Structure, Organization, and Expression of the lct Gene for Lacticin 481, a Novel Lantibiotic Produced by Lactococcus lactis

The structural gene for the lactococcal lantibiotic lacticin 481 (lct) has been identified and cloned using a degenerated 20-mer DNA oligonucleotide based on the amino-terminal 7 amino acid residues of the purified protein. The transcription of the lct gene was analyzed, and its promoter was mapped. DNA sequence analysis of the lct gene revealed an open reading frame encoding a peptide of 51 amino acids. Comparison of its deduced amino acid sequence with the amino-terminal sequence and the amino acid composition of lacticin 481 indicates that the 61-residue peptide is prelacticin 481, containing a 27-residue carboxyl-terminal propeptide and a 24-residue amino-terminal leader peptide which lacks the properties of a typical signal sequence and which is significantly different from the leaders of other lantibiotics. The predicted amino acid sequence of prolacticin 481 contains 3 cysteines, 2 serines, and 2 threonines which were not detectable in amino acid analyses of mature lacticin 481. Based on these results and on characterization by two-dimensional NMR techniques, a structural model is proposed in which 2 cysteine residues are involved in lanthionine and one in β-methyllanthionine formation, and a 4th threonine residue is dehydrated. This model predicts a molecular mass for lacticin 481 of 2,901, which is in excellent agreement with that obtained from mass spectrometry.

Semiclassical expansion of parametric correlation functions of the quantum time delay

We derive semiclassical periodic orbit expansions for a correlation function of the Wigner time delay. We consider the Fourier transform of the two-point correlation function, the form factor $K(\tau,x,y,M)$, that depends on the number of open channels $M$, a non-symmetry breaking parameter $x$, and a symmetry breaking parameter $y$. Several terms in the Taylor expansion about $\tau=0$, which depend on all parameters, are shown to be identical to those obtained from Random Matrix Theory.Comment: 21 pages, no figure

Engineering Dehydrated Amino Acid Residues in the Antimicrobial Peptide Nisin

The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering. Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L. lactis as a host. Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR. Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used. In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis. This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties.

Comparison of fibre optical measurements and discrete element simulations for the study of granulation in a spout fluidized bed

Spout fluidized beds are frequently used for the production of granules or particles through granulation. The products find application in a large variety of applications, for example detergents, fertilizers, pharmaceuticals and food. Spout fluidized beds have a number of advantageous properties, such as a high mobility of the particles, which prevents undesired agglomeration and yields excellent heat transfer properties. The particle growth mechanism in a spout fluidized bed as function of particle-droplet interaction has a profound influence on the particle morphology and thus on the product quality. Nevertheless, little is known about the details of the granulation process. This is mainly due to the fact that the granulation process is not visually accessible. In this work we use fundamental, deterministic models to enable the detailed investigation of granulation behaviour in a spout fluidized bed. A discrete element model is used describing the dynamics of the continuous gas-phase and the discrete droplets and particles. For each element momentum balances are solved. The momentum transfer among each of the three phases is described in detail at the level of individual elements. The results from the discrete element model simulations are compared with local measurements of particle volume fractions as well as particle velocities by using a novel fibre optical probe in a fluidized bed of 400 mm I.D. Simulations and experiments were carried out for three different cases using Geldart B type aluminium oxide particles: a freely bubbling fluidized bed; a spout fluidized bed without the presence of droplets and a spout fluidized bed with the presence of droplets. It is demonstrated how the discrete element model can be used to obtain information about the interaction of the discrete phases, i.e. the growth zone in a spout fluidized bed. Eventually this kind of information can be used to obtain closure information required in more coarse grained models

MOTIFATOR: detection and characterization of regulatory motifs using prokaryote transcriptome data

Summary: Unraveling regulatory mechanisms (e.g. identification of motifs in cis-regulatory regions) remains a major challenge in the analysis of transcriptome experiments. Existing applications identify putative motifs from gene lists obtained at rather arbitrary cutoff and require additional manual processing steps. Our standalone application MOTIFATOR identifies the most optimal parameters for motif discovery and creates an interactive visualization of the results. Discovered putative motifs are functionally characterized, thereby providing valuable insight in the biological processes that could be controlled by the motif.

