Dechlorination of Chloral Hydrate by Pseudomonas putida LF54 which Possesses Biofilm Adhesin Protein LapA

Because of the lack of enzymes in critical steps of catabolic pathways, low-molecular-weight halogenated compounds are often recalcitrant to biodegradation. In our previous study, we isolated Pseudomonas sp. LF54 (LF54), the first bacterium that has been shown to use chloral hydrate (CH) as sole carbon source by an assimilation pathway in which dechlorination is the critical step. In this study, we identified a transposon (Tn) mutant that can render LF54 defective in CH dechlorination. The molecular characterization of Tn mutants revealed that the transposon insertion sites map to lapA. Sequence analyses verified the existence of lapA in LF54. Additionally, induced expression of lapA in the conditional lapA mutant of LF54 further verified that defective lapA expression renders LF54 defective in dechlorination. Recent studies have revealed that the largest cell-surface-associated protein LapA, a biofilm adhesin, is able to initiate biofilm formation. This function was also verified in the induced conditional lapA mutant and in LF54. Furthermore, we also found out that the defective lapA mutant rendered the variation of bacterial motility. LapA, the largest biofilm adhesin protein of P. putida, which influences CH dechlorination and flagella motility, is a novel discovery not previously reported

A c-di-GMP Effector System Controls Cell Adhesion by Inside-Out Signaling and Surface Protein Cleavage

In Pseudomonas fluorescens Pf0-1 the availability of inorganic phosphate (Pi) is an environmental signal that controls biofilm formation through a cyclic dimeric GMP (c-di-GMP) signaling pathway. In low Pi conditions, a c-di-GMP phosphodiesterase (PDE) RapA is expressed, depleting cellular c-di-GMP and causing the loss of a critical outer-membrane adhesin LapA from the cell surface. This response involves an inner membrane protein LapD, which binds c-di-GMP in the cytoplasm and exerts a periplasmic output promoting LapA maintenance on the cell surface. Here we report how LapD differentially controls maintenance and release of LapA: c-di-GMP binding to LapD promotes interaction with and inhibition of the periplasmic protease LapG, which targets the N-terminus of LapA. We identify conserved amino acids in LapA required for cleavage by LapG. Mutating these residues in chromosomal lapA inhibits LapG activity in vivo, leading to retention of the adhesin on the cell surface. Mutations with defined effects on LapD's ability to control LapA localization in vivo show concomitant effects on c-di-GMP-dependent LapG inhibition in vitro. To establish the physiological importance of the LapD-LapG effector system, we track cell attachment and LapA protein localization during Pi starvation. Under this condition, the LapA adhesin is released from the surface of cells and biofilms detach from the substratum. This response requires c-di-GMP depletion by RapA, signaling through LapD, and proteolytic cleavage of LapA by LapG. These data, in combination with the companion study by Navarro et al. presenting a structural analysis of LapD's signaling mechanism, give a detailed description of a complete c-di-GMP control circuit—from environmental signal to molecular output. They describe a novel paradigm in bacterial signal transduction: regulation of a periplasmic enzyme by an inner membrane signaling protein that binds a cytoplasmic second messenger

TET2 inhibits differentiation of embryonic stem cells but does not overcome methylation-induced gene silencing

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5′-methylcytosine to 5′-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5′-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5′-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5′-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented

