Regulation of uptake and processing of the quorum-sensing autoinducer AI-2 in Escherichia coli

Abstract

AI-2 is a quorum-sensing signaling molecule proposed to be involved in interspecies communication. In Escherichia coli and Salmonella enterica serovar Typhimurium, extracellular AI-2 accumulates in exponential phase, but the amount decreases drastically upon entry into stationary phase. In S. enterica serovar Typhimurium, the reduction in activity is due to import and processing of AI-2 by the Lsr transporter. We show that the Lsr transporter is functional in E. coli, and screening for mutants defective in AI-2 internalization revealed lsrK and glpD. Unlike the wild type, lsrK and glpD mutants do not activate transcription of the lsr operon in response to AI-2. lsrK encodes the AI-2 kinase, and the lsrK mutant fails to activate lsr expression because it cannot produce phospho-AI-2, which is the lsr operon inducer. glpD encodes the glycerol-3-phosphate (G3P) dehydrogenase, which is involved in glycerol and G3P metabolism. G3P accumulates in the glpD mutant and represses lsr transcription by preventing cyclic AMP (cAMP)-catabolite activator protein (CAP)-dependent activation. Dihydroxyacetone phosphate (DHAP) also accumulates in the glpD mutant, and DHAP represses lsr transcription by a cAMP-CAP-independent mechanism involving LsrR, the lsr operon repressor. The requirement for cAMP-CAP in lsr activation explains why AI-2 persists in culture fluids of bacteria grown in media containing sugars that cause catabolite repression. These findings show that, depending on the prevailing growth conditions, the amount of time that the AI-2 signal is present and, in turn, the time that a given community of bacteria remains exposed to this signal can vary greatly.

FIG. 1.

Model for AI-2 production and internalization in S. enterica serovar Typhimurium. (A) AI-2 (pentagons) is synthesized by LuxS and accumulates extracellularly. AI-2 is internalized by the Lsr ABC-type transporter, and internalized AI-2 is phosphorylated by the LsrK kinase. Phospho-AI-2 is the inducer of transcription of the lsr operon and is proposed to act by binding to LsrR, the repressor of the lsr operon, inactivating it. LsrF and LsrG are required for further processing of internalized AI-2. The dotted lines indicate hypothetical processes. (B) E. coli b1513 operon is homologous to the S. enterica serovar Typhimurium lsr operon. lsr gene designations are indicated under the annotations. b1516 (lsrB) encodes the periplasmic AI-2 binding protein. b1514 (lsrC) and b1515 (lsrD) encode the channel proteins, and b1513 (lsrA) encodes the ATPase that provides energy for AI-2 transport. b1517 (lsrF) is similar to genes specifying aldolases, and b1518 (lsrG) encodes a protein with an unknown function. There is no lsrE in the E. coli lsr operon. ydeV and ydeW encode proteins homologous to the AI-2 kinase LsrK and the lsr repressor LsrR, respectively.

FIG. 2.

Extracellular AI-2 accumulation in E. coli. WT E. coli strain MG1655 was inoculated into LB medium at time zero, and at various times aliquots were taken. (A) Cell growth was monitored by measuring the optical density (•), and AI-2 activity in cell-free culture fluids was measured using the V. harveyi bioluminescence assay (▪). (B) LuxS production was determined by Western blotting using anti-LuxS antibodies.

FIG. 3.

Extracellular AI-2 accumulation in E. coli mutants. AI-2 activity in cell-free culture fluids was measured using the V. harveyi bioluminescence bioassay. The following strains were analyzed: MG1655 (WT), KX17 (glpD), and KX11 (lsrK, annotated ydeV).

FIG. 4.

Expression of the lsr operon in E. coli mutants in the presence and absence of luxS. The β-galactosidase activity of the lsr-lacZ (b1513-lacZ) fusion was determined in strains KX1123 (WT), KX1218 (luxS), KX1186 (lsrK), KX1372 (lsrK luxS), KX1304 (glpD), and KX1306 (glpD luxS) after 5 h of growth.

FIG. 5.

Extracellular AI-2 accumulation in E. coli Lsr transporter mutants. AI-2 activity in cell-free culture fluids was measured using the V. harveyi bioluminescence bioassay. The following strains were analyzed: MG1655 (WT), KX1382 (lsrCDB), and KX11 (lsrK).

FIG. 6.

Aerobic glycerol and G3P metabolism in E. coli. Glycerol enters the cytoplasm through the glycerol facilitator (GlpF) and can be phosphorylated to G3P by the glycerol kinase (GlpK). In the presence of oxygen, G3P is oxidized by the G3P dehydrogenase (GlpD) to DHAP, which is further metabolized through the glycolytic pathway. Intracellular glycerol can also be oxidized to DHA by GldA. DHA is converted to DHAP by the DHA kinase (DhaK), which uses DhaM as a phosphoryl donor protein. G3P is required for phospholipid biosynthesis, and in the absence of extracellular glycerol, intracellular G3P is formed from DHAP by the G3P synthase (GpsA). In a glpD mutant G3P accumulates due to conversion of glycerol to G3P by GlpK. Intracellular G3P accumulation prevents cAMP formation by inhibiting the stimulation of adenylate cyclase via phospho-EIIA^Glc (16). As a consequence, cAMP-CAP activation of the lsr operon is inhibited significantly. G3P accumulation also inhibits GpsA by a negative feedback mechanism, which leads to DHAP accumulation. We hypothesize that DHAP represses the lsr operon by a mechanism independent of cAMP-CAP that involves LsrR, the repressor of the lsr operon. The solid lines indicate enzymatic reactions, the dashed lines indicate regulatory interactions, and the dotted lines indicate the newly proposed regulatory interaction resulting from this work. FAD, flavin adenine dinucleotide; FADH₂, reduced flavin adenine dinucleotide.

FIG. 7.

Effect of catabolite repression and the GldA pathway on the expression of lsr transcription. The β-galactosidase activity of the lsr-lacZ fusion was measured in strains KX1123 (WT), KX1481 (Δcya), KX1468 (Δcya crp*), KX1304 (glpD), KX1483 (glpD Δcya crp*), KX1536 (glpD glpK), KX1541 (glpD glpK cya crp*), KX1547 (glpD glpK gldA), and KX1549 (glpD glpK gldA cya crp*) after 5 h of growth in LB medium.

FIG. 8.

lsrR is epistatic to glpD. (A) β-Galactosidase activity of the lsr-lacZ fusion in strains KX1123 (WT), KX1304 (glpD), KX1328 (lsrR), and KX1374 (glpD lsrR) at different times during growth in LB medium. (B) AI-2 activity in cell-free culture fluids of strains MG1655 (WT), KX17 (glpD), KX1328 (lsrR), and KX1374 (glpD lsrR) as determined by the V. harveyi bioluminescence bioassay.