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A considerable body of evidence links together mitochondrial dysfunctions, toxic action of metalloid oxyanions, and system and neurodegenerative disorders. In this study we have used the model yeast Saccharomyces cerevisiae to investigate the genetic determinants associated with tellurite resistance/sensitivity. Nitrosoguanidine-induced K 2 TeO 3 -resistant mutants were isolated, and one of these mutants, named Sc57-Te 5 R , was characterized. Both random spore analysis and tetrad analysis and growth of heterozygous (Te S /Te 5 R ) diploid from Sc57-Te 5 R mutant revealed that nuclear and recessive mutation(s) was responsible for the resistance. To get insight into the mechanisms responsible for K 2 TeO 3 -resistance, RNA microarray analyses were performed with K 2 TeO 3 -treated and untreated Sc57-Te 5 R cells. A total of 372 differentially expressed loci were identified corresponding to 6.37% of the S. cerevisiae transcriptome. Of these, 288 transcripts were up-regulated upon K 2 TeO 3 treatment. About half of up-regulated transcripts were associated with the following molecular functions: oxidoreductase activity, structural constituent of cell wall, transporter activity. Comparative whole-genome sequencing allowed us to identify nucleotide variants distinguishing Sc57-Te 5 R from parental strain Sc57. We detected 15 CDS-inactivating mutations, and found that 3 of them affected genes coding mitochondrial ribosomal proteins (MRPL44 and NAM9) and mitochondrial ribosomal biogenesis (GEP3) pointing out to alteration of mitochondrial ribosome as main determinant of tellurite resistance.

Mitochondria are eukaryotic organelles which contain the own genetic material and evolved from free-living Eubacteria, namely hydrogen-producing Alphaproteobacteria. Since 1965, biologists provided, by research at molecular level, evidence for the prokaryotic origins of mitochondria. However, determining the precise origins of mitochondria is challenging due to inherent difficulties in phylogenetically reconstructing ancient evolutionary events. The use of new tools to evidence the prokaryotic origin of mitochondria could be useful to gain an insight into the bacterial endosymbiotic event that resulted in the permanent acquisition of bacteria, from the ancestral cell, that through time were transformed into mitochondria. Electron microscopy has shown that both proteobacterial and yeast cells during their growth in the presence of increasing amount of tellurite resulted in dose-dependent blackening of the culture due to elemental tellurium (Te(0)) that formed large deposits either along the proteobacterial membrane or along the yeast cell wall and mitochondria. Since the mitochondrial inner membrane composition is similar to that of proteobacterial membrane, in the present work we evidenced the black tellurium deposits on both, cell wall and mitochondria of ρ(+) and respiratory deficient ρ(-) mutants of yeast. A possible role of tellurite in studying the evolutionary origins of mitochondria will be discussed., (Copyright © 2015. Published by Elsevier B.V.)

Crenothrix polyspora Cohn 1870 and Clonothrix fusca Roze 1896 are two filamentous, sheathed microorganisms exhibiting complex morphological differentiation, whose phylogeny and physiology have been obscure for a long time due to the inability to cultivate them. Very recently, DNA sequencing data from uncultured C. polyspora-enriched material have suggested that Crenothrix is a methane-oxidizing gamma-proteobacterium (39). In contrast, the possible ecological function of C. fusca, originally considered a developmental stage of C. polyspora, is unknown. In this study, temporal succession of two filamentous, sheathed microorganisms resembling Cohn's Crenothrix and Roze's Clonothrix was observed by analyzing the microbial community of an artesian well by optical microscopy. Combined culture-based and culture-independent approaches enabled us to assign C. fusca to a novel subgroup of methane-oxidizing gamma-proteobacteria distinct from that of C. polyspora. This assignment was supported by (i) methane uptake and assimilation experiments, (ii) ultrastructural data showing the presence in C. fusca cytoplasm of an elaborate membrane system resembling that of methanotrophic gamma-proteobacteria, and (iii) sequencing data demonstrating the presence in its genome of a methanol dehydrogenase alpha subunit-encoding gene (mxaF) and a conventional particulate methane mono-oxygenase alpha subunit-encoding gene (pmoA) that is different from the unusual pmoA (u-pmoA) of C. polyspora.

