The divisome is a self-enhancing machine in Escherichia coli and Caulobacter crescentus.

During bacterial cytokinesis, polymers of the bacterial tubulin FtsZ coalesce into the Z ring to orchestrate divisome assembly and septal cell wall synthesis. Previous studies have found that Z ring condensation and stability is critical for successful cell division. However, how FtsZ filaments condense into a Z ring remains enigmatic and whether septal cell wall synthesis can feedback to the Z ring has not been investigated. Here, we show that FtsZ-associated proteins (Zaps) play important roles in Z ring condensation and stability, and discover septal cell wall synthesis as a novel player for Z ring condensation and stabilization in Escherichia coli and Caulobacter crescentus. Moreover, we find that the interaction between the Z ring membrane anchor, FtsA, and components of the septal cell wall synthetic complex are critical for septal cell wall synthesis-mediated Z ring condensation. Altogether, these findings suggest that the divisome is a self-enhancing machine in these two gram-negative bacteria, where the Z ring and the septal cell wall synthetic complex communicate with and reinforce each other to ensure robustness of cell division.

OpgH is an essential regulator of Caulobacter morphology.

Bacterial growth and division rely on intricate regulation of morphogenetic complexes to remodel the cell envelope without compromising envelope integrity. Significant progress has been made in recent years towards understanding the regulation of cell wall metabolic enzymes. However, other cell envelope components play a role in morphogenesis as well. A primary factor required to protect envelope integrity in low osmolarity environments is OpgH, the synthase of osmoregulated periplasmic glucans (OPGs). Here, we demonstrate that OpgH is essential in the α-proteobacterium Caulobacter crescentus. Unexpectedly, depletion of OpgH or attempted complementation with a catalytically dead OpgH variant results in striking asymmetric bulging and cell lysis. These shape defects are accompanied by reduced cell wall synthesis and mislocalization of morphogenetic complexes. Interestingly, overactivation of the CenKR two-component system that has been implicated in cell envelope stress homeostasis in α-proteobacteria phenocopies the morphogenetic defects associated with OpgH depletion. Each of these perturbations leads to an increase in the levels of the elongasome protein, MreB, and decreases in the levels of divisome proteins FtsZ and MipZ as well as OpgH, itself. Constitutive production of OpgH during CenKR overactivation prevents cell bulging, but cells still exhibit morphogenetic defects. We propose that OPG depletion activates CenKR, leading to changes in the expression of cell envelope-related genes, but that OPGs also exert CenKR-independent effects on morphogenesis. Our data establish a surprising function for an OpgH homolog in morphogenesis and reveal an essential role of OpgH in maintaining cell morphology in Caulobacter.IMPORTANCEBacteria must synthesize and fortify the cell envelope in a tightly regulated manner to orchestrate growth and adaptation. Osmoregulated periplasmic glucans (OPGs) are important, but poorly understood, constituents of Gram-negative cell envelopes that contribute to envelope integrity and protect against osmotic stress. Here, we determined that the OPG synthase OpgH plays a surprising, essential role in morphogenesis in Caulobacter crescentus. Loss of OpgH causes asymmetric cell bulging and lysis via misregulation of the localization and activity of morphogenetic complexes. Overactivation of the CenKR two-component system involved in envelope homeostasis phenocopies OpgH depletion, suggesting that depletion of OpgH activates CenKR. Because cell envelope integrity is critical for bacterial survival, understanding how OpgH activity contributes to morphogenesis and maintenance of envelope integrity could aid in the development of antibiotic therapies.

Regulation of potassium uptake in Caulobacter crescentus.

