Natural competence, the ability of bacteria to produce proteins that mediate the uptake of extracellular dsDNA, is widely distributed among the bacterial domain of life (1
). DNA imported via natural competence is highly advantageous to cells and can be utilized in numerous, non-mutually exclusive ways. Once in the cytosol, the DNA can be metabolized to supply additional nucleotides and/or cellular carbon (2 - 4
). If naturally competent cells encounter chromosomal DNA damage, the internalized DNA can be used for repair either through excision-mediated mechanisms or can be integrated into the resident chromosome via homologous recombination (5 - 8
). Finally, the internalized DNA can confer novel genetic elements into the competent cell’s genome (9 - 11
). The process wherein DNA is internalized and subsequently integrated into the competent cell’s chromosome is known as natural transformation and is one of the major drivers of horizontal gene transfer in bacteria (12
The mechanisms governing Gram-positive natural transformation have been well characterized owing to the work performed on model organisms such as Streptococcus pneumoniae
and Bacillus subtilis
). Transcription of genes involved in the production of DNA uptake and transformation proteins is activated through varied environmental cues (1
). Once produced, a subset of these proteins must mediate the translocation of extracellular DNA across the negatively charged, 30–40 nm thick Gram-positive cell wall (15
). The comG
operon includes seven genes, each of which is homologous to components present in both type IV pilus (T4P) and type II secretion pseudopilus (T2SS) systems (16
). These evolutionarily related systems employ a conserved set of proteins including at least one ATPase, at least one polytopic integral membrane protein, and a set of structural proteins known as pilins that work together to dynamically assemble membrane-bound, filamentous protein helices composed of repeating pilin subunits (17
). These filaments are known as either pili for T4P or pseudopili for T2SS, with the main discriminating characteristic being filament length. T4P pili can be multiple microns long and readily observed in the extracellular space, while T2SS pseudopili are typically not long enough to be visible in the extracellular space (19
). Crucially, both systems can produce filaments that span the cell envelope and reach the extracellular space [T2SS pilin genes must be overexpressed for this to occur (19
operon mediates the production of seven proteins that work together to form a T4P filament, composed of hundreds to thousands of individual ComGC pilin subunits, which extends through the cell wall into the extracellular space (20 - 22
). Once extended, the ComGC pilus binds to free dsDNA in the environment (20
). Dynamic retraction of the ComGC subunits back into the membrane shortens the pilus, transporting bound DNA through the gap formed in the cell wall, and into contact with the membrane-bound DNA receptor/binding protein ComEA (23 - 26
). After ComEA binding, DNA is brought into contact with the ComEC membrane channel (27
). The cytosolic protein ComFA aids in DNA entry across this channel via ATP hydrolysis (29 - 31
). One strand of the incoming DNA is hydrolyzed, resulting in ssDNA in the cytosol (32 - 34
). Through the action of various ssDNA binding proteins and the competence-specific RecA-loading protein DprA, RecA binds to the ssDNA and directs homologous recombination of the incoming ssDNA into the chromosome to complete natural transformation (35
Although this mechanism has been thoroughly researched, an interesting discrepancy remains regarding DNA translocation across the cell wall. While S. pneumoniae
undoubtedly produces type IV pili to mediate initial DNA reception and translocation across the cell wall, all attempts to identify such a structure in B. subtilis
have yielded negative results, despite B. subtilis
carrying a comG
operon that is both essential for natural transformation and is homologous to the S. pneumoniae comG
). Biochemical data indicate that B. subtilis
produces multimeric ComGC structures associated with the cell wall (22
). However, no microscopy data or structural studies have confirmed the existence of bona fide
pili or pseudopili, so the exact nature of B. subtilis
translocation of transforming DNA across the cell wall is unclear.
