Performance of Lactobacillus helveticus Spontaneous Phage-resistant Mutants in Hard Cheese Production

From the strain Lactobacillus helveticus ATCC 15807, a total of 66 clones were isolated using the lytic phages hv and ATCC 15807-B1. Phenotypic parameters related to their phage resistance capacity (efficiency of plaquing, phage resistance stability, lysogeny and adsorption rates) were determined. The morphological, biochemical (sugar fermentation patterns) and technological (acidifying power, proteolytic activity and slime production) characteristics of the isolates were also studied. Phage resistance stability was a variable property among the isolates but a high level of resistance was exhibited as quantified by the efficiency of plaquing. Furthermore, a total absence of acquired lysogeny was demonstrated. The phage-resistant variants were completely or partially unable to bind phages and did not show differences with Lb. helveticus ATCC 15807 in their biochemical characteristics. However, the technological properties (acidifying powers and proteolytic activities) were heterogeneously distributed among the mutants. The variant RB1-28 (isolated under selective pressure of the phage ATCC 15807-B1) was evaluated for the technological performance in Sardo cheese production in comparison with Lb. helveticus ATCC 15807. There were no significant differences between the two types of cheeses, taking in consideration physicochemical parameters (proteolysis level, dry matter, total and soluble proteins, moisture), microbiological counts or sensory analysis. These phage-resistant variants could be employed in starter strain rotation programs for cheesemaking.     

Antagonism, non-native interactions and non-two-state folding in S6 revealed by double-mutant cycle analysis

When folding to the native state N in the presence of salt, the apparent two-state folder S6 transiently forms a transient off-pathway state C with substantial secondary and tertiary structure. Fifteen double mutant cycles were analysed to compare side-chain interaction energies DeltaDeltaG(int) in C, N and TS (the transition state between N and the denatured state). The kinetic signatures of these destabilizing mutants suggest folding scenarios involving unfolding intermediates and even alternative unfolding pathways. However, restricting the kinetic data to linear parts of the chevron plot allows reliable extrapolation to zero molar denaturant of rate constants of folding, unfolding and misfolding. Side-chain interactions appear to contribute to the stability of C, but in a substantially non-native environment, as shown by changes in the sign of DeltaDeltaG(int) between C and N. Remarkably, there appear to be significant (0.7-2 kcal/mol) antagonistic interactions between the two residues Leu30 and Leu75 in N and TS, which may be linked to subtle structural changes seen in the crystal structures of the mutants. A small number of overlapping residues are involved in these kinds of antagonistic interactions in N, TS and C, suggesting that repulsive interactions are coded into the protein topology whether the protein folds or misfolds. Destabilizing double mutants indicate that apparent two-state folders can be induced to behave in more complex ways provided that the native state is suitably destabilized.     

大观霉素高产菌的诱变选育和发酵条件研究

大观霉素产生菌Streptomyces spectabilisDG8#经紫外线诱变(30W、40cm、60s),氮离子注入(50keV,5×1014ion/cm2)后分离,在含有大观霉素的浓度梯度平板上筛选大观霉素抗性突变株,得到大观霉素抗性突变株SP0403,其发酵效价较出发菌株提高了69.4%,并且遗传特性稳定。对其进行了接种量、种龄、通气量和补料方面的探索。     

Metabolic regulation in the yeast Hansenula polymorpha. Growth of dihydroxyacetone kinase/glycerol kinase-negative mutants on mixtures of methanol and xylose in continuous cultures

