AcrR and Rex Control Mannitol and Sorbitol Utilization through Their Cross-Regulation of Aldehyde-Alcohol Dehydrogenase (AdhE) in Lactobacillus plantarum

Abstract

Lactobacillus plantarum is a versatile bacterium that occupies a wide range of environmental niches. In this study, we found that a bifunctional aldehyde-alcohol dehydrogenase-encoding gene, adhE, was responsible for L. plantarum being able to utilize mannitol and sorbitol through cross-regulation by two DNA-binding regulators. In L. plantarum NF92, adhE was greatly induced, and the growth of an adhE-disrupted (ΔadhE) strain was repressed when sorbitol or mannitol instead of glucose was used as a carbon source. The results of enzyme activity and metabolite assays demonstrated that AdhE could catalyze the synthesis of ethanol in L. plantarum NF92 when sorbitol or mannitol was used as the carbon source. AcrR and Rex were two transcriptional factors screened by an affinity isolation method and verified to regulate the expression of adhE DNase I footprinting assay results showed that they shared a binding site (GTTCATTAATGAAC) in the adhE promoter. Overexpression and knockout of AcrR showed that AcrR was a novel regulator to promote the transcription of adhE The activator AcrR and repressor Rex may cross-regulate adhE when L. plantarum NF92 utilizes sorbitol or mannitol. Thus, a model of the control of adhE by AcrR and Rex during L. plantarum NF92 utilization of mannitol or sorbitol was proposed.IMPORTANCE The function and regulation of AdhE in the important probiotic genus Lactobacillus are rarely reported. Here we demonstrated that AdhE is responsible for sorbitol and mannitol utilization and is cross-regulated by two transcriptional regulators in L. plantarum NF92, which had not been reported previously. This is important for L. plantarum to compete and survive in some harsh environments in which sorbitol or mannitol could be used as carbon source. A novel transcriptional regulator AcrR was identified to be important to promote the expression of adhE, which was unknown before. The cross-regulation of adhE by AcrR and Rex is important to balance the level of NADH in the cell during sorbitol or mannitol utilization.

Keywords: DNA-binding regulator; Lactobacillus plantarum; aldehyde-alcohol dehydrogenase (AdhE); mannitol; sorbitol.

FIG 1

The importance of AdhE in sorbitol and mannitol utilization. (A) Downstream gene clusters of adhE. rbsR, LacI family transcriptional regulator, ribose; rbsK, ribokinase; rbsD, d-ribose pyranase; rbsU, ribose transport protein; srlD, sorbitol-6-phosphate 2-dehydrogenase; srlR, sorbitol operon transcription antiterminator, BglG family; srlM, sorbitol operon activator; ptsC, PTS, glucitol/sorbitol-specific EIIC component; ptsBC, PTS sorbitol transporter subunit IIB; ptsA, PTS sorbitol transporter subunit IIA. (B to E) Effects of adhE disruption on growth in glucose (B), d-ribose (C), sorbitol (D), and mannitol (E). (F) qRT-PCR transcriptional analysis of adhE when L. plantarum NF92 was grown in glucose, mannitol, or sorbitol. Relative values were obtained using the transcription of the 16S rRNA-coding gene as a reference. The cDNA used as the template in this experiment was diluted 10-fold for adhE but diluted 10,000-fold for the 16S rRNA-coding gene. The x axis represents the different carbon sources. Mean values and standard deviations were calculated from three independent experiments. **, P < 0.01.

FIG 2

Effects of adhE disruption on production of ethanol or lactic acid and consumption of sugar or acetate. (A to C) Ethanol production by the WT, ΔadhE, and complemented P-adhE strains grown in glucose (A)-, sorbitol (B)-, and mannitol (C)-containing media. (D to F) Glucose (D), sorbitol (E), and mannitol (F) consumption by the WT, ΔadhE, and complemented P-adhE strains. (G to I) Lactic acid production by the WT, ΔadhE, and complemented P-adhE strains grown in glucose (G)-, sorbitol (H)-, and mannitol (I)-containing media. (J to L) Acetate consumption by the WT, ΔadhE, and complemented P-adhE strains grown in glucose (J)-, sorbitol (K)-, and mannitol (L)-containing media. Mean values and standard deviations were calculated from experiments performed in triplicate.

