From isotopically labeled DNA to fluorescently labeled dynamic pili: building a mechanistic model of DNA transport to the cytoplasmic membrane

SUMMARY

Natural competence, the physiological state wherein bacteria produce proteins that mediate extracellular DNA transport into the cytosol and the subsequent recombination of DNA into the genome, is conserved across the bacterial domain. DNA must successfully translocate across formidable permeability barriers during import, including the cell membrane(s) and the cell wall, that are normally impermeable to large DNA polymers. This review will examine the mechanisms underlying DNA transport from the extracellular space to the cytoplasmic membrane. First, the challenges inherent to DNA movement through the cell periphery will be discussed to provide context for DNA transport during natural competence. The following sections will trace the development of a comprehensive model for DNA translocation to the cytoplasmic membrane, highlighting the crucial studies performed over the last century that have contributed to building contemporary DNA import models. Finally, this review will conclude by reflecting on what is still unknown about the process and the possible solutions to overcome these limitations.

INTRODUCTION

Natural competence refers to the physiological state wherein bacteria produce proteins that mediate exogenous DNA transport into the cytosol and subsequent recombination of that DNA into the genome (1–3). The conservation of natural competence across the bacterial domain suggests that the trait is useful under diverse circumstances (4). Three primary, non-mutually exclusive potential benefits to importing DNA during natural competence have been identified (2). First, imported DNA could aid in DNA damage repair via homologous recombination and/or the establishment of new replication origins (5–7). Second, imported DNA could be catabolized to obtain vital nutrients and/or energy (2, 8, 9). Alternatively, nucleotides derived from imported DNA could be recycled for DNA synthesis, bypassing energetically expensive de novo nucleotide synthesis (8). Finally, imported heterologous DNA can be integrated into the genome, introducing novel genetic elements (2). Useful traits, such as antibiotic resistance, can be transferred between cells in this manner (10, 11).

Realization of these potential benefits requires the successful translocation of extracellular DNA across the cell periphery. Numerous cellular barriers prevent entry of DNA into the cytosol under most circumstances. In Gram-negative species, the outer membrane first prevents DNA internalization. This lipid bilayer is composed of densely packed phospholipids and lipopolysaccharide (LPS) (12, 13). DNA, a large macromolecular polymer which bears negative charge along its entire length, cannot normally cross the outer membrane barrier. Charge repulsion from negatively charged LPS molecules and phospholipid head groups, as well as physical blockage by the tightly packed arrangement of LPS and phospholipids comprising the membrane, renders the outer membrane impermeable to DNA. The cell wall, a thin (less than 10 nm thick) mono- or dual-layered meshwork of glycan strands cross-linked together by short peptide chains, exists immediately internal to the outer membrane (14–17). The cross-linking of glycan strands results in cell wall pores with an estimated diameter of ~4 nm, meaning that an unmodified cell wall could physically allow passage of ~2.4-nm-wide, extended B-form DNA (18–20).

For Gram positives, the cell wall first prevents DNA entry into the cell. Gram-positive cell walls are thicker (between 50 and 60 nm thick) than Gram-negative cell walls and are composed of 40 or more stacked peptidoglycan layers (17, 21–24). The glycan subunits of the Gram-positive cell wall are extensively modified with wall teichoic acids (WTA) composed of repeating polyol-phosphate monomers (25). The phosphates within the WTA impart a significant negative charge throughout and beyond the cell wall (25, 26). Imported DNA must therefore navigate tens of layers of Gram-positive cell wall, with each layer being negatively charged. Because the estimated diameter of Gram-positive cell wall pores is also ~4 nm, charge repulsion is most likely responsible for preventing DNA from passing through the Gram-positive cell wall (20).

Additional challenges to DNA transport can exist at the outer edge of the cell periphery if a given strain is encapsulated. The bacterial capsule, a dense network of carbohydrates or peptides formed exterior to the outer membrane of Gram negatives or the cell wall of Gram positives, impedes DNA transfer during both phage transduction and plasmid conjugation (27–29). Lastly, all bacteria must mediate DNA crossing the cytoplasmic membrane during transformation. The cytoplasmic membrane is another lipid bilayer composed of roughly equal amounts of protein and phospholipid that maintains a net negative charge (30, 31). DNA normally cannot cross the cytoplasmic membrane, for the same reasons DNA cannot cross the Gram-negative outer membrane.

Significant resources have been devoted to characterizing natural competence and transformation. A fundamental question lies at the heart of this work: how do the proteins produced during natural competence function to translocate large DNA polymers across the normally impermeable cell periphery? In this review, we will address the first half of this question, namely, how naturally competent cells transport DNA across the outer membrane and/or cell wall. This review is intended to be a chronological and comprehensive examination of the seminal studies that contributed to the modern model of DNA translocation during natural competence, with a focus on how these studies contemporaneously added to existing models. With that said, let us rewind to the year 1928 in London, England, where Frederick Griffith would unexpectedly discover the “transformation” of pneumococcal strains.

