E. coli grown in LB medium releases and accumulates AI-2 in culture fluids during exponential growth. Maximal AI-2 activity is observed at the transition from exponential phase to stationary growth phase, after which the AI-2 activity rapidly disappears from the culture fluids. In S. enterica serovar Typhimurium, the disappearance of AI-2 from culture fluids is due to AI-2 uptake by the Lsr transport system, which is induced by the presence of AI-2. E. coli possesses an operon (previously designated the b1513 operon) homologous to the S. enterica serovar Typhimurium lsr operon. Transcriptional analysis of the E. coli lsr-lacZ fusion showed that AI-2 induces the expression of the lsr operon, and examination of mutants showed that the lsr operon is required for import and processing of AI-2. We concluded that the E. coli lsr operon functions analogously to the lsr operon of S. enterica serovar Typhimurium.
A screen for E. coli mutants defective in AI-2 internalization allowed us to identify lsrK and glpD. In these mutants, the lsr operon is uninducible, and so they display only low-level expression of the lsr operon both in the presence and in the absence of AI-2. We suspect that repression of transcription of the lsr operon in lsrK and glpD mutants results in their inability to assemble the Lsr transport apparatus, which in turn impairs their ability to internalize AI-2.
The E. coli lsrK homolog (annotated ydeV) is 79% identical to the S. enterica serovar Typhimurium lsrK gene. We previously showed that S. enterica serovar Typhimurium lsrK mutants do not internalize AI-2 like the WT does, and the lsr operon is not inducible by AI-2. Our characterization of the E. coli lsrK mutant showed it has phenotypes identical to those of the S. enterica serovar Typhimurium lsrK mutant (Fig. and ), suggesting that these mutants are functionally equivalent. We have shown explicitly that in S. enterica serovar Typhimurium LsrK phosphorylates AI-2. Therefore, lsrK mutants accumulate extracellular AI-2 because they cannot sequester AI-2 (as phospho-AI-2) in the cell. We propose that in E. coli, lsrK mutants do not induce lsr transcription in response to AI-2 because phospho-AI-2 is the antirepressor of lsr transcription, and this molecule is not produced in lsrK mutants.
A mutation in glpD
causes a defect in AI-2 internalization, and, similar to the lsrK
mutant, the glpD
mutant does not induce transcription of lsr
in response to AI-2. In a glpD
mutant, G3P metabolism is blocked, and intracellular G3P accumulates as a consequence of phospholipid metabolism (11
). Our results show that the lsr
operon is repressed by G3P via a cAMP-CAP-dependent mechanism. However, catabolite repression by G3P is not sufficient to explain lsr
repression in the glpD
mutant since this repression is not fully relieved by introduction of the Δcya crp
* double mutation or by preventing G3P accumulation through introduction of a glpK
mutation. Thus, an additional G3P-independent, cAMP-CAP-independent mechanism of lsr
transcriptional repression must also be involved. When G3P metabolism is blocked, in addition to accumulation of G3P, DHAP can accumulate because G3P feedback inhibits GpsA-catalyzed conversion of DHAP to G3P (4
) (Fig. ). Thus, increased G3P levels can promote increased DHAP levels. Therefore, the repression that we observed in the glpD
mutant could have been due to DHAP or a metabolite derived from DHAP. Consistent with this hypothesis, when we eliminated glycerol metabolism to DHAP in the glpK glpD
double mutant via inactivation of the GldA-DhaK pathway, repression of lsr
was fully relieved.
Our results show that LsrR is required for the repression that we observed in the glpD mutant (Fig. ). We propose that the cAMP-CAP-independent mechanism of lsr repression involves the interaction of DHAP (or possibly a metabolite derived from it) with the LsrR protein. Consistent with this, exogenous addition of DHA (which is converted to DHAP intracellularly) also causes LsrR-dependent lsr repression (data not shown). DHAP could act as an anti-inducer of the lsr operon by inhibiting the binding of phospho-AI-2 to LsrR, which could cause LsrR to remain locked in its active, repressing state. To validate this hypothesis, we are currently purifying LsrR for binding and competition assays with phospho-AI-2 and DHAP.
