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loren anderson
Court Anker
Rachel Berardinelli
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Roland Windsor Vincent

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Rachel Berardinelli

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  1. Cellular communication is necessary for multicellular organisms, but in the case of several types of bacteria, signaling systems have been characterized in which bacterial cell communication is carried out to the detriment of the multicellular organisms infected with or in the vicinity of these sometimes pathogenic strains. Such bacteria are through the excretion of small diffusible molecules able to communicate with other of their own kind and even with bacteria of other species by way of these autoinducer–type signaling molecules.
    In the case of Vibrio fischeri, in which this signaling system termed “quorum sensing” has been characterized and fairly well understood for several years, the signaling molecules are acylated homoserine lactones (AHLs).1 As a matter of fact , there are multiple autoinducer systems which employ positive feedback loops leading to up regulation of various target genes. One such autoinducer molecule is manufactured and excreted ito the environment by V. fischeri through the action of the product of the luxI gene, a LuxI-like AHL synthase, which produces the autoinducer signaling molecule. The other component of the quorum sensing circuit is the LuxR protein which is equipped with a receptor which is capable of detecting levels of AHLs manufactured by other V. fischeri. Once the bacteria involved in the quorum sensing circuit reach a threshold population density, the LuxR-type receptor senses the elevated AHL levels in the environment, the LuxR protein binds to the DNA of the bacteria, causing the expression of genes whose products are in the case of V. fischeri bioluminescent proteins. To understand this background information is to have a good foundation in understanding how Dialkylresorcinols (DARs) and cyclohexanediones (CHDs) function in pathogenic bacteria such as Photorhabdus asymbiotica.
    Recent research by some German scientists had shown the presence of a quorum sensing circuit involving Pyrones as the bacterial signaling molecules in Photorhabdus luminescens.4 In the journal article, Dialkylresorcinols as bacterial signaling molecules, 4 the researchers characterized another quorum sensing circuit this time in Photorhabdus asymbiotica. The quorum sensing proteins DarABC and PauR manufactured by P. asymbiotica were shown to function analogously to LuxI and LuxR of V. fischeri respectively. The researchers tested the hypothesis that if the DarABC synthesis pathway could produce the molecules necessary for its existence as a quorum sensing circuit, these components which would effect the signaling should be manufactured by the bacteria, thus discoverable and empirically tested and analyzed. They went about to test the byproducts of P. asymbiotica gene expression as well as the extant products of gene expression of over 90 different Photorhabdus strains utilizing HPLC/MS technology and discovered that DARs and CHDs are linked to P. asymbiotica, the human pathogen in that theyse chemicals were produced by the bacteria and readily available in sufficient quantities to measure and characterize in terms of structure, binding affinities and the like. They reasoned that these compounds might in fact be the quorum sensing circuit components they were seeking, and upon further testing discovered that this was the case.
    They grew a strain of P. asymbiotica bearing a plasmid for a red fluorescent protein, mCherry and plated it, and found that fluorescence and cell clumping were combined effects of exposure to Dialkylresorcinol (DAR) or bacteria that was producing DAR, as well as the effluent thereof. Further tests led them to conclude that the darABC operon, whose transcription along with the pcfABCDEF gene results in the production of Photorhabdus clumping factor, essential to the virulence of the bacteria. These tests included growing ΔpauR deletion mutants of P. asymbiotica to ascertain whether or not PpcfA (promoter) activation is actuated by the presence of PauR protein. In this case neither cell clumping nor fluorescence were observed, leading to the conclusion that the darABC operon is muted in the absence of the PauR protein. As well, E. coli expressing darABC, protein coding gene ngrA and the bkd operon, which itself encodes at least 4 proteins, were added to an E. coli strain overproducing the darABC operon resulting in the release of DARs and CHDs from the bacteria into the supernatant. The accompanying fluorescence and cell clumping in P. aymbiotica Ppcfa-mCherry reporter strain which was grown alongside the E. coli was understood to be clear indication that DarABC/PauR is indeed a type of quorum sensing circuit in which bacterial cells are receiving information and expressing certain genes when prompted by chemical cues. E coli with an empty plasmid was used as a control in this part of the experiment, in which the DARs and CHDs were shown to be the cues necessary for production of virulence genes. These experiments were performed in triplicate.
    Sophisticated computer programs were also employed by the research group. It having been established that the darABC operon encodes a pathway for the production of several CHDs and DARs (the former is a chemical precursor of the latter), and that these compounds have to do with quorum sensing and initiation of density-dependent behaviors in Photorhabdus strains, further analysis in silico by way of docking experiments also demonstrated that certain conserved amino acids play key roles in ligand binding with regards to DAR and the active binding site of PauR, the signal receptor protein. When these two crucial amino acids were replaced by other amino acids, binding of DAR to PauR was greatly reduced, likely due to steric hindrance in the binding site molecules. Other statistical analyses as well as comparative genomics were employed to further remove doubt that Dialkylresorcinols are indeed the signaling molecule used by P. asymbiotica to initiate density-dependent behaviors.
    This research built upon over a decade of previous research and actually it was an honor to familiarize myself with some of these new concepts. The fact that the experiments were done at least three times in several cases shows replicability of the results. Also, it would seem that adequate controls were in place, demonstrating the accuracy of the logic employed in this research, and filling in the blanks, answering the research questions with convincing evidence. Further, sophisticated computer programs made it possible to understand not only the what and how related to the particular quorum sensing circuit employed by P. asymbiotica but the why related to the autoinducer ligands binding to their receptors in this simple signaling mechanism. It would be difficult not to respect the work put in buy these researchers and I look forward to presenting this topic once I have worked out a better understanding of some new terminology. Finally, I found the logic in the paper to be compelling evidence that the claims of the researchers merit further investigation.

    1. Churchill, M.E.; Chen, L. “Structural basis of acyl-homoserine lactone-dependent signaling.”. Chem. Rev. 111 (1): 68–85. doi:10.1021/cr1000817. PMC 3494288. PMID 21125993. [Internet]. 2011 [cited 2015 Feb 11] Available from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494288/

    2. Miller ST, Xavier KB, Campagna SR, Taga ME, Semmelhack MF, Bassler BL, Hughson FM. almonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2.
    [Internet]. 2004 [cited 2015 Feb 11] Available from http://www.ncbi.nlm.nih.gov/pubmed/15350213

    3 Alexander O Brachmann, Sophie Brameyer, Darko Kresovic, Ivana Hitkova, Yannick Kopp, Christian Manske, Karin Schubert, Helge B Bode, & Ralf Heermann Pyrones as bacterial signaling molecules Nature Chemical Biology 9, 573–578 doi:10.1038/nchembio.1295 [Internet]. 2013 [cited 2015 February 11] Available from: http://www.nature.com/nchembio/ journal/v9/n9/abs/nchembio.1295.html
    4. Sophie Brameyer, Darko Kresovic, Helge B. Bode, and Ralf Heermann Dialkylresorcinols as bacterial signaling molecules [Internet]. 2014 [cited 2015 Feb 11] Available from: http://www.pnas.org/content/112/2/572.abstract


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