Physical and genetic analysis of IS110, a transposable element of Streptomyces coelicolor A3(2)

Summary On at least three independent occasions a 1.6 kb segment of Streptomyces coelicolor DNA was detected in apparently the same location in an attP-deleted derivative of the temperature phage C31 that carried a selectable viomycin resistance gene. This sequence (termed IS110) allowed integration of the phage (giving viomycin-resistant transductants) at homologous sequences (detected by Southern hybridisation) at several locations in the S. coelicolor genome. The inserted prophages facilitated genetic mapping of two IS110 copies in the chromosomal linkage map. A third copy did not exhibit simple segregation with chromosomal markers, and there appeared to be a frequent DNA rearrangement close to this copy. Some variation in the number of copies of IS110 and their location has taken place in the pedigree of S. coelicolor derivatives. IS110 did not hybridise to any known S. coelicolor plasmid, nor to any of several other IS-like elements previously described in other Streptomyces plasmids or phages. It hybridised strongly to DNA from only a small minority of other Streptomyces species and was absent from S. lividans, a close relative of S. coelicolor.     

Cloning and analysis of DNA sequences from Streptomyces hygroscopicus encoding geldanamycin biosynthesis

A gene library constructed from large (20 kb) fragments of total DNA from the geldananmycin-producing strain Streptomyces hygroscopicus 3602 cloned in the plasmid vector pIJ61 were used to transform S. lividans TK24. Three transformants of about 800 tested were found to have acquired the ability to produce an antibiotic lethal to a geldanamycin-sensitive strain of Bacillus subtilis. The plasmids isolated from these transformants, pIA101, pIA102 and pIA103, each contained an insert of 15 kb. A 4.5 kb DNA fragment from the insert in pIA102 hybridised to DNA from S. hygroscopicus 3602 and to DNA encoding part of the erythromycin polyketide synthase but not to S. lividans TK24 DNA. The integration-defective phage vector C31 KC515 containing this 4.5 kb fragment was able to lysogenise S. hygroscopicus 3602 to produce lysogens defective in geldanamycin production. Loss of the prophage restored the ability to produce geldanamycin. Extracts of fermentation broth cultures of S. lividans containing pIA101, pIA102 and pIA102 and pIA103 analysed by thin-layer chromatography (TLC) contained compounds identical or very similar to purified geldanamycin, which were not present in S. lividans. These compounds showed a mass spectrum indistinguishable from geldanamycin. The evidence suggests that the clones contain DNA sequences encoding functions required for geldanamycin biosynthesis including components of the polyketide synthase.     

Simplified typing of mouse hemoglobin (Hbb) phenotypes using cystamine

Cellulose acetate electrophoresis of mouse hemoglobins modified with the disulfide reagent cystamine permits rapid, unequivocal discrimination of all combinations of the codominant mouse hemoglobin single (Hbb ^s ) and diffuse (Hbb ^d and Hbb ^p ) alleles. The single, diffuse major, diffuse d-minor, and diffuse p-minor adult hemoglobins are all resolved by this method, which depends on the presence of a cysteine in the chains of diffuse mice which is not found in the chain of single mice.This work was supported by research grants ACS-VC58 and NIH CA-01074. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

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.     

The glycerol utilization operon of Streptomyces coelicolor: genetic mapping of gyl mutations and the analysis of cloned gylDNA

Summary Glycerol-3-phosphate dehydrogenase (gylB) mutations (which cause glycerol sensitivity), and presumed gylcerol kinase (gylA) and/or regulatory mutations eliminating both glycerol-3-phosphate dehydrogenase and glcerol kinase activities, map close to the argA locus of Streptomyces coelicolor A3(2). Using the plasmid vector pIJ702 and restriction enzymes Bg/II and SstI, extensively overlapping S. coelicolor DNA fragments of 2.74 kb and 2.84 kb were isolated, either of which could restore the wild-type phenotype to gylB and some gylA mutants. Genetic and biochemical analyses of mutants carrying the cloned gylDNA suggested that a functional gyl promoter had not been cloned, and that restoration of the Gyl⁺ phenotype was achieved by recombination between the cloned and chromosomal gyl DNA sequences. After subcloning parts of this DNA into the phage vector C31 KC400, gene disruption analysis was carried out, which confirmed the absence of the gyl promoter, and indicated that a polycistronic mRNA traverses gylA and then gylB.     

