Molecular design of a signaling system influences noise in protein abundance under acid stress in different γ-Proteobacteria

Abstract

Bacteria have evolved different signaling systems to sense and adapt to acid stress. One of these systems, the CadABC-system, responds to a combination of low pH and lysine availability. In Escherichia coli, the two signals are sensed by the pH sensor and transcription activator CadC and the co-sensor LysP, a lysine-specific transporter. Activated CadC promotes the transcription of the cadBA operon, which codes for the lysine decarboxylase CadA and the lysine/cadaverine antiporter CadB. The copy number of CadC is controlled translationally. Using a bioinformatics approach, we identified the presence of CadC with ribosomal stalling motifs together with LysP in species of the Enterobacteriaceae family. In contrast, we identified CadC without stalling motifs in species of the Vibrionaceae family, but the LysP co-sensor was not identified. Therefore, we compared the output of the Cad system in single cells of the distantly related organisms E. coli and V. campbellii using fluorescently-tagged CadB as the reporter. We observed a heterogeneous output in E. coli, and all the V. campbellii cells produced CadB. The copy number of the pH sensor CadC in E. coli was extremely low (≤4 molecules per cell), but it was 10-fold higher in V. campbellii An increase in the CadC copy number in E. coli correlated with a decrease in heterogeneous behavior. This study demonstrated how small changes in the design of a signaling system allow a homogeneous output and, thus, adaptation of Vibrio species that rely on the CadABC-system as the only acid resistance system.Importance Acid resistance is an important property of bacteria, such as Escherichia coli, to survive acidic environments like the human gastrointestinal tract. E. coli possess both passive and inducible acid resistance systems to counteract acidic environments. Thus, E. coli evolved sophisticated signaling systems to sense and appropriately respond to environmental acidic stress by regulating the activity of its three inducible acid resistance systems. One of these systems is the Cad system that is only induced under moderate acidic stress in a lysine-rich environment by the pH-responsive transcriptional regulator CadC. The significance of our research is in identifying the molecular design of the Cad systems in different Proteobacteria and their target expression noise at single cell level during acid stress conditions.

FIG 1

Phylogenetic tree of CadC and cooccurrence with LysP and/or stalling XPP or PPX motifs in gammaproteobacteria. (a) The protein sequences of 3,223 CadC homologs were aligned, and a phylogenetic tree was generated and displayed as a circular cladogram. The branches of the tree are colored according to the family of the organisms containing a CadC homolog. The presence of an XPP or PPX motif is displayed in the outer blue/dark gray ring, and the presence/absence of LysP is displayed in the inner salmon/light gray ring. The CadC homolog sequences were examined for XPP or PPX motifs, which were grouped according to their stalling strength as follows: strongest PPP, dark blue; other strong XPP or PPX, blue; moderate/weak XPP or PPX, light blue; or no stalling motif, dark gray. (b) Percentage of CadC sequences containing strong stalling XPP or PPX, moderate/weak XPP or PPX, or no XPP or PPX stalling motif. Sequences and XPP or PPX motifs are summarized in Data Set S1 in the supplemental material. Arrows indicate the occurrence of CadC in the E. coli and V. campbellii clades.

FIG 2

Acid resistance of V. campbellii and CadA decarboxylase activity. (a) Survival of E. coli and V. campbellii at pH 3.0 in complex medium (LB or LM, respectively). At the indicated times, the samples were collected, and the number of CFU was analyzed. (b) Lysine decarboxylase activity of purified CadA of V. campbellii (1 μg/ml) was tested in the presence of increasing lysine concentrations and fitted to the Michaelis-Menten equation (gray line) by nonlinear regression calculations (K_m = 4.7 ± 2.7 mM).

FIG 3

Heterogenous CadB production in E. coli in response to acid stress. At time zero, E. coli was shifted from pH 7.6 to pH 5.8 in the presence of lysine. CadB-eGFP intensity was quantified in single E. coli cells over time (upper graph; the gray line indicates background fluorescence). Representative fluorescence and phase-contrast images of E. coli cells containing CadB-eGFP are shown. As a control, E. coli cells containing CadB-eGFP were analyzed under nonstress conditions (pH 7.6, 2.5 h, first column). The noise and mean relative fluorescence intensity (RF) were calculated for 1,000 cells per condition and time point. Single-cell fluorescence intensity was quantified by microscopy and the use of the ImageJ software. The inset graph illustrates the distribution of CadB-eGFP (percentage of fluorescence) among single cells (gray, cells in the off state; dark green, cells with a high CadB level [on state]) (see Table S1 in the supplemental material). Noise, standard deviation/mean of log-transformed values; PH, phase contrast; GFP, green fluorescent channel. Scale bar, 5 μm. Arrows mark exemplary cells in the off state.

