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PubMed2004

Laryngeal abductor muscle reinnervation in a pig model.

OBJECTIVE: To develop a large animal model for studies of laryngeal abductor reinnervation. MATERIAL AND METHODS: Six minipigs underwent unilateral anastomosis of the phrenic nerve-abductor branch of the recurrent laryngeal nerve (RLN). Polyhydroxybutyrate (PHB) conduits were used for repair. At each of 30, 60 and 120 days, 2 animals underwent video laryngeal endoscopy (VLE) and were then killed. VLE was also performed in the 120-day pair at 60 days. Nerve-conduit-nerve-muscle samples were fixed for light and immunofluorescence (pan-neurofilaments, S-100) microscopy. Laryngeal muscles were harvested (myosin heavy chain analysis). RESULTS: VLE showed recovery of abductor function in 1 animal at 60 days and in 1 at 120 days. Haematoxylin-eosin staining demonstrated a complex inflammatory response. Eosinophil recruitment was observed. Stepwise regeneration and reorganization of the distal nerve between 30 and 120 days was observed with pan-NF staining. The mean minimum diameter in the reinnervated posterior crico-arytenoids tended to increase for up to 120 days. CONCLUSIONS: Anastomosis of the phrenic nerve-abductor branch of the RLN with a PHB conduit in a pig can result in functional and histological recovery within 2-4 months and appears to at least sustain abductor muscle fibre morphology. Recovery occurs despite a complex inflammatory response, which may be an essential part of healing rather than inhibitory.

Anastomosis, Surgical↗
PubMed2000

Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis.

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2004

Alternative materials to acrylic bone cement for delivery of depot antibiotics in orthopaedic infections.

Acrylic bone cement has considerable laboratory and clinical data validating it as a delivery material for depot administration of antibiotics. However, an alternate material that does not require a secondary procedure for removal is desired. Many biodegradable materials have been evaluated as alternatives including protein-based materials (collagen, fibrin, thrombin, clotted blood), bone-graft, bone-graft substitutes and extenders (hydroxyapatite, beta-tricalcium phosphate, calcium sulfate, bioglass), and synthetic polymers (polyhanhydride, polylactide, polyglycolide, polyhydroxybutyrate-co-hydroxyvalerate, polyhydroxyalkanoate). Various forms and combinations of these materials have been investigated worldwide, characterizing their elution properties and performance in treating osteomyelitis in animal models. Many of these have had limited clinical evaluation. Outside the United States, some of these materials are used clinically. In the United States, none have been approved. None are commercially available for clinical use. Morselized cancellous bone and calcium sulfate are the two materials that have been used clinically in the United States on a physician-prescribed, hand-mixed, basis. Considering the limited clinical data that currently are available, the use of these materials still is experimental. Clinical application should be cautious, limiting the total antibiotic load. Until definitive data are available, a prudent dose would be no higher than one that would have acceptable toxicity risk if administered intravenously over 24 hours.

Animals↗
PubMed2002

Characterization of a novel halophilic archaeon, Halobiforma haloterrestris gen. nov., sp. nov., and transfer of Natronobacterium nitratireducens to Halobiforma nitratireducens comb. nov.

Strain 135(T), a novel red-pigmented, aerobic, extremely halophilic member of the Archaea showing rod, coccus and slightly pleomorphic morphology, was isolated from hypersaline soil close to Aswan (Egypt). This organism is neutrophilic, motile and requires at least 2.2 M NaCl, but no MgCl2, for growth and exhibits optimal growth at 42 degrees C. Polar lipid analysis revealed the presence of sulfated triglycosyl diether and triglycosyl diether as the sole glycolipids as well as the absence of the glycerol diether analogue of phosphatidyl glycerosulfate. C20:C20 and C20:C25 core lipids are present in almost equal proportions. The G+C content of the DNA is 66.9 mol%. 16S rDNA analysis revealed that strain 135(T) was a member of the phyletic group defined by the family Halobacteriaceae, but there was a low degree of similarity to other members of this family. Highest similarity values of 96.4 and 93.8-94.3% were obtained to the 16S rDNA of Natronobacterium nitratireducens and Natronobacterium gregoryi, Natronococcus occultus and Natronococcus amylolyticus. Strain 135(T) is able to accumulate polyhydroxybutyrate as intracellular reserve material. On the basis of the data presented, strain 135(T) should be placed in a new genus, Halobiforma gen. nov. as Halobiforma haloterrestris sp. nov. The type strain is strain 135(T) (= DSM 13078(T) = JCM 11627(T)). Moreover, the transfer of Natronobacterium nitratireducens to Halobiforma nitratireducens comb. nov. is proposed.

