Comparative genomics of the pIPO2/pSB102 family of environmental plasmids: sequence, evolution, and ecology of pTer331 isolated from Collimonas fungivorans Ter331

Plasmid pTer331 from the bacterium Collimonas fungivorans Ter331 is a new member of the pIPO2/pSB102 family of environmental plasmids. The 40 457-bp sequence of pTer331 codes for 44 putative ORFs, most of which represent genes involved in replication, partitioning and transfer of the plasmid. We confirmed that pTer331 is stably maintained in its native host. Deletion analysis identified a mini-replicon capable of replicating autonomously in Escherichia coli and Pseudomonas putida. Furthermore, plasmid pTer331 was able to mobilize and retromobilize IncQ plasmid pSM1890 at typical rates of 10(-4) and 10(-8), respectively. Analysis of the 91% DNA sequence identity between pTer331 and pIPO2 revealed functional conservation of coding sequences, the deletion of DNA fragments flanked by short direct repeats (DR), and sequence preservation of long DRs. In addition, we experimentally established that pTer331 has no obvious contribution in several of the phenotypes that are characteristic of its host C. fungivorans Ter331, including the ability to efficiently colonize plant roots. Based on our findings, we hypothesize that cryptic plasmids such as pTer331 and pIPO2 might not confer an individual advantage to bacteria, but, due to their broad-host-range and ability to retromobilize, benefit bacterial populations by accelerating the intracommunal dissemination of the mobile gene pool.     

PCR-Denaturing Gradient Gel Electrophoresis Profiling of Inter- and Intraspecies 18S rRNA Gene Sequence Heterogeneity Is an Accurate and Sensitive Method To Assess Species Diversity of Arbuscular Mycorrhizal Fungi of the Genus Gigaspora

Despite the importance of arbuscular mycorrhizal fungi in the majority of terrestrial ecosystems, their ecology, genetics, and evolution are poorly understood, partly due to difficulties associated with detecting and identifying species. We explored the inter- and intraspecies variations of the 18S rRNA genes of the genus Gigaspora to assess the use of this marker for the discrimination of Gigaspora isolates and of Gigasporaceae populations from environmental samples. Screening of 48 Gigaspora isolates by PCR-denaturing gradient gel electrophoresis (DGGE) revealed that the V3-V4 region of the 18S rRNA gene contained insufficient variation to discriminate between different Gigaspora species. In contrast, the patterns of 18S ribosomal DNA (rDNA) heterogeneity within the V9 region of this marker could be used for reliable identification of all recognized species within this genus. PCR-DGGE patterns provided insight into some putative misidentifications and could be used to differentiate geographic isolates of G. albida, G. gigantea, and G. margarita but not G. rosea. Two major clusters were apparent based upon PCR-DGGE ribotype patterns, one containing G. albida, G. candida, G. ramisporophora, and G. rosea and the other containing G. decipiens and G. margarita. Dissection of the DGGE patterns by cloning, DGGE screening, and sequencing confirmed these groupings and revealed that some ribotypes were shared across species boundaries. Of the 48 isolates examined, only two displayed any spore-to-spore variation, and these exceptions may be indicative of coisolation of more than one species or subspecies within these cultures. Two Brazilian agricultural soils were also analyzed with a Gigasporaceae-specific nested PCR approach, revealing a dominance of G. margarita within this family.     

Lysosome mediated Kir2.1 breakdown directly influences inward rectifier current density

The inward rectifier current generated by Kir2.1 ion channel proteins is primarily responsible for the stable resting membrane potential in various excitable cell types, like neurons and myocytes. Tight regulation of Kir2.1 functioning prevents premature action potential formation and ensures optimal repolarization times. While Kir2.1 forward trafficking has been addressed in a number of studies, its degradation pathways are thus far unknown. Using three different lysosomal inhibitors, NH₄Cl, chloroquine and leupeptin, we now demonstrate involvement of the lysosomal degradation pathway in Kir2.1 breakdown. Upon application of the inhibitors, increased steady state protein levels are detectable within few hours coinciding with intracellular granular Kir2.1 accumulation. Treatment for 24 h with either chloroquine or leupeptin results in increased plasmamembrane originating inward rectifier current densities, while current-voltage characteristics remain unaltered. We conclude that the lysosomal degradation pathway contributes to Kir2.1 mediated inward rectifier current regulation.     

cGMP and glutathione-conjugate transport in human erythrocytes.