FIG 3

Identification of regulators for adhE during sorbitol and mannitol utilization. (A) Secondary structure of P_adhE, which was predicted by using DNAMAN. (B to D) EMSAs of LysR (B), AcrR (C), and Rex (D) binding to the upstream region of adhE. Each lane contained 20 ng of DNA probe.

FIG 4

Identification of the binding sites for AcrR and Rex on P_adhE. (A) Determination of AcrR-binding sites on P_adhE by DNase I footprinting. Each lane contained 1.6 μg of DNA probe, which was incubated with increasing concentrations of AcrR protein (0.1, 0.5, 1, and 5 μM). DNA probe incubated with 7 μM bovine serum albumin (BSA) was used as the negative control. (B) Determination of Rex-binding sites on P_adhE by DNase I footprinting. The concentrations of Rex protein were 0.5, 1, and 5 μM. (C) Binding of AcrR to the 41-bp sequence (site I) containing the 14-bp repeat sequence GTTCATTAATGAAC. FB contained the 41-bp sequence. FA, nonspecific DNA as a negative control; P_adhE, positive control. Each lane contained 20 ng DNA fragment and either 0 or 200 nM AcrR, as indicated. (D) Binding of Rex to site I or site II. FC, insertion of the 41-bp sequence in FA, containing site II.

FIG 5

qRT-PCR transcriptional analysis of adhE in the WT, AcrR⁺, △acrR, and P-acrR strains. (A) Effects of acrR overexpression on adhE transcription when sorbitol or mannitol was used as a carbon source. (B) Effects of acrR disruption on adhE transcription when sorbitol or mannitol was used as a carbon source. Relative values were obtained using the transcription of the 16S rRNA-coding gene as a reference. The cDNA used as the template in this experiment was diluted 10-fold for adhE but diluted 10,000-fold for the 16S rRNA-coding gene. The x axis represents the different carbon sources. Mean values and standard deviations were calculated from three independent experiments. **, P < 0.01.

FIG 6

Function of Rex in regulating adhE. (A) qRT-PCR transcriptional analysis of adhE in the WT and Rex⁺ strains when sorbitol or mannitol was used as a carbon source. Relative values were obtained using the transcription of the 16S rRNA-coding gene as a reference. The cDNA used as the template in this experiment was diluted 10-fold for adhE but diluted 10,000-fold for the 16S rRNA-coding gene. The x axis represents the different carbon sources. Mean values and standard deviations were calculated from three independent experiments. **, P < 0.01. (B) Effects of NADH on binding of Rex to P_adhE as determined by EMSA. The concentrations of NADH were 0, 0.5, 1, and 2 mM, and 200 nM Rex was used.

FIG 7

Proposed pathway of sorbitol or mannitol utilization in L. plantarum NF92. S6PDH, sorbitol-6-phosphate 2-dehydrogenase; Mtl1PDH, mannitol-1-phosphate dehydrogenase; LDH, lactate dehydrogenase; PFL, pyruvate formate-lyase; ACK, acetate kinase; PTA, phosphate acetyltransferase. Red arrows and words show the process of acetate utilization during the later stage of growth. Dashed arrow, pyruvate may not be converted to acetyl-CoA by PFL.

FIG 8

Proposed model of AdhE regulation during sorbitol or mannitol metabolism in L. plantarum NF92. When L. plantarum NF92 is grown in glucose-containing medium, Rex binds to P_adhE and the expression of adhE is repressed. When sorbitol or mannitol is used as the carbon source, much more NADH is formed and accumulates in cells. Excess NADH binds to Rex, causing its release from P_adhE and releasing repression of adhE, and then the activator AcrR binds to the same binding site as Rex in P_adhE and promotes the expression of adhE. This might be the reason why the transcriptional level of adhE is largely increased when L. plantarum NF92 utilizes sorbitol or mannitol.