In this investigation, we report that ectopic expression of select comGC
cysteine substitution alleles (comGCCys
) in parallel with comGCWT
allows for natural transformation to occur in B. subtilis
. Expressing comGCCys
results in extracellular filaments that are labelable with a fluorescent maleimide-dye conjugate. We interpret these filaments to be pili composed mainly of ComGC. The pili can retract back toward the cell body and can bind to extracellular DNA. These data all support a model of DNA translocation across naturally competent B. subtilis
cell walls by binding of DNA to pili, followed by pili retracting to transport bound DNA across the cell wall. We also find the localization of pilus biogenesis and DNA binding strongly biased to the periphery of the cell long axis. In addition, GFP-ComEA and GFP-ComFA also reside predominantly along the long axis of the cell. Together, the data suggest that DNA translocation across the cell wall is biased away from the poles, and even later steps in transformation may not be localized exclusively to or near the cell poles as has been previously reported (40
In Bacillus subtilis
, the comG
operon is essential for natural transformation (37
). The ComG proteins of B. subtilis
have been assumed to be involved in the production of a pilus capable of conveying extracellular DNA across the cell wall. This idea stems from multiple lines of evidence: each protein coded for in the operon has homology to components of either type IV pilus or type II secretion pseudopilus systems; there is biochemical evidence supporting multimerization of ComGC associated with the cell wall; ComGC pili have been demonstrated in Streptococcus pneumoniae
). However, direct evidence of ComG-mediated pilus production in B. subtilis
has been lacking, with no microscopy or structural studies supporting pilus biogenesis. Here, we demonstrate that B. subtilis
in the naturally competent state produces ComGC-based pili. These pili are capable of stable binding of DNA, and they can retract dynamically back toward the cell membrane after assembly. ComGC pili were also found to localize along the long axis of the cell at the cell periphery, mirroring the localization of bound DNA and the downstream essential competence proteins ComEA and ComFA we observed.
We demonstrated that comGCE56C
alleles were functional by complementation of ΔcomGC
for transformation efficiency (Fig. S1). These comGCCys
variants complemented almost as well as comGCWT
, further supporting their functionality. The comGC
complementation construct itself caused massive decreases in transformability regardless of the comGC
allele used, and no pili were ever observed in either of the ΔcomGC
complementation strains (Fig. S4). The most likely explanation for the poor transformability of these strains is a polar effect due to the introduction of a sub-promoter into the comG
operon during native comGC
). There are four additional pilin gene homologues downstream of comGC
in the operon (47
). Overexpression of certain pilins can be inhibitory for pilus production, so it is feasible that the sub-promoter could cause overexpression of comGDEFG
, and at least one gene product could inhibit pilus biogenesis (48
). Regardless of the exact reason, the failure of the complementation strains to produce pili spurred us to try another approach to produce biologically relevant, visualizable pili.
Rather than alter the endogenous comG
promoter, we instead took a less perturbative approach and expressed comGCCys
alongside native comGC
. In stark contrast to the comGC
complementation strains, we demonstrated that comGCE56C
merodiploid strains closely approximated wild-type transformation efficiencies (within twofold, Fig. 1A
). The levels of total ComGC in both comGCE56C
were variable and slightly lower than for the isogenic control (Fig. 1B
). While this may suggest that the ComGC pili we observed were artifactually shortened due to a smaller ComGC pool, it must be noted that the ComGC peptide used to raise the ComGC antiserum we employed for Western blotting included both positions E56 and S65 (38
). It is, therefore, possible that the ComGC antiserum may not recognize ComGCE56C
as efficiently as ComGCWT
since the binding epitope could have been altered. Total ComGC levels in comGCE56C
may, therefore, be roughly equivalent to the isogenic control, making the pili we observe reflective of those found in wild type cells, or possibly slightly shorter.