The nature of cGMP transport in human erythrocytes, its relationship to glutathione conjugate transport, and possible mediation by multidrug resistance-associated proteins (MRPs) have been investigated. MRP1, MRP4 and MRP5 are detected in immunoblotting studies with erythrocytes. MRP1 and MRP5 are also detected in multidrug resistant COR-L23/R and MOR/R cells but at greatly reduced levels in the parent, drug sensitive COR-L23/P cells. MRP4 is detected in MOR/R but not COR-L23/R cells. Uptake of cGMP into inside-out membrane vesicles prepared by a spontaneous, one-step vesiculation process is shown to be by a low affinity system that accounts for more than 80% of the transport at all concentrations above 3 micro m. This transport is reduced by MRP inhibitors and substrates including MK-571, methotrexate, estradiol 17-beta-d-glucuronide, and S(2,4-dinitrophenyl)glutathione (DNP-SG) and also by glibenclamide and frusemide but not by the monoclonal Ig QCRL-3 that inhibits high-affinity transport of DNP-SG by MRP1. It is concluded that the cGMP exporter is distinct from MRP1 and has properties similar to those reported for MRP4. Furthermore the evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes.     

Microbial Community Composition Affects Soil Fungistasis

Most soils inhibit fungal germination and growth to a certain extent, a phenomenon known as soil fungistasis. Previous observations have implicated microorganisms as the causal agents of fungistasis, with their action mediated either by available carbon limitation (nutrient deprivation hypothesis) or production of antifungal compounds (antibiosis hypothesis). To obtain evidence for either of these hypotheses, we measured soil respiration and microbial numbers (as indicators of nutrient stress) and bacterial community composition (as an indicator of potential differences in the composition of antifungal components) during the development of fungistasis. This was done for two fungistatic dune soils in which fungistasis was initially fully or partly relieved by partial sterilization treatment or nutrient addition. Fungistasis development was measured as restriction of the ability of the fungi Chaetomium globosum, Fusarium culmorum, Fusarium oxysporum, and Trichoderma harzianum to colonize soils. Fungistasis did not always reappear after soil treatments despite intense competition for carbon, suggesting that microbial community composition is important in the development of fungistasis. Both microbial community analysis and in vitro antagonism tests indicated that the presence of pseudomonads might be essential for the development of fungistasis. Overall, the results lend support to the antibiosis hypothesis.     

Soil structural aspects of decomposition of organic matter by micro-organisms

Soil architecture is the dominant control over microbially mediated decomposition processes in terrestrial ecosystems. Organic matter is physically protected in soil so that large amounts of well-decomposable compounds can be found in the vicinity of largely starving microbial populations. Among the mechanisms proposed to explain the phenomena of physical protection in soil are adsorption of organics on inorganic clay surfaces and entrapment of materials in aggregates or in places inaccessible to microbes. Indirect evidence for the existence of physical protection in soil is provided by the occurrence of a burst of microbial activity and related increased decomposition rates following disruption of soil structures, either by natural processes such as the remoistening of a dried soil or by human activities such as ploughing. In contrast, soil compaction has only little effect on the transformation of ¹⁴C-glucose. Another mechanism of control by soil structure and texture on decomposition in terrestrial ecosystems is through their impact on microbial turnover processes. The microbial population is not only the main biological agent of decomposition in soil, it is also an important, albeit small, pool through which most of the organic matter in soil passes. Estimates on the relative importance of different mechanisms controlling decomposition in soil could be derived from results of combined tracer and modelling studies. However, suitable methodology to quantify the relation between soil structure and biological processes as a function of different types and conditions of soils is still lacking.     

