Surface Attachment of Ammonia-Oxidizing Bacteria in Soil

Abstract Indigenous ammonia-oxidizing bacteria (AOB) in a clay loam soil were extremely difficult to release from soil particles compared to most heterotrophic bacteria; less than 1% of indigenous AOB (estimated as potential ammonia oxidation rate) were extractable by the dispersion-density-gradient centrifugation technique. This is at least 10-fold less than the extractability of heterotrophic bacteria. Urea applications to the same soil induced a 5-fold increase in the potential ammonia oxidation rate, and this resulted in a much higher percentage (8%) extractability of AOB. Thus, the newly grown AOB in the urea-treated soil were less strongly attached to the soil particles. The contrast suggests that the strong attachment of indigenous AOB is a gradual process taking place due to a long residence time (infrequent/slow cell division) compared to heterotrophic organisms. However, the contrast could also reflect differences in species composition of the original AOB community and those growing in response to urea inputs. Specific detection of AOB in extinction dilution cultures was done by PCR and sequencing of the products. Considerable diversity was found within the genus Nitrosospira, but severe problems with the specificity of the primers were observed. Two allegedly AOB specific PCR primers pairs were used: one specific for Nitrosospira (SPIRA) and one which should encompass all AOB within the β-Proteobacteria (GAOB). Only 33% of the cultures that gave PCR products with GAOB also gave products with the SPIRA primer pair, suggesting the presence of AOB other than Nitrosospira. However, the phylogeny based on the sequencing placed all the cultures in various clusters of the Nitrosospira clade, suggesting that the SPIRA primers do not match all members of the Nitrosospira genus. The cultures obtained from the urea-treated soil were different from the others in giving PCR products only with the SPIRA primers and not with the GAOB. Since sequencing also here confirmed the presence of Nitrosospira, these observations suggest that the GAOB primers do not match all AOB species. Received: 15 September 1999; Accepted: 8 November 1999; Online Publication: 28 April 2000     

Insulin analog with additional disulfide bond has increased stability and preserved activity

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα‐Cα distances, solvent exposure, and side‐chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C‐terminus of the B‐chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild‐type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R‐state conformation and thus showing that the R‐state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.     

Temperature sensitivity and substrate quality in soil organic matter decomposition: results of an incubation study with three substrates

Kinetic theory suggests that the temperature sensitivity of decomposition of soil organic matter should increase with increasing recalcitrance. This ‘temperature–quality hypothesis’ was tested in a laboratory experiment. Microcosms with wheat straw, spruce needle litter and mor humus were initially placed at 5, 15 and 25 °C until the same cumulative amount of CO₂ had been respired. Thereafter, microcosms from each single temperature were moved to a final set of incubation temperatures of 5, 15 and 25 °C. Straw decomposed faster than needle litter at 25 and 15 °C, but slower than needle litter at 5 °C, and showed a higher temperature sensitivity (expressed as Q₁₀) than needle litter at low temperatures. When moved to the same temperature, needle litter initially incubated at 5 and 15 °C had significantly higher respiration rates in the final incubation than litters initially placed at 25 °C. Mor humus placed at equal temperatures during the initial and final incubations had higher cumulative respiration during the final incubation than humus experiencing a shift in temperature, both up‐ and downwards. These results indicate that other factors than substrate quality are needed to fully explain the temperature dependence. In agreement with the hypothesis, Q₁₀ was always higher for the temperature step between 5 and 15 °C than between 15 and 25 °C. Also in agreement with the temperature–quality hypothesis, Q₁₀ significantly increased with increasing degree of decomposition in five out of the six constant temperature treatments with needle litter and mor humus. Q₁₀s for substrates moved between temperatures tended to be higher than for substrates remaining at the initial temperature and an upward shift in temperature increased Q₁₀ more than a downward shift. This study largely supports the temperature–quality hypothesis. However, other factors like acclimation and synthesis of recalcitrant compounds can modify the temperature response.     

Pore water phosphorus and iron concentrations in a shallow,eutrophic lake — indications of bacterial regulation

Peak pore water SRP and iron(II) concentrations were found during summer in surface sediments in the shallow and eutrophic L. Finjasjön, Sweden, and the concentrations generally increased with water depth. The SRP variation in surface sediments (0–2 cm) was correlated with temperature (R² = 0.82–0.95) and iron(II) showed a correlation with sedimentary carbon on all sites (R² = 0.42–0.96). In addition, sedimentary Chla, bacterial abundances and production rates in surface sediments (0–2 cm) varied seasonally, with peaks during spring and fall sedimentation. Bacterial production rates were correlated with phosphorus and carbon in the sediment (R² = 0.90–0.95 and R² = 0.31–0.95, respectively), indicating a coupling with algal sedimentation. A general increase in sediment Chla and bacterial abundances towards sediments at greater water depth was found. Further, data from 1988–90 reveal that TP and TFe concentrations in the lake were significantly correlated during summer (R² = 0.81 and 0.76, in the hypolimnion and epilimnion, respectively). The results indicate that the increase in pore water SRP and Fe(II) in surface sediments during summer is regulated by bacterial activity and the input of organic matter. In addition, spatial and temporal variations in pore water composition are mainly influenced by temperature and water depth and the significant correlation between TP and TFe in the water suggests a coupled release from the sediment. These findings support the theory of anoxic microlayer formation at the sediment-water interface.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Summary In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using³⁵S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using³⁵S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Effects of donor plant environment and light during incubation on anther cultures of some spring wheat (Triticum aestivum L.) cultivars

Four different growth environments (field, two phytotron greenhouses and one growth chamber) were compared, using two genotypes of spring wheat, one recalcitrant and one responsive. Field-grown plants gave inferior results. Large improvements could be made by improving the conditions, embryoid frequencies in the two genotypes reaching 77.1% and 183.9% per 100 anthers, respectively. High light intensity during the induction phase strongly suppressed induction in both genotypes, but stimulated regeneration of green plants in the recalcitrant genotype, which had the lowest regeneration ability. Weak, diffuse light did not inhibit induction while the positive effect on regeneration was maintained. Also, another recalcitrant genotype was grown in the field, together with two F₁-hybrids (recalcitrant x recalcitrant and recalcitrant x responsive). Evidence for a three-factor system was obtained.     

Ipsdienol-specific receptor cells in bark beetles: structure-activity relationships of various analogues and of deuterium-labelled ipsdienol

Summary The specificity of receptor cells specialized for the bark beetle pheromone component, ipsdienol, was studied electrophysiologically (extracellular recordings) in species ofIps. Single cells were tested with both ipsdienol enantiomers, their racemate, and racemates of structurally similar compounds. The two cell types, keyed to (+)- and to (–)-ipsdienol, respectively, showed much weaker responses to the analogues, demonstrating the high specialization of the cells. The relationships between the dose-response curves of the two types were similar, favoring the concept of particular membrane receptor types (acceptors) in both cell types.The stimulatory efficacy of the various analogues suggested three key features of ipsdienol: (i) the free hydroxyl group at C-4, (ii) the C-2-C-3 double bond, and (iii) the diene system attached to C-5 which may be in acis-conformation.Substituting the C-4 hydrogen by deuterium did not affect the stimulatory effect of ipsdienol.