Potassium (K+) is an essential physiological element determining membrane potential, intracellular pH, osmotic/turgor pressure, and protein synthesis in cells. Here, we describe the regulation of potassium uptake systems in the oligotrophic α-proteobacterium Caulobacter crescentus known as a model for asymmetric cell division. We show that C. crescentus can grow in concentrations from the micromolar to the millimolar range by mainly using two K+ transporters to maintain potassium homeostasis, the low-affinity Kup and the high-affinity Kdp uptake systems. When K+ is not limiting, we found that the kup gene is essential while kdp inactivation does not impact the growth. In contrast, kdp becomes critical but not essential and kup dispensable for growth in K+-limited environments. However, in the absence of kdp, mutations in kup were selected to improve growth in K+-depleted conditions, likely by increasing the affinity of Kup for K+. In addition, mutations in the KdpDE two-component system, which regulates kdpABCDE expression, suggest that the inner membrane sensor regulatory component KdpD mainly works as a phosphatase to limit the growth when cells reach late exponential phase. Our data therefore suggest that KdpE is phosphorylated by another non-cognate histidine kinase. On top of this, we determined the KdpE-dependent and independent K+ transcriptome. Together, our work illustrates how an oligotrophic bacterium responds to fluctuation in K+ availability.IMPORTANCEPotassium (K+) is a key metal ion involved in many essential cellular processes. Here, we show that the oligotroph Caulobacter crescentus can support growth at micromolar concentrations of K+ by mainly using two K+ uptake systems, the low-affinity Kup and the high-affinity Kdp. Using genome-wide approaches, we also determined the entire set of genes required for C. crescentus to survive at low K+ concentration as well as the full K+-dependent regulon. Finally, we found that the transcriptional regulation mediated by the KdpDE two-component system is unconventional since unlike Escherichia coli, the inner membrane sensor regulatory component KdpD seems to work rather as a phosphatase on the phosphorylated response regulator KdpE~P.

Bidirectional pilus processing in the Tad pilus system motor CpaF.

The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.

Genes required for phosphosphingolipid formation in Caulobacter crescentus contribute to bacterial virulence.

Sphingolipids are ubiquitous in membranes of eukaryotes and are associated with important cellular functions. Although sphingolipids occur scarcely in bacteria, for some of them they are essential and, in other bacteria, they contribute to fitness and stability of the outer membrane, such as in the well-studied α-proteobacterium Caulobacter crescentus. We previously defined five structural genes for ceramide synthesis in C. crescentus, among them the gene for serine palmitoyltransferase, the enzyme that catalyzes the committed step of sphingolipid biosynthesis. Other mutants affected in genes of this same genomic region show cofitness with a mutant deficient in serine palmitoyltransferase. Here we show that at least two phosphosphingolipids are produced in C. crescentus and that at least another six gene products are needed for the decoration of ceramide upon phosphosphingolipid formation. All eleven genes participating in phosphosphingolipid formation are also required in C. crescentus for membrane stability and for displaying sensitivity towards the antibiotic polymyxin B. The genes for the formation of complex phosphosphingolipids are also required for C. crescentus virulence on Galleria mellonella insect larvae.

Upstream CtrA-binding sites both induce and repress pilin gene expression in Caulobacter crescentus.

Pili are bacterial surface structures important for surface adhesion. In the alphaproteobacterium Caulobacter crescentus, the global regulator CtrA activates transcription of roughly 100 genes, including pilA which codes for the pilin monomer that makes up the pilus filament. While most CtrA-activated promoters have a single CtrA-binding site at the - 35 position and are induced at the early to mid-predivisional cell stage, the pilA promoter has 3 additional upstream CtrA-binding sites and it is induced at the late predivisional cell stage. Reporter constructs where these additional sites were disrupted by deletion or mutation led to increased activity compared to the WT promoter. In synchronized cultures, these mutations caused pilA transcription to occur approximately 20 min earlier than WT. The results suggested that the site overlapping the - 35 position drives pilA gene expression while the other upstream CtrA-binding sites serve to reduce and delay expression. EMSA experiments showed that the - 35 Site has lower affinity for CtrA∼P compared to the other sites, suggesting binding site affinity may be involved in the delay mechanism. Mutating the upstream inhibitory CtrA-binding sites in the pilA promoter caused significantly higher numbers of pre-divisional cells to express pili, and phage survival assays showed this strain to be significantly more sensitive to pilitropic phage. These results suggest that pilA regulation evolved in C. crescentus to provide an ecological advantage within the context of phage infection.

