Multicopy single-stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli

Retrons are genetic elements that encode multicopy single-stranded DNAs called msONAs. They are clonally distributed in Escherichia coli and retrons in different clones produce DNAs with different nucleotide sequences. msDNAs consist of an RNA molecule covalently linked to a single-stranded DNA molecule. The latter contains an inverted repeat, resulting in a stem-loop structure. In two retrons, Ec83 and Ec78, the DNA is cleaved off from the RNA. All known retrons except Ec78, have one or more mismatched base pairs in the stem-loop structure. We found that two retrons, Ec86 and Ec83, when present in high copy numbers are mutagenic. The ratios of mutation frequencies observed in Lac indicator strains were similar to the ratios observed for a mutant defective in mismatch repair. It is known that some proteins required for mismatch repair bind to mismatched base pairs prior to carrying out repair. The similarity in the mutation frequency ratios suggested that the mutagenesis caused by msDNAs of retrons Ec86 and Ec83 might be due to seqestration of a mismatch repair protein by msDNA. Strong support for this interpretation was obtained from the finding that the msDNA produced by retron Ec78 is not mutagenic.     

Multicopy single-stranded DNA of Escherichia coli enhances mutation and recombination frequencies by titrating MutS protein

Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. In Escherichia coli , such molecules are encoded by genetic elements called retrons. The DNA moieties of msDNAs have characteristic stem-loop structures, and most of these structures contain mismatched base pairs. Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids. In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E. coli . To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs. We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination. We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein.     

Bird community energetics in a boreal coniferous forest

The annual minimum energy consumption of the bird community was 2524 × 10³ kcal km⁻², of which 44% was consumed by wintering species and 73% by passerines. The daily energy consumption was in summer 14–16 × 10³ and in winter 1–2 × 10³ kcal km⁻². In spruce forests and in afforested swamps birds required approximately 0.12% of the net primary production. Their total annual energy consumption was covered by invertebrates (59%), vertebrates (2%) and vegetable matter (39%); the food derived from the ground (55%), from trees (44%) and from the air (1%). Arboreal insectivorous passerines, ground passerines and gallinaceous birds were the most important ecological guilds. Among passerines existence metabolism accounted for 73% of the annual energy consumption, extra activity for 17%, breeding activity for 1%, moult for 4% and nestlings for 4%.     

Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon

Abstract A plasmid-based food-grade vector system was developed for Lactococcus lactis by exploiting the genes for lactose metabolism. L. lactis MGS267 is a plasmid-free strain containing the entire lactose operon as a chromosomal insertion. The lacF gene was deleted from this strain by a double cross-over homologous recombination event. The lacF -deficient strain produced a Lac⁻ phenotype on indicator agar. A cloned copy of the lacF gene expressed on a plasmid was capable of complementing the lacF -deficient strain resulting in a Lac⁺ phenotype. This stably maintained system fits the requirements of a self-selecting vector system and has the potential to be exploited in the food industry.     

Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon, Salmo salar

Blood samples from healthy adult Atlantic salmon fed an optimal diet in net sea pens were collected at intervals from October to May. Haematological determinations and biochemical serum analyses were carried out on 20 fish in each of seven samples. The ranges of haemato-logical values for sample means were: haematocrit 44–49%, haemoglobin 8.9–10.4 g 100ml⁻¹, red blood cell count 0.85–1.10 × 10¹² l⁻¹, MCV 441–553 × 10⁻¹⁵ 1, MCH 94–106 × 10⁻⁶ g, MCHC 19.4–21.7 g 100ml⁻¹ and leucocrit 0.43–0.96%. The ranges of enzyme activities in serum, for sample means, were: alkaline phosphatase 647–988Ul⁻¹, aspartate aminotrans-ferase 202–351 Ul⁻¹ and alanine aminotransferase 4–8 Ul⁻¹. The ranges of the other parameters analyzed in serum were: total protein 41.6–56.6 gl⁻¹, albumin 18.3–24.3 gl⁻¹, albumin/total protein ratio 39.3–44.0%, creatinine 26–46 μmol, triglycerides 2.53–4.98 μmol and cholesterol 9.3–12.8 μmol. These values are considered to be the normal ranges in healthy fish. Variations due to seasonal changes, and the clinical significance of the selected parameters, are discussed. Data showing the reproducibility of the biochemical analyses in serum are presented.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Key Residues Defining the μ-Opioid Receptor Binding Pocket: A Site-Directed Mutagenesis Study