Base Composition↗
PubMed1992

Physiology and morphology of Legionella pneumophila in continuous culture at low oxygen concentration.

Two strains of Legionella pneumophila serogroup 1 monoclonal subgroup Pontiac were grown for the first time in continuous culture using a chemically defined medium. The influence of temperature on physiology and morphology was investigated by fixing the growth rate (equal to the dilution rate, D) at 0.08 h-1 and controlling the pH and dissolved oxygen concentration of the culture. Serine provided the principal source of carbon and energy but growth was limited by tyrosine. The bacterium behaved as a microaerophile in this medium, with maximal growth occurring at 0.31 (mg O2)I-1 (equivalent to a dissolved oxygen tension of 4% (v/v) air saturation at 30 degrees C). The cultures consisted of flagellated, short rods at 24 degrees C, but exhibited an increased level of pleomorphism and the loss of flagella as the temperature was increased to 37 degrees C. The presence of intracellular granules was noted, and their abundance was temperature-dependent. Polyhydroxybutyrate was present in L. pneumophila, and the proportion of the cell dry weight that it accounted for varied with temperature, being maximal at 24 degrees C. The ratio of saturated to unsaturated fatty acids in the cells decreased as the temperature was reduced towards 24 degrees C, so as to maintain membrane fluidity at low growth temperature.

Anaerobiosis↗
PubMed2005

Thalassobacter stenotrophicus Macian et al. 2005 is a later synonym of Jannaschia cystaugens Adachi et al. 2004, with emended description of the genus Thalassobacter.

The type strains of Jannaschia cystaugens (LMG 22015(T)) and Thalassobacter stenotrophicus (CECT 5294(T)) were analysed by means of genomic DNA-DNA hybridization, comparison of 16S rRNA gene sequences and phenotypic properties determined under the same methodological conditions. J. cystaugens LMG 22015(T) showed DNA-DNA relatedness levels of 72% when hybridized with the genomic DNA of T. stenotrophicus CECT 5294(T). Sequence comparisons revealed that the 16S rRNA genes of the two strains had a similarity of 99.8%. The cellular fatty acid and polar lipid compositions of the two strains and their DNA mol% G+C contents were almost identical. Bacteriochlorophyll a (Bchl a) and polyhydroxybutyrate were produced by both strains under the same culture conditions. Their closest phylogenetic neighbours were Jannaschia helgolandensis and Jannaschia rubra; however, the low sequence similarity values (95.7-95.9%) and several important differences in phenotypic traits (ionic requirements, Bchl a production and polar lipids) support the distinction between the genera Thalassobacter and Jannaschia. Thus, we propose the unification of J. cystaugens (LMG 22015(T)) and T. stenotrophicus (CECT 5294(T)) as Thalassobacter stenotrophicus (type strain, CECT 5294(T)=DSM 16310(T)). An emended description of the genus Thalassobacter is also presented.

Bacterial Typing Techniques↗
PubMed2005

Biochemical and molecular characterization of a periplasmic hydrolase for oxidized polyvinyl alcohol from Sphingomonas sp. strain 113P3.

