No two clones are alike: characterization of heterologous subpopulations in a transgenic cell line of the model diatom Phaeodactylum tricornutum.

BACKGROUND: Conjugation-based episome delivery is a highly efficient method used to transfer DNA into the diatom Phaeodactylum tricornutum, facilitating the production of recombinant proteins and high-value metabolites. However, previous reports have indicated phenotypic heterogeneity among individual cells from clonally propagated exconjugant cell lines, potentially affecting the stability of recombinant protein production in the diatom. RESULTS: Here, we characterized the differences between subpopulations with distinct fluorescence intensity phenotypes derived from a single exconjugant colony of P. tricornutum expressing the enhanced green fluorescent protein (eGFP). We analyzed the expression cassette sequence integrity, plasmid copy number, and global gene expression. Our findings reveal that lower copy numbers and the deletion of the expression cassette in part of the population contributed to low transgene expression. Gene co-expression analysis identified a set of genes with similar expression pattern to eGFP including a gene encoding a putative Flp recombinase, which may be related to variations in fluorescence intensity. These genes thus present themselves as potential candidates for increasing recombinant proteins production in P. tricornutum episomal expression system. CONCLUSIONS: Overall, our study elucidates genetic and transcriptomic differences between distinct subpopulations in a clonally propagated culture, contributes to a better understanding of heterogeneity in diatom expression systems for synthetic biology applications.

Plasmid-free production of the plant lignan pinoresinol in growing Escherichia coli cells.

BACKGROUND: The high-value aryl tetralin lignan (+)-pinoresinol is the main precursor of many plant lignans including (-)-podophyllotoxin, which is used for the synthesis of chemotherapeutics. As (-)-podophyllotoxin is traditionally isolated from endangered and therefore limited natural sources, there is a particular need for biotechnological production. Recently, we developed a reconstituted biosynthetic pathway from (+)-pinoresinol to (-)-deoxypodophyllotoxin, the direct precursor of (-)-podophyllotoxin, in the recombinant host Escherichia coli. However, the use of the expensive substrate (+)-pinoresinol limits its application from the economic viewpoint. In addition, the simultaneous expression of multiple heterologous genes from different plasmids for a multi-enzyme cascade can be challenging and limits large-scale use. RESULTS: In this study, recombinant plasmid-free E. coli strains for the multi-step synthesis of pinoresinol from ferulic acid were constructed. To this end, a simple and versatile plasmid toolbox for CRISPR/Cas9-assisted chromosomal integration has been developed, which allows the easy transfer of genes from the pET vector series into the E. coli chromosome. Two versions of the developed toolbox enable the efficient integration of either one or two genes into intergenic high expression loci in both E. coli K-12 and B strains. After evaluation of this toolbox using the fluorescent reporter mCherry, genes from Petroselinum crispum and Zea mays for the synthesis of the monolignol coniferyl alcohol were integrated into different E. coli strains. The product titers achieved with plasmid-free E. coli W3110(T7) were comparable to those of the plasmid-based expression system. For the subsequent oxidative coupling of coniferyl alcohol to pinoresinol, a laccase from Corynebacterium glutamicum was selected. Testing of different culture media as well as optimization of gene copy number and copper availability for laccase activity resulted in the synthesis of 100 mg/L pinoresinol using growing E. coli cells. CONCLUSIONS: For efficient and simple transfer of genes from pET vectors into the E. coli chromosome, an easy-to-handle molecular toolbox was developed and successfully tested on several E. coli strains. By combining heterologous and endogenous enzymes of the host, a plasmid-free recombinant E. coli growing cell system has been established that enables the synthesis of the key lignan pinoresinol.

Swarms of Enzymatic Nanobots for Efficient Gene Delivery.

This study investigates the synthesis and optimization of nanobots (NBs) loaded with pDNA using the layer-by-layer (LBL) method and explores the impact of their collective motion on the transfection efficiency. NBs consist of biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles and are powered by the urease enzyme, enabling autonomous movement and collective swarming behavior. In vitro experiments were conducted to validate the delivery efficiency of fluorescently labeled NBs, using two-dimensional (2D) and three-dimensional (3D) cell models: murine urothelial carcinoma cell line (MB49) and spheroids from human urothelial bladder cancer cells (RT4). Swarms of pDNA-loaded NBs showed enhancements of 2.2- to 2.6-fold in delivery efficiency and 6.8- to 8.1-fold in material delivered compared to inhibited particles (inhibited enzyme) and the absence of fuel in a 2D cell culture. Additionally, efficient intracellular delivery of pDNA was demonstrated in both cell models by quantifying and visualizing the expression of eGFP. Swarms of NBs exhibited a >5-fold enhancement in transfection efficiency compared to the absence of fuel in a 2D culture, even surpassing the Lipofectamine 3000 commercial transfection agent (cationic lipid-mediated transfection). Swarms also demonstrated up to a 3.2-fold enhancement in the amount of material delivered in 3D spheroids compared to the absence of fuel. The successful transfection of 2D and 3D cell cultures using swarms of LBL PLGA NBs holds great potential for nucleic acid delivery in the context of bladder treatments.