Abstract: Structural elements of the rat μ-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn¹⁵⁰ to Ala, His²⁹⁷ to Ala, and Tyr³²⁶ to Phe) and two designed to test for μ/δ selectivity (Ile¹⁹⁸ to Val and Val²⁰² to Ile). Mutation of His²⁹⁷ in transmembrane domain 6 (TM6) resulted in no detectable binding with [³H]DAMGO (³H-labeled d -Ala², N -Me-Phe⁴,Gly-ol⁵-enkephalin), [³H]bremazocine, or [³H]ethylketocyclazocine. Mutation of Asn¹⁵⁰ in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β-endorphin_1–31, JOM-13, deltorphin II, dynorphin_1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr³²⁶ mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val²⁰² to Ile in TM4 produced no change on ligand affinity, but Ile¹⁹⁸ to Val resulted in a four- to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.     

The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein

MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell division site of Escherichia coli . We have examined the self-association properties of MinE by analytical ultracentrifugation and by studies of hetero-oligomer formation in non-denaturing polyacrylamide gels. The self-association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C-terminal MinE^22–88 fragment forms hetero-oligomers with MinE⁺ when the proteins are co-expressed. In contrast, the MinE^36–88 fragment does not form MinE⁺/MinE^36–88 hetero-oligomers, although MinE^36–88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co-expressed with MinE⁺ in wild-type cells. Therefore, hetero-oligomer formation is not necessary for the induction of minicelling by expression of MinE^36–88 in wild-type cells. The interference with normal septal placement is ascribed to competition between MinE^36–88 and the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.     

Estimation of the starvation losses of nitrogen and energy in the rainbow trout (Oncorhynchus mykiss) with special regard to protein and energy maintenance requirements

Literature data are analysed regarding losses of body substances occurring during a period of food deprivation in rainbow trout ( Oncorhynchus mykiss ). Nitrogen (protein) and energy losses show a distinct dependence on fish mass (FM [g]) and water temperature (T [°C]). Several regression models for this relationship were compared with best testing estimates as follows:
Nitrogen loss [mg N 2 fish⁻¹ 2 d⁻¹] = 0.0658 e^(1.037) 2 FM^0.739
( n = 49, 9–20°C, 5–400g fish mass, P < 0.001, B = 0.826)
Nitrogen-corrected energy loss [J 2 fish⁻¹ 2 d⁻¹] = 22.09 e^(1.034) 2 FM^0.833
( n = 63, 9–25°C, 8–400 g fish mass, P < 0.001, B = 0.887).
For nitrogen loss as well as for nitrogen-corrected energy loss, the metabolic rate shows a progressive increase with rising water temperature. The temperature coefficient increases in magnitude as temperature increases. The introduction of a general common exponent (0.8 instead of 0.739 for nitrogen loss and 0.833 for energy loss) for fish mass decreases the precision of the estimate. The equations could serve as a base for estimating net protein and net energy maintenance requirements of rainbow trout. Possible limitations, caused by uncertainities in estimating the elevated metabolic rate by food intake and ingestion, are discussed.     

Mutations that affect production of branched RNA-linked msDNA in Myxococcus xanthus.

