Flagellar Motility is Mutagenic.

Flagella are highly complex rotary molecular machines that enable bacteria to not only migrate to optimal environments but to also promote range expansion, competitiveness, virulence, and antibiotic survival. Flagellar motility is an energy-demanding process, where the sum of its production (biosynthesis) and operation (rotation) costs has been estimated to total ∼10% of the entire energy budget of an E. coli cell. The acquisition of such a costly adaptation process is expected to secure short-term benefits by increasing competitiveness and survival, as well as long-term evolutionary fitness gains. While the role of flagellar motility in bacterial survival has been widely reported, its direct influence on the rate of evolution remains unclear. We show here that both production and operation costs contribute to elevated mutation frequencies. Our findings suggest that flagellar movement may be an important player in tuning the rate of bacterial evolution.

A heritable iron memory enables decision-making in Escherichia coli.

The importance of memory in bacterial decision-making is relatively unexplored. We show here that a prior experience of swarming is remembered when Escherichia coli encounters a new surface, improving its future swarming efficiency. We conducted >10,000 single-cell swarm assays to discover that cells store memory in the form of cellular iron levels. This "iron" memory preexists in planktonic cells, but the act of swarming reinforces it. A cell with low iron initiates swarming early and is a better swarmer, while the opposite is true for a cell with high iron. The swarming potential of a mother cell, which tracks with its iron memory, is passed down to its fourth-generation daughter cells. This memory is naturally lost by the seventh generation, but artificially manipulating iron levels allows it to persist much longer. A mathematical model with a time-delay component faithfully recreates the observed dynamic interconversions between different swarming potentials. We demonstrate that cellular iron levels also track with biofilm formation and antibiotic tolerance, suggesting that iron memory may impact other physiologies.

Flagellar motor remodeling during swarming requires FliL.

FliL is an essential component of the flagellar machinery in some bacteria, but a conditional one in others. The conditional role is for optimal swarming in some bacteria. During swarming, physical forces associated with movement on a surface are expected to exert a higher load on the flagellum, requiring more motor torque to move. FliL was reported to enhance motor output in several bacteria and observed to assemble as a ring around ion-conducting stators that power the motor. In this study we identify a common new function for FliL in diverse bacteria-Escherichia coli, Bacillus subtilis, and Proteus mirabilis. During swarming, all these bacteria show increased cell speed and a skewed motor bias that suppresses cell tumbling. We demonstrate that these altered motor parameters, or "motor remodeling," require FliL. Both swarming and motor remodeling can be restored in an E. coli fliL mutant by complementation with fliL genes from P. mirabilis and B. subtilis, showing conservation of a swarming-associated FliL function across phyla. In addition, we demonstrate that the strong interaction we reported earlier between FliL and the flagellar MS-ring protein FliF is confined to the RBM-3 domain of FliF that links the periplasmic rod to the cytoplasmic C-ring. This interaction may explain several phenotypes associated with the absence of FliL.

Iron Memory in E. coli.

The importance of memory in bacterial decision-making is relatively unexplored. We show here that a prior experience of swarming is remembered when E. coli encounters a new surface, improving its future swarming efficiency. We conducted >10,000 single-cell swarm assays to discover that cells store memory in the form of cellular iron levels. This memory pre-exists in planktonic cells, but the act of swarming reinforces it. A cell with low iron initiates swarming early and is a better swarmer, while the opposite is true for a cell with high iron. The swarming potential of a mother cell, whether low or high, is passed down to its fourth-generation daughter cells. This memory is naturally lost by the seventh generation, but artificially manipulating iron levels allows it to persist much longer. A mathematical model with a time-delay component faithfully recreates the observed dynamic interconversions between different swarming potentials. We also demonstrate that iron memory can integrate multiple stimuli, impacting other bacterial behaviors such as biofilm formation and antibiotic tolerance.

Flagellar motor remodeling during swarming requires FliL.

