Bacteriophage Mu-1: A tool to transpose and to localize bacterial genes

In a previous publication (Faelen et al., 1975), it was predicted that the temperate phage Mu-1 would mediate transposition of bacterial genes. Here we show that this is indeed the case. By mating either induced F′ strains (which carry a thermoinducible Mu prophage in the bacterial chromosome), or sensitive F′ infected with Mu, with appropriate recipients, we were able to isolate new F′ episomes which carry various lengths of bacterial DNA. The frequency of transposition of a given marker can be as high as 10^?4. The episomes which carry the transposed DNA always carry Mu as well. When this is coupled with the fact that induction or infection with Mu is necessary for transposition to occur, it is probable that both Mu enzymes and Mu DNA are required by the transposition process. Episomes selected for the presence of a given marker were analyzed for the presence of unselected markers. It was found that: (1) only markers linked to the selected marker can be cotransposed with it; (2) when two markers are simultaneously transposed, all markers lying between them on the chromosome are also transposed; (3) the frequency at which an unselected marker is cotransposed is in some way related to the distance between that marker and the selected marker; (4) the transposition process occurs in both Rec⁺ and Rec^? strains. Mu-mediated transposition offers a new way to isolate F′ episomes and to localize and order bacterial genes as far apart as three minutes.     

Chromosome mobilization and integration of F-factors in the chromosome of RecA strains of E. coli under the influence of bacteriophage Mu-1

Summary Transfer of chromosome promoted by an F⁺ or an F needs a recombinational event between episome and chromosome and is therefore very low in RecA strains (Wilkins, 1969).When F⁺ or F RecA cells are mated with F^- cells and simultaneously infected with bacteriophage Mu-1 the transfer of chromosome is greatly stimulated and seems to take place on any site of the chromosome even when homology is present between the F and the chromosome.A spot-test based on Mu-1 promoted chromosome-mobilization was developed to search for mutants of Mu-1 that had lost the ability to promote chromosome-mobilization. Two conditional lethal amber mutants falling in different complementation groups were found to have lost this property.The integration of an Ftslac ⁺ in the chromosome of a RecA strain is also strongly stimulated by Mu-1 and not site-specific. The resulting Hfr's are very stable and of the clockwise and counterclockwise types. Some of the integrated F-primes are sterile and not able to transfer chromosome or do not form F-pilli.Also the Mu-1 stimulated integration of an Fts(nadA-chlA)⁺ in the chromosome of a RecA strain which has a deletion on the chromosome from nadA-chlA was studied. It was found that when the F is integrated under the influence of Mu-1 also episomal genes carried by that F can be inactivated.     

Vegetative recombination of bacteriophage Mu-1 in Escherichia coli

Summary Twenty-two amber mutants of the thermoinducible mutator phage Mu-c4ts were isolated. These mutants fall into 11 complementation groups. The data obtained by crossing these amber mutants suggest that bacteriophage Mu-1 has a linear vegetative linkage map. In a recombination deficient host of the RecA type the recombination frequencies are extremely low, indicating that Mu-1, in contrast to many other E. coli phages, is dependent on the recombination system of its host. With as a helper phage, recombination between Mu phages in a RecA host is restored to about 1/3 of the frequency in a Rec⁺ host. Although Mu-1 is able to integrate efficiently into the chromosome of a RecA strain, it seems that its integration system does not contribute to vegetative recombination.The survival of UV-irradiated Mu-1 was measured on different radiation sensitive mutants of E. coli. The survival on a UvrB strain was very low as compared to the wild-type; the survival on a RecA strain was almost the same as on the wild-type.Research Fellow from the Laboratory of Genetics, State University, Leiden, The Netherlands.     

O0 and strong-polar mutations in the gal operon are insertions

Summary Three dg phages carrying strong-polar mutations in the gal operon are denser than the corresponding phages carrying the wildtype gal operon or reversions of the mutations to the Gal ⁺ phenotype. The latter phages have the same density. It is concluded that these strong-polar mutations are insertions of DNA into the gal operon.The amount of inserted DNA is different in the three mutations and is calculated to be 450, 1,080 and 1,800 nucleotide pairs respectively.The strong-polar phenotype is also found in a mutant supplied by A. Taylor which carries a Mu-1 phage integrated into the transferase gene.     

