxyalkanoate synthase of Ralstonia eutropha was incubated with 14C-labeled PHB and CoASH. The reaction mixture was fractionated by HPLC and then analyzed with a scintillation counter. The analysis revealed 3-hydroxybutyryl-CoA to be a product of the reaction. When NADP+ and acetoacetyl-CoA reductase were added to the reaction mixture, an increase in absorbance at 340 nm was observed. Native PHB inclusion bodies from R. eutropha also showed thiolytic activity. This is the first indication that polyhydroxyalkanoate synthase catalyzes both the synthesis and degradation of PHB, and that native PHB inclusion bodies has thiolytic activity.

Acyltransferases↗
PubMed1984

Proton correlation NMR studies of metabolism in Rhodopseudomonas palustris.

A 1H correlation NMR study is reported, on the metabolism of a photosynthetic bacterium, Rhodopseudomonas palustris, in dark and light anaerobic conditions. Alkali treatment as well as sonication of the cells were employed to follow the process of accumulation and decomposition of poly-beta-hydroxybutyrate (PHB) which is the reserve material for the bacterium. It was shown that synthesis of PHB from trans-crotonate proceeds in the granules of the cells. It was also demonstrated that under anaerobic light conditions photometabolism and glycolysis generally compete with concomitant synthesis and decomposition of PHB, respectively, and that glycolysis gradually replaces photometabolism with aging of the cells. In contrast, glycolysis is always predominant in the dark and PHB is primarily used as the carbon source. It was observed that photo-induced transport of beta-hydroxybutyrate through the membrane occurs when photometabolism and glycolysis are equally active in the light. The implications of this observation are briefly discussed.

Anaerobiosis↗
PubMed2002

Synthesis of a novel class of polyhydroxyalkanoates in Arabidopsis peroxisomes, and their use in monitoring short-chain-length intermediates of beta-oxidation.

The poly[(R)-3-hydroxyalkanoate] (PHA) synthase gene (phaC(Ac)) of Aeromonas caviae FA440 was modified by adding a peroxisome targeting signal encoding the last 10 amino acids at the carboxyl-terminal of spinach glycolate oxidase. The modified gene was introduced into Arabidopsis thaliana plants by Agrobacterium-mediated transformation. The transgenic Arabidopsis plant expressed the introduced gene and its protein, and it accumulated PHA in its tissues. Gas chromatography-mass spectrometry analysis demonstrated the accumulation of a novel type of PHA, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate). This strongly suggests that short-chain-length (R)-3-hydroxyacyl-CoAs were generated from intermediates of peroxisomal beta-oxidation. It was revealed by using this transgenic plant that Tween-20 can activate peroxisomal beta-oxidation of short-chain-length fatty acids.

Acyltransferases↗
PubMed1992

Supply of O2 regulates O2 demand during utilization of reserves of poly-beta-hydroxybutyrate in N2-fixing soybean bacteroids.

A liquid reaction medium containing dissolved air and oxyleghaemoglobin, but no energy-yielding substrate, was supplied to bacteroids confined in a stirred flow reaction chamber. The relative oxygenation of the leghaemoglobin in the chamber was determined automatically by spectrophotometry of the effluent solution, and the concentrations of free, dissolved O2 ([O2]) and rates of O2 consumption were calculated. Dissolved CO2 and NH3 from N2 fixation were determined in fractions of the effluent solution. Bacteroids utilized endogenous reserves of poly-beta-hydroxybutyrate (PHB), which were depleted by 9.2% during a typical 5 h-long experiment. Stepwise increases in flow rate (increasing supply of O2) initially produced a drop in O2 demand and resulted in a rise in [O2] and a decline in N2 fixation. Subsequently, O2 demand rose (presumably because of increased mobilization of substrate from PHB) and [O2] declined to a low level. N2 fixation was fully restored, or even enhanced, within 15-20 min of establishment of a new, steady [O2]. This pattern of regulation by O2 supply was completely eliminated by adding low concentrations (20-50 microM) of oxidizable substrate (succinate, malate, ethanol) to the reaction medium. During endogenous activity, rates of CO2 evolution were proportional to, but less than, rates of O2 consumption up to 5.4 nmol O2 min-1 mg-1, above which CO2 evolution exceeded O2 consumption. These and other features of endogenous activity are discussed in relation to sustaining N2 fixation by nodules in vivo.

