放线菌Streptomyces sp．FJ3的核糖体工程改良与活性产物的分离

[目的]利用核糖体工程抗性筛选技术,获得有抗菌活性突变株,并对突变株新产生活性物质进行研究.[方法]以三峡库区筛选出的无抗菌活性放线菌野生株为出发菌,通过单菌落挑选与平板划线培养,分离筛选具有链霉素和利福平抗性突变株;通过摇瓶发酵和对发酵液进行纸片法活性测定,获得抗金葡菌活性突变株;采用高效液相色谱法(HPLC)分析其发酵液组分,通过LC-MS对变化峰进行分析;进行16S rDNA及形态学鉴定.[结果]链霉素和利福平对放线菌菌株FJ3的MIC分别为0.5μg/mL和110μg/mL;在FJ3突变菌株中,共获得24株链霉素突变菌株和20株利福平突变菌株,抗菌活性筛选显示6株具有抗菌活性,其中2株链霉素突变菌株对金葡菌有强抑菌活性,采用Doskochilova溶剂系统纸层析结果表明,该活性物质为一种核酸类抗生素,HPLC和LC-MS显示该活性物质可能为硫藤黄菌素.[结论]利用核糖体工程技术可以改变放线菌的次级代谢,获得具有生物活性的突变株,拓展药源放线菌活性菌株新资源.     

Isolation and characterization of autolysis-defective mutants of Staphylococcus aureus created by Tn917-lacZ mutagenesis.

Two autolysis-defective mutants (Lyt-1 and Lyt-2) of Staphylococcus aureus have been isolated by transposon Tn917-lacZ mutagenesis. The mutants exhibited normal growth rate, cell division, cell size, and adaptive responses to environmental changes. No autolytic activities were detected in a crude autolytic enzyme preparation from the Lyt- mutants. The rate of autolysis of whole cells and cell walls in the mutants were negligible, but mutant cell wall preparations were degraded by crude enzyme preparations from the wild-type strain. Zymographic analyses of enzyme extracts from the mutants showed a single autolytic enzyme band, compared with more than 10 autolytic enzyme bands from the parent strain. Analyses of intracellular and exoprotein fractions gave results similar to those in experiments with total-cell extracts. Southern blot analysis indicated the insertion of a single copy of the transposon into the chromosome of Lyt mutants. Isogenic Lyt mutants constructed by phage phi 11 transduction showed similar phenotypes. Because both Lyt- mutants had Tn917-lacZ inserted in the appropriate orientation, it was possible to determine gene activity under various conditions by measuring beta-galactosidase activity. The gene activity was found to be induced by low pH, low temperature, and high sucrose and high sodium chloride concentrations. From these data, we propose that the mutation lies in either a master regulatory gene or a structural gene which is responsible for the synthesis or processing of a majority of the autolytic enzyme bands.     

Possible involvement of bacterial autolytic enzymes in flagellar morphogenesis.

Autolytic enzymes were found to be required for flagellar morphogenesis in Bacillus subtilis 168 and Bacillus licheniformis 6346. Two previously characterized, poorly lytic, chain-forming mutants of B. subtilis 168, strains FJ3 (temperature conditional) and FJ6, each 90 to 95% deficient in the production of N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase, were observed to be nonmotile at 35 degrees C in a variety of liquid and semisolid meida. In contrast, cells of the isogenic wild-type strain were motile and fully separated. Electron microscopy revealed the complete absence of flagella on the mutant cells. Similar observations were made with another poorly lytic strain of B. subtilis 168 (Nil5) and with two poorly lytic, phosphoglucomutase-deficient mutants of B. licheniformis 6346 (MH-3, MH-5). In minimal media lacking galactose (restrictive conditions), the B. licheniformis mutants failed to form flagella, or had serious abnormalities in flagellar morphogenesis and motility. Under permissive conditions, mutants FJ3 (grown at 17 degrees C) and MH-5 (grown with addend galactose) showed increased autolytic activities, grew in the dechained form, and regained their capacities to synthesize functional flagella. Examination of several classes of spontaneous revertants derived from the various mutant strains further demonstrated a close relationship between autolysin acttivity and flagellation in the two Bacillus spp.     

