Transfer and expression of PCB-degradative genes into heavy metal resistantAlcaligenes eutrophus strains

Sites polluted with organic compounds frequently contain inorganic pollutants such as heavy metals. The latter might inhibit the biodegradation of the organics and impair bioremediation. Chromosomally located polychlorinated biphenyl (PCB) catabolic genes ofAlcaligenes eutrophus A5,Achromobacter sp. LBS1C1 andAlcaligenes denitrificans JB1 were introduced into the heavy metal resistantAlcaligenes eutrophus strain CH34 and related strains by means of natural conjugation. Mobile elements containing the PCB catabolic genes were transferred fromA. eutrophus A5 andAchromobacter sp. LB51C1 intoA. eutrophus CH34 after transposition onto their endogenous IncP plasmids pSS50 and pSS60, respectively. The PCB catabolic genes ofA. denitrificans JB1 were transferred intoA. eutrophus CH34 by means of RP4::Mu3A mediated prime plasmid formation. TheA. eutrophus CH34 transconjugant strains expressed both catabolic and metal resistance markers. Such constructs may be useful for the decontamination of sites polluted by both organics and heavy metals.     

Recovery of Soil Ammonia Oxidation After Long-Term Zinc Exposure Is Not Related to the Richness of the Bacterial Nitrifying Community

A soil sterilization–reinoculation approach was used to manipulate soil microbial diversity and to assess the effect of the diversity of the ammonia-oxidizing bacteria (AOB) on the recovery of the nitrifying community to metal stress (zinc). Gamma-irradiated soil was inoculated with 13 different combinations of up to 22 different soils collected worldwide to create varying degrees of AOB diversity. Two months after inoculation, AOB amoA DGGE based diversity (weighted richness) varied more than 10-fold among the 13 treatments, the largest value observed where the number of inocula had been largest. Subsequently, the 13 treatments were either or not amended with ZnCl₂. Initially, Zn amendment completely inhibited nitrification. After 6 months of Zn exposure, recovery of the potential nitrification activity in the Zn amended soils ranged from <10 % to >100 % of the potential nitrification activity in the corresponding non-amended soils. This recovery was neither related to DGGE-based indices of AOB diversity nor to the AOB abundance assessed 2 months after inoculation (p?>?0.05). However, recovery was significantly related (r?=?0.75) to the potential nitrification rate before Zn amendment and only weakly to the number of soil inocula used in the treatments (r?=?0.46). The lack of clear effects of AOB diversity on recovery may be related to an inherently sufficient diversity and functional redundancy of AOB communities in soil. Our data indicate that potential microbial activity can be a significant factor in recovery.     

Environmental Dissolved Organic Matter Governs Biofilm Formation and Subsequent Linuron Degradation Activity of a Linuron-Degrading Bacterial Consortium

It was examined whether biofilm growth on dissolved organic matter (DOM) of a three-species consortium whose members synergistically degrade the phenylurea herbicide linuron affected the consortium''s integrity and subsequent linuron-degrading functionality. Citrate as a model DOM and three environmental DOM (eDOM) formulations of different quality were used. Biofilms developed with all DOM formulations, and the three species were retained in the biofilm. However, biofilm biomass, species composition, architecture, and colocalization of member strains depended on DOM and its biodegradability. To assess the linuron-degrading functionality, biofilms were subsequently irrigated with linuron at 10 mg liter⁻¹ or 100 μg liter⁻¹. Instant linuron degradation, the time needed to attain maximal linuron degradation, and hence the total amount of linuron removed depended on both the DOM used for growth and the linuron concentration. At 10 mg liter⁻¹, the final linuron degradation efficiency was as high as previously observed without DOM except for biofilms fed with humic acids which did not degrade linuron. At 100 μg liter⁻¹ linuron, DOM-grown biofilms degraded linuron less efficiently than biofilms receiving 10 mg liter⁻¹ linuron. The amount of linuron removed was more correlated with biofilm species composition than with biomass or structure. Based on visual observations, colocalization of consortium members in biofilms after the DOM feed appears essential for instant linuron-degrading activity and might explain the differences in overall linuron degradation. The data show that DOM quality determines biofilm structure and composition of the pesticide-degrading consortium in periods with DOM as the main carbon source and can affect subsequent pesticide-degrading activity, especially at micropollutant concentrations.     

