Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (or) [ and ]. Inactivation or deletion of or results in SegA^– plasmids. Better than random segregation requires an active segB region. The segB region contains two ors (or and or). Inactivation of either of the orfs does not lead to an increase in cell death, but or^– plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.     

Construction of stable lactose-utilizing Xanthomonas campestris by chromosomal integration of cloned lac genes using filamentous phage ϕLf DNA

Summary Previously, we constructed a lactose-utilizing strain of Xanthomonas campestris, Xc17 (pKMLT), by cloning lacZY genes with the RK2-derived vector pLAFR1. In this study, the narrow-host-range, -galactosidase expression plasmid pKM was fused with an integration vector pS19 to form pSF14. Following insertion into Xc17, pSF14 was integrated into the host chromosome. The integration function was provided by the 0.85-kb EcoRI-PstI fragment from the filamentous phage Lf. The integration caused no adverse effect to the cells and was stable for at least 66 generations without selection. The engineered strain, Xc17::pSF14, was able to grow as well and produce as much xanthan gum in lactose medium as the wild-type cells did in glucose medium, and the Xc17(pKMLT) in lactose medium. Therefore, Xc17::pSF14 is potentially useful for xanthan production by direct use of whey lactose as the fermentation substrate. This study has advanced one more step our efforts to contruct lactose-utilizing X. campestris and confirmed the feasibility of using pS19 as an integration vector.     

Modulation of formation of tumor metastases by peritoneal macrophages elicited by various agents

Summary We have studied the formation of experimental B16 melanoma metastases in the lungs of mice inoculated IV with tumoricidal or nontumoricidal peritoneal macrophages elicited by various agents. IV inoculation of peritoneal M elicited by Brewer's thioglycollate medium (TG-M) 1 day before the injection of B16 melanoma cells dramatically increased the number of metastatic foci in the lungs. NIH thioglycollate broth and proteose peptone each elicited a relatively low number of M, which were morphologically distinguishable from TG-M and did not influence the yield of B16 melanoma colonies in the lungs. Resident or C. pravum-elicited M also did not augment metastatis formation. TG-M became highly tumoricidal after IP stimulation with poly I: C. However, tumoricidal TG-M inoculated IV 1 day before IV inoculation of B16 melanoma cells did not have an antimetastatic effect. On the contrary, both tumoricidal and nontumoricidal TG-M augmented metastasis formation. Poly I: C treatment had a substantial antimetastatic effect in the normal mice, but not in mice with adoptively transferred TG-M. Histological analysis revealed that IV-inoculated TG-M (tumoricidal or nontumoricidal, either viable or disrupted) induced severe intravascular reaction in the lungs, but not in the liver or kidney. This reaction manifested in the aggregation of the various blood cells, preferentially neutrophils. These reactions were not observed after IV inoculation of PM or NIH TG-M.Intravascular inflammatory reactions induced by TG-M may be responsible for the augmentation of metastasis formation, partly by suppression of NK reactivity and mostly by the acceleration of the processes of tumor cell extravasation. These data may provide some insight into the failure to achieve systemic adoptive immunotherapy using activated peritoneal TG-M. Abbreviations used in this paper are: TG-M, thioglycollate-elicited macrophages; PM, proteose-peptone-elicited macrophages; NIH TG-M, macrophages elicited with NIH thioglycollate broth; CP-M, macrophages; elicited with C. parvum; poly I: C, polyinosinic: polycytidylic acid; TGM, thioglycollate medium; NIHTGB, NIH thioglycollate broth     

Exploration and exploitation of soil by apple,kiwifruit, peach,Asian pear and grape roots