Influence of Amino Acid Substitutions in the Nisin Leader Peptide on Biosynthesis and Secretion of Nisin by Lactococcus lactis

Structural genes for small lanthionine-containing antimicrobial peptides, known as lantibiotics, encode N-terminal leader sequences which are not present in the mature peptide, but are cleaved off at some stage in the maturation process. Leader sequences of the different lantibiotics share a number of identical amino acid residues, but they are clearly different from sec-dependent protein export signal sequences. We studied the role of the leader sequence of the lantibiotic nisin, which is produced and secreted by Lactococcus lactis, by creating site-directed mutations at various positions in the leader peptide sequence. Mutations at Arg-1 and Ala-4, but not at the conserved Pro-2, strongly affected the processing of the leader sequence and resulted in the extracellular accumulation of a biologically inactive precursor peptide. Amino acid analysis and 1H NMR studies indicated that the precursor peptide with an Ala-4 → Asp mutation contained a modified nisin structural part with the (mutated) unmodified leader sequence still attached to it. The Ala-4 → Asp precursor peptide could be activated in vitro by enzymatic cleavage with trypsin, liberating nisin. These results confirmed that cleavage of the leader peptide is the last step in nisin maturation and is necessary to generate a biologically active peptide. Several mutations, i.e. Pro-2 → Gly, Pro-2 → Val, Asp-7 → Ala, Lys-9 → Leu, Ser-10 → Ala/Ser-12 → Ala and Val-11 → Asp/Val-13 → Glu in the leader peptide did not have any detectable effect on nisin production and secretion, although some of them affected highly conserved residues. When mutations were created in the -18 to -15 region of the nisin leader peptide (i.e. Phe-18 → Leu, Leu-16 → Lys, Asp-15 → Ala), no secretion or intracellular accumulation could be detected of nisin or its precursors. This suggested that these conserved residues are involved in the maturation process and may interact with lantibiotic-specific modifying enzymes.

Maturation pathway of nisin and other lantibiotics:post-translationally modified antimicrobial peptides exported by Gram-positive bacteria

Lantibiotics form a family of highly modified peptides which are secreted by several Gram-positive bacteria. They exhibit antimicrobial activity, mainly against other Gram-positive bacteria, by forming pores in the cellular membrane. These antimicrobial peptides are ribosomally synthesized and contain leader peptides which do not show the characteristics of signal sequences. Several amino acid residues of the precursor lantibiotic are enzymatically modified, whereafter secretion and processing of the leader peptide takes place, yielding the active antimicrobial substance. For several lantibiotics the gene clusters encoding biosynthetic enzymes, translocator proteins, self-protection proteins, processing enzymes and regulatory proteins have been identified. This MicroReview describes the current knowledge about the biosynthetic, immunity and regulatory processes leading to lantibiotic production. Most of the attention is focused on the lantibiotic nisin, which is produced by the food-grade bacterium Lactococcus lactis and is widely used as a preservative in the food industry

Semiclassical universality of parametric spectral correlations

We consider quantum systems with a chaotic classical limit that depend on an external parameter, and study correlations between the spectra at different parameter values. In particular, we consider the parametric spectral form factor $K(\tau,x)$ which depends on a scaled parameter difference $x$. For parameter variations that do not change the symmetry of the system we show by using semiclassical periodic orbit expansions that the small $\tau$ expansion of the form factor agrees with Random Matrix Theory for systems with and without time reversal symmetry.Comment: 18 pages, no figure

Molecular Dynamics Simulation of Solvent-Polymer Interdiffusion. I. Fickian diffusion

The interdiffusion of a solvent into a polymer melt has been studied using large scale molecular dynamics and Monte Carlo simulation techniques. The solvent concentration profile and weight gain by the polymer have been measured as a function of time. The weight gain is found to scale as t^{1/2}, which is expected for Fickian type of diffusion. The concentration profiles are fit very well assuming Fick's second law with a constant diffusivity. The diffusivity found from fitting Fick's second law is found to be independent of time and equal to the self diffusion constant in the dilute solvent limit. We separately calculated the diffusivity as a function of concentration using the Darken equation and found that the diffusivity is essentially constant for the concentration range relevant for interdiffusion.Comment: 17 pages and 7 figure