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Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur- and PerR-transcriptomes.

Butcher J, Handley RA, van Vliet AH, Stintzi A - BMC Genomics (2015)

Bottom Line: It was found that 202 genes were differentially expressed in at least one mutant under iron-replete conditions and 331 genes were differentially expressed in at least one mutant under iron-limited conditions.The CjFur and CjPerR transcriptomes characterized in this study were compared to those previously identified using microarray profiling and found to be more extensive than previously understood.Moreover, subsets of genes were found which are only differentially expressed when both CjFur and CjPerR are deleted and includes genes that appear to be simultaneously activated by CjFur and repressed by CjPerR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada. jbutc076@uottawa.ca.

ABSTRACT

Background: The genome of Campylobacter jejuni contains two iron activated Fur-family transcriptional regulators, CjFur and CjPerR, which are primarily responsible for regulating iron homeostasis and oxidative stress respectively. Both transcriptional regulators have been previously implicated in regulating diverse functions beyond their primary roles in C. jejuni. To further characterize their regulatory networks, RNA-seq was used to define the transcriptional profiles of C. jejuni NCTC11168 wild type, Δfur, ΔperR and ΔfurΔperR isogenic deletion mutants under both iron-replete and iron-limited conditions.

Results: It was found that 202 genes were differentially expressed in at least one mutant under iron-replete conditions and 331 genes were differentially expressed in at least one mutant under iron-limited conditions. The CjFur and CjPerR transcriptomes characterized in this study were compared to those previously identified using microarray profiling and found to be more extensive than previously understood. Interestingly, our results indicate that CjFur/CjPerR appear to co-regulate the expression of flagellar biogenesis genes in an opposing and iron-independent fashion. Moreover the ΔfurΔperR isogenic deletion mutant revealed that CjFur and CjPerR can compensate for each other in certain cases, suggesting that both regulators may compete for binding to specific promoters.

Conclusions: The CjFur and CjPerR transcriptomes are larger than previously reported. In particular, deletion of perR results in the differential expression of a large group of genes in the absence of iron, suggesting that CjPerR may also regulate genes in an iron-independent manner, similar to what has already been demonstrated with CjFur. Moreover, subsets of genes were found which are only differentially expressed when both CjFur and CjPerR are deleted and includes genes that appear to be simultaneously activated by CjFur and repressed by CjPerR. In particular the iron-independent co-regulation of flagellar biogenesis by CjFur/CjPerR represents a potentially novel regulatory function for these proteins. These findings represent additional modes of co-regulation by these two transcriptional regulators in C. jejuni.

No MeSH data available.


Related in: MedlinePlus

RT-qPCR validation of genes under apparent inverse regulation by CjFur/CjPerR under iron limitation. RT-qPCR of selected genes in the Δfur (Panel a), ΔperR (Panel b) and ΔfurΔperR (Panel c) strains under iron limitation. Genes colored in blue show reduced expression in the Δfur mutant, increased expression in the ΔperR mutant and no change in expression in the ΔfurΔperR mutant in both the RNA-seq data and the RT-qPCR validation. Genes in green are differentially expressed in the ΔperR/ΔfurΔperR mutants via RT-qPCR but only the ΔperR under RNA-seq. Cj1386 was found to be differentially expressed in the ΔperR/ΔfurΔperR mutants under both techniques. AhpC and chuA are negative controls for metal dependant CjFur/CjPerR regulation. All expression values are relative to wild-type and normalized to slyD. Asterisks denote fold changes >1.5 with a p value <0.05
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Fig4: RT-qPCR validation of genes under apparent inverse regulation by CjFur/CjPerR under iron limitation. RT-qPCR of selected genes in the Δfur (Panel a), ΔperR (Panel b) and ΔfurΔperR (Panel c) strains under iron limitation. Genes colored in blue show reduced expression in the Δfur mutant, increased expression in the ΔperR mutant and no change in expression in the ΔfurΔperR mutant in both the RNA-seq data and the RT-qPCR validation. Genes in green are differentially expressed in the ΔperR/ΔfurΔperR mutants via RT-qPCR but only the ΔperR under RNA-seq. Cj1386 was found to be differentially expressed in the ΔperR/ΔfurΔperR mutants under both techniques. AhpC and chuA are negative controls for metal dependant CjFur/CjPerR regulation. All expression values are relative to wild-type and normalized to slyD. Asterisks denote fold changes >1.5 with a p value <0.05

Mentions: The results from the iron-independent RNA-seq experiments were confirmed using RT-qPCR (Fig. 4). Specifically, genes that appeared to show inverse CjFur/CjPerR regulation with no difference in the ΔfurΔperR mutant were chosen for validation. As shown in Fig. 4, flgE1, flgE2, flgH, and fliK all showed the same expression pattern as identified using the RNA-seq analysis (flgG was the lone exception). Moreover ahpC and chuA, two well characterized metal dependent CjPerR and CjFur targets, were not differentially expressed in any of the strains under iron limitation asserting the experimental condition. In addition cj0414, cj0415 and cj1386 were confirmed to be differentially expressed in the ΔperR mutant. Interestingly, all three were also differentially expressed in the ΔfurΔperR mutant while only the cj1386 was identified using RNA-seq (although the cj0414/cj0415 do show reduced expression in the ΔfurΔperR mutant).Fig. 4


Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur- and PerR-transcriptomes.