The mean pilus length of 0.33 µm measured in B. subtilis
with simultaneous expression of comGCWT
) is notably shorter than the mean lengths of competence pili identified in V. cholerae
or S. pneumoniae
of 1 µm and 0.5 µm, respectively (20
). The somewhat smaller difference between B. subtilis
and S. pneumoniae
ComGC pili may be attributable to pilus measurement error. Exact pilus start and end points were manually assigned based on the phase-contrast and epifluorescence images taken, which could lead to length differences based on user interpretation. Additionally, we determined pilus start points to be at the border of the phase-contrast (cell body) signal and not at the border of the epifluorescence (ComGC) signal. There was generally a gap between the internal ComGC signal and the external cell body signal, so pilus lengths would have been increased had pilus start points been marked at the ComGC signal border. Another source of variation could be the presence of endogenous cysteine residues in B. subtilis
ComGC. An off-target, BdbDC-mediated disulfide bond between an endogenous cysteine on one ComGC monomer and the mutant cysteine of another ComGCCys
monomer might result in early termination of pilus elongation due to conformational changes at the pilus base, which inhibit proper coordination of the pilus biogenesis apparatus. S. pneumoniae
ComGC, in contrast, contains no endogenous cysteine residues that could improperly form disulfide bonds with the ComGCCys
cysteine (see PDB 5NCA), negating this possibility. And finally, the observed length difference may simply stem from intrinsic differences in the protein sequences of each ComG protein comprising the two systems.
The larger difference between V. cholerae
and B. subtilis
pilus lengths may stem from the differences in how force for pilus biogenesis is generated. V. cholerae
employs both a dedicated extension (PilB) and a retraction (PilT) ATPase, whereas B. subtilis
only has one identifiable pilus ATPase homologue (ComGA) (16
). PilB ATPase activity may simply be faster and/or more processive than that of ComGA, which would grant PilB greater ability to incorporate new pilin subunits into a growing pilus prior to disassembly than ComGA, making the average pilus longer for PilB-polymerized pili. Further investigation of pilin levels and extension/retraction ATPase activities is warranted to produce a complete picture of how competence pilus lengths are established.
Our work provides evidence of direct B. subtilis
ComGC pilus-DNA interactions with physiological levels of ComGC proteins (Fig. 3
). Such interactions are consistent with the data from a diverse set of naturally competent bacteria, including V. cholerae
, S. pneumoniae
, Neisseria gonorrhoeae
, and Thermus thermophilus
that demonstrate either direct binding of pili to DNA or individual pilins to DNA (20
). We also demonstrate that B. subtilis
ComGC pili are retractile in nature, as has been observed for V. cholerae
and S. pneumoniae
competence pili (Fig. 4
). Intriguingly, we observed two distinct populations of retracting pili with variable retraction rates: a slow population that retracts with a median rate of 3 nm/second and a fast population that retracts with a median rate of 12 nm/second (Fig. 4C
). The slow population may be retracting spontaneously, whereas the fast population is most likely actively retracting via ComGA activity. Spontaneous retraction of V. cholerae
competence pili has been observed with a notably slower retraction rate compared to active retraction, strengthening this idea (42
). Moreover, the clear distinction in retraction rates between the two populations suggests that a specific process increases the retraction rate of the fast-retracting pili. The most parsimonious explanation is, of course, that the pilus ATPase homologue ComGA is promoting active retraction events within the fast population.
The retraction rate for the fastest ComGC pili observed (median = 12 nm/second) is much slower than for V. cholerae
(median ~100 nm/second) and S. pneumoniae
(median ~80 nm/second). We consider two possible explanations for this difference. First, the rate differences could be reflective of differences in the enzymatic activities of the ATPases utilized for retraction across these systems (Fig. 4B
). Alternatively, the disulfide isomerization involved in assembly and disassembly of ComGC pili in B. subtilis
may impact the retraction rate. Dissection of the contribution of the disulfide is not trivial, as disulfide bond formation is necessary for ComGC pilus biogenesis (22
), but future mechanistic studies to address this question will be valuable.