Measurements of root-length density (RLD) in a range of 31 apple, kiwifruit, peach, Asian pear and grape orchards were used to derive indices to describe the exploration and exploitation of rooting volumes. Orchards were of various ages and located on a range of soil types, geographic regions, management systems etc. Data were obtained from core samples of volume 1.66×10^-4 m³ randomly taken within a standard volume, determined by average planting grids, of 2 m radius centred on tree stems, and 1 m depth. Root systems were described using an exploitation index, E(), and an exploration index, E(0). E() is defined as the proportion of the soil volume which contains roots at RLD greater than or equal to some specified value, . E(0) is defined as the proportion of the soil volume which contains roots at any RLD greater than zero. These indices are dependent on sample size, as are all volumetric or soil-coring data.Estimates of E(0) for each orchard were obtained as the proportions of cores containing any RLD>O and assessed for dependence on species. Peach trees had a significantly higher value of E(0), equal to almost 1.0, compared to the other four species where E(0) was approximately 0.8 (p0.01) or less. There was also some variation with age. E(0) was lower for very young plants which had not fully occupied the sampled soil volume. Exploration indices for woody roots increased with rootstock age but otherwise did not explain large differences in E() between species for given values.For example at =0.05×10⁴ m.m^-3, E() was approximately 0.45 for peach and kiwifruit, and 0.05 for apple, Asian pear and grape, whereas at =0.5×10⁴ m.m^-3 the corresponding values were 0.1 and almost zero. Negative exponential curves relating E(), scaled by dividing by E(0), to were fitted for each of the 31 orchards. Exponents for these curves, k, were significantly smaller for kiwifruit and peaches than apples, grapes and Asian pears (p0.05), and smaller for apples than grapes and Asian pears (p0.05). A larger k implies a rapid fall-off in E() as increases. Although all five species contained zero and low RLD samples, only kiwifruit and peaches contained higher RLD values and consequently have higher mean RLD. This trend was consistent across all soils, regions, sampling dates, and plant ages.The analyses demonstrate that core sampling can give useful insights into macro-scale root-system distribution, such as the proportion of a soil volume explored and how it is exploited. If positions of core samples are noted during sampling using angular direction, depth and radial distance as spatial coordinates the method can be used to describe root-system structures.     

Intracellular effects of phage phi W-14 DNA on transformation of Bacillus subtilis

Summary Uptake of transforming DNA by competent Bacillus subtilis cells in the presence of phage W-14 DNA (in which half the thymine residues are replaced by -putrescinyl-thymine) is accompanied by a decrease in the amount of trichloracetic acid-precipitable label of the former retained by recipient cells during subsequent incubation. Fractionation of lysates of cells incubated for 0.5 min at 37°C after DNA uptake at 30°C in the presence of low concentrations of W-14 DNA (0.1 g/ml) demonstrated the presence of single-stranded transforming DNA molecules, typical for DNA taken up by B. subtilis. The intracellular effect of W-14 DNA was enhanced by an increase in its concentration (to 0.5–1 g/ml), or by increasing the temperature of uptake (to 37°C). With either of these treatments transforming DNA taken up was found in the form of a broad asymmetric band, indicative of degradation, and partially located at the density characteristic for single-stranded molecules. Fractionation of lysates of cells treated (0.1 g/ml) or untreated with W-14 DNA, and incubated for 20 min at 37°C after DNA uptake, showed disappearance of the single-stranded band. Donor DNA label was then found exclusively in the recipient DNA band, its amount being lower in samples treated with W-14 DNA. The influence of a high concentration of W-14 DNA on retention of transforming DNA label was correlated with its effect on transformation. On exposure to low concentrations of phage DNA, such a correlation was observed only after longer periods of incubation, due to slower intracellular degradation of homologous DNA taken up. The results are consistent with the proposal that W-14 DNA-induced reduction in efficiency of transformation is due to intracellular stimulation of transforming DNA degradation, leading to a decrease in the number of donor molecules available for recombination with the recipient chromosome.     

Die optischen Übertragungseigenschaften der Komplexaugen von Drosophila

Summary The transfer properties of the optical system in the arthropod compound eye are determined by the interommatidial angle , influencing the resolving power, and by the width of the visual fields of single ommatidia , influencing the response at high spatial frequencies of brightness distributions in the object space. The energy transfer/ receptor is proportional to ( )² and decreases with in-inreasing approximation of the perfect-imaging condition: gD 0; 0. However, a value > 0 has to be maintained in order to overcome the threshold of nervous excitation at a certain minimum-brightness level. Theoretical treatment yields /=0.62 to 0.88 as the corresponding optimum-imaging relation. The actual ratio can be derived from measurements of the optomotor reactions to the movement of periodic brightness patterns. The approximate value 0.76 is obtained from the fruitfly Drosophila with normal and mutant eye pigmentation. As a result, the parameters of this imaging system are found to be established in a way that enables optimum performance at sufficient illumination. An dieser Stelle möchte ich Dr. W. Reichardt für sein eingehendes Interesse und manche anregende Diskussion über die Sehvorgänge im Komplexauge meinen Dank sagen. Dr. K. Kirschfeld verdanke ich ebenfalls wertvolle Hinweise. Herrn E. Freiberg bin ich für die Anfertigung der Abbildungen sehr verbunden.     