A deletion mutation of the gene (msd-msr) for the branched RNA-linked msDNA of Myxococcus xanthus was constructed by replacing the chromosomal 0.7-kilobase (kb) SmaI-XhoI fragment encompassing msd-msr with a 1.4-kb fragment carrying a gene for kanamycin resistance. It was found that this deletion strain (delta msSX) could not produce msDNA, although it still contained another species of msDNA, mrDNA (msDNA, reduced size). No apparent differences between delta msSX and the wild-type strain were observed in terms of cell growth, morphogenesis, fruiting-body formation, or motility. Both a deletion mutation at the region 100 base pairs upstream of msd and an insertion mutation at a site 500 base pairs upstream of msd showed a significant reduction of msDNA production, indicating that there is a cis- or trans-acting positive element in this region. When the 3.5-kb BamHI fragment carrying msd-msr from Stigmatella aurantiaca was inserted into the M. xanthus chromosome, the S. aurantiaca msDNA was found to be produced in M. xanthus.     

Comparison of epilithic and epixylic biofilm development in a boreal river

SUMMARY. 1. We assessed substratum effects on lotic biofilm development by placing glass and white pine sampling units in a fourth-order boreal river, and analysing, at 6-week intervals, upper-surface biofilms for ATP, chlorophyll, ergosterol, and the activities of nine exoenzymes.
2. All parameters, except chlorophyll standing stock (range 80–320 μg dm⁻²) and β-xylosidase activity (range 0.4–4.8 μmol h⁻¹ dm⁻²), were significantly greater for epixylic biofilms than for epilithic ones, but the magnitude of the increases varied from 2 to 5 fold, showing that, even under similar hydrodynamic conditions, epilithic and epixylic biofilms are structurally and functionally distinct. For example, ergosterol concentrations ranged from undetectable to 0.93 μg dm⁻² for epilithon and from 11–49 μg dm⁻² for epixylon; corresponding ranges for ATP were 1.6–3.7 (epilithon) and 4.2–7.7 μg dm⁻² (epixylon), for acid phosphatase activity: 2.3–4.9 and 20–41 μmolh⁻¹dm⁻², and for alkaline phosphatase activity: 1.9–8.1 and 29–150 μmol h⁻¹dm⁻², respectively.
3. The more extensive epixylic development was attributed to utilization of the wood substratum as a supplemental carbon source and to a higher density of microbial attachment sites.     

Electrical signalling and cytokinins mediate effects of light and root cutting on ion uptake in intact plants

Nutrient acquisition in the mature root zone is under systemic control by the shoot and the root tip. In maize, exposure of the shoot to light induces short-term (within 1–2 min) effects on net K⁺ and H⁺ transport at the root surface. H⁺ efflux decreased (from −18 to −12 nmol m⁻² s⁻¹) and K⁺ uptake (∼2 nmol m⁻² s⁻¹) reverted to efflux (∼−3 nmol m⁻² s⁻¹). Xylem probing revealed that the trans-root (electrical) potential drop between xylem vessels and an external electrode responded within seconds to a stepwise increase in light intensity; xylem pressure started to decrease after a ∼3 min delay, favouring electrical as opposed to hydraulic signalling. Cutting of maize and barley roots at the base reduced H⁺ efflux and stopped K⁺ influx in low-salt medium; xylem pressure rapidly increased to atmospheric levels. With 100 m m NaCl added to the bath, the pressure jump upon cutting was more dramatic, but fluxes remained unaffected, providing further evidence against hydraulic regulation of ion uptake. Following excision of the apical part of barley roots, influx changed to large efflux (−50 nmol m⁻² s⁻¹). Kinetin (2–4  µ m ), a synthetic cytokinin, reversed this effect. Regulation of ion transport by root-tip-synthesized cytokinins is discussed.     

Histone HMf from the hyperthermophilic archaeon Methanothermus fervidus binds to DNA in vitro using physiological conditions

Abstract The concentration of HMf (histone Methanothermus fervidus ) in vivo has been shown to be between 1 and 2 × 10⁴ molecules per genome. At this mass ratio the amount of HMf bound to pUC19 DNA in vitro was found to be dependent on the topology of the plasmid DNA. M. fervidus grows optimally between 80 and 85°C and contains approx. 1 M K⁺ plus 300 mM 2'3'(cyclic) diphosphoglycerate. (Hensel, R. and König, H. 1988. FEMS Microbiol. Lett. 49, 75–79). HMf binding to DNA in vitro under these conditions has been demonstrated.     

