Azoarcus sp. CIB,an Anaerobic Biodegrader of Aromatic Compounds Shows an Endophytic Lifestyle

Background

Endophytic bacteria that have plant growth promoting traits are of great interest in green biotechnology. The previous thought that the Azoarcus genus comprises bacteria that fit into one of two major eco-physiological groups, either free-living anaerobic biodegraders of aromatic compounds or obligate endophytes unable to degrade aromatics under anaerobic conditions, is revisited here.

Methodology/Principal Findings

Light, confocal and electron microscopy reveal that Azoarcus sp. CIB, a facultative anaerobe β-proteobacterium able to degrade aromatic hydrocarbons under anoxic conditions, is also able to colonize the intercellular spaces of the rice roots. In addition, the strain CIB displays plant growth promoting traits such nitrogen fixation, uptake of insoluble phosphorus and production of indoleacetic acid. Therefore, this work demonstrates by the first time that a free-living bacterium able to degrade aromatic compounds under aerobic and anoxic conditions can share also an endophytic lifestyle. The phylogenetic analyses based on the 16S rDNA and nifH genes confirmed that obligate endophytes of the Azoarcus genus and facultative endophytes, such as Azoarcus sp. CIB, locate into different evolutionary branches.

Conclusions/Significance

This is the first report of a bacterium, Azoarcus sp. CIB, able to degrade anaerobically a significant number of aromatic compounds, some of them of great environmental concern, and to colonize the rice as a facultative endophyte. Thus, Azoarcus sp. CIB becomes a suitable candidate for a more sustainable agricultural practice and phytoremediation technology.     

Rab8 Binding to Immune Cell-Specific Adaptor LAX Facilitates Formation of trans-Golgi Network-Proximal CTLA-4 Vesicles for Surface Expression

Despite playing a central role in tolerance, little is known regarding the mechanism by which intracellular CTLA-4 is shuttled from the trans-Golgi network to the surfaces of T cells. In this context, Ras-related GTPase Rab8 plays an important role in the intracellular transport, while we have previously shown that CTLA-4 binds to the immune cell adaptor TRIM in T cells. In this study, we demonstrate that CTLA-4 forms a multimeric complex comprised of TRIM and related LAX that in turn binds to GTP bound Rab8 for post-Golgi transport to the cell surface. LAX bound via its N terminus to active GTP-Rab8, as well as the cytoplasmic tail of CTLA-4. TRIM required LAX for binding to Rab8 in a complex. Wild-type LAX or its N terminus (residues 1 to 77) increased CTLA-4 surface expression, whereas small interfering RNAs of Rab8 or LAX or disruption of LAX/Rab8 binding reduced numbers of CTLA-4-containing vesicles and its coreceptor surface expression. LAX also promoted the polarization of CTLA-4 and the reorientation of the microtubule-organizing center to the site of T-cell receptor engagement. Our results identify a novel CTLA-4/TRIM/LAX/Rab8 effector complex in the transport of CTLA-4 to the surfaces of T cells.     

The Avena geo-curvature test

Summary The Avena geo-curvature test is a bioassay for auxin-type growth regulators. Etiolated oat coleoptiles are used and the test is conducted in special perspex trays under diffuse daylight. The coleoptiles, with the primary leaves intact, are arranged in grooves on the trays in 4 series of 6 coleoptiles each, and cut to a size of 10 mm. The test substance is applied on an agar strip covering only the lower halves of the apical cut surfaces of each series. After a 4-hour stay in the dark in moist Petri dishes the coleoptile cylinders are shadowgraphed on a 35-mm photographic film. The curvatures are measured from an enlarged projection (10 times natural size) of the shadowgraphs.The lowest IAA concentration which can be determined is around 30 to 60 g/l, i.e. about 1 to 2 ng IAA. The concentration response curves follow a logarithmic course up to 1,000 g/l. The standard error of the mean in a test series comprising 6 coleoptiles is on an average ± 7%. The simple and quick procedures make it possible for a worker to test 60 to 80 fractions a day.Dedicated to Professor Hans Söding on the occasion of his seventieth birthday. The senior author expresses his special gratitude for having been initiated by Professor Söding in the study of auxins.present address: Dpt. of Biology Princeton Univ. Princeton, N. J. 08540, U.S.A.     

Allogeneic and autogenous transplantations of MSCs in treatment of the physeal bone bridge in rabbits

Background

Serum and high ionic strength solutions constitute important barriers to cationic lipid-mediated intravenous gene transfer. Preparation or incubation of lipoplexes in these media results in alteration of their biophysical properties, generally leading to a decrease in transfection efficiency. Accurate quantification of these changes is of paramount importance for the success of lipoplex-mediated gene transfer in vivo.

Results

In this work, a novel time-resolved fluorescence resonance energy transfer (FRET) methodology was used to monitor lipoplex structural changes in the presence of phosphate-buffered saline solution (PBS) and fetal bovine serum. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA lipoplexes, prepared in high and low ionic strength solutions, are compared in terms of complexation efficiency. Lipoplexes prepared in PBS show lower complexation efficiencies when compared to lipoplexes prepared in low ionic strength buffer followed by addition of PBS. Moreover, when serum is added to the referred formulation no significant effect on the complexation efficiency was observed. In physiological saline solutions and serum, a multilamellar arrangement of the lipoplexes is maintained, with reduced spacing distances between the FRET probes, relative to those in low ionic strength medium.