Long Range Force between Pre-Replication Complexes (Pre-RC) in DNA Controls Replication and Cell Cycle Progression

A nonstationary interaction, that controls DNA replication and the cell cycle, is derived from a manybody physics model in a chemically open T cell. The model predicts a long range force F()=-(/2) (1-)(2-) between the pre-replication complexes (pre-RCs) bound by DNA, =/N being the relative displacement of preRCs, the number of pre-RCs, N the threshold for initiation, and the compressibility modulus in thelattice of pre-RCs which behaves like an elastically braced string. Initiation of DNA replication is induced by a switch of sign of F(), from attraction (-)and assembly in the G ₁ phase (0 < < N), to repulsion (+) and partialdisassembly in the S phase (N < < 2N), with release of licensing factors from the pre-RCs, thus explaining prevention of re-replication. Replication is terminated by a switch of sign of F at = 2N, when all primed replicons are duplicated once, and F(0)=0 corresponds to a resting cell in absence of driving force at = 0. The switch of sign of force at = N also explains the dynamic instability in growing microtubules (MTs), as well as switch in the interleukin-2 (IL2) interaction with its receptor in late G ₁, at the restriction point. Shape, slope and scale of the response curves derived agree well with experimental data from dividing T cells and polymerizing MTs, the variable length of which is due to anonlinear dependence of the growth amplitude on the initial concentrations of tubulin dimers and guanosine-tri-phosphate (GTP).     

On maturation of the bacteriophage lambda chromosome

Summary Repressed lambda chromosomes that possess a duplication of the cohesive end site (cos site) are matured during lytic growth of 80. This result is in contrast to repressed non-duplication lambda chromosomes that are not matured by 80. DNA molecules matured by 80 are unreplicated and lack the duplication: both cos sites are cleaved. These results indicate that in normal lambda development, mature, unit-length chromosomes are generated from a multichromosomal length of lambda DNA by cleavage of two cos sites.     

Bacteriophage phi 1 as a gene-cloning vector in Bacillus subtilis

Summary We attempted to use Bacillus subtilis phage 1 as a gene-cloning vector since the 1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A 1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, 1E1 and 1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant 1E21 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage 11 DNA, that 1E21 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten 1 clones isolated from independent transfectants and found that six of them carried 11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases at the 11 DNA portion, whereas the parental 1E21 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases.     

Transfection of protoplasts of Bacillus subtilis with ?29 DNA

Summary The protoplast-polyethyleneglycol(PEG) transformation procedure of Chang and Cohen (2) can be used for 29 DNA transfection. 29 DNA without terminal proteins is not transfectious in the protoplast-PEG procedure.     

Transductional mapping of nine linked chromosomal genes in Bacillus thuringiensis

Summary We have previously described a phage (63) for generalized transduction in Bacillus thuringiensis and used it for mapping of four chromosomal antibiotic resistance markers, namely nalA-rifA-strA-spcA (Landén et al. 1981). From 63 we have now isolated a host range mutant called 64 which contains 52–56 megadalton of DNA. Phage 64 was found to be a more efficient transducing vector than 63. The host range of 64 is wide, with good growth on subspecies gelechiae, kurstaki, galleriae, thuringiensis and thompsoni, restriction on some derivatives of finitimus and ostrinae and no growth on alesti, israelensis and aizawai.Using 64 and a series of new mutants of subspecies gelechiae we have no added five new genes to the antibiotic resistance group described before. The gene order found was guaB-purB-metA-novA-(purA-nalA)-rifA-strA-spcA. Linkage was also demonstrated between hisA and lysA.     

