Measurement of the Isolated Nuclear Two-Photon Decay in

The nuclear two-photon or double-gamma (2γ) decay is a second-order electromagnetic process whereby a nucleus in an excited state emits two gamma rays simultaneously. To be able to directly measure the 2γ decay rate in the low-energy regime below the electron-positron pair-creation threshold, we combined the isochronous mode of a storage ring with Schottky resonant cavities. The newly developed technique can be applied to isomers with excitation energies down to ∼100 keV and half-lives as short as ∼10 ms. The half-life for the 2γ decay of the first-excited 0^{+} state in bare ^{72}Ge ions was determined to be 23.9(6) ms, which strongly deviates from expectations.

Alkane-degrading properties of Dietzia sp. H0B, a key player in the Prestige oil spill biodegradation (NW Spain).

AIMS: Investigation of the alkane-degrading properties of Dietzia sp. H0B, one of the isolated Corynebacterineae strains that became dominant after the Prestige oil spill. METHODS AND RESULTS: Using molecular and chemical analyses, the alkane-degrading properties of strain Dietzia sp. H0B were analysed. This Grampositive isolate was able to grow on n-alkanes ranging from C12 to C38 and branched alkanes (pristane and phytane). 8-Hexadecene was detected as an intermediate of hexadecane degradation by Dietzia H0B, suggesting a novel alkane-degrading pathway in this strain. Three putative alkane hydroxylase genes (one alkB homologue and two CYP153 gene homologues of cytochrome P450 family) were PCR-amplified from Dietzia H0B and differed from previously known hydroxylase genes, which might be related to the novel degrading activity observed on Dietzia H0B. The alkane degradation activity and the alkB and CYP153 gene expression were observed constitutively regardless of the presence of the substrate, suggesting additional, novel pathways for alkane degradation. CONCLUSIONS: The results from this study suggest novel alkane-degrading pathways in Dietzia H0B and a genetic background coding for two different putative oil-degrading enzymes, which is mostly unexplored and worth to be subject of further functional analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the scarce information available about the genetic background of alkane degradation in genus Dietzia and suggests new pathways and novel expression mechanisms of alkane degradation.

ICB database: the gyrB database for identification and classification of bacteria.

The Identification and Classification of Bacteria (ICB) database (http:/www.mbio.co.jp/icb) contains currently available information about the DNA gyrase subunit B (gyrB) gene in bacteria. The database is designed to provide the scientific community with a reference point for using gyrB as an evolutionary and taxonomic marker. Nucleic and amino acid sequence data are currently available for over 850 strains, along with alignments at several different taxonomic levels and an exhaustive review of primer selection and background information.

Polycyclic aromatic hydrocarbon bioremediation design.

Many polycyclic aromatic hydrocarbons (PAHs) are known to be mutagenic or carcinogenic, and their contamination in soil and aquifer is of great environmental concern. Limited numbers of microorganisms including mycobacteria, Sphingomonas and white rot fungi were found to be capable of degrading PAHs with four or more fused aromatic rings. In white rot fungi, lignin peroxidases are believed to be involved in the degradation of PAHs. In addition to these enzymes, P450 monooxygenases in some fungi were implicated in the degradation of PAHs. The stimulation of PAH biodegradation by the addition of surfactants was observed with some of these microorganisms although the agents were inhibitory on biodegradation with some other microorganisms. Mathematical models were constructed to explain the effect of surfactants on biodegradation. Further studies should be carried out to select the best microorganisms and surfactants for applications to PAH bioremediation.

Artificial evolution by DNA shuffling.

Improvement of enzymes is one of the important objectives of biotechnology. In vitro evolution of enzymes using DNA shuffling involves the assembly of two or more DNA segments into a full-length gene by homologous, or site-specific, recombination. Before the assembly, the segments are often subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods. Many useful enzymes and peptides have been isolated following the artificial evolution.

