Rasika M. Harshey

We show in this study that Salmonella cells, which do not upregulate flagellar gene expression during swarming, also do not increase flagellar numbers per μm of cell length as determined by systematic counting of both flagellar filaments and hooks. Instead, doubling of the average length of a swarmer cell by suppression of cell division effectively doubles the number of flagella per cell. The highest agar concentration at which Salmonella cells swarmed increased from the normal 0.5% to 1%, either when flagella were overproduced or when expression of the FliL protein was enhanced in conjunction with stator proteins MotAB. We surmise that bacteria use the resulting increase in motor power to overcome the higher friction associated with harder agar. Higher flagellar numbers also suppress the swarming defect of mutants with changes in the chemotaxis pathway that were previously shown to be defective in hydrating their colonies. Here we show that the swarming defect of these mutants can also be suppressed by application of osmolytes to the surface of swarm agar. The “dry” colony morphology displayed by che mutants was also observed with other mutants that do not actively rotate their flagella. The flagellum/motor thus participates in two functions critical for swarming, enabling hydration and overriding surface friction. We consider some ideas for how the flagellum might help attract water to the agar surface, where there is no free water.

Movement over an agar surface via swarming motility is subject to formidable challenges not encountered during swimming. Bacteria display a great deal of flexibility in coping with these challenges, which include attracting water to the surface, overcoming frictional forces, and reducing surface tension. Bacteria that swarm on “hard” agar surfaces (robust swarmers) display a hyperflagellated and hyperelongated morphology. Bacteria requiring a “softer” agar surface (temperate swarmers) do not exhibit such a dramatic morphology. For polarly flagellated robust swarmers, there is good evidence that restriction of flagellar rotation somehow signals the induction of a large number of lateral flagella, but this scenario is apparently not relevant to temperate swarmers. Swarming bacteria can be further subdivided by their requirement for multiple stators (Mot proteins) or a stator-associated protein (FliL), secretion of essential polysaccharides, cell density-dependent gene regulation including surfactant synthesis, a functional chemotaxis signaling pathway, appropriate cyclic (c)-di-GMP levels, induction of virulence determinants, and various nutritional requirements such as iron limitation or nitrate availability. Swarming strategies are as diverse as the bacteria that utilize them. The strength of these numerous designs stems from the vantage point they offer for understanding mechanisms for effective colonization of surface niches, acquisition of pathogenic potential, and identification of environmental signals that regulate swarming. The signature swirling and streaming motion within a swarm is an interesting phenomenon in and of itself, an emergent behavior with properties similar to flocking behavior in diverse systems, including birds and fish, providing a convenient new avenue for modeling such behavior.

Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5′ flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli—the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps. Transposon activity shapes genome structure and evolution. The movement of these elements generates target site duplications as a result of staggered cuts in the target made initially by the transposase. For replicative transposons, the single-stranded gaps generated after the initial strand transfer event are filled by target-primed replication. However, the majority of known transposable elements transpose by a non-replicative mechanism. Despite a wealth of information available for the mechanism of transposase action, little is known about how the cell repairs gaps left in the wake of transposition of these majority elements. Phage Mu is unique in using both replicative and non-replicative modes of transposition. Our study finds that during its non-replicative pathway, the gaps created by Mu insertion are repaired by the primary machinery for double-strand break repair in E. coli, not by gap-filling polymerases as previously thought. This first report of specific host processes involved in repair of transposon insertions in bacteria is likely to have a broad significance, given also that double-strand break repair pathways have been implicated in repair of the retroviral and Line retroelement insertions.

Summary: Chemoreceptors McpB and McpC in Salmonella enterica have been reported to promote chemotaxis in LB motility‐plate assays. Of the chemicals tested as potential effectors of these receptors, the only response was towards L‐cysteine and its oxidized form, L‐cystine. Although enhanced radial migration in plates suggested positive chemotaxis to both amino acids, capillary assays failed to show an attractant response to either, in cells expressing only these two chemoreceptors. In vivo fluorescence resonance energy transfer (FRET) measurements of kinase activity revealed that in wild‐type bacteria, cysteine and cystine are chemoeffectors of opposing sign, the reduced form being a chemoattractant and the oxidized form a repellent. The attractant response to cysteine was mediated primarily by Tsr, as reported earlier for Escherichia coli. The repellent response to cystine was mediated by McpB/C. Adaptive recovery upon cystine exposure required the methyl‐transferase/‐esterase pair, CheR/CheB, but restoration of kinase activity was never complete (i.e. imperfect adaptation). We provide a plausible explanation for the attractant‐like responses to both cystine and cysteine in motility plates, and speculate that the opposing signs of response to this redox pair might afford Salmonella a mechanism to gauge and avoid oxidative environments.