Oxidized polyvinyl alcohol hydrolase (OPH) and polyvinyl alcohol dehydrogenase were found to be constitutively present in the periplasm of Sphingomonas sp. strain 113P3 (formerly Pseudomonas sp. 113P3). The OPH was purified to homogeneity with a yield of 40 % and a 5.9-fold increase in specific activity. The enzyme was a homodimer consisting of 35 kDa subunits. Its activity was inhibited by PMSF, Hg(2+) and Zn(2+). The enzyme hydrolysed oxidized polyvinyl alcohol (oxidized PVA) and p-nitrophenyl acetate (PNPA), but did not hydrolyse any of the mono- or diketones tested. K(m) and V(max) values for oxidized PVA and PNPA were 0.2 and 0.3 mM, and 0.1 and 3.4 micromol min(-1) mg(-1), respectively. The gene for OPH was cloned and sequenced. Sequencing analysis revealed that the open reading frame consisted of 1095 bp, corresponding to a protein of 364 amino acids residues, encoding a signal peptide and a mature protein of 34 and 330 amino acids residues, respectively. The presence of a serine-hydrolase motif (a lipase box; Gly-X-Ser-X-Gly) strongly suggested that the enzyme belongs to the serine-hydrolase family. The protein exhibited homology with OPH of the Pseudomonas sp. strain VM15C (63 % identity) and the polyhydroxybutyrate depolymerases from Mesorhizobium loti, Rhizobium sp. and Sinorhizobium meliloti (29-32 % identity). The oph gene was expressed in Escherichia coli under the control of the lac promoter. The recombinant protein had the same molecular mass and N-terminal amino acid sequence as the purified OPH from strain 113P3.

Alcohol Oxidoreductases↗
PubMed2005

A framework for multidimensional modelling of activity and structure of multispecies biofilms.

Concepts from previous biofilm models were integrated to create a framework for the implementation of multidimensional (2D and 3D) multispecies biofilm models. The framework is here described at three levels: (i) mathematical representation of the processes involved in biofilm formation, (ii) numerical implementation into a computer program (freely available from our website http://www.biofilms.bt.tudelft.nl/frameworkMaterial) and (iii) using the program for the creation of biofilm models with multiple bacterial and solute species. An improved version of the individual-based modelling (IbM) that allows structured biomass was used. In this approach biomass composition may be discriminated into any number of particulate species, including extracellular polymeric substances (EPS) for which specific functionality was included. Detachment is also included, described as occurring at the biofilm surface with variable local rates derived from functions of state variables. The application of this modelling framework to a multispecies system with structured biomass is illustrated in a case study where the competition between an organism capable of accumulating polyhydroxybutyrate (PHB, an internal storage compound) and an EPS-producing organism in a two-species biofilm is analysed. Results illustrate that biofilms enriched in PHB-producing organisms may be obtained by supplying substrate intermittently in feast/famine cycles.

Bacteria↗
PubMed2000

Sinorhizobium meliloti strain 1021 bioS and bdhA gene transcriptions are both affected by biotin available in defined medium.

Sinorhizobium meliloti 1021 responds to external biotin signals from alfalfa plants through the bioS regulatory locus. Immunogold labeling and electron microscopy revealed that the BioS protein is located within the S. meliloti cytoplasm. Under biotin-limiting conditions the S. meliloti cell lumen was filled with polyhydroxybutyrate (PHB) granules suggesting that either PHB synthesis or degradation are influenced by biotin. To test this hypothesis a 3-hydroxybutyrate-dehydrogenase-lacZ (bdhA-lacZ) fusion was mobilized into S. meliloti. beta-galactosidase tests revealed an overall 3.6-5.2-fold higher bdhA transcription in the presence of added biotin. Comparison of the bdhA and the bioS promoter regions identified several common motifs.

Bacterial Proteins↗
PubMed2002

PCR cloning of polyhydroxyalkanoate biosynthesis genes from Burkholderia caryophylli and their functional expression in recombinant Escherichia coli.