Fis suurendab Pseudomonas putida biofilmi hulka, tõstes lapA ekspressiooni

Väitekirja elektrooniline versioon ei sisalda publikatsiooneBakterid elavad looduses valdavalt biofilmis, pinnale kinnituvate bakterite kogumis. Biofilmis elamisel on bakterite jaoks mitmeid eeliseid. Biofilmis elavad bakterid on kahjulike keskkonnamõjude eest paremini kaitstud ja heades tingimustes aitab pinnale kinnitumine nendesse tingimusesse jäädagi. Biofilm ei ole kasulik mitte ainult bakteritele, vaid paljud biofilmi moodustavad bakterid on vajalikud ka taimedele. Üheks selliseks bakteriks on Pseudomonas putida, mida uurin oma doktoritöös. P. putida moodustab biofilmi taime juurtel ja kaitseb taimi, ka põllumajanduslikult olulisi kultuure, haigustekitajate eest ning soodustab nende kasvu. Käesoleva töö käigus leiti juhuslikult, et P. putida biofilmi mõjutab bakteri keskne regulaatorvalk Fis. Kui bakterid satuvad headesse tingimustesse, siis Fis-i roll on suunata nad kiirelt kasvama ja kui toitained saavad otsa, siis aeglustada kasv jälle maha. Me nägime, et liiga palju Fis-i tootvad bakterid ei suuda ringi liikuda. Sealt tekkis kahtlus, et äkki nad ei liigu sellepärast, et jäävad hoopis paigale ja moodustavad biofilmi. See teooria osutus tõeseks ja püstitas kohe järgmise küsimuse, kuidas Fis suunab bakterid biofilmi moodustama? Me leidsime, et Fis soodustab ühe väga suure kleepvalgu tootmist. See kleepvalk, LapA, on biofilmi tekkeks väga oluline, kuna võimaldab bakteritel nii pinnale kui teineteisega kleepuda. Käesolevas töös näitasime, et Fis seondub kahes kohas lapA geeni ette ja just neid kohti kasutades soodustab LapA tootmist. Lisaks kirjeldasime need kohad (promootorid), kust algab lapA mRNA tootmine. Kui tavaliselt on ühel geenil üks või kaks promootorit, siis lapA geenil on neid lausa kuus. Kokkuvõttes lõi see töö uusi teadmisi P. putida biofilmi regulatsioonist ja aitab seega paremini mõista selle kasuliku mullabakteri elu väga olulist etappi.Tartu Ülikool. Loodus- ja täppisteaduste valdkondBiofilms are the prevailing lifestyle of bacteria in most natural environments. This is because living in a biofilm gives bacteria a number of advantages. When the environment is hazardous, residing in a biofilm protects them; and when conditions are favourable, biofilm is a way to settle down and not be carried away from the good life. Biofilm is not only useful for bacteria but many biofilm-forming bacteria are also beneficial for plants. One of those beneficial bacteria is Pseudomonas putida, the focus of this thesis. P. putida forms biofilm on roots of plants, including agriculturally important crops, protecting them against disease-causing microorganisms and promoting plant growth. During this work, it was discovered that P. putida’s biofilm is regulated by the central regulator protein Fis. Fis is the regulator that starts fast growth in good conditions and when nutrients deplete, decelerates growth. We saw that bacteria producing elevated levels of Fis were unable to move around. This prompted the idea that maybe they are not moving because they are settling down and forming biofilm. This idea proved true and raised the next question, how does Fis lead the bacteria to form biofilm? We found that Fis upregulates the production of a huge adhesion protein. This protein, LapA, is a key factor for biofilm formation as it enables bacteria to stick both to surfaces and to one another. In this work, we show that Fis binds upstream of the lapA gene in two positions and utilizes these sites to boost the production of LapA. We also described the location of the promoter sites where the production of lapA mRNA is initiated. While many genes have one or two promoter, lapA has surprisingly many – six. Altogether, this work shed light onto P. putida biofilm regulation and thus helps us better understand a very important stage in this beneficial soil bacterium’s life