Conclusion

The time-resolved FRET methodology described in this work allowed us to monitor stability and characterize quantitatively the structural changes (variations in interchromophore spacing distances and complexation efficiencies) undergone by DOTAP/DNA complexes in high ionic strength solutions and in presence of serum, as well as to determine the minimum amount of potentially cytotoxic cationic lipid necessary for complete coverage of DNA. This constitutes essential information regarding thoughtful design of future in vivo applications.     

Alterations in metabolism and gene expression in brain regions during cuprizone-induced demyelination and remyelination

Exposure of mice to the copper chelator, cuprizone, results in CNS demyelination. There is remyelination after removal of the metabolic insult. We present brain regional studies identifying corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6 weeks of exposure. We examined recovery of cerebroside and ability to synthesize cerebroside and cholesterol following removal of the toxicant. The temporal pattern for concentration of myelin basic protein resembled that of cerebroside. We applied Affymetrix GeneChip technology to corpus callosum to identify temporal changes in levels of mRNAs during demyelination and remyelination. Genes coding for myelin structural components were greatly down-regulated during demyelination and up-regulated during remyelination. Genes related to microglia/macrophages appeared in a time-course (peaking at 6 weeks) correlating with phagocytosis of myelin and repair of lesions. mRNAs coding for many cytokines had peak expression at 4 weeks, compatible with intercellular signaling roles. Of interest were other genes with temporal patterns correlating with one of the three above patterns, but of function not obviously related to demyelination/remyelination. The ability to correlate gene expression with known pathophysiological events should help in elucidating further function of such genes as related to demyelination/remyelination.     

Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease

The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram‐positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl‐leucyl‐leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP‐activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP‐dependent protein degradation.     

High-throughput single-nucleotide polymorphism genotyping by fluorescent competitive allele-specific polymerase chain reaction (SNiPTag)

Single nucleotide polymorphisms (SNPs) are becoming widely recognized as the new currency for gene mapping as increasing numbers are discovered. Here we outline a method for their rapid analysis based on an allele-specific polymerase chain reaction (PCR) which employs a competitive approach, whereby both allele-specific primers are present in the same reaction and carry different fluorescent labels. This procedure is simple and amenable to high-throughput genotyping using conventional automated sequencing equipment, and no post-PCR modifications are required. Verification of the procedure was carried out by comparison of results derived by this method with those from restriction enzyme digestion of the ALDH2 exon 12 functional polymorphism (Glu-487-Lys) in 109 individuals. Additionally, we have examined all combinations of nucleotide substitutions and shown them to be differentiated by this method. As proof of concept, several assays were combined and loaded on a single gel lane/capillary to substantially improve throughput. This was made possible by designing the PCR products to be of different lengths and no interference was observed between products differing in size by only six nucleotides. We outline a number of test assays for well-characterized SNPs in human candidate genes for behavioral disorders.     

Safety in numbers: multiple occurrences of highly similar homologs among Azotobacter vinelandii carbohydrate metabolism proteins probably confer adaptive benefits

Background

Gene duplication and horizontal gene transfer are common processes in bacterial and archaeal genomes, and are generally assumed to result in either diversification or loss of the redundant gene copies. However, a recent analysis of the genome of the soil bacterium Azotobacter vinelandii DJ revealed an abundance of highly similar homologs among carbohydrate metabolism genes. In many cases these multiple genes did not appear to be the result of recent duplications, or to function only as a means of stimulating expression by increasing gene dosage, as the homologs were located in varying functional genetic contexts. Based on these initial findings we here report in-depth bioinformatic analyses focusing specifically on highly similar intra-genome homologs, or synologs, among carbohydrate metabolism genes, as well as an analysis of the general occurrence of very similar synologs in prokaryotes.

Results

Approximately 900 bacterial and archaeal genomes were analysed for the occurrence of synologs, both in general and among carbohydrate metabolism genes specifically. This showed that large numbers of highly similar synologs among carbohydrate metabolism genes are very rare in bacterial and archaeal genomes, and that the A. vinelandii DJ genome contains an unusually large amount of such synologs. The majority of these synologs were found to be non-tandemly organized and localized in varying but metabolically relevant genomic contexts. The same observation was made for other genomes harbouring high levels of such synologs. It was also shown that highly similar synologs generally constitute a very small fraction of the protein-coding genes in prokaryotic genomes. The overall synolog fraction of the A. vinelandii DJ genome was well above the data set average, but not nearly as remarkable as the levels observed when only carbohydrate metabolism synologs were considered.

Conclusions

Large numbers of highly similar synologs are rare in bacterial and archaeal genomes, both in general and among carbohydrate metabolism genes. However, A. vinelandii and several other soil bacteria harbour large numbers of highly similar carbohydrate metabolism synologs which seem not to result from recent duplication or transfer events. These genes may confer adaptive benefits with respect to certain lifestyles and environmental factors, most likely due to increased regulatory flexibility and/or increased gene dosage.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-192) contains supplementary material, which is available to authorized users.     

Tolerance of the resting cysts of Colpoda inflata (Ciliophora,Colpodea) and Meseres corlissi (Ciliophora,Spirotrichea) to desiccation and freezing

The survival of ciliate resting cysts, in the presence and absence of soil, was studied under two environmental stresses: desiccation and freezing. Laboratory strains of the common species Colpoda inflata and the rare species Meseres corlissi were used in these experiments, which yielded the following results: 1) Freezing of cysts in soil with a residual moisture level exceeding ～30% was destructive for both species. 2) Survival of Meseres corlissi cysts depended largely on the presence of soil. 3) In the absence of soil, Colpoda inflata cysts had greater tolerance to desiccation and freezing than Meseres corlissi cysts. Possible consequences for the distribution of natural populations are discussed.