Systematic mutagenesis of potential glycosylation sites of lysosomal acid lipase

Lysosomal acid lipase (LAL; EC 3.1.1.13) is a key enzyme in the intracellular lipid metabolism. It hydrolyzes exogenous triglycerides and cholesterol esters taken up by various cell types. LAL has six potential N-glycosylation sites and one potential O-glycosylation site. Elimination of each of the six Asn-(X)-Ser/Thr sites by site-directed mutagenesis and expression in baculovirus-infected Spodoptera frugiperda cells resulted in two single-mutant enzymes without lipolytic activities (N134Q and N246Q) and four mutants with preserved activities. The two inactive mutants were not detectable on immunoblot analysis, indicating that they were not secreted. Six double mutants in all possible combinations except for the two inactive single mutants were produced and expressed. Double mutants in combination with the N9 glycosylation site showed reduced activities as compared to the other mutants or the wild-type enzyme. Kinetic data of LAL glycosylation mutants indicate that substrate affinity of N9Q was not changed, but k (cat) of N9 mutants was reduced distinctly compared to the wild-type enzyme. Peanut agglutinin lectin did not recognize LAL, demonstrating that the protein has no core1 structure (Galbeta 1-3 GalNAc) of O-glycosylation. These data indicate that at least two of the six N-glycosylation sites are used in native lipase. N134 and N246 were found to be essential for LAL activity. We conclude that glycosylation plays an important role in the formation of functional LAL.     

IL 2 production by mitogen-stimulated T cell subsets: helper-precursors are predominantly Lyt-2-

Although there is strong evidence that IL 2 can be secreted by at least some cells in both the Lyt-2- and Lyt-2+ T cell subsets, it is unclear whether lymphokine-secreting cells are present in equal frequencies in each population. We addressed this question by using a T cell mitogen, concanavalin A (Con A), that activates a large proportion of the cells of both subsets, as tested by their ability to grow in the presence of added conditioned medium. We found that, although Con A-responsive precursors of proliferating cells are at least as frequent in the Lyt-2+ set as in the Lyt-2- set, the frequency of Con A-responsive precursors of IL 2-secreting cells is much higher in the Lyt-2- population. Expressed as precursors per cell, lymphokine-secreting helpers are 17-fold more frequent in the Lyt-2- population. After adjusting for relative cell recoveries and for contamination, the data suggest that at least 97% (and more likely 99%) of the precursors for Con A-responsive IL 2-producing cells are in the Lyt-2- set. Thus, although the correlation between Lyt-2 surface antigen and helper function may not be absolute, our data support the idea that Lyt-2 does mark a functional distinction among T cells independent of antigen specificity.     

Insertional inactivation of the major autolysin gene of Streptococcus pneumoniae.

The lytA gene encoding the major pneumococcal autolysin (N-acetylmuramoyl-L-alanine amidase) was inactivated by inserting the 2-kilobase MspI fragment of pE194 containing the staphylococcal ermC gene. Stable autolysis-deficient (Lyt-) mutants and their isogenic Lyt+ parents were used in experiments designed to test possible physiological functions of the amidase. No autolysis could be induced in the mutants grown at 37 degrees C by deoxycholate, by incubation in stationary phase, or by treatment with penicillin. On the other hand, the Lyt- mutants exhibited normal growth rates and yields and normal adaptive responses during shifts from one growth temperature or nutritional condition to another. There was no evidence for impeded cell separation (chain formation). Colonies of Lyt- insertional mutants produced normal hemolytic zones on blood agar; they showed normal (high) levels of competence for genetic transformation. Lyt- mutants were also able to produce type 3 and 6 capsular polysaccharides, and such strains showed the same degree of virulence in mice as did the isogenic Lyt+ parent. The physiological function(s) of the amidase remains a puzzle.     