NATURAL COMPETENCE AND TRANSFORMATION IN THE EARLY-MID 20TH CENTURY

In 1928, Frederick Griffith elegantly demonstrated that murine pneumococcal virulence could be transferred between strains (32). By heat-killing a virulent pneumococcus strain and mixing the resulting lysate with an avirulent strain prior to mouse injection, the avirulent strain was “transformed” with productive murine infection capabilities in vivo (32). How such transformation had occurred remained poorly understood until 1944, when Oswald Avery et al. used a much more controllable in vitro transformation system to find that DNA within pneumococcal lysates was responsible for transformation (33). The broader scientific community remained unconvinced of Avery’s findings until the early 1950s, when Hershey and Chase definitively demonstrated DNA as the biological hereditary material, and Watson and Crick published the molecular structure of DNA and its potential for information encoding by base sequence (34–37).

The discovery of antibiotic-resistant and auxotrophic strains of numerous species provided convenient selectable markers for use in the earliest, detailed studies of natural competence and transformation from the mid-1950s to mid-1960s [Bacillus subtilis (38–41), Streptococcus pneumoniae (42–44), Haemophilus influenza (45–48), and Neisseria meningitidis (49, 50)]. Antibiotic-sensitive or auxotrophic strains could be transformed with DNA conferring antibiotic resistance or prototrophy, respectively, and then plated on selective medium to determine the number of cells transformed in an experiment. This strategy allowed for the rapid optimization of methods used for the transformation of the model organisms noted above by screening parameters such as which strain, growth medium and nutritional supplementation, growth temperature, growth phase, DNA concentration, and time of DNA exposure would result in maximum transformation. These studies were often performed concurrently with ³²P-labeled transforming DNA to track DNA uptake temporally, giving early insight into DNA uptake kinetics during transformation (40, 41, 44, 51, 52). DNA uptake in these examples is defined as irreversible DNA binding exhibited by naturally competent cells, wherein DNA becomes resistant to exogenous DNase digestion. Uptake occurred rapidly (within 5 minutes) when competent cells were exposed to DNA and would mostly be completed within 15 minutes.

During the early 1960s–mid-1970s, the application of density gradient ultracentrifugation techniques, as well as some chromatographic separations, to radiolabeled or density-labeled donor DNA isolated during transformation led to the development of the overarching DNA uptake model still in use today [B. subtilis (53–57), S. pneumoniae (51, 58–60), and H. influenza (61, 62)]. In these experiments, naturally competent cultures were exposed to labeled donor DNA for a short time to initiate DNA uptake. Further DNA uptake would then be terminated by DNase treatment while still allowing the already-initiated uptake events to proceed. At time intervals after termination, DNA would be extracted from the cultures, and the various DNA species would be separated by the methods described above, followed by spectrophotometric or radiographic analysis. Extracted DNA species containing labeled nucleotides from the donor DNA would separate differentially, depending on multiple factors, including the species’ molecular weight or density, form of DNA (single vs double stranded, nucleotide vs nucleoside), and association with the recipient chromosome (free or associated, covalently or reversibly bound). Crucially, this information could be used to quantitatively determine the fate of all donor DNA molecules through time after exposure of recipient cells to the donor DNA. This would grant a deep understanding of the sequence of events occurring during transformation to ultimately integrate donor DNA into the genome.

Together, the studies above suggested the following conserved mechanism of transformation: shortly after exposure of naturally competent cells to exogenous DNA, DNA would breach the outer membrane and/or cell wall, then be fragmented and stably bound to the cell surface. Once bound, one strand of the transforming DNA would be hydrolyzed, while the other strand would be transported through the cytoplasmic membrane into the cytosol (though which step came first was not resolvable). Finally, homologous single-stranded DNA in the cytoplasm would pair with the recipient chromosome and eventually be covalently joined to the chromosome to complete the transformation process. Elucidating this sequence of events during transformation was a huge step forward in understanding the process, but an understanding of the proteins involved in transformation and how they worked together mechanistically to achieve each step remained unknown.

Shortly after this transformation model was established, the first S. pneumoniae essential transformation protein was identified in 1975 (63, 64). This protein, an endonuclease responsible for the breakdown of the non-transforming DNA strand observed during DNA uptake, was identified by screening a mutagenized S. pneumoniae population for loss of external DNase capabilities (63, 65). Several mutants were isolated, each mapping to S. pneumoniae’s major endonuclease, end (63). Within the next decade, several transformation-deficient strains of B. subtilis would be identified, genetically mapped, and partially characterized (66–70). It would not be until the implementation of transposon mutagenesis that the bulk of transformation-associated proteins would be identified and characterized, including those comprising a putative pilus production system likely responsible for translocation of DNA across the outer membrane and/or cell wall.

PILI: ESSENTIAL MEDIATORS OF DNA TRANSPORT TO THE CYTOPLASMIC MEMBRANE (LATE 20TH CENTURY TO PRESENT)

By the mid 1970s, numerous independent studies observing bacterial surfaces by electron microscopy had discovered long (up to multiple micrometers in length), thin (multiple nanometers in width) filaments attached to the cell surface of diverse bacteria (71–73). Multiple classes of such filaments were identified, differing substantially in average length, width, cell surface localization, number emitting from a given cell, curvature, and function (71, 73). Among these filaments were the flagella, known to propel bacteria through liquid media, as well as a diverse set of non-flagellar filaments designated as “pili” due to their hair-like appearances in electron micrographs (73, 74). Both flagella and pili were found to be proteinaceous, with one pilus type from Escherichia coli found to be composed entirely of a single low-molecular-weight protein monomer known as “pilin” (71). Due to the ubiquity of pili, their widely varying physical attributes, and their seeming inessentiality for cell viability under laboratory culture conditions, researchers would devote much effort in uncovering pilus function (71, 73).

