Membrane recognition and dynamics of the RNA degradosome.

RNase E, which is the central component of the multienzyme RNA degradosome, serves as a scaffold for interaction with other enzymes involved in mRNA degradation including the DEAD-box RNA helicase RhlB. Epifluorescence microscopy under live cell conditions shows that RNase E and RhlB are membrane associated, but neither protein forms cytoskeletal-like structures as reported earlier by Taghbalout and Rothfield. We show that association of RhlB with the membrane depends on a direct protein interaction with RNase E, which is anchored to the inner cytoplasmic membrane through an MTS (Membrane Targeting Sequence). Molecular dynamics simulations show that the MTS interacts with the phospholipid bilayer by forming a stabilized amphipathic α-helix with the helical axis oriented parallel to the plane of the bilayer and hydrophobic side chains buried deep in the acyl core of the membrane. Based on the molecular dynamics simulations, we propose that the MTS freely diffuses in the membrane by a novel mechanism in which a large number of weak contacts are rapidly broken and reformed. TIRFm (Total Internal Reflection microscopy) shows that RNase E in live cells rapidly diffuses over the entire inner membrane forming short-lived foci. Diffusion could be part of a scanning mechanism facilitating substrate recognition and cooperativity. Remarkably, RNase E foci disappear and the rate of RNase E diffusion increases with rifampicin treatment. Control experiments show that the effect of rifampicin is specific to RNase E and that the effect is not a secondary consequence of the shut off of E. coli transcription. We therefore interpret the effect of rifampicin as being due to the depletion of RNA substrates for degradation. We propose a model in which formation of foci and constraints on diffusion arise from the transient clustering of RNase E into cooperative degradation bodies.

Dual role of transcription and transcript stability in the regulation of gene expression in Escherichia coli cells cultured on glucose at different growth rates.

Microorganisms extensively reorganize gene expression to adjust growth rate to changes in growth conditions. At the genomic scale, we measured the contribution of both transcription and transcript stability to regulating messenger RNA (mRNA) concentration in Escherichia coli. Transcriptional control was the dominant regulatory process. Between growth rates of 0.10 and 0.63 h(-1), there was a generic increase in the bulk mRNA transcription. However, many transcripts became less stable and the median mRNA half-life decreased from 4.2 to 2.8 min. This is the first evidence that mRNA turnover is slower at extremely low-growth rates. The destabilization of many, but not all, transcripts at high-growth rate correlated with transcriptional upregulation of genes encoding the mRNA degradation machinery. We identified five classes of growth-rate regulation ranging from mainly transcriptional to mainly degradational. In general, differential stability within polycistronic messages encoded by operons does not appear to be affected by growth rate. We show here that the substantial reorganization of gene expression involving downregulation of tricarboxylic acid cycle genes and acetyl-CoA synthetase at high-growth rates is controlled mainly by transcript stability. Overall, our results demonstrate that the control of transcript stability has an important role in fine-tuning mRNA concentration during changes in growth rate.

Repeated Exposure of Escherichia coli to High Ciprofloxacin Concentrations Selects gyrB Mutants That Show Fluoroquinolone-Specific Hyperpersistence.

Recent studies have shown that not only resistance, but also tolerance/persistence levels can evolve rapidly in bacteria exposed to repeated antibiotic treatments. We used in vitro evolution to assess whether tolerant/hyperpersistent Escherichia coli ATCC25922 mutants could be selected under repeated exposure to a high ciprofloxacin concentration. Among two out of three independent evolution lines, we observed the emergence of gyrB mutants showing an hyperpersistence phenotype specific to fluoroquinolones, but no significant MIC increase. The identified mutation gives rise to a L422P substitution in GyrB, that is, outside of the canonical GyrB QRDR. Our results indicate that mutations in overlooked regions of quinolone target genes may impair the efficacy of treatments via an increase of persistence rather than resistance level, and support the idea that, in addition to resistance, phenotypes of tolerance/persistence of infectious bacterial strains should receive considerations in the choice of antibiotic therapies.

Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress.

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including the ß-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

SpxA1, a novel transcriptional regulator involved in X-state (competence) development in Streptococcus pneumoniae.