A prokaryotic Argonaute protein recruits a helicase-nuclease to degrade invading plasmids.

DdmDE is a novel plasmid defense system that was discovered in the seventh pandemic Vibrio cholerae strain of the biotype O1 EI Tor. In this issue of Cell, Yang and coworkers reveal the mechanisms underlying the assembly and activation of the DdmDE defense system.

A Photothermal Polymeric Platform for Efficient and Safe Gene Transfection: When Polyethylenimine Collaborates with Indocyanine Green.

Gene transfection, defined by the delivery of nucleic acids into cellular compartments, stands as a crucial procedure in gene therapy. While branched polyethylenimine (PEI) is widely regarded as the "gold standard" for nonviral vectors, its cationic nature presents several issues, including nonspecific protein adsorption and notable cytotoxicity. Additionally, it often fails to achieve high transfection efficiency, particularly with hard-to-transfect cell types. To overcome these challenges associated with PEI as a vector for plasmid DNA (pDNA), the photothermal agent indocyanine green (ICG) is integrated with PEI and pDNA to form the PEI/ICG/pDNA (PI/pDNA) complex for more efficient and safer gene transfection. The negatively charged ICG serves a dual purpose: neutralizing PEI's excessive positive charges to reduce cytotoxicity and, under near-infrared irradiation, inducing local heating that enhances cell membrane permeability, thus facilitating the uptake of PI/pDNA complexes to boost transfection efficiency. Using pDNA encoding vascular endothelial growth factor as a model, our system shows enhanced transfection efficiency in vitro for hard-to-transfect endothelial cells, leading to improved cell proliferation and migration. Furthermore, in vivo studies reveal the therapeutic potential of this system in accelerating the healing of infected wounds by promoting angiogenesis and reducing inflammation. This approach offers a straightforward and effective method for gene transfection, showing potentials for tissue engineering and cell-based therapies.

Following plasmid propagation in complex bacterial communities.

In this issue of Cell Reports, Ma et al.1 identify causative regulatory links between self-organization in surface-attached bacterial colonies and the rate of horizontal gene transfers (conjugations) and subsequent selection of the newly arising population of recipient bacteria (transconjugants).

Metabolic interactions control the transfer and spread of plasmid-encoded antibiotic resistance during surface-associated microbial growth.

Surface-associated microbial systems are hotspots for the spread of plasmid-encoded antibiotic resistance, but how surface association affects plasmid transfer and proliferation remains unclear. Surface association enables prolonged spatial proximities between different populations, which promotes plasmid transfer between them. However, surface association also fosters strong metabolic interactions between different populations, which can direct their spatial self-organization with consequences for plasmid transfer and proliferation. Here, we hypothesize that metabolic interactions direct the spatial self-organization of different populations and, in turn, regulate the spread of plasmid-encoded antibiotic resistance. We show that resource competition causes populations to spatially segregate, which represses plasmid transfer. In contrast, resource cross-feeding causes populations to spatially intermix, which promotes plasmid transfer. We further show that the spatial positionings that emerge from metabolic interactions determine the proliferation of plasmid recipients. Our results demonstrate that metabolic interactions are important regulators of both the transfer and proliferation of plasmid-encoded antibiotic resistance.

A Strategy of Killing Two Birds With One Stone for Blocking Drug Resistance Spread With Engineered Bdellovibrio bacteriovorus.

