Microbial consortia that degrade 2,4-DNT by interspecies metabolism: isolation and characterisation

Two consortia, isolated by selective enrichment from a soil sample of anitroaromatic-contaminated site, degraded 2,4-DNT as their sole nitrogensource without accumulating one or more detectable intermediates. Thoughoriginating from the same sample, the optimised consortia had no commonmembers, indicating that selective enrichment resulted in different end points.Consortium 1 and consortium 2 contained four and six bacterial speciesrespectively, but both had two members that were able to collectivelydegrade 2,4-DNT. Variovorax paradoxus VM685 (consortium 1)and Pseudomonas sp. VM908 (consortium 2) initiate the catabolismof 2,4-DNT by an oxidation step, thereby releasing nitrite and forming4-methyl-5-nitrocatechol (4M5NC). Both strains contained a gene similarto the dntAa gene encoding 2,4-DNT dioxygenase. They subsequentlymetabolised 4M5NC to 2-hydroxy-5-methylquinone (2H5MQ) and nitrite,indicative of DntB or 4M5NC monooxygenase activity. A second consortiummember, Pseudomonas marginalis VM683 (consortium 1) and P.aeruginosa VM903, Sphingomonas sp. VM904, Stenotrophomonasmaltophilia VM905 or P. viridiflava VM907 (consortium 2), was foundto be indispensable for efficient growth of the consortia on 2,4-DNT and forefficient metabolisation of the intermediates 4M5NC and 2H5MQ. Knowledgeabout the interactions in this step of the degradation pathway is rather limited.In addition, both consortia can use 2,4-DNT as sole nitrogen and carbon source.A gene similar to the dntD gene of Burkholderia sp. strain DNT that catalyses ring fission was demonstrated by DNA hybridisation in the secondmember strains. To our knowledge, this is the first time that consortia are shownto be necessary for 2,4-DNT degradation.     

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.     

Effects of Ionic Strength on Bacterial Adhesion and Stability of Flocs in a Wastewater Activated Sludge System

The success of biological wastewater treatment is to a large extent governed by the ability of bacteria to induce floc formation, thereby facilitating the separation of particles from the treated water. We performed studies on the dynamics of floc stability, the desorption of cells from the flocs, and the reflocculation of detached material. The floc stability was affected by the ionic strength of the medium in a way that strongly suggests that the interactions between the floc components can be explained by the theory of Derjaugin, Landau, Verwey, and Overbeek (DLVO theory). At increasing concentrations of electrolytes, the stability of the flocs increased. However, above an ionic strength of about 0.1 the floc stability decreased, and it seems that at this high electrolyte concentration the DLVO theory cannot be applied. The reversibility of the electrostatic double-layer effects was experimentally shown by treating the sludge repeatedly with a low-ionic-strength solution until parts of the flocs detached. When salt was added at this point, flocs re-form, resulting in a dramatic decrease in the turbidity of the supernatant liquid. Both reflocculation and detachment of floc material were seen with calcium as well as with potassium. This finding clearly indicates that the reflocculation and destabilization of flocs were due to changes in double-layer thickness rather than bridging effects of multivalent ions such as calcium. The results indicate that the ionic strength may well be an important factor for the floc stability in wastewater in situ.     

Multifunctional floodplain management and biodiversity effects: a knowledge synthesis for six European countries

Floodplain ecosystems are biodiversity hotspots and supply multiple ecosystem services. At the same time they are often prone to human pressures that increasingly impact their intactness. Multifunctional floodplain management can be defined as a management approach aimed at a balanced supply of multiple ecosystem services that serve the needs of the local residents, but also those of off-site populations that are directly or indirectly impacted by floodplain management and policies. Multifunctional floodplain management has been recently proposed as a key concept to reconcile biodiversity and ecosystem services with the various human pressures and their driving forces. In this paper we present biophysics and management history of floodplains and review recent multifunctional management approaches and evidence for their biodiversity effects for the six European countries Ireland, the Netherlands, Germany, Slovakia, Hungary and the Ukraine. Multifunctional use of floodplains is an increasingly important strategy in some countries, for instance in the Netherlands and Hungary, and management of floodplains goes hand in hand with sustainable economic activities resulting in flood safety and biodiversity conservation. As a result, biodiversity is increasing in some of the areas where multifunctional floodplain management approaches are implemented. We conclude that for efficient use of management resources and ecosystem services, consensual solutions need to be realized and biodiversity needs to be mainstreamed into management activities to maximize ecosystem service provision and potential human benefits. Multifunctionality is more successful where a broad range of stakeholders with diverse expertise and interests are involved in all stages of planning and implementation.     