Perhaps unsurprisingly, the diversity in pilus function mirrored the diversity of pilus types. Pili were found to mediate adhesion to a wide range of surfaces, from the abiotic surfaces of glass microscope slides to the biotic surfaces of epithelial cells and blood cells (73). Such adhesive properties also extended in some cases to cell-cell contact, with piliated cells of many species forming pellicles on the liquid-air interface of cultures and characteristic cell aggregates visible by light microscopy (73). Of interest for this review is the finding that horizontal gene transfer was facilitated by pilus filaments, with pilus production necessary for successful DNA transfer between mating pairs via conjugation (73, 75). Finally, by the early 1980s, the role of pili in a specific type of bacterial motility became clear. This mechanism of motility on surfaces, deemed “twitching motility” due to the short and intermittent movements of observed bacteria, was determined to be the result of pili adhering to surfaces and subsequently shortening or retracting (76, 77). The dynamic nature of some pilus systems will be of significant importance for assessing the role of pili in natural transformation going forward.

While this review has so far followed a chronological telling of events, we will briefly discuss the modern model for type IV pilus (T4P) biogenesis and retraction to provide additional context for the development of models concerning DNA translocation across the outer cell periphery. An understanding of T4P molecular composition and production dynamics is helpful for understanding how and why pili could be so crucial for DNA transport during natural transformation [reviewed in references (78, 79)]. Each T4P filament is an oligomer of pilin proteins mentioned above, which are small 10- to 25-kDa single-pass integral cytoplasmic membrane proteins. Pilins are composed of a conserved N-terminal alpha helical domain which anchors the pilin in the cytoplasmic membrane and a globular C-terminal domain situated outside the cytoplasmic membrane (Fig. 1A). Dedicated proteases cleave the pilins at their extreme N-termini, converting the pilins to peripherally associated membrane proteins that reside on the outer face of the cytoplasmic membrane.

Fig 1.

Type IV pilus structure and production models. (A) (Left) Cartoon structural model of a pilin subunit from a cryogenic electron microscopy (cryoEM) reconstruction of the Neisseria gonorrhoeae PilE type IV pilus (PDB 2HIL) (80). The pilin is composed of two domains, including the extended N-terminal alpha helix in red, and the C-terminal globular domain in blue. (Right) Space-filling structural model of the same PilE pilin subunit (80). Structural models created with PyMOL version 2.4.0. (B) (Left) Space-filling structural model of a cryoEM reconstructed PilE type IV pilus filament (PDB 2HIL) (80), with individual pilins colored according to the same scheme in panel A; (right) The same structural model rotated 90° toward the reader so that the filament axis is perpendicular to the reader. Notice in both images that the blue pilin C-terminal domains are facing outside the filament, whereas the red N-terminal domains are buried in the filament core. Structural models were created with PyMOL version 2.4.0. (C) Simplified cartoon model of type IV pilus biogenesis and retraction in Gram-negative bacteria. (Left) First, pilin subunits are translated and inserted into the cytoplasmic membrane as pre-proteins. Dedicated pilin peptidases proteolyze the pilins at their extreme N-termini, producing mature pilins capable of polymerizing into a pilus filament. (Middle left) Through the concerted actions of an integral membrane platform protein and a cytosolic extension ATPase, pilin monomers are added sequentially to an actively extending pilus filament, resulting in filament elongation. (Middle right) The filament will eventually encounter a gated outer membrane secretin pore complex, passing through the pore into the extracellular space. (Right) A cytosolic retraction ATPase will then provide energy for disassembly of the pilus, resulting in pilin monomers at the pilus base flowing back into the cytoplasmic membrane, where they can be reused in subsequent pilus extension events. OM = outer membrane; CW = cell wall; CM = cytoplasmic membrane.

A cytosolic ATPase and a polytopic membrane protein then cooperate to couple ATP hydrolysis to the extrusion of pilins into a 6- to 9-nm-wide, multiple micrometers-long helical filament (the pilus) held together by protein-protein interactions between pilin monomers. The conserved N-terminal alpha helices are buried in the core of the filament, while the variable C-terminal domains are solvent-exposed along the length of the pilus (Fig. 1B). This cytoplasmic membrane-anchored pilus extends across the cell wall and outer membrane, through a protein channel (secretin), into the extracellular space where the pilin C-terminal domains can interact with the environment. The pilus can be retracted back into the cytoplasmic membrane if a retraction ATPase or a single bifunctional ATPase is present in the cytoplasm (see Fig. 1C for a graphical depiction of this process) (81). The homologous type II secretion systems (T2SS) likely assemble pili in a similar fashion to drive protein secretion, although these structures do not extend beyond the cell envelope, leading to their designation as “pseudopili” [reviewed in reference (82)].

Pili are uniquely positioned to solve the problem of DNA transport across the outer cell periphery. Their ability to penetrate the cell wall and outer membrane provides a theoretical entryway for extracellular DNA to travel through to eventually reach the cytoplasmic membrane, while their proteinaceous composition and solvent-exposed C-terminal pilin domains allow for potential interactions with extracellular DNA. Finally, the ability for pili to retract back into the cytoplasmic membrane after production could aid in pulling DNA through the gap left in the cell periphery during pilus production if the pilus had bound the DNA in the extracellular space. Alternatively, pilus extension and retraction could clear a pathway for DNA to enter the cell through other mechanisms. These points will be relevant as we proceed through this review.

