DNA supercoiling is a key regulator of all DNA metabolic processes including replication, transcription, and recombination, yet a reliable genomic assay for supercoiling is lacking. Here, we present a robust and flexible method (Psora-seq) to measure whole-genome supercoiling at high resolution. Using this tool in Escherichia coli, we observe a supercoiling landscape that is well correlated to transcription. Supercoiling twin-domains generated by RNA polymerase complexes span 25 kb in each direction – an order of magnitude farther than previous measurements in any organism. Thus, ribosomal and many other highly expressed genes strongly affect the topology of about 40 neighboring genes each, creating highly integrated gene circuits. Genomic patterns of supercoiling revealed by Psora-seq could be aptly predicted from modeling based on gene expression levels alone, indicating that transcription is the major determinant of chromosome supercoiling. Large-scale supercoiling patterns were highly symmetrical between left and right chromosome arms (replichores), indicating that DNA replication also strongly influences supercoiling. Skew in the axis of symmetry from the natural ori-ter axis supports previous indications that the rightward replication fork is delayed several minutes after initiation. Implications of supercoiling on DNA replication and chromosome domain structure are discussed.

The well-conserved genes surrounding the E. coli replication origin, mioC and gidA, do not normally affect chromosome replication and have little known function. We report that mioC and gidA mutants exhibit a moderate cell division inhibition phenotype. Cell elongation is exacerbated by a fis deletion, likely owing to delayed replication and subsequent cell cycle stress. Measurements of replication initiation frequency and origin segregation indicate that mioC and gidA do not inhibit cell division through any effect on oriC function. Division inhibition is also independent of the two known replication/cell division checkpoints, SOS and nucleoid occlusion. Complementation analysis indicates that mioC and gidA affect cell division in trans, indicating their effect is at the protein level. Transcriptome analysis by RNA sequencing showed that expression of a cell division septum component, YmgF, is significantly altered in mioC and gidA mutants. Our data reveal new roles for the gene products of gidA and mioC in the division apparatus, and we propose that their expression, cyclically regulated by chromatin remodeling at oriC, is part of a cell cycle regulatory program coordinating replication initiation and cell division.

Summary Although it has been recognized for several decades that chromosome structure regulates the capacity of replication origins to initiate, very little is known about how or if cells actively regulate structure to direct initiation [1–3]. We report that a localized inducible protein tether between the chromosome and cell membrane in E. coli cells imparts a rapid and complete block to replication initiation. Tethers, composed of a trans-membrane and transcription repressor fusion protein bound to an array of operator sequences, can be placed up to one megabase from the origin with no loss of penetrance. Tether-induced initiation blocking has no effect on elongation at pre-existing replication forks and does not cause cell or DNA damage. Whole-genome and site-specific fluorescent DNA labeling in tethered cells indicates that global nucleoid structure and chromosome organization are disrupted. Gene expression patterns, assayed by RNA sequencing shows that tethering induces global supercoiling changes, which are likely incompatible with replication initiation. Parallels between tether-induced initiation blocking and rifampicin treatment, and the role of programmed changes in chromosome structure in replication control are discussed.

The phosphoenopyruvate:carbohydrate phosphotransferase system (PTS) enables Vibrio cholerae – and other bacteria – to recognize and transport exogenous carbon sources for energy, including the six-carbon sugar alcohol, mannitol. The mannitol-specific PTS transporter is encoded by mtlA and its expression is expected to be regulated by the putative repressor encoded by the mtlR gene. Here, we show that mtlR overexpression inhibits V. cholerae growth in medium supplied with mannitol as the sole carbon source and represses MtlA-mediated biofilm formation. We demonstrate that when V. cholerae is grown in non-mannitol medium, knocking out mtlR leads to both increased MtlA protein and mtlA mRNA levels, with these increases being especially pronounced in non-glucose sugars. We propose that in non-mannitol, non-glucose growth conditions, MtlR is a major regulator of mtlA transcription. Surprisingly, with regard to mtlR expression, transcript and protein levels are highest in mannitol medium, conditions where mtlA expression should not be repressed. We further show that MtlR levels increase during growth of the bacteria and linger in cells switched from mannitol to non-mannitol medium. Our data suggests an expression paradigm for mtlA where MtlR acts as a transcriptional repressor responsible for calibrating MtlA levels during environmental transitions.

A green organic laboratory experiment was developed in which students synthesize a sensor for thiols using a microscale, solventless Diels–Alder reaction at room temperature or 37 °C. The molecular probe is easily purified by column chromatography in a Pasteur pipet and characterized by thin-layer chromatography and NMR spectroscopy. The thiol-reactive sensor becomes intensely fluorescent upon exposure to thiols from N-acetylcysteine, bovine serum albumin, or human hair (pretreated with a reducing agent to reveal cysteine thiols in α-keratin). This fluorescence is observable even with micrograms of probe.