IncP-1-beta plasmid pGNB1 isolated from a bacterial community from a wastewater treatment plant mediates decolorization of triphenylmethane dyes

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1beta plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the alpha-proteobacterium Sinorhizobium meliloti and the gamma-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1beta plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.     

Elsinochrome A production by the bindweed biocontrol fungus Stagonospora convolvuli LA39 does not pose a risk to the environment or the consumer of treated crops

Biological control as an alternative to chemical pesticides is of increasing public interest. However, to ensure safe use of biocontrol methods, strategies to assess the possible risks need to be developed. The production of toxic metabolites is an aspect which has so far largely been neglected in the risk assessment and the registration process for biocontrol products. We have evaluated the risks of elsinochrome A (ELA) and leptosphaerodione production by the fungus Stagonospora convolvuli LA39, an effective biocontrol agent used against bindweeds. The toxicity of the two metabolites to bacteria, protozoa, fungi and plants was evaluated in in vitro assays. The most sensitive bacteria and fungi were already affected at 0.01-0.07 microM ELA, whereas plants were far less sensitive. Leptosphaerodione was less toxic than ELA. Subsequently, it was investigated whether ELA is present in the applied biocontrol product or LA39-treated bindweed and crop plants. In plants ELA was never detected and in the biocontrol product the ELA concentration was far too low to have toxic effects even on the most sensitive organisms. We conclude that the production of ELA by biocontrol strain LA39 does not pose a risk to the environment or to the consumer.     

Cold responsive EARLI1 type HyPRPs improve freezing survival of yeast cells and form higher order complexes in plants

Plants have large families of proteins sharing a conserved eight-cysteine-motif (8CM) domain. The biological functions of these proteins are largely unknown. EARLI1 is a cold responsive Arabidopsis gene that encodes a hybrid proline-rich protein (HyPRP) with a three-domain architecture: a putative signal peptide at the N-terminus, a proline-rich domain (PRD) in the middle, and an 8CM domain at the C-terminus. We report here that yeast cells expressing different EARLI1 genes had significantly higher rates of freezing survival than empty-vector transformed controls. Arabidopsis plants with knocked down EARLI1 genes had an increased tendency for freezing-induced cellular damage. EARLI1-GFP fluorescence in transgenic plants and immunoblot analyses using protoplasts suggested cell wall localization for EARLI1 proteins. Immunoblot analyses showed that EARLI1 proteins form higher order complexes in plants, and that the PRD is a soluble and the 8CM an insoluble protein domain. We propose that EARLI1 proteins have a bimodular architecture in which the PRD may interact with the cell wall and the 8CM domain with the plasma membrane to protect the cells during freezing stress.     

K(ATP)-dependent neurotransmitter release in the neuronal network of the rat caudate nucleus

K(ATP) channels can couple the bioenergetic metabolism of the cell to membrane excitability. Here, we show gamma-aminobutyric acid (GABA) mediated inhibition of dopamine outflow from slices of the rat caudate nucleus that is regulated by extracellular glucose via high- and low-affinity K(ATP) channels. During glucose reduction, a biphasic dopamine effect could be observed with first a dopamine increase followed by a decline at low glucose concentrations. Both phases were inhibited by glibenclamide. Pinacidil decreased DA outflow without an effect of glucose reduction implying an overall activation of K(ATP) channels. The first phase with dopamine increase was related to reduced GABAergic activity and could be blocked by bicuculline. Our results may be explained by different types of K(ATP) channels with low affinity of ATP and glibenclamide on inhibitory GABAergic and high-affinity on excitatory DAergic neurons. This led us to suggest a biological principle through which neuronal networks are functioning.     

A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene

Obligatory homologous recombination (HR) is required for chiasma formation and chromosome segregation in meiosis I. Meiotic HR is initiated by DNA double-strand breaks (DSBs), generated by Spo11, a homologue of the archaebacterial topoisomerase subunit Top6A. In Saccharomyces cerevisiae, Rad50, Mre11 and Com1/Sae2 are essential to process an intermediate of the cleavage reaction consisting of Spo11 covalently linked to the 5' termini of DNA. While Rad50 and Mre11 also confer genome stability to vegetative cells and are well conserved in evolution, Com1/Sae2 was believed to be fungal-specific. Here, we identify COM1/SAE2 homologues in all eukaryotic kingdoms. Arabidopsis thaliana Com1/Sae2 mutants are sterile, accumulate AtSPO11-1 during meiotic prophase and fail to form AtRAd51 foci despite the presence of unrepaired DSBs. Furthermore, DNA fragmentation in AtCom1 is suppressed by eliminating AtSPO11-1. In addition, AtCOM1 is specifically required for mitomycin C resistance. Interestingly, we identified CtIP, an essential protein interacting with the DNA repair machinery, as the mammalian homologue of Com1/Sae2, with important implications for the molecular role of CtIP.     

