Environmentally directed mutations in the dehalogenase system of Pseudomonas putida strain PP3

Favourable mutations involving the two dehalogenases (DehI and DehII) of Pseudomonas putida PP3 and derivative strains containing the cloned gene for DehI (dehI) occurred in response to specific environmental conditions, namely: starvation conditions; the presence of dehalogenase substrates (halogenated alkanoic acids — HAAs) which were toxic to P. putida; and/or the presence of a potential growth substrate. Fluctuation tests showed that these mutations were environmentally directed by the presence of HAAs. the mutations were associated with complex DNA rearrangements involving the movement of dehI located on a transposon DEH. Some mutations resulted in switching off the expression of either one or both of the dehalogenases, events which were effective in protecting P. putida from toxic compounds in its growth environment. Other mutations partially restored P. putida's dehalogenating capability under conditions where toxic substrates were absent. Restoration of the capability to untilize HAAs was favoured when normal growth substrates were present in the environment.     

DIVERSITY OF HALOGENATED SECONDARY METABOLITES IN THE RED ALGA LAURENCIA NIPPONICA (RHODOMELACEAE,CERAMIALES)1,2

Many morphologically similar, but chemically distinct, populations have been found in the marine red alga Laurencia nipponica Yamada (Rhodomelaceae, Ceramiales) growing in Japan. Each chemical type is characterized by a specific end-product of halogenated secondaly metabolite synthesis: chamigrane-type sesquiterpenoids such as prepacifenol and halochamigrene epoxide and C₁₅ bromoethers such as laurencin, laureatin, isoprelaurefucin, epilaurallene, and kumausallene. These seven types of secondary metabolite syntheses remained the same in the wild and under various culture conditions. Because bromoethers and terpenoids are probably synthesized by different metabolic pathways, it is virtually certain that different sets of enzymes participate in their synthesis. Prepacifenol- and laureatin-producing populations were selected as representatives of terpenoid and bromoether groups, respectively. F₁ tetrasporophytes derived from crosses between reciprocal, female and male gametophytes of prepacifenol- and laureatin-producing strains bore both types of metabolites, suggesting that the genes Producing these enzyme systems are encoded by nuclear genomes. The F₁ gametophytes resulting from the reciprocal crosses produced either prepacifenol or laureatin, and the four individuals derived from spore tetrads (a set of tetraspores derived from a single tetrasporangium) produced either prepacifenol or laureatin in a 1:1 ratio, indicating that genes participating in terpenoid synthsis and those participating in bromoether synthesis are on different loci of homologous chromosomes and are segregated at meiosis (tetrasporogenesis). One individual of this interpopulational F₁ gamtophyte produced both parental types of metabolite, perhaps indicating the occurrence of a recombination type. Natural hybrid individuals, including such recombination-type gametophytes, were found in a sympatric locality at which these two chemical types occur. F₁ tetrasporophytes derived from crosses between respective prepacifenol- and laureatin-producing strains and their F₁ gametohytes produced only parental-type metabolite-producing plants. These results indicate that the diverse chemical types can be referred to as races (chemical races).     

Effect of 2,6-Diisopropylphenol and Halogenated Anesthetics on Tetraphenylphosphonium Uptake by Rat Brain Synaptosomes: Determination of Membrane Potential

The effect of 2,6-diisopropylphenol (propofol) in comparison to that of the halogenated anesthetics enflurane, isoflurane, and halothane on tetrapenylphosphonium uptake by rat brain synaptosomes was studied. A direct method to separately measure the synaptosomal and the mitochondrial transmembrane potential by using the tetraphenylphosphonium cation (TPP⁺) was utilized. The latter is a lipophylic charged molecule which distributes between two compartments according to the transmembrane electrical potential in the presence or absence of 60 mM KCl as a synaptosomal membrane depolarizing agent. After previously reporting the damages induced by general anesthetics on isolated mitochondria, the aim of this paper was to study their possible action on the synaptosomal membrane potential and whether or not drugs concentrations damaging isolated mitochondria are also effective on synaptosomal mitochondria. The results indicated that, in the presence of glucose, mitochondria included in synaptosomes were able to maintain a transmembrane potential of 202 ± 8 mV (mean ± SD) while the synaptosomal membrane showed a potential of 78 ± 8 mV (mean ± SD). When anesthetic concentrations (0.6–1 mM propofol, 10–40 M enflurane, 30–50 M isoflurane, 8–15 M halothane) that impair mitochondrial energy metabolism were used, the synaptosomal transmembrane potential was maintained and, in addition, a slight increase of the TPP⁺ taken up was observed as the anesthetic concentration was increased.     

