Research ArticleBIOCHEMISTRY

Evidence of link between quorum sensing and sugar metabolism in Escherichia coli revealed via cocrystal structures of LsrK and HPr

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Science Advances  01 Jun 2018:
Vol. 4, no. 6, eaar7063
DOI: 10.1126/sciadv.aar7063
  • Fig. 1 Crystal structure of the HisLsrK/HPr/ATP complex.

    (A) The crystal asymmetric unit contained two molecules of the HisLsrK/HPr complex, in which the contact between two HisLsrK/HPr units is maintained by the ionic interaction mediated by two phosphate and LsrK-K204 residues (boxed region). (B) The ATP molecule seems to bind LsrK mainly via the hydrophobic interaction mediated by the adenine base, and the phosphate groups participate in additional hydrogen bonds and ionic interactions. The presence of hydrogen bonds is indicated with cyan lines, and the slightly longer distances than the limit of a hydrogen bond are indicated with orange lines. (C) The base of ATP fits into the hydrophobic pocket of LsrK. (D) The residues of HPr that participate in the interaction with LsrK are represented in the green ribbon model. F48 fits into the deep hydrophobic pocket of LsrK, and the neighboring L47 also participates in this hydrophobic interaction. HPr-H15 is located in the interface of the HisLsrK/HPr complex. (E) A structural overlay of free HPr and that in the HisLsrK/HPr complex suggests that H15 has structural flexibility, and the steric hindrance induced by the phosphorylated H15 may not be stringent. The phosphate group is simply attached to the H15-ND1 atom of the free HPr form for more clear comparison. The residues of LsrK that are located close to HPr-H15 are labeled yellow (R118, 4.15 Å; E122, 2.65 Å; E125, 4.50 Å).

  • Fig. 2 Structure-based alignments of various LsrK and HPr sequences.

    Various LsrK (A) and HPr (B) sequences of Gram-negative [Escherichia coli (E. coli), Salmonella typhimurium LT2 (S. typh), Yersinia pestis (Y. pest), and Klebsiella pneumoniae subsp. pneumoniae (K. pneu)] and Gram-positive [Bacillus subtilis (B. subt), Bacillus thuringiensis (B. thur), and Streptococcus sp. oral taxon 056 (Str. sp)] bacteria were aligned on the basis of the determined structure of the HisLsrK/HPr/ATP complex. The E. coli LsrK residues that contact with ATP (marked with A) and HPr (marked with numbers) are indicated in the bottom of the aligned sequences. The letters and numbers are colored following the characteristics of the interactions (hydrophobic, red; hydrogen bond, green; ionic interaction, blue), and the other neighboring residues remain as black.

  • Fig. 3 Enzyme activity of LsrK is inhibited by HPr.

    An enzymatic model of the LsrK and HPr interactions is summarized at the top. (A) Enzyme kinetic studies of LsrK were performed with a fixed ATP concentration (0.75 mM). The Lineweaver-Burk plots of LsrK activities in the presence of the HPr protein show that HPr inhibited LsrK activity via a combined manner of competitive and uncompetitive inhibition, as noted. Competitive inhibition (Ki1) was more substantive than uncompetitive inhibition (Ki2). (B) The relative activity of LsrK was measured as increasing the concentration of the wild-type (WT) and mutant HPr proteins. The concentrations of (S)-DPD and ATP were fixed at 3 and 1 mM, respectively. The reaction reference already contained the copurified HPr at a molar equivalent of LsrK (0.1 μM). Although the inhibition curves were not completely fitted by a simple inhibition equation (Eq. 3), inhibition activities of the HPrH15E and HPrH15A mutants were about five times less than and similar to that of the WT HPr, respectively. (C) The relative inhibition of LsrK activity by the native HPr and p-HPr proteins was assessed with 0.75 mM (S)-DPD and ATP. The relative inhibition of LsrK activity by the native HPr protein was described using the parameters determined in (A) (Eq. 2). The comparison of the relative inhibition activities between HPr and p-HPr was analyzed by using a simple inhibition equation (Eq. 3). The inhibition activity of p-HPr was much lower (more than 25 times) than that of the native HPr. The higher LsrK activity in the case of the p-HPr inhibition (marked with an asterisk) likely resulted from the phosphorylation of the copurified HPr with the LsrK protein by the EI enzyme that was retained in the p-HPr solution.

  • Fig. 4 E. coli lsr promoter activity during cell growth (indirect measurement of LsrK activity).