Nitrogen and phosphorus addition differentially enhance seed production of dominant species in a temperate steppe

Previous studies have demonstrated changes in plant growth and reproduction in response to nutrient availability, but responses of plant growth and reproduction to multiple levels of nutrient enrichment remain unclear. In this study, a factorial field experiment was performed with manipulation of nitrogen (N) and phosphorus (P) availability to examine seed production of the dominant species, Stipa krylovii, in response to N and P addition in a temperate steppe. There were three levels of N and P addition in this experiment, including no N addition (0 g N m⁻² year⁻¹), low N addition (10 g N m⁻² year⁻¹), and high N addition (40 g N m⁻² year⁻¹) for N addition treatment, and no P addition (0 g P m⁻² year⁻¹), low P addition (5 g P m⁻² year⁻¹), and high P addition (10 g P m⁻² year⁻¹) for P addition treatment. Low N addition enhanced seed production by 814%, 1371%, and 1321% under ambient, low, and high P addition levels, respectively. High N addition increased seed production by 2136%, 3560%, and 3550% under ambient, low, and high P addition levels, respectively. However, P addition did not affect seed production in the absence of N addition, but enhanced it under N addition. N addition enhanced seed production mainly by increasing the tiller number and inflorescence abundance per plant, whereas P addition stimulated it by decreasing the plant density yet stimulating height of plants and their seed number per inflorescence. Our results indicate seed production is not limited by P availability but rather by N availability in the temperate steppe, whereas seed production will be increased by P addition when N availability is improved. These findings enable a better understanding of plant reproduction dynamics in the temperate steppe under intensified nutrient enrichment and can inform their improved management in the future.     

Head length determination in bacteriophage T4: The role of the core protein P22

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail.Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1¹, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII¹ is present in reduced amounts as compared to wild type. The precursor to B1¹ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved.The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).     

Chromosomal rearrangements by an IS2 insertion in phage Mu-1

We have isolated and characterized a mutant of temperate phage Mu-1 carrying an IS2 insertion in the middle of its β region. This mutant gives rise spontaneously to secondary mutants which have deletions of different sizes adjacent to IS2. One particular derivative however, was found to have acquired an additional insertion sequence adjacent to IS2. This derivative gave rise to tertiary mutants carryinh a deletion next to the tandem insertion. The tandem insertion was located at the same place in the Mu β region as another 2.6 kb insertion independently isolated by Chow et al. (1977) and was found to be homologous to that insertion. The properties of this particular secondary mutant show that Mu phage particles lacking their S end are defective for growth and lysogenisation.     

Litter decomposition in a Cerrado savannah stream is retarded by leaf toughness, low dissolved nutrients and a low density of shredders

1. To assess whether the reported slow breakdown of litter in tropical Cerrado streams is due to local environmental conditions or to the intrinsic leaf characteristics of local plant species, we compared the breakdown of leaves from Protium brasiliense, a riparian species of Cerrado (Brazilian savannah), in a local and a temperate stream. The experiment was carried out at the time of the highest litter fall in the two locations. An additional summer experiment was conducted in the temperate stream to provide for similar temperature conditions. 2. The breakdown rates (k) of P. brasiliense leaves in the tropical Cerrado stream ranged from 0.0001 to 0.0008 day⁻¹ and are among the slowest reported. They were significantly (F = 20.12, P < 0.05) lower than in the temperate stream (0.0046–0.0055). The maximum ergosterol content in decomposing leaves in the tropical Cerrado stream was 106 μg g⁻¹, (1.9% of leaf mass) measured by day 75, which was lower than in the temperate stream where maximum ergosterol content of 522 μg g⁻¹ (9.5% of leaf mass) was achieved by day 30. The ATP content, as an indicator of total microbial biomass, was up to four times higher in the tropical Cerrado than in the temperate stream (194.0 versus 49.4 nmoles g⁻¹). 3. Unlike in the temperate stream, leaves in the tropical Cerrado were not colonised by shredder invertebrates. However, in none of the experiments did leaves exposed (coarse mesh bags) and unexposed (fine mesh bags) to invertebrates differ in breakdown rates (F = 1.15, P > 0.05), indicating that invertebrates were unable to feed on decomposing P. brasiliense leaves. 4. We conclude that the slow breakdown of P. brasiliense leaves in the tropical Cerrado stream was because of the low nutrient content in the water, particularly nitrate (0.05 mgN L⁻¹), which slows down fungal activity and to the low density of invertebrates capable of using these hard leaves as an energy source.     

