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Next generation sequencing analysis reveals that the ribonucleases RNase II, RNase R and PNPase affect bacterial motility and biofilm formation in E. coli.

Pobre V, Arraiano CM - BMC Genomics (2015)

Bottom Line: We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase.Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms.Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal. vaniapobre@itqb.unl.pt.

ABSTRACT

Background: The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome.

Results: In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms.

Conclusions: In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.

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Exoribonucleases affect biofilm formation. Effect of the deletion of the exoribonuclease on biofilm production. The different strains (wt, ∆rnb, ∆rnr and ∆pnp) were inoculated into the wells of a fresh microtiter plate and left at 37°C for 24 h. The biofilms formation was measured by determining the OD550 after staining with crystal violet. The biofilm formation values were normalized with the OD600 of the cultures measured after the 24 h inoculation. The wild-type was used as reference and all other values were obtained by the formula: normalised OD (mutant)/normalised OD (wt). Error bars represent standard deviations.
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Fig5: Exoribonucleases affect biofilm formation. Effect of the deletion of the exoribonuclease on biofilm production. The different strains (wt, ∆rnb, ∆rnr and ∆pnp) were inoculated into the wells of a fresh microtiter plate and left at 37°C for 24 h. The biofilms formation was measured by determining the OD550 after staining with crystal violet. The biofilm formation values were normalized with the OD600 of the cultures measured after the 24 h inoculation. The wild-type was used as reference and all other values were obtained by the formula: normalised OD (mutant)/normalised OD (wt). Error bars represent standard deviations.

Mentions: When analysing more closely the lists of transcripts that were being differentially expressed in the different mutants, we found that both RNase II and PNPase affected antigen-43 expression that, as mentioned previously, is known to promote aggregation of the cells and impair motility [36]. Antigen-43 has also been found to affect biofilm formation in E. coli [39]. We also found that there were other biofilm related transcripts being affected by the exoribonuclease deletion besides the antigen-43 (Additional file 1: Tables S1, S2 and S3). Because biofilm formation is inversely correlated with cell motility we hypothesized that the motility impairment could be an indirect effect due to an increase in biofilm formation. We have performed biofilm formation assays to determine if the exoribonucleases mutants did affect the biofilm formation. The RNA-Seq data analysis indicated that RNase II and RNase R mutants were probably able to form more biofilms than the wild-type, and our experimental results confirmed this fact (Figure 5). Surprisingly the PNPase mutant did not formed biofilms. This result was initially unexpected because several transcripts related with biofilm formation were significantly affected in the ∆pnp mutant (Additional file 1: Table S3). Similarly ∆pnp mutant in Salmonella also formed less biofilms then the wild-type control [40]. When analysing more closely our RNA-Seq data we could also find some evidences, corroborating our results for the lack of biofilm formation in the ∆pnp mutant. For example, the bssR gene that is known to be induced during biofilm formation [41] is significantly down-regulated in the ∆pnp mutant. These results show how complex the biofilm formation pathway is and that the RNA-Seq data should be experimentally validated when we are predicting a phenotype.Figure 5


Next generation sequencing analysis reveals that the ribonucleases RNase II, RNase R and PNPase affect bacterial motility and biofilm formation in E. coli.

Pobre V, Arraiano CM - BMC Genomics (2015)

Exoribonucleases affect biofilm formation. Effect of the deletion of the exoribonuclease on biofilm production. The different strains (wt, ∆rnb, ∆rnr and ∆pnp) were inoculated into the wells of a fresh microtiter plate and left at 37°C for 24 h. The biofilms formation was measured by determining the OD550 after staining with crystal violet. The biofilm formation values were normalized with the OD600 of the cultures measured after the 24 h inoculation. The wild-type was used as reference and all other values were obtained by the formula: normalised OD (mutant)/normalised OD (wt). Error bars represent standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4335698&req=5

Fig5: Exoribonucleases affect biofilm formation. Effect of the deletion of the exoribonuclease on biofilm production. The different strains (wt, ∆rnb, ∆rnr and ∆pnp) were inoculated into the wells of a fresh microtiter plate and left at 37°C for 24 h. The biofilms formation was measured by determining the OD550 after staining with crystal violet. The biofilm formation values were normalized with the OD600 of the cultures measured after the 24 h inoculation. The wild-type was used as reference and all other values were obtained by the formula: normalised OD (mutant)/normalised OD (wt). Error bars represent standard deviations.
Mentions: When analysing more closely the lists of transcripts that were being differentially expressed in the different mutants, we found that both RNase II and PNPase affected antigen-43 expression that, as mentioned previously, is known to promote aggregation of the cells and impair motility [36]. Antigen-43 has also been found to affect biofilm formation in E. coli [39]. We also found that there were other biofilm related transcripts being affected by the exoribonuclease deletion besides the antigen-43 (Additional file 1: Tables S1, S2 and S3). Because biofilm formation is inversely correlated with cell motility we hypothesized that the motility impairment could be an indirect effect due to an increase in biofilm formation. We have performed biofilm formation assays to determine if the exoribonucleases mutants did affect the biofilm formation. The RNA-Seq data analysis indicated that RNase II and RNase R mutants were probably able to form more biofilms than the wild-type, and our experimental results confirmed this fact (Figure 5). Surprisingly the PNPase mutant did not formed biofilms. This result was initially unexpected because several transcripts related with biofilm formation were significantly affected in the ∆pnp mutant (Additional file 1: Table S3). Similarly ∆pnp mutant in Salmonella also formed less biofilms then the wild-type control [40]. When analysing more closely our RNA-Seq data we could also find some evidences, corroborating our results for the lack of biofilm formation in the ∆pnp mutant. For example, the bssR gene that is known to be induced during biofilm formation [41] is significantly down-regulated in the ∆pnp mutant. These results show how complex the biofilm formation pathway is and that the RNA-Seq data should be experimentally validated when we are predicting a phenotype.Figure 5

Bottom Line: We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase.Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms.Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal. vaniapobre@itqb.unl.pt.

ABSTRACT

Background: The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome.

Results: In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms.

Conclusions: In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.

Show MeSH
Related in: MedlinePlus