Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals where it can be both a commensal and a pathogen1–3. Intricate regulatory mechanisms ensure bacteria have the right complement of β-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat4,5. Yet no mechanism is known for replacing OMPs in the outer membrane (OM), a biological enigma further confounded by the lack of an energy source and the high stability6 and abundance of OMPs5. Here, we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature wherein old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics (MD) simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form islands of ~0.5 μm diameter where their diffusion was restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the OM7,8. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence the OM of a Gram-negative bacterium is a spatially and temporally organised structure and this organisation lies at the heart of how OMPs are turned over in the membrane.

A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k off ‫؍‬ 2.9 s ؊1 ) of T7 RNAP and promoter DNA, the transition to the elongation mode (k for ‫؍‬ 0.36 s ؊1 ), and the processive synthesis (k pol ‫؍‬ 43 nt s ؊1 ) and release of a gene-length RNA transcript (ϳ1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k off > k for ), identifying a rate-limiting step between promoter DNA binding and promoter escape. Transcription initiation by RNAP1 is an important regulatory step for gene expression in vivo. However, the details of transcription initiation are difficult to elucidate using conventional solution methods because (i) initiation consists of a series of transient intermediate steps between promoter binding and elongation, and (ii) within a population of actively transcribing RNAP molecules, only a small fraction is engaged in initiation at any given time. Most biochemical studies of transcription initiation utilize solution conditions which allow only a single enzymatic turnover, e.g. RNAP halted at a known position in the DNA sequence by ribonucleotide starvation or rapid mixing/quenching of the transcription assay. An attempt has been made to synchronize a population of T7 RNAP molecules in solution (1); however, synchrony is rapidly lost as the transitions between states are probabilistic events. The challenges associated with synchronizing a population of molecules can be avoided by making measurements on a single molecule. Previous single-molecule studies of transcription by Escherichia coli RNAP have only allowed DNA binding (2), elongation (artificially halted by ribonucleotide starvation) (3-8), and termination (9) to be observed in isolation from the other transcriptional states. No previous single-molecule transcription assay has allowed promoter recognition and the transition from initiation to elongation to be observed.T7 RNAP is a common paradigm for studies of transcription initiation, as it is a single-subunit enzyme sharing many of the biochemical characteristics of the more complex multi-subunit RNAPs from prokary...

Two recent antifungal agents, miconazole and ketoconazole, were combined with three tissue conditioners and tested in vitro for their effects on the growth of Candida albicans. Studies for comparison were carried out using the earlier antifungal agents, amphotericin B and nystatin. Miconazole and ketoconazole were as effective as nystatin in completely inhibiting the growth of Candida albicans. The ineffectiveness of amphotericin B when combined with tissue conditioners as an antifungal agent was confirmed. (See Addendum p.181.).

Summary In some mild haemophilia A patients (discrepant haemophilia), factor VIII coagulant activity (FVIII:C) levels, by one‐stage assay are more than double than those by two‐stage assay. This may be due to the longer incubation times (10–12 min) in the two‐stage assay. This study aimed to determine the time course of the activation phase of the two‐stage assay, using both classical coagulation and chromogenic detection methods. In both systems, for equivalent patients (equivalent FVIII:C levels by one‐stage and two‐stage assays, n = 6, all different mutations), similar FVIII:C results were obtained with short‐ or long‐incubation times. In contrast, plasma from discrepant patients (n = 8, five different mutations) showed higher FVIII:C at shorter incubation times than after longer incubation times. In the chromogenic assay, FVIII:C levels were higher after incubation for 2 min (23–56%, mean 41%) than after 10 min (19–41%, mean 29%). In the classical coagulation assay, FVIII:C levels were higher at shorter incubation times (21–64%, mean 37%) than with the longer incubation times usually used (13–29%, mean 23%). These time‐course experiments have verified that the longer incubation time used in the two‐stage assay is at least partly responsible for the lower FVIII:C measured by that assay in discrepant haemophilia.