Genetics of subtilin and nisin biosyntheses

Several peptide antibiotics have been described as potent inhibitors of bacterial growth. With respect to their biosynthesis, they can be devided into two classes: (i) those that are synthesized by a non-ribosomal mechanism and (ii) those that are ribosomally synthesized. Subtilin and nisin belong to the ribosomally synthesized peptide antibiotics. They contain the rare amino acids dehydroalanine, dehydrobutyrine, meso-lanthionine, and 3-methyl-lanthionine. They are derived from prepeptides which are post-translationally modiffied and have been termed lantibiotics because of their characteristic lanthionine bridges (Schnell et al. 1988). Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain gram-positive bacteria (Mattick & Hirsch 1944, 1947; Rayman & Hurst 1984). It is produced by Lactococcus lactis strains belonging to serological group N. The potent bactericidal activities of nisin and other lantibiotics are based on depolarization of energized bacterial cytoplasmic membranes. Breakdown of the membrane potential is initiated by the formation of pores through which molecules of low molecular weight are released. A trans-negative membrane potential of 50 to 100 mV is necessary for pore formation by nisin (Ruhr & Sahl 1985; Sahl et al. 1987). Nisin occurs as a partially amphiphilic molecule (Van de Ven et al. 1991). Apart from the detergent-like effect of nisin on cytoplasmic membranes, an inhibition of murein synthesis has also been discussed as the primary effect (Reisinger et al. 1980). In several countries nisin is used to prevent the growth of clostridia in cheese and canned food. The nisin peptide structure was first described by Gross & Morall (1971), and its structural gene was isolated in 1988 (Buchman et al. 1988; Kaletta & Entian 1989). Nisin has two natural variants, nisin A and nisin Z, which differ in a single amino acid residue at position 27 (histidin in nisin A is replaced by asparagin in nisin Z (Mulders et al. 1991; De Vos et al. 1993). Subtilin is produced by Bacillus subtilis ATCC 6633. Its chemical structure was first unravelled by Gross & Kiltz (1973) and its structural gene was isolated in 1988 (Banerjee & Hansen 1988). Subtilin shares strong similarities to nisin with an identical organization of the lanthionine ring structures (Fig. 1), and both lantibiotics possess similar antibiotic activities. Due to its easy genetic analysis B. subtilis became a very suitable model organism for the identification and characterization of genes and proteins involved in lantibiotic biosynthesis. The pathway by which nisin is produced is very similar to that of subtilin, and the proteins involved share significant homologies over the entire proteins (for review see also De Vos et al. 1995b). The respective genes have been identified adjacent to the structural genes, and are organized in operon-like structures (Fig. 2). These genes are responsible for post-translational modification, transport of the modified prepeptide, proteolytic cleavage, and immunity which prevents toxic effects on the producing bacterium. In addition to this, biosynthesis of subtilin and nisin is strongly regulated by a two-component regulatory system which consists of a histidin kinase and a response regulator protein.     

High-throughput RNA extraction from plant samples based on homogenisation by reciprocal shaking in the presence of a mixture of sand and glass beads

We describe here a reliable high-throughput method for extraction of RNA from fresh or frozen plant tissue that obviates laborious and time-consuming homogenisation by mortar and pestle. The method is based on homogenisation by high-speed reciprocal shaking in presence of a mixture of inexpensive abrasive materials; i.e., quartz sand and glass beads. After homogenisation, the method follows a standard procedure for RNA extraction by phenol/LiCl. Yield and quality of RNA obtained by homogenisation with the sand/glass bead mix are identical to those obtained by mortar and pestle.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Catecholamine stimulated lipolysis in differentiated human preadipocytes in a serum-free,defined medium

Adipocyte precursors from the stromal vascular fraction of human adipose tissue were allowed to differentiate in serum-free defined medium, whereafter their catecholamine stimulated lipolytic response was compared to that of mature isolated human adipocytes. Seventy-five to ninety percent of the fibroblast-like cells accumulated lipid droplets and glycerol-3-phosphate dehydrogenase activities of 1,000–2,800 mU/mg protein were measured in cell homogenates of differentiated cells. Lipolysis could be stimulated by both isoproterenol and norepinephrine in both differentiated preadipocytes as well as mature adipocytes. The results obtained with β-adrenergic agents suggested the presence of a higher affinity receptor in differentiated preadipocytes as compared to mature adipocytes. Mature adipocytes responded well to β-adrenergic agents, but no antilipolytic α₂-adrenergic response was observed in the differentiated preadipocytes. The presence of Gi proteins in the differentiated preadipocytes was suggested by the antilipolytic effect of adenosine as well as the lipolytic activity generated by pertussis toxin. In conclusion, our medium supported the differentiation of a very high percentage of human preadipocytes which developed a sensitive β-adrenergic lipolytic response but which lacked an α₂-adrenergic antilipolytic response.     

Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous α-amylase

The ups45 gene encodes the major extracellular protein from Lactococcus lactis. The deduced sequence of the 27 residue leader peptide revealed the tripartite characteristics of a signal peptide. This leader peptide directed the efficient secretion of the homologous proteinase (PrtP) in L. lactis, indicating that the putative signal peptide of PrtP can be replaced by the 27 residue Usp45 leader peptide. In addition, the 27 residue leader peptide could be used to secrete the Bacillus stearothermophilus α-amylase, encoded by the amyS gene. Fusion of the usp45 promoter region and various parts of the leader sequence to an amyS gene devoid of its signal sequence, showed that in Escherichia coli the first 19, 20, and 27 residues of the Usp45 leader are able to direct α-amylase secretion. In L. lactis the shorter signal peptides did not result in secretion of α-amylase, providing experimental evidence for the hypothesis that gram-positive bacteria require a longer signal peptide for secretion than gram-negative organisms.     

Photo-induction of electrical current with the cyanobacteriumAgmenellum quadruplicatum PR-6

We investigated the possibility of eliciting a measurable photoinduced electrical current from the cyanobacteriumAgmenellum quadruplicatum PR-6 (Synechococcus PCC 7002). This proved virtually impossible for intact cells. However, treated PR-6 cells fixed in an alginate matrix on tin oxide as the active electrode in a three electrode electrochemical cell gave rise to a significant light response. Cell treatments involving toluene, chloroform or detergents were effective and gave current responses up to 250 nA. Drying the cyanobacterial matrix increased the current yield at least fifty-fold. These effects were observed for light wavelengths > 650 nm and were not influenced by inhibitors or enhancers of photosynthesis nor by sustained argon bubbling of the electrolyte.French pressure cell lysates facilitated distinction between two light induced current components. Lysates prepared without CaCl₂ gave current induction kinetics that were indistinguishable from those on chemically treated cells i.e. slowly rising to a stable maximum in 10–15 min. When CaCl₂ was present during lysis, a rapidly induced (<1 s) unstable component was observed. Toluenization of PR-6 either prior to or post lysing abolished the CaCl₂ related effect. CaCl₂ had no effect on current induction in strain PR-6008, which lacked the and subunits of phycocyanin and exhibited slow current induction kinetics.The observed effects are interpreted as responses of components of the photosystems of PR-6 rather than in terms of an integrated photosynthetic process.     

Transpiration and drought resistance of Douglas-fir seedlings exposed to excess ammonium

 In a pot trial growth and transpiration of 3-year-old Douglas-fir seedlings on an acid, sandy soil was examined at a deficient (30 kg N ha^{–  1} year^{–  1}) and an excessive level (120 kg N ha^{–  1} year^{–  1}) of NH₄ application. Dissolved ammonium sulphate was applied to the pots weekly for two growing seasons. In half of the pots a complete set of other nutrients was applied in optimal proportions to the applied nitrogen. Water supply was optimal and transpiration was recorded. At the end of the second treatment season irrigation was stopped for 2 weeks during dry and sunny weather. Both high application of NH₄ and additional nutrients increased shoot growth and transpiration demand in the first treatment year. The root system was smaller at higher N level and this reduced water uptake accordingly. In the second year the combination of high NH₄ ⁺ and additional nutrients affected root functioning predominantly due to salinity effects and this seriously decreased water uptake capacity and shoot water potentials, finally resulting in tree death. Without addition of other nutrients the high NH₄ ⁺ application resulted in a high degree of soil acidification, which damaged the roots, that showed a decrease in water uptake capacity. At the low NH₄ supply level soil acidification was lower, and root functioning was not affected, and the trees recovered quickly from the imposed drought. Higher needle K and P status depressed transpiration rates at the low NH₄ application rate. Received: 9 January 1995 / Accepted: 18 September 1995     

Applications of DNA shuffling to pharmaceuticals and vaccines

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.     

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae