Exploiting heterozygosity in industrial yeasts to create new and improved baker's yeasts.

The main aim of the work was to utilize heterozygosity of industrial yeast strains to construct new baker's yeast strains. Commercial baker's yeast strain ALKO 743, its more ethanol tolerant descendant ALKO 554 selected initially for growth over 300 generations in increasing ethanol concentrations in a glucose medium, and ALKO 3460 from an old domestic sour dough starter were used as starting strains. Isolated meiotic segregants of the strains were characterized genetically for sporulation ability and mating type, and the ploidy was determined physically. Heterozygosity of the segregant strains was estimated by a variety of molecular characterizations and fermentation and growth assays. The results showed wide heterozygosity and that the segregants were clustered into subgroups. This clustering was used for choosing distantly or closely related partners for strain construction crosses. Intrastrain hybrids made with segregants of ALKO 743 showed 16-24% hybrid vigour or heterosis. Interstrain hybrids with segregants of ALKO 743 and ALKO 3460 showed a wide variety of characteristics but also clear heterosis of 27-31% effects as assayed by lean and sugar dough raising. Distiller's yeast ALKO 554 turned out to be a diploid genetic segregant and not just a more ethanol tolerant mutant of the tetraploid parent strain ALKO 743.

Effects of yeasts and bacteria on the levels of folates in rye sourdoughs.

Fermentation of rye dough is often accompanied with an increase in folate content. In this study, three sourdough yeasts, Candida milleri CBS 8195, Saccharomyces cerevisiae TS 146, and Torulaspora delbrueckii TS 207; a control, baker's yeast S. cerevisiae ALKO 743; and four Lactobacillus spp., L. acidophilus TSB 262, L. brevis TSB 307, L. plantarum TSB 304, and L. sanfranciscensis TSB 299 originally isolated from rye sourdough were examined for their abilities to produce or consume folates. The microorganisms were grown in yeast extract-peptone-d-glucose medium as well as in small-scale fermentations that modelled the sourdough fermentation step used in rye baking. Total folate contents were determined using Lactobacillus rhamnosus (ATCC 7469) as the growth indicator organism. The microorganisms studied did not excrete folates into the media in significant amounts. Yeasts increased the folate contents of sterilised rye flour-water mixtures from 6.5 microg/100 g to between 15 and 23 microg/100 g after 19-h fermentation, whereas lactic acid bacteria decreased it to between 2.9 and 4.2 microg/100 g. Strains of Lactobacillus bulgaricus, L. casei, L. curvatus, L. fermentum, L. helveticus, Pediococcus spp., and Streptococcus thermophilus that were also tested gave folate contents after fermentation that varied between 2 and 10.4 microg/100 g. Although the four Lactobacillus spp. from sourdough consumed folates their effect on folate contents in co-cultivations was minimal. It was concluded that the increase of folate content during fermentation was mainly due to folate synthesis by yeasts. Fermentation of non-sterilised flour-water mixtures as such resulted in three-fold increases in the folate contents. Two folate producing bacteria were isolated from the non-sterilised flour and identified as Enterobacter cowanii and Pantoea agglomerans.

Species' identification and microarray-based comparative genome analysis of Streptomyces species isolated from potato scab lesions in Norway.