The Italian cigar manufacturing process includes a fermentation step that leads to accumulation of nitrite and tobacco-specific nitrosamines (TSNA), undesirable by-products due to their negative impact on health. In this study, growth and biochemical properties of Debaryomyces hansenii TOB-Y7, a yeast strain that predominates during the early phase of fermentation, have been investigated. With respect to other D. hansenii collection strains (Y7426, J26, and CBS 1796), TOB-Y7 was characterized by the ability to tolerate very high nitrite levels and to utilize nitrite, but not nitrate, as a sole nitrogen source in a chemically defined medium, a property that was enhanced in microaerophilic environment. The ability to assimilate nitrite was associated to the presence of YNI1, the gene encoding the assimilatory NAD(P)H:nitrite reductase (NiR), absent in Y7426, J26, and CBS 1796 by Southern blot data. YNI1 from TOB-Y7 was entirely sequenced, and its expression was analyzed in different media by Northern blot and reverse transcriptase polymerase chain reaction. The evidence that, in D. hansenii TOB-Y7, YNI1 was transcriptional active also in the presence of high ammonia concentration typical of tobacco fermentation, stimulated the development of an improved process that, on a laboratory scale, was proved to be effective in minimizing nitrite and TSNA accumulation.

The transposition of the Ty mobile genetic element of Saccharomyces cerevisiae is induced by carcinogens. While the molecular background of spontaneous Ty1 transposition is well understood, the detailed mechanism of carcinogen induced Ty1 transposition is not clear. We found that mitochondrial functions participate in the Ty induced transposition induced by carcinogens. Contrary to the parental rho(+) cells rho(-) mutants (spontaneous or induced by ethidium bromide) do not increase the rate of Ty1 transposition upon treatment with carcinogens. Preliminary results strongly suggest that the absence of oxidative phosphorylation in rho(-) mutants is the reason for the inhibited Ty transposition. The lack of carcinogen induced Ty1 transposition in rho(-) cells is not specific for a particular carcinogen and represents a general feature of different carcinogenic substances inducing rho(-). It is concluded that carcinogen induced Ty1 transposition depends on the functional state of mitochondria and cannot take place in cells with compromised mitochondrial function (rho(-)).

Some nuclear genes in Saccharomyces cerevisiae (S. cerevisiae) respond to signals from the mitochondria in a process called by Butow (Cell Death Differ. 9 (2002) 1043-1045) retrograde regulation. Expression of these genes is activated in cells lacking mitochondrial function by involvement of RTG1, RTG2 and RTG3 genes whose protein products bind to "R-boxes" in the promoter region; RTG2p is a cytoplasmic protein. Since S. cerevisiae rho0 strains, lacking the entire mitochondrial genome, are resistant to lycorine, an alkaloid extracted from Amaryllis plants, it could be hypothesized that in rho0 cells the dysfunctional mitochondrial status stimulates overexpression of nuclear genes very likely involved in both nuclear and mitochondrial DNA replication. In this report we show that the resistance of rho0 cells to lycorine is affected by the deletion of RTG genes.

Actinomadura sp. ATCC 39727 produces the glycopeptide antibiotic A40926, structurally similar to teicoplanin. Production of A40926 is governed by the stringent response at the transcriptional level. In fact, addition of an amino acid pool prevented the transcription of dbv cluster genes involved in the A40926 biosynthesis and the antibiotic production in chemically defined media, and a thiostrepton-resistant relaxed mutant was severely impaired in its ability to produce the antibiotic. The derivative strain rif19, highly resistant to rifampicin (minimal inhibitory concentration, MIC > 200 microg ml(-1)), was isolated from the wild type strain that exhibited low resistance to rifampicin (MIC < 25 microg ml(-1)). In this strain A40926 production started earlier than in the wild type, and reached higher final levels. Moreover, the antibiotic production was not subjected to the stringent control. Molecular analysis led to the identification of two distinct rpoB alleles, rpoBS and rpoBR, in both the wild type and the rif19. rpoBR harboured the H426N missense which is responsible for rifampicin-resistance in bacteria, in addition to other nucleotide substitutions affecting the primary structure of the RNA polymerase beta-chain. Transcript analysis revealed that rpoBR was expressed at a very low level in the wild type strain during the pseudo-exponential growth phase, and that the amount of rpoBR mRNA increased during the transition to the stationary phase. In contrast, expression of rpoBR was constitutive in the rif19. The results of mRNA half-life analysis did not support the hypothesis that post-transcriptional events are responsible for the different rpoB expression patterns in the two strains, suggesting a role of transcriptional mechanisms.