Caulobacter crescentus RNase E condensation contributes to autoregulation and fitness.

RNase E is the most common RNA decay nuclease in bacteria, setting the global mRNA decay rate and scaffolding formation of the RNA degradosome complex and BR-bodies. To properly set the global mRNA decay rate, RNase E from Escherichia coli and neighboring γ-proteobacteria were found to autoregulate RNase E levels via the decay of its mRNA's 5' untranslated region (UTR). While the 5' UTR is absent from other groups of bacteria in the Rfam database, we identified that the α-proteobacterium Caulobacter crescentus RNase E contains a similar 5' UTR structure that promotes RNase E autoregulation. In both bacteria, the C-terminal intrinsically disordered region (IDR) of RNase E is required for proper autoregulation to occur, and this IDR is also necessary and sufficient for RNase E to phase-separate, generating BR-bodies. Using in vitro purified RNase E, we find that the IDR's ability to promote phase separation correlates with enhanced 5' UTR cleavage, suggesting that phase separation of RNase E with the 5' UTR enhances autoregulation. Finally, using growth competition experiments, we find that a strain capable of autoregulation rapidly outcompetes a strain with a 5' UTR mutation that cannot autoregulate, suggesting autoregulation promotes optimal cellular fitness.

Proteolytic control of FixT by the Lon protease impacts FixLJ signaling in Caulobacter crescentus.

Responding to changes in oxygen levels is critical for aerobic microbes. In Caulobacter crescentus, low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including those involved in heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease when the oxygen levels are sufficient, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address whether degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that ∆lon strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of fixT suppresses this defect. Transcriptomics validate this result as, along with heme biosynthesis, there is diminished expression of many FixL-activated genes in ∆lon. However, stabilization of FixT in ∆lon strains does not contribute to restoring any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during growth in standard aerobic conditions. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient Caulobacter. IMPORTANCE: The Lon protease shapes protein quality control, signaling pathways, and stress responses in many bacteria species. Loss of Lon often results in multiple phenotypic consequences. In this work, we found a connection between the Lon protease and deficiencies in heme accumulation that then led to our finding of a global change in gene expression due in part to degradation of a regulator of the hypoxic response. However, loss of degradation of this regulator did not explain other phenotypes associated with Lon deficiencies demonstrating the complex and multiple pathways that this highly conserved protease can impact.

Control of a gene transfer agent cluster in Caulobacter crescentus by transcriptional activation and anti-termination.

Gene Transfer Agents (GTAs) are phage-like particles that cannot self-multiply and be infectious. Caulobacter crescentus, a bacterium best known as a model organism to study bacterial cell biology and cell cycle regulation, has recently been demonstrated to produce bona fide GTA particles (CcGTA). Since C. crescentus ultimately die to release GTA particles, the production of GTA particles must be tightly regulated and integrated with the host physiology to prevent a collapse in cell population. Two direct activators of the CcGTA biosynthetic gene cluster, GafY and GafZ, have been identified, however, it is unknown how GafYZ controls transcription or how they coordinate gene expression of the CcGTA gene cluster with other accessory genes elsewhere on the genome for complete CcGTA production. Here, we show that the CcGTA gene cluster is transcriptionally co-activated by GafY, integration host factor (IHF), and by GafZ-mediated transcription anti-termination. We present evidence that GafZ is a transcription anti-terminator that likely forms an anti-termination complex with RNA polymerase, NusA, NusG, and NusE to bypass transcription terminators within the 14 kb CcGTA cluster. Overall, we reveal a two-tier regulation that coordinates the synthesis of GTA particles in C. crescentus.

The cytoplasmic phosphate level has a central regulatory role in the phosphate starvation response of Caulobacter crescentus.