The physiological responses of Hansenula polymorpha wild-type and mutant strains 17B (dihydroxyacetone kinase-negative) and 17BG51 (dihydroxyacetone kinase- and glycerol kinase-negative) to growth on mixtures of xylose and methanol in chemostats were investigated. Increasing methanol concentrations (0–110 mM ) in the feed of the wild-type culture resulted in increasing cell densities and a gradual switch towards methanol metabolism. At the lower methanol feed concentrations the mutant cultures used methanol and xylose to completion and changes in enzyme patterns comparable to the wild type were observed. This was not reflected in significant changes in cell densities. Instead, formaldehyde assimilation resulted in dihydroxyacetone (DHA) production, which was proportional to the amount of methanol added. At intermediate methanol concentration the cultures showed a strong variation in DHA levels and cell densities. Further increased in the methanol feed concentrations resulted in a drop in DHA accumulation rates, repression of alcohol oxidase synthesis and accumulation of residual methanol. The phenomena were studied in more detail in transition experiments and with gradients of methanol. The results indicate that xylulose-5-phosphate (Xu5P) generated in xylose metabolism served as acceptor molecule for formaldehyde assimilation by the peroxisomal enzyme DHA synthase. Accumulation of DHA in the mutant cultures, however, further diminished the availability of carbon for growth. The data suggest that with increasing methanol concentrations Xu5P eventually became growth rate limiting. This resulted in an unstable situation but wash-out of the culture did not occur to a significant extent. Instead, DHA accumulation ceased and cell densities, and enzymes specifically involved in xylose metabolism increase, indicating that the organism resumed its xylose metabolism. The molecular mechanisms controlling the partitioning of Xu5P over xylose (pentose phosphate pathway) and methanol (peroxisome) metabolism under these conditions remain to be elucidated.     

Assembly of amine oxidase and D-amino acid oxidase in the cytosol of peroxisome-deficient mutants of the yeast Hansenula polymorpha during growth of cells on glucose in the presence of primary amines or D-alanine as the sole nitrogen source

We have studied growth of two peroxisome-deficient mutant strains of Hansenula polymorpha on glucose in the presence of different organic nitrogen sources (methylamine, ethylamine and D -alanine), the metabolism of which is mediated by peroxisome-borne oxidases in wild-type (WT) cells. Both strains grew well on each of these substrates with growth rates comparable to WT cells. Growth on both methylamine and ethylamine was associated with enhanced levels of catalase and amine oxidase in the cells; in D -alanine-grown cells D -amino acid oxidase activity and increased. In WT cells of H-polymorpha the activities of these enzymes were confined to the peroxisomal matrix; however, in both peroxisome-deficient strains their activities were localized in the cytosol. Electron microscopy indicated that, dependent on the stage of growth, the enzymes may form large protein aggregates. The molecular masses of both amine oxidase and D -amino acid oxidase in the mutant strains were identical to their respective counterparts in WT cells, indicating that both proteins were correctly assembled and active in the cytosol.     

Urban Interactive

Valentina Croci profiles the work of entertainment design office Area/Code which foregrounds urban locations in its games. As Croci explains the approach of this NewYork-based partnership, which delivers sat nav-inspired gaming to the mobile phone, demonstrates an inherent understanding of the importance of the physical in enhancing the imaginary. Copyright © 2010 John Wiley & Sons, Ltd.     

Mutations in five loci affecting GAP1-independent uptake of neutral amino acids in yeast

In order to identify genes involved in uptake of isoleucine, leucine and valine in Saccharomyces cerevisiae we isolated mutants that, on a complex medium, were sensitive to an inhibitor of the biosynthesis of the branched-chain amino acids. Mutants that in a secondary screen showed reduced uptake of isoleucine, leucine and valine when growing in synthetic complete medium were further characterized. Genetic analysis identified five loci, named ssy1 through ssy5. ssy2 corresponds to the previously characterized bap1 mutation, which we recently have found to be allelic to stp1. ssy1, ssy3 and ssy5 exhibit a reduced uptake of phenylalanine, methionine and threonine, as well. Furthermore, they are resistant to several neutral amino acid analogs. ssy4 only affects uptake of few neutral amino acids and is as sensitive as the wild type to the amino acid analogs tested. It was previously found that a C-terminal truncation of 29 codons of BAP2, which encodes a branched-chain amino acid permease, results in increased uptake of the branched-chain amino acids. We find epistasis of the C-terminally truncated BAP2 gene over the ssy4 mutation, while the other ssy mutations are epistatic over the truncated BAP2 gene. SSY1, SSY3 and SSY5 were cloned from a low-copy genomic library by complementation of the mutants. The SSY3 gene and the SSY5 gene show no significant homology to any sequence in the databases. SSY1 is a member of the major family of genes encoding amino acid permeases in yeast. We discuss possible roles of Ssy1p in amino acid uptake. © 1998 John Wiley & Sons, Ltd.