Inline-tandem purification of viruses from cell lysate by agarose-based chromatography.

An efficient chromatography-based virus purification method has been developed and validated for the non-pathogenic infectious virus PRD1. Compared to the conventional method that consists of relatively time-consuming and labour-intensive precipitation and density gradient ultracentrifugation steps, the method developed here is performed in a single flow using tandem-coupled anion exchange and size exclusion chromatography (AIEX-SEC) columns. This inline approach helps to minimize the loss of virus in the process and streamlines time consumption, since no physical transfer of the sample is required between purification steps. In the development process, sample feed composition, dynamic binding capacity and elution conditions for the AIEX resin as well as different exclusion limits for SEC resins were optimized to achieve maximal yield of pure infectious viruses. Utilizing this new approach, a high-quality virus sample was produced from a lysate feed in 320 min with a total yield of 13 mg purified particles per litre of cell lysate, constituting a 3.5-fold yield increase as compared to the conventional method, without compromising the high specific infectivity of the product (6 × 1012 to 7 × 1012 pfu/mg of protein). The yield of infectious viruses of the lysate feed was 54%. The easy scalability of chromatography-based methods provide a direct route to industrial usage without any significant changes needed to be made to the purification regime. This is especially interesting as the method has high potential to be used for purification of various viruses and nanoparticles, including adenovirus.

Kinetics of dCas9 target search in Escherichia coli.

How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.

Extracting intracellular diffusive states and transition rates from single-molecule tracking data.

We provide an analytical tool based on a variational Bayesian treatment of hidden Markov models to combine the information from thousands of short single-molecule trajectories of intracellularly diffusing proteins. The method identifies the number of diffusive states and the state transition rates. Using this method we have created an objective interaction map for Hfq, a protein that mediates interactions between small regulatory RNAs and their mRNA targets.

The toxin-antitoxin system tisB-istR1: Expression, regulation, and biological role in persister phenotypes.

Chromosomally encoded toxin-antitoxin (TA) systems are abundantly present in bacteria and archaea. They have become a hot topic in recent years, because-after many frustrating years of searching for biological functions-some are now known to play roles in persister formation. Persister cells represent a subset of a bacterial population that enters a dormant state and thus becomes refractory to the action of antibiotics. TA modules come in several different flavors, regarding the nature of their gene products, their molecular mechanisms of regulation, their cellular targets, and probably their role in physiology. This review will primarily focus on the SOS-associated tisB/istR1 system in Escherichia coli and discuss its nuts and bolts as well as its effect in promoting a subpopulation phenotype that likely benefits long-term survival of a stressed population.

A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp.

Roughly 10% of all genes in Escherichia coli are controlled by the global transcription factor Lrp, which responds to nutrient availability. Bioinformatically, we identified lrp as one of several putative targets for the sRNA MicF, which is transcriptionally downregulated by Lrp. Deleting micF results in higher Lrp levels, while overexpression of MicF inhibits Lrp synthesis. This effect is by antisense; mutations in the predicted interaction region relieve MicF-dependent repression of Lrp synthesis, and regulation is restored by compensatory mutations. In vitro, MicF sterically interferes with initiation complex formation and inhibits lrp mRNA translation. In vivo, MicF indirectly activates genes in the Lrp regulon by repressing Lrp, and causes severely impaired growth in minimal medium, a phenotype characteristic of lrp deletion strains. The double negative feedback between MicF and Lrp may promote a switch for adequate Lrp-dependent adaptation to nutrient availability. Lrp adds to the growing list of transcription factors that are targeted by sRNAs, thus indicating that perhaps the majority of all bacterial genes may be directly or indirectly controlled by sRNAs.

A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli.

We previously reported on an SOS-induced toxin, TisB, in Escherichia coli and its regulation by the RNA antitoxin IstR-1. Here, we addressed the mode of action of TisB. By placing the tisB reading frame downstream of a controllable promoter on a plasmid, toxicity could be analysed in the absence of the global SOS response. Upon induction of TisB, cell growth was inhibited and plating efficiency decreased rapidly. The onset of toxicity correlated with a drastic decrease in transcription, translation and replication rates. Cellular RNA was degraded, but in vitro experiments showed that TisB did not affect translation or transcription directly. Thus, these effects are downstream consequences of membrane damage: TisB is predicted to be hydrophobic and membrane spanning, and Western analyses demonstrated that this peptide was strictly localized to the cytoplasmic membrane fraction. Membrane damage and cell killing under tisB multicopy expression are also seen by live/death staining and the formation of ghost cells. This is reminiscent of another toxin, Hok of plasmid R1, which also targets the membrane. The biological significance of the istR/tisB locus is still elusive; deletion of the entire locus gave no fitness phenotype in competition experiments.

Dealing with stable structures at ribosome binding sites: bacterial translation and ribosome standby.

Bacterial ribosomes have great difficulties to initiate translation on stable structures within mRNAs. Translational coupling and induced structure changes are strategies to open up inhibitory RNA structures encompassing ribosome binding sites (RBS). There are, however, mRNAs in which stable structures are not unfolded, but that are nevertheless efficiently initiated at high rates. de Smit and van Duin(1) proposed a "ribosome standby" model to theoretically solve this paradox: the 30S ribosome binds nonspecifically to an accessible site on the mRNA (standby site), waiting for a transient opening of a stable RBS hairpin. Upon unfolding, the 30S subunit relocates to form a productive initiation complex. Recent reports have provided experimental support for this model. This review will describe and compare two different flavors of standby sites, their properties, and their likely implications. We also discuss the possibility that ribosome standby may be a more general strategy to obtain high translation rates.

An antisense RNA inhibits translation by competing with standby ribosomes.

Most antisense RNAs in bacteria inhibit translation by competing with ribosomes for translation initiation regions (TIRs) on nascent mRNA. We propose a mechanism by which an antisense RNA inhibits translation without binding directly to a TIR. The tisAB locus encodes an SOS-induced toxin, and IstR-1 is the antisense RNA that counteracts toxicity. We show that full-length tisAB mRNA (+1) is translationally inactive and endonucleolytic processing produces an active mRNA (+42). IstR-1 binding inhibits translation of this mRNA, and subsequent RNase III cleavage generates a truncated, inactive mRNA (+106). In vitro translation, toeprinting, and structure mapping suggest that active, but not inactive, tisAB mRNAs contain an upstream ribosome loading or "standby" site. Standby binding is required for initiation at the highly structured tisB TIR. This may involve ribosome sliding to a transiently open tisB TIR. IstR-1 competes with ribosomes by base pairing to the standby site located approximately 100 nucleotides upstream.