In bacteria, the availability of environmental inorganic phosphate is typically sensed by the conserved PhoR-PhoB two-component signal transduction pathway, which uses the flux through the PstSCAB phosphate transporter as a readout of the extracellular phosphate level to control phosphate-responsive genes. While the sensing of environmental phosphate is well-investigated, the regulatory effects of cytoplasmic phosphate are unclear. Here, we disentangle the physiological and transcriptional responses of Caulobacter crescentus to changes in the environmental and cytoplasmic phosphate levels by uncoupling phosphate uptake from the activity of the PstSCAB system, using an additional, heterologously produced phosphate transporter. This approach reveals a two-pronged response of C. crescentus to phosphate limitation, in which PhoR-PhoB signaling mostly facilitates the utilization of alternative phosphate sources, whereas the cytoplasmic phosphate level controls the morphological and physiological adaptation of cells to growth under global phosphate limitation. These findings open the door to a comprehensive understanding of phosphate signaling in bacteria.

Comparison of CcrM-dependent methylation in Caulobacter crescentus and Brucella abortus by nanopore sequencing.

Bacteria rely on DNA methylation for restriction-modification systems and epigenetic control of gene expression. Here, we use direct detection of methylated bases by nanopore sequencing to monitor global DNA methylation in Alphaproteobacteria, where use of this technique has not yet been reported. One representative of this order, Caulobacter crescentus, relies on DNA methylation to control cell cycle progression, but it is unclear whether other members of this order, such as Brucella abortus, depend on the same systems. We addressed these questions by first measuring CcrM-dependent DNA methylation in Caulobacter and showing excellent correlation between nanopore-based detection and previously published results. We then directly measure the impact of Lon-mediated CcrM degradation on the epigenome, verifying that loss of Lon results in pervasive methylation. We also show that the AlkB demethylase has no global impact on DNA methylation during normal growth. Next, we report on the global DNA methylation in B. abortus for the first time and find that CcrM-dependent methylation is reliant on Lon but impacts the two chromosomes differently. Finally, we explore the impact of the MucR transcription factor, known to compete with CcrM methylation, on the Brucella methylome and share the results with a publicly available visualization package. Our work demonstrates the utility of nanopore-based sequencing for epigenome measurements in Alphaproteobacteria and reveals new features of CcrM-dependent methylation in a zoonotic pathogen.IMPORTANCEDNA methylation plays an important role in bacteria, maintaining genome integrity and regulating gene expression. We used nanopore sequencing to directly measure methylated bases in Caulobacter crescentus and Brucella abortus. In Caulobacter, we showed that stabilization of the CcrM methyltransferase upon loss of the Lon protease results in prolific methylation and discovered that the putative methylase AlkB is unlikely to have a global physiological effect. We measured genome-wide methylation in Brucella for the first time, revealing a similar role for CcrM in cell-cycle methylation but a more complex regulation by the Lon protease than in Caulobacter. Finally, we show how the virulence factor MucR impacts DNA methylation patterns in Brucella.

Time-resolved cryogenic electron tomography for the study of transient cellular processes.

Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling subnanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second before freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 s of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a subsecond temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level.

GTPase activity regulates FtsZ ring positioning in Caulobacter crescentus.

Bacterial cell division is crucial for replication and requires careful coordination via proteins collectively called the divisome. The tubulin-like GTPase FtsZ is the master regulator of this process and serves to recruit downstream divisome proteins and regulate their activities. Upon assembling at mid-cell, FtsZ exhibits treadmilling motion driven by GTP binding and hydrolysis. Treadmilling is proposed to play roles in Z-ring condensation and in distribution and regulation of peptidoglycan (PG) cell wall enzymes. FtsZ polymer superstructure and dynamics are central to its function, yet their regulation is incompletely understood. We addressed these gaps in knowledge by evaluating the contribution of GTPase activity to FtsZ's function in vitro and in Caulobacter crescentus cells. We observed that a lethal mutation that abrogates FtsZ GTP hydrolysis impacts FtsZ dynamics and Z-ring positioning, but not constriction. Aberrant Z-ring positioning was due to insensitivity to the FtsZ regulator MipZ when GTPase activity is reduced. Z-ring mislocalization resulted in DNA damage, likely due to constriction over the nucleoid. Collectively, our results indicate that GTP hydrolysis serves primarily to position the Z-ring at mid-cell in Caulobacter. Proper Z-ring localization is required for effective coordination with chromosome segregation to prevent DNA damage and ensure successful cell division.

