高静水压诱导水稻变异的初步研究

以高静水压处理水稻粤香占、粤丰占、998、999和毕粳38种子后的当代群体(M1),以及从中筛选到的突变株粤变长、巨无霸、粤丰压变1号-5号、毕籼为材料,研究了静水高压对水稻生长发育及其农艺性状的影响,结果发现：(1)与对照相比,粤香占和998处理当代群体的株高和有效穗数明显增加,但株叶形态没有明显变化;粤丰占、999和毕粳38的处理群体没有明显变化;(2)粤变长和巨无霸从第二代(M2)到第四代(M4)性状稳定,未发现明显分离,但在株高、有效穗数、穗粒数、粒长和千粒重等方面,与对照相比明显不同;(3)在M,代,粤丰压变1号一5号中除粤丰压变2号外的株高都发生了分离,而且粤丰压变4号分离出了大粒型和长芒型的植株;(4)毕籼从Mz代到lVt,代,性状稳定,但其株高、穗长、粒长和穗粒数均比对照显著增加,结实率和千粒重则明显降低;(5)经静水高压处理后的粤丰占种子发芽试验中,出现了双苗和3苗现象。这些结果表明静水高压能够影响水稻的生长发育,并能诱导水稻产生变异。     

Kinases in Leuconostoc mesenteroides

Enzyme extracts of Leuconostoc mesenteroides were found to contain at least four separate kinases: one active with glucose, glucosamine, and N-acetylglucosamine; one active with fructose and mannose; and two active with gluconate, one constitutive and one inducible. The molecular sizes of all the kinases, estimated from sucrose gradient centrifugation, are about the same, 37,000 to 50,000 daltons, except the constitutive gluconate kinase, which is about 100,000 daltons. Apparent Michaelis constants were calculated for all of the substrates mentioned. The kinases are separable on triethylaminoethyl cellulose.     

Evaluation of structural changes induced by high hydrostatic pressure in Leuconostoc mesenteroides

Scanning electron microcopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC) were used to evaluate structural changes in Leuconostoc mesenteroides cells as a function of high-hydrostatic-pressure treatment. This bacterium usually grows in chains of cells, which were increasingly dechained at elevated pressures. High-pressure treatments at 250 and 500 MPa also caused changes in the external surface and internal structure of cells. Dechaining and blister formation on the surface of cells increased with pressure, as observed in SEM micrographs. TEM studies showed that cytoplasmic components of the cells were affected by high-pressure treatment. DSC studies of whole cells showed increasing denaturation of ribosomes with pressure, in keeping with dense compacted regions in the cytoplasm of pressure-treated cells observed in TEM micrographs. Apparent reduction of intact ribosomes observed in DSC thermograms was related to the reduction in number of viable cells. The results indicate that inactivation of L. mesenteroides cells is mainly due to ribosomal denaturation observed as a reduction of the corresponding peak in DSC thermograms and condensed interior regions of cytoplasm in TEM micrographs.     

Structural Characteristics of Dextrans Synthesized by Dextransucrases from Leuconostoc mesenteroides NRRL B-1299

Four major dextransucrase (EC 2.4.1.5) preparations from Leuconostoc mesenteroides were studied in relation to their reaction products. The extracellular enzyme II, a highly aggregated form of enzyme I, synthesized the largest amount of dextran per 1 unit of enzyme. Moreover, this dextran emerged at the void volume by Sepharose 6B chromatography. Dextran produced by the enzyme I was composed almost exclusively of water-soluble form having a molecular weight (MW) smaller than that of the product with enzyme II. Although soluble dextran produced by the intracellular enzyme (enzyme III or IV) had a low MW, ratio of insoluble dextran to total dextran was higher than that of the products with extracellular enzyme. Dextran produced by the enzyme II contained a large amount of non-α-l,6-linkages whereas dextran produced by the enzyme I was rich in linear α-l,6-linked structure. The structural analyses of various dextrans showed that each enzyme seemed to be responsible for the synthesis of both α-1,6 and non-α-l,6-linkages. Difference in the amounts and structures of dextrans suggests that the extracellular enzymes may play a major role for the dextran synthesis in vivo.     

Structural analysis and properties of dextran produced by Leuconostoc mesenteroides NRRL B-640

A water-soluble dextran was produced by purified dextransucrase from Leuconostoc mesenteroides NRRL B-640. The dextran was purified by alcohol precipitation. The structure of dextran was determined by FT-IR, ¹H NMR, ¹³C NMR and 2-dimensional NMR spectroscopic techniques. NMR techniques (1D ¹H, ¹³C and 2D HMQC) were used to fully assign the ¹H and ¹³C spectra. All the spectral data showed that the dextran contains d-glucose residues in a linear chain with consecutive α(1 → 6) linkages. No branching was observed in the dextran structure. The viscosity of dextran solution decreased with the increase in shear rate exhibiting a typical non-Newtonian pseudoplastic behavior. The surface morphology of dried and powdered dextran studied using Scanning electron microscopy revealed the cubical porous structure.     

