We show that several interacting environmental factors influence the topology of intracellular DNA. Negative supercoiling of DNA in vivo is increased by anaerobic growth and is also influenced by growth phase. The tonB promoter of Escherichia coli and Salmonella typhimurium was found to be highly sensitive to changes in DNA supercoiling. Expression was increased by novobiocin, an inhibitor of DNA gyrase, and was decreased by factors which increase DNA superhelicity. Expression of the plasmid-encoded tonB gene was enhanced by yB insertions in cis in a distance-and orientation-independent fashion. Both the res site and the TnpR protein of ,y8, which is known to function as a type I topoisomerase, were required for this activation. tonB expression increased during the growth cycle and was reduced by anaerobiosis. There was excellent correlation between tonB expression from a plasmid and the level of supercoiling of that plasmid under a wide range of conditions. The chromosomal tonB gene was regulated in a manner identical to that of the plasmid-encoded gene. Thus, the physiological regulation of tonB expression in response to anaerobiosis and growth phase appears to be mediated by environmentally induced changes in DNA superhelicity.Chromosomal DNA isolated from bacteria is negatively supercoiled. In vivo supercoiling is determined, at least in part, by a balance between the relaxing activity of topoisomerase I and the ATP-dependent supercoiling activity of DNA gyrase (16,43,44,51,71). The degree of supercoiling can influence a variety of processes, including promoter function and the initiation of transcription (reviewed in references 17, 18, and 72). The possibility that a specific environmental stimulus might alter DNA supercoiling in vivo and consequently contribute to the regulation of transcription of a subset of genes is an attractive idea. In this paper we present evidence that environmentally induced changes in DNA supercoiling are an important factor in the regulation of tonB gene expression in response to both anaerobiosis and growth phase.The tonB gene is located near trp at 27 min on the Escherichia coli genetic map and at 34 min in Salmonella typhimurium. TonB plays a key role in cellular physiology, being required for many energy-dependent outer membrane processes. Mutants lacking TonB are deficient in all highaffinity iron transport systems and vitamin B12 uptake and are resistant to many bacteriophages and colicins (reviewed in references 34, 46, and 48). Although the precise role of TonB is not known, it seems likely that the protein is required to couple energy to outer membrane processes (21,48,58
The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC−MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.
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