Streptomyces strains were isolated from scab lesions on potatoes collected from different parts of Norway. Twenty-eight plant-pathogenic strains, as tested on seedlings of radish and on potato, were identified on the basis of physiological and molecular criteria. Polymerase chain reaction (PCR) analysis, using species-specific primers, and sequencing of the 16S rRNA gene identified 14 nonmelanin-producing strains to S. turgidiscabies. Fourteen melanin-producing strains were detected with primers specific to S. scabies, but whole-genome microarray analysis, based on 12 766 probes designed for 8848 predicted open reading frames (ORFs) of S. scabies, showed that the 14 strains were different from S. scabies. They were subsequently identified to be S. europaeiscabiei based on the internal transcribed spacer (ITS) sequences of the rRNA genes. This is the first report of the occurrence of S. turgidiscabies and S. europaeiscabiei in Norway. The putative 762 genes exhibiting the highest sequence differences between strains of S. europaeiscabiei and S. scabies according to microarray analysis were concentrated in relatively few gene ontology (GO) categories, including 'symbiosis and mutualism through parasitism', 'cell death' and 'responses to biotic stimulus', whereas genes related to primary metabolism appeared to be more conserved. Microarray data and 16S rRNA gene phylogeny showed, consistently, that there were two genetically distinguishable groups of S. europaeiscabiei on the basis of differences in 131 genes. The results provide novel information about the genetic variability of S. europaeiscabiei and the gene-specific variability between the genomes of S. europaeiscabiei and S. scabies. The usefulness of a custom-designed, whole-genome oligonucleotide microarray in a survey of bacterial plant pathogens was demonstrated.

Microarray-based comparison of genetic differences between strains of Streptomyces turgidiscabies with focus on the pathogenicity island.

The areas of the pathogenicity island (PAI) designated as 'colonization region' (CR) and 'toxicogenic region' (TR) [Lerat et al. (2009) Mol. Plant Pathol. 10, 579-585] contain genes required for virulence and phytoxin production, respectively, in Streptomyces spp. causing common scab on potatoes. The PAI was tested for genetic variability by microarray analysis in strains of S. turgidiscabies isolated from potatoes in Finland. The data revealed four types of PAI based on divergent CR and TR which occurred in different combinations. Only one PAI type was highly similar to S. scabies (strains 87.22 and ATTC49173). Using probes designed for the predicted genes of S. scabies, two gene clusters in S. scabies appeared to be similar to most strains of S. turgidiscabies and contained PAI genes corresponding to CR and TR. They were located approximately 5 Mb apart in the S. scabies genome, as compared with only 0.3 Mb in S. turgidiscabies Car8. Data from comparative genomic hybridization with probes designed for S. scabies genes and for the PAI of S. turgidiscabies were compared by multilocus cluster analysis, which revealed two strains of S. turgidiscabies that were very closely related at the whole-genome level, but contained distinctly different PAIs. The type strain of S. reticuliscabiei (DSM41804; synonymous to S. turgidiscabies) was clustered with S. turgidiscabies. Taken together, the data indicate wide genetic variability of PAIs among strains of S. turgidiscabies, and demonstrate that PAI is made up of a mosaic of regions which may undergo independent evolution.

Microarray profiling of host-extract-induced genes and characterization of the type VI secretion cluster in the potato pathogen Pectobacterium atrosepticum.

Pectobacterium atrosepticum is a Gram-negative plant-pathogenic bacterium that rots potato stems and tubers. Microarray analysis was used to identify genes that were differentially expressed when host extracts were added to the growth medium. Potato extracts downregulated the expression of ribosomal genes and genes related to uptake and metabolism of nutrients, and upregulated genes needed for nitrate or phosphonate use. Some of the observed changes in gene expression in host-extract-induced cultures are similar to those during attachment of the bacterium to host tissues. Other responses indicated defence against toxic metabolites in the extract. Tuber extract induced a large gene cluster having homology to type VI secretion genes shown to be virulence determinants in many, but not all, animal and human pathogens. Two of the genes in the type VI cluster were found to be expressed during infection in potato tubers and stems, and mutants with knockouts of the corresponding genes had increased virulence on potato. One of the type VI secretion mutants was further characterized and found to grow to higher cell density in culture in the presence of host extract and to produce slightly more extracellular tissue-macerating enzymes than the wild-type strain. Analysis of secreted proteins showed that this type VI mutant was affected in the production of haemolysin-coregulated proteins (Hcps), which have been suggested to be secreted by the type VI pathway in other bacteria. The results suggest that the type VI secretion system of P. atrosepticum is needed for secretion of Hcps but not for virulence on its host plant, potato.