Under certain in vitro (salt and temperature) conditions rRNA aggregation occurs in female inflorescences but not in leaves or pollen RNA preparations from hazelnut (Corylus avellana L.), a species of economic interest. This paper describes experiments addressing an explanation of this phenomenon. The experiments demonstrate that: (i) trans-acting factors induce rRNA aggregate formation in female inflorescences RNA preparations; (ii) these factors support aggregation also of heterologous rRNA; (iii) aggregation is a function of temperature pre-treatment of rRNA and not of source 18S rRNA; (iv) the factors inducing rRNA aggregates are sensitive to RNase; (v) antisense small nucleolar RNAs (snoRNAs) participate in rRNA aggregate formation. snoRNAs are involved in pre-rRNA spacer cleavages, and are required for the two most common types of rRNA modifications: 2'-O-ribose methylation and pseudouridylation. Even though it is questionable whether rRNA aggregation really happens in female inflorescence in vivo, the phenomenon observed in vitro may reflect the abundance of snoRNAs in these reproductive structures. In fact the level of accumulation of three tested snoRNAs, R1, U14 and U3, is much higher in female inflorescence than in leaves or pollen of hazelnut. This finding opens the possibility of studying the role of snoRNAs in tissue development in plants.

Neisseria meningitidis strains belonging to the hypervirulent lineage ET-37 and several unrelated strains are extremely UV sensitive. The phenotype is consequent to the presence of a nonfunctional recB(ET-37) allele carrying multiple missense mutations. Phenotypic analysis has been performed with congenic meningococcal strains harboring either the wild-type recB allele or the recB(ET-37) allele. Congenic recB(ET-37) meningococci, in addition to being sensitive to UV, were defective both in repair of DNA lesions induced by UV treatment and, partially, in recombination-mediated transformation. Consistently, the wild-type, but not the recB(ET-37), allele was able to complement the Escherichia coli recB21 mutation to UV resistance and proficiency in recombination. recB(ET-37) meningococci did not exhibit higher frequencies of spontaneous mutation to rifampin resistance than recB-proficient strains. However, mutation rates were enhanced following UV treatment, a phenomenon not observed in the recB-proficient counterpart. Interestingly, the results of PCR-based assays demonstrated that the presence of the recB(ET-37) allele considerably increased the frequency of recombination at the pilin loci. The main conclusion that can be drawn is that the presence of the defective recB(ET-37) allele in N. meningitidis isolates causes an increase in genetic diversity, due to an ineffective RecBCD-dependent DNA repair and recombination pathway, and an increase in pilin antigenic variation.

In this paper we report the isolation and preliminary characterisation of nuclear mutants with increased mitochondrial mutability in fission yeast. Screening of about 2000 clones after nitrosoguanidine mutagenesis led to the isolation of ten mutator mutants. For one of them (mut-1) we show that the mutation is chromosomally encoded. The activity of the mutator is restricted to the mitochondrial genome, since it increases the mutation rate to mitochondrially encoded drug resistance considerably, whereas the mutability of nuclear genes is not altered.

Expression of serogroup B meningococcal capsular polysaccharide undergoes frequent phase variation involving reversible frameshift mutations within a homopolymeric repeat in the siaD gene. A high rate of phase variation is the consequence of a biochemical defect in methyl-directed mismatch repair. The mutator phenotype is associated to the absence of DNA adenine methyltransferase (Dam) activity in all pathogenic isolates and in 50% of commensal strains. Analysis of the meningococcal dam gene region revealed that in all Dam- strains a gene encoding a putative restriction endonuclease (drg) that cleaves only the methylated DNA sequence 5'-GmeATC-3' replaced the dam gene. Insertional inactivation of the dam and/or drg genes indicated that high rates of phase variation and hypermutator phenotype are caused by absence of a functional dam gene.

In vivo analysis of expression of the chloroplast rDNA cluster during somatic embryogenesis of Daucus carota (D.carota) was performed by Northern-blot analysis with different DNA probes, spanning both the 16S rRNA gene, the 16S-23S rRNA spacer, which contains the two mosaic tRNA genes tRNA(Ile) and tRNA(Ala), and the region upstream of the 16S rRNA gene, where a tRNA(Val) maps. We show that expression both of the spacer tRNAs tRNA(Ile) and tRNA(Ala) is not significantly regulated during development whereas the amount of the transcript corresponding to tRNA(Val) is not detectable during early embryonic stages and progressively accumulates during late phases. Multiple transcription start sites have been identified upstream of the tRNA(Val) gene by S1 mapping analysis, which are activated late during the embryogenesis. These data indicate that developmental control mechanisms act on plastid gene expression during embryogenesis in carrot.