Hydroxybutyrates↗
PubMed2000

Taxonomic implications of synthesis of poly-beta-hydroxybutyrate and other poly-beta-hydroxyalkanoates by aerobic pseudomonads.

Whereas poly-beta-hydroxybutyrate (PHB) production by Pseudomonas species is rare, synthesis of medium-chain-length poly-beta-hydroxyalkanoates (mcl-PHAs) other than PHB, has been observed in fluorescent and non-fluorescent species. Contrary to original reports, Pseudomonas corrugata and Pseudomonas ficuserectae accumulate mcl-PHAs and not PHB. The taxonomic implications of these characteristics are discussed.

Bacteria, Aerobic↗
PubMed2002

Caldimonas manganoxidans gen. nov., sp. nov., a poly(3-hydroxybutyrate)-degrading, manganese-oxidizing thermophile.

A poly(3-hydroxybutyrate) (PHB)-degrading, gram-negative, aerobic bacterium, strain HS(T), was isolated from a hot spring and chemotaxonomically and phylogenetically characterized. The oxidase-positive, weakly catalase-positive, non-pigmented cells (0.6 x 2.6 microm) exhibited a single polar flagellum and accumulated PHB granules. Strain HS(T) was capable of manganese oxidation. Highest growth rate was attained at 50 degrees C. The optimum pH for growth was 7-8. The major respiratory quinone was ubiquinone-8 and major cellular fatty acids were C16:0, C16:1 and C18:1. The G+C content of the DNA was 66.2 mol%. Comparative 16S rDNA analysis indicated that strain HS(T) is related to the Rubrivivax subgroup and the family Comamonadaceae. The nearest phylogenetic relatives were Ideonella dechloratans (92.1% similarity), Leptothrix discophora (93.6%), Roseateles depolymerans (92.4%) and Rubrivivax gelatinosus (92.2%). On the basis of its phylogenetic and phenotypic properties, it is proposed that this isolate be designated Caldimonas manganoxidans gen. nov., sp. nov.; the type strain is HS(T) (= JCM 10698T = IFO 16448T = ATCC BAA-369T).

Bacterial Typing Techniques↗
PubMed1989

Anaerobic degradation of acetone and higher ketones via carboxylation by newly isolated denitrifying bacteria.

Five strains of Gram-negative denitrifying bacteria that used various ketones as sole carbon and energy sources were isolated from activated sludge from a municipal sewage plant. Three strains are related to the genus Pseudomonas; two non-motile species have not yet been affiliated. All strains grew well with ketones and fatty acids (C2 to C7), but sugars were seldom utilized. The physiology of anaerobic acetone degradation was studied with strain BunN, which was originally enriched with butanone. Bicarbonate was essential for growth with acetone under anaerobic and aerobic conditions, but not if acetate or 3-hydroxybutyrate were used as substrates. An apparent Ks value of 5.6 mM-bicarbonate was determined for growth with acetone in batch culture. The molar growth yield was 24.8-29.8 g dry cell matter (mol acetone consumed)-1, with nitrate as the electron acceptor in batch culture; it varied slightly with the extent of poly-beta-hydroxybutyric acid (PHB) formation. During growth with acetone, 14CO2 was incorporated mainly into the C-1 atom of the monomers of the storage polymer PHB. With 3-hydroxybutyrate as substrate, 14CO2 incorporation into PHB was negligible. The results provide evidence that acetone is channelled into the intermediary metabolism of this strain via carboxylation to acetoacetate.

Anaerobiosis↗
PubMed1997

Colonial opacity variations among the choleragenic vibrios.