Comparisons of the Genomes of New Giant Phages Isolated from Environmental Pseudomonas aeruginosa Strains of Different Regions

To study the genome diversity of bacteriophages from geographically distant natural populations, new giant KZ-like Pseudomonas aeruginosa phages isolated in two different regions were compared with earlier known phages of three species (KZ, Lin68, EL). A broad spectrum of lytic activity was demonstrated for all KZ-like phages. Phages of the KZ species proved to be common in natural populations of various regions, while EL- and Lin68-related phages were extremely rare. Most KZ-related phages had unique DNA restriction patterns, but the differences between these were only minor, and the genomes did not contain nonhomologous fragments. The spectrum of capsid polypeptides proved to be conserved in each species, and was proposed as a character necessary and sufficient for express classification of phages with an accuracy of species. Phages isolated in different geographical regions showed no substantial difference. Some phages only slightly differing in DNA restriction pattern from KZ may be used to study the origin of KZ genes coding for orthologs of proteins of unrelated species (other phages, pathogenic bacteria, eukaryotes).     

Plasmid transduction in Streptococcus thermophilus

Summary Streptococcus thermophilus is a common dairy starter for which very few genetic exchange systems have been described. Here we report plasmid transduction by 17 and 56, two virulent phages of industrial yogurt starter strains. Several replicons could be transduced, independently of their size, with efficiencies which are high enough to allow gene transfer from a transformable intermediary strain of S. thermophilus to other hosts of the bacteriophages studied.     

Secondary structural effects on protein NMR chemical shifts

For an amino acid in protein, its chemical shift, (, )_s, is expressed as a function of its backbone torsion angles ( and ) and secondary state (s): (, )_{s=, )_coil+(, )_s}, where (, )_coil represents its chemical shift at coil state (s=coil); (, )_s (s=sheet or helix) is herein defined as secondary structural effect correction factor, which are quantitatively determined from Residue-specific Secondary Structure Shielding Surface (RSS) for ¹³CO, ¹³C, ¹³C,¹H, ¹⁵N, and ¹HN nuclei. The secondary structural effect correction factors defined in this study differ from those in earlier investigations by separating out the backbone conformational effects. As a consequence, their magnitudes are significantly smaller than those earlier reported. The present (, )_sheet and (, )_helix were found varying little with backbone conformation and the 20 amino acids, specifically for ¹³CO, ¹³C, and ¹H nuclei. This study also carries out some useful investigations on other chemical shift prediction approaches – the traditional shielding surfaces, SHIFTS, SHIFTX, PROSHIFT, and identifies some unexpected shortcomings with these methods. It provides some useful insights into understanding protein chemical shifts and suggests a new route to improving chemical shifts prediction. The RSS surfaces were incorporated into the program PRSI [Wang and Jardetzky, J. Biomol. NMR, 28: 327–340 (2004)], which is available for academic users at http://www.pronmr.com or by sending email to the author (yunjunwang@yahoo.com).     

Catalase in salivary gland striated and excretory duct cells. II. ? Body: an ellipsoidal peroxisomal organelle with crystalloid axial projections

Synopsis A new distinctive and unique peroxisomal organelle with a spindle shape has been observed in luminal epithelial cells of striated and excretory ducts of mouse salivary glands. Light microscopic studies indicate it has an ellipsoidal centre from which catalase-positive filamentous or rod-like processes protrude along its major axis; hence, it is called a body. A role for this specialized peroxisome in the formation of nearby free filaments and rods is suggested by the frequent observation of segmentation of its axial processes. Complementary ultrastructural studies of osmium-fixed preparations show that the deformation to an oval shape results from the pressure of the extruding crystalloid coincident with the major axis of the ellipsoidal body. The size range and conformation of body axial processes are comparable to those of free catalase-positive rods and filaments observed in the same cells. The periodic substructure of the crystalloid in the body core is identical with that of nearby cytoplasmic rods. These observations are consistent with the view that the rods and filaments observed free in the cytoplasm are formed by extrusion from the crystalloid core of the body. Bodies could also be responsible for the Aver rods of leukemic leukocytes.     