The PCR cloning strategy for type II polyhydroxyalkanoate (PHA) biosynthesis genes established previously for Pseudomonas was successfully applied to Burkholderia caryophylli strain AS 1.2741. The whole pha locus containing PHA synthase genes phaC1, phaC2 and PHA depolymerase gene phaZ was cloned. The complete open reading frames of phaC1(Bc), phaC2(Bc) and phaZ(Bc) were identified. Sequence analyses of the phaC1(Bc), phaZ(Bc) and phaC2(Bc) showed more than 77.7%, 73.7% and 68.5% identities compared with the corresponding pha loci of the known Pseudomonas strains, respectively. The functional expression of the phaC1(Bc) or phaC2(Bc) in Escherichia coli strain KM32B (fadB deleted mutant) showed the abilities of PHA production by the estimated PHA synthase genes. Over 1% PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) was detected from cells of recombinant E. coli KM32B (pHXM11) harboring phaC1(Bc), grown on octanoate. At the same time over 3% of PHA consisting of 3HO and 3HD was produced from cells of recombinant E. coli KM32B (pHXM21) harboring phaC2(BC), grown on decanoate. Results showed the PCR cloning strategy developed previously can be applied to non-Pseudomonas strains such as Burkholderia in this case. This result also provided evidence for the presumption that the Burkholderia strain possesses not only polyhydroxybutyrate synthase genes, but also synthase for medium-chain-length polyhydroxyalkanoates consisting of 3HHx, 3HO and 3HD.

Acyltransferases↗
PubMed2002

Polyhydroxyalkanoate biosynthesis in Pseudomonas pseudoalcaligenes YS1.

Pseudomonas pseudoalcaligenes strain YS1 isolated from oil contaminated soil was able to produce polyhydroxybutyrate blended with medium-chain-length polyhydroxyalkanoates (mcl PHA). PHA synthesis genes were cloned from this strain. A fadB (gene for fatty acid degradation) deleted mutant Escherichia coli KM32B (FADB::Tet) was constructed to express the cloned PHA synthesis gene phaC1(Pp) or phaC2(Pp). The fadB deleted mutant KM32B harboring phaC1(Pp) or phaC2(Pp) showed mcl PHA accumulation while the intact E. coli KM32 did not. The results demonstrated that P. pseudoalcaligenes YS1 possessed at least two PHA synthesis pathways; one of them was responsible for production of mcl PHA.

Acyltransferases↗
PubMed1989

Use of strips containing tetracycline hydrochloride or metronidazole for the treatment of advanced periodontal disease.

Strips containing tetracycline hydrochloride or metronidazole 25% in polyhydroxybutyric acid as a biodegradable polymer matrix, showed sustained release in simulated gingival fluid pH 6.6 at 37 degrees C. When evaluated in patients suffering from advanced periodontal disease, the greatest response to therapy was observed with tetracycline hydrochloride strips inserted into periodontal pockets at four-day intervals for 16 days, compared with an untreated control group. A reduction in plaque index, gingival index and pocket depth was observed. A favourable alteration occurred in the microbial flora of treated pockets with an increase in the proportion of cocci and decrease in gram-negative rods, fusiforms and spirochetes. Metronidazole strips or root-planning tended not to be as effective. The clinical improvement produced by each treatment was not maintained when treatment was terminated.

Administration, Topical↗
PubMed1996

Biosynthesis of Poly(3-Hydroxyalkanoic Acid) Copolymer from CO(inf2) in Pseudomonas acidophila through Introduction of the DNA Fragment Responsible for Chemolithoautotrophic Growth of Alcaligenes hydrogenophilus.

Pseudomonas acidophila is a bacterial strain producing a poly(3-hydroxyalkanoic acid) (PHA) copolymer from low-molecular-weight organic compounds such as formate and acetate. The genes responsible for PHA production were cloned in cosmid pIK7 containing a 14.8-kb HindIII fragment of P. acidophila DNA. With the aim of developing a means of producing a PHA copolymer from CO(inf2), cosmid pIK7 was introduced into a polymer-negative mutant of the chemolithoautotrophic bacterium Alcaligenes eutrophus PHB(sup-)4. However, the recombinant strain produced a homopolymer of 3-hydroxybutyric acid (polyhydroxybutyric acid) from CO(inf2). Since it was thought that the composition of the accumulated polymer might depend not on the PHA biosynthetic genes but on the metabolism of the host strain, a recombinant plasmid, pFUS, containing the genes for chemolithoautotrophic growth of the hydrogen-oxidizing bacterium A. hydrogenophilus was introduced into P. acidophila by conjugation. The recombinant plasmid pFUS was stably maintained in P. acidophila in the absence of chemolithoautotrophic or antibiotic selection. This pFUS-harboring strain possessed the ability to grow under a gas mixture of H(inf2), O(inf2), and CO(inf2) in a mineral salts medium, and PHA copolymer accumulation was confirmed by nuclear magnetic resonance spectral analysis. A gas chromatogram obtained by gas chromatography-mass spectrometry showed the composition of the polymer to be 52.8% 3-hydroxybutyrate, 41.1% 3-hydroxyoctanoate, and 6.1% 3-hydroxydecanoate. This is the first report of the production of a PHA copolymer from CO(inf2) as sole carbon source.