Cultures of Vibrio cholerae 01, biotype El Tor, from the current epidemic of cholera in the Western Hemisphere, and of the new V. cholerae serogroup O139, from the current outbreak in India and Bangladesh, revealed marked colonial heterogeneity when received by the authors. By comparison with reference colony types, using a stereoscope and transmitted oblique illumination, colonies of approximately 10 different degrees of opacity could be distinguished. In contrast, strains freshly isolated from patients and rapidly and carefully preserved were more homogeneous although still differentiable by this technique. These (and older) observations prompted the questions: (1) why is a V. cholerae colony opaque or translucent? and (2) what benefit is it to the vibrios to vary their colonial appearance? The observed changes in colonial opacity, which are reversible, are sometimes (rarely) accompanied by changes in virulence for infant rabbits and, more frequently, by other phenotypic variations including the ability to produce poly-beta-hydroxybutyrate inclusion bodies on glycerol-containing medium, the degree of encapsulation in 0139, changes in outer-membrane proteins, alteration in lipopolysaccharide structure, changes in expression of glycolytic pathways, and differences in ability to survive under adverse conditions. Colonial variations in choleragenic vibrios are phenotypically multifactorial. The genetic mechanisms(s) underlying the observed phenotypic changes remain to be defined.

Americas↗
PubMed2001

Multiple evidence for widespread and general occurrence of type-III PHA synthases in cyanobacteria and molecular characterization of the PHA synthases from two thermophilic cyanobacteria: Chlorogloeopsis fritschii PCC 6912 and Synechococcus sp. strain MA19.

Eleven different cyanobacteria were investigated with respect to their capabilities to synthesize poly-3-hydroxybutyrate [poly(3HB)] and the type of poly-beta-hydroxyalkanoic acid (PHA) synthase accounting for the synthesis of this polyester. Several methods, including (i) Southern blot analysis using a phaC-specific DNA probe, (ii) Western blot analysis using specific polyclonal anti-PhaE antibodies raised in this study against PhaE of Synechocystis sp. strain PCC 6803, (iii) generation and sequence analysis of PCR products using phaC-specific oligonucleotides as primers, and/or (iv) cloning and sequence analysis of PHA synthase structural genes, were used to provide evidence for the presence of a type-III PHA synthase in the following cyanobacteria: Synechococcus sp. strains MA19 and PCC 6715, Chlorogloeopsis fritschii PCC 6912, Anabaena cylindrica SAG 1403-2, Cyanothece sp. strains PCC 7424, PCC 8303 and PCC 8801, and Gloeocapsa sp. strain PCC 7428. The screening was compared with corresponding studies using crude protein extracts and genomic DNA of Synechocystis sp. strain PCC 6803, as a positive control, which is so far the only cyanobacterium for which molecular data of the PHA synthase genes are available. No evidence for the presence of a type-III PHA synthase could be obtained for only three of the eleven investigated cyanobacteria (Stanieria sp. strain PCC 7437, Cyanothece sp. strain PCC 8955 and Gloeothece sp. strain PCC 6501). The entire PHA synthase structural genes of the two thermophilic cyanobacteria Synechococcus sp. strain MA19 and Chlorogloeopsis fritschii PCC 6912, and in addition a central region of the phaC gene of Cyanothece sp. strain PCC 8303, were cloned, sequenced and also heterologously expressed in Escherichia coli.

Acyltransferases↗
PubMed2001

Alginate formation in Azotobacter vinelandii UWD during stationary phase and the turnover of poly-beta-hydroxybutyrate.