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.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

The effect of signal sequences on the efficiency of secretion of a heterologous phosphotriesterase by Streptomyces lividans

Summary A heterologous phosphotriesterase (parathion hydrolase) containing the native Flavobacterium species signal sequence was previously shown to be secreted by Streptomyces lividans. Western blot analysis of the recombinant phosphotriesterase produced by S. lividans demonstrated only the mature form extracellular but both processed and unprocessed forms in cell-associated samples. To investigate the efficiency of secretion in Streptomyces, a construction was made that substituted a native Streptomyces -galactosidase signal sequence for the Flavobacterium signal sequence. This resulted in a higher proportion of hydrolase in the extracellular fluid and a lower proportion of parathion hydrolase remaining cell-associated. These results suggest that use of a native Streptomyces signal sequence may result in more efficient secretion of heterologous proteins.Correspondence to: M. K. Speedie     

Increased yield of a lysozyme after self-cloning of the gene in Streptomyces coelicolor “Müller”

Summary Streptomyces coelicolor Müller DSM3030 excretes a lysozyme comprising both -1,4-N-acetyl-and -1,4-N,6-O-diacetyl muramidase activities. The lysozyme is named Cellosyl. Gene libraries have been established using genomic DNA from the wild-type strain, S. coelicolor DSM3030, and from an overproducing mutant, S. coelicolor HP1, which exhibits about a twofold increase in lysozome production. The lysozyme-encoding genes (cel) from both strains were detected by oligodeoxynucleotide hybridization. The nucleotide sequence of the cel genes isolated from both strains was shown to be identical. The different levels of lysozyme production could not be correlated with any mutations at the cel gene locus. The cel gene isolated from the wild-type strain could not be expressed in some other species of Streptomyces. However, self-cloning of the cel gene into S. coelicolor DSM3030 and HP1 resulted in a 2.5-fold increase in lysozyme production.     

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.     

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.     

Properties of Streptomyces sp. endo-β-xylanases in relation to their applicability in kraft pulp bleaching

A screening of a collection of Streptomyces sp. strains has shown that Streptomyces achromogenes 5028 (S1), Streptomyces longisporus ruber 4–167 (S2) and Streptomyces sp. 8812 (S3) degraded efficiently beechwood xylan. The -xylanase activities present in the culture filtrate of the strains were purified to electrophoretic homogeneity and found to be typical non-debranching endo--xylanases (1,4--D-xylan xylanohydrolases: E.C. 3.2.1.8) with respective molecular weights of 25,000 (S1), 45,000 (S2) and 22,000 (S3). The enzymes were characterized with respect to their temperature–pH relationship and kinetic profile. Immunological experiments suggested that the enzyme produced by S1 belonged to family 11 of glycanases and the S3 enzyme to family 10. The three xylanases adsorbed onto crystalline cellulose but were catalytically inert towards this material, indicating a possible application of these enzymes in biobleaching processes. With respect to its effect on and brightness values in a DEDED bleaching sequence, the xylanase produced by the S1 strain appeared as comparable to a Trichoderma longibrachiatum commercial enzyme preparation (Novozym 431). Streptomyces sp. xylanases may find applications in elemental-chlorine-free bleaching procedures.     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.     

Unusual suppression properties of lysogens containing derivatives of ?80 psu3+

Summary Nonsense codon suppressing lysogens of E. coli have been made using 80 psu ₃ ⁺ -A2 and 80 psu _oc ⁺ -A2, heat-sensitive amber and ochre suppressing derivatives, respectively, of bacteriophage 80. The various lysogens selected differ in strength of suppression as well as in heat sensitivity of suppressor function. Heat-resistant derivatives, some still carrying the A2 mutation, can be selected from the heat sensitive parents. Mapping expreiments indicate that the 80 derivatives integrate at the tyrTV locus, which contains two copies of tRNA ₁ ^Tyr . The origin of the various suppressor phenotypes appears to be related to the great variety of distinctive recombination events possible either between the incoming tRNA ₁ ^Tyr gene and the host copies, or among the three copies of this gene in the lysogens.     