B. subtilis transposon mutagenesis screen reveals a putative pilus essential for transformation

In 1987, a transposon mutagenesis screen of B. subtilis for transformation-deficient mutants was performed that not only identified many novel genes associated with transformation but also gave early insight into their potential functions (83). One group of mutants mapping to the same locus was completely non-transformable and unable to bind exogenous DNA during natural competence. Further characterization of this locus revealed an operon (comG) composed of seven genes, each homologous to essential genes in Gram-negative T4P and T2SS, as discussed above (Fig. 2A) (3, 84, 85). The protein product of comG ORF 3 (now known as comGC) was found to be homologous to major structural pilin proteins from a diverse set of T4P and T2SS, suggesting that ComGC may oligomerize into a pilus capable of interacting with environmental DNA (Fig. 2B) (3). Instead of mediating protein secretion through the cell wall and outer membrane as seen in homologous T2SS, the ComG proteins could be mediating DNA transport from outside of the cell through the cell wall (3). This idea was further supported by the presence of another essential competence protein (now known as ComC) in B. subtilis that is homologous to pre-pilin proteases essential for pilus biogenesis in other systems (3, 83). ComGC proteolytic processing was, indeed, later found to be dependent on ComC (86)

Fig 2.

B. subtilis comG operon and comC gene organization and homology to T2SS and T4P essential genes (A) (Top) Cartoon model depicting the core essential proteins of T2SS pseudopilus and T4P systems; (Bottom) to scale graphical depiction of the B. subtilis comG operon and comC gene, with ORFs shown as arrows. Each ORF is color-coded to display, after translation, which essential pilus component is homologous to the cognate protein. comG + comC encode the minimal set of proteins necessary for pilus biogenesis: An ATPase (ComGA) that powers pilus assembly, a polytopic membrane protein (ComGB) that promotes pilus assembly, pilin protein (ComGC-G) constituents of a pilus, and a pre-pilin peptidase (ComC) needed to activate pilins for assembly into pilus filaments. (B) Alignment of B. subtilis ComGC major pilin to homologous major pilins from diverse T2SS and T4P systems. The boxed/colored residues highlight the region of homology found at pilin N-termini, which includes the conserved G/A-X-X-X-X-E/D pre-pilin peptidase cleavage signal motif. The coloring follows Clustal X conventions, and the numeric residue conservation scores are calculated using the Analysis of Multiply Aligned Sequences (AMAS) method (87). The scissors icon displays the site of peptidase cleavage for each pilin. Each pilin homolog sequence was obtained from UniProt (ComGC_Bs, P25955; PilA_Pa, P04739; PilA_Mx, Q59589; PilA4_Tt, Q72JC0; PilA5_Tt, Q72GL2; PilE_Ng, P11764; PilE_Nm, P57039; XcpT_Pa, Q00514; GspG_Ec, P41442; ExeG_Ah, P31733; PulG_Kp, P15746; OutG_Dc, P31585)

ComGC oligomerization facilitates DNA translocation across the cell wall

Despite the strong bioinformatic data supporting the production of ComGC pili during B. subtilis natural competence, these putative structures had not been observed via imaging techniques such as electron microscopy, so their existence remained in question (88). To address this discrepancy, a 2006 investigation sought to biochemically characterize cell envelope-associated protein complexes containing ComGC that were produced during natural competence (88). Multiple ComGC-containing species were discovered within the cell wall fraction of competent cells upon Western blotting for ComGC following non-reducing SDS-PAGE. The apparent molecular weight of these proteins ranged from ~10 kDa (corresponding to ComGC monomer) up to the resolution limit of the gel, increasing by ~10 kDa along the entire lane. These data indicated that competent cells produce disulfide-linked ComGC oligomers that extend outward past the cell membrane, suggesting ComGC oligomerizes into pili.

With strong evidence for ComGC pilus biogenesis in hand, this same study assessed the genetic factors responsible for pilus biogenesis. Expression of comG and comC alone, in a strain background incapable of producing other competence-associated proteins, was sufficient for production of ComGC oligomers. The ability to induce ComGC pilus production in otherwise non-competent cells then allowed for a controlled investigation of the specific role of these pili during transformation.

Irreversible DNA binding to cells was achieved by expression of comEA, which encodes the integral membrane protein responsible for DNA binding at the cell membrane, alongside comG and comC (89). Because ComEA localizes to the cell membrane and exerts its activity there, this heavily implied that the function of the ComGC pilus was to mediate translocation of DNA across the normally impermeable cell wall (89, 90). comG is dispensable for DNA binding to naturally competent protoplasts, i.e., cells lacking cell walls, lending additional support for ComG facilitating DNA translocation across the cell wall (90). The mechanisms underlying this translocation step, however, remained unclear.

