Differential expression of “Fe-repressible” and “growth-rate-sensitive” proteins in Neisseria meningitidis and Neisseria gonorrhoeae

By varying growth conditions, we were able to differentiate (SDS-PAGE) between true Fe-repressible proteins and growth-rate-sensitive proteins (GSP's) (60-110 Kd) in outer membranes of iron-starved meningococci and gonococci. Immunological characterization with monoclonal antibodies raised against the GSP's revealed (GIRA) crossreactivity between different GSP's. On the basis of common epitopes, two groups of proteins could be distinguished. Immuno-electron-microscopy showed that proteins of both groups were exposed at the cell surface.     

Opposite roles of domains 2 + 3 of Escherichia coli EF-Tu and Bacillus stearothermophilus EF-Tu in the regulation of EF-Tu GTPase activity

The effect of noncatalytic domains 2 + 3 on the intrinsic activity and thermostability of the EF-Tu GTPase center was evaluated in experiments with isolated domains 1 and six chimeric variants of mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) EF-Tus. The isolated catalytic domains 1 of both EF-Tus displayed similar GTPase activities at their optimal temperatures. However, noncatalytic domains 2 + 3 of the EF-Tus influenced the GTPase activity of domains 1 differently, depending on the domain origin. Ecdomains 2 + 3 suppressed the GTPase activity of the Ecdomain 1, whereas those of BstEF-Tu stimulated the Bstdomain 1 GTPase. Domain 1 and domains 2 + 3 of both EF-Tus positively cooperated to heat-stabilize their GTPase centers to attain optimal activity at a temperature close to the optimal growth temperature of either organism. This can be explained by a stabilization effect of domains 2 + 3 on α-helical regions of the G-domain as revealed by CD spectroscopy.     

Coexpression of β-mannanase and xylanase genes in Escherichia coli

[目的]β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域.构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价.[方法]通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl.[结果]菌株诱导21h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍.[结论]SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外.此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好.菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础.     

Plasmolysis bays in Escherichia coli: are they related to development and positioning of division sites?

Plasmolysis bays, induced in Escherichia coli by hypertonic treatment, are flanked by zones of adhesion between the plasma membrane and the cell wall. To test the proposition of Cook et al. (W. R. Cook, F. Joseleau-Petit, T. J. MacAlister, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 84:7144-7148, 1987) that these zones, called periseptal annuli, play a role in determining the division site, we analyzed the positions of these zones by phase-contrast and electron microscopy. In situ treatment of cells grown in agar showed that the youngest cell pole was the most susceptible to plasmolysis, whereas the constriction site was resistant. Lateral bays occurred only at some distance from a polar bay or a resistant constriction site. Orienting cells with their most prominently plasmolyzed polar bay in one direction showed that the lateral bays were always displaced away from the polar bay at about half the distance to the other cell pole. If no poles were plasmolyzed, lateral bays occurred either in the centers of nonconstricting cells or at the 1/4 or 3/4 position of cell length in constricting cells. The asymmetric positions of lateral plasmolysis bays, caused by their abrupt displacement in the presence of polar bays or constriction sites, does not confirm the periseptal annulus model (Cook et al.), which predicts a gradual and symmetric change in the position of lateral bays with increasing cell length. Our analysis indicates that plasmolysis bays have no relation to the development and positioning of the future division site.     

Optimization of recombinant β-NGF expression in Escherichia coli using response surface methodology

Human nerve growth factor a member of the neurotrophin family can be used to treat neurodegenerative diseases. As it has disulfide bonds in its structure, periplasmic expression of it using appropriate signal sequence is beneficial. Therefore, in this work β-nerve growth factor (β-NGF) was expressed in Escherichia coli using pET39b expression vector containing DsbA signal sequence. In an initial step, the effect of isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose concentration as inducer on protein production was investigated using response surface methodology. Then the effect of different postinduction time and temperature on protein production was studied. Our results indicated that the highest β-NGF production was achieved with 1?mM IPTG and low concentrations of lactose (0–2% w/v), low cultivation temperature of 25°C and postinduction time of 2?hr. Also following β-NGF purification, bioassay test using PC12 cell line was done. The biological activity of the purified β-NGF showed a similar cell proliferation activity with the standard recombinant human β-NGF. In conclusion, the results indicated an optimized upstream process to obtain high yields of biologically active β-NGF.     

Secretion of β-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin

The effect of cerulenin on the production of -lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 g/ml) had almost no effect on the growth rate of E. coli but it decreased the amount of -lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 g/ml)decreased turbidity and almost completely prevented synthesis of -lactamase and other periplasmic proteins. Cerulenin decreased incorporation of l-[³⁵S]methionine into membranes during growth as well. Spheroplasts secreted -lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 g/ml) this secretion was prevented by more than 90%. -Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5% of the total amount of pre--lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 g/ml) did not catalyze processing of pre--lactamase at all. Membrane preparations from Bacillus subtilis did not process pre--lactamase either in the absence or in the presence of cerulenin.     