Peritoneal adhesions after laparoscopic gastrointestinal surgery

Although laparoscopy has the potential to reduce peritoneal trauma and post-operative peritoneal adhesion formation, only one randomized controlled trial and a few comparative retrospective clinical studies have addressed this issue. Laparoscopy reduces de novo adhesion formation but has no efficacy in reducing adhesion reformation after adhesiolysis. Moreover, several studies have suggested that the reduction of de novo post-operative adhesions does not seem to have a significant clinical impact. Experimental data in animal models have suggested that CO2 pneumoperitoneum can cause acute peritoneal inflammation during laparoscopy depending on the insufflation pressure and the surgery duration. Broad peritoneal cavity protection by the insufflation of a low-temperature humidified gas mixture of CO2, N2O and O2 seems to represent the best approach for reducing peritoneal inflammation due to pneumoperitoneum. However, these experimental data have not had a significant impact on the modification of laparoscopic instrumentation. In contrast, surgeons should train themselves to perform laparoscopy quickly, and they should complete their learning curves before testing chemical anti-adhesive agents and anti-adhesion barriers. Chemical anti-adhesive agents have the potential to exert broad peritoneal cavity protection against adhesion formation, but when these agents are used alone, the concentrations needed to prevent adhesions are too high and could cause major post-operative side effects. Anti-adhesion barriers have been used mainly in open surgery, but some clinical data from laparoscopic surgeries are already available. Sprays, gels, and fluid barriers are easier to apply in laparoscopic surgery than solid barriers. Results have been encouraging with solid barriers, spray barriers, and gel barriers, but they have been ambiguous with fluid barriers. Moreover, when barriers have been used alone, the maximum protection against adhesion formation has been no greater than 60%. A recent small, randomized clinical trial suggested that the combination of broad peritoneal cavity protection with local application of a barrier could be almost 100% effective in preventing post-operative adhesion formation. Future studies should confirm the efficacy of this global strategy in preventing adhesion formation after laparoscopy by focusing on clinical end points, such as reduced incidences of bowel obstruction and abdominal pain and increased fertility

Management of the Loco (Concholepas concholepas) as a Driver for Self-governance of Small-scale Benthic Fisheries in Chile

WRI led the "Reefs at Risk Revisited" analysis in collaboration with a broad partnership of more than 25 research, conservation, and educational organizations. Partners have provided data, offered guidance on the analytical approach, contributed to the report, and served as critical reviewers of the maps and findings.

The Vulnerable State and Technical Fixes: An Analysis of Official Climate Change Discourses in Nepal

I conduct discourse analysis of seven selected official climate change policies and documents of Nepal. In the first part of my analysis, I draw from international climate justice discourses to analyze how policy makers construct Nepal’s position in the global arena, in relation to the issue of climate change. In the second part, I draw from political ecology and anthropological understandings of ‘vulnerability’ and ‘adaptation’ to analyze how policy makers construct those terms in the context of Nepal. The result shows that Nepal has adhered to the ‘vulnerability’ and ‘transition’ discourses, which serve as important tools to advocate for financial support from the international climate change regime. Driven primarily by international processes and guidelines, the climate change policies and documents in Nepal project a heavily technocratic approach with little socio-cultural considerations. Vulnerability is understood as a static property and assessed based on sectors and geographic areas, while adaptation is understood as series of actions to be implemented. Overall, the policies are at risk of perpetuating the existing systemic ills, as well as impeding imaginaries to pursue more radical socio-political and cultural change as effective adaptation measures

Isotope and Organic Geochemistry of a Unique Proterozoic, Postglacial Succession: The Lapa Formation, Vazante Group, Brazil

This study is the second to investigate biological characteristics associated with Proterozoic glaciation via molecular fossils from organic matter preserved in shale. In the Vazante Group, Minas Gerais, Brazil, the Serra da Lapa Formation unconformably overlies a formation recently dated to ca. 1.13 Ga. Lithologic and isotopic data suggest the Lapa represents deposition immediately after either an early Neoproterozoic "snowball Earth" ice age or a possible regional, but still low-latitude, late Mesoproterozoic ice age. The relative abundances of biomarkers and other organic molecules show variations that match lithologic and isotopic changes observed in 40 meters of exploration drill core studied. Inconsistencies among biomarker abundances as well as differences between the organic matter of the Lapa Formation and that of the underlying formation hint at heterogeneity among and within formations. A more complete picture of the Vazante Group is warranted before characteristics of preserved organic matter can be interpreted in the context of depositional environments or postdepositional processes