A rearranged chamigrene derivative and its potential biogenetic precursor from a new species of the marine red algal genus Laurencia (rhodomelaceae)

The structures of a novel rearranged sesquiterpenoid and a biogenetically-related chamigrene derivative have been determined by combined spectral and chemical methods. These sesquiterpenoids were components of an undescribed Laurencia species, and each was toxic toward the damselfish Pomacentrus coeruleus.     

Metabolism of mono- and dihalogenated C1 and C2 compounds by <Emphasis Type="Italic">Xanthobacter autotrophicus</Emphasis> growing on 1,2-dichloroethane

The conversion of and toxic effects exerted by several mono- and dihalogenated C1 and C2 compounds on cultures of Xanthobacter autotrophicus GJ10 growing on 1,2-dichloroethane were investigated. Bromochloromethane, dibromomethane and 1-bromo-2-chloroethane were utilized by strain GJ10 in batch culture as a cosubstrate and sole carbon source. The rate of degradation of dihalomethanes by whole cells was lower than that of 1,2-dichloroethane, but a significant increase of the rate of dihalomethane biodegradation was observed when methanol or ethanol were added as a cosubstrate. Products of the degradation of several tested compounds by haloalkane dehalogenase were analyzed and a new metabolic pathway based on hydrolytic conversion to formaldehyde was proposed for the dihalomethanes. Strain GJ10 growing on 1,2-dichloroethane converted 2-fluoroethanol and 1-chloro-2-fluoroethane to 2-fluoroacetate, which was tolerated up to a concentration of 2.5 mM. On the basis of the results from batch cultures an inert (dichloromethane), a growth-supporting (dibromomethane) and a toxic (1,2-dibromoethane) compound were selected for testing their effects on a continuous culture of strain GJ10 growing on 1,2-dichloroethane. The compounds were added as pulses to a steady-state chemostat and the response of the culture was followed. The effects varied from a temporary decrease in cell density for dibromomethane to severe toxicity and culture washout with 1,2-dibromoethane. Our results extend the spectrum of halogenated C1 and C2 compounds that are known to be degraded by strain GJ10 and provide information on toxic effects and transformation of compounds not serving as a carbon source for this bacterium.     

Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide series on the terminal sequence of XpYp telomere in HT1080 cells

Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A–Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A–Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A–Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A–Br in HT1080 cells are discussed.     

Volatile halogenated organic compounds in European estuaries

Sources, sinks, and distribution patterns ofvolatile halogenated organic compounds (VHOC)in estuaries were investigated during 5 cruises within the BIOGEST programme. Due to their chemical and physical properties (e.g. toxicity, persistence, mobility) these compounds are of considerable environmental concern. A wide range of compounds has been identified and quantified generally ranging from 0.1 ng l^–1 to 350 ng l^–1. Insome samples extraordinarily high values up to4700 ng l^–1 were observed indicatingcontribution from anthropogenic sources.Generally, concentrations of halogenatedcompounds of anthropogenic origin dominatedthose of prevalent natural origin. Data ofselected VHOC are presented in relation tosalinity, particular organic carbon, and totalsuspended matter. Furthermore the observedconcentrations are compared with establishedwater quality regulations. Distributionpatterns of VHOC along the estuary indicatedcommon sources for specific halogenatedcompounds. Decreasing concentrations of mostVHOC along the estuary confirm that degassingto the atmosphere and dilution with sea waterare the dominating processes controlling thefate of these compounds in estuaries.     

