Pathways for AI-2 biosynthesis and SAM utilization in E. coli.

Effects of glucose on extracellular AI-2 activity. Overnight cultures of E. coli W3110 were diluted in LB or LB plus 0.8% glucose to an OD₆₀₀ below 0.03. At different time points during cell growth, aliquots were collected for measurement of the OD₆₀₀ (triangles and squares) and ΑI-2 activity (bars). AI-2 activity in the culture fluids was measured using the V. harveyi BB170 AI-2 bioassay, and the values shown are representative of three independent experiments (some values were very small, but measured, as indicated). Replicate assays agreed to within 10%.

crp and cya mutations increase extracellular AI-2 activity. Overnight cultures of E. coli W3110 (wild type) and strains containing deletion of crp and cya were diluted in LB to an OD₆₀₀ below 0.03. At different time points, aliquots were collected for measurement of the OD₆₀₀ (diamonds, triangles, and squares) and ΑI-2 activity (bars). Plasmids pHA7E and pIT302 carry wild-type crp and cya genes, respectively. AI-2 activities shown are representative of three independent experiments. Replicate assays agreed to within 10%.

Effects of cAMP and CRP on the transcription of luxS and pfs. Conditions for cell growth and β-galactosidase activity are described in Materials and Methods. E. coli ZK126 (wild type) and isogenic crp mutant carrying plasmid pLW10 (luxS-lacZ) (A) and pYH10 (pfs-lacZ) (B) were grown in LB, LB plus 0.8% glucose, or LB plus 0.8% glucose plus 10 mM cAMP. At different time points during cell growth, aliquots were collected for measurement of the OD₆₀₀ (triangles and squares) and β-galactosidase activity (bars).

AI-2 activity profiles of E. coli lsr mutants. Overnight cultures of E. coli W3110 (wild type) and strains containing deletion of lsrR, lsrK, or lsrACDBFG were diluted in LB (A) or LB plus 0.8% glucose (B) to an OD₆₀₀ below 0.03. At different time points during cell growth, aliquots were collected for measurement of the OD₆₀₀ (triangles and squares) and ΑI-2 activity (bars). AI-2 activities shown are representative of three independent experiments. Replicate assays agreed to within 10%.

Transcriptional regulation of the E. coli lsr operon. E. coli ZK126 (wild type) and strains containing deletions of luxS, lsrK, lsrR and lsrACDBFG carry plasmid pLW11 (lacZ fusion containing wild type lsrA promoter region). ZK126 (WT*) carries plasmid pLW12 (lacZ fusion containing mutated lsrA promoter region with base substitutions in CRP binding motif). Cells were grown in LB medium. At different time points during cell growth, aliquots were collected for measurement of the OD₆₀₀ (triangles, squares, and diamonds) and β-galactosidase activity (bars).

cAMP-CRP binds to an upstream region of the lsr promoter. (A) CRP consensus sequence and DNA fragments used for the CRP binding assay. Consensus and potential CRP recognition sites are shown in capital letters. The underlined bases in seq4 show substitutions eliminating CRP binding. The numbers indicate the nucleotide position relative to the predicted lsrA transcription start site. (B and C) Gel mobility shift assays were performed as described in Materials and Methods. Digoxigenin-labeled DNA fragments of seq1, seq2, seq3, and seq4 were incubated with 0 to 80 nM purified CRP, as indicated. cAMP was included in all reaction mixtures at a final concentration of 100 μM. The arrow denotes the CRP-DNA complex.

Conceptual model of AI-2 synthesis and uptake in E. coli. In the presence of glucose, low levels of cAMP and CRP result in almost no expression of the lsr operon. Indirect upregulation of luxS, and likely increased precursor flux, increases AI-2 synthesis. Both enable rapid accumulation of AI-2 in the extracellular medium. In the absence of glucose, cAMP-CRP is needed to stimulate lsr expression, while LsrR represses its expression in the absence of the inducer phospho-AI-2. As AI-2 accumulates, lsr transcription is de-repressed, enabling more AI-2 uptake. In addition, σ^s negatively affects lsr expression, especially during the late exponential phase. As noted above, the expression of pfs is negatively influenced by the presence of glucose; the effects of this are unclear but might be complicated by the polyamine pathways also utilizing Pfs and SAM. Transcriptional regulation is shown by solid arrows (direct) or dashed arrows (indirect or unclear mechanisms). Plus and minus signs indicate positive and negative regulations, respectively. DPD, 4,5-dihydroxy-2,3-pentanedione. See text for additional details.