Inversion of helix orientation in Bacillus subtilis macrofibers

The ability of helical macrofibers of Bacillus subtilis to convert from left- to right-handed structures or vice versa has been known to be controlled by the nutritional environment (N. H. Mendelson, Proc. Natl. Acad. Sci. U.S.A., 75:2478-2482, 1978). lyt mutants (Ni15, FJ3, FJ6, and FJ7) and also lyt phenocopies of wild-type strain FJ8 were able to undergo helix hand inversion as a function of temperature. The transition between right- and left-handed structures was in a very narrow range (about 2.5 degrees C) in the low to mid-40 degrees C. The helix orientation of these strains was also influenced by the concentration of divalent ions. Macrofiber handedness is governed, therefore, by at least four factors: genetic composition, temperature, and nutritional and ionic environments. Conditions normally used for growth fall, within this matrix, in the region favoring right-handed structures. Inhibition studies suggest that cell growth must occur for helix hand inversion.     

Levels of enzymes of the pentose phosphate pathway in Pachysolen tannophilus Y-2460 and selected mutants

The compositions of intracellular pentose phosphate pathway enzymes have been examined in mutants of Pachysolen tannophilus NRRL Y-2460 which possessed enhanced D-xylose fermentation rates. The levels of oxidoreductive enzymes involved in converting D-xylose to D-xylulose via xylitol were 1.5–14.7-fold higher in mutants than in the parent. These enzymes were still under inductive control by D-xylose in the mutants. The D-xylose reductase activity (EC 1.1.1.21) which catalyses the conversion of D-xylose to xylitol was supported with either NADPH or NADH as coenzyme in all the mutant strains. Other enzyme specific activities that generally increased were: xylitol dehydrogenase (EC 1.1.1.9), 1.2–1.6-fold; glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 1.9–2.6-fold; D-xylulose-5-phosphate phosphoketolase (EC 4.1.2.9), 1.2–2.6-fold; and alcohol dehydrogenase (EC 1.1.1.1), 1.5–2.7-fold. The increase of enzymatic activities, 5.3–10.3-fold, occurring in D-xylulokinase (EC 2.7.1.17), suggested a pivotal role for this enzyme in utilization of D-xylose by these mutants. The best ethanol-producing mutant showed the highest ratio of NADH- to NADPH-linked D-xylose reductase activity and high levels of all other pentose phosphate pathway enzymes assayed.     

Cryptic Plasmids in Glutamic Acid-producing Bacteria

Seven filamentous (fil) mutants were isolated from B. subtilis, and the mutations were mapped by means of lysed-protoplast transformation. Five of the mutations were linked to aroD and the others to pyrD. rgn mutations, which lead to a decrease in autolysin(s) and the formation of filaments, were also linked to aroD, and the mapping order was rgn-dnaE-aroD. On comparison with other reported filamentous mutations (lyt-1, lyt-2 and lyt-152), fil-1, fil-3 to -6, rgn and the above lyt mutations were determined to be in the same locus. All of the seven fil strains lacked flagella and showed decreased aμtolysin activity. Among them, only mutants having arod- linked mutations showed low competency. Protease assay results indicated that rgn mutants produce a several times higher amount of the enzyme than the parent strain, and the initiation time for the production in rgn mutants was two hours earlier than in the parent strain.     

The participation of glucose-1,6-diphosphate in the regulation of hexokinase and phosphoglucomutase activities in brains of young and adult rats

1. The level of glucose-1,6-diphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was found to be strikingly decreased in brains of adult rats (5 months of age) as compared to young (10-14 days of age). 2. This age-related decrease in Glc-1,6-P2, the potent inhibitor of hexokinase and activator of phosphoglucomutase, was accompanied by a correlated increase in the activity of hexokinase and a reduction in phosphoglucomutase. 3. Evidence is provided showing that Glc-1,6-P2 participates in the regulation of these enzymes' activities with age. 4. The age-related changes in Glc-1,6-P2 and in the enzymes' activities in brain were opposite to those which we previously found in skeletal muscle. 5. These results suggest that Glc-1,6-P2 is involved in the regulation of carbohydrate metabolism during growth in both brain and muscle, as well as in the interrelationship between these two tissues.     

