Project description:Transcriptional profiling of an El Tor biotype crp mutant The virulent V. cholerae El Tor Ogawa strain C7258 (Peru isolate 1991) and an isogenic deletion mutant (WL7258) lacking DNA sequences encoding the cAMP receptor protein were grown in LB medium to optical density at 600 nm of 1.5. The cultures were chilled in ice, cells quickly collected by centrifugation and total RNA imediately extracted. RNAwas extracted and purified using the Trizol plus RNA purification system (Invitrogen) followed RNEasy miniElute cleanup (Qiagen). RNA samples were conserved at - 80 C and used within a week. 4 replicates
Project description:This study assessed the development of disseminated candidiasis within Galleria mellonella larvae and characterised the proteomic responses of Candida albicans to larval hemolymph. Infection of larvae with an inoculum of 1 × 106 yeast cell reduced larval viability 24 (53.33 ± 3.33%), 48 (33.33 ± 3.33%) and 72 (6.66 ± 3.33%) hour post infection. C. albicans infection quickly disseminated from the site of inoculation and the presence of yeast and hyphal forms were found in nodules extracted from infected larvae at 6 and 24 hours. A range of proteins secreted during infection of G. mellonella were detected in larval hemolymph and these were enriched for biological processes such as interaction with host and pathogenesis. The candicidal activity of hemolymph after immediate incubation of yeast cells resulted in a decrease in yeast cell viability (0.23 ± 0.03 × 106, p < 0.05) as compared to control (0.99 ± 0.01 × 106). extracellular (in vivo) proteome of C. albicans in larval hemolymph were assessed. C. albicans responds to incubation in hemolymph ex vivo by the induction of an oxidative stress response, a decrease in proteins associated with protein synthesis and an increase in glycolytic proteins.
Project description:The transcription of the cldEFGC gene cluster of Bifidobacterium breve UCC2003 was shown to be induced upon growth on cellodextrins, implicating these genes in the metabolism of these sugars. Phenotypic analysis of a B. breve UCC2003::cldE insertion mutant confirmed that the cld gene cluster is exclusively required for cellodextrin utilization by this bacterium. HPAEC-PAD analysis of medium samples obtained during growth of B. breve UCC2003 on a mixture of cellodextrins revealed its ability to utilize cellobiose, cellotriose, cellotetraose and cellopentaose, with cellotriose representing the preferred substrate. The cldC gene of the cld operon of B. breve UCC2003 was shown to be the first described bifidobacterial β-glucosidase exhibiting hydrolytic activity towards various cellodextrins. In order to investigate differences in gene expression patterns of B. breve UCC2003 when grown on cellobiose or cellodextrins as compared to growth on glucose, DNA microarray experiments were performed. Total RNA was isolated from B. breve UCC2003 cultures grown on cellobiose, cellodextrins, or glucose (see Materials and Methods). The cultures were harvested at the time points that ensured that B. breve UCC2003 was metabolizing cellobiose or cellodextrins as opposed to the residual glucose present in the cellodextrin preparation. Analysis of the DNA microarray data was obtained from two independent biological replicates.
