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The small RNA GlmY acts upstream of the sRNA GlmZ in the activation of glmS expression and is subject to regulation by polyadenylation in Escherichia coli.

Reichenbach B, Maes A, Kalamorz F, Hajnsdorf E, Görke B - Nucleic Acids Res. (2008)

Bottom Line: Expression of glmS is activated by the unprocessed form of GlmZ, which accumulates when the intracellular GlcN-6-P concentration decreases.When the intracellular GlcN-6-P concentration decreases, GlmY accumulates and causes in turn accumulation of full-length GlmZ, which finally activates glmS expression.In glmZ mutants, GlmY has no effect on glmS, whereas artificially expressed GlmZ can activate glmS expression also in the absence of GlmY.

View Article: PubMed Central - PubMed

Affiliation: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, Grisebachstrasse 8, D-37077 Göttingen, Germany.

ABSTRACT
In Escherichia coli the glmS gene encoding glucosamine 6-phosphate (GlcN-6-P) synthase GlmS is feedback regulated by GlcN-6-P in a pathway that involves the small RNA GlmZ. Expression of glmS is activated by the unprocessed form of GlmZ, which accumulates when the intracellular GlcN-6-P concentration decreases. GlmZ stabilizes a glmS transcript that derives from processing. Overexpression of a second sRNA, GlmY, also activates glmS expression in an unknown way. Furthermore, mutations in two genes, yhbJ and pcnB, cause accumulation of full-length GlmZ and thereby activate glmS expression. The function of yhbJ is unknown and pcnB encodes poly(A) polymerase PAP-I known to polyadenylate and destabilize RNAs. Here we show that GlmY acts indirectly in a way that depends on GlmZ. When the intracellular GlcN-6-P concentration decreases, GlmY accumulates and causes in turn accumulation of full-length GlmZ, which finally activates glmS expression. In glmZ mutants, GlmY has no effect on glmS, whereas artificially expressed GlmZ can activate glmS expression also in the absence of GlmY. Furthermore, we show that PAP-I acts at the top of this regulatory pathway by polyadenylating and destabilizing GlmY. In pcnB mutants, GlmY accumulates and induces glmS expression by stabilizing full-length GlmZ. Hence, the data reveal a regulatory cascade composed of two sRNAs, which responds to GlcN-6-P and is controlled by polyadenylation.

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GlmY requires GlmZ for the activation of glmS expression. (A) Overexpression of glmY induces expression of the glmS’-lacZ reporter fusion in the wild-type but not in the ΔglmZ mutant. Strains Z8 (wild-type), Z38 (ΔglmZ) and Z28 (ΔyhbJ) were grown in the absence (grey bars) or presence of the glmY overproducing plasmid pBGG149 (black bars) and the β-galactosidase activities were determined. (B) Northern blot analysis of glmS and GlmZ RNAs in strains overproducing GlmY. Total RNAs were isolated from strains R1279 (wild-type), Z37 (ΔyhbJ), Z45 (ΔglmZ) and Z116 (ΔyhbJ, ΔglmZ), which were either untransformed (lanes 1, 3, 5 and 7) or transformed with plasmid pBGG149 overproducing GlmY (lanes 2, 4, 6 and 8). The RNAs were hybridized with a glmS probe (upper panel) and a GlmZ probe (second panel).
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Figure 3: GlmY requires GlmZ for the activation of glmS expression. (A) Overexpression of glmY induces expression of the glmS’-lacZ reporter fusion in the wild-type but not in the ΔglmZ mutant. Strains Z8 (wild-type), Z38 (ΔglmZ) and Z28 (ΔyhbJ) were grown in the absence (grey bars) or presence of the glmY overproducing plasmid pBGG149 (black bars) and the β-galactosidase activities were determined. (B) Northern blot analysis of glmS and GlmZ RNAs in strains overproducing GlmY. Total RNAs were isolated from strains R1279 (wild-type), Z37 (ΔyhbJ), Z45 (ΔglmZ) and Z116 (ΔyhbJ, ΔglmZ), which were either untransformed (lanes 1, 3, 5 and 7) or transformed with plasmid pBGG149 overproducing GlmY (lanes 2, 4, 6 and 8). The RNAs were hybridized with a glmS probe (upper panel) and a GlmZ probe (second panel).