Gracilaria and its epiphytes: 4. The response of two Gracilaria species to Ulva lactuca in a bacteria-limited environment

The responses of Gracilaria lemaneiformis, an easily epiphytized host,and the relatively resistant G. cornea mutant, to the green alga Ulva lactuca were studied using biculture experiments with and withoutantibiotics. Both Gracilaria species grown with and without U.lactuca showed different levels of growth rate, release of hydrogenperoxide and of halogenated hydrocarbons. These quantitative differencesled to a successful response against Ulva lactuca in the case of G.cornea mutant and to a failure in response in the case of G.lemaneiformis. The response of each Gracilaria species to U.lactuca was qualitatively similar to its response to bacteria. This suggeststhe involvement of oligosaccharide elicitors produced in the presence ofepiphytes and bacteria. A clear Gracilaria inhibition was demonstratedwith extracts of the culture medium. It appears that hydrogen peroxide,halogenated hydrocarbons and oligosaccharides may be components of theinhibitory activity of the extracts. The responses of Gracilaria speciesto the presence of U. lactuca suggest the characterization of adefence response.     

Characterization of an α-haloalkanoic acid–degrading Pseudomonas aeruginosa MX1 isolated from contaminated seawater

Halogenated compounds such as α-halocarboxylix acids (αHAs) are widely liberated into the ecosystem through the prevailing xenobiotic activities that involve the use of herbicides for weed management in the agricultural sector and mass production of various commercial halogenated chemical intermediates. Since such compounds exert stress on the environment owing to their recalcitrance and are not easily degraded, the study aimed to isolate, identify, and characterize dehalogenase-producing bacteria with the purpose of bioremediation. The MX1 bacterium was successfully isolated from seawater samples off the coast of Desaru, Malaysia, using an enrichment technique supplemented with 2,2-dichloropropionic acid (2,2-DCP). Interestingly, the MX1 strain grew best in a 20 mM 2,2-DCP minimal medium as the sole carbon source and illustrated a 44 ± 0.2 h cell-doubling time as well as a 38 μmol Cl^?/ml maximum rate of chloride ion release. However, 2,2-DCP–containing medium with concentrations that exceeded 30 mM inhibited the growth of the MX1, possibly attributable to the increased toxicity of the compound on the bacteria. Biochemical examinations and 16S rDNA sequence analysis revealed that the MX1 strain shares high identity to Pseudomonas aeruginosa, and the gene sequence was deposited into GenBank as Pseudomonas aeruginosa MX1 under accession number KP336490. The presence of the putative dehalogenase gene in the MX1 strain was established by polymerase chain reaction (PCR) analysis, which proved the presence of conserved amino acid residues belonging to the Group I dehalogenase. This is the first report detailing a P. aeruginosa strain capable of degrading the recalcitrant 2,2-DCP and its functional amino acid residues.     

Chemical races in the red alga Laurencia nipponica (Rhodomelaceae, Ceramiales)

Genetic variation in the synthesis of halogenated secondary metabolites in the Japanese marine red alga Laurencia nipponica Yamada (Rhodomelaceae, Ceramiales) has been investigated in laboratory crossing experiments and chemical analyses, F₁ tetrasporophytes and F₁ gametophytes resulting from crosses within chemical races produced major metabolites characteristic of these races. F₁ tetrasporophytes derived from reciprocal interracial crosses produced: (i) both parental types of secondary metabolites; (ii) either of the parental types; or (iii) a further major compound in addition to both parental types or in addition to either of the parental types. The latter cases suggest that hybrid-specific products were formed by the combined enzymatic complements of the parents, as F₁ gametophytes derived from these interracial F₁ tetrasporophytes yielded one or other of their parental products in an approximate 1:1 ratio. The population structure was analyzed at localities in Hokkaido, where two of the chemical races occur sympatrically. At Usujiri (Minami-kayabe), where the prepacifenol race and the laureatin race were sym-patric, hybrid gametophytes (recombination type) were found in high frequency in addition to hybrid tetra sporophytes, which strongly suggests that a new, pre-pacifenol/laureatm race is beginning to be produced by natural hybridization and recombination. By contrast, at Oshoro Bay, where the laurencin race and the epi-lauraliene race grew together, the interracial hybrids were rare: only a few tetrasporophytes (probably F₁ generation) were found, suggesting that racial integrity may be retained by habitat segregation and/or the absence of recombination-type gametophytes.