Pectin degradation in plant material by Leuconostoc mesenteroides

A strain of Leuconostoc mesenteroides that was able to reduce the viscosity of tomato juice serum and of a growth medium containing pectin was isolated and the pectolytic activity of its cell-free culture supernatant (CFS) was studied. In vitro tests showed that the CFS was active on high methoxyl pectin but almost inactive on low methoxyl pectin and polygalacturonic acid. It was most active in the pH range 4mD5–5mD5 and had no detectable pectinesterase activity. In vivo tests showed that the CFS degraded pectin in the walls of tomato fruit cells and caused degradation of cucumber fruit tissues.     

Acetoin production in growing Leuconostoc mesenteroides

Leuconostoc mesenteroides NCDO 518, provided with oxygen and pyruvate, preferentially used oxygen as accessory electron acceptor and converted pyruvate to acetoin. With glucose, 5.6 mM, as sole energy source only small amounts of acetoin were formed (0.08–0.21 mM). With glucose, 5.6 mM, and pyruvate, 20 mM, substantial amounts of acetoin were produced in growing, aerated cultures at pH 5 (2.8 mM, equivalent to 0.5 mol [mol glucose fermented]^–1). On exhaustion of glucose, growth ceased but metabolism of pyruvate continued with the formation of acetate and a little acetoin. In aerated cultures at pH 6 the general pattern was similar to that at pH 5 but less acetoin (0.6 mM) was formed during the growth phase and, after the exhaustion of glucose, pyruvate was converted very slowly to acetate only. Leuc. mesenteroides did not grow with pyruvate as sole energy source.     

Influence of lactose-citrate co-metabolism on the differences of growth and energetics in Leuconostoc lactis, Leuconostoc mesenteroides ssp. mesenteroides and Leuconostoc mesenteroides ssp. cremoris

The biodiversity of growth and energetics in Leuconostoc sp. has been studied in MRS lactose medium with and without citrate. On lactose alone, Ln. lactis has a growth rate double that of Ln. cremoris and Ln. mesenteroides. The pH is a more critical parameter for Ln. mesenteroides than for Ln. lactis or Ln. cremoris; without pH control Ln. mesenteroides is unable to acidify the medium under pH 4.5, while with pH control and as a consequence of a high Y(ATP) its growth is greater than Ln. lactis and Ln. cremoris. In general, lactose-citrate co-metabolism increases the growth rate, the biomass synthesis, the lactose utilisation ratio, and the production of lactate and acetate from lactose catabolism. The combined effect of the pH and the co-metabolism lactose-citrate on the two components of the proton motive force (deltap = deltapsi - ZdeltapH) has been studied using resting-cell experiments. At neutral pH deltap is nearly entirely due to the deltapsi, whereas at acidic pH the deltapH is the major component. On lactose alone, strains have a different aptitude to regulate their intracellular pH value, for Ln. mesenteroides it drastically decreases at acidic pH values (pH, = 5.2 for pH 4), while for Ln. lactis and Ln. cremoris it remains above pH 6. Lactose-citrate co-metabolism allows a better control of pH homeostasis in Ln. mesenteroides, consequently the pHi becomes homogeneous between the three strains studied, for pH 4 it is in an interval of 0.3 pH unit (from pHi = 6.4 to pHi = 6.7). In this metabolic state, and as a consequence of the variation in deltapH, and to some extent in the deltapsi, the difference of deltap between the three strains is restricted to an interval of 20 mV.     

Peptide utilization by Lactococcus lactis and Leuconostoc mesenteroides

To explain the competition for nitrogenous nutrients observed in mixed strain cultures of Lactococcus lactis and Leuconostoc mesenteroides, the utilization of peptides as a source of essential amino acids for growth in a chemically defined medium was compared in 12 strains of dairy origin. Both species were multiple amino acid auxotrophs and harboured a large set of intracellular peptidases. Lactococcus lactis can use a wide variety of peptides up to 13 amino acid residues whereas Leuc. mesenteroides assimilated only shorter peptides containing up to seven amino acids. Growth was limited by the transport of peptides and not by their hydrolysis. The nutritional value of peptides varied with the strains and the composition of the peptides, L. lactis being advantaged over Leuc. mesenteroides.     

Structural studies of the capsular polysaccharide produced by Leuconostoc mesenteroides ssp. cremoris PIA2

The structure of the capsular polysaccharide (CPS) produced by Leuconostoc mesenteroides ssp. cremoris PIA2 has been determined using component analysis and NMR spectroscopy. (1)H and (13)C resonances were assigned using 2D NMR experiments, and sequential information was obtained by (1)H,(1)H-NOESY and (1)H,(13)C-HMBC experiments. The CPS consists of linear pentasaccharide repeating units with the following structure: →3)-β-D-Galf-(1→6)-β-D-Galf-(1→2)-β-D-Galf-(1→6)-β-D-Galf-(1→3)-β-D-Galp-(1→, in which four out of the five sugar residues have the furanoid ring form, a structural entity found in bacteria but not in mammals. The analysis of the magnitude of the homonuclear three-bond coupling constants of the anomeric protons for the five-membered sugar rings indicates that the sugar residues substituted at a primary carbon atom show one kind of conformational preferences, whereas those substituted at a secondary carbon atom show another kind of conformational preferences.     