Mentions: Next, we investigated the effect of GlmY overexpression. For this purpose, the glmY gene was cloned on a plasmid under control of the strong constitutively active λPL promoter. The resulting plasmid was introduced into the wild-type strain that carried a glmS′–lacZ reporter fusion expressed from a constitutive promoter on the chromosome (4). This fusion is perfectly regulated by GlmZ and YhbJ. The presence of the glmY expression plasmid led to induction of glmS′–lacZ expression (Figure 3A), whereas no increase in β-galactosidase activity was detectable when the empty expression vector was present (data not shown). To confirm these results we performed northern experiments using probes specific for glmS and GlmZ. In the wild-type strain, overexpression of glmY caused the strong accumulation of the glmS transcript and concomitantly of full-length GlmZ sRNA (Figure 3B, lanes 1 and 2). Hence, it can be concluded that GlmY positively regulates the glmS mRNA, which is in agreement with a recent publication demonstrating that GlmY overexpression causes overproduction of GlmS protein (9). Our additional observation that GlmY overproduction stabilizes full-length GlmZ, raises the possibility that GlmY acts on glmS indirectly via GlmZ. To test this idea, we repeated the experiments described above in ΔglmZ and ΔglmZ ΔyhbJ mutants. In these strains, GlmY overproduction had no stimulatory effect, neither on expression of the glmS′–lacZ reporter fusion (Figure 3A) nor on the glmS transcript level as detected by northern analysis (Figure 3B, lanes 5–8). Next, we tested the effect of GlmY overexpression in the yhbJ mutant. In this strain glmS strongly accumulates and the glmS′–lacZ reporter fusion is highly expressed (Figure 3A and B, lane 3). Additional overexpression of the glmY construct, however, had no additive effect on the GlmZ and glmS RNA levels (Figure 3B, lanes 3 and 4) and on the expression of the glmS′–lacZ fusion (Figure 3A).Figure 3.


The small RNA GlmY acts upstream of the sRNA GlmZ in the activation of glmS expression and is subject to regulation by polyadenylation in Escherichia coli.

Reichenbach B, Maes A, Kalamorz F, Hajnsdorf E, Görke B - Nucleic Acids Res. (2008)

GlmY requires GlmZ for the activation of glmS expression. (A) Overexpression of glmY induces expression of the glmS’-lacZ reporter fusion in the wild-type but not in the ΔglmZ mutant. Strains Z8 (wild-type), Z38 (ΔglmZ) and Z28 (ΔyhbJ) were grown in the absence (grey bars) or presence of the glmY overproducing plasmid pBGG149 (black bars) and the β-galactosidase activities were determined. (B) Northern blot analysis of glmS and GlmZ RNAs in strains overproducing GlmY. Total RNAs were isolated from strains R1279 (wild-type), Z37 (ΔyhbJ), Z45 (ΔglmZ) and Z116 (ΔyhbJ, ΔglmZ), which were either untransformed (lanes 1, 3, 5 and 7) or transformed with plasmid pBGG149 overproducing GlmY (lanes 2, 4, 6 and 8). The RNAs were hybridized with a glmS probe (upper panel) and a GlmZ probe (second panel).
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Related In: Results  -  Collection