Streptococcus pneumoniae is a naturally transformable human pathogen. Genome and phylogenetic analyses uncovered two Spx-like global transcriptional regulators, SpxA1 and SpxA2, encoded by S. pneumoniae. spxA1 and spxA2 are not essential, but their simultaneous inactivation is lethal. SpxA1 represses transcription of the early competence operon comCDE and thereby negatively regulates the initiation of the X-state (competence). The molecular basis of this repression could be similar to that of SpxA of Bacillus subtilis, involving a specific interaction with the alpha subunit of RNA polymerase. S. pneumoniae lacks an SOS-like stress response and the X-state is proposed to be a general stress response mechanism in this species. In light of this, SpxA1-dependent repression could act to sense environmental or metabolic stresses and prevent launching of the X-state in the absence of stress.

Addressing the role of centromere sites in activation of ParB proteins for partition complex assembly.

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.

Single-particle tracking for DNA tether length monitoring.

We describe a simple single-particle tracking approach for monitoring the length of DNA molecules in tethered particle motion experiments. In this method, the trajectory of a submicroscopic bead tethered by a DNA molecule to a glass surface is determined by videomicroscopy coupled to image analysis. The amplitude of motion of the bead is measured by the standard deviation of the distribution of successive positions of the bead in a given time interval. We were able to describe theoretically the variation of the equilibrium value of the amplitude of the bead motion with the DNA tether length for the entire applicable DNA length range (up to approximately 3500 bp). The sensitivity of the approach was illustrated by the evidence obtained for conformational changes introduced into a Holliday junction by the binding of the Escherichia coli RuvA protein. An advantage of this method is that the trajectory of the tethered bead, rather than its averaged motion, is measured, allowing analysis of the conformational dynamics of DNA chains at the single-molecule level.

The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

Host processing of branched DNA intermediates is involved in targeted transposition of IS911.

A simplified system using bacterial insertion sequence IS911 has been developed to investigate targeted insertion next to DNA sequences resembling IS ends. We show here that these IR-targeted events occur by an unusual mechanism. In the circular IS911 transposition intermediate the two IRs are abutted to form an IR/IR junction. IR-targeted insertion involves transfer of a single end of the junction to the target IR to generate a branched DNA structure. The single-end transfer (SET) intermediate, but not the final insertion product, can be detected in an in vitro reaction. SET intermediates must be processed by the bacterial host to obtain the final insertion products. Sequence analysis of these IR-targeted insertion products and of those obtained in vivo revealed high levels of DNA sequence conversion in which mutations from one IR were transferred to another. These sequence changes cannot be explained by the classic transposition pathway. A model is presented in which the four-way Holliday-like junction created by SET is processed by host-mediated branch migration, resolution, repair and replication. This pathway resembles those described for processing other branched DNA structures such as stalled replication forks.

IS911 partial transposition products and their processing by the Escherichia coli RecG helicase.

Insertion of bacterial insertion sequence IS911 can often be directed to sequences resembling its ends. We have investigated this type of transposition and shown that it can occur via cleavage of a single end and its targeted transfer next to another end. The single end transfer (SET) events generate branched DNA molecules that contain a nicked Holliday junction and can be considered as partial transposition products. Our results indicate that these can be processed by the Escherichia coli host independently of IS911-encoded proteins. Such resolution depends on the presence of homologous DNA regions neighbouring the cross-over point in the SET molecule. Processing is often accompanied by sequence conversion between donor and target sequences, suggesting that branch migration is involved. We show that resolution is greatly reduced in a recG host. Thus, the branched DNA-specific helicase, RecG, involved in processing of potentially lethal DNA structures such as stalled replication forks, also intervenes in the resolution of partial IS911 transposition products.

Functional domains of the IS1 transposase: analysis in vivo and in vitro.

The IS1 bacterial insertion sequence family, considered to be restricted to Enterobacteria, has now been extended to other Eubacteria and to Archaebacteria, reviving interest in its study. To analyse the functional domains of the InsAB' transposase of IS1A, a representative of this family, we used an in vivo system which measures IS1-promoted rescue of a temperature-sensitive pSC101 plasmid by fusion with a pBR322::IS1 derivative. We also describe the partial purification of the IS1 transposase and the development of several in vitro assays for transposase activity. These included a DNA band shift assay, a transposase-mediated cleavage assay and an integration assay. Alignments of IS family members (http://www-is.biotoul.fr) not only confirmed the presence of an N-terminal helix-turn-helix and a C-terminal DDE motif in InsAB', but also revealed a putative N-terminal zinc finger. We have combined the in vitro and in vivo tests to carry out a functional analysis of InsAB' using a series of site-directed InsAB' mutants based on these alignments. The results demonstrate that appropriate mutations in the zinc finger and helix-turn-helix motifs result in loss of binding activity to the ends of IS1 whereas mutations in the DDE domain are affected in subsequent transposition steps but not in end binding.