FliL is an essential component of the flagellar machinery in some bacteria, but a conditional one in others. The conditional role is for optimal swarming in some bacteria. During swarming, physical forces associated with movement on a surface are expected to exert a higher load on the flagellum, requiring more motor torque to move. Bacterial physiology and morphology are also altered during swarming to cope with the challenges of surface navigation. FliL was reported to enhance motor output in several bacteria and observed to assemble as a ring around ion-conducting stators that power the motor. In this study we identify a common new function for FliL in diverse bacteria - Escherichia coli, Bacillus subtilis and Proteus mirabilis . During swarming, all these bacteria show increased cell speed and a skewed motor bias that suppresses cell tumbling. We demonstrate that these altered motor parameters, or 'motor remodeling', require FliL. Both swarming and motor remodeling can be restored in an E. coli fliL mutant by complementation with fliL genes from P. mirabilis and B. subtilis , showing conservation of swarming-associated FliL function across phyla. In addition, we demonstrate that the strong interaction we reported earlier between FliL and the flagellar MS-ring protein FliF is confined to the RBM-3 domain of FliF that links the periplasmic rod to the cytoplasmic C-ring. This interaction may explain several phenotypes associated with the absence of FliL.

A Second Role for the Second Messenger Cyclic-di-GMP in E. coli: Arresting Cell Growth by Altering Metabolic Flow.

c-di-GMP primarily controls motile to sessile transitions in bacteria. Diguanylate cyclases (DGCs) catalyze the synthesis of c-di-GMP from two GTP molecules. Typically, bacteria encode multiple DGCs that are activated by specific environmental signals. Their catalytic activity is modulated by c-di-GMP binding to autoinhibitory sites (I-sites). YfiN is a conserved inner membrane DGC that lacks these sites. Instead, YfiN activity is directly repressed by periplasmic YfiR, which is inactivated by redox stress. In Escherichia coli, an additional envelope stress causes YfiN to relocate to the mid-cell to inhibit cell division by interacting with the division machinery. Here, we report a third activity for YfiN in E. coli, where cell growth is inhibited without YfiN relocating to the division site. This action of YfiN is only observed when the bacteria are cultured on gluconeogenic carbon sources, and is dependent on absence of the autoinhibitory sites. Restoration of I-site function relieves the growth-arrest phenotype, and disabling this function in a heterologous DGC causes acquisition of this phenotype. Arrested cells are tolerant to a wide range of antibiotics. We show that the likely cause of growth arrest is depletion of cellular GTP from run-away synthesis of c-di-GMP, explaining the dependence of growth arrest on gluconeogenic carbon sources that exhaust more GTP during production of glucose. This is the first report of c-di-GMP-mediated growth arrest by altering metabolic flow. IMPORTANCE The c-di-GMP signaling network in bacteria not only controls a variety of cellular processes such as motility, biofilms, cell development, and virulence, but does so by a dizzying array of mechanisms. The DGC YfiN singularly represents the versatility of this network in that it not only inhibits motility and promotes biofilms, but also arrests growth in Escherichia coli by relocating to the mid-cell and blocking cell division. The work described here reveals that YfiN arrests growth by yet another mechanism in E. coli, changing metabolic flow. This function of YfiN, or of DGCs without autoinhibitory I-sites, may contribute to antibiotic tolerant persisters in relevant niches such as the gut where gluconeogenic sugars are found.

Swarming Motility Assays in Salmonella.

Salmonella enterica has six subspecies, of which the subspecies enterica is the most important for human health. The dispersal and infectivity of this species are dependent upon flagella-driven motility. Two kinds of flagella-mediated movements have been described-swimming individually in bulk liquid and swarming collectively over a surface substrate. During swarming, the bacteria acquire a distinct physiology, the most significant consequence of which is acquisition of adaptive resistance to antibiotics. Described here are protocols to cultivate, verify, and study swimming and swarming motility in S. enterica, and an additional "border-crossing" assay, where cells "primed" to swarm are presented with an environmental challenge such as antibiotics to assess their propensity to handle the challenge.

Efflux-linked accelerated evolution of antibiotic resistance at a population edge.

Efflux is a common mechanism of resistance to antibiotics. We show that efflux itself promotes accumulation of antibiotic-resistance mutations (ARMs). This phenomenon was initially discovered in a bacterial swarm where the linked phenotypes of high efflux and high mutation frequencies spatially segregated to the edge, driven there by motility. We have uncovered and validated a global regulatory network connecting high efflux to downregulation of specific DNA-repair pathways even in non-swarming states. The efflux-DNA repair link was corroborated in a clinical "resistome" database: genomes with mutations that increase efflux exhibit a significant increase in ARMs. Accordingly, efflux inhibitors decreased evolvability to antibiotic resistance. Swarms also revealed how bacterial populations serve as a reservoir of ARMs even in the absence of antibiotic selection pressure. High efflux at the edge births mutants that, despite compromised fitness, survive there because of reduced competition. This finding is relevant to biofilms where efflux activity is high.

