Aromatic compounds degradation plays a role in colonization of Arabidopsis thaliana and Acacia caven by Cupriavidus pinatubonensis JMP134

Plant rhizosphere and internal tissues may constitute a relevant habitat for soil bacteria displaying high catabolic versatility towards xenobiotic aromatic compounds. Root exudates contain various molecules that are structurally related to aromatic xenobiotics and have been shown to stimulate bacterial degradation of aromatic pollutants in the rhizosphere. The ability to degrade specific aromatic components of root exudates could thus provide versatile catabolic bacteria with an advantage for rhizosphere colonization and growth. In this work, Cupriavidus pinatubonensis JMP134, a well-known aromatic compound degrader (including the herbicide 2,4-dichlorophenoxyacetate, 2,4-D), was shown to stably colonize Arabidopsis thaliana and Acacia caven plants both at the rhizoplane and endorhizosphere levels and to use root exudates as a sole carbon and energy source. No deleterious effects were detected on these colonized plants. When a toxic concentration of 2,4-D was applied to colonized A. caven, a marked resistance was induced in the plant, showing that strain JMP134 was both metabolically active and potentially beneficial to its host. The role for the β-ketoadipate aromatic degradation pathway during plant root colonization by C. pinatubonensis JMP134 was investigated by gene inactivation. A C. pinatubonensis mutant derivative strain displayed a reduced ability to catabolise root exudates isolated from either plant host. In this mutant strain, a lower competence in the rhizosphere of A. caven was also shown, both in gnotobiotic in vitro cultures and in plant/soil microcosms.     

Functions of flavin reductase and quinone reductase in 2,4,6-trichlorophenol degradation by Cupriavidus necator JMP134

The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH₂)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH₂, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for the functions of TcpX and TcpB, the two corresponding genes (tcpX and tcpB) were cloned, overexpressed, and purified in Escherichia coli. TcpX was purified as a C-terminal His tag fusion (TcpX_H) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpX_H had no activity toward NADPH or riboflavin. Coupling of TcpX_H and TcpA demonstrated that TcpX_H provided FADH₂ for TcpA catalysis. Among several substrates tested, TcpB showed the best activity for quinone reduction, with FMN or FAD as the cofactor and NADH as the reductant. TcpB could not replace TcpX_H in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB could enhance TcpA activity. Further, we showed that TcpB was more effective in reducing 6-chlorohydroxyquinone than chemical reduction alone, using a thiol conjugation assay to probe transitory accumulation of the quinone. Thus, TcpB was acting as a quinone reductase for 6-chlorohydroxyquinone reduction during 2,4,6-TCP degradation.     

Characterization of MnpC, a Hydroquinone Dioxygenase Likely Involved in the meta-Nitrophenol Degradation by Cupriavidus necator JMP134

Cupriavidus necator JMP134 utilizes meta-nitrophenol (MNP) as the sole source of carbon, nitrogen, and energy. The metabolic reconstruction of MNP degradation performed in silico suggested that MnpC might have played an important role in MNP degradation. In order to experimentally confirm the prediction, we have now characterized the mnpC-encoded (amino)hydroquinone dioxygenase involved in the ring-cleavage reaction of MNP degradation. Real-time PCR analysis indicated that mnpC played an essential role in MNP degradation. MnpC was purified to homogeneity as an N-terminal six-His-tagged fusion protein, and it was proved to be a dimer as demonstrated by gel filtration. MnpC was a Fe²⁺- and Mn²⁺-dependent dioxygenase, catalyzing the ring-cleavage of hydroquinone to 4-hydroxymuconic semialdehyde in vitro and proposed as an aminohydroquinone dioxygenase involved in MNP degradation in vivo. Phylogenetic analysis suggested that MnpC diverged from the other (chloro)hydroquinone dioxygenases at an earlier point, which might result in the preference for its physiological substrate.     

A FANCD2 domain activates Tip60‐dependent apoptosis

The FA (Fanconi anaemia) FANCD2 protein is pivotal in the cellular response to DNA interstrand cross‐links. Establishing cells expressing exogenous FANCD2 has proven to be difficult compared with other DNA repair genes. We find that in transformed normal human fibroblasts, exogenous nuclear expression of FANCD2 induces apoptosis, dependent specifically on exons 10–13. This is the same region required for interaction with the histone acetyltransferase, Tip60. Deletion of exons 10–13 from FANCD2 N‐terminal constructs (nucleotides 1–1100) eliminates the binary interaction with Tip60 and the cellular apoptotic response; moreover, cells can stably express FANCD2 at high levels if Tip60 is depleted. The results indicate that FANCD2‐sponsored apoptosis requires an interaction with Tip60 and depends on Tip60.     

Chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis

Psychological stress affects different physiological processes including haematopoiesis. However, erythropoietic effects of chronic psychological stress remain largely unknown. The adult spleen contains a distinct microenvironment favourable for rapid expansion of erythroid progenitors in response to stressful stimuli, and emerging evidence suggests that inappropriate activation of stress erythropoiesis may predispose to leukaemic transformation. We used a mouse model to study the influence of chronic psychological stress on erythropoiesis in the spleen and to investigate potential mediators of observed effects. Adult mice were subjected to 2 hrs daily restraint stress for 7 or 14 consecutive days. Our results showed that chronic exposure to restraint stress decreased the concentration of haemoglobin in the blood, elevated circulating levels of erythropoietin and corticosterone, and resulted in markedly increased number of erythroid progenitors and precursors in the spleen. Western blot analysis revealed significantly decreased expression of both erythropoietin receptor and glucocorticoid receptor in the spleen of restrained mice. Furthermore, chronic stress enhanced the expression of stem cell factor receptor in the red pulp. Moreover, chronically stressed animals exhibited significantly increased expression of bone morphogenetic protein 4 (BMP4) in the red pulp as well as substantially enhanced mRNA expression levels of its receptors in the spleen. These findings demonstrate for the first time that chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis and leads to the prolonged activation of stress erythropoiesis pathways. Prolonged activation of these pathways along with an excessive production of immature erythroid cells may predispose chronically stressed subjects to a higher risk of leukaemic transformation.     

MiR‐134‐dependent regulation of Pumilio‐2 is necessary for homeostatic synaptic depression

Neurons employ a set of homeostatic plasticity mechanisms to counterbalance altered levels of network activity. The molecular mechanisms underlying homeostatic plasticity in response to increased network excitability are still poorly understood. Here, we describe a sequential homeostatic synaptic depression mechanism in primary hippocampal neurons involving miRNA‐dependent translational regulation. This mechanism consists of an initial phase of synapse elimination followed by a reinforcing phase of synaptic downscaling. The activity‐regulated microRNA miR‐134 is necessary for both synapse elimination and the structural rearrangements leading to synaptic downscaling. Results from miR‐134 inhibition further uncover a differential requirement for GluA1/2 subunits for the functional expression of homeostatic synaptic depression. Downregulation of the miR‐134 target Pumilio‐2 in response to chronic activity, which selectively occurs in the synapto‐dendritic compartment, is required for miR‐134‐mediated homeostatic synaptic depression. We further identified polo‐like kinase 2 (Plk2) as a novel target of Pumilio‐2 involved in the control of GluA2 surface expression. In summary, we have described a novel pathway of homeostatic plasticity that stabilizes neuronal circuits in response to increased network activity.