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Figure 3: GlmY requires GlmZ for the activation of glmS expression. (A) Overexpression of glmY induces expression of the glmS’-lacZ reporter fusion in the wild-type but not in the ΔglmZ mutant. Strains Z8 (wild-type), Z38 (ΔglmZ) and Z28 (ΔyhbJ) were grown in the absence (grey bars) or presence of the glmY overproducing plasmid pBGG149 (black bars) and the β-galactosidase activities were determined. (B) Northern blot analysis of glmS and GlmZ RNAs in strains overproducing GlmY. Total RNAs were isolated from strains R1279 (wild-type), Z37 (ΔyhbJ), Z45 (ΔglmZ) and Z116 (ΔyhbJ, ΔglmZ), which were either untransformed (lanes 1, 3, 5 and 7) or transformed with plasmid pBGG149 overproducing GlmY (lanes 2, 4, 6 and 8). The RNAs were hybridized with a glmS probe (upper panel) and a GlmZ probe (second panel).
Mentions: Next, we investigated the effect of GlmY overexpression. For this purpose, the glmY gene was cloned on a plasmid under control of the strong constitutively active λPL promoter. The resulting plasmid was introduced into the wild-type strain that carried a glmS′–lacZ reporter fusion expressed from a constitutive promoter on the chromosome (4). This fusion is perfectly regulated by GlmZ and YhbJ. The presence of the glmY expression plasmid led to induction of glmS′–lacZ expression (Figure 3A), whereas no increase in β-galactosidase activity was detectable when the empty expression vector was present (data not shown). To confirm these results we performed northern experiments using probes specific for glmS and GlmZ. In the wild-type strain, overexpression of glmY caused the strong accumulation of the glmS transcript and concomitantly of full-length GlmZ sRNA (Figure 3B, lanes 1 and 2). Hence, it can be concluded that GlmY positively regulates the glmS mRNA, which is in agreement with a recent publication demonstrating that GlmY overexpression causes overproduction of GlmS protein (9). Our additional observation that GlmY overproduction stabilizes full-length GlmZ, raises the possibility that GlmY acts on glmS indirectly via GlmZ. To test this idea, we repeated the experiments described above in ΔglmZ and ΔglmZ ΔyhbJ mutants. In these strains, GlmY overproduction had no stimulatory effect, neither on expression of the glmS′–lacZ reporter fusion (Figure 3A) nor on the glmS transcript level as detected by northern analysis (Figure 3B, lanes 5–8). Next, we tested the effect of GlmY overexpression in the yhbJ mutant. In this strain glmS strongly accumulates and the glmS′–lacZ reporter fusion is highly expressed (Figure 3A and B, lane 3). Additional overexpression of the glmY construct, however, had no additive effect on the GlmZ and glmS RNA levels (Figure 3B, lanes 3 and 4) and on the expression of the glmS′–lacZ fusion (Figure 3A).Figure 3.

Bottom Line: Expression of glmS is activated by the unprocessed form of GlmZ, which accumulates when the intracellular GlcN-6-P concentration decreases.When the intracellular GlcN-6-P concentration decreases, GlmY accumulates and causes in turn accumulation of full-length GlmZ, which finally activates glmS expression.In glmZ mutants, GlmY has no effect on glmS, whereas artificially expressed GlmZ can activate glmS expression also in the absence of GlmY.

View Article: PubMed Central - PubMed

Affiliation: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, Grisebachstrasse 8, D-37077 Göttingen, Germany.

ABSTRACT
In Escherichia coli the glmS gene encoding glucosamine 6-phosphate (GlcN-6-P) synthase GlmS is feedback regulated by GlcN-6-P in a pathway that involves the small RNA GlmZ. Expression of glmS is activated by the unprocessed form of GlmZ, which accumulates when the intracellular GlcN-6-P concentration decreases. GlmZ stabilizes a glmS transcript that derives from processing. Overexpression of a second sRNA, GlmY, also activates glmS expression in an unknown way. Furthermore, mutations in two genes, yhbJ and pcnB, cause accumulation of full-length GlmZ and thereby activate glmS expression. The function of yhbJ is unknown and pcnB encodes poly(A) polymerase PAP-I known to polyadenylate and destabilize RNAs. Here we show that GlmY acts indirectly in a way that depends on GlmZ. When the intracellular GlcN-6-P concentration decreases, GlmY accumulates and causes in turn accumulation of full-length GlmZ, which finally activates glmS expression. In glmZ mutants, GlmY has no effect on glmS, whereas artificially expressed GlmZ can activate glmS expression also in the absence of GlmY. Furthermore, we show that PAP-I acts at the top of this regulatory pathway by polyadenylating and destabilizing GlmY. In pcnB mutants, GlmY accumulates and induces glmS expression by stabilizing full-length GlmZ. Hence, the data reveal a regulatory cascade composed of two sRNAs, which responds to GlcN-6-P and is controlled by polyadenylation.

Show MeSH
Related in: MedlinePlus