Microbial Response to Experimentally Controlled Redox Transitions at the Sediment Water Interface

The sediment–water interface of freshwater lakes is characterized by sharp chemical gradients, shaped by the interplay between physical, chemical and microbial processes. As dissolved oxygen is depleted in the uppermost sediment, the availability of alternative electron acceptors, e.g. nitrate and sulfate, becomes the limiting factor. We performed a time series experiment in a mesocosm to simulate the transition from aerobic to anaerobic conditions at the sediment–water interface. Our goal was to identify changes in the microbial activity due to redox transitions induced by successive depletion of available electron acceptors. Monitoring critical hydrochemical parameters in the overlying water in conjunction with a new sampling strategy for sediment bacteria enabled us to correlate redox changes in the water to shifts in the active microbial community and the expression of functional genes representing specific redox-dependent microbial processes. Our results show that during several transitions from oxic-heterotrophic condition to sulfate-reducing condition, nitrate-availability and the on-set of sulfate reduction strongly affected the corresponding functional gene expression. There was evidence of anaerobic methane oxidation with NO_x. DGGE analysis revealed redox-related changes in microbial activity and expression of functional genes involved in sulfate and nitrite reduction, whereas methanogenesis and methanotrophy showed only minor changes during redox transitions. The combination of high-frequency chemical measurements and molecular methods provide new insights into the temporal dynamics of the interplay between microbial activity and specific redox transitions at the sediment–water interface.     

Efficient Transduction by an Amphotropic Retrovirus Vector Is Dependent on High-Level Expression of the Cell Surface Virus Receptor

Transduction by murine leukemia virus-based retrovirus vectors is limited in certain cell types, particularly in nondividing cells. But transduction can be inefficient even in cells that divide rapidly. For example, exposure of 208F rat embryo fibroblasts to an excess of an amphotropic retrovirus vector encoding alkaline phosphatase results in a transduction efficiency of only about 10%, even though these cells divide rapidly. Here we show that transduction of 208F cells is limited by cell surface retrovirus receptor levels; overexpression of the amphotropic retrovirus receptor Pit2 markedly improved the transduction efficiency to 50%. To characterize receptor levels and binding affinity, we synthesized a fusion protein that joins the amino terminus of the amphotropic envelope protein to the Fc region of a human immunoglobulin G1 molecule for use in binding assays. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These results provide the rationale for further examination of the role of receptor levels in inefficient transduction, especially with regard to target cells for gene therapy, where a high transduction rate is often crucial.     

The protein-import apparatus of plant mitochondria

The import and assembly of mitochondrial proteins synthesized in the cytosol is mediated by the protein-import apparatus which plays a central role in mitochondrial biogenesis. Ten years ago only some components of the protein-import apparatus from fungi and mammals were characterized, but today its major components have been analyzed at the molecular level also in plants. Interestingly there are specific features which distinguish the protein-import apparatus of plants from that of fungi and mammals. Here we give an overview of all known components of the protein-import apparatus from plants and focus on its differences in comparison to heterotrophic eukaryotes. Received: 29 March 1999 / Accepted: 14 May 1999     

Metabotropic glutamate receptor expression in olfactory receptor neurons from the channel catfish,Ictalurus punctatus

Metabotropic glutamate receptors (mGluRs) were identified in olfactory receptor neurons of the channel catfish, Ictalurus punctatus, by polymerase chain reaction. DNA sequence analysis confirmed the presence of two subtypes, mGluR1 and mGluR3, that were coexpressed with each other and with the putative odorant receptors within single olfactory receptor neurons. Immunocytochemical data showed that both mGluR subtypes were expressed in the apical dendrites and some cilia of olfactory neurons. Pharmacological analysis showed that antagonists to each mGluR subtype significantly decreased the electrophysiological response to odorant amino acids. α-Methyl-L -CCG1/(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a known antagonist to mGluR3, and (S)-4-carboxyphenylglycine (S-4CPG), a specific antagonist to mGluR1, each significantly reduced olfactory receptor responses to L -glutamate. S-4CPG and MCCG reduced the glutamate response to 54% and 56% of control, respectively, which was significantly greater than their effect on a neutral amino acid odorant, methionine. These significant reductions of odorant response by the antagonists, taken with the expression of these receptors throughout the dendritic and ciliated portions of some olfactory receptor neurons, suggest that these mGluRs may be involved in olfactory reception and signal transduction. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 94–104, 1998     

Functional strain redundancy and persistent phage infection in Swiss hard cheese starter cultures

Undefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics.Subject terms: Microbial ecology, Applied microbiology     

Cloning and expression of the Campylobacter jejuni lon gene detected by RNA arbitrarily primed PCR

Fingerprinting of RNA by arbitrarily primed PCR was used to identify a heat-inducible gene in Campylobacter jejuni. Comparing RNA fingerprints from C. jejuni cells before and after 20 min of heat shock at 48°C, a differentially amplified PCR product was identified which displayed a high degree of homology to bacterial lon genes. By screening C. jejuni genomic libraries, the entire lon gene was cloned and sequenced. It encodes a protein of 791 amino acids with a calculated molecular mass of 90.2 kDa. Alignment of the Lon amino acid sequence with that of other bacterial species revealed an overall identity of up to 56.6% (Helicobacter pylori). Northern and RNA dot blot experiments confirmed heat induction of the C. jejuni lon gene, revealing a maximum 6–8-fold increase in the level of specific mRNA.     

β-Lactamase export across the outer membrane of Acinetobacter calcoaceticus:perturbation of the outer membrane lipopolysaccharide leaflet by enzyme overproduction

Acinetobacter calcoaceticus is able to produce a β-lactamase which was found in the periplasm and to be released into the extracellular culture medium. β-Lactamase export was dependent on enzyme over-production in a cooperative manner. Furthermore, it was accompanied by a steadily increasing release of lipopolysaccharide, an outer membrane constituent, and by an increase in the susceptibility to hydrophobic antibiotics. The data point towards a self-promoted perturbation of the outer membrane by overproduction of the enzyme, leading to a semi-selective increase in membrane permeability.     

Evidence for a specific mechanism of laminin assembly

The specificity of laminin chain assembly was investigated using fragments E8 and C8-9, derived from the long arm of the molecule, whose rod-like domain consists of the alpha-helical regions of the A, B1 and B2 chains. Urea-induced chain separation and unfolding were monitored by transverse urea/polyacrylamide gel electrophoresis (PAGE) and circular dichroism. Separation of the A and disulphide-linked B1-B2 chains occurred at 3.5-4.0 M urea and by 7.0 M urea all residual alpha-helicity was lost. Removal of urea by dialysis resulted in high recoveries (87-100%) of renatured protein which in its apparent molecular mass, alpha-helix content, chain composition, degree of association and ultrastructural appearance was indistinguishable from native E8. Reduction or reduction and alkylation of the chains did not lead to a decrease in their ability to reassemble specifically. Reformation of the single interchain disulphide, linking the B1 and B2 chains, clearly demonstrates that these chains are correctly aligned in parallel and in register in E8 renatured from its reduced chains.Renaturation of E8 from its reduced and alkylated chains precludes a role for disulphide formation in determining chain alignment but suggests rather than it is involved in the stabilisation of the correctly assembled molecule. These results, together with recent sequence data, provide evidence for the interaction of the alpha-helical regions of the A, B1 and B2 chains in the formation of a triple coiled-coil within the long arm of the molecule. The highly specific nature of this interaction suggests that it is the mechanism by which laminin is assembled in vivo.