Subcellular localization analysis of ComGC pili and associated DNA produced a localization pattern quite distinct from the previously reported polar localization patterns of other late competence gene products, including ComGA and ComEC (40
). We found that ComGC pili and associated DNA both predominantly localize across the long axis of the cell (Fig. 5A and B
), with little clustering of ComGC pilus biogenesis. Additionally, ComEA and ComFA were seen to localize along the long axis of competent cells (Fig. 5C and D
). These localization patterns are consistent with the predominant distributions reported for DNA bound at the cell membrane (25
). These observations suggest that the initial step of mobilizing extracellular DNA across the peptidoglycan layer for ComEA binding at the cell membrane may occur throughout the surface of the cell, predominantly along the long axis, and may even be excluded from the cell poles. One possible explanation for this is that during natural competence, the lateral cell wall is mostly static, as the cells have ceased growth and elongation, and any remaining peptidoglycan remodeling is likely occurring at the cell poles at sites of cell separation (53 - 55
). This may facilitate ComGC pilus formation at this region, since steric clashes with active cell wall remodeling systems could potentially be reduced, and any channels formed in the cell wall may be more likely to remain open for extended periods of time.
While our data for ComEA localization are largely consistent with previous reports, our analysis differs significantly for ComFA localization (25
). We observed GFP-ComFA puncta throughout the long axis of the cell (Fig. 5D
), whereas polar ComFA-YFP localization had been observed previously. This difference is of critical importance since the localization of ComFA to the cell poles, as well as that of other late competence proteins such as ComEC and ComGA, led to the conclusion that DNA translocation across the cell membrane occurred at or near the cell poles (40
). Our data, on the other hand, are consistent with DNA entry points distributed along the lateral cell membrane.
To address the disparities, we consider DNA uptake in the context of the two-stage model for transformation (56
). First, our data may best represent the location of the first step of DNA uptake, where DNA crosses the cell well. The localization of pilus biogenesis and retractile pili that interact with DNA provides a direct view of active competence pili. In contrast, the polar localization of ComGA holds the implicit assumption that the foci are coincident with active protein (40
). Given the necessity of ComGA for pilus assembly, functional ComGA should localize to the sites of pilus biogenesis (i.e., along the cell long axis) but that has not been observed (39
). This raises questions as to the biological relevance of the observed localization patterns of the late competence protein fluorescent fusions used in previous studies. Subpopulations of proteins are often sufficient for biological activity, and active fractions do not always correspond to the visualized population (58
). This could be the case for the prior results with ComGA fusions.
The location of the second stage of DNA import, across the cell membrane as assessed by ComEC and ComFA localization, remains less clear. Studies from multiple groups have come to the same conclusion regarding polar localization of ComFA (40
). The constructs used in the respective localization experiments differ from ours in placement of the fluorescent tag. The data presented here use a complementing amino-terminal fluorescent fusion to ComFA. Consistent with prior published work, we observe predominantly polar localization when the fluorescent protein is at the carboxy-terminal end of ComFA. However, there is significant proteolysis of the fluorescent tag and free ComFA in these cells, reducing confidence in these fusions as reporters of active protein. Thus, we favor the amino-terminal fusion presented here as reporting on functional localization. However, from these data alone we cannot rule out that DNA translocation across the cell membrane could occur at cell poles. In such a system, DNA that had been captured by ComEA after translocation across the cell wall would most likely be transported to the cell pole, where the DNA would then be internalized (59
Critically, our results expand on the mechanism of DNA reception and translocation across the cell wall during Gram-positive natural competence, where models have thus far relied on observations made solely using S. pneumoniae as a model organism. Our observations are most consistent with the following model of DNA translocation across the cell wall. During natural competence, the protein products of the comG operon work together to generate an extracellular pilus that is comprised primarily of ComGC along the long axis of the cell. These pili are dynamic and retract stochastically. At some point, dsDNA in the environment binds to the pilus surface. At some point after DNA binding, the pilus will retract back into the membrane, which will consequently pull the bound DNA through the gap left in the cell wall. Once across the cell wall, the DNA will bind to ComEA at the cell membrane, and translocation into the cytoplasm will commence, either at a cell pole or along the long axis of the cell.
