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Escherichia coli YmdB regulates biofilm formation independently of its role as an RNase III modulator.

Kim T, Lee J, Kim KS - BMC Microbiol. (2013)

Bottom Line: Of these, ten are involved in biofilm formation.Moreover, biofilm formation is interdependently regulated by RpoS, a known stress response regulator and biofilm inhibitor, and by YmdB.This is the first global profile of target genes modulated by YmdB-induced RNase III inhibition in E. coli, and the data reveal a novel, hitherto unrecognized regulatory role for YmdB in biofilm modulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Superbacteria Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Korea. sunny06@kribb.re.kr.

ABSTRACT

Background: Ribonuclease III (RNase III) activity modulates hundreds of genes in Escherichia coli (E. coli). YmdB, a member of the macrodomain protein family, is one of known trans-acting regulators of RNase III activity; however, the significance of its regulatory role in specific bacterial cellular processes and related genes has not been determined. YmdB overexpression was used to model YmdB-induced RNase III inhibition in vivo, and microarray analysis identified gene targets and cellular processes related to RNase III inhibition.

Results: The expression of >2,000 E. coli genes was modulated by YmdB induction; 129 genes were strongly regulated, of which 80 have not been reported as RNase III targets. Of these, ten are involved in biofilm formation. Significantly, YmdB overexpression also inhibited biofilm formation via a process that is not uniquely dependent upon RNase III inhibition. Moreover, biofilm formation is interdependently regulated by RpoS, a known stress response regulator and biofilm inhibitor, and by YmdB.

Conclusions: This is the first global profile of target genes modulated by YmdB-induced RNase III inhibition in E. coli, and the data reveal a novel, hitherto unrecognized regulatory role for YmdB in biofilm modulation.

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Regulation of RpoS levels and activity by YmdB. (A) Effect of YmdB on in vivo expression levels of RpoS. KS004 [SG30013 (λRpoS750::LacZ] [31] strains containing either pCA24N (−gfp) or ASKA-ymdB (−) were grown to OD600 = 0.2, induced by IPTG (0.1 mM final), and further grown to OD600 = 1.0. Aliquots were then assayed for β-galactosidase activity. Data represent the mean values from n = 3 experiments (p = 0.05). (B) Expression level of RpoS. Total lysates prepared from the cell described in (A) and from Keio-∆rpoS cells were immunoblotted antibodies against RpoS and S1. The Keio-∆rpoS strain is included to show the specificity of the antibody. The relative levels of RpoS normalized against S1 protein are shown. ND, not determined. (C) Determination of steady-state levels of rpoS transcript induced by YmdB. cDNA synthesized from total RNA obtained from BW25113, KSK002 (∆ymdB), KSK001 (rnc14) or BW25113 cells containing either pCA24N (−gfp) or ASKA-ymdB (−) were qPCR amplified using the rpoS- or 16S RNA-specific primer sets listed in Additional file 1: Table S2 and then compared. Data represent the mean values from triplicate experiments.
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Figure 4: Regulation of RpoS levels and activity by YmdB. (A) Effect of YmdB on in vivo expression levels of RpoS. KS004 [SG30013 (λRpoS750::LacZ] [31] strains containing either pCA24N (−gfp) or ASKA-ymdB (−) were grown to OD600 = 0.2, induced by IPTG (0.1 mM final), and further grown to OD600 = 1.0. Aliquots were then assayed for β-galactosidase activity. Data represent the mean values from n = 3 experiments (p = 0.05). (B) Expression level of RpoS. Total lysates prepared from the cell described in (A) and from Keio-∆rpoS cells were immunoblotted antibodies against RpoS and S1. The Keio-∆rpoS strain is included to show the specificity of the antibody. The relative levels of RpoS normalized against S1 protein are shown. ND, not determined. (C) Determination of steady-state levels of rpoS transcript induced by YmdB. cDNA synthesized from total RNA obtained from BW25113, KSK002 (∆ymdB), KSK001 (rnc14) or BW25113 cells containing either pCA24N (−gfp) or ASKA-ymdB (−) were qPCR amplified using the rpoS- or 16S RNA-specific primer sets listed in Additional file 1: Table S2 and then compared. Data represent the mean values from triplicate experiments.

