A bacteriophage tubulin harnesses dynamic instability to center DNA in infected cells.

Dynamic instability, polarity, and spatiotemporal organization are hallmarks of the microtubule cytoskeleton that allow formation of complex structures such as the eukaryotic spindle. No similar structure has been identified in prokaryotes. The bacteriophage-encoded tubulin PhuZ is required to position DNA at mid-cell, without which infectivity is compromised. Here, we show that PhuZ filaments, like microtubules, stochastically switch from growing in a distinctly polar manner to catastrophic depolymerization (dynamic instability) both in vitro and in vivo. One end of each PhuZ filament is stably anchored near the cell pole to form a spindle-like array that orients the growing ends toward the phage nucleoid so as to position it near mid-cell. Our results demonstrate how a bacteriophage can harness the properties of a tubulin-like cytoskeleton for efficient propagation. This represents the first identification of a prokaryotic tubulin with the dynamic instability of microtubules and the ability to form a simplified bipolar spindle

Phenotypic consequences of RNA polymerase dysregulation in Escherichia coli

Many bacterial adaptive responses to changes in growth conditions due to biotic and abiotic factors involve reprogramming of gene expression at the transcription level. The bacterial RNA polymerase (RNAP), which catalyzes transcription, can thus be considered as the major mediator of cellular adaptive strategies. But how do bacteria respond if a stress factor directly compromises the activity of the RNAP? We used a phage-derived small protein to specifically perturb bacterial RNAP activity in exponentially growing Escherichia coli. Using cytological profiling, tracking RNAP behavior at single-molecule level and transcriptome analysis, we reveal that adaptation to conditions that directly perturb bacterial RNAP performance can result in a biphasic growth behavior and thereby confer the ‘adapted’ bacterial cells an enhanced ability to tolerate diverse antibacterial stresses. The results imply that while synthetic transcriptional rewiring may confer bacteria with the intended desirable properties, such approaches may also collaterally allow them to acquire undesirable traits

Transposon Assisted Gene Insertion Technology (TAGIT): A Tool for Generating Fluorescent Fusion Proteins

We constructed a transposon (transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses KanR to select for insertions on the chromosome or plasmid, β-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. The resulting gfp insertions maintain target gene reading frame (to the 5′ and 3′ of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins

Systematic study of the low-lying electric dipole strength in Sn isotopes and its astrophysical implications

The $\gamma$-ray strength functions (GSF) and nuclear level densities (NLD) below the neutron threshold have been extracted for $^{111-113,116-122,124}$Sn from particle-$\gamma$ coincidence data with the Oslo method. The evolution of bulk properties of the low-lying electric dipole response has been investigated on the basis of the Oslo GSF data and results of a recent systematic study of electric and magnetic dipole strengths in even-even Sn isotopes with relativistic Coulomb excitation. The obtained GSFs reveal a resonance-like peak on top of the tail of the isovector giant dipole resonance, centered at $\approx$8 MeV and exhausting $\approx$2\% of the classical Thomas-Reiche-Kuhn (TRK) sum. In contrast to predictions of the relativistic quasiparticle random-phase and time-blocking approximation calculations (RQRPA and RQTBA), no monotonous increase in the total low-lying $E1$ strength was observed in the experimental data from $^{111}$Sn to $^{124}$Sn, demonstrating rather similar strength distributions in these nuclei. The Oslo GSFs and NLDs were further used as inputs to constrain the cross sections and Maxwellian-averaged cross sections of $(n,\gamma)$ reactions in the Sn isotopic chain using TALYS. The obtained results agree well with other available experimental data and the recommended values from the JINA REACLIB, BRUSLIB, and KADoNiS libraries. Despite relatively small exhausted fractions of the TRK sum rule, the low-lying electric dipole strength makes a noticeable impact on the radiative neutron-capture cross sections in stable Sn isotopes. Moreover, the experimental Oslo inputs for the $^{121,123}$Sn$(n,\gamma)$$^{122,124}$Sn reactions were found to affect the production of Sb in the astrophysical $i$-process, providing new constraints on the uncertainties of the resulting chemical abundances from multi-zone low-metallicity Asymptotic Giant Branch stellar models.Comment: 27 pages, 14 pages. Submitted to Physical Review C journal on 13 November 202

Realization of the farad from the dc quantum Hall effect with digitally-assisted impedance bridges

A new traceability chain for the derivation of the farad from dc quantum Hall effect has been implemented at INRIM. Main components of the chain are two new coaxial transformer bridges: a resistance ratio bridge, and a quadrature bridge, both operating at 1541 Hz. The bridges are energized and controlled with a polyphase direct-digital-synthesizer, which permits to achieve both main and auxiliary equilibria in an automated way; the bridges and do not include any variable inductive divider or variable impedance box. The relative uncertainty in the realization of the farad, at the level of 1000 pF, is estimated to be 64E-9. A first verification of the realization is given by a comparison with the maintained national capacitance standard, where an agreement between measurements within their relative combined uncertainty of 420E-9 is obtained.Comment: 15 pages, 11 figures, 3 table

High-throughput, quantitative analyses of genetic interactions in E. coli.

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli

Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC(50) to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules

"Dangerous to Themselves and Others, and of Public Scandal": The Internment Procedure

Abstract Through G.'s admission and medical files, this chapter illustrates internment laws and procedures, highlighting how Fascism pushed pre-existing legislation to its extreme consequences. In reconstructing internment's bureaucratic and legal practices, the chapter emphasises how the law could be bent to accommodate the regime's need to isolate those perceived as "different" and how psychiatry acquiesced in offering to "correct" individuals considered "non-conforming", "amoral", "immoral", "deviant", rebellious and, among them, homosexuals, in exchange for an increase of power and status