Die Kohlehydratverdauung bei Astacus fluviatilis

Zusammenfassung Im Flußkrebsmagensaft fanden wir Amylase, Maltase, sehr wenig In vertase und keine Lactase.Das p_H-Optimum der Amylase ist bei p_H 5,6, der Invertase bei p_H 5,7–6,0. Maltase hat kein scharfes Optimum, sondern ein flaches Optimum zwischen p_H 5 und 6.Die Maltase und die Amylase des Saftes haben ungefähr dieselbe Wirkungsgeschwindigkeit.Die Temperatur verschiebt das p_H-Optimum der Amylase nicht oder nur sehr wenig.Aus der Tatsache, daß die Amylase und die Maltase die gleiche Wirkungsgeschwindigkeit haben, ergibt sich, daß dem Flußkrebs die für die Wirbeltiere charakteristische Regulierung der Ernährung fehlt. Das gleiche dürfte nach Untersuchung von Shinoda und Krueger auch für Eiweißverdauung gelten. Eine Spekulation über die Möglichkeit der Überschwemmung des Krebsorganismus mit Zucker können wir uns sparen, da wir noch zu wenig davon wissen. Nur so viel sei gesagt, daß eine Zuckerresorption im Magen nicht stattfindet, und daß die Menge des Zuckers, die in die Mitteldarmdrüse gelangt, in der Zeiteinheit äußerst gering sein muß, aus Gründen, die wir in der Einleitung besprachen. Schon deswegen muß eine Überschwemmung, wie sie bei den Wirbeltieren stattfinden könnte, hier ausgeschlossen sein. Die Frage, inwieweit überhaupt bei diesen. Tieren eine Regulierung der Kohlehydrate des Blutes lebensnotwendig ist, kann nicht beantwortet werden. Sicherlich aber haben nach Henningsen (11) die Tiere keinen festen Zuckerspiegel, wie ihn die Säugetiere haben, sie können ohne jeden Zuckergehalt des Blutes leben. Nach reichlichen Mahlzeiten steigt die Zuckermenge des Blutes allerdings schnell an, bis etwa 0,030%. Eine Regulation gibt es nur insoweit, als nach Einspritzung größerer Zuckermengen in das Blut der Zuckergehalt des Blutes bald sinkt, bis er gleich demjenigen wird, den man beim gefütterten Tiere findet, solange der Vorrat reicht, wird er dann konstant erhalten. Die große Empfindlichkeit des Säugetieres gegen Schwankungen des Zuckergehaltes im Blute dürfte hier also fehlen. Wie in so vielen Fällen, ist also das niedere Tier Schwankungen der Lebensbedingungen ausgesetzt, es fehlt ihm eine Regulation, wie sie für den Zucker beim Säugetier neben der Leber und den bekannten Hormonen auch durch die beschriebene Eigenartigkeit der Verdauung gewährleistet wird. Wir wollen noch darauf hinweisen, daß Jordan und Begemann (12) gezeigt haben, daß durch den Darm der Schnecken (Helix pomatia) Disaccharide ungefähr ebenso leicht diffundieren, als Monosaccharide (Rohrzucker fast ebenso leicht als Traubenzucker).Dem International Educational sind wir zu großem Dank verpflichtet, da es uns in den Stand gesetzt hat, die benötigte Apparatur anzuschaffen.     

A simple method for determining the sensitivity of micro-organisms to serial dilutions of antibiotics

Summary At this laboratory the routine sensitivity tests of micro-organisms against sulfathiazol and antibiotics have been performed in plastic dishes for half a year. The dishes are cleaned and sterilized in the same way as bacteriological glassware. The use of these dishes offers some advantages over other conventional dilution methods employing tubes or Petri dishes with solid media: the sensitivity to serial dilutions of one antibiotic may be measured in one and the same plate, and in this way a considerable amount of glassware and of space in the incubator may be saved; the readings are easily done and distinct. It has been demonstrated that sensitivity tests with two standard strains against sulfathiazol and various antibiotics may be reproduced with the same endpoints.     

Performance of aneuploid backcross hybrids between the crop Brassica napus and its wild relative B. rapa

• Crossings between the diploid wild Brassica rapa (AA , 2n = 20) and the tetraploid cultivar B. napus (AACC , 2n = 38) can readily be made. Backcrosses to the wild B. rapa (BC ₁) produce aneuploids with variable chromosome numbers between 20 and 29. How does survival and performance relate to DNA content of plants?
• Growth of the BC ₁ plants was measured in the lab. One plant in the F₁ self‐pollinated spontaneously and produced abundant F₂ seeds that were also examined. The number of C‐chromosomes was estimated from DNA values obtained with flow cytometry.
• Average DNA value of the BC ₁ was similar to that of the parents, which shows that C‐chromosomes do not reduce success of pollen or embryos. The average DNA value in the F₂ was 13% higher than in the F₁, suggesting that extra C‐chromosomes facilitated gamete success and/or embryo survival. Under both optimal and drought stress conditions growth and survival of BC ₁ hybrids was similar to that of B. rapa . No significant correlations existed between growth or survival and DNA value.
• Aneuploid plants were not inferior under the conditions of the growth room and may persist in nature. We discuss other factors, such as herbivory, that could prevent hybrid establishment in the field.