Evidence for pilus-DNA binding capabilities found both in vitro and in vivo

By the early 2000s, a correlation between T4P production and natural competence had been described for numerous Gram-negative bacteria, including Neisseria and Pseudomonas species (91, 92). In these same species, the competence defect in pilus biogenesis mutants appeared to arise from an inability to bind extracellular DNA, as was observed for B. subtilis comG transposon insertion mutants (83, 92, 93). From the mid 2000s to early 2010s, further studies into Gram-negative T4P demonstrated unambiguous binding of DNA to T4P. Purified, intact Pseudomonas aeruginosa T4P (noted above) was shown to bind both prokaryotic and eukaryotic DNA sequence non-specifically in vitro, and this interaction was disrupted significantly by pre-incubation of the T4P with a monoclonal antibody targeting the pilus tip, suggesting that the pilus tip binds DNA (94). Interestingly, purified pilin was unable to bind DNA, which pointed to DNA binding being an emergent property of the pilus quaternary structure. N. meningitidis T4P has also been found to bind DNA in vitro in the context of intact pili (95). In contrast to the P. aeruginosa T4P, the DNA binding of N. meningitidis T4P is greatly enhanced by the presence of a specific 12-bp sequence known as the DNA uptake sequence, and this specific binding is achieved by the presence of a low-abundance pilin, ComP, in the pilus fiber. Purified ComP contained intrinsic DNA binding capabilities, seen in electrophoretic mobility shift assays, which suggested that the pilus quaternary structure may not always be crucial for DNA binding.

A 2013 investigation into the comG operon of S. pneumoniae demonstrated, for the first time, direct in vivo evidence of a competence-specific pilus binding to extracellular DNA. The S. pneumoniae comG operon encodes seven genes, each of which shares extensive homology with those in the B. subtilis comG operon noted above (96). Both immunofluorescence and electron microscopy revealed that thin, extended structures were produced at the surface of competent cells and that these structures were composed, at least in part, of ComGC. The flexibility, length, and width of these structures was consistent with pili from T4P systems. Mass spectrometry analysis of affinity-purified pili confirmed that these pili were composed primarily of ComGC. Co-incubation of competent cells with bacteriophage lambda DNA, followed by electron microscopy, showed pili on the surface of S. pneumoniae cells in close association with the DNA both at the pili tips and along the fibers. These data together led to the conclusions that a dedicated type IV competence pilus composed primarily of ComGC is produced by S. pneumoniae and that this competence pilus directly binds extracellular DNA to begin the internalization process. The latter conclusion is complicated by the potential for the cell fixation process, which was necessary for electron microscopy at the time, to artifactually place T4P and DNA in close proximity. How DNA would be translocated into the periplasm once bound to competence pili, if binding was occurring, remained uncertain.

DNA binding and translocation by dynamic pili is confirmed via fluorescence microscopy

The next breakthrough in this area came in 2017 from the development of a method to label dynamic, transformation-specific T4P from live cells (97, 98). This method works by first introducing a cysteine substitution into the main pilin protein, at a solvent-accessible residue, to provide the pilin with a free thiol group. Upon production of this cysteine-substituted pilin, a maleimide-fluorophore conjugate is added to the growth medium. Maleimide is a thiol-reactive compound capable of forming covalent bonds with the free thiols found on each pilin monomer. The maleimide-fluorophore conjugate reacts with the available pool of pilin monomers, thereby labeling the pilin with whichever fluorophore is conjugated to the maleimide. The active assembly and disassembly of T4P composed of these labeled pilins can then be observed in real time using widefield fluorescence microscopy.

This technique was successfully applied to the T4P of the Gram-negative Vibrio cholerae by introducing a cysteine into the major pilin PilA to make PilA^Cys (99). Upon activation of natural competence and subsequent addition of a maleimide dye to the growth medium, dynamic extension (up to 2.5 µm in length) and retraction of fluorescent T4P filaments were observed via widefield fluorescence microscopy (Fig. 3A). When a fluorescently labeled PCR product was added to the medium, these T4P filaments co-localized DNA at their tips (Fig. 4A), with pilus retraction bringing the DNA in close proximity to the cell periphery (Fig. 5A). Rates of natural transformation in V. cholerae mutants that produced DNA-binding-deficient T4P were severely reduced, indicating that DNA binding was crucial for transformation. Physical obstruction of T4P retraction, achieved by linking maleimide-biotin to the PilA^Cys monomers and adding neutravidin to the medium to form bulky T4P-neutravidin complexes, resulted in a similar decrease in transformation rate, indicating that T4P retraction was also critical for transformation.

Fig 3.

Examples of natural competence pili fluorescently labeled via the maleimide labeling method. Widefield fluorescence micrographs of naturally competent cells producing pili (false-colored green) composed of cysteine substitution mutant pilin monomers, labeled by exposure to maleimide-conjugated Alexa Fluor 488. Labels indicate the length of each pilus shown. Organisms shown include (A) V. cholerae [courtesy of Ankur B. Dalia (Indiana University), reprinted with permission; see additional images in reference (97)]; (B) S. pneumoniae [adapted from reference (98) with permission of the publisher (copyright 2021 John Wiley & Sons Ltd.)]; (C) B. subtilis [adapted from reference (100)]. B. subtilis/V. cholerae green false coloring and S. pneumoniae/B. subtilis/V. cholerae pilus measurement bars were added to this figure.

Fig 4.