Cell wall constituents of Leuconostoc citrovorum and Leuconostoc mesenteroides

The cell wall constituents of Leuconostoc citrovorum 8082, L. mesenteroides 10830a, and L. mesenteroides 11449 have been ascertained. All three strains contained glycerol. Glucose and rhamnose were the major reducing sugar constituents. Alanine, glutamic acid, lysine, glucosamine, and muramic acid were the principal amino acids and amino sugars in all three strains. In addition, strain 10830a contained l-serine as a major cell wall component. Quantitative amino acid analyses indicate that glutamic acid, lysine, glucosamine, muramic acid, and serine may be present in the cell walls in equimolar amounts and that alanine is present in three to four times these quantities. The similarities and differences between the cell wall constituents of the leuconostocs and those of the lactobacilli and streptococci are discussed.     

Synthesis of Oligosaccharides by Growing Culture of Leuconostoc mesenteroides

When Leuconostoc mesenteroides NRRL B-1299 was grown on a medium containing sucrose and lactose, a trisaccharide named ‘Lactsucrose’ (O-β-d-galactopyranosyl- (l→4) -O-α-d-glucopyranosyl- (l→2) -β-d-fructofuranoside) was produced. This sugar was obtained as a crystalline acetyl derivative and its structure was identified by the methods of paper chromatography, paper ionophoresis, hydrolysis with acid or enzymes and periodate oxidation.     

Insoluble Glucan Formation by Leuconostoc mesenteroides B-1355

Leuconostoc mesenteroides B-1355 produced at least three glucosyltransferases (GTFs). We previously identified GTF-2 as alternansucrase and GTF-3 as fraction L dextransucrase. We here show that GTF-1 is a previously unreported sucrase that synthesized water-insoluble dextran. Our evidence consisted of the following. (i) GTF-1 was a major component and GTF-2 was a minor component of culture supernatant fractions, but supernatant fractions actively synthesized water-insoluble glucan. (ii) GTF-1 and culture supernatants produced an unusual high-pressure liquid chromatography pattern of malto-oligosaccharides that was not reproduced by GTF-2-GTF-3 mixtures. (iii) GTF-2, GTF-3, and GTF-2-GTF-3 mixtures did not synthesize insoluble glucan from sucrose. Nearly all of the alternansucrase in young (less than 17-h) cultures was associated with the cells.     

Synthesis of Oligosaccharides by Growing Culture of Leuconostoc mesenteroides

With all glucobioses (eleven types) as acceptors, Leuconostoc mesenteroides (NRRL B-512) was grown on a sucrose medium. The trisaccharides produced were analyzed for their yields, and the trisaccharide structures were determined after separation on a column.

The yield of the tri- and higher-saccharides indicated that isomaltose (28%) was the most efficient acceptor and maltose (24%) was next. With the other eight glucobioses, oligosaccharides were obtained in 3~15% yield. Among these sugars, α,β-trehalose (15%) and β,β-trehalose (11%) were efficient acceptors next to maltose, but α,α-trehalose was inert.

In every case, except for cellobiose, α-glucosyl transfer occurred to the position 6 of non-reducing moiety of glucobiose.

The sugars produced contained five new trisaccharides which were isolated as pure compounds.     

Morphological and Physiological Changes Induced by High Hydrostatic Pressure in Exponential- and Stationary-Phase Cells of Escherichia coli: Relationship with Cell Death

The relationship between a loss of viability and several morphological and physiological changes was examined with Escherichia coli strain J1 subjected to high-pressure treatment. The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min. Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells. In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium. Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments. The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells.     

Acetoin production in Leuconostoc mesenteroides NCDO 518

Abstract Cell suspensions of Leuconostoc mesenteroides NCDO 518 converted pyruvate to acetoin and a small amount of lactate and acetate. Acetoin was not produced from mixtures of pyruvate and glucose unless the ratio of pyruvate to glucose was greater than 2:1. In the presence of glucose, external pyruvate was first used as an electron acceptor, being reduced to lactate, and was converted to acetoin only after the exhaustion of glucose. Use of added pyruvate as an electron acceptor suppressed ethanol formation and the products of glucose fermentation were then lactate and acetate; 2 mol of pyruvate per mol of glucose were required to completely suppress ethanol formation. It is suggested that acetoin is produced by heterofermentative organisms when available pyruvate is in excess of that required for reoxidation of all NADH produced during glucose fermentation.