Drug-resistant pathogens significantly threaten human health and life. Simply killing drug-resistant pathogens cannot effectively eliminate their threat since the drug-resistant genes (DRGs) released from dead drug-resistant pathogens are difficult to eliminate and can further spread via horizontal gene transfer, leading to the spread of drug resistance. The development of antibacterial materials with sterilization and DRGs cleavage activities is highly crucial. Herein, a living system, Ce-PEA@Bdello, is fabricated with bacterial killing and DRGs cleavage activities for blocking bacterial drug resistance dissemination by engineered Bdellovibrio bacteriovorus (Bdello). Ce-PEA@Bdello is obtained by engineering Bdello with dopamine and a multinuclear cerium (IV) complex. Ce-PEA@Bdello can penetrate and eliminate kanamycin-resistant P. aeruginosa (KanR) biofilms via the synergistic effect of predatory Bdello and photothermal polydopamine under near-infrared light. Additionally, the DNase-mimicking ability of Ce-PEA@Bdello endows it with genome and plasmid DNA cleavage ability. An in vivo study reveals that Ce-PEA@Bdello can eliminate P. aeruginosa (KanR) and cleave DRGs in scald/burn infected wounds to block the spread of drug resistance and accelerate wound healing. This bioactive system constructed from natural living materials offers a promising means for blocking the spread of drug resistance.

Synonymous codon substitutions modulate transcription and translation of a divergent upstream gene by modulating antisense RNA production.

Synonymous codons were originally viewed as interchangeable, with no phenotypic consequences. However, substantial evidence has now demonstrated that synonymous substitutions can perturb a variety of gene expression and protein homeostasis mechanisms, including translational efficiency, translational fidelity, and cotranslational folding of the encoded protein. To date, most studies of synonymous codon-derived perturbations have focused on effects within a single gene. Here, we show that synonymous codon substitutions made far within the coding sequence of Escherichia coli plasmid-encoded chloramphenicol acetyltransferase (cat) can significantly increase expression of the divergent upstream tetracycline resistance gene, tetR. In four out of nine synonymously recoded cat sequences tested, expression of the upstream tetR gene was significantly elevated due to transcription of a long antisense RNA (asRNA) originating from a transcription start site within cat. Surprisingly, transcription of this asRNA readily bypassed the native tet transcriptional repression mechanism. Even more surprisingly, accumulation of the TetR protein correlated with the level of asRNA, rather than total tetR RNA. These effects of synonymous codon substitutions on transcription and translation of a neighboring gene suggest that synonymous codon usage in bacteria may be under selection to both preserve the amino acid sequence of the encoded gene and avoid DNA sequence elements that can significantly perturb expression of neighboring genes. Avoiding such sequences may be especially important in plasmids and prokaryotic genomes, where genes and regulatory elements are often densely packed. Similar considerations may apply to the design of genetic circuits for synthetic biology applications.

BsuMI regulates DNA transformation in Bacillus subtilis besides the defense system and the constructed strain with BsuMI-absence is applicable as a universal transformation platform for wild-type Bacillus.

BACKGROUND: To effectively introduce plasmids into Bacillus species and conduct genetic manipulations in Bacillus chassis strains, it is essential to optimize transformation methods. These methods aim to extend the period of competence and enhance the permeability of the cell membrane to facilitate the entry of exogenous DNA. Although various strategies have been explored, few studies have delved into identifying metabolites and pathways associated with enhanced competence. Additionally, derivative Bacillus strains with non-functional restriction-modification systems have demonstrated superior efficiency in transforming exogenous DNA, lacking more explorations in the regulation conducted by the restriction-modification system to transformation process. RESULTS: Transcriptomic comparisons were performed to discover the competence forming mechanism and the regulation pathway conducted by the BsuMI methylation modification group in Bacillus. subtilis 168 under the Spizizen transformation condition, which were speculated to be the preferential selection of carbon sources by the cells and the preference for specific metabolic pathway when utilizing the carbon source. The cells were found to utilize the glycolysis pathway to exploit environmental glucose while reducing the demand for other phosphorylated precursors in this pathway. The weakening of these ATP-substrate competitive metabolic pathways allowed more ATP substrates to be distributed into the auto-phosphorylation of the signal transduction factor ComP during competence formation, thereby increasing the expression level of the key regulatory protein ComK. The expression of ComK upregulated the expression of the negative regulator SacX of starch and sucrose in host cells, reinforcing the preference for glucose as the primary carbon source. The methylation modification group of the primary protein BsuMI in the restriction-modification system was associated with the functional modification of key enzymes in the oxidative phosphorylation pathway. The absence of the BsuMI methylation modification group resulted in a decrease in the expression of subunits of cytochrome oxidase, leading to a weakening of the oxidative phosphorylation pathway, which promoted the glycolytic rate of cells and subsequently improved the distribution of ATP molecules into competence formation. A genetic transformation platform for wild-type Bacillus strains was successfully established based on the constructed strain B. subtilis 168-R-M- without its native restriction-modification system. With this platform, high plasmids transformation efficiencies were achieved with a remarkable 63-fold improvement compared to the control group and an increased universality in Bacillus species was also obtained. CONCLUSIONS: The enhanced competence formation mechanism and the regulation pathway conducted by the functional protein BsuMI of the restriction-modification system were concluded, providing a reference for further investigation. An effective transformation platform was established to overcome the obstacles in DNA transformations in wild-type Bacillus strains.