    (A) E. coli PH02 strain (ΔptsH and ΔluxS) carrying the reporter plasmid pLW11 (lacZ gene under lsr operon) and the pSkunk-empty, pSkunk-HPr, or pSkunk-HPrH15A plasmid. Cells were cultivated in LB medium in the presence or absence of 40 μM AI-2. The culture aliquots were collected for the measurement of β-galactosidase activity at 0.8 and 4.0 OD600 nm. The data showed representative experiments performed independently. Data are means ± SDs of technical triplicates. (B) Growth curves of the E. coli PH02 strains cotransformed with pLW11 and pSkunk-empty (circle), pSkunk-HPr (triangle), or pSkunk-HPrH15A (square) were measured in the absence or presence of 40 μM AI-2, as indicated. Aliquots were collected for the measurement of OD at 600 nm at different time points during cell growth. The growth rates of the E. coli strains in the absence of AI-2 were only slightly higher than those in the presence of 40 μM AI-2. (C) E. coli PH01 (ΔptsH) strains cotransformed with pLW11 and either pSkunk-empty or pSkunk-HPr were inoculated into LB medium with or without 0.8% glucose at t = 0. Samples were taken every 2 hours for the measurement of the extracellular AI-2 activity. Cultures with pSkunk-empty or pSkunk-HPr are indicated as “− HPr” or “+ HPr,” respectively. Data are the average of technical duplicates. (D) Growth curves of the E. coli PH01 strains cotransformed with pLW11 and either pSkunk-empty or pSkunk-HPr in the presence or absence of 0.8% glucose.

  • Fig. 5 1D STD NMR experiments of ATPγS and DPD in the presence of LsrK and HPr proteins.

    (A) The peak intensities of the 1D NMR spectrum reflect the relative concentrations of ATPγS (1 mM) and DPD (10 mM racemic mixture). (B) A small molecule displaying a binding exchange process with target proteins produces positive peaks in the 1D STD spectra. An incomplete cancellation due to slightly different peak shapes between two 1D spectra obtained with on- and off-saturation at 0.5 and 30 ppm, respectively, resulted in the peak spikes with both positive and negative signs (marked with an asterisk). Overall integration of the peak spike is zero, and thus, the molecule corresponding to the peak spikes is shown not to bind to LsrK. The residual protein peaks in the 1D STD spectrum are indicated with bold gray lines. The saturation transfer efficiency from the LsrK/HPr protein to DPD was much lower than that of ATPγS (top), which means that the LsrK/HPr protein exhibited much less binding to DPD than to ATPγS. The presence of an additional HPr further decreased the peak intensities of DPD (middle). The 1D STD spectrum in the absence of the proteins did not produce any positive peaks (bottom).

  • Fig. 6 Structural comparison of HisLsrK with ecGK and ecXK.

    GK and XK are the members of the sugar kinases/Hsp70/actin superfamily and consist of two domains, in which the substrate sugar and ATP molecules bind to domain I (D-I) and domain II (D-II), respectively. ADP and ATP molecules in GK and HisLsrK are shown in the ball-and-stick model, and the glycerol and xylulose in GK and XK are represented as spheres. The structure of ecGK (PDB code, 1GLB) corresponds to the closed form, which allows the reaction of the phosphate transfer from the ATP to the glycerol. The native EIIAGlc binds to domain I and has been proposed to inhibit the enzyme activity through long-range conformational changes. On the other hand, the structures of HisLsrK and ecXK (PDB code, 2ITM) are open form, in which the distance between the ATP and the substrate molecules is too far for the enzyme reaction. HPr binds domain I of HisLsrK, instead of domain II.

  • Fig. 7 Summarized model for the role of HPr between two distinct bacterial processes: QS and PTS.

    QS signaling should be strongly related to a bacterial metabolic status, because QS accompanies a transition from planktonic to sessile growth. General transcriptional regulation is under control of σ70 and σS during the exponential growth and stationary phase, respectively. In the PTS sugar transport system, HPr delivers the activated phosphate group from EI to EIIA and then the phosphate is eventually transferred to imported glucose (PTS sugars) via the transporter (EIIB and EIIC). The native HPr is dominant during the exponential growth, but p-HPr is accumulated in the stationary phase largely due to the absence of sugar import. The Ki1 (0.08 μM) and Ki2 (0.27 μM) values of the HPr protein were very low, and thus, LsrK inhibition by HPr is likely to be released during the late stationary phase when the accessible HPr is extremely limited. In addition, at these later times, AI-2 is taken up by the Lsr transporter and more LsrK is expressed. In the current scenario, LsrK activity is modulated directly via glucose metabolism and by transcriptional regulation via AI-2 QS. The former is presumably far quicker than the latter, indicating that QS activity and sugar metabolism are tightly linked, and hence, population-scale behavior can be influenced rapidly by rapid changes in nutrient availability within a particular niche.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/6/eaar7063/DC1

    fig. S1. Identification of copurified protein with LsrK.

    fig. S2. SEC-MALS analysis of the purified LsrK/HPr protein complex.

    fig. S3. Electron density maps in the region of the bound HPr, ATP, and ADP.

    fig. S4. Monitoring the stability of the synthesized p-15NHPr using HSQC experiment.

    table S1. X-ray data collection and refinement statistics.

    table S2. Strains and plasmids used for Miller assay.

    table S3. Primer sequences for the in vivo studies of the wild-type and mutant HPr proteins.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Identification of copurified protein with LsrK.
    • fig. S2. SEC-MALS analysis of the purified LsrK/HPr protein complex.
    • fig. S3. Electron density maps in the region of the bound HPr, ATP, and ADP.
    • fig. S4. Monitoring the stability of the synthesized p-15NHPr using HSQC experiment.
    • table S1. X-ray data collection and refinement statistics.
    • table S2. Strains and plasmids used for Miller assay.
    • table S3. Primer sequences for the in vivo studies of the wild-type and mutant HPr proteins.

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