Stimulation of sulfate reduction rates in Mediterranean fish farm sediments inhabited by the seagrass <Emphasis Type="Italic">Posidonia oceanica</Emphasis>

Sulfate reduction rates and biogeochemical parameters of fish farm sediments across the Mediterranean were investigated in the order to evaluate the potential effects of organic matter inputs on habitat quality for the common seagrass Posidonia oceanica. Four study sites were selected in Spain, Italy, Greece and Cyprus to represent the Mediterranean basin. P. oceanica was found in immediate vicinity of all the farms, which were located at physically exposed sites about 1 km from the shore lines. Organic matter accumulation, sulfate reduction rates and sulfur pools were measured in depth profiles along transects from the farms in both bare and vegetated sediments. Results show that although the organic matter accumulation was minor at the sites (POC < 2.8% DW), the sulfate reduction rates were high, in particular at the largest farm in Italy (up to 212 mmol m⁻² d⁻¹), similar to rates found at shallower, temperate fish farm sites, where higher sedimentation rates can be expected. Sulfate reducing bacteria in these low-organic, carbonate-rich Mediterranean sediments respond strongly to organic matter loadings and cause habitat degradation. Sulfate reduction rates measured in the P. oceanica sediments were among the highest recorded (7.8–42.0 mmol m⁻² d⁻¹) similar to rates found in degrading meadows impacted by organic matter loadings. As sulfate reduction rates were correlated with the sedimentation rates along the transects rather than organic matter pools this suggests mineralization processes were controlled by organic matter loading in fish farm sediments. The vegetated sediments near the net cages were more reduced due to accumulation of sulfides compared to control sites, which is a possible contributing factor to the observed seagrass decline in the farm surroundings. It is recommended that Mediterranean fish farms are placed in areas with rapid dispersal of particulate waste products to minimize organic matter loading of the sediments and thereby preserve habitat quality for benthic fauna and flora.     

Localization by whole-body autoradiography of intact and fragmented radiolabeled antibodies in a metastatic human colonic cancer model

In this report, we have employed macroautoradiography to compare the tumor targeting of ¹²⁵I-labeled anti-carcinoembryonic antigen (CEA) MAb (NP-4) to ¹²⁵I-labeled anti-colon-specific antigen-p (CSAp) MAb (Mu-9) and their labeled F(ab′)₂ and Fab′ fragments, in nude mice each bearing large dorsal human colonic tumor xenografts, and small nodular tumors in the liver and lungs. Using intact MAbs (NP-4 and Mu-9), clearance of background radioactivity was delayed to 3–7 days post-treatment. Treatment with F(ab′)₂ and Fab′ fragments of both NP-4 and Mu-9 MAbs, however, promoted clearance of background ¹²⁵I-radioactivity which was well advanced by 6–24 h and complete by 24–48 h after injection. Localization of ¹²⁵I-radioactivity in large and micrometastatic tumor perimeters was the most characteristic uptake pattern observed for both intact and fragmented MAbs. Qualitative analysis of macroautoradiographic images and quantitative densitometry indicated that the higher tumor-to-blood ratios achieved with labeled F(ab′)₂ and Fab′ fragments at early time points, compared to labeled whole immunoglobulin, appeared to be more a function of rapid plasma clearance, tumor mass, location of xenografts and specific tumor growth patterns than increased tumor penetrance by lower molecular weight univalent and bivalent immune fragments.     

