Two nuclear genes, Nicl and Nic2, regulate nicotine levels in tobacco. nicl and nic2 are semidominant mutations in Burley 21 that reduce leaf nicotine levels and the activities of multiple enzymes in the nicotine pathway and simultaneously increase polyamine levels in cultured roots. Cultured roots homozygous for both mutations were used to isolate two cDNAs by subtraction hybridization; the transcript levels of these two cDNAs were much lower in the mutant roots than in the wild-type roots. The A411 gene encodes a 41-kD protein with considerable homology to mammalian spermidine synthase, whereas the A622 gene encodes a 35-kD protein with high homology to isoflavone reductase. When these genes were expressed in Escherichia coli, A411 had no spermidine synthase activity but did show putrescine N-methyltransferase activity, which is the first enzyme committed to the nicotine biosynthetic pathway, and A622 did not show isoflavone reductase activity. Both the methyltransferase and A622 genes are predominantly expressed in the root, and their expression levels in cultured roots are coordinately decreased by the nic mutations in the order of wild type > nic2 > nicl > nicl nic2. Removal of tobacco flower heads and young leaves rapidly and coordinately induced both genes in the root. Further, exogenous supply of auxin down-regulated both genes in cultured tobacco roots. These results suggest that N i c l and Nic2 are regulatory genes for nicotine biosynthesis.

The antimicrobial mechanism and structural changes of hen egg white lysozyme irreversibly inactivated at 80 °C and at different pHs were investigated. We found that heat denaturation of lysozyme at increasing temperatures for 20 min at pH 6.0 results in progressive loss of enzyme activity while greatly promotes its antimicrobial action to Gram-negative bacteria. Interestingly, lysozyme devoid of enzyme activity (heated at 80 °C and pH 7.0 or at pH 6.0 over 90 °C) exhibited strong bactericidal activity against Gram-negative and -positive bacteria, suggesting action independent of catalytic function. The most potent antimicrobial lysozyme to either Gram-negative or -positive bacteria was that heated at 80 °C and pH 6.0 (HLz80/6), retaining 50% of the native enzymatic activity, which exhibited a 14-fold increase in surface hydrophobicity, with two exposed thiol groups. HLz80/6-induced agglutination coincided with severe reduction in colony-forming ability of the susceptible bacteria in a dose-dependent manner. Denatured lysozyme HLz80/6 showed promoted binding capacity to peptidoglycan of Staphylococcus aureus and lipopolysaccharide of Escherichia coli as assessed by ELISA. Addition of HLz80/6 to E. coli phospholipid vesicles resulted in a blue shift in the intrinsic tryptophan fluorescence accompanied by an increase in the size of the vesicles, indicating enhanced protein−membrane binding and subsequent fusion of liposomes. Direct membrane damage of E. coli membrane by HLz80/6 was revealed by electron microscopy observation. Thus, the results introduce an interesting finding that partial unfolding of lysozyme with the proper acquisition of the hydrophobic pocket to the surface can switch its antimicrobial activity to include Gram-negative bacteria without a detrimental effect on the inherent bactericidal effect against Gram-positive ones. The data suggest that the unique antimicrobial action of unfolded lysozyme attributes to membrane binding and subsequent perturbation of its functions. Keywords: Lysozyme; conformational changes; antimicrobial action; agglutination; membrane interaction and fusion

Two nuclear genes, Nic1 and Nic2, regulate nicotine levels in tobacco. nic1 and nic2 are semidominant mutations in Burley 21 that reduce leaf nicotine levels and the activities of multiple enzymes in the nicotine pathway and simultaneously increase polyamine levels in cultured roots. Cultured roots homozygous for both mutations were used to isolate two cDNAs by subtraction hybridization; the transcript levels of these two cDNAs were much lower in the mutant roots than in the wild-type roots. The A411 gene encodes a 41-kD protein with considerable homology to mammalian spermidine synthase, whereas the A622 gene encodes a 35-kD protein with high homology to isoflavone reductase. When these genes were expressed in Escherichia coli, A411 had no spermidine synthase activity but did show putrescine N-methyltransferase activity, which is the first enzyme committed to the nicotine biosynthetic pathway, and A622 did not show isoflavone reductase activity. Both the methyltransferase and A622 genes are predominantly expressed in the root, and their expression levels in cultured roots are coordinately decreased by the nic mutations in the order of wild type > nic2 > nic1 > nic1 nic2. Removal of tobacco flower heads and young leaves rapidly and coordinately induced both genes in the root. Further, exogenous supply of auxin down-regulated both genes in cultured tobacco roots. These results suggest that Nic1 and Nic2 are regulatory genes for nicotine biosynthesis.

It was recently found that partial denaturation of hen egg white lysozyme at 80 °C for 20 min at pH 6.0 produces a strong bactericidal lysozyme (HLz) not only against Gram-positive bacteria but also against the Gram-negative ones. The novel antimicrobial action of the mildly denatured lysozyme was found to operate through a membrane-disrupting mechanism independent of its muramidase activity. To evaluate the role of Ca2+ and Mg2+, which are known to stabilize the structure of bacterial membranes, in the antimicrobial activity of HLz, the antimicrobial activity in the presence of divalent cations and chelator EDTA was tested. The antimicrobial activity of HLz against test microorganisms Staphylococcus aureus and Escherichia coli K12 progressively decreased with an increase in Ca2+ or Mg2+ concentration, whereas 1 mM cation produced nonbactericidal lysozyme, either the native (NLz) or the heated enzyme (HLz). However, the inhibitory effect of divalent cations on the antimicrobial activity was more pronounced on HLz than on NLz. The antimicrobial activity of HLz against Gram-negative E. coli, which has been abolished in the presence of free Ca2+ (1 mM), was recovered by the addition of 1 mM EDTA, indicating the competition between free and membrane-bound Ca2+ on the part of HLz molecule. Addition of increasing concentration of NaCl up to 10 mM had no inhibitory effect on the strong antimicrobial activity of HLz. Thus, the inhibitory effect of divalent cations on the activity of HLz is unlikely to be simply due to charge suppression. Intrinsic fluorescence analysis revealed that Ca2+ induces conformational change of the HLz molecules, thus providing evidence that HLz exerts its antimicrobial action against Gram-negative bacteria by disrupting the normal electrostatic interactions between divalent cations and components of the outer membrane. Structural changes relevant to divalent cations sensitization of HLz are discussed. Keywords: Lysozyme; conformational changes; cyclic imide; antimicrobial action; calcium binding; tryptic peptides

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