Fine and coarse regulation of reactive oxygen species in the salt tolerant mutants of barnyard grass and their wild-type parents under salt stress

The growth of the wild-type and three salt tolerant mutants of barnyard grass ( Echinochloa crusgalli L.) under salt stress was investigated in relation to oxidative stress and activities of the antioxidant enzymes superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), phenol peroxidase (POD: EC 1.11.1.7), glutathione reductase (GR: EC 1.8.1.7) and ascorbate peroxidase (APX: EC 1.11.1.1). The three mutants ( fows B17, B19 and B21) grew significantly better than the wild-type under salt stress (200 m M NaCl) but some salt sensitive individuals were still detectable in the populations of the mutants though in smaller numbers compared with the wild-type. The salt sensitive plants had slower growth rates, higher rates of lipid peroxidation and higher levels of reactive oxygen species (ROS) in their leaves compared with the more tolerant plants from the same genotype. These sensitivity responses were maximized when the plants were grown under high light intensity suggesting that the chloroplast could be a main source of ROS under salt stress. However, the salt sensitivity did not correlate with reduced K ⁺/Na ⁺ ratios or enhanced Na ⁺ uptake indicating that the sensitivity responses may be mainly because of accumulation of ROS rather than ion toxicity. SOD activities did not correlate to salt tolerance. Salt stress resulted in up to 10-fold increase in CAT activity in the sensitive plants but lower activities were found in the tolerant ones. In contrast, the activities of POD, APX and GR were down regulated in the sensitive plants compared with the tolerant ones. A correlation between plant growth, accumulation of ROS and differential modulation of antioxidant enzymes is discussed. We conclude that loss of activities of POD, APX and GR causes loss of fine regulation of ROS levels and hence the plants experience oxidative stress although they have high CAT activities.     

Enzymatic studies of high stearic acid sunflower seed mutants

Three high stearic acid sunflower (Helianthus annuus L.) mutants, CAS-3, CAS-4 and CAS-8, accumulating 28, 15 and 14 % of stearic acid in the seed lipids have been biochemically characterised. In vivo conversion rate of palmitic acid into stearic acid is not altered in the mutants but the conversion rate of stearic acid into oleic acid shows a reduction that correlated with the total stearic acid content of seed lipid mutants. Two enzymatic activities are found to be involved in the mutant phenotype, the acyl-ACP thioesterase (EC 3.1.2.14) and the stearoyl-ACP desaturase (EC 1.12.99.6). Our data suggest that the high stearic phenotype is due to the combined effect of a reduced stearoyl-ACP desaturase activity and an acyl-ACP thioesterase with higher activity on stearoyl-ACP. The same thioesterase activity increment, found on stearoyl-ACP, was also found on palmitoyl-ACP, suggesting that the affected thioesterase activity could be a FatB type.     

Clonal heterogeneity in colony stimulating factor production by murine T lymphocytes

A panel of 55 alloreactive murine T-lymphocyte clones was screened for the production of granulocyte-macrophage colony stimulating factor (GM-CSF), multilineage CSF (multi-CSF), human-active eosinophil CSF (human-active EO-CSF), and interleukin 2 (IL-2) in response to stimulation with the lectin concanavalin A. Many clones were also characterized for cytolytic specificity and expression of the T-cell antigen receptor-associated surface markers Lyt-2 and L3T4, which reflect their specificity for Class I (H-2K, H-2D) or Class II (H-2l, Mls) histocompatibility antigens, respectively. Eighty percent of the clones secreted detectable quantities of at least one of the four factors measured. Of the factor-producing clones, all appeared to secrete GM-CSF and half also secreted multi-CSF. A subpopulation of multi-CSF producers also released human-active EO-CSF. More than half of the factor-producing clones secreted detectable IL-2; whereas the IL-2-producing clones included some that did not secrete multi-CSF, IL-2 production was always associated with concomitant synthesis of GM-CSF. Comparison of the range and quantities of factors secreted by Lyt-2+ and L3T4+ clones indicated that more L3T4+ clones produced measurable titers of the four factors; on average, this group also secreted 10- to 100-fold higher titers of both the hemopoietic regulators and IL-2 than Lyt-2+ clones. Cells of the L3T4+ phenotype would therefore be expected to account for the majority of CSF and IL-2 secretion by polyclonal populations of activated T lymphocytes.     

Organization of a multifunctional protein in pyrimidine biosynthesis. A domain hypersensitive to proteolysis.