Project description:The differentiation of cells into distinct cell types, each of which is heritable for many generations, underlies many biological phenomena. White and opaque cells of the fungal pathogen Candida albicans are two such heritable cell types, each thought to be adapted to unique niches within their human host. To systematically investigate the differences between the two cell types, we performed strand-specific massively-parallel sequencing of RNA from C. albicans white and opaque cells. Combining the resulting data from both cell types, we first substantially re-annotated the C. albicans transcriptome, finding 1443 novel coding and non-coding transcriptionally active regions. Using the new annotation, we compared differences in transcript abundance between the two cell types with the genomic regions bound by the master regulator of the white-opaque switch (Wor1). We found that the revised transcriptional landscape considerably alters our understanding of the circuit governing differentiation. In particular, we can now resolve the poor concordance between binding of the master regulator and the differential expression of adjacent genes, a discrepancy observed in many other studies of cell differentiation. More than one third of the Wor1-bound differentially-expressed transcripts were previously unannotated, which explains the formerly puzzling presence of Wor1 at these positions along the genome. Indeed, many of these newly identified Wor1-regulated genes are non-coding and transcribed antisense to coding transcripts. We also found that 5' and 3' untranslated regions (UTRs) of mRNAs in the circuit are unusually long and that 5' UTRs often differ in length between white and opaque cells. These observations suggest that the use of alternative promoters is widespread in the circuit and that important regulatory information is carried in the long UTRs. Further analysis revealed that the revised Wor1 circuit bears several striking similarities to the Oct4 circuit that specifies the pluripotency of mammalian embryonic stem cells. Additional characteristics shared with the Oct4 circuit suggest a set of general hallmarks characteristic of heritable differentiation states in eukaryotes. RNA-Seq was applied to Candida albicans white and opaque cells to identify novel transcripts and UTRs that are differentially regulated between the two cell types. Two biological replicates each of white and opaque cell cultures. One of the white cell RNA samples was split just after isolation to allow a comparison of the poly(A)-selection and ribo-depletion sample preparation strategies.
Project description:Comparative phenotype and transcriptome analyses were performed with Bacillus cereus ATCC 14579 exposed to acid down-shock to pH 5.5 set with different acidulants. When acidified with hydrochloric acid (HCl), growth was diminished, whereas 2 mM undissociated lactic acid (HL) or acetic acid (HAc) stopped growth without inactivation (bacteriostatic condition), and 15 mM undissociated HAc caused growth arrest and, finally, cell death, as reflected by a 3 to 4 log inactivation (bactericidal condition). Within the first 60 min after pH down-shock, the intracellular ATP levels of cultures shocked with HCl were increased. The bacteriostatic pH shocks did not result in increased nor decreased intracellular ATP levels, indicating that the high energy status within the stressed aerobically grown B. cereus cells could be maintained. In contrast, exposure to 15 mM undissociated HAc resulted in significant lower ATP levels, which was in accordance with the observed inactivation. The transcriptomic responses pH down-shocked cultures were studied in the same time frame. The analyses revealed general and specific responses coupled to the different phenotypes and the acidulant used. The general acid stress response, shown in all different pH shocks, involves modulation of pyruvate metabolism and an oxidative stress response. The shifts in pyruvate metabolism include induction dehydrogenases of a butanediol fermentation pathway under non-lethal acid stress conditions and of lactate, formate, and ethanol fermentation pathways under 15 mM HAc stress. Other 15 mM HAc-specific responses were induction of the alternative electron-transport systems, including cydAB, and fatty acid biosynthesis genes. Differences in gene expression for the bacteriostatic organic acid stress conditions compared to the growth-retarded inorganic stress condition indicated a more stringent oxidative stress response, including induction of an additional catalase gene and a gene encoding a Dps-like protein. Moreover, modulations in amino acid and oligopeptide transport were also found for the 2 mM HAc and HL shocks. HL-specific and HAc-specific responses both involve amino acid metabolism. Our study on the genome-wide responses of aerobically grown B. cereus pH 5.5 shocks provides a unique overview of the different responses induced by three acidulants relevant for food preservation. Per acid down-shock three exposure times (i.e., 10, 30 and 60 min) were each compared with non-exposed cells (i.e., t0). In total 4 different pH 5.5 acid down-shocks were applied. pH 5.5 was reached by adding different acidulants i.e., hydrochloric acid (HCl), lactic acid (HL) resulting in 2 mM undissociated HL, acetic acid (HAc) resulting in 15 mM undissociated HAc, and a combination of acetic acid and hydrochloric acid (HAc/HCl) resulting in 2 mM undissociated HAc. The experiments were performed in duplicate and the duplicate samples were hybridised with a dye-swap.