Yeast ribosomal proteins

Summary Homology maps between bacteriophages 81, 80 and were constructed on the basis of electron microscope observation of DNA heteroduplexes. In 81/80 heteroduplex, the left half and the right terminal region of 13% the total molecular length were highly homologous, while the remaining region covering the early gene cluster was entirely nonhomologous. In 81/ heteroduplex, high-degree homologies were detected at the left 14% terminal region covering the head gene cluster, the central 3.8% region covering the att-int-xis region and the 1.3% Q homology region. Low-degree homologies of shorter length were scattered at the tail gene cluster, b2 region, cIII region, PQ region and SR region. Comparing our results with the homology maps of other lambdoid phages reported by Simon et al. (1971) and Fiandt et al. (1971), a phylogenic relation of 81 to other lambdoid phages and the role of recombination in the course of divergence of lambdoid phages are discussed.     

Purification and characterization of β-xylosidase from <Emphasis Type="Italic">Streptomyces</Emphasis>sp. CH7 and its gene sequence analysis

A thermostable -xylosidase was extracted and purified from Streptomyces sp. CH7 mycelium. The apparent molecular weight of the native enzyme estimated by gel filtration was around 173 and 87 kDa for the two subunits estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Its optimal pH and temperature were 6.5 and 55 °C, respectively. The enzyme was stable at pH 6–9 and at 50 °C after 30 min. The K _m values for p-nitrophenyl---xylopyranoside and o-nitrophenyl---xylopyranoside were 0.56 and 0.94 mM with the V _m values of 26.3 and 6.6 U/mg protein, respectively. The enzyme was inhibited strongly by Hg²⁺, Fe²⁺, Cu²⁺ and Zn²⁺. It was inhibited by xylose competitively for p-nitrophenyl---xylopyranoside with the K _i value of 40 mM. Characterization of the nucleotide sequence of pCH7-1 carrying the -xylosidase gene from Streptomyces sp. CH7 revealed 3 open reading frames (ORF). The first truncated ORF, bxlI, encodes a putative ABC-type sugar transport system, permease component. The second ORF, bxl2, encodes -xylosidase, while the third truncated ORF, bxl3, encodes a putative oxidoreductase. The deduced 791 amino acid sequence of Bxl2 showed 84, 71 and 66% identity to those of Streptomyces coelicolor, Streptomyces lividansand Streptomyces thermoviolaceus, respectively. The calculated molecular mass of the deduced amino acid sequence revealed close similarity to that of the purified enzyme.     

Delivery of foreign genes to intact barley cells by high-velocity microprojectiles

Summary Foreign DNA was introduced through the cell walls of intact suspension culture cells of barley (Hordeum vulgare L.) by utilizing the particle acceleration approach. DNA-coated microscopic tungsten particles were accelerated to velocities that permitted their penetration of intact cells. Chimaeric constructs of -glucuronidase and neomycin phosphotransferase II under the control of the dual Agrobacterium T_R 12 promoter or the cauliflower mosaic virus 35S promoter served as reporter genes. Three days after particle delivery, high-level expression of both reporter genes was observed. That plasmid size could be critical for stabilizing DNA in the course of particle delivery will be discussed.     

Localization of the Laevigatum powdery mildew resistance gene to barley chromosome 2 by the use of RFLP markers

Summary The powdery mildew disease resistance gene Ml(La) was found to belong to a locus on barely chromosome 2. We suggest that this locus be designated MlLa. Linkage analysis was carried out on 72 chromosome-doubled, spring-type progeny lines from a cross between the winter var Vogelsanger Gold and the spring var Alf. A map of chromosome 2 spanning 119cM and flanked by two peroxidase gene loci was constructed. In addition to the Laevigatum resistance locus the map includes nine RFLP markers, the two peroxidase gene loci and the six-row locus in barley.     

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae

Summary A 2 m circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in high-copy and low-copy number cells was determined. High-copy number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.     

X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.