Mentions: Since YmdB is transcriptionally activated by RpoS [18] and the level of rpoS transcripts was increased by YmdB overexpression, it is possible that YmdB modulates RpoS expression. YmdB could affect this change in rpoS transcript levels by either acting as an as yet unknown transcription factor or by acting as an effector protein for the factor(s) involved in rpoS transcription. We found that YmdB overexpression had no effect on rpoS promoter activity (data not shown), thereby excluding any role as a transcription factor. A linear relationship between rpoS transcript levels and RpoS protein levels was then investigated following YmdB induction, and similar increases (~2.5-fold) in the induced β-galactosidase activity of the rpoS’-‘lacZ protein fusion and the RpoS protein level were observed (Figures 4A,B). Moreover, the steady-state level of rpoS transcript (Figure 4C) was oppositely regulated in the absence of chromosomal ymdB. Additionally, the level of rpoS transcript following YmdB overexpression was lower than that in the RNase III mutant strain. These data suggest that YmdB-mediated regulation of RNase III activity alone cannot fully regulate the processing of the 5′ UTR of rpoS mRNA. Because RpoS can negatively regulate biofilm formation by itself (Figure 3B) and is also required for complete YmdB function (Figure 3B), it is a matter of debate whether YmdB can modulate RpoS activity. When the RpoS protein was overexpressed in a wild-type and in an ymdB knockout strain, RpoS-mediated inhibition of biofilm formation was decreased from 70% to 43% (Figure 3B). This, when taken together with the other data, suggests that the regulation of RpoS function during biofilm formation is dependent upon YmdB. Moreover, RpoS overexpression phenotype on biofilm inhibition was not dependent upon the presence of RNase III activity (Additional file 1: Figure S3). Thus, YmdB is a novel post-transcriptional regulator of RpoS levels that acts independently of RNase III.


Escherichia coli YmdB regulates biofilm formation independently of its role as an RNase III modulator.

Kim T, Lee J, Kim KS - BMC Microbiol. (2013)

