Publications

• Mechanistic insights into the LsrK kinase required for autoinducer-2 quorum sensing activation.

  Zhu J., Hixon M.S., Globisch D., Kaufmann G.F., Janda K.D.

  In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm an ... >> More

  In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm and subsequent derepression of AI-2-related genes necessary for quorum development. While LsrK is a critical enzyme within the DPD quorum sensing relay system, kinetic details of this kinase have yet to be reported. A continuous UV-vis spectrophotometric assay was developed that allowed steady-state kinetic analysis of LsrK to be undertaken with the substrates ATP and DPD. The data was most consistent with a rapid equilibrium ordered mechanism with ATP binding first: kcat (7.4 ± 0.6 s(-1)), Km,ATP (150 ± 30 μM) and Km(app),DPD (1.0 ± 0.2 mM). The assay also allowed a DPD substrate profile to be conducted, which provided an unexpected biochemical disconnect between the previous agonist/antagonist cell-based reporter assay and the LsrK assay presented herein. Together these findings raise the importance of LsrK and lay the foundation not only for further understanding of this enzyme and its critical biological role but also for the rational design of regulatory molecules targeting AI-2 quorum sensing in pathogenic bacteria. << Less

  J. Am. Chem. Soc. 135:7827-7830(2013) [PubMed] [EuropePMC]

• Cross species quorum quenching using a native AI-2 processing enzyme.

  Roy V., Fernandes R., Tsao C.Y., Bentley W.E.

  Bacterial quorum sensing (QS) is a cell-cell communication process, mediated by signaling molecules, that alters various phenotypes including pathogenicity. Methods to interrupt these communication networks are being pursued as next generation antimicrobials. We present a technique for interruptin ... >> More

  Bacterial quorum sensing (QS) is a cell-cell communication process, mediated by signaling molecules, that alters various phenotypes including pathogenicity. Methods to interrupt these communication networks are being pursued as next generation antimicrobials. We present a technique for interrupting communication among bacteria that exploits their native and highly specific machinery for processing the signaling molecules themselves. Specifically, our approach is to bring native intracellular signal processing mechanisms to the extracellular surroundings and "quench" crosstalk among a variety of strains. In this study, the QS system based on the interspecies signaling molecule autoinducer-2 (AI-2) is targeted because of its prevalence among prokaryotes (it functions in over 80 bacterial species). We demonstrate that the Escherichia coli AI-2 kinase, LsrK, can phosphorylate AI-2 in vitro, and when LsrK-treated AI-2 is added ex vivo to E. coli populations, the native QS response is significantly reduced. Further, LsrK-mediated degradation of AI-2 attenuates the QS response among Salmonella typhimurium and Vibrio harveyi even though the AI-2 signal transduction mechanisms and the phenotypic responses are species-specific. Analogous results are obtained from a synthetic ecosystem where three species of bacteria (enteric and marine) are co-cultured. Finally, the addition of LsrK and ATP to growing co-cultures of E. coli and S. typhimurium exhibits significantly reduced native "cross-talk" that ordinarily exists among and between species in an ecosystem. We believe this nature-inspired enzymatic approach for quenching QS systems will spawn new methods for controlling cell phenotype and potentially open new avenues for controlling bacterial pathogenicity. << Less

  ACS Chem. Biol. 5:223-232(2010) [PubMed] [EuropePMC]

• Phosphorylation and processing of the quorum-sensing molecule autoinducer-2 in enteric bacteria.

  Xavier K.B., Miller S.T., Lu W., Kim J.H., Rabinowitz J., Pelczer I., Semmelhack M.F., Bassler B.L.

  Quorum sensing is a process of chemical communication that bacteria use to assess cell population density and synchronize behavior on a community-wide scale. Communication is mediated by signal molecules called autoinducers. The LuxS autoinducer synthase produces 4,5-dihydroxy-2,3-pentanedione (DP ... >> More

  Quorum sensing is a process of chemical communication that bacteria use to assess cell population density and synchronize behavior on a community-wide scale. Communication is mediated by signal molecules called autoinducers. The LuxS autoinducer synthase produces 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor to a set of interconverting molecules that are generically called autoinducer-2 (AI-2). In enteric bacteria, AI-2 production induces the assembly of a transport apparatus (called the LuxS regulated (Lsr) transporter) that internalizes endogenously produced AI-2 as well as AI-2 produced by other bacterial species. AI-2 internalization is proposed to be a mechanism enteric bacteria employ to interfere with the signaling capabilities of neighboring species of bacteria. We have previously shown that Salmonella enterica serovar Typhimurium binds a specific cyclic derivative of DPD. Here we show that following internalization, the kinase LsrK phosphorylates carbon-5 of the open form of DPD. Phosphorylated DPD (P-DPD) binds specifically to the repressor of the lsr operon, LsrR, consistent with P-DPD being the inducer of the lsr operon. Subsequently, LsrG catalyzes the cleavage of P-DPD producing 2-phosphoglycolic acid. This series of chemical events is proposed to enable enteric bacteria to respond to the presence of competitor bacteria by sequestering and destroying AI-2, thereby eliminating the competitors' intercellular communication capabilities. << Less

  ACS Chem. Biol. 2:128-136(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.