Heat resistance and genomics of spoilage Alicyclobacillus spp. Isolated from fruit juice and fruit-based beverages.

Alicyclobacillus acidoterrestris is a spore-forming bacterium of importance to the fruit juice industry due to its remarkable heat resistance and production of guaiacol taint. Whole genome sequencing analysis reveals species demarcation corresponds to the two major genotypic groups to which A. acidoterrestris isolates belong. Heat resistance was significantly different between genotypic groups 1 and 2 with D90 values of 15.5 and 9.3 min, respectively (p < 0.01). Comparison of squalene-hopene cyclase (shc) encoding sequences reveals non-synonymous changes and the alteration of glutamine residues. Glutamine absence may link to the stability reinforcement of the enzyme structure against thermal denaturation. Genomic islands harbouring heavy metal resistance genes are found in the majority of genotypic group 1 genomes (63%) but occurs in only one genome (5%) of genotypic group 2. Distribution of the genomic islands in the genotypic groups 1 and 2 is also consistent with phylogenetic trees and ANI and dDDH values. Subsequently, we propose genotypic group 1 as a new species closely related to A. acidoterrestris that possesses enhanced heat resistance.

Diversity and guaiacol production of Alicyclobacillus spp. from fruit juice and fruit-based beverages.

Alicyclobacillus acidoterrestris is an acido-thermophilic, spore-forming bacterial species that can spoil acidic fruit juice and fruit-based beverages. The metabolism of taint compounds by this bacterial species has led to its status as a targeted microorganism in the fruit juice industry. This study aims to assess the genetic diversity of Alicyclobacillus spp. including A. acidoterrestris and its correlation to spoilage taint metabolism. Alicyclobacillus cultures, which were previously isolated from a wide range of domestic and international products including fruit juice, fruit drinks and fruit juice concentrates, were subjected to DNA fingerprint analysis by using randomly amplified polymorphic DNA (RAPD) - polymerase chain reaction. Isolates were classified on the basis of their RAPD profile and the results were used to select representative strains to undergo taint production assessment. The taint guaiacol produced by Alicyclobacillus spp. was measured by headspace gas chromatography and mass spectrometry. From produced RAPD profiles, two genotypic groups and two sub-groups were identified. The groups were independent of product types and geographical origins. A significant number of isolates were clustered in genotypic group I, including A. acidoterrestris ATCC 49025. These isolates produced significant levels of guaiacol, 8.7 mg/L on average. A smaller number of isolates was found in genotypic group II including A. acidocaldarius and they produced no guaiacol. Primer F-64 was useful to identify Alicyclobacillus at the species level, and permitted rapid identification of strains producing fruit juice taint compounds such as guaiacol.

Concordance between RNA-sequencing data and DNA microarray data in transcriptome analysis of proliferative and quiescent fibroblasts.

DNA microarrays and RNA sequencing (RNA-seq) are major technologies for performing high-throughput analysis of transcript abundance. Recently, concerns have been raised regarding the concordance of data derived from the two techniques. Using cDNA libraries derived from normal human foreskin fibroblasts, we measured changes in transcript abundance as cells transitioned from proliferative growth to quiescence using both DNA microarrays and RNA-seq. The internal reproducibility of the RNA-seq data was greater than that of the microarray data. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarray values were moderate. The two technologies had good agreement when considering probes with the largest (both positive and negative) fold change (FC) values. An independent technique, quantitative reverse-transcription PCR (qRT-PCR), was used to measure the FC of 76 genes between proliferative and quiescent samples, and a higher correlation was observed between the qRT-PCR data and the RNA-seq data than between the qRT-PCR data and the microarray data.

Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq) in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq) of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A)-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.

Dissecting the involvement of formins in Bud6p-mediated cortical capture of microtubules in S. cerevisiae.

In S. cerevisiae, spindle orientation is linked to the inheritance of the ;old' spindle pole by the bud. A player in this asymmetric commitment, Bud6p, promotes cortical capture of astral microtubules. Additionally, Bud6p stimulates actin cable formation though the formin Bni1p. A relationship with the second formin, Bnr1p, is unclear. Another player is Kar9p, a protein that guides microtubules along actin cables organised by formins. Here, we ask whether formins mediate Bud6p-dependent microtubule capture beyond any links to Kar9p and actin. We found that both formins control Bud6p localisation. bni1 mutations advanced recruitment of Bud6p at the bud neck, ahead of spindle assembly, whereas bnr1Delta reduced Bud6p association with the bud neck. Accordingly, bni1 or bnr1 mutations redirected microtubule capture to or away from the bud neck, respectively. Furthermore, a Bni1p truncation that can form actin cables independently of Bud6p could not bypass a bud6Delta for microtubule capture. Conversely, Bud6(1-565)p, a truncation insufficient for correct actin organisation via formins, supported microtubule capture. Finally, Bud6p or Bud6(1-565)p associated with microtubules in vitro. Thus, surprisingly, Bud6p may promote microtubule capture independently of its links to actin organisation, whereas formins would contribute to the program of Bud6p-dependent microtubule-cortex interactions by controlling Bud6p localisation.

Inhibition, Injury, and Inactivation of Four Psychrotrophic Foodborne Bacteria by the Preservatives Methyl ρ-Hydroxybenzoate and Potassium Sorbate.

The minimum inhibitory concentrations (MICs) of methyl ρ-hydroxybenzoate (methyl paraben) and potassium sorbate for four psychrotrophic bacteria were compared at pH 5 and 6 and at 5 and 30°C. The bacteria tested were Listeria monocytogenes , Pseudomonas putida , Yersinia enterocolitica , and Aeromonas hydrophila . L. monocytogenes was generally the most resistant and A. hydrophila the least resistant to the preservatives. The differences between the bacteria were substantial. The MICs of the two preservatives were similar at pH 5, but at pH 6 the MICs of paraben were well below those of sorbate, except in the case of A. hydrophila . The MICs at 5°C were much lower than those observed at 30°C for all of the bacteria except P. putida . All four bacteria were inhibited by 1000 mg methyl paraben per L at 5°C. Exposure of the bacteria to concentrations of preservative that permitted growth at 30°C did not lead to adaptation to the preservative. The death rates of the bacteria in media containing 1000 mg methyl paraben per L varied over a wide range. At 5°C, a 3 log10 decrease in viable counts of L. monocytogenes and A. hydrophila took >4 months and a few days, respectively. Injury of L. monocytogenes , Y. enterocolitica , and A. hydrophila was detected under these conditions. Repair of the injury was demonstrated, with up to 24 h required for complete recovery. The type of buffer in which the test medium was prepared affected the preservative MICs and rate of injury of L. monocytogenes .