Selection of independent plasmids determining phenol degradation in Pseudomonas putida and the cloning and expression of genes encoding phenol monooxygenase and catechol 1,2-dioxygenase

Long-term cultivation of the Pseudomonas putida multiplasmid strain EST1020 on phenol resulted in the formation of individual PHE plasmids determining phenol degradation. Four types of PHE plasmids, pEST1024, pEST1026, pEST1028, and pEST1029, are characterized. They all contain a transferrable replicon similar to pWWO-8 with a partly duplicated DNA sequence of the 17-kb transposable element of this plasmid and include various amounts of DNA that carry genes encoding phenol degradation (phe genes). We cloned the genes determining phenol monooxygenase and catechol 1,2-dioxygenase from the Pseudomonas sp. parent strain plasmid DNA into the broad host range vector pAYC32 and studied the expression of the cloned DNA. The formation of a new hybrid metabolic plasmid, pEST1354, was demonstrated in P. putida PaW85 as the result of transposition of the 17-kb genetic element from the chromosome of PaW85 into the plasmid carrying cloned phe genes. The target site for the 17-kb transposon was localized in the vector DNA, just near the cloning site. In subcloning experiments we found two regions in the 17-kb DNA stretch that are involved in the expression of the cloned phe genes.     

Methane emissions reduce the radiative cooling effect of a subtropical estuarine mangrove wetland by half

The role of coastal mangrove wetlands in sequestering atmospheric carbon dioxide (CO₂) and mitigating climate change has received increasing attention in recent years. While recent studies have shown that methane (CH₄) emissions can potentially offset the carbon burial rates in low‐salinity coastal wetlands, there is hitherto a paucity of direct and year‐round measurements of ecosystem‐scale CH₄ flux (F_CH4) from mangrove ecosystems. In this study, we examined the temporal variations and biophysical drivers of ecosystem‐scale F_CH4 in a subtropical estuarine mangrove wetland based on 3 years of eddy covariance measurements. Our results showed that daily mangrove F_CH4 reached a peak of over 0.1 g CH₄‐C m^?2 day^?1 during the summertime owing to a combination of high temperature and low salinity, while the wintertime F_CH4 was negligible. In this mangrove, the mean annual CH₄ emission was 11.7 ± 0.4 g CH₄‐C m^–2 year^?1 while the annual net ecosystem CO₂ exchange ranged between ?891 and ?690 g CO₂‐C m^?2 year^?1, indicating a net cooling effect on climate over decadal to centurial timescales. Meanwhile, we showed that mangrove F_CH4 could offset the negative radiative forcing caused by CO₂ uptake by 52% and 24% over a time horizon of 20 and 100 years, respectively, based on the corresponding sustained‐flux global warming potentials. Moreover, we found that 87% and 69% of the total variance of daily F_CH4 could be explained by the random forest machine learning algorithm and traditional linear regression model, respectively, with soil temperature and salinity being the most dominant controls. This study was the first of its kind to characterize ecosystem‐scale F_CH4 in a mangrove wetland with long‐term eddy covariance measurements. Our findings implied that future environmental changes such as climate warming and increasing river discharge might increase CH₄ emissions and hence reduce the net radiative cooling effect of estuarine mangrove forests.     

Soluble CD44 interacts with intermediate filament protein vimentin on endothelial cell surface

CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12-37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells.     

Dual Function of UNC-51-like Kinase 3 (Ulk3) in the Sonic Hedgehog Signaling Pathway

The Sonic hedgehog (Shh) signaling pathway controls a variety of developmental processes and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Recently, we identified Ulk3 as an active kinase able to positively regulate Gli proteins, mediators of the Shh signaling in mammals. Here, we provide several lines of evidence that Ulk3 participates in the transduction of the Shh signal also independently of its kinase activity. We demonstrate that Ulk3 through its kinase domain interacts with Suppressor of Fused (Sufu), a protein required for negative regulation of Gli proteins. Sufu blocks Ulk3 autophosphorylation and abolishes its ability to phosphorylate and positively regulate Gli proteins. We show that Shh signaling destabilizes the Sufu-Ulk3 complex and induces the release of Ulk3. We demonstrate that the Sufu-Ulk3 complex, when co-expressed with Gli2, promotes generation of the Gli2 repressor form, and that reduction of the Ulk3 mRNA level in Shh-responsive cells results in higher potency of the cells to transmit the Shh signal. Our data suggests a dual function of Ulk3 in the Shh signal transduction pathway and propose an additional way of regulating Gli proteins by Sufu, through binding to and suppression of Ulk3.     

Quantifying an overlooked aspect of partial migration using otolith microchemistry

For the first time, an overlooked aspect of partial migration was quantified using otolith microchemistry and brown trout, Salmo trutta, as a model species. Relative contributions of freshwater resident and anadromous female brown trout to mixed-stock sea trout populations in the Baltic Sea were estimated. Out of 236 confirmed wild sea trout sampled around the coast of Estonia 88% were of anadromous maternal origin and 12% were of resident maternal origin. This novel finding underscores the importance of the resident contingent in maintaining the persistence and resilience of the migratory contingent.     

Sequence of the gene (pheA) encoding phenol monooxygenase from Pseudomonas sp. EST1001: expression in Escherichia coli and Pseudomonas putida.

The plasmid pEST1412 contains the genes, pheA and pheB, encoding phenol monooxygenase (PMO) and catechol 1,2-dioxygenase (C12]), respectively. Thse were originally cloned from the plasmid DNA of Pseudomonas sp. EST1001 [Kivisaar et al., Plasmid 24 (1990) 25-36]. Although pheA and pheB are cotranscribed using the promoter sequences derived from Tn4652 and the level of expression of C120 activities from pEST1412 was equal both in Escherichia coli and in Pseudomonas putida, the level of PMO activity measured in the cell-free extracts of E. coli was lower than that in P. putida. The nucleotide sequence of the 2.0-kb PstI-HindIII fragment of pEST1412 carrying pheA was determined. A 1821-bp ORF was found in this DNA. The structural gene (tfdB) encoding 2,4-dichlorophenol hydroxylase from pJP4 has been sequenced [Perkins et al., J. Bacteriol. 172 (1990) 2351-2359]. Comparison of the deduced amino acid sequences of tfdB and pheA revealed highly conserved regions in the protein products of these genes.