Maleimide-labeled natural competence pili binding to fluorescently labeled DNA. Widefield fluorescence micrographs of naturally competent cells producing maleimide-labeled pili (false-colored green or cyan), binding to Cy3- or Cy5-labeled PCR products (false-colored red or magenta). White arrows show the location of pili involved in DNA binding. Organisms shown include (A) V. cholerae [courtesy of Ankur B. Dalia (Indiana University), reprinted with permission; see additional images in reference (97)]; (B) S. pneumoniae [adapted from reference (98) with permission of the publisher (copyright 2021 John Wiley & Sons Ltd.)]; (C) B. subtilis [adapted from reference (100)]. The top images in each panel are colored green/red for maximum contrast, while the bottom images in each panel are colored cyan/magenta for enhanced color blindness accessibility. The cyan/magenta color scheme was added for S. pneumoniae/V. cholerae, and the green/red color scheme was added for B. subtilis/V. cholerae for this figure.

Fig 5.

Extension and retraction of natural competence pili. Widefield fluorescence micrographs of naturally competent cells extending and retracting maleimide-labeled pili (false-colored green). White arrows show the location of pili. The earliest timepoints are displayed in the top images, progressing through time to the latest timepoints displayed in the bottom images. Timescales vary for each organism shown. Organisms shown include (A) V. cholerae [courtesy of Ankur B. Dalia (Indiana University), reprinted with permission; see additional images in reference (97)]; (B) S. pneumoniae [adapted from reference (98) with permission of the publisher (copyright 2021 John Wiley & Sons Ltd.)]; (C) B. subtilis [adapted from reference (100)]. B. subtilis/V. cholerae green false coloring was added for this figure.

The same general strategy has been successfully employed for the Gram-positive organisms S. pneumoniae and B. subtilis. S. pneumoniae also produced dynamic pilus filaments (Fig. 3B) that were capable of DNA binding (Fig. 4B), with retraction being similarly crucial for transformation (Fig. 5B) (100). Multiple groups, including our own, have recently found B. subtilis capable of producing retractile, DNA-binding pili (Fig. 3C, 4C and 5C) (101–103). Although both organisms employ a homologous comG operon for pilus biogenesis, numerous differences exist between the two systems. The observed pilus length distribution of B. subtilis pili is notably shorter than that of S. pneumoniae pili, and the retraction rate of B. subtilis pili is slower than that of S. pneumoniae pili (100, 102). These results could reflect true biological differences between the systems, potentially explained by variable enzymatic activity of the predicted assembly ATPase ComGA, or they may be artifacts of the genetic manipulations necessary for pilus imaging. Critically, regardless of the observed differences between these pilus systems, all available evidence points to a conserved mechanism of DNA translocation across the outer layers of the cell periphery during natural competence: naturally competent cells produce pili that can access the extracellular space; these pili bind to DNA in the extracellular space; pilus retraction brings the bound DNA across the outer membrane and/or the cell wall into association with proteins essential for the next steps in uptake (Fig. 6).

Fig 6.

Model of pilus-mediated translocation of DNA across the outer cell periphery. The far-left and middle-right columns show a cartoon model of the outer cell periphery of naturally competent Gram-negative and Gram-positive cells, respectively, during a given step in the DNA translocation process. The middle-left and far-right columns show examples of widefield fluorescence micrographs of cyan, maleimide-labeled naturally competent Gram-negative (V. cholerae) and Gram-positive (S. pneumoniae) cells, respectively, during translocation of a Cy3-labeled PCR product (false-colored magenta) (99, 100). For the fluorescence micrographs, white arrows show the location of assembled pili. The cartoon model component identities are as follows: the purple rounded trapezoids in the outer membrane represent outer membrane secretins; the orange cytoplasmic membrane cylinder represents pilus assembly platform protein; the green rounded rectangles in the cytoplasmic membrane represent pilins; the blue/red six-pointed stars represent pilus extension/retraction dedicated ATPases, respectively. For the Gram-positive cartoons, a single ATPase is present, which presumably powers both extension and retraction. (A) Naturally competent cells produce proteins required for pilus biogenesis. (B) Pilin monomers are oligomerized into pilus filaments through the concerted actions of platform proteins and extension ATPases, which breach into the extracellular space. (C) Pili bind to free DNA molecules at their tips, most likely via minor pilins. (D) Retraction ATPases facilitate the disassembly of pilin monomers from the pilus base, which causes the pilus to retract and simultaneously pull bound DNA through the pores created in the outer cell periphery. S. pneumoniae fluorescence micrographs (rightmost column) were adapted from reference (98) with permission of the publisher (copyright 2021 John Wiley & Sons Ltd.), and the V. cholerae fluorescence micrographs (second column from the left) are courtesy of Ankur B. Dalia (Indiana University), reprinted with permission. S. pneumoniae false colors changed from green to cyan/red to magenta, and the same false colors were added to V. cholerae images for enhanced color blindness accessibility.

Helicobacter pylori: translocation of DNA by a type IV secretion system

So far, each of the organisms discussed either employs a bona fide T4P (V. cholerae) or a ComG pilus (S. pneumoniae and B. subtilis) for the reception and translocation of extracellular DNA across the outer cell periphery. The only known exception, H. pylori, uses a type IV secretion system (T4SS) to translocate DNA into the periplasmic space (104–106). T4SS, much like T4P and ComG systems, produce pili that extend into the extracellular space (107). These filaments normally attach to neighboring cells in the environment and function to deliver either DNA or protein into those cells, in the case of conjugation systems or protein secretion systems, respectively (107). During conjugation, recent evidence suggests that ssDNA-relaxase nucleoprotein complexes transfer between donor and recipient cells by traveling through a channel within T4SS filaments (108). Such an observation raises an interesting question: does the H. pylori T4SS pilus operate similarly to competence T4P or ComG pili to pull DNA into the periplasm, or does it rather provide a convenient conduit for extracellular DNA to travel through into the periplasm? Maleimide labeling studies on H. pylori T4SS pili could potentially distinguish between these possibilities. Evidence for either model could be generated by observing maleimide-labeled T4SS pilus interactions with fluorescently labeled DNA. Whatever the outcome may be, we are confident that such experiments will shed light on this unique system.