Numerous questions remain unresolved regarding the mechanism of DNA translocation across the cell wall. Are each of the five pilin homologues in the comG
operon present in the competence pilus, and if so, where are they located? Each gene in the comG
operon is essential for transformation, so presumably each pilin is involved to some degree with pilus biogenesis and/or function (37
). In S. pneumoniae
, it was recently discovered that ComGC, ComGF, and ComGG were present throughout pili, although no other pilins were detected (60
). It is possible that the other pilins (ComGD and ComGE) are located at the tip of the pilus in single copy and are responsible for DNA binding. This is consistent with the role of low abundance “minor pilins” in the initiation of pilus assembly and interactions with the environment (17
). Identification of minor pilin point mutations that allow for DNA-binding-deficient pili to be produced, as was achieved for V. cholera
competence pili, would support this hypothesis (42
). With successful methods to visualize ComGC pili and DNA capture in B. subtilis
, several of these open questions now become accessible.
MATERIALS AND METHODS
General methods for strain construction were performed according to published protocols (62
). Molecular cloning was performed using the Gibson assembly method with HiFi assembly enzyme mix (NEB) (64
). PCR amplification templates were derived from either chromosomal DNA isolated from the prototrophic domesticated B. subtilis
strain PY79 or plasmid pKRH83. Introduction of DNA into PY79 derivatives was conducted by transformation (65
). The bacterial strains, plasmids, and oligonucleotide primers used in this study are listed in Tables S1 to S3.
Media and growth conditions
For general propagation, B. subtilis
strains were grown at 37°C in Lennox lysogeny broth (LB) medium (10 g tryptone per liter, 5 g yeast extract per liter, 5 g NaCl per liter) or on LB plates containing 1.5% Bacto agar. Where indicated, B. subtilis
strains were grown in the nutrient-limiting medium, medium for competence, with 2% fructose [MC-Fru; 61.5 mM K2
, 38.2 mM KH2
, 2% (wt/vol; 110 mM) D-fructose, 3 mM Na3
O, 80 µM ferric ammonium citrate, 0.1% (w/v) casein hydrolysate, 11 mM L-Glutamic acid potassium salt monohydrate] substituted for 2% glucose to prevent catabolite repression of Pxyl
). When appropriate, antibiotics were included in the growth medium as follows: 100 µg mL−1
spectinomycin, 5 µg mL−1
chloramphenicol, 5 µg mL−1
kanamycin, 10 µg mL−1
tetracycline, and 1 µg mL−1
erythromycin plus 25 µg mL−1
lincomycin (mls). When required, 0.5% (wt/vol; ~30 mM) D-xylose was added to the cultures to induce protein expression.
Producing lysed protoplasts for B. subtilis transformation
A single colony of the B. subtilis strain bBB050 (CmR) was inoculated into LB medium and incubated at 37°C with 250 rpm shaking for 3 hours. The OD600 of a 1:10 dilution of the culture was measured, and then 1 mL of culture was pelleted at 21,000 × g for 2 minutes and the supernatant removed completely. The pellet was resuspended to an OD600 = 10 in Bacillus protoplasting buffer (50 mM tris pH 8.0, 50 mM NaCl, 5 mM MgCl2, 25% (wt/vol) sucrose, 0.2 mg/mL lysozyme) and incubated in a 37°C water bath for 30 minutes to protoplast cells. The sample was removed from the water bath and left at room temperature until transformation, at which time protoplasts were pelleted at 10,000 × g for 5 minutes, the supernatant was completely removed, and then an equal volume of ddH2O was added. The protoplasts were lysed by resuspension in the ddH2O by repeated pipetting.
Transformation efficiency assays
Single colonies of B. subtilis
strains of interest were inoculated into MC-Fru and cultured at 37°C with 250 rpm shaking until an OD600
= 0.2–0.5 was reached. Cells were pelleted at 10,000 × g
for 2 minutes and resuspended in ~15% of residual supernatant to concentrate cells, OD600
of a 1:20 dilution of resuspended cells was measured, and the resuspensions were diluted into fresh MC-Fru to an OD600
= 0.05. Cultures were incubated at 37°C with 250 rpm shaking for 2 hours (1 hour prior to max natural competence induction in these conditions), and strains containing Pxyl-comK
were induced with 0.5% xylose to maximize the proportion of competent cells in the populations. The strains were continued to be cultured at 37°C with 250 rpm shaking for 1 hour to allow for maximal natural competence induction, and then ~105
protoplasts per µL competent cells were added to the cultures (31
). Transformation was allowed to proceed under the same culturing conditions for 2 hours. Tenfold serial dilutions of each sample were made down to 106
-fold diluted in PBS, and appropriate dilutions were plated onto LB and LB–Cm5
agar plates and incubated for 16–20 hours at 37°C to allow for colony growth. The number of transformants and total cells were calculated from the single colonies on LB–Cm5
and LB, respectively, and the transformation efficiency was calculated as the ratio of transformants to total cells in a given sample.