Microbial sulfate-reducing activities in anoxic sediment from Marine Lake Grevelingen: screening of electron donors and acceptors

Sulfate-reducing bacteria in marine sediments mainly utilize sulfate as a terminal electron acceptor with different organic compounds as electron donors. This study investigated microbial sulfate-reducing activity of coastal sediment from Marine Lake Grevelingen (MLG), the Netherlands using different electron donors and electron acceptors. All four electron donors (ethanol, lactate, acetate and methane) showed sulfate-reducing activity with sulfate as electron acceptor, suggesting the presence of an active sulfate-reducing bacterial population in the sediment, even at dissolved sulfide concentrations exceeding 12 mM. Ethanol showed the highest sulfate reduction rate of 55 µmol g _VSS ^?1 day^?1 compared to lactate (32 µmol g _VSS ^?1 day^?1), acetate (26 µmol g _VSS ^?1 day^?1) and methane (4.7 µmol g _VSS ^?1 day^?1). Sulfide production using thiosulfate and elemental sulfur as electron acceptors and methane as the electron donor was observed, however, mainly by disproportionation rather than by anaerobic oxidation of methane coupled to sulfate reduction. This study showed that the MLG sediment is capable of performing sulfate reduction by using diverse electron donors, including the gaseous and cheap electron donor methane.     

New short cationic antibacterial peptides. Synthesis,biological activity and mechanism of action

We report a theoretical and experimental study on a new series of small-sized antibacterial peptides. Synthesis and bioassays for these peptides are reported here. In addition, we evaluated different physicochemical parameters that modulate antimicrobial activity (charge, secondary structure, amphipathicity, hydrophobicity and polarity). We also performed molecular dynamic simulations to assess the interaction between these peptides and their molecular target (the membrane). Biophysical characterization of the peptides was carried out with different techniques, such as circular dichroism (CD), linear dichroism (LD), infrared spectroscopy (IR), dynamic light scattering (DLS), fluorescence spectroscopy and TEM studies using model systems (liposomes) for mammalian and bacterial membranes. The results of this study allow us to draw important conclusions on three different aspects. Theoretical and experimental results indicate that small-sized peptides have a particular mechanism of action that is different to that of large peptides. These results provide additional support for a previously proposed four-step mechanism of action. The possible pharmacophoric requirement for these small-sized peptides is discussed. Furthermore, our results indicate that a net +4 charge is the adequate for 9 amino acid long peptides to produce antibacterial activity. The information reported here is very important for designing new antibacterial peptides with these structural characteristics.     

Activation of Delta F508 CFTR in an epithelial monolayer

The F508 mutation leads to retention of cystic fibrosistransmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems.In stably transfected LLC-PK₁(porcine kidney) epithelial cells, F508 CFTR conforms to thisparadigm and is not present at the plasma membrane. WhenLLC-PK₁ cells or human nasal polyp cells derived from a F508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2%for 4 days) or when LLC-PK₁ cellsare grown as polarized monolayers on permeable supports, plasmamembrane F508 CFTR is significantly increased. Moreover, whenconfluent LLC-PK₁ cells expressingF508 CFTR were treated with DMSO and mounted in an Ussing chamber, afurther increase in cAMP-activated short-circuit current (i.e., ~7µA/cm²;P < 0.00025 compared with untreatedcontrols) was observed. No plasma membrane CFTR was detected after DMSOtreatment in nonepithelial cells (mouse L cells) expressing F508CFTR. The experiments describe a way to augment F508 CFTR maturationin epithelial cells that appears to act through a novel mechanism andallows insertion of functional F508 CFTR in the plasma membranes oftransporting cell monolayers. The results raise the possibility thatincreased epithelial differentiation might increase the delivery ofF508 CFTR from the endoplasmic reticulum to the Golgi, where theF508 protein is shielded from degradative pathways such as theproteasome and allowed to mature.