A simple procedure for transferring genes cloned in Escherichia coli vectors into other gram-negative bacteria: phenotypic analysis and mapping of TOL plasmid gene xylK.

A simple method to transfer non-conjugative Escherichia coli plasmids to other Gram-negative bacteria and their maintenance is described. This method involves generation of inverse transposition-mediated cointegrates of the non-conjugative E. coli plasmid with a conjugative IncW broad-host-range plasmid, R388, carrying Tn10. Isolation of such cointegrates was readily effected by conjugal transfer from an E. coli donor containing the two plasmids to an E. coli recipient, with selection for transconjugants expressing a marker of the E. coli plasmid. This method is particularly useful when large series of E. coli vector-based clones need to be expressed in other Gram-negative bacteria to be functionally analysed, either by complementation or recombination. Utility of the method is shown by a functional analysis in Pseudomonas putida of pBR322 hybrid plasmids containing catabolic genes of TOL plasmid pWW0.

PCR isolation of catechol 2,3-dioxygenase gene fragments from environmental samples and their assembly into functional genes.

A method was developed to isolate central segments of catechol 2, 3-dioxygenase (C23O) genes from environmental samples and to insert these C23O gene segments into nahH (the structural gene for C23O encoded by catabolic plasmid NAH7) by replacing the corresponding nahH sequence with the isolated segments. To PCR-amplify the central C23O gene segments, a pair of degenerate primers was designed from amino acid sequences conserved among C23Os. Using these primers, central regions of the C23O genes were amplified from DNA isolated from a mixed culture of phenol-degrading or crude oil-degrading bacteria. Both the 5' and 3' regions of nahH were also PCR-amplified by using appropriate primers. These three PCR products, the 5'-nahH and 3'-nahH segments and the central C23O gene segments, were mixed and PCR-amplified again. Since the primers for the amplification of the central C23O gene segments were designed so that the 20 nucleotides at both ends of the segments are identical to the 3' end of the 5'-nahH segment and the 5' end of the 3'-nahH segment, respectively, the central C23O gene segments could anneal to both the 5'- and 3'-nahH segments. After the second PCR, hybrid C23O genes in the form of (5'-nahH segment-central C23O gene segment-3'-nahH segment) were amplified to full length. The resulting products were cloned into a vector and used to transform Escherichia coli. This method enabled divergent C23O sequences to be readily isolated, and more than 90% of the hybrid plasmids expressed C23O activity. Thus, the present method is useful to create, without isolating bacteria, a library of functional hybrid genes.

Novel family shuffling methods for the in vitro evolution of enzymes.

It has recently been shown that shuffling of the amino acid sequences of family enzymes allows the generation of improved enzymes. Family shuffling is generally achieved by a DNase I treatment and then by PCR. Shuffling of the xylE and nahH genes, both encoding catechol 2,3-dioxygenases, was carried out by the published method. However, nahH-xylE hybrids were only formed at a very low frequency (less than 1%). Therefore, we developed improved methods for family shuffling by which DNA was cleaved by restriction enzymes instead of by DNase I. With the first improved method, five nahH fragments and five xylE fragments that had been generated by restriction enzyme digestion were subjected to the PCR reactions in two steps, the first being without a primer and the second with a set of primers. This method enabled nahH-xylE hybrid genes to be formed at a high frequency (almost 100%). With the second improved method, nahH and xylE were cleaved by several sets of restriction enzymes, and these digests were then reassembled in two steps. The nahH and xylE DNAs were each cleaved by two (or three) sets of restriction enzymes, and one type of nahH digest and one type of xylE digest were mixed, thus making four (or nine) different mixtures of the nahH and xylE digests. These mixtures were used as templates to carry out PCR without a primer. After the first PCR reaction, all the mixtures were combined, and a second PCR reaction was carried out without a primer. Following these two PCR assembly steps, a third PCR reaction was carried out with two primers to amplify the full-length nahH-xylE hybrid genes. This second method also yielded nahH-xylE hybrids at a frequency of 100%. The degree of recombination of the products with the second method was higher than that with the first method. These methods were used to isolate catechol 2,3-dioxygenases exhibiting relatively high stability at high temperature, one of them being respectively 13- and 26-fold more thermostable than XylE and NahH at 50 degrees C.