Host cells subdivide nutrient niches into discrete biogeographical microhabitats for gut microbes.

Changes in the microbiota composition are associated with many human diseases, but factors that govern strain abundance remain poorly defined. We show that a commensal Escherichia coli strain and a pathogenic Salmonella enterica serovar Typhimurium isolate both utilize nitrate for intestinal growth, but each accesses this resource in a distinct biogeographical niche. Commensal E. coli utilizes epithelial-derived nitrate, whereas nitrate in the niche occupied by S. Typhimurium is derived from phagocytic infiltrates. Surprisingly, avirulent S. Typhimurium was shown to be unable to utilize epithelial-derived nitrate because its chemotaxis receptors McpB and McpC exclude the pathogen from the niche occupied by E. coli. In contrast, E. coli invades the niche constructed by S. Typhimurium virulence factors and confers colonization resistance by competing for nitrate. Thus, nutrient niches are not defined solely by critical resources, but they can be further subdivided biogeographically within the host into distinct microhabitats, thereby generating new niche opportunities for distinct bacterial species.

Goodbye PAM: Phage λ's Red recombination system cripples PAMs and helps dodge CRISPR attacks.

Ever wondered how the phage λ Red recombination system resembles the Red Queen? Hossain et al. (2021) provide an answer in this issue of Cell Host & Microbe. They show that Red debilitates PAM sequences by mutagenic repair of CRISPR-targeted DNA breaks in infecting λ, thus shaping the phage-CRISPR arms race.

Dead cells release a 'necrosignal' that activates antibiotic survival pathways in bacterial swarms.

Swarming is a form of collective bacterial motion enabled by flagella on the surface of semi-solid media. Swarming populations exhibit non-genetic or adaptive resistance to antibiotics, despite sustaining considerable cell death. Here, we show that antibiotic-induced death of a sub-population benefits the swarm by enhancing adaptive resistance in the surviving cells. Killed cells release a resistance-enhancing factor that we identify as AcrA, a periplasmic component of RND efflux pumps. The released AcrA interacts on the surface of live cells with an outer membrane component of the efflux pump, TolC, stimulating drug efflux and inducing expression of other efflux pumps. This phenomenon, which we call 'necrosignaling', exists in other Gram-negative and Gram-positive bacteria and displays species-specificity. Given that adaptive resistance is a known incubator for evolving genetic resistance, our findings might be clinically relevant to the rise of multidrug resistance.

Deep sequencing reveals new roles for MuB in transposition immunity and target-capture, and redefines the insular Ter region of E. coli.

BACKGROUND: The target capture protein MuB is responsible for the high efficiency of phage Mu transposition within the E. coli genome. However, some targets are off-limits, such as regions immediately outside the Mu ends (cis-immunity) as well as the entire ~ 37 kb genome of Mu (Mu genome immunity). Paradoxically, MuB is responsible for cis-immunity and is also implicated in Mu genome immunity, but via different mechanisms. This study was undertaken to dissect the role of MuB in target choice in vivo. RESULTS: We tracked Mu transposition from six different starting locations on the E. coli genome, in the presence and absence of MuB. The data reveal that Mu's ability to sample the entire genome during a single hop in a clonal population is independent of MuB, and that MuB is responsible for cis-immunity, plays a minor role in Mu genome immunity, and facilitates insertions into transcriptionally active regions. Unexpectedly, transposition patterns in the absence of MuB have helped extend the boundaries of the insular Ter segment of the E. coli genome. CONCLUSIONS: The results in this study demonstrate unambiguously the operation of two distinct mechanisms of Mu target immunity, only one of which is wholly dependent on MuB. The study also reveals several interesting and hitherto unknown aspects of Mu target choice in vivo, particularly the role of MuB in facilitating the capture of promoter and translation start site targets, likely by displacing macromolecular complexes engaged in gene expression. So also, MuB facilitates transposition into the restricted Ter region of the genome.

Tumble Suppression Is a Conserved Feature of Swarming Motility.