ComGC Western blotting
strains of interest were cultured according to the same protocol noted for transformation efficiency assays, but 1 mL of the culture was centrifuged at 21,000 × g
for 2 minutes to pellet cells after 3-hour incubation post-dilution, and supernatant was completely removed. The OD600
of a 1:10 dilution of each culture was measured, and each cell pellet was resuspended in cell lysis buffer [25 mM Tris pH 8.0, 25 mM NaCl, 3 mM MgCl2
, 1 mM CaCl2
, 0.2 mg/mL lysozyme, 0.1 mg/mL DNase I] to an OD600
= 10 based on the previous measurements. Resuspended cells were incubated in a 37°C water bath for 20 minutes to lyse cells and degrade genomic DNA. Cell lysates were mixed with an equal volume of 2× reducing tricine sample buffer [200 mM tris pH 6.8, 40% (vol/vol) glycerol, 2% (wt/vol) SDS, 0.04% (wt/vol) Coomassie Blue G-250, 2% (vol/vol) beta-mercaptoethanol] and heated to 37°C for 30 minutes for protein denaturation. Five microliters of each preparation were added to the wells of a tris-tricine mini gel [stacking gel: 1M tris pH 8.45, 4% (wt/vol) acrylamide/bis-acrylamide (29:1); resolving gel: 1M tris pH 8.45, 15% (wt/vol) glycerol, 10% (wt/vol) acrylamide/bis-acrylamide (29:1)] and electrophoresed [running buffer: 0.1 M tris-Cl, 0.1 M tricine, 0.1% (wt/vol) SDS] at 100 V until loading dye exited the gel (typically 1.75–2 hours). Separated proteins were Western transferred to a 0.2 µm PVDF membrane using the semi-dry transfer method at 15 V for 20 minutes with Towbin transfer buffer (66
). The membrane was washed 3× in ddH2
O for 5 minutes with gentle shaking to remove transfer buffer; then, the membrane was stained with 0.1% Ponceau S solution to detect total protein for loading normalization. The membrane was blocked with 5% (wt/vol) milk in TBS-T (Tris-buffered saline with Tween 20) for 1 hour with gentle shaking, and then the membrane was incubated with 1:2,000 rabbit-derived antisera containing 1° ComGC antibody [TBS-T + 1% (wt/vol) milk] overnight at 4°C (37
). The membrane was washed 3× in TBS-T for 5 minutes with gentle shaking, and then the membrane was incubated with 1:20,000 Abcam goat anti-rabbit 2° HRP antibody [TBS-T + 1% (wt/vol) milk] for 1 hour at room temperature with gentle shaking. The membrane was washed as described previously and then developed using Clarity ECL substrate (Bio-Rad) and imaged for chemiluminescence using a Bio-Rad ChemiDoc Touch imaging system.
Preparing cover glass and agar pads for use in microscopy
All cover glasses used in microscopy experiments were pre-cleaned prior to use. 22 mm × 22 mm #1.5 borosilicate coverslips (DOT Scientific) were placed in a Wash-N-Dry coverslip rack (Sigma) and submerged in ~80 mL of 1 M NaOH in a 100-mL glass beaker. The beaker was then placed into an ultrasonic cleaning bath (frequency = 40 kHz, power = 120 W) and sonicated for 30 minutes to remove the thin grease layer present on the coverslips. The coverslip rack was submerged into a fresh 250-mL glass beaker filled with ddH2O, the ddH2O was removed, and then the coverslips were washed 3× with 250 mL of ddH2O. The coverslips were then either air-dried overnight or dried immediately with compressed air.