An effective family shuffling method using single-stranded DNA.

Family shuffling, which is one of the most powerful techniques for in vitro protein evolution, always involves the problem of reassembling the gene fragments into parental gene sequences, because such a process prevents the formation of chimeric sequences. In order to improve the efficiency of hybrid formation in family shuffling, single-stranded DNAs (ssDNAs) were used as templates. The ssDNAs of two catechol 2,3-dioxygenase genes, nahH and xylE, were prepared, the xylE strand being complementary to the nahH strand. When these ssDNAs were digested by DNase I and reassembled, chimeric genes were obtained at a rate of 14%, which was much higher than the rate of less than 1% obtained by shuffling with double-stranded DNAs. Chimeric catechol 2,3-dioxygenases that were more thermally stable than the parental enzymes, XylE and NahH, were obtained by this ssDNA-based DNA shuffling.

Divergent evolution of chloroplast-type ferredoxins.

The TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes required for the oxidation of toluene to Krebs cycle intermediates. The structural genes for these enzymes are encoded in two operons which comprise the xylCMABN and xylXYZLTEGFJQKIH genes, respectively. The function of the xylT gene has not yet been identified. The nucleotide sequence of xylT was determined in this study and putative gene product was shown to contain a sequence characteristic for chloroplast-type ferredoxins. The nahT gene, the homologue of xylT, present on NAH plasmid NAH7 encoding naphthalene-degrading enzymes, was also sequenced. The sequence conservation between xylT and nahT strongly suggests that both gene products have some physiological function. Chloroplast-type ferredoxins have been discovered in photosynthetic organisms (plants, algae, cyanobacteria and Rhodobacter) and Halobacterium species. Furthermore, chloroplast-type ferredoxin-like sequences have been found in the electron-transfer components of some oxygenases. The sequences of XylT and NahT were compared with those of the previously identified chloroplast-type ferredoxins, in order to examine their evolutionary relationships.

Purification and characterization of Acinetobacter calcoaceticus 4-hydroxybenzoate 3-hydroxylase after its overexpression in Escherichia coli.

4-Hydroxybenzoate 3-hydroxylase [EC 1.14.13.2] from Acinetobacter calcoaceticus was purified to homogeneity following the 40-fold overexpression of this gene (pobA) in Escherichia coli. Overexpression was accomplished by placing the folA gene (encoding trimethoprim-resistant dihydrofolate reductase) directly downstream of the pobA gene, and demanding growth of recombinants on elevated concentration of trimethoprim. Presumably, the surviving variants have undergone a genetic alteration which allowed the overexpression of both folA and pobA. 4-Hydroxybenzoate 3-hydroxylase was purified in two chromatographic steps, characterized biochemically, and its properties were compared to those of its homolog from Pseudomonas fluorescens. The two enzymes differ in their response to Cl- ion inhibition. A single amino acid change in the putative NADPH-binding site is proposed to account for this difference. The inhibitory and catalytic properties of substrate analogs were also examined.

Biochemical characterization of a Ca(2+)-dependent lectin from the hemolymph of a photosymbiotic marine bivalve, Tridacna derasa (Röding).