Differential growth regulates asymmetric size partitioning in Caulobacter crescentus.

Cell size regulation has been extensively studied in symmetrically dividing cells, but the mechanisms underlying the control of size asymmetry in asymmetrically dividing bacteria remain elusive. Here, we examine the control of asymmetric division in Caulobacter crescentus, a bacterium that produces daughter cells with distinct fates and morphologies upon division. Through comprehensive analysis of multi-generational growth and shape data, we uncover a tightly regulated cell size partitioning mechanism. We find that errors in division site positioning are promptly corrected early in the division cycle through differential growth. Our analysis reveals a negative feedback between the size of daughter cell compartments and their growth rates, wherein the larger compartment grows slower to achieve a homeostatic size partitioning ratio at division. To explain these observations, we propose a mechanistic model of differential growth, in which equal amounts of growth regulators are partitioned into daughter cell compartments of unequal sizes and maintained over time via size-independent synthesis.

Structural mechanisms of Tad pilus assembly and its interaction with an RNA virus.

Caulobacter crescentus Tad (tight adherence) pili, part of the type IV pili family, are crucial for mechanosensing, surface adherence, bacteriophage (phage) adsorption, and cell-cycle regulation. Unlike other type IV pilins, Tad pilins lack the typical globular ß sheet domain responsible for pilus assembly and phage binding. The mechanisms of Tad pilus assembly and its interaction with phage ΦCb5 have been elusive. Using cryo-electron microscopy, we unveiled the Tad pilus assembly mechanism, featuring a unique network of hydrogen bonds at its core. We then identified the Tad pilus binding to the ΦCb5 maturation protein (Mat) through its ß region. Notably, the amino terminus of ΦCb5 Mat is exposed outside the capsid and phage/pilus interface, enabling the attachment of fluorescent and affinity tags. These engineered ΦCb5 virions can be efficiently assembled and purified in Escherichia coli, maintaining infectivity against C. crescentus, which presents promising applications, including RNA delivery and phage display.

Lifecycle of a predatory bacterium vampirizing its prey through the cell envelope and S-layer.

Predatory bacteria feed upon other bacteria in various environments. Bdellovibrio exovorus is an obligate epibiotic predator that attaches on the prey cell surface, where it grows and proliferates. Although the mechanisms allowing feeding through the prey cell envelope are unknown, it has been proposed that the prey's proteinaceous S-layer may act as a defensive structure against predation. Here, we use time-lapse and cryo-electron microscopy to image the lifecycle of B. exovorus feeding on Caulobacter crescentus. We show that B. exovorus proliferates by non-binary division, primarily generating three daughter cells. Moreover, the predator feeds on C. crescentus regardless of the presence of an S-layer, challenging its assumed protective role against predators. Finally, we show that apparently secure junctions are established between prey and predator outer membranes.

Cell cycle dependent coordination of surface layer biogenesis in Caulobacter crescentus.