A BRIEF EXAMINATION OF NATURAL TRANSFORMATION AFTER DNA RECEPTION AND INITIAL TRANSLOCATION

The next steps in DNA uptake, while not the focus of this review, warrant at least a brief examination. These steps begin with binding of incoming DNA by proteins generally localized either in the periplasm for Gram-negative cells or in the cytoplasmic membrane for Gram-positive cells (1, 2, 109). These proteins (designated ComEA in B. subtilis) bind DNA sequence non-specifically and are thought to act as a ratchet to prevent DNA from moving out of the cell while simultaneously capturing additional DNA segments as the molecule diffuses into the cell (2, 90, 109). Next, the bound DNA is fragmented by non-specific endonucleases, presumably to generate a free DNA end that will be used to start the cytosolic internalization process (109). In B. subtilis, endonucleolytic digestion is facilitated by the endonuclease NucA, though NucA homologs are not universal (109, 110).

At this stage, DNA is transported into the cytosol through a conserved cytoplasmic membrane channel composed of multiple ComEC protein subunits (109, 111). The mechanism of DNA transport through the ComEC channel is not well understood. A fascinating consequence of DNA translocation into the cytosol is the seemingly concomitant exonucleolytic digestion of one DNA strand that results in only a single DNA strand entering the cytosol (109). Recent evidence suggests that ComEC may possess intrinsic exonucleolytic capability, and degradation of the non-transforming DNA strand by ComEC may facilitate DNA entry (112). A cytoplasmic ATPase, ComFA, is thought to provide energy for DNA transport into the cytosol (109, 113).

Once the transforming DNA strand enters the cytosol, it has the potential to be integrated into the recipient genome to complete the transformation process. As the DNA strand enters, single-stranded DNA binding proteins, including the conserved DprA, begin to coat and protect the incoming DNA (109, 114). DprA, along with SsbA and SsbB, mediates RecA binding to the ssDNA. After RecA binding occurs, the imported DNA can be integrated into the recipient genome via homologous recombination, thereby completing natural transformation (109).

OUTSTANDING QUESTIONS AND FUTURE DIRECTIONS

Although real-time fluorescence imaging, combined with maleimide labeling, has drastically improved the field’s understanding of DNA translocation across the outer cell periphery, a few major questions remain unresolved. First, is there a conserved mechanism of DNA binding to competence T4P, and how is this binding achieved? The necessity of a specific minor pilin (ComP) for DNA binding to Neisseria T4P both in vivo and in vitro, the existence of V. cholerae minor pilin point mutants that significantly reduce DNA binding and natural transformation while still allowing for wild type piliation, and the known localization of minor pilins to the tips of T4P (where DNA appears to bind in maleimide labeling experiments) all point to a significant contribution of minor pilins in the binding of T4P to DNA (95, 99, 115, 116). Binding DNA at the pilus tip minimizes the overall width of a pilus-DNA complex, which could reduce steric hindrance when DNA ultimately passes through the outer membrane secretin and/or the pore formed in the cell wall from pilus extrusion, making DNA entry more feasible. However, DNA binding only at the tip would imply that a U-shaped DNA molecule might trail the pilus tip across the cell wall, raising a host of biophysical questions about this process. The lack of homology between known DNA-binding minor pilins, including a recently characterized minor pilin (ComZ) from Thermus thermophilus, and the known preference for Neisseria ComP to bind DNA sequence specifically, suggests that the evolution of DNA binding properties by a minor pilin may have occurred independently across numerous clades, and therefore the underlying mechanism of DNA binding may not be conserved (95, 117).

Recent advancements in both pilus purification and cryogenic electron microscopy techniques have set the stage to understand the competence pilus-DNA binding interaction more comprehensively. Morais et al. demonstrated that pili from Lacticaseibacillus rhamnosus could be efficiently purified in high concentration by a simple protocol consisting of cell wall digestion in an osmotically protective buffer to liberate pili without significant cell lysis, followed by two chromatography steps to remove impurities (118). The underlying principles at work in this purification should be widely applicable to other pilus systems. Therefore, intact competence pili from a diverse array of competent bacteria should now be readily purifiable for downstream in vitro DNA interaction studies. This is especially important for any pilus system that depends on the quaternary structure of the pilus or a pilin complex for DNA binding, as purification of individual pilus components would be insufficient for DNA binding studies in these cases.