Summary: flhE belongs to the flhBAE flagellar operon in Enterobacteria, whose first two members function in Type III secretion (T3S). In Salmonella enterica, absence of FlhE affects swarming, but not swimming, motility. Based on a chance observation of a ‘green’ colony phenotype of flhE mutants on pH indicator plates containing glucose, we have established that this phenotype is associated with lysis of flagellated cells in an acidic environment created by glucose metabolism. The flhE mutant phenotype of Escherichia coli is similar overall to that of S. enterica but is seen in the absence of glucose and, unlike in S. enterica, causes a substantial growth defect. flhE mutants have a lowered cytoplasmic pH in both bacteria, indicative of a proton leak. GFP reporter assays indicate that the leak is dependent on the flagellar system, is present before the T3S system switches to secretion of late substrates, and gets worse after the switch and upon filament assembly, leading to cell lysis. We show that FlhE is a periplasmic protein that co‐purifies with flagellar basal bodies. FlhE may act as a plug or a chaperone to regulate proton flow through the flagellar T3S system.

This article traces the pioneering contributions of phage Mu to our current knowledge of how movable elements move/transpose. Mu provided the first molecular evidence of insertion elements in E. coli, postulated by McClintock to control gene activity in maize in the pre-DNA era. An early Mu-based model successfully explained all the DNA rearrangements associated with transposition, providing a blueprint for navigating the deluge of accumulating reports on transposable element activity. Amplification of the Mu genome via transposition meant that its transposition frequencies were orders of magnitude greater than any rival, so it was only natural that the first in vitro system for transposition was established for Mu. These experiments unraveled the chemistry of the phosphoryl transfer reaction of transposition, and shed light on the nucleoprotein complexes within which they occur. They hastened a similar analysis of other transposons and ushered in the structural era where many transpososomes were crystallized. While it was a lucky break that the mechanism of HIV DNA integration turned out to be similar to that of Mu, it is no accident that current drugs for HIV integrase inhibitors owe their discovery to trailblazing experiments done with Mu. Shining the light on how movable elements restructure genomes, Mu has also given of itself generously to understanding the genome.

Of all known transposable elements, phage Mu exhibits the highest transposition efficiency and the lowest target specificity. In vitro, MuB protein is responsible for target choice. In this work, we provide a comprehensive assessment of the genome-wide distribution of MuB and its relationship to Mu target selection using high-resolution Escherichia coli tiling DNA arrays. We have also assessed how MuB binding and Mu transposition are influenced by chromosome-organizing elements such as AT-rich DNA signatures, or the binding of the nucleoid-associated protein Fis, or processes such as transcription. The results confirm and extend previous biochemical and lower resolution in vivo data. Despite the generally random nature of Mu transposition and MuB binding, there were hot and cold insertion sites and MuB binding sites in the genome, and differences between the hottest and coldest sites were large. The new data also suggest that MuB distribution and subsequent Mu integration is responsive to DNA sequences that contribute to the structural organization of the chromosome.

Swarming bacteria move in multicellular groups and exhibit adaptive resistance to multiple antibiotics. Analysis of this phenomenon has revealed the protective power of high cell densities to withstand exposure to otherwise lethal antibiotic concentrations. We find that high densities promote bacterial survival, even in a nonswarming state, but that the ability to move, as well as the speed of movement, confers an added advantage, making swarming an effective strategy for prevailing against antimicrobials. We find no evidence of induced resistance pathways or quorum-sensing mechanisms controlling this group resistance, which occurs at a cost to cells directly exposed to the antibiotic. This work has relevance to the adaptive antibiotic resistance of bacterial biofilms.