Methane Consumption in Temperate and Subarctic Forest Soils: Rates, Vertical Zonation, and Responses to Water and Nitrogen

Rates of methane consumption were measured in subarctic coniferous and temperate mixed-hardwood forest soils, using static chambers and intact soil cores. Rates at both sites were generally between 1 and 3 mg of CH₄ m^-2 day^-1 and decreased with increasing soil water contents above 20%. Addition of ammonium (1 μmol g of soil^-1) strongly inhibited methane oxidation in the subarctic soils; a lesser inhibition was observed for temperate forest samples. The response to nitrogen additions occurred within a few hours and was probably due to physiological changes in the active methane-consuming populations. Methane consumption in soils from both sites was stratified vertically, with a pronounced subsurface maximum. This maximum was coincident with low levels of both nitrate and ammonium in the mixed-hardwood forest soil.     

Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli.

The prophages of the related temperate bacteriophages P1 and P7, which normally exist as plasmids, suppress Escherichia coli dnaA (ts) mutants by integrating into the host chromosome. The locations of the sites on the prophage used for integrative recombination were identified by restriction nuclease analysis and DNA-DNA hybridization techniques. The integration of P1 and P7 often involves a specific site on the host DNA and a specific site on the phage DNA; the latter is probably the end of the phage genetic map. When this site is utilized, the host Rec⁺ function is not required. In Rec⁺ strains, P1 and P7 may also recombine with homologous regions on the host chromosome; at least one of these regions is an IS1 element. In some integration events, prophage deletions are observed which are often associated with inverted repeat structures on the phage DNA. Thus, P1 and P7 may employ one of several different mechanisms for integration.     

Mutations in domain V of the 23S ribosomal RNA of Bacillus subtilis that inactivate its protein folding property in vitro

The active site of a protein folding reaction is in domain V of the 23S rRNA in the bacterial ribosome and its homologs in other organisms. This domain has long been known as the peptidyl transferase center. Domain V of Bacillus subtilis is split into two segments, the more conserved large peptidyl transferase loop (RNA1) and the rest (RNA2). These two segments together act as a protein folding modulator as well as the complete domain V RNA. A number of site-directed mutations were introduced in RNA1 and RNA2 of B.subtilis, taking clues from reports of these sites being involved in various steps of protein synthesis. For example, sites like G2505, U2506, U2584 and U2585 in Escherichia coli RNA1 region are protected by deacylated tRNA at high Mg²⁺ concentration and A2602 is protected by amino acyl tRNA when the P site remains occupied already. Mutations A2058G and A2059G in the RNA1 region render the ribosome Ery^r in E.coli and Lnc^r in tobacco chloroplast. Sites in P loop G2252 and G2253 in E.coli are protected against modification by the CCA end of the P site bound tRNA. Mutations were introduced in corresponding nucleotides in B.subtilis RNA1 and RNA2 of domain V. The mutants were tested for refolding using unfolded protein binding assays with unfolded carbonic anhydrase. In the protein folding assay, the mutants showed partial to complete loss of this activity. In the filter binding assay for the RNA–refolding protein complex, the mutants showed an extent of protein binding that agreed well with their protein folding activity.     

δ<Superscript>15</Superscript>N constraints on long-term nitrogen balances in temperate forests