Activity of methanotrophic bacteria in Green Bay sediments

Abstract Sediment pore water samples obtained from a 19 m station in Green Bay in Lake Michigan were examined for levels of ambient dissolved methane and copper, and for the potential for in situ methane oxidation by methanotrophs found within surface sediments. The in situ methane concentration in the upper oxic sediment layer ranged from 20–150 μmol · 1⁻¹ at this station. The activity of methanotrophs and the kinetics of methane oxidation in these sediments were demonstrated by the uptake of radiolabeled methane. K_s values varied between 4.1–9.6 nmol · cm³ of sediment slurry. High V_max values (12.7–35.2 nmol · cm⁻³ · h⁻¹) suggest a large population of methanotrophs in the sediments. An average methane flux to the oxic sediments of 0.24 mol · m⁻² · year⁻¹ was calculated from the pore water methane gradients. Pore water concentrations of copper in the upper sediment layer ranged from 10–120 nmol · 1⁻¹. Based upon the copper concentration, other measured parameters, and equilibrium conditions defined by WATEQF4, an estimate for dissolved free Cu²⁺ concentration of 5–38 nmol · 1⁻¹ pore water was obtained. Several factors control the rate of methane oxidation, including oxygen, methane, and the bioavailability of free Cu²⁺.     

Bactericidal activity of catechin-copper (II) complexes on Escherichia coli ATCC11775 in the absence of hydrogen peroxide

Washed Escherichia coli ATCC11775 cells were killed by (–)-epigallocatechin (EGC) in the presence of a non- lethal concentration of Cu²⁺ (1 μmol l⁻¹) without additional H₂O₂, but not by (–)-epicatechin (EC). EGC alone (< 0·1 mmol l⁻¹) did not reduce the viability of the cells. The survival curve obtained in the presence of EGC and Cu²⁺ was similar to that obtained in the presence of (–)-adrenaline (EN) and Cu²⁺.     

The effect of beet cyst nematode, Heterodera schachtii, on the yield of sugar beet in organic soils

In ten experiments on commercial sugar-beet crops grown on organic soils in 1984–86, a Genstat programme was used to examine the relationship between the initial population of Heterodera schachtii and sugar-beet root yield using the equation
Y = Ymin + (Ymax - Ymin) Z^pi-T
Fixing T = 200 eggs + juveniles 100 g^-1 soil and Z^T= 0.95, estimated values of Ymax varied from 49.2–67.1 t ha^-1 (129– 155% of the national average root yield for the years in which the experiments were carried out) and estimates of Ymin varied from 14.5–53.9 t ha^-1 (27–94% of Ymax). The estimated average root yield loss caused by the nematode was 6.95 t ha^-1.     

A light intensity threshold for schooling in the Atlantic mackerel, Scomber scombrus

The schooling behaviour of Atlantic mackerel was studied in a large tank at different light intensities in the range 12.6–1.8 × 10⁻¹⁰μEs⁻¹ m⁻². Variable light intensity was produced by accurately controlling the current to a green light-emitting diode (LED) 3 m above the experimental tank. Under high light levels (1.8 × 10⁻⁶μEs⁻¹ m⁻²) mackerel always formed a single school, whereas at lower levels (1.8 × 10⁻⁸μEs⁻¹ m⁻²) they swam as individuals. At light levels down to 1.0 × 10⁻⁶μEs⁻¹ m⁻² the mean nearest neighbour distance in a school remained relatively constant (0.3–0.9 body lengths), and individual mackerel swam along a path which deviated from the position of their nearest neighbours by less than 14°. As light dropped below 1.8 × 10⁻⁷μEs⁻¹ m⁻², both nearest neighbour distance and heading angle between nearest neighbours increased, with mean values of 1–1.8 body lengths and 23–92°, respectively, at 1.8 × 10⁻⁹μEs⁻¹ m⁻². The results are discussed in terms of ambient light conditions in the sea.