Existence and uniqueness of a sharp travelling wave in degenerate non-linear diffusion Fisher-KPP equations

In this paper we use a dynamical systems approach to prove the existence of a unique critical value c ^* of the speed c for which the degenerate density-dependent diffusion equation u _ct = [D(u)u _x ] _x + g(u) has: 1. no travelling wave solutions for 0 < c < c ^*, 2. a travelling wave solution u(x, t) = (x - c ^* t) of sharp type satisfying (– ) = 1, () = 0 ^*; '(^*–) = – c ^*/D'(0), '(^*+) = 0 and 3. a continuum of travelling wave solutions of monotone decreasing front type for each c > c ^*. These fronts satisfy the boundary conditions (– ) = 1, '(– ) = (+ ) = '(+ ) = 0. We illustrate our analytical results with some numerical solutions.     

O-Glycosylhydrolases of Marine Filamentous Fungi: β-1,3-Glucanases of Trichoderma aureviride

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from the bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl--D-glucosaminidase, -D-glucosidase, -D-galactosidase, -1,3-glucanase, amylase, and pustulanase. -1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that -1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.     

Isolation and characterization of ?80dgal transducing phages that carry gal operator-promoter insertion mutations

Summary 80dgal transducing bacteriophages have been isolated by the F-fusion technique of Press et al. (1971) and gal-operator-promoter insertion mutations have been introduced by homogenote formation.Five different 80dgal isolates have been studied in more detail. One of the 80 phages transduces the gal operon and gene aroG as well as at least part of the trp-operon; the gal operon of another 80dgal transducing phage is inverted with respect to the 80dgal sequences. Heteroduplex DNA mapping indicates that one of the 80dgal isolates in addition to the gal operon and a portion of the adjacent chromosomal region carries an IS2-element which is derived from the F'gal episome.The isolated 80dgal phages may be utilized for preparing pure gal mRNA and insertion-RNA as well as pure gal operon DNA.     

Host genes involved in the replication of single-stranded DNA phage ?K

Summary Using various replication mutants of E. coli, the host genes that participate in the replication of some K12-specific single-stranded DNA phages have been determined. Functional products of dnaE,-F,-G and -Z genes are required for the multiplication of K, whereas dnaA,-B,-C(D), H,-I and -P are dispensable for viral replication. In contrast with polB, recA, B, C, or xth functions, host rep activity is essential for K. At the restrictive temperature, the yield of K was markedly reduced in the ligts7 mutant and partially decreased in a polA ^ts strain. The phage K is thus less dependent on the host cells than X174 and A which require additionally the dnaB,-C(D) and -H functions. Replication of phage St-1 depends on dnaG and -Z gene products, but not on dnaP function. Although not much affected in polA ^ts host, growth of St-1 was significantly diminished in dnaF or ligts7 mutants.     

The distribution of Heterotrissocladius oliveri Saether (Diptera: Chironomidae) in Lake Michigan

Fifty one chironomid species were identified from 504 samples collected at depths ranging 8 to 267 m in Lake Michigan, U.S.A. Heterotrissocladius oliveri Saether occurred in 32% of these samples and had an average abundance of 22 m^–2 which was similar to other estimates from the Great Lakes. Maximum average lake-wide density was at 30 to 60 m (41 m^–2). At depths 60 m, H. oliveri was the dominant chironomid species comprising 75% of total Chironomidae. The substrate preference of H. oliveri differed within each depth regime considered: at 30–60 m, 2–3 ; at 60–120 m, 3–5 , 7–9 ; and at 120–180 m, 6–8 . Abundance was notably reduced at all depths in substrates characterized as medium silt (5–6 ). On a lake-wide basis, the distribution pattern suggested H. oliveri was most numerous from 30 to 60 m along the southwestern, eastern, and northern shorelines and at 60–120 m depths along the southern and eastern shorelines. Increased abundance in the South Basin was concurrent with evidence of increased sedimentation at 60 to 100 m. However, in several other areas of the lake, high densities were associated with medium to very fine sands relatively free of silts and clays. This observation suggested occurrence of H. oliveri was minimally affected by sediment type.Widely variable, but generally elevated water temperatures likely prevent H. oliveri from establishing a substantial population density at depths < 30 m. With increased depth, temperature fluctuation is negligible and food is more stable, though the source is variable. Factors limiting abundance of H. oliveri at depths 30 m were related to decreased food supply due to distance from shore, food sources of lower value (clays), and, most importantly, to reproductive replenishment.Although still oligotrophic in nature, high density occurrences in both high and low sedimentation areas of the lake suggest the trophic indicator status of H. oliveri might be broader than previously thought.