Roles of rpoS-activating small RNAs in pathways leading to acid resistance of Escherichia coli

Abstract

Escherichia coli and related enteric bacteria can survive under extreme acid stress condition at least for several hours. RpoS is a key factor for acid stress management in many enterobacteria. Although three rpoS-activating sRNAs, DsrA, RprA, and ArcZ, have been identified in E. coli, it remains unclear how these small RNA molecules participate in pathways leading to acid resistance (AR). Here, we showed that overexpression of ArcZ, DsrA, or RprA enhances AR in a RpoS-dependent manner. Mutant strains with deletion of any of three sRNA genes showed lowered AR, and deleting all three sRNA genes led to more severe defects in protecting against acid stress. Overexpression of any of the three sRNAs fully rescued the acid tolerance defects of the mutant strain lacking all three genes, suggesting that all three sRNAs perform the same function in activating RpoS required for AR. Notably, acid stress led to the induction of DsrA and RprA but not ArcZ.

Keywords: Acid resistance; Escherichia coli; RpoS; small noncoding RNA..

Figure 1

Protein expression profile of ArcZ overexpressing cells. (A) Equal amounts of total protein from Escherichia coli MG1655 cells containing pG-ArcZ and the pGEM3 vector, respectively, were subjected to electrophoresis on a 10% acrylamide SDS gel and compared. Increased or decreased protein bands are indicated with arrows. (B) Protein bands were excised and identified using Q-TOF2 MS/MS. As peptides containing the N-terminal end regions of p50 were not detected by Q-TOF MS/MS, GadA and GadB were indistinguishable so that p50 was assigned to GadA or GadB (GadA/GadB).

Figure 2

Effect of ArcZ overexpression on acid resistance of E. coli cells. (A) Survival rates of MG1655 transformed with IPTG-inducible RNA expression plasmid overexpressing ArcZ. Exponentially growing Escherichia coli cells were exposed to pH 2.0 for 1 h. Expression of ArcZ was increased with increasing IPTG concentrations. The RNA expression vector, pHMB1, was used as the control plasmid. Before and after acid exposure, cells were serially diluted in LB media and plated on LB agar plates. The survival rate was calculated based on the ratio of CFU of cells after acid exposure to CFU before acid exposure. Tenfold serial dilutions of cultures exposed to acid challenge were additionally spotted on LB agar and grown overnight. Cells containing the vector plasmid grown in the presence of 1 mmol/L were used as a control, instead of cells containing RNA expression plasmids grown at zero concentrations of IPTG. (B) IPTG-induced overexpression of ArcZ was analyzed by Northern blot analysis. A ³²P-labeled antisense oligonucleotide for ArcZ was used as the probe.

Figure 3

Effect of overexpression of rpoS-activating sRNAs on acid resistance of Escherichia coli. (A) Acid resistance of E. coli cells overexpressing DsrA, RprA, and ArcZ, respectively, was examined. Survival rates of exponentially growing or stationary-phase cells after exposure to pH 2.0 for 1 h were determined. Overexpression of sRNAs was induced by 1.0 mmol/L IPTG. Values are averaged from at least three independent experiments. Tenfold serial dilutions of cultures exposed to acid challenge were additionally spotted on LB agar and grown overnight. (B) β-Galactosidase activities from cells containing a rpoS-lacZ translational fusion (SG30013) were measured upon sRNA overexpression at the exponential or stationary phase. (C) Overexpression of rpoS-activating sRNAs from the corresponding RNA expression plasmids was confirmed using Northern blot analysis. Antisense oligonucleotides for ArcZ, DsrA, and RprA were mixed, labeled with ³²P, and used as probes.

Figure 4

Role of RpoS in enhanced acid resistance by rpoS-activating sRNAs. (A) Survival rates of ΔrpoS mutant cells overexpressing DsrA, RprA, and ArcZ, respectively, after exposure to pH 3.0 were determined. As ΔrpoS cells did not survive during acid challenge at pH 2.0, cells were challenged at pH 3.0 instead. Overexpression of sRNAs was induced by 1.0 mmol/L IPTG. (B) Wild-type MG1655 cells overexpressing RpoS were exposed to pH 2.0. Overexpression of RpoS was induced from pBAD-RpoS by 0.002% arabinose in exponentially growing cells. Acid resistance was evaluated by analysis of colony-forming abilities of serial 10-fold dilutions of the cultures. (C) Glutamate dependency of acid resistance. Exponentially growing wild-type MG1655 cells were exposed to pH 2.0, and their colony-forming abilities analyzed by 10-fold serial dilution.

Figure 5

Role of GadX in enhanced acid resistance by rpoS-activating sRNAs. Expression of each rpoS-activating sRNA was induced with 1 mmol/L IPTG in gadX mutant cells, and exponentially growing cells exposed to pH 2.0. Acid resistance was evaluated based on the colony-forming abilities of serial 10-fold dilutions of the cultures. The results are representative of at least three independent experiments.

Figure 6

Acid resistance of cells lacking rpoS-activating sRNA genes. (A) Survival rates of Escherichia coli mutant cells with deletion of each rpoS-activating sRNA gene under conditions of acid shock (pH 2.0) were determined using stationary-phase cells (shown in linear scale). Values are averaged from at least three independent experiments. (B) Acid resistance of mutant cells was evaluated based on colony-forming ability. (C) Acid resistance of MG1655Δ3 cells lacking all three rpoS-activating sRNA genes was evaluated based on the colony-forming abilities of stationary-phase cells.

Figure 7

Recovery of acid resistance of mutant cells lacking all three rpoS-activating sRNA genes induced by single sRNAs. Each rpoS-activating sRNA was overexpressed in the triple mutant cells, and acid resistance evaluated based on colony-forming abilities of both exponentially growing and stationary-phase cells. Overexpression of sRNAs was induced by 1.0 mmol/L IPTG.

Figure 8

Induction of rpoS-activating sRNAs and activation of rpoS translation upon acid challenge. Overnight cultures of wild-type and ΔrpoS cells were diluted 1:100 in LB medium and grown for 2 h at 37°C. Cultures were split into two and either maintained or subjected to pH 5.0 (LB buffered by 100 mmol/L MES), then further incubated at 37°C. Aliquots were sampled at specific time intervals for Northern blot analysis (A), and for growth curves (B). Cells containing a rpoS-lacZ translational fusion (SG30013) was also shifted to pH 5.0 and aliquots were sampled for β-galactosidase assay (C).

Figure 9

Model for acid resistance acquisition by rpoS-activating sRNAs in Escherichia coli cells. All three sRNAs are upregulated in the stationary phase. Expression of ArcZ and DsrA is induced under aerobic conditions and low temperatures, respectively. Expression of RprA is activated by Rcs phosphorelay. Expression of DsrA and RprA, but not ArcZ, is induced under acid stress. RpoS is activated by all three sRNAs in a Hfq-dependent manner. RpoS promotes gadX expression, and subsequently, GadX activates expression of GadE, which is a transcriptional activator for gadA and gadBC encoding key components in the AR2 system. RpoS-dependent, GadX-independent pathways of GadE induction also exist. RcsB, a response regulator of Rcs phosphorelay system, interacts with GadE to activate GadE.