Phylogeographic patterns in Drosophila montana

The Drosophila virilis species group offers valuable opportunities for studying the roles of chromosomal re-arrangements and mating signals in speciation. The 13 species are divided into two subgroups, the montana and virilis 'phylads'. There is greater differentiation among species within the montana phylad in both karyotype and acoustic signals than exists among members of the virilis phylad. Drosophila montana is a divergent species which is included in the montana phylad. Here, we analyse the phylogeography of D. montana to provide a framework for understanding divergence of acoustic signals among populations. We analysed mitochondrial sequences corresponding to the cytochrome oxidase I and cytochrome oxidase II genes, as well as 16 microsatellite loci, from 108 lines of D. montana covering most of the species' range. The species shows a clear genetic differentiation between North American and Scandinavian populations. Microsatellite allele frequencies and mitochondrial DNA haplotypes gave significant FST values between populations from Canada, USA and Finland. A Bayesian analysis of population structure based on the microsatellite frequencies showed four genetically distinct groups, corresponding to these three populations plus a small sample from Japan. A network based on mitochondrial haplotypes showed two Finnish clades of very different shape and variability, and another clade with all sequences from North America and Japan. All D. montana populations showed evidence of demographic expansion but the patterns inferred by coalescent analysis differed between populations. The divergence times between Scandinavian and North American clades were estimated to range from 450,000 to 900,000 years with populations in Canada and the USA possibly representing descendants of different refugial populations. Long-term separation of D. montana populations could have provided the opportunity for differentiation observed in male signal traits, especially carrier frequency of the song, but relaxation of sexual selection during population expansion may have been necessary.     

Sequence analysis of the 181-kb accessory plasmid pSmeSM11b, isolated from a dominant Sinorhizobium meliloti strain identified during a long-term field release experiment

The 181 251 bp accessory plasmid pSmeSM11b of Sinorhizobium meliloti strain SM11, belonging to a dominant indigenous S. meliloti subpopulation identified during a long-term field release experiment, was sequenced. This plasmid has 166 coding sequences (CDSs), 42% of which encode proteins with homology to proteins of known function. Plasmid pSmeSM11b is a member of the repABC replicon family and contains a large gene region coding for a conjugation system similar to that of other self-transmissible plasmids in Rhizobium and Agrobacterium. Another pSmeSM11b gene region, possibly involved in sugar metabolism and polysaccharide catabolism, resembled a region of S. meliloti 1021 megaplasmid pSymB and in the genome of Sinorhizobium medicae WSM419. Another module of plasmid pSmeSM11b encodes proteins similar to those of the nitrogen-fixing actinomycete Frankia CcI3, and which are likely to be involved in the synthesis of a secondary metabolite. Several ORFs of pSmeSM11b were predicted to play a role in nonribosomal peptide synthesis. Plasmid pSmeSM11b has many mobile genetic elements, which contribute to the mosaic composition of the plasmid.     

Biodegradation of chlorobenzene under hypoxic and mixed hypoxic-denitrifying conditions

Pseudomonas veronii strain UFZ B549, Acidovorax facilis strain UFZ B530, and a community of indigenous groundwater bacteria, adapted to oxygen limitation, were cultivated on chlorobenzene and its metabolites 2-chloro-cis,cis-muconate and acetate/succinate under hypoxic and denitrifying conditions. Highly sensitive approaches were used to maintain defined low oxygen partial pressures in an oxygen-re-supplying headspace. With low amounts of oxygen available all cultures converted chlorobenzene, though the pure strains accumulated 3-chlorocatechol and 2-chloro-cis,cis-muconate as intermediates. Under strictly anoxic conditions no chlorobenzene transformation was observed, while 2-chloro-cis,cis-muconate, the fission product of oxidative ring cleavage, was readily degraded by the investigated chlorobenzene-degrading cultures at the expense of nitrate as terminal electron acceptor. Hence, we conclude that oxygen is an obligatory reactant for initial activation of chlorobenzene and fission of the aromatic ring, but it can be partially replaced by nitrate in respiration. The tendency to denitrify in the presence of oxygen during growth on chlorobenzene appeared to depend on the oxygen availability and the efficiency to metabolize chlorobenzene under oxygen limitation, which is largely regulated by the activity of the intradiol ring fission dioxygenase. Permanent cultivation of a groundwater consortium under reduced oxygen levels resulted in enrichment of a community almost exclusively composed of members of the β-Proteobacteria and Bacteroidetes. Thus, it is deduced that these strains can still maintain high activities of oxygen-requiring enzymes that allow for efficient CB transformation under hypoxic conditions.