Biogeochemical theory emphasizes nitrogen (N) limitation and the many factors that can restrict N accumulation in temperate forests, yet lacks a working model of conditions that can promote naturally high N accumulation. We used a dynamic simulation model of ecosystem N and δ¹⁵N to evaluate which combination of N input and loss pathways could produce a range of high ecosystem N contents characteristic of forests in the Oregon Coast Range. Total ecosystem N at nine study sites ranged from 8,788 to 22,667 kg ha⁻¹ and carbon (C) ranged from 188 to 460 Mg ha⁻¹, with highest values near the coast. Ecosystem δ¹⁵N displayed a curvilinear relationship with ecosystem N content, and largely reflected mineral soil, which accounted for 96–98% of total ecosystem N. Model simulations of ecosystem N balances parameterized with field rates of N leaching required long-term average N inputs that exceed atmospheric deposition and asymbiotic and epiphytic N₂-fixation, and that were consistent with cycles of post-fire N₂-fixation by early-successional red alder. Soil water δ¹⁵NO₃ ⁻ patterns suggested a shift in relative N losses from denitrification to nitrate leaching as N accumulated, and simulations identified nitrate leaching as the primary N loss pathway that constrains maximum N accumulation. Whereas current theory emphasizes constraints on biological N₂-fixation and disturbance-mediated N losses as factors that limit N accumulation in temperate forests, our results suggest that wildfire can foster substantial long-term N accumulation in ecosystems that are colonized by symbiotic N₂-fixing vegetation.     

Spatial variability in macroinvertebrate assemblages along and among neighbouring equatorial glacier‐fed streams

1. During the past two decades, understanding of the structure and function of glacier‐fed stream ecosystems at temperate latitudes has increased substantially. In contrast, information on their tropical counterparts is very limited. We studied three neighbouring glacier‐fed streams in the tropical Andes of Ecuador. Our main goals were (i) to determine overall longitudinal patterns in density, taxon richness and the composition of macroinvertebrate assemblages and driving factors in equatorial glacial streams and (ii) to examine variability among replicate streams in faunal metrics and assemblages, and stream‐specific effects of supposed environmental key factors. 2. We measured four geographical and 17 environmental factors and collected five Surber samples (500 cm²) of macroinvertebrates at each of nine sites, three sites along three streams. The streams were located 1–5 km apart. In each stream, the three sites were placed at comparable distances from the glacier and were grouped as ‘upper’ (50–200 m), ‘middle’ (1.5 km) and ‘lower’ sites (3.5–5.6 km). 3. In total, 2200 individuals (64% chironomids) were collected and 47 taxa (30 dipterans, 18 of these Chironomidae) identified. Density ranged from 176 to 372 ind. m^?2, and the number of taxa ranged from 2 to 6 at the upper sites and 868–3044 ind. m^?2 and 21–27 taxa at the lower sites. Density, number of taxa, rarefied richness and axis‐1 coordinates from a MDS ordination increased logarithmically with distance from the glacier. These faunal metrics were equally related to altitude and glacier per cent of catchment and correlated with maximum conductivity, mean temperature, mean daily maximum temperature and a channel stability index. As expected, the mean difference in distance decay in similarity was higher at the upper (47% km^?1) than at the lower reaches (20% km^?1) of the streams. 4. The number of taxa varied among sites within the upper and middle groups, but not among the lower sites. In contrast, but in accordance with our expectation, assemblage composition did not differ among upper sites but did so at middle and lower sites, following a supposed decrease in environmental harshness along the streams. Relationships between faunal metrics and the four environmental variables mean temperature, the stability index, chlorophyll a and coarse particulate organic matter also varied among the three streams. Generalised linear model analyses revealed that temperature interacted with stream on macroinvertebrate density, while chlorophyll a had a significant effect on the number of taxa in interaction with stream and stability. 5. The basic predictions of the Milner et al. (2001a) , model regarding longitudinal faunal patterns and temperature and stability as main driving factors were met by our three replicate equatorial glacial streams. Qualitative departures from the model were mainly because of zoogeographical differences. We demonstrated that variability in assemblages between comparable sites in closely situated streams was considerable, and the effect of key environmental factors varied among streams and interacted with other factors. Quantifying spatial variation in benthic assemblages may help us foresee possible consequences for biodiversity as a result of glacial retreat.     

