It is generally assumed that antibiotics can promote horizontal gene transfer (HGT). However, because of a variety of confounding factors that complicate the interpretation of previous studies, the mechanisms by which antibiotics modulate HGT remain poorly understood. In particular, it is unclear whether antibiotics directly regulate the efficiency of HGT, serve as a selection force to modulate population dynamics after HGT has occurred, or both. Here, we address this question by quantifying conjugation dynamics in the presence and absence of antibiotic-mediated selection. Surprisingly, we find that sub-lethal concentrations of antibiotics from the most widely used classes do not significantly increase the conjugation efficiency. Instead, our modeling and experimental results demonstrate that conjugation dynamics are dictated by antibiotic-mediated selection, which can both promote and suppress conjugation dynamics. Our findings suggest that the contribution of antibiotics to the promotion of HGT may have been overestimated. These findings have implications for designing effective antibiotic treatment protocols and for assessing the risks of antibiotic use.

SUMMARY The NtrC-like AAA+ ATPases control virulence and other important bacterial activities through delivering mechanical work to σ54-RNA polymerase to activate transcription from σ54-dependent genes. We report the first crystal structure for such an ATPase, NtrC1 of Aquifex aeolicus, in which the catalytic arginine engages the gamma-phosphate of ATP. Comparing the new structure with those previously known for apo and ADP-bound states supports a novel rigid-body displacement model that is consistent with large-scale conformational changes observed by low-resolution methods. First, the arginine-finger induces rigid-body roll, extending surface loops above the plane of the ATPase ring to bind σ54. Second, ATP hydrolysis permits Pi release and retraction of the arginine with a reversed roll, remodeling σ54-RNAP. This model provides a fresh perspective on how ATPase subunits interact within the ring-ensemble to promote transcription, directing attention to structural changes on the arginine-finger side of an ATP-bound interface.

Protein secretion typically involves translocation of unfolded polypeptides or transport of monomeric folded proteins. Here we provide, to our knowledge, the first experimental evidence for secretion of an intact multimeric complex requiring a signal formed by both members of the complex. Using systematic mutagenesis of a substrate involved in early secretory antigen 6 kDa (ESX) secretion in Bacillus subtilis, we demonstrate that export of the substrate requires two independent motifs. Using mixed dimers, we show that these motifs must form a composite secretion signal in which one motif is contributed by each subunit of the dimer. Finally, through targeted crosslinking we show that the dimer formed in the cell is likely secreted as a single unit. We discuss implications of this substrate recognition mechanism for the biogenesis and quality control of secretion substrates and describe its likely conservation across ESX systems.WXG protein | type VII secretion system | protein translocation | YukE P rotein secretion is critical for protein targeting in any living cell and for its communication with the environment. Bacteria use a wide range of secretion mechanisms to export proteins out of the cytoplasm. Signals for secretion are most commonly primary amino acid sequences, but in some cases also may be formed through interacting surfaces of a substrate and its delivery effector. Some secretion systems unfold their substrates to translocate them across the membrane and cell wall. Other systems export folded proteins, sometimes in complex with bound cofactors. For example, the general secretory machinery (Sec) denatures the tertiary and secondary structure of its substrates to thread the polypeptide through the narrow opening of the integral membrane translocon complex, SecYEG (1). Type III secretion system (T3SS) machinery is thought to unfold the tertiary structure of its substrates, while preserving the secondary structure elements for the substrate recognition (2, 3). In contrast, the twin-arginine transport (Tat) system exports folded substrates (4) and is hypothesized to be able to translocate protein oligomers and complexes via a "hitchhiking" mechanism (5). Overall, these and other secretion types differ in the nature of substrate recognition signal and the mode of substrate translocation.Early secretory antigen 6 kDa (ESX, or type VII) secretion systems are widespread in actinomycetes and Gram-positive bacteria and affect a range of bacterial processes including sporulation, conjugation, and cell wall stability (6-10). In two notorious human pathogens, Mycobacterium tuberculosis and Staphylococcus aureus, ESX secretion was found to be crucial for establishing and maintaining the infection (11-15). Despite the importance of the ESX secretion for human health, the mechanism of this type of secretion is still largely unknown.Recent characterization of the ESX system in Bacillus subtilis confirmed that a functional system is encoded by the yuk/yue operon (16,17). Importantly, the B. subtilis system codes for a...

Pseudomonas aeruginosa is an opportunistic pathogen that often infects open wounds or patients with cystic fibrosis. Once established, P. aeruginosa infections are notoriously difficult to eradicate. This difficulty is in part due to the ability of P. aeruginosa to tolerate antibiotic treatment at the individual-cell level or through collective behaviors. Here, we describe a new phenomenon by which P. aeruginosa tolerates antibiotic treatment. In particular, treatment of P. aeruginosa with sublethal concentrations of antibiotics covering all major classes promoted accumulation of the redox-sensitive phenazine pyocyanin (PYO). PYO in turn conferred general tolerance against diverse antibiotics for both P. aeruginosa and other gram-negative and gram-positive bacteria. This property is shared by other redox-active phenazines produced by P. aeruginosa. Our discovery sheds new insights into the physiological functions of phenazines and has implications for designing effective antibiotic treatment protocols.

It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with s54-RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of s54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.