A Ca(2+)-dependent lectin was purified from the hemolymph of a photosymbiotic bivalve, Tridacna derasa. An electrophoretically homogeneous form was obtained by using affinity chromatography with Sepharose 4B. More than 80% of the hemolymph protein was accounted for by this lectin. The apparent molecular mass of the lectin, in its native form exhibiting hemagglutinating activity, was estimated by gel filtration analysis to be approximately 480 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in the absence of reductants, it migrated as a single band corresponding to a very large size, while in the presence of 2-mercaptoethanol, it migrated as two distinct bands of 23 and 46 kDa. These results indicate that the subunits were linked by disulfide bridges to form the native protein. After reducing pyridylethylation, each of the 23- and 46-kDa polypeptides was isolated by gel filtration in a mobile phase containing guanidine-HCl. The two polypeptides had the same amino-terminal sequence and a similar amino-acid composition, and in the presence of 2-mercaptoethanol gave a single band on isoelectric focusing at a pH of 6.0. The results suggested that the 46-kDa peptide is a homodimer of 23-kDa subunits held together by a covalent bond other than a disulfide linkage. This lectin required calcium ions for its activity. By ultraviolet spectrophotometry the association constant for the calcium ion was determined to be 0.88 mM. The hemagglutinating activity decreased dramatically below pH 6.5, but re-increased to the original level when the solution was neutralized. Such a pH-dependent alteration of the ligand-binding activity was similar to that found in vertebrate asialoglycoprotein receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The predatory soil flagellate Heteromita globosa stimulates toluene biodegradation by a Pseudomonas sp.

A model food chain was established to investigate the influence of grazing by flagellates on bacteria degrading toluene in batch culture. The rate of toluene consumed by a Pseudomonas sp. strain PS+ (max. 0.37 fmol cell(-1) h(-1)) was significantly higher in the presence of the bacterivorous flagellate Heteromita globosa (max. 1.38 fmol cell(-1) h(-1)). A maximum increase of up to 7.5 times was observed in the rate of toluene consumed by these bacteria during exponential growth of this flagellate. Carbon conversion efficiency (CCE) of bacteria to flagellate biomass was estimated to be 33.4% based on measured biovolumes and published values for carbon contents. However, the CCE for toluene-derived carbon was lower (max. 4.9%) when calculations were based on incorporation of [ring-U-(14)C]toluene into biomass of flagellates grazing on labelled bacteria. The findings suggest a potential role for flagellates in bioremediation processes.

Regulation of the rpoN, ORF102 and ORF154 genes in Pseudomonas putida.

The DNA sequence downstream of the Pseudomonas putida rpoN gene and the adjacent ORF102 was determined. This region encodes an ORF (ORF154) whose gene product was found to be homologous to a family of phosphotransferases. Insertional mutagenesis and analysis of mRNA transcripts showed that the rpoN gene is transcribed separately from the two downstream genes. The rpoN promoter was localized to an 86 nucleotide-long region upstream of the rpoN gene by examination of the expression of a series of rpoN::lacZ fusions. The expression of rpoN in P. putida was independent of the nitrogen status of the cell but was 5 times higher in the rpoN mutant than in the wild-type strain, suggesting that the expression of the rpoN gene in this organism is autoregulated.

Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting.

Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGGE). In this study, sequences of bacterial universal primers frequently used in PCR/DGGE were compared with 16S rDNA sequences that represent recently proposed divisions in the domain Bacteria. We found mismatches in 16S rDNA sequences from some groups of bacteria. Inosine residues were then introduced into the bacterial universal primers to reduce amplification biases caused by these mismatches. Using the improved primers, phylotypes affiliated with Verrucomicrobia and candidate division OP11, were detected in DGGE fingerprints of groundwater populations, which have not been detected by PCR/DGGE with conventional universal primers.

Interspecific variations in adhesive protein sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus.