When the multifunctional protein that catalyses the first three steps of pyrimidine biosynthesis in hamster cells is treated with staphylococcal V8 proteinase, a single cleavage takes place. The activities of carbamoyl-phosphate synthetase (EC 6.3.5.5), aspartate carbamoyltransferase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3) and the allosteric inhibition by UTP are unaffected. One fragment, of Mr 182000, has the first and third enzyme activities, whereas the other fragment, of Mr 42000, has aspartate carbamoyltransferase activity and an aggregation site. A similar small fragment is observed in protein digested with low concentrations of trypsin. A similar large fragment is seen after digestion with trypsin and as the predominating form of this protein in certain mutants defective in pyrimidine biosynthesis. These results indicate that a region located adjacent to the aspartate carbamoyltransferase domain is hypersensitive to proteinase action in vitro and may also be sensitive to proteolysis in vivo.     

Structural requirements for dimerization,glycosylation, secretion,and biological function of VPF/VEGF

Vascular permeability factor (VPF) also known as vascular endothelial growth factor (VEGF), is a dimeric protein that affects endothelial cell (EC) and vascular functions including enhancement of microvascular permeability and stimulation of EC growth. To investigate the structural features of VPF/VEGF necessary for efficient dimerization, secretion, and biological activities, we employed site-directed mutagenesis with a Cos-1 cell expression system. Several cysteine residues essential for VPF dimerization were identified by mutation analysis of the Cys-25, Cys-56, and Cys-67 residues. Mutant VPF isoforms lacking either of these cysteines were secreted as monomers and were completely inactive in both vascular permeability and endothelial cell mitotic assays. VPF Cys-145 mutant protein was efficiently secreted as a glycosyaated, dimeric polypeptide, but had a reduction in biological activities. The site of N-linked glycosylation was directly identified as Asn-74, which, when mutated produced an inefficiently secreted dimeric protein without post-translational glycosylation, yet maintained full vascular permeability activity. Finally, we found that one VPF mutant isoform Cys-101 was not secreted and this mutant functioned as a dominant-negative suppressor of wild-type VPF secretion as demonstrated by co-expression assays in Cos-1 cells.     

Diet and carbohydrate digestion in the yellow-spotted longicorn beetle Psacothea hilaris

Larval and adult Psacothea hilaris feed on mulberry wood and leaves, respectively. High levels of endogenous activity against the major dietary carbohydrates, cellulose, hemicellulose, starch and soluble sugars were secreted in the gut of larvae and adults. Activity against pectin was also high and multiple polygalacturonase (EC 3.2.1.15) components were secreted in the gut of larvae. One glycanase component, beta-EG1, which was primarily an endo-beta-1,4-glucanase (EC 3.2.1.4) and another, beta-EG2, which was mostly an endo-beta-1,4-xylanase (EC 3.2.1.8), were also secreted, while at least four additional components hydrolysed laminarin, lichenin and crystalline cellulose. The beta-glycosidase component beta-GD1 was associated with most of the beta-mannosidase (EC 3.2.1.25) and beta-xylosidase (EC 3.2.1.37) activity secreted in the gut of larvae, while another, beta-GD2, was a beta-glucosidase (EC 3.2.1.21), the activity of which was directed against cellobiose and other beta-linked disaccharides, and a beta-fucosidase (EC 3.2.1.38). A beta-galactosidase (EC 3.2.1.23), which did not hydrolyse lactose, was also secreted, as were distinct beta-N-acetylhexosaminidase (EC 3.2.1.52), trehalase (EC 3.2.1.28), alpha-L-arabinosidase (EC 3.2.1.55), alpha-galactosidase (EC 3.2.1.22) and a minimum of four alpha-glucosidase (EC 3.2.1.20) components, one of which was also likely to be associated with a peak of alpha-mannosidase (EC 3.2.1.24) activity. The alpha-glucosidase components varied in their specificity for alpha-linked disaccharides, but none was active against sucrose, which was hydrolysed by a beta-fructofuranosidase (EC 3.2.1.26) component. Overall average levels of activity in larvae were twice those of adults, but the secretion of individual carbohydrases in both was not regulated in response to the relative abundance of particular carbohydrate components in their respective diets.     