Azotobacter vinelandii UWD is a mutant of strain UW that is defective in the respiratory oxidation of NADH. This mutation causes an overproduction of polyhydroxyalkanoates (PHAs), as polyester synthesis is used as an alternative electron sink. Since PHAs have potential for use as natural, biodegradable plastics, studies of physiology related to their production are of interest. Alginate production by this strain is limited to < 11 microg (mg cell protein)(-1), which permits high efficiency conversion of carbon source into PHA. However, < or = 400 microg (mg cell protein)(-1) was formed when UWD cells were oxygen-limited and in the stationary phase of growth. Alginate formation was fuelled by PHA turnover, which was coincident with the synthesis of alkyl resorcinols, under conditions of exogenous glucose limitation. However, alginate production was a phenotypic and reversible change. Alginate production was stopped by interruption of algD with Tn5lacZ. LacZ activity in UWD was shown to increase in stationary phase, while LacZ activity in a similarly constructed mutant of strain UW did not. Transcription of algD in strain UWD started from a previously identified RpoD promoter and not from the AlgU (RpoE) promoter. This is because strain UWD has a natural insertion element in algU. Differences between strain UW and UWD may reside in the defective respiratory oxidation of NADH, where the NADH surplus in strain UWD may act as a signal of stationary phase. Indeed, a backcross of UW DNA into UWD generated NADH-oxidase-proficient cells that failed to form alginate in stationary phase. Evidence is also presented to show that the RpoD promoter may be recognized by the stationary phase sigma factor (RpoS), which may mediate alginate production in strain UWD.

Alginates↗
PubMed2002

Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16.

Regulation of expression of the phasin PhaP, which is the major protein at the surface of polyhydroxyalkanoate (PHA) granules in Ralstonia eutropha H16, was studied and analysed at the molecular level. The regulation of PhaP expression is achieved by an autoregulated repressor, which is encoded by phaR in R. eutropha. The occurrence of PhaR homologues and the organization of phaR genes was analysed in detail in 29 different bacteria. Three kinds of molecule to which PhaR binds were identified in cells of R. eutropha, as revealed by gel-mobility-shift assays, DNaseI footprinting, cell fractionation, immunoelectron microscopy studies employing anti-PhaR antibodies raised against purified N-terminal hexahistidine-tagged PhaR and in vitro binding studies employing artificial PHA granules. PhaR binds upstream of phaP at two sites comprising the transcriptional start site plus the -10 region and a region immediately upstream of the -35 region of the sigma(70) promoter of phaP, where two imperfect 12 bp repeat sequences (GCAMMAAWTMMD) were identified on the sense and anti-sense strands. PhaR also binds 86 bp upstream of the phaR translational start codon, where the sigma(54)-dependent promoter was identified. PhaR also binds to the surface of PHA granules. In the cytoplasm of a phaROmegaKm mutant of R. eutropha H16, increased quantities of PhaP were detected and the cells formed by this strain were much smaller and had many more PHA granules present than the wild-type. These data support the following model for the regulation of phaP expression. Under cultivation conditions not permissive for PHA biosynthesis or in mutants defective in PHA biosynthesis, PhaR binds to the phaP promoter region and represses transcription of this gene. After the onset of PHA biosynthesis, under conditions that are permissive for the formation of nascent granules, PhaR binds to PHA granules and phaP is transcribed. At the later stages of PHA accumulation, PhaR no longer binds to the granules and the transcription of phaP is again repressed. In addition to this, phaR expression is subject to autoregulation. Excess PhaR that has not bound to the phaP upstream region or to PHA granules binds to the phaR upstream region, thereby repressing its own transcription.

Bacterial Proteins↗
PubMed1995

Poly-beta-hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41.

Genes encoding beta-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORF1 were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB- Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB- strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORF1 with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.

Acetyl-CoA C-Acyltransferase↗
PubMed2003

Schlegelella thermodepolymerans gen. nov., sp. nov., a novel thermophilic bacterium that degrades poly(3-hydroxybutyrate-co-3-mercaptopropionate).

A novel thermophilic bacterium, strain K14T, capable of degrading poly(3-hydroxybutyrate) as well as copolymers containing 3-hydroxybutyrate and 3-mercaptopropionate linked by thioester bonds, was isolated. 16S rDNA sequence analysis showed that strain DhA-71, a dehydroabietic acid-degrading bacterium, was the nearest phylogenetic neighbour and that both strains should be placed as members of a newly created genus, Schlegelella gen. nov., in the Rubrivivax subgroup of the beta-Proteobacteria. Strain K14T (= LMG 21644T = DSM 15344T) is proposed as the type strain of Schlegelella thermodepolymerans gen. nov., sp. nov. Its phylogenetic, morphological, biochemical and chemotaxonomic characteristics are described in detail.

3-Mercaptopropionic Acid↗