For the preparation of agar pads, pre-cleaned borosilicate glass microscope slides were first rinsed free of detritus using ddH2
O and were then either air-dried overnight or dried immediately with compressed air. Working in a fume hood, half of the total rinsed slides were dipped into a 2% (vol/vol) solution of dichlorodimethylsilane (in chloroform) so that their entire surface was contacted by the solution. The solution was allowed to drip off the slides into the original container, and the remaining organic solvent on the slides was allowed to evaporate in the fume hood for 30 minutes. The slides were then thoroughly rinsed in ddH2
O and dried as mentioned previously to generate dry glass slides with extremely hydrophobic surfaces. Two pieces of lab tape (VWR #89098-074) were placed on top of one another, running lengthwise across an unrinsed microscope slide. For every agar pad to be produced, two of these taped slides were made. Approximately 15 minutes prior to imaging, conditioned MC-Fru medium (0.22 µM PES filter sterilized) from the cultures grown to competence via the transformation efficiency assay protocol was heated to 37°C, added in the ratio of 1:1 to 90°C 2% (wt/vol) molten LE agarose (SeaKem) in ddH2
O, and then vortexed to make molten 1% (wt/vol) agarose in 0.5× conditioned MC-Fru medium. Twenty microliters of this mixture were applied to the surface of a hydrophobic glass slide, and a solidified pad was produced according to a previously established protocol, using a rinsed (but untreated and hydrophilic) glass slide to form the top of the pad (67
). This specific setup allows for the agar pad to stick to the bottom slide and easily slide out from the top slide, leaving an unmarred and flat surface for imaging.
Fluorescent labeling—ComGC pili
For all experiments involving ComGC pilus labeling, B. subtilis strains of interest were cultured according to the same protocol noted for transformation efficiency assays. After 3-hour incubation post-dilution, 100 µL of each culture was transferred to a 37°C pre-warmed 13 mm glass test tube, and 25 µg/mL Alexa Fluor 488-maleimide was added to each culture aliquot. The aliquots were incubated in the dark at 37°C on a rolling drum for 20 minutes to allow for ComGCCys pilin labeling. The aliquots were transferred to centrifuge tubes, centrifuged at 5,000 × g for 30 seconds to gently pellet the cells, and all supernatant was removed. The cell pellets were washed by gently resuspending in conditioned MC-Fru medium (0.22 µM PES filter sterilized) via gentle pipetting. The cells were centrifuged again at 5,000 × g for 30 seconds, the supernatant removed, and the cells gently resuspended in one-tenth the original volume of conditioned MC-Fru medium.
Fluorescent labeling—PCR product
An ~4.5 kb PCR product was amplified from genomic DNA of a B. subtilis strain bearing Physp-bdbDC at the ycgO locus (bJZ185) using LongAmp Taq DNA Polymerase (NEB) and oligonucleotide primer pair (oJZ436 + oJZ437). This PCR product was purified using the E.Z.N.A. Cycle Pure Kit (Omega Bio-Tek) following the manufacturer’s instructions. One microgram of the PCR product was fluorescently labeled using the Label IT-Cy5 Nucleic Acid Labeling Kit (Mirus) following the manufacturer’s instructions, with a sufficient quantity of Label IT-Cy5 reagent to covalently link a fluorophore to approximately 1.5% to 5% of basepairs (75–230 Cy5 molecules per DNA molecule). The Cy5-labeled PCR product was purified using the E.Z.N.A. Cycle Pure Kit as mentioned previously. The final product was electrophoresed on a 1% agarose in TAE mini gel at 100 V for 45 min, stained using SYBR Safe dye (Invitrogen) according to the manufacturer’s instructions, and quantified by densitometric analysis of the product band compared to a reference band of known quantity.