Understanding the physiology underlying the Glp-Lsr connection requires further analysis of the fate of internalized phospho-AI-2. In S. enterica serovar Typhimurium, the LsrF and LsrG proteins are involved in modifying phospho-AI-2, but the specific reactions that each carries out have not been characterized, nor are the products of these modifications known. It is possible that one of these products is DHAP since pentose phosphates are often converted to DHAP in order to be channeled to the glycolytic pathway for further metabolism. We are currently focusing on characterizing these biochemical reactions in both E. coli and S. enterica serovar Typhimurium.
Interestingly, the glpD mutant phenotype more closely mimics the lsrK mutant phenotype than the lsrCDB transporter mutant phenotypes. In transporter mutants, the presence of AI-2 in culture fluids is prolonged; however, AI-2 eventually disappears, presumably due to internalization by some low-affinity transporter. In contrast, AI-2 persists in culture fluids indefinitely in lsrK mutants, and we attribute this to a lack of phosphorylation or sequestration of internalized AI-2. Specifically, in an lsrK mutant, any AI-2 internalized by a secondary transporter does not get phosphorylated, and thus it cannot be sequestered. However, we do not believe that sequestration (i.e., AI-2 phosphorylation) is affected in the glpD mutant because in an lsrR glpD double mutant AI-2 is rapidly imported and remains sequestered (Fig. ), whereas in an lsrR lsrK double mutant extracellular AI-2 accumulates to wild-type levels (data not shown). We propose instead that the defect in AI-2 internalization is more severe in a glpD mutant than in the lsr transporter mutants because the secondary AI-2 transporter(s) is also subject to G3P catabolite repression in the glpD mutant. Consistent with this idea, in a glpD Δcya crp* triple mutant, although the lsr transporter is greatly repressed, most of the AI-2 is internalized by 10 h (data not shown).
Previous reports showed that high levels of extracellular AI-2 are detected when E. coli
is grown on glucose, whereas no AI-2 can be detected in cell-free culture fluids when E. coli
is grown in the absence of glucose (46
). The present results explain both of these observations. First, in the presence of glucose, the lsr
operon is not transcribed due to catabolite repression. Thus, AI-2 cannot be imported, and it accumulates in cell-free culture fluids. Second, in the absence of glucose, AI-2 is produced, but its presence is extremely transient due to rapid internalization by the Lsr transporter. DeLisa et al. (13
) used DNA microarrays to identify genes controlled by AI-2 in E. coli
. These experiments were performed with E. coli
grown in the presence of glucose, and catabolite repression of transcription of the lsr
operon by glucose could explain why none of the lsr
genes was identified in this study. Similarly, it was reported that glucose, by an unknown mechanism, caused AI-2 to persist in cell-free culture fluids of E. coli
). We show here that this mechanism is in fact cAMP-CAP-mediated repression of AI-2 import primarily through the Lsr apparatus.
At present, we do not understand the benefit that enteric bacteria derive from producing and releasing AI-2, only to internalize it later. Further work is necessary to determine if the physiological function of AI-2 as a signal in these bacteria is more significant under conditions in which AI-2 is imported and processed or under conditions in which the lsr transporter is not produced and AI-2 accumulates in the medium. In the first case, internalization of AI-2 could be used as a mechanism to terminate AI-2-controlled behaviors in E. coli or in other species in the vicinity. Alternatively, AI-2 internalization and modification could be used to transform the AI-2 signal into a different cytoplasmic signal. In the second case, in which the genes encoding the Lsr transport apparatus are repressed, cells that encounter AI-2 are exposed to this signal for a longer period of time than when the transporter is produced. Prolonged exposure to the signal could be useful for controlling other AI-2-dependent behaviors. There could be AI-2 receptors on the surface that, rather than internalize the signal, transduce the AI-2 sensory information to the cytoplasm to alter target gene expression. Studying the lsrK and lsrR mutants, in which the lsr operon is constitutively repressed and constitutively derepressed, respectively, should enable us to examine the role of AI-2 under these two conditions.