DdmDE eliminates plasmid invasion by DNA-guided DNA targeting.

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgos) and the DNA defense module DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions.

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics.

Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.

Modulating efficacy and cytotoxicity of lipoamino fatty acid nucleic acid carriers using disulfide or hydrophobic spacers.

Double pH-responsive xenopeptides comprising polar ionizable succinoyl tetraethylene pentamine (Stp) motifs and lipophilic ionizable lipoamino fatty acids (LAFs) were recently found to efficiently transfect mRNA and pDNA at low doses. However, potency was often accompanied with cytotoxicity at higher doses. Insertion of bioreducible disulfide building blocks (ssbb) or non-reducible hydrophobic spacers between polar and apolar ionizable domains of LAF-Stp carriers should mitigate toxicity of xenopeptides. Carriers showed stable nucleic acid complexation and endosomal pH-dependent lytic activities, both of which were abolished after reductive cleavage of ssbb-containing carriers. For pDNA, U-shaped carriers with one Stp and two LAF units or bundle carriers with two Stps and four LAFs displayed highest potency. For mRNA, best transfection was achieved with bundle carriers with one Stp and four LAFs. Both the ssbb and hydrophobic spacer containing analogs displayed improved metabolic activity, reduced membrane damage, and improved cell growth. The ssbb carriers were most beneficial regarding living cell count and low apoptosis rates. Mechanistically, inserted spacers decelerated the transfection kinetics and altered the requirement of endosomal protonation. Overall, mRNA and pDNA carriers with improved biocompatibility have been designed, with their high potency illustrated in transfection of various cell lines including low passage number colon carcinoma cells.

Balancing the AspC and AspA Pathways of Escherichia coli by Systematic Metabolic Engineering Strategy for High-Efficient l-Homoserine Production.

l-Homoserine is a promising C4 platform compound used in the agricultural, cosmetic, and pharmaceutical industries. Numerous works have been conducted to engineer Escherichia coli to be an excellent l-homoserine producer, but it is still unable to meet the industrial-scale demand. Herein, we successfully engineered a plasmid-free and noninducible E. coli strain with highly efficient l-homoserine production through balancing AspC and AspA synthesis pathways. First, an initial strain was constructed by increasing the accumulation of the precursor oxaloacetate and attenuating the organic acid synthesis pathway. To remodel the carbon flux toward l-aspartate, a balanced route prone to high yield based on TCA intensity regulation was designed. Subsequently, the main synthetic pathway and the cofactor system were strengthened to reinforce the l-homoserine synthesis. Ultimately, under two-stage DO control, strain HSY43 showed 125.07 g/L l-homoserine production in a 5 L fermenter in 60 h, with a yield of 0.62 g/g glucose and a productivity of 2.08 g/L/h. The titer, yield, and productivity surpassed the highest reported levels for plasmid-free strains in the literature. The strategies adopted in this study can be applied to the production of other l-aspartate family amino acids.

PUFFFIN: an ultra-bright, customisable, single-plasmid system for labelling cell neighbourhoods.

Cell communication coordinates developmental processes, maintains homeostasis, and contributes to disease. Therefore, understanding the relationship between cells in a shared environment is crucial. Here we introduce Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a cell neighbour-labelling system based upon secretion and uptake of positively supercharged fluorescent protein s36GFP. We fused s36GFP to mNeonGreen or to a HaloTag, facilitating ultra-bright, sensitive, colour-of-choice labelling. Secretor cells transfer PUFFFIN to neighbours while retaining nuclear mCherry, making identification, isolation, and investigation of live neighbours straightforward. PUFFFIN can be delivered to cells, tissues, or embryos on a customisable single-plasmid construct composed of interchangeable components with the option to incorporate any transgene. This versatility enables the manipulation of cell properties, while simultaneously labelling surrounding cells, in cell culture or in vivo. We use PUFFFIN to ask whether pluripotent cells adjust the pace of differentiation to synchronise with their neighbours during exit from naïve pluripotency. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour.

DdmABC-dependent death triggered by viral palindromic DNA sequences.