HylA,an Alternative Hydrolase for Initiation of Catabolism of the Phenylurea Herbicide Linuron in Variovorax sp. Strains

Variovorax sp. strain WDL1, which mineralizes the phenylurea herbicide linuron, expresses a novel linuron-hydrolyzing enzyme, HylA, that converts linuron to 3,4-dichloroaniline (DCA). The enzyme is distinct from the linuron hydrolase LibA enzyme recently identified in other linuron-mineralizing Variovorax strains and from phenylurea-hydrolyzing enzymes (PuhA, PuhB) found in Gram-positive bacteria. The dimeric enzyme belongs to a separate family of hydrolases and differs in K_m, temperature optimum, and phenylurea herbicide substrate range. Within the metal-dependent amidohydrolase superfamily, HylA and PuhA/PuhB belong to two distinct protein families, while LibA is a member of the unrelated amidase signature family. The hylA gene was identified in a draft genome sequence of strain WDL1. The involvement of hylA in linuron degradation by strain WDL1 is inferred from its absence in spontaneous WDL1 mutants defective in linuron hydrolysis and its presence in linuron-degrading Variovorax strains that lack libA. In strain WDL1, the hylA gene is combined with catabolic gene modules encoding the downstream pathways for DCA degradation, which are very similar to those present in Variovorax sp. SRS16, which contains libA. Our results show that the expansion of a DCA catabolic pathway toward linuron degradation in Variovorax can involve different but isofunctional linuron hydrolysis genes encoding proteins that belong to evolutionary unrelated hydrolase families. This may be explained by divergent evolution and the independent acquisition of the corresponding genetic modules.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Stroma Cell-Derived Factor-1α Signaling Enhances Calcium Transients and Beating Frequency in Rat Neonatal Cardiomyocytes

Stroma cell-derived factor-1α (SDF-1α) is a cardioprotective chemokine, acting through its G-protein coupled receptor CXCR4. In experimental acute myocardial infarction, administration of SDF-1α induces an early improvement of systolic function which is difficult to explain solely by an anti-apoptotic and angiogenic effect. We wondered whether SDF-1α signaling might have direct effects on calcium transients and beating frequency.Primary rat neonatal cardiomyocytes were culture-expanded and characterized by immunofluorescence staining. Calcium sparks were studied by fluorescence microscopy after calcium loading with the Fluo-4 acetoxymethyl ester sensor. The cardiomyocyte enriched cellular suspension expressed troponin I and CXCR4 but was vimentin negative. Addition of SDF-1α in the medium increased cytoplasmic calcium release. The calcium response was completely abolished by using a neutralizing anti-CXCR4 antibody and partially suppressed and delayed by preincubation with an inositol triphosphate receptor (IP₃R) blocker, but not with a ryanodine receptor (RyR) antagonist. Calcium fluxes induced by caffeine, a RyR agonist, were decreased by an IP₃R blocker. Treatment with forskolin or SDF-1α increased cardiomyocyte beating frequency and their effects were additive. In vivo, treatment with SDF-1α increased left ventricular dP/dtmax.These results suggest that in rat neonatal cardiomyocytes, the SDF-1α/CXCR4 signaling increases calcium transients in an IP₃-gated fashion leading to a positive chronotropic and inotropic effect.     

Deletions of mob and tra pJP4 transfer functions after mating of Ralstonia eutropha JMP134 (pJP4) with Escherichia coli harboring F'::Tn10

One-tenth of Escherichia coli transconjugants resulting from the transfer of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 to E. coli XL1Blue, contained pJP4 derivatives with deletions (approximately 15-30 kb). The occurrence of these deletions is probably associated with the presence of Tn10 in the recipient. DNA endonuclease restriction analysis of the pJP4 deletion derivatives showed the absence of SphI and EcoRI fragments previously reported to hybridize with IncP Tra DNA probes. Moreover, these pJP4 deletion derivatives are not able to self-transfer, nor are they able to be mobilized. Accordingly, these pJP4 deletion derivatives lack transfer functions.     

Chromosomal Integration, Tandem Amplification, and Deamplification in Pseudomonas putida F1 of a 105-Kilobase Genetic Element Containing the Chlorocatechol Degradative Genes from Pseudomonas sp. Strain B13

Analysis of chlorobenzene-degrading transconjugants of Pseudomonas putida F1 which had acquired the genes for chlorocatechol degradation (clc) from Pseudomonas sp. strain B13 revealed that the clc gene cluster was present on a 105-kb amplifiable genetic element (named the clc element). In one such transconjugant, P. putida RR22, a total of seven or eight chromosomal copies of the entire genetic element were present when the strain was cultivated on chlorobenzene. Chromosomal integrations of the 105-kb clc element occurred in two different loci, and the target sites were located within the 3′ end of glycine tRNA structural genes. Tandem amplification of the clc element was preferentially detected in one locus on the F1 chromosome. After prolonged growth on nonselective medium, transconjugant strain RR22 gradually diverged into subpopulations with lower copy numbers of the clc element. Two nonadjacent copies of the clc element in different loci always remained after deamplification, but strains with only two copies could no longer use chlorobenzene as a sole substrate. This result suggests that the presence of multiple copies of the clc gene cluster was a prerequisite for the growth of P. putida RR22 on chlorobenzene and that amplification of the element was positively selected for in the presence of chlorobenzene.     

Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase'' Npr1'' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.