Butcher J, Handley RA, van Vliet AH, Stintzi A - BMC Genomics (2015)

RT-qPCR validation of genes under apparent inverse regulation by CjFur/CjPerR under iron limitation. RT-qPCR of selected genes in the Δfur (Panel a), ΔperR (Panel b) and ΔfurΔperR (Panel c) strains under iron limitation. Genes colored in blue show reduced expression in the Δfur mutant, increased expression in the ΔperR mutant and no change in expression in the ΔfurΔperR mutant in both the RNA-seq data and the RT-qPCR validation. Genes in green are differentially expressed in the ΔperR/ΔfurΔperR mutants via RT-qPCR but only the ΔperR under RNA-seq. Cj1386 was found to be differentially expressed in the ΔperR/ΔfurΔperR mutants under both techniques. AhpC and chuA are negative controls for metal dependant CjFur/CjPerR regulation. All expression values are relative to wild-type and normalized to slyD. Asterisks denote fold changes >1.5 with a p value <0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491227&req=5

Fig4: RT-qPCR validation of genes under apparent inverse regulation by CjFur/CjPerR under iron limitation. RT-qPCR of selected genes in the Δfur (Panel a), ΔperR (Panel b) and ΔfurΔperR (Panel c) strains under iron limitation. Genes colored in blue show reduced expression in the Δfur mutant, increased expression in the ΔperR mutant and no change in expression in the ΔfurΔperR mutant in both the RNA-seq data and the RT-qPCR validation. Genes in green are differentially expressed in the ΔperR/ΔfurΔperR mutants via RT-qPCR but only the ΔperR under RNA-seq. Cj1386 was found to be differentially expressed in the ΔperR/ΔfurΔperR mutants under both techniques. AhpC and chuA are negative controls for metal dependant CjFur/CjPerR regulation. All expression values are relative to wild-type and normalized to slyD. Asterisks denote fold changes >1.5 with a p value <0.05
Mentions: The results from the iron-independent RNA-seq experiments were confirmed using RT-qPCR (Fig. 4). Specifically, genes that appeared to show inverse CjFur/CjPerR regulation with no difference in the ΔfurΔperR mutant were chosen for validation. As shown in Fig. 4, flgE1, flgE2, flgH, and fliK all showed the same expression pattern as identified using the RNA-seq analysis (flgG was the lone exception). Moreover ahpC and chuA, two well characterized metal dependent CjPerR and CjFur targets, were not differentially expressed in any of the strains under iron limitation asserting the experimental condition. In addition cj0414, cj0415 and cj1386 were confirmed to be differentially expressed in the ΔperR mutant. Interestingly, all three were also differentially expressed in the ΔfurΔperR mutant while only the cj1386 was identified using RNA-seq (although the cj0414/cj0415 do show reduced expression in the ΔfurΔperR mutant).Fig. 4

Bottom Line: It was found that 202 genes were differentially expressed in at least one mutant under iron-replete conditions and 331 genes were differentially expressed in at least one mutant under iron-limited conditions.The CjFur and CjPerR transcriptomes characterized in this study were compared to those previously identified using microarray profiling and found to be more extensive than previously understood.Moreover, subsets of genes were found which are only differentially expressed when both CjFur and CjPerR are deleted and includes genes that appear to be simultaneously activated by CjFur and repressed by CjPerR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada. jbutc076@uottawa.ca.

ABSTRACT

Background: The genome of Campylobacter jejuni contains two iron activated Fur-family transcriptional regulators, CjFur and CjPerR, which are primarily responsible for regulating iron homeostasis and oxidative stress respectively. Both transcriptional regulators have been previously implicated in regulating diverse functions beyond their primary roles in C. jejuni. To further characterize their regulatory networks, RNA-seq was used to define the transcriptional profiles of C. jejuni NCTC11168 wild type, Δfur, ΔperR and ΔfurΔperR isogenic deletion mutants under both iron-replete and iron-limited conditions.

Results: It was found that 202 genes were differentially expressed in at least one mutant under iron-replete conditions and 331 genes were differentially expressed in at least one mutant under iron-limited conditions. The CjFur and CjPerR transcriptomes characterized in this study were compared to those previously identified using microarray profiling and found to be more extensive than previously understood. Interestingly, our results indicate that CjFur/CjPerR appear to co-regulate the expression of flagellar biogenesis genes in an opposing and iron-independent fashion. Moreover the ΔfurΔperR isogenic deletion mutant revealed that CjFur and CjPerR can compensate for each other in certain cases, suggesting that both regulators may compete for binding to specific promoters.

Conclusions: The CjFur and CjPerR transcriptomes are larger than previously reported. In particular, deletion of perR results in the differential expression of a large group of genes in the absence of iron, suggesting that CjPerR may also regulate genes in an iron-independent manner, similar to what has already been demonstrated with CjFur. Moreover, subsets of genes were found which are only differentially expressed when both CjFur and CjPerR are deleted and includes genes that appear to be simultaneously activated by CjFur and repressed by CjPerR. In particular the iron-independent co-regulation of flagellar biogenesis by CjFur/CjPerR represents a potentially novel regulatory function for these proteins. These findings represent additional modes of co-regulation by these two transcriptional regulators in C. jejuni.

No MeSH data available.


Related in: MedlinePlus