FIG 4

Homogeneous CadB production in V. campbellii in response to acid stress. At time zero, V. campbellii was shifted from pH 7.6 to pH 5.8 in tryptone-containing (LM) medium. The CadB-eGFP intensity was quantified in single V. campbellii cells over time (upper graph; the gray line indicates background fluorescence). Representative fluorescence and phase-contrast images of V. campbellii cells containing CadB-eGFP are shown. As a control, V. campbellii cells containing CadB-eGFP were analyzed under nonstress conditions (pH 7.6, 2.5 h, first column). The noise and mean relative fluorescence intensity (RF) were calculated for 1,000 cells per condition and time point. Single-cell fluorescence intensity was quantified by microscopy and the use of the ImageJ software. The inset graph illustrates the distribution of CadB-eGFP (percentage of fluorescence) among single cells (gray, cells in the off state; dark green, cells with a high CadB level [on state]) (see Table S2 in the supplemental material). Noise, standard deviation/mean of log-transformed values; PH, phase contrast; GFP, green fluorescent channel. Scale bar, 5 μm.

FIG 5

CadC copy number influences the degree of heterogenous production of CadB within single E. coli cells. (a) Quantification of the CadB-eGFP intensity of single E. coli cells at 2.5 h after a shift to acid stress conditions (pH 5.8 plus lysine) and various levels of CadC protein copy number due to plasmid-carried cadC (upper graph; the gray line indicates background fluorescence). Representative fluorescence and phase-contrast images of quantified E. coli cells containing CadB-eGFP are shown. The noise and mean relative fluorescence intensity (RF) were calculated for 1,000 cells per condition and time point. Single-cell fluorescence intensity was quantified by microscopy and the use of the ImageJ software. The inset graph illustrates the distribution CadB-eGFP (percentage of fluorescence) among single cells (gray, cells in the off state; dark green, cells with a high CadB level [on state]) (see Table S1 in the supplemental material). The arrows indicate the cells with low CadB content. (b) Quantification of mCherry-LysP intensity of single E. coli cells at 2.5 h after a shift to acid stress conditions (pH 5.8 plus lysine) and nonstress conditions (pH 7.6) (upper graph; the gray line indicates background fluorescence). The noise and mean relative fluorescence intensity (RF) were calculated for 1,000 cells per condition and time point. Noise, standard deviation/mean of log-transformed values; PH, phase contrast; GFP, green fluorescent channel; mCH, red fluorescent channel (mCherry). Scale bar, 5 μm.

FIG 6

CadC and LysP copy numbers in E. coli and V. campbellii. Dot blot analysis was used to determine the CadC copy number in the indicated strains of E. coli and V. campbellii under nonstress (pH 7.6, absence of lysine) conditions and acid stress conditions (pH 5.8 plus 10 mM lysine or LM medium at pH 5.8). The copy number of fluorescently tagged LysP was quantified in E. coli under nonstress (pH 7.6) conditions and acid stress conditions (pH 5.8 plus 10 mM lysine). The numbers below the dot blot images indicate the quantified protein copy numbers per strain and condition. The E. coli ΔcadC and V. campbellii ΔcadC mutants were used as background controls. The E. coli ΔcadC strain carrying the pET-mCherry-cadC plasmid (24, 25) was used as the reference for quantification using ImageJ (47).

FIG 7

The molecular design of a signaling system influences gene expression noise. E. coli (left panels) contains the three inducible AR systems, namely, Gad, Adi, and Cad, whereas V. campbellii (right panels) contains only the Cad system. The main components of the Cad system, including (i) the membrane-integrated pH-responsive regulator CadC, (ii) the lysine decarboxylase CadA, and (iii) the lysine/cadaverine antiporter CadB, are similar in E. coli and V. campbellii. However, E. coli has additional regulatory elements. CadC activity is regulated by the cosensor LysP, which is a lysine transporter, and CadC has a low copy number due to the presence of the strong stalling PPP motif. Due to the differences in the molecular design of the Cad systems, the output reflected by the distribution of CadB-eGFP can be either heterogeneous, such as in E. coli, or homogeneous, such as in V. campbellii. PP, periplasm; CM, cytoplasmic membrane; CP, cytoplasm.