Microarray-based transposon mapping analysis of Enterococcus faecium E1162 in a rat endocarditis model or control medium
Ontology highlight
ABSTRACT:
ORGANISM(S): Enterococcus faecium
SUBMITTER: Fernanda Paganelli
PROVIDER: E-MTAB-4523 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Mapping of transposon mutant library in Enterococcus faecium during growth in Brain Heart Infusion (BHI) broth and in a semi-static biofilm model. The goal of this study was to identify factors that play a role in E. faecium biofilm formation by selection of transposon insertion mutants that lost the capacity to form biofilm in vitro.
2016-05-01 | E-MTAB-4524 | biostudies-arrayexpress
Project description:S. aureus has a propensity to cause endocarditis; diabetes mellitus is a frequent underlying comorbitity in patents with S. aureus endocarditis. S. aureus Affymetrix GeneChips were used to compare S. aureus expression properties in cardiac vegatations isolated from diabetic and nondiabetic rats. S. aureus Affymetrix GeneChips were also used to compare the S. aureus expression properties of cardiac vegatations (both diabetic and nondiabetic) in comparsions to planktonic cells. Few differences were observed between the expression properties of S. aureus harvested from diabetic vs. nondiabetic cardiac vegatations. Significant differences were observed between the expression properties of S. aureus harvested from cardiac vegetations in comparison to exponential and/or stationary phase planktonically grown cells. S. aureus strain COL was used to establish cardiac vegetations in diabetic and nondiabetic rats or grown in laboratory medium to exponential or stationary phase, total bacterial RNA was isolated, labeled and applied to Affymetrix GeneChips. We sought to determine whether the transcriptional profiles of S. aureus differed in diabetic vs. nondiabetic rats and whether vegetations differed from that of planktonic S. aureus.
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Project description:Transcriptional profiling of the spxA1-null mutant of Streptococcus sanguinis SK36 compared with wild type. The spxA1 gene was inactivated in Streptococcus sanguinis SK36, and the mutant demonstrated opaque colony morphology, reduced hydrogen peroxide (H2O2) production, and reduced antagonistic activity against Streptococcus S. mutans UA159 both on plates and in liquid media. The mutant also showed decreased tolerance to high temperature, and acidic and oxidative stresses. Complementation of the ΔspxA1 mutant with spxA1 restored colony morphology, H2O2 production and stress tolerance to the ΔspxA1 mutant. The mutant also exhibited an ~5-fold reduction in competitiveness in an animal model of endocarditis, indicating the involvement of SpxA1 in endocarditis virulence. Microarray studies revealed that a number of SpxA1-upregulated genes are involved in oxidative stress. The expression of spxB and nox (which encode pyruvate oxidase and NADH oxidase, respectively, and are involved in H2O2 production and nox involved virulence) significantly decreased in ΔspxA1 compared with the wild type. This may be at least partly responsible for the decreased H2O2 production and reduced virulence in the ΔspxA1 mutant because spxB and nox were involved in H2O2 production and nox involved virulence. One-condition experiment: ΔspxA1 vs. S. sanguinis SK36 cells. Biological replicates: 3 wild type, 3 ΔspxA1, independently grown and harvested. One replicate (one wild type and one ΔspxA1 mixture) per array.
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Project description:The human opportunistic pathogen Staphylococcus aureus has developed multiple strategies to adapt to various environments, to fight and escape the immune system, to spread and persist in host tissues. It is responsible for numerous diseases ranging from benign skin infections to more serious such as endocarditis or septicemia. The pathogenicity is due to the production of a multitude of virulence factors, whose synthesis is finely regulated by a combination of regulatory proteins and small non-coding RNAs (sRNAs). Our study reveals an unexpected function of an atypical sRNA, RsaC, which is at the heart of networks controlling defence responses to oxidative stress, manganese import and nutrition immunity. This work highlights a novel mechanism required for S. aureus to survive into its host.
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Project description:In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also harbor a proteasome, suggesting fates for pupylated proteins other than degradation via a proteasome or degradation at all. In the present study we set out to study pupylation in the proteasome-lacking non-pathogenic model microorganism and biotechnological workhorse Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew as the control indicating that pupylation seems to be dispensable under the conditions tested. By expression of homologous Pup carrying a poly-histidine tag in C. glutamicum ATCC 13032 we purified the first pupylome of a microorganism lacking a proteasome. Multidimensional Protein Identification Technology (MudPIT) unraveled 54 proteins being pupylated in this organism. Similar to mycobacteria, the majority of pupylated proteins in C. glutamicum can be classified as enzymes of the metabolism or as involved in translation. These results help to elucidate the common target pathways of pupylation in bacteria. Sample 1: For growth in CGXII minimal media, a preculture 1 was grown in 5 ml BHI medium inoculated with a single colony from a fresh BHI agar plate and incubated at 170 rpm for 8 hours. Then 500 M-BM-5l of preculture 1 were used to inoculate preculture 2 in 100 ml shake flasks with 20 ml CGXII minimal medium containing 4 % (w/v) glucose and incubated over night (140 rpm , 30 M-BM-0C ). Subsequent main cultures (50 ml CGXII medium with 4 % glucose) were inoculated with cells from preculture 2 to an OD600 of about 0.4. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done. Sample 2-4: For growth in CGXII minimal media, a preculture 1 was grown in 5 ml BHI medium inoculated with a single colony from a fresh BHI agar plate and incubated at 170 rpm for 8 hours. Then 500 M-BM-5l of preculture 1 were used to inoculate preculture 2 in 100 ml shake flasks with 20 ml CGXII minimal medium containing 4 % (w/v) glucose and incubated over night (140 rpm , 30 M-BM-0C ). Subsequent main cultures (50 ml CGXII medium with 4 % glucose) were inoculated with cells from preculture 2 to an OD600 of about 0.4. For growth experiments in complex BHI medium, 20 ml brain heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) were inoculated with a single colony from a fresh BHI agar plate and incubated at 140 rpm as overnight cultures. Subsequent main cultures (50 ml BHI medium) were inoculated with cells from overnight cultures to an optical density at 600 nm (OD600) of about 1. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done for each growth medium. Sample 5-6: For growth experiments in complex BHI medium, 20 ml brain heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) were inoculated with a single colony from a fresh BHI agar plate and incubated at 140 rpm as overnight cultures. Subsequent main cultures (50 ml BHI medium) were inoculated with cells from overnight cultures to an optical density at 600 nm (OD600) of about 1. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done.
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