Surface layers (S-layers) are proteinaceous, two-dimensional paracrystalline arrays that constitute a major component of the cell envelope in many prokaryotic species. In this study, we investigated S-layer biogenesis in the bacterial model organism Caulobacter crescentus. Fluorescence microscopy revealed localised incorporation of new S-layer at the poles and mid-cell, consistent with regions of cell growth in the cell cycle. Light microscopy and electron cryotomography investigations of drug-treated bacteria revealed that localised S-layer insertion is retained when cell division is inhibited, but is disrupted upon dysregulation of MreB or lipopolysaccharide. We further uncovered that S-layer biogenesis follows new peptidoglycan synthesis and localises to regions of high cell wall turnover. Finally, correlated cryo-light microscopy and electron cryotomographic analysis of regions of S-layer insertion showed the presence of discontinuities in the hexagonal S-layer lattice, contrasting with other S-layers completed by defined symmetric defects. Our findings present insights into how C. crescentus cells form an ordered S-layer on their surface in coordination with the biogenesis of other cell envelope components.

Chromosome organization shapes replisome dynamics in Caulobacter crescentus.

DNA replication in bacteria takes place on highly compacted chromosomes, where segregation, transcription, and repair must occur simultaneously. Within this dynamic environment, colocalization of sister replisomes has been observed in many bacterial species, driving the hypothesis that a physical linker may tether them together. However, replisome splitting has also been reported in many of the same species, leaving the principles behind replisome organization a long-standing puzzle. Here, by tracking the replisome ß-clamp subunit in live Caulobacter crescentus, we find that rapid DNA segregation can give rise to a second focus which resembles a replisome, but does not replicate DNA. Sister replisomes can remain colocalized, or split apart to travel along DNA separately upon disruption of chromosome inter-arm alignment. Furthermore, chromosome arm-specific replication-transcription conflicts differentially modify replication speed on the two arms, facilitate the decoupling of the two replisomes. With these observations, we conclude that the dynamic chromosome organization flexibly shapes the organization of sister replisomes, and we outline principles which can help to reconcile previously conflicting models of replisome architecture.

Spatial organization of bacterial sphingolipid synthesis enzymes.

Sphingolipids are produced by nearly all eukaryotes where they play significant roles in cellular processes such as cell growth, division, programmed cell death, angiogenesis, and inflammation. While it was previously believed that sphingolipids were quite rare among bacteria, bioinformatic analysis of the recently identified bacterial sphingolipid synthesis genes suggests that these lipids are likely to be produced by a wide range of microbial species. The sphingolipid synthesis pathway consists of three critical enzymes. Serine palmitoyltransferase catalyzes the condensation of serine with palmitoyl-CoA (or palmitoyl-acyl carrier protein), ceramide synthase adds the second acyl chain, and a reductase reduces the ketone present on the long-chain base. While there is general agreement regarding the identity of these bacterial enzymes, the precise mechanism and order of chemical reactions for microbial sphingolipid synthesis is more ambiguous. Two mechanisms have been proposed. First, the synthesis pathway may follow the well characterized eukaryotic pathway in which the long-chain base is reduced prior to the addition of the second acyl chain. Alternatively, our previous work suggests that addition of the second acyl chain precedes the reduction of the long-chain base. To distinguish between these two models, we investigated the subcellular localization of these three key enzymes. We found that serine palmitoyltransferase and ceramide synthase are localized to the cytoplasm, whereas the ceramide reductase is in the periplasmic space. This is consistent with our previously proposed model wherein the second acyl chain is added in the cytoplasm prior to export to the periplasm where the lipid molecule is reduced.

The type IVc pilus: just a Tad different.

Bacteria utilize type IV pili (T4P) to interact with their environment, where they facilitate processes including motility, adherence, and DNA uptake. T4P require multisubunit, membrane-spanning nanomachines for assembly. The tight adherence (Tad) pili are an Archaea-derived T4P subgroup whose machinery exhibits significant mechanistic and architectural differences from bacterial type IVa and IVb pili. Most Tad biosynthetic genes are encoded in a single locus that is widespread in bacteria due to facile acquisition via horizontal gene transfer. These loci experience extensive structural rearrangements, including the acquisition of novel regulatory or biosynthetic genes, which fine-tune their function. This has permitted their integration into many different bacterial lifestyles, including the Caulobacter crescentus cell cycle, Myxococcus xanthus predation, and numerous plant and mammalian pathogens and symbionts.