Defense systems that recognize viruses provide important insights into both prokaryotic and eukaryotic innate immunity mechanisms. Such systems that restrict foreign DNA or trigger cell death have recently been recognized, but the molecular signals that activate many of these remain largely unknown. Here, we characterize one such system in pandemic Vibrio cholerae responsible for triggering cell density-dependent death (CDD) of cells in response to the presence of certain genetic elements. We show that the key component is the Lamassu DdmABC anti-phage/plasmid defense system. We demonstrate that signals that trigger CDD were palindromic DNA sequences in phages and plasmids that are predicted to form stem-loop hairpins from single-stranded DNA. Our results suggest that agents that damage DNA also trigger DdmABC activation and inhibit cell growth. Thus, any infectious process that results in damaged DNA, particularly during DNA replication, can in theory trigger DNA restriction and death through the DdmABC abortive infection system.

Specificity of DNA damage formation induced by femtosecond near-infrared laser filamentation in water.

We investigated the deoxyribonucleic acid (DNA) damage induced by laser filamentation, which was generated by focusing femtosecond near-infrared Ti:Sapphire laser light in water at several repetition rates ranging from 1000 Hz to 10 Hz. Using plasmid DNA (pUC19), the single-strand break, double-strand break, nucleobase lesions, and the fragmented DNA were analyzed and quantified by agarose gel electrophoresis. Additionally, the H2O2 concentration after irradiation was determined. We observed that (1) the DNA damage per laser shot and (2) the enzyme-sensitive base lesions per total DNA damage decreased as the laser repetition rate increased. Furthermore, (3) extraordinarily short DNA fragments were likely to be produced, compared with those produced using X-rays, and (4) most OH radicals could be eliminated by recombination to generate H2O2, preventing them from damaging the DNA. The Monte-Carlo simulation of the strand break formation implies that the observed dependency of strand break efficiency on the laser repetition rate is mainly due to diffusion of DNA molecules. These findings quantitatively and qualitatively revealed that an intense laser pulse induces a specific DNA damage profile that is not induced by X-rays, a sparsely ionizing radiation source.

Establishing a High-Yield Chloroplast Cell-Free System for Prototyping Genetic Parts.

Plastid engineering offers the potential to carry multigene traits in plants; however, it requires reliable genetic parts to balance expression. The difficulty of chloroplast transformation and slow plant growth makes it challenging to build plants just to characterize genetic parts. To address these limitations, we developed a high-yield cell-free system from Nicotiana tabacum chloroplast extracts for prototyping genetic parts. Our cell-free system uses combined transcription and translation driven by T7 RNA polymerase and works with plasmid or linear template DNA. To develop our system, we optimized lysis, extract preparation procedures (e.g., runoff reaction, centrifugation, and dialysis), and the physiochemical reaction conditions. Our cell-free system can synthesize 34 ± 1 µg/mL luciferase in batch reactions and 60 ± 4 µg/mL in semicontinuous reactions. We apply our batch reaction system to test a library of 103 ribosome binding site (RBS) variants and rank them based on cell-free gene expression. We observe a 1300-fold dynamic range of luciferase expression when normalized by maximum mRNA expression, as assessed by the malachite green aptamer. We also find that the observed normalized gene expression in chloroplast extracts and the predictions made by the RBS Calculator are correlated. We anticipate that chloroplast cell-free systems will increase the speed and reliability of building genetic systems in plant chloroplasts for diverse applications.

dMAD7 is a promising tool for targeted gene regulation in the methylotrophic yeast Komagataella phaffii.

The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant "dead" MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in K. phaffii to regulate the expression of the enhanced green fluorescent protein (eGFP). A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of eGFP in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in K. phaffii through successfully regulating eGFP expression.

Cryo-EM structure of the R388 plasmid conjugative pilus reveals a helical polymer characterized by an unusual pilin/phospholipid binary complex.

Bacterial conjugation is a process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It is the main means by which antibiotic resistance genes spread among bacterial populations. It is crucially dependent upon the elaboration of an extracellular appendage, termed "pilus," by a large double-membrane-spanning secretion system termed conjugative "type IV secretion system." Here we present the structure of the conjugative pilus encoded by the R388 plasmid. We demonstrate that, as opposed to all conjugative pili produced so far for cryoelectron microscopy (cryo-EM) structure determination, the conjugative pilus encoded by the R388 plasmid is greatly stimulated by the presence of recipient cells. Comparison of its cryo-EM structure with existing conjugative pilus structures highlights a number of important differences between the R388 pilus structure and that of its homologs, the most prominent being the highly distinctive conformation of its bound lipid.