Regulation of RpoS levels and activity by YmdB. (A) Effect of YmdB on in vivo expression levels of RpoS. KS004 [SG30013 (λRpoS750::LacZ] [31] strains containing either pCA24N (−gfp) or ASKA-ymdB (−) were grown to OD600 = 0.2, induced by IPTG (0.1 mM final), and further grown to OD600 = 1.0. Aliquots were then assayed for β-galactosidase activity. Data represent the mean values from n = 3 experiments (p = 0.05). (B) Expression level of RpoS. Total lysates prepared from the cell described in (A) and from Keio-∆rpoS cells were immunoblotted antibodies against RpoS and S1. The Keio-∆rpoS strain is included to show the specificity of the antibody. The relative levels of RpoS normalized against S1 protein are shown. ND, not determined. (C) Determination of steady-state levels of rpoS transcript induced by YmdB. cDNA synthesized from total RNA obtained from BW25113, KSK002 (∆ymdB), KSK001 (rnc14) or BW25113 cells containing either pCA24N (−gfp) or ASKA-ymdB (−) were qPCR amplified using the rpoS- or 16S RNA-specific primer sets listed in Additional file 1: Table S2 and then compared. Data represent the mean values from triplicate experiments.
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Figure 4: Regulation of RpoS levels and activity by YmdB. (A) Effect of YmdB on in vivo expression levels of RpoS. KS004 [SG30013 (λRpoS750::LacZ] [31] strains containing either pCA24N (−gfp) or ASKA-ymdB (−) were grown to OD600 = 0.2, induced by IPTG (0.1 mM final), and further grown to OD600 = 1.0. Aliquots were then assayed for β-galactosidase activity. Data represent the mean values from n = 3 experiments (p = 0.05). (B) Expression level of RpoS. Total lysates prepared from the cell described in (A) and from Keio-∆rpoS cells were immunoblotted antibodies against RpoS and S1. The Keio-∆rpoS strain is included to show the specificity of the antibody. The relative levels of RpoS normalized against S1 protein are shown. ND, not determined. (C) Determination of steady-state levels of rpoS transcript induced by YmdB. cDNA synthesized from total RNA obtained from BW25113, KSK002 (∆ymdB), KSK001 (rnc14) or BW25113 cells containing either pCA24N (−gfp) or ASKA-ymdB (−) were qPCR amplified using the rpoS- or 16S RNA-specific primer sets listed in Additional file 1: Table S2 and then compared. Data represent the mean values from triplicate experiments.
Mentions: Since YmdB is transcriptionally activated by RpoS [18] and the level of rpoS transcripts was increased by YmdB overexpression, it is possible that YmdB modulates RpoS expression. YmdB could affect this change in rpoS transcript levels by either acting as an as yet unknown transcription factor or by acting as an effector protein for the factor(s) involved in rpoS transcription. We found that YmdB overexpression had no effect on rpoS promoter activity (data not shown), thereby excluding any role as a transcription factor. A linear relationship between rpoS transcript levels and RpoS protein levels was then investigated following YmdB induction, and similar increases (~2.5-fold) in the induced β-galactosidase activity of the rpoS’-‘lacZ protein fusion and the RpoS protein level were observed (Figures 4A,B). Moreover, the steady-state level of rpoS transcript (Figure 4C) was oppositely regulated in the absence of chromosomal ymdB. Additionally, the level of rpoS transcript following YmdB overexpression was lower than that in the RNase III mutant strain. These data suggest that YmdB-mediated regulation of RNase III activity alone cannot fully regulate the processing of the 5′ UTR of rpoS mRNA. Because RpoS can negatively regulate biofilm formation by itself (Figure 3B) and is also required for complete YmdB function (Figure 3B), it is a matter of debate whether YmdB can modulate RpoS activity. When the RpoS protein was overexpressed in a wild-type and in an ymdB knockout strain, RpoS-mediated inhibition of biofilm formation was decreased from 70% to 43% (Figure 3B). This, when taken together with the other data, suggests that the regulation of RpoS function during biofilm formation is dependent upon YmdB. Moreover, RpoS overexpression phenotype on biofilm inhibition was not dependent upon the presence of RNase III activity (Additional file 1: Figure S3). Thus, YmdB is a novel post-transcriptional regulator of RpoS levels that acts independently of RNase III.

Bottom Line: Of these, ten are involved in biofilm formation.Moreover, biofilm formation is interdependently regulated by RpoS, a known stress response regulator and biofilm inhibitor, and by YmdB.This is the first global profile of target genes modulated by YmdB-induced RNase III inhibition in E. coli, and the data reveal a novel, hitherto unrecognized regulatory role for YmdB in biofilm modulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Superbacteria Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Korea. sunny06@kribb.re.kr.

ABSTRACT

Background: Ribonuclease III (RNase III) activity modulates hundreds of genes in Escherichia coli (E. coli). YmdB, a member of the macrodomain protein family, is one of known trans-acting regulators of RNase III activity; however, the significance of its regulatory role in specific bacterial cellular processes and related genes has not been determined. YmdB overexpression was used to model YmdB-induced RNase III inhibition in vivo, and microarray analysis identified gene targets and cellular processes related to RNase III inhibition.

Results: The expression of >2,000 E. coli genes was modulated by YmdB induction; 129 genes were strongly regulated, of which 80 have not been reported as RNase III targets. Of these, ten are involved in biofilm formation. Significantly, YmdB overexpression also inhibited biofilm formation via a process that is not uniquely dependent upon RNase III inhibition. Moreover, biofilm formation is interdependently regulated by RpoS, a known stress response regulator and biofilm inhibitor, and by YmdB.

Conclusions: This is the first global profile of target genes modulated by YmdB-induced RNase III inhibition in E. coli, and the data reveal a novel, hitherto unrecognized regulatory role for YmdB in biofilm modulation.

Show MeSH
Related in: MedlinePlus