Cryogenic electron microscopy (cryoEM) can be leveraged to deduce the key molecular interactions stabilizing competence pilus-DNA binding. Molecular structure determination via cryoEM involves the rapid freezing of samples in amorphous ice (vitrification), collecting anywhere from 10⁴ to 10⁶ images of the relevant molecule(s) in the sample in various orientations using electron microscopy, and then computationally reconstructing a three-dimensional model of the molecule(s) from the electron micrographs [reviewed in references (119, 120)]. Improvements in cryoEM technology (mainly in electron detection and image processing) have allowed for electron density map resolutions of <4 Å, which are generally sufficient for generating atomic models (121). Notably, cryoEM does not require the crystallization of target molecules or any fixation procedure prior to vitrification. Therefore, any purified competence pilus can be incubated with substrate DNA in vitro to allow binding to occur, and the sample can then be directly vitrified and subjected to electron microscopy for structural determination. As long as a high-enough resolution is achieved, an atomic model should be possible to build that depicts the critical binding interactions between pilus and bound DNA. This process can be applied to numerous bacterial competence pilus systems, and a comparative analysis of the pilus-DNA binding interactions could be performed to assess the diversity of these interactions. Structural determination via cryoEM was recently used to determine how MutS dimers bind and respond to DNA mismatches to subsequently initiate the mismatch repair pathway, which supports the utility of cryoEM for studying interactions within nucleoproteins (122).

The step immediately downstream of DNA binding, pilus retraction, remains enigmatic for Gram-positive organisms. For competent Gram-negative model organisms, such as V. cholerae and Neisseria gonorrhoeae, a dedicated retraction ATPase is known to provide the energy for competence pilus retraction (99, 123). The well-characterized and competent Gram-positive model organisms, B. subtilis and S. pneumoniae, possess only a single putative ATPase gene (comGA) homologous to those in other T4P systems (3, 96). In B. subtilis, non-polar deletion of comGA ablates production of high-molecular-weight pilin complexes, as do point mutations in the comGA Walker A and Walker B motifs, which suggests that ComGA powers competence pilus extension (124). The protein players and mechanisms responsible for competence pilus retraction, on the other hand, are currently unknown.

There are two known pilus retraction mechanisms consistent with a single ATPase. In the first case, the lone ATPase is bifunctional, able to both extend and retract the pilus. The Caulobacter crescentus ATPase CpaF exhibits bifunctionality for extending and retracting tight adherence pili that are essential for surface colonization (81). In the second case, pilus retraction has been shown to be spontaneous. V. cholerae competence pili retract at greatly reduced rates in the absence of the retraction dedicated ATPase pair PilTU, and this spontaneous retraction appears to stem from the preference of the terminal pilin subunit to associate with the membrane over binding to the next pilin in the filament, which leads to dissociation of the filament over time in the absence of active extension (125).

Differentiating between these two mechanisms in the competent Gram positives will be difficult. The elucidation of CpaF’s bifunctionality was greatly facilitated by a phage whose infectivity is dependent on pilus retraction, which allowed for the selection of piliated, yet retraction-deficient, cpaF mutants (81, 125). To our knowledge, no such phage has been characterized for either of the Gram-positive models, and the transformation frequencies of both models (~10⁻⁵ to 10⁻⁴ transformants/CFU) are far too low to rely on transformation of a counter-selectable marker to cull retractile comGA mutants from an otherwise piliated comGA mutant library. For the time being, this essential step in the Gram-positive transformation process remains poorly understood.

While it may be easy to fixate on the unknowns of DNA transport across the outer cell periphery, we should reflect positively on the discoveries made within the last century of study. The genesis of this field predates the discovery of DNA as the hereditary material within bacteria, and the field has rapidly evolved alongside the organisms we seek to study. From rudimentary radiolabeled DNA fractionation experiments to sophisticated time-lapse fluorescence microscopy experiments involving targeted pilin mutants and multiple labeling chemistries, the field has dramatically improved in its understanding of the process. All these years of research have culminated in our understanding of the extensive usage of pili across the bacterial domain for initial DNA capture and subsequent transport to the cell membrane. We look to the next generation of methods to reveal the finer molecular details of how competence pili are assembled and disassembled, how competence pili bind to DNA substrates, and how competence pili interact with the rest of the natural competence proteins to mediate the handoff of DNA for transport into the cytosol (80, 87).

Biographies

Jason D. Zuke graduated from Washington University in St. Louis in 2015, receiving a B.A. in biology. From 2015-2017, he worked as a research technician in Dr. Petra Anne Levin’s laboratory at the same institution, where he studied the post-translational regulation of a critical cell size regulatory protein (UgtP) in response to nutrient availability. Afterwards, he joined Briana M. Burton’s laboratory at the University of Wisconsin-Madison where he pursued his Ph.D. in microbiology within the Microbiology Doctoral Training Program. From 2017-2023, he studied the mechanisms underlying DNA transport across the cell wall of naturally competent Bacillus subtilis cells, leveraging advances made in live cell epifluorescence microscopy to study the role of pili during this process. Jason received his Ph.D. in microbiology in 2023 and is now seeking employment in private industry.

Briana M. Burton is an associate professor of bacteriology at the University of Wisconsin-Madison. She received a Ph.D. in Biology from the Massachusetts Institute of Technology in 2003, where she studied the role of Clp proteases in remodeling Mu transpososomes. She performed post-doctoral research at Harvard Medical School, examining chromosome translocation during Bacillus subtilis sporulation. From 2008-2015, she was an assistant/associate professor at Harvard University. In 2015 she moved to the University of Wisconsin-Madison where her laboratory continues to focus on mechanisms of macromolecular transport across Gram-positive cellular barriers with an emphasis in natural genetic transformation.