Variation in the adhesive protein gene sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus collected in Delaware, Kamaishi (Japan), and Alaska, respectively, was analyzed by the polymerase chain reaction (PCR) using two sets of oligonucleotide primers. The first set, Me 13 and Me 14, was designed to amplify the repetitive region. The length of the amplified fragments was highly variable, even among samples of the same species. Another set, Me 15 and Me 16, was designed to amplify a part of the nonrepetitive region. The length of the amplified fragments was uniform in each species and differed interspecifically; 180, 168, and 126 bp for M. edulis, M. trossulus, and M. galloprovincialis, respectively. The amplified sequence of M. trossulus resembled that of M. edulis. Mussels from other sites were also examined by PCR using Me 15 and Me 16. Wild mussels from Tromsö (Norway) and cultured mussels from Brittany (France) were identified as M. edulis. Cultured mussels from the Mediterranean coast of France and wild mussels from Shimizu (Japan) were identified as M. galloprovincialis. Some wild mussels from Hiura (Japan) were identified as a hybrid between M. galloprovincialis and M. trossulus. Thus, the length of this part (variable region) of the sequence is proposed as a diagnostic marker for these three morphologically similar species and their hybrids.

Characterization of genes for enzymes involved in the phenanthrene degradation in Nocardioides sp. KP7.

The nucleotide sequence of the gene cluster, phdEFABGHCD, encoding enzymes responsible for the transformation of phenanthrene to 1-hydroxy-2-naphthoate in Nocardioides sp. strain KP7 was determined. This gene cluster, which may constitute a single operon, resided at 6.1-kb downstream of the phdIJK gene cluster encoding the enzymes for the transformation of 1-hydroxy-2-naphthoate to o-phthalate. In general, the phd products exhibited moderate degrees of homology with isofunctional enzymes found in pathways for the degradation of other aromatic compounds. Remarkably, the phdC gene product had features of the [3Fe-4S] type ferredoxin, which has not been found so far as a component of the ring-hydroxylating dioxygenase. Escherichia coli carrying the genes for phenanthrene dioxygenase, phdABCD, was capable to oxidize phenanthrene.

Photo-oxidation of biodegraded crude oil and toxicity of the photo-oxidized products.

We investigated the physicochemical changes resulting from irradiation by sunlight of biodegraded crude oil. An Arabian light crude oil sample was first subjected to microbial degradation. n-Alkanes and aromatic compounds such as naphthalenes, fluorenes, dibenzothiophenes and phenanthrenes possessing short, alkyl side chain(s) were almost completely degraded, while the contents of the saturated and aromatic fractions were reduced by 70% and 40%, respectively. This biodegraded oil was then suspended in seawater and exposed to sunlight irradiation for several weeks. The most remarkable change caused by the irradiation was a substantial decline in the aromatic fraction with a concomitant increase in the resin and asphaltene fractions. A 13C-nuclear magnetic resonance (NMR) spectroscopic analysis showed that the aromaticity of the biodegraded oil was significantly lower in the irradiated sample. A field desorption-mass spectrometric (FD-MS) analysis showed that sunlight irradiation reduced the average molecular weight of the oil components and formed oxygenated compounds. Consistent with this observation is that the oxygen content in the oil increased as the irradiation was prolonged. The bioavailability of the biodegraded oil was increased by the photo-oxidation: the growth of seawater microbes was minimal when the non-irradiated biodegraded oil was used as the source of carbon and energy; however, growth was significant when irradiated biodegraded oil was used. The concentration of dissolved organic carbon (DOC) increased linearly during the sunlight irradiation of the biodegraded oil, and this increase was matched by an increase in ultraviolet-absorptive materials in the seawater. The photochemically formed, water-soluble fraction (WSF) showed acute toxicity against the halophilic crustacean, Artemia.

Enhanced mineralization of benzo[a]pyrene in the presence of nonaqueous phase liquids.

Bacterial mineralization of [7-14C]benzo[a]pyrene (BaP) to 14CO2 was enhanced by the presence of nonaqueous phase liquids (NAPLs). Mineralization of BaP was affected differently by different NAPLs, and the mode of enhancement of mineralization by a NAPL most likely occurred by a combination of cometabolic and physical effects. Mineralization was enhanced to the greatest extent when BaP was dissolved in a high-boiling distillation product of diesel fuel.