Many bacteria use flagellum-driven motility to swarm or move collectively over a surface terrain. Bacterial adaptations for swarming can include cell elongation, hyperflagellation, recruitment of special stator proteins, and surfactant secretion, among others. We recently demonstrated another swarming adaptation in Escherichia coli, wherein the chemotaxis pathway is remodeled to decrease tumble bias (increase run durations), with running speeds increased as well. We show here that the modification of motility parameters during swarming is not unique to E. coli but is shared by a diverse group of bacteria we examined-Proteus mirabilis, Serratia marcescens, Salmonella enterica, Bacillus subtilis, and Pseudomonas aeruginosa-suggesting that increasing run durations and speeds are a cornerstone of swarming.IMPORTANCE Bacteria within a swarm move characteristically in packs, displaying an intricate swirling motion in which hundreds of dynamic rafts continuously form and dissociate as the swarm colonizes an increasing expanse of territory. The demonstrated property of E. coli to reduce its tumble bias and hence increase its run duration during swarming is expected to maintain and promote side-by-side alignment and cohesion within the bacterial packs. In this study, we observed a similar low tumble bias in five different bacterial species, both Gram positive and Gram negative, each inhabiting a unique habitat and posing unique problems to our health. The unanimous display of an altered run-tumble bias in swarms of all species examined in this investigation suggests that this behavioral adaptation is crucial for swarming.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series.

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The "border-crossing" assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

A Well-Mixed E. coli Genome: Widespread Contacts Revealed by Tracking Mu Transposition.

The three-dimensional structures of chromosomes are increasingly being recognized as playing a major role in cellular regulatory states. The efficiency and promiscuity of phage Mu transposition was exploited to directly measure in vivo interactions between genomic loci in E. coli. Two global organizing principles have emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation.

Under Elevated c-di-GMP in Escherichia coli, YcgR Alters Flagellar Motor Bias and Speed Sequentially, with Additional Negative Control of the Flagellar Regulon via the Adaptor Protein RssB.

In Escherichia coli and Salmonella, the c-di-GMP effector YcgR inhibits flagellar motility by interacting directly with the motor to alter both its bias and speed. Here, we demonstrate that in both of these bacteria, YcgR acts sequentially, altering motor bias first and then decreasing motor speed. We show that when c-di-GMP levels are high, deletion of ycgR restores wild-type motor behavior in E. coli, indicating that YcgR is the only motor effector in this bacterium. Yet, motility and chemotaxis in soft agar do not return to normal, suggesting that there is a second mechanism that inhibits motility under these conditions. In Salmonella, c-di-GMP-induced synthesis of extracellular cellulose has been reported to entrap flagella and to be responsible for the YcgR-independent motility defect. We found that this is not the case in E. coli Instead, we found through reversion analysis that deletion of rssB, which codes for a response regulator/adaptor protein that normally directs ClpXP protease to target σS for degradation, restored wild-type motility in the ycgR mutant. Our data suggest that high c-di-GMP levels may promote altered interactions between these proteins to downregulate flagellar gene expression.IMPORTANCE Flagellum-driven motility has been studied in E. coli and Salmonella for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in E. coli, we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σS regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated.

Escherichia coli Remodels the Chemotaxis Pathway for Swarming.

Many flagellated bacteria "swarm" over a solid surface as a dense consortium. In different bacteria, swarming is facilitated by several alterations such as those corresponding to increased flagellum numbers, special stator proteins, or secreted surfactants. We report here a change in the chemosensory physiology of swarming Escherichia coli which alters its normal "run tumble" bias. E. coli bacteria taken from a swarm exhibit more highly extended runs (low tumble bias) and higher speeds than E. coli bacteria swimming individually in a liquid medium. The stability of the signaling protein CheZ is higher in swarmers, consistent with the observed elevation of CheZ levels and with the low tumble bias. We show that the tumble bias displayed by wild-type swarmers is the optimal bias for maximizing swarm expansion. In assays performed in liquid, swarm cells have reduced chemotactic performance. This behavior is specific to swarming, is not specific to growth on surfaces, and persists for a generation. Therefore, the chemotaxis signaling pathway is reprogrammed for swarming.IMPORTANCE The fundamental motile behavior of E. coli is a random walk, where straight "runs" are punctuated by "tumbles." This behavior, conferred by the chemotaxis signaling system, is used to track chemical gradients in liquid. Our study results show that when migrating collectively on surfaces, E. coli modifies its chemosensory physiology to decrease its tumble bias (and hence to increase run durations) by post-transcriptional changes that alter the levels of a key signaling protein. We speculate that the low tumble bias may contribute to the observed Lévy walk (LW) trajectories within the swarm, where run durations have a power law distribution. In animals, LW patterns are hypothesized to maximize searches in unpredictable environments. Swarming bacteria face several challenges while moving collectively over a surface-maintaining cohesion, overcoming constraints imposed by a physical substrate, searching for nutrients as a group, and surviving lethal levels of antimicrobials. The altered chemosensory behavior that we describe in this report may help with these challenges.

Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase.

The Gam protein of transposable phage Mu is an ortholog of eukaryotic and bacterial Ku proteins, which carry out nonhomologous DNA end joining (NHEJ) with the help of dedicated ATP-dependent ligases. Many bacteria carry Gam homologs associated with either complete or defective Mu-like prophages, but the role of Gam in the life cycle of Mu or in bacteria is unknown. Here, we show that MuGam is part of a two-component bacterial NHEJ DNA repair system. Ensemble and single-molecule experiments reveal that MuGam binds to DNA ends, slows the progress of RecBCD exonuclease, promotes binding of NAD+-dependent Escherichia coli ligase A, and stimulates ligation. In vivo, Gam equally promotes both precise and imprecise joining of restriction enzyme-digested linear plasmid DNA, as well as of a double-strand break (DSB) at an engineered I-SceI site in the chromosome. Cell survival after the induced DSB is specific to the stationary phase. In long-term growth competition experiments, particularly upon treatment with a clastogen, the presence of gam in a Mu lysogen confers a distinct fitness advantage. We also show that the role of Gam in the life of phage Mu is related not to transposition but to protection of genomic Mu copies from RecBCD when viral DNA packaging begins. Taken together, our data show that MuGam provides bacteria with an NHEJ system and suggest that the resulting fitness advantage is a reason that bacteria continue to retain the gam gene in the absence of an intact prophage.

The 3D architecture of a bacterial swarm has implications for antibiotic tolerance.

Swarming bacteria are an example of a complex, active biological system, where high cell density and super-diffusive cell mobility confer survival advantages to the group as a whole. Previous studies on the dynamics of the swarm have been limited to easily observable regions at the advancing edge of the swarm where cells are restricted to a plane. In this study, using defocused epifluorescence video imaging, we have tracked the motion of fluorescently labeled individuals within the interior of a densely packed three-dimensional (3D) region of a swarm. Our analysis reveals a novel 3D architecture, where bacteria are constrained by inter-particle interactions, sandwiched between two distinct boundary conditions. We find that secreted biosurfactants keep bacteria away from the swarm-air upper boundary, and added antibiotics at the lower swarm-surface boundary lead to their migration away from this boundary. Formation of the antibiotic-avoidance zone is dependent on a functional chemotaxis signaling system, in the absence of which the swarm loses its high tolerance to the antibiotics.

A Diguanylate Cyclase Acts as a Cell Division Inhibitor in a Two-Step Response to Reductive and Envelope Stresses.

UNLABELLED: Cell division arrest is a universal checkpoint in response to environmental assaults that generate cellular stress. In bacteria, the cyclic di-GMP (c-di-GMP) signaling network is one of several signal transduction systems that regulate key processes in response to extra-/intracellular stimuli. Here, we find that the diguanylate cyclase YfiN acts as a bifunctional protein that produces c-di-GMP in response to reductive stress and then dynamically relocates to the division site to arrest cell division in response to envelope stress in Escherichia coli YfiN localizes to the Z ring by interacting with early division proteins and stalls cell division by preventing the initiation of septal peptidoglycan synthesis. These studies reveal a new role for a diguanylate cyclase in responding to environmental change, as well as a novel mechanism for arresting cell division. IMPORTANCE: While the major role of c-di-GMP signaling is to control the decision to move freely or settle in a biofilm, recent studies show a broader range of output functions for c-di-GMP signaling. This work reports an unexpected second role for YfiN, a conserved diguanylate cyclase in Gram-negative bacteria, known to contribute to persistence in the host. We find that YfiN acts as a cell division inhibitor in response to envelope stress. Unlike known cell division inhibitors, the interaction of YfiN with cell division proteins retains the Z ring at the midcell but prevents septal invagination. The new function of YfiN not only emphasizes the versatility of c-di-GMP signaling but describes a novel mechanism for a cell division checkpoint.