Transient and dynamic DNA supercoiling potently stimulates the leu-500 promoter in Escherichia coli.

The inactive prokaryotic leu-500 promoter (Pleu-500) contains a single A-to-G point mutation in the -10 region of the leucine operon promoter, which causes leucine auxotrophy. This promoter can be activated by (-) DNA supercoiling in Escherichia coli topA strains. However, whether this activation arises from global, permanent, or transient, dynamic supercoiling is still not fully understood. In this article, using a newly established in vivo system carrying a pair of divergently coupled promoters, i.e. an IPTG-inducible promoter and Pleu-500 that control the expression of lacZ and luc (the firefly luciferase gene), respectively, we demonstrate that transient, dynamic (-) DNA supercoiling provided by divergent transcription in both wild-type and topA strains can potently activate Pleu-500 We found that this activation depended on the promoter strength and the length of RNA transcripts, which are functional characteristics of transcription-coupled DNA supercoiling (TCDS) precisely predicted by the twin-supercoiled domain model of transcription in which a (+) supercoiled domain is produced ahead of the RNA polymerase and a (-) supercoiled domain behind it. We also demonstrate that TCDS can be generated on topologically open DNA molecules, i.e. linear DNA molecules, in Escherichia coli, suggesting that topological boundaries or barriers are not required for the production of TCDS in vivo This work demonstrates that transient, dynamic TCDS by RNA polymerases is a major chromosome remodeling force in E. coli and greatly influences the nearby, coupled promoters/transcription.

Substituted lactam and cyclic azahemiacetals modulate Pseudomonas aeruginosa quorum sensing.

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence that is critical for establishing infection. The most common QS signaling molecule used by Gram-negative bacteria are acylhomoserine lactones. The development of non-native acylhomoserine lactone (AHL) ligands has emerged as a promising new strategy to inhibit QS in Gram-negative bacteria. In this work, we have synthesized a set of optically pure γ-lactams and their reduced cyclic azahemiacetal analogues, bearing the additional alkylthiomethyl substituent, and evaluated their effect on the AHL-dependent Pseudomonas aeruginosa las and rhl QS pathways. The concentration of these ligands and the simple structural modification such as the length of the alkylthio substituent has notable effect on activity. The γ-lactam derivatives with nonylthio or dodecylthio chains acted as inhibitors of las signaling with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent was found to strongly inhibit both las and rhl signaling at higher concentrations while the propylthio analogue strongly stimulated the las QS system at lower concentrations.

Simple electrospray mass spectrometry detection of acylhomoserine lactones.

Simple (non-tandem) electrospray mass spectrometry (ESMS) can detect acyl homoserine lactones (AHL) in bacteriological media in picomole amounts. The chemical reactivity of AHLs and their extraction behaviour into ethyl acetate, coupled with detection in the ESMS, has shown that these lactones can be detected as the protonated pseudomolecular ions themselves as well as solvent and ammonium adducts, and as dimers. ESMS detects and identifies these molecules, utilizing simple chemical properties of AHLs.