Extracellular hydrolase profiles of fungi isolated from koala faeces invite biotechnological interest

The extracellular enzymes of seven fungal strains isolated from koala faeces have been comprehensively characterised for the first time, revealing potential for biotechnological applications. The fungal isolates were grown in a hydrolase-inducing liquid medium and the supernatants were analysed using enzyme assays and zymogram gels. Temperature and pH profiles were established for xylanase (EC 3.2.1.8 endo-1,4-β-xylanase), mannanase (EC 3.2.1.78 mannan endo-1,4-β-mannosidase), endoglucanase (EC 3.2.1.4 cellulase), β-glucosidase (EC 3.2.1.21 β-glucosidase), amylase (EC 3.2.1.1 α-amylase), lipase (EC 3.1.1.3 triacylglycerol lipase) and protease (EC 3.4 peptidase) activities. Comparisons were made to the high-secreting hypercellulolytic mutant strain Trichoderma reesei RUT-C30 and the wild-type T. reesei QM6a. The isolates from koala faeces Gelasinospora cratophora A10 and Trichoderma atroviride A2 were good secretors of total protein and heat-tolerant enzymes. Doratomyces stemonitis C8 secreted hemicellulase(s), endoglucanase(s) and β-glucosidase(s) with neutral to alkaline pH optimums. A cold-tolerant lipase was secreted by Mariannaea camptospora A11. The characteristics displayed by the enzymes are highly sought after for industrial processes such as the manufacture of paper, detergents and food products. Furthermore, the enzymes were produced at good starting levels that could be increased further by strain improvement programs.     

Characterization and virulence properties of Erwinia chrysanthemi lipopolysaccharide-defective, phi EC2-resistant mutants.

Outer membrane alterations were characterized in spontaneous mutants of the Erwinia chrysanthemi 3937jRH, which were selected for resistance to bacteriophage phi EC2. All but one of the mutants analyzed were affected in their lipopolysaccharide (LPS) structure, lacking the entire heterogeneous region of apparent high molecular weight present in the wild-type E. chrysanthemi LPS. At least two 3937jRH mutants, one selected as phi EC2 resistant (RH6065) and the other previously selected (D. Expert and A. Toussaint, J. Bacteriol. 163:221-227, 1985) as bacteriocin resistant (R1456), were cross-resistant to bacteriophage Mu and had rough LPSs with an altered core structure. Two phi EC2r mutants (RH6053 and RH6065) were most severely affected in their outer membrane integrity and also lost their virulence on saintpaulia plants, although they still possessed normal extracellular levels of pectinolytic and cellulolytic activities. The two Mur mutants RH6065 and R1456 were also able to induce systemic resistance in the challenged plant. All the other phi EC2r mutants retained the virulence of 393jRH.     

The global regulatory proteins LetA and RpoS control phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of Legionella pneumophila JR32

Legionella pneumophila possesses a variety of secreted and cell-associated hydrolytic activities that could be involved in pathogenesis. The activities include phospholipase A, lysophospholipase A, glycerophospholipid:cholesterol acyltransferase, lipase, protease, phosphatase, RNase, and p-nitrophenylphosphorylcholine (p-NPPC) hydrolase. Up to now, there have been no data available on the regulation of the enzymes in L. pneumophila and no data at all concerning the regulation of bacterial phospholipases A. Therefore, we used L. pneumophila mutants in the genes coding for the global regulatory proteins RpoS and LetA to investigate the dependency of hydrolytic activities on a global regulatory network proposed to control important virulence traits in L. pneumophila. Our results show that both L. pneumophila rpoS and letA mutants exhibit on the one hand a dramatic reduction of secreted phospholipase A and glycerophospholipid:cholesterol acyltransferase activities, while on the other hand secreted lysophospholipase A and lipase activities were significantly increased during late logarithmic growth phase. The cell-associated phospholipase A, lysophospholipase A, and p-NPPC hydrolase activities, as well as the secreted protease, phosphatase, and p-NPPC hydrolase activities were significantly decreased in both of the mutant strains. Only cell-associated phosphatase activity was slightly increased. In contrast, RNase activity was not affected. The expression of plaC, coding for a secreted acyltransferase, phospholipase A, and lysophospholipase A, was found to be regulated by LetA and RpoS. In conclusion, our results show that RpoS and LetA affect phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of L. pneumophila in a similar way, thereby corroborating the existence of the LetA/RpoS regulation cascade.