Anomalous effect of uncouplers on respiratory chain-linked transhydrogenation in Escherichia coli membranes: evidence for a localized proton pathway?

Energization of the pyridine nucleotide transhydrogenase in everted membrane vesicles from Escherichia coli JM83 was compared with the process in vesicles of the same strain transformed with the plasmid pDC21 overexpressing this enzyme. Proton translocation was assayed by the quenching of the fluorescence of the probe quinacrine. Agents able to discharge transmembrane proton gradients such as nigericin and the uncouplers 3,3',4',5-tetrachlorosalicylanilide and carbonyl cyanide m-chlorophenylhydrazone inhibited ATP-dependent transhydrogenation of NADP by NADH and discharged transmembrane proton gradients generated by transhydrogenation of AcNAD by NADPH, by oxidation of NADH, and by hydrolysis of ATP. This was observed in everted membrane vesicles of both strains JM83 and JM83pDC21. These strains differed significantly in the response of the NADH oxidation-dependent transhydrogenase. This reaction was inhibited by nigericin and uncouplers in membrane vesicles of JM83 but there was little inhibition or the reaction was stimulated in JM83pDC21, in spite of the discharge of the NADH oxidation-generated proton gradient measured by quinacrine fluorescence in the latter strain. It is proposed that the transhydrogenase is energized by direct or local (nonbulk phase) proton translocation in membranes of this strain. Uncouplers might facilitate these routes but would not discharge them. The generality of these observations was shown using other strains. NADH oxidase activity was severalfold lower in membrane vesicles of JM83pDC21 compared with JM83. The levels of ubiquinone and cytochromes, and the activities of NADH dehydrogenases I and II, and of cytochrome oxidase, were similar in the two strains. It is concluded that the NADH oxidase activity of JM83pDC21 is low because of the reduced rate of collision between electron-transferring complexes of the respiratory chain due to the large amount of transhydrogenase protein in the membranes of this strain. The large amount of transhydrogenase favors direct, nonbulk phase proton transfer. Transhydrogenase activity was stimulated by Ca2+, Mg2+, or Mn2+.     

Unique mechanistic features of post-translational regulation of glutamine synthetase activity in Methanosarcina mazei strain Gö1 in response to nitrogen availability

PII-like signal transduction proteins are found in all three domains of life and have been shown to play key roles in the control of bacterial nitrogen assimilation. This communication reports the first target protein of an archaeal PII-like protein, representing a novel PII receptor. The GlnK(1) protein of the methanogenic archaeon Methanosarcina mazei strain Go1 interacts and forms stable complexes with glutamine synthetase (GlnA(1)). Complex formation with GlnK(1) in the absence of metabolites inhibits the activity of GlnA(1). On the other hand, the activity of this enzyme is directly stimulated by the effector molecule 2-oxoglutarate. Moreover, 2-oxoglutarate antagonized the inhibitory effects of GlnK(1) on GlnA(1) activity but did not prevent GlnK(1)/GlnA(1) complex formation. On the basis of these findings, we hypothesize that besides the dominant effector molecule 2-oxoglutarate, the nitrogen sensor GlnK(1) allows finetuning control of the glutamine synthetase activity under changing nitrogen availabilities and propose the following model. (i) Under nitrogen limitation, increasing concentrations of 2-oxoglutarate stimulate maximal GlnA(1) activity and transform GlnA(1) into an activated conformation, which prevents inhibition by GlnK(1). (ii) Upon a shift to nitrogen sufficiency after a period of nitrogen limitation, GlnA(1) activity is reduced by decreasing internal 2-oxoglutarate concentrations through diminished direct activation and by GlnK(1) inhibition.     

Multi-level metabolic engineering of Escherichia coli for high-titer biosynthesis of 2′-fucosyllactose and 3-fucosyllactose

Fucosyllactoses (FL), including 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL), have garnered considerable interest for their value in newborn formula and pharmaceuticals. In this study, an engineered Escherichia coli was developed for high-titer FL biosynthesis by introducing multi-level metabolic engineering strategies, including (1) individual construction of the 2′/3-FL-producing strains through gene combination optimization of the GDP-L-fucose module; (2) screening of rate-limiting enzymes (α-1,2-fucosyltransferase and α-1,3-fucosyltransferase); (3) analysis of critical intermediates and inactivation of competing pathways to redirect carbon fluxes to FL biosynthesis; (4) enhancement of the catalytic performance of rate-limiting enzymes by the RBS screening, fusion peptides and multi-copy gene cloning. The final strains EC49 and EM47 produced 9.36 g/L for 2′-FL and 6.28 g/L for 3-FL in shake flasks with a modified-M9CA medium. Fed-batch cultivations of the two strains generated 64.62 g/L of 2′-FL and 40.68 g/L of 3-FL in the 3-L bioreactors, with yields of 0.65 mol 2′-FL/mol lactose and 0.67 mol 3-FL/mol lactose, respectively. This research provides a viable platform for other high-value-added compounds production in microbial cell factories.