Rate of Carbon Sequestration at Two Thicket Restoration Sites in the Eastern Cape, South Africa

Ecosystem carbon storage in intact thicket in the Eastern Cape, South Africa exceeds 20 kg/m², which is an unusually large amount for a semiarid ecosystem. Heavy browsing by goats transforms the thicket into an open savanna and can result in carbon losses greater than 8.5 kg/m². Restoration of thicket using cuttings of the dominant succulent shrub Portulacaria afra could return biodiversity to the transformed landscape, earn carbon credits on international markets, reduce soil erosion, increase wildlife carrying capacity, improve water infiltration and retention, and provide employment to rural communities. Carbon storage in two thicket restoration sites was investigated to determine potential rates of carbon sequestration. At the farm Krompoort, near Kirkwood, 11 kg C/m² was sequestered over 27 years (average rate of 0.42 ± 0.08 kg C m^?2 yr^?1). In the Andries Vosloo Kudu Nature Reserve, near Grahamstown, approximately 2.5 kg C/m² was sequestered over 20 years (0.12 ± 0.03 kg C m^?2 yr^?1). Slower sequestration in the Kudu Reserve was ascribed to browsing by black rhinoceros and other herbivores, a shallower soil and greater stone volumes. Planting density and P. afra genotype appeared to affect sequestration at Krompoort. Closely‐packed P. afra planting may create a positive feedback through increased infiltration of rainwater. The rate of sequestration at Krompoort is comparable to many temperate and tropical forests. Potential earnings through carbon credits are likely to rival forest‐planting schemes, but costs are likely to be less due to the ease of planting cuttings, as opposed to propagating forest saplings.     

Physical control of Eurasian watermilfoil in an oligotrophic lake

The introduction of Eurasian watermilfoil (Myriophyllum spicatum) into oligotrophic waters of high water clarity in temperate zones of North America has produced growth in excess of 6 m depth and yearly biomass approaching 1000 g m^–2 dry weight. From its initial observation in Lake George, New York, USA in 1985, by 1993 milfoil had spread to 106 discrete locations within the lake. A 7-year study of one site having no management showed milfoil to grow expansively, suppressing native plant species from 20 in 1987 to 6 in 1993 with the average number of species m^–2 quadrat declining from 5.5 in 1987 to less than 2 in 1993. Management of milfoil by means of hand harvesting, suction harvesting and benthic barrier has reduced the number of unmanaged sites from 106 in 1993 to 11. One year post-treatment at sites utilizing suction harvesting, showed a greater number of native species at all sites than pretreatment with a substantial reduction in milfoil biomass. At sites where benthic barrier was removed 1–2 years after installation, milfoil had recolonized 44% of grid squares within 30 days. Ninety days after barrier removal 74% of grid squares contained milfoil and one year later 71% of the grids supported milfoil. During the first year following mat removal, the average number of species m^–2 peaked at 4.7 and stabilized at 4.5 during the second year. Hand harvesting by SCUBA in areas of limited milfoil growth (new sites of infestation and sites of former treatment) was found to reduce the number of milfoil plants present in subsequent years. Hand harvesting did not eliminate milfoil at any of the sites and regrowth/colonization necessitated reharvesting every 3 or more years. Results of evaluations of physical plant management techniques indicate that (1) an integrated program utilizing different techniques based on plant density reduced the growth of milfoil and (2) long term commitment to aquatic plant management is necessary since none of the techniques employed singly were found to eliminate milfoil.     

UVB-induced experimental carcinogenesis: dysregulation of apoptosis and p53 signalling pathway

Abstract

UVB irradiation is a major cause of human skin cancer. In addition to inducing neoplastic changes, UVB irradiation also causes immune suppression which may impair local defence reactions allowing abnormal cells to proliferate and develop into skin tumours.¹ The immuno-suppressive events induced by UVB irradiation are mediated by a number of pathways involving altered DNA,² cis-urocanic acid (cis-UCA),³ IL-10,⁴ and mast cells.⁵ UVB-damaged DNA can result in mutations including those of p53, allowing the proliferation of aberrant cells and, ultimately, local tumour invasion. In p53 gene knockout mice, development of skin tumours increases more rapidly after exposure to UVB irradiation.⁶ p53 as a target for UV-induced mutations in developing murine and human skin cancer involves C→T and CC→TT transitions at dipyrimidine sites.⁷ This paper summarises the adaptive responses in mouse skin following acute and chronic UV irradiation.