Journal Article↗
PubMed1996

Degradation of Trichloroethylene by Methanol-Grown Cultures of Methylosinus trichosporium OB3b PP358.

A soluble methane monooxygenase-constitutive mutant strain of Methylosinus trichosporium OB3b, strain PP358, was grown with methanol as the carbon source, and the kinetics of trichloroethylene (TCE) degradation were determined. PP358 exhibited high TCE degradation rates under both oxygen- and carbon-limiting conditions. The optimal pseudo first-order rate constant for TCE was comparable to the values measured for cells grown with methane. We found that growth under oxygen-limiting conditions results in increased accumulation of polyhydroxybutyrate, which in turn correlates with higher transformation capacities for TCE. It was also shown that methanol inhibits TCE degradation only at high concentrations. Thus, methanol-grown cultures of PP358 represent an efficient system for the biodegradation of chlorinated hydrocarbons.

Journal Article↗
PubMed2002

Morphological changes of rhizobia in peat cultures.

Morphological changes that take place in peat cultures of several species of rhizobia were examined. These changes seemed to be associated with enhanced survival of cells in peat and after inoculation onto plastic beads, which were used as a model system for seeds. Cell wall changes, in which the periplasmic space appeared to be occluded with electron-dense material, were observed in Rhizobium sp. strain SU343 and Bradyrhizobium lupini WU425 cells after 7 and 14 days in peat, respectively. Nutrient limitation and low O(2) concentration in peat are suggested to be factors involved in the induction of the morphological changes. Polyhydroxybutyrate reserves, which were present in broth-cultured cells of both species of rhizobia, were mobilized after transfer into peat but did not appear to influence survival after inoculation onto beads. Enhanced expression of an iron-manganese superoxide dismutase was also observed after the cells were transferred into peat. We conclude that cell wall thickening in rhizobia after transfer from broth cultures into peat is an adaptive response for long-term survival under nutrient-limited conditions in peat. Cells with thickened walls may also be more resistant to other types of stress, such as that encountered on a seed surface.

Colony Count, Microbial↗
PubMed2002

Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthase.

By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC(Ac), was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC(Ac) gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) [by LS5218 [fadR601 atoC(Con)] strain]. The expression vector for the phaC(Ac) gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).

Acyltransferases↗
PubMed2005

Integrated recombinant protein expression and purification platform based on Ralstonia eutropha.

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own "affinity matrix," thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active beta-galactosidase was obtained in a single step using the above described method.

Bacterial Proteins↗
PubMed1978

Effect of R-plasmid RP1 and nutrient depletion on the gross cellular composition of Escherichia coli and its resistance to some uncoupling phenols.

The resistance of Escherichia coli batch cultures depleted of carbon (C-dep), magnesium (Mg-dep), or phosphate (P-dep) against low concentrations of 3-chlorophenol, 4-chlorophenol, or 2-phenoxyethanol varied. C-dep cultures were always significantly more sensitive than Mg-dep or P-dep cultures. The presence of R-plasmid RP1 increased the sensitivity of C-dep cultures to 3- and 4-chlorophenol, yet had little effect on those cultured depleted in magnesium or phosphate ions. Cultures with R-plasmid RP1 had increased levels of beta-polyhydroxybutyrate irrespective of the nature of the depleting nutrient. P-dep bacteria had less than one-third of the phospholipid of other cell types, this deficiency being compensated for by increases in fatty acid and neutral lipid content. The reduction in phospholipid content of P-dep cultures was entirely accounted for by decreased diphosphatidylglycerol and phosphatidylethanolamine levels in these cells.

Carbon↗