Microscopy—Alexa Fluor 488-maleimide-labeled ComGC pili and Label IT-Cy5-labeled PCR product
For the initial identification of ComGC pili, quantification of pilus production, pilus length measurements, and the determination of ComGC pilus retraction rates, cells were labeled with Alexa Fluor 488-maleimide as previously described. Resuspended, labeled cells of 0.5 µL volume were applied to the center of a conditioned MC-Fru agar pad, and a pre-cleaned 22 mm × 22 mm #1.5 borosilicate coverslip was applied to the drop of culture. The coverslip was gently compressed with a gloved finger to ensure cells made contact with the agar pad, and then the space between the coverslip and glass slide was sealed by applying molten (60°C) VaLAP sealant (1:1:1 petroleum jelly:lanolin:paraffin) to the coverslip edges. The cells were imaged using a Zeiss Axio Observer.Z1 inverted epifluorescence microscope equipped with a Zeiss Plan-Apochromat 100×/1.4 Oil PH3 objective lens and a Teledyne Photometrics CoolSNAP HQ2 CCD camera. Cells were typically exposed to light from a Zeiss Colibri 469 nm LED module with Zeiss filter set 38 HE for 2 seconds at 100% LED power to observe ComGC pili. Cell bodies were imaged using phase-contrast microscopy (50 ms exposures). Imaging was performed at 37°C, achieved by a PECON Heater S objective heater calibrated via thermocouple. Time lapse microscopy was performed with the above conditions, with exposures occurring every 5–11 seconds depending on the experiment.
For co-localization experiments on ComGC pili and PCR products, cells were labeled with Alexa Fluor 488-maleimide as previously described. Just prior to the deposition of labeled cells onto a conditioned MC-Fru agar pad, Cy5-labeled PCR product was added to the cell suspension to a final concentration of 120 pg/µL. The new mixture of Alexa Fluor 488-labeled cells and Cy5-labeled PCR product was applied to a conditioned MC-Fru agar pad as described above. Imaging was performed using a Zeiss Axio Imager.Z2 upright epifluorescence microscope equipped with a Zeiss Plan-Apochromat 100×/1.4 Oil PH3 objective lens and a Teledyne Photometrics Prime 95B sCMOS camera. Cells were typically exposed to light from a Zeiss Colibri 469 nm LED module with Zeiss filter set 38 HE for 200 ms at 20% LED power to observe ComGC pili, while Label IT-Cy5-labeled PCR product was observed using light from a Zeiss Colibri 631 nm LED module with Zeiss filter set 90 HE for 500 ms at 20% LED power. Cell bodies were imaged using phase-contrast microscopy (100 ms exposures). Z-stacks were acquired in 0.2 µm increments starting 1 µm above the midcell plane and progressing until 1 µm below the midcell plane. Due to a fortuitous imaging delay during phase-contrast acquisition, the time interval between epifluorescent image acquisition was ~5 s, producing a time lapse.
Microscopy—localization of GFP-ComEA and GFP-ComFA
Fluorescence microscopy was performed as previously described (68
). Exposure times were typically 500 ms for GFP-ComEA and 250 ms for GFP-ComFA. Membranes of gfp-comEA
cells were stained with TMA-DPH [1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate] (Molecular Probes), at a final concentration of 0.01 mM, and imaged with exposure times of 200 ms. Cell bodies of gfp-comFA
cells were imaged using phase-contrast microscopy (20 ms exposures). Fluorescence images were analyzed, adjusted, and cropped using Metamorph v 6.1 software (Molecular Devices).
Microscopy—post-processing of Alexa Fluor 488-maleimide-labeled ComGC pili and Label IT-Cy5-labeled PCR product images
All epifluorescence microscopy images presented in Fig. 2
were deconvoluted prior to publication. First, point-spread-functions (PSFs) were computationally estimated for Alexa Fluor 488 and Cy5 signals using the PSF Generator plugin (EPFL Biomedical Imaging Group) on Fiji (NIH) (70
). For the Alexa Fluor 488 signal captured on the Zeiss Axio Observer.Z1 microscope configured as described above, the following parameters were entered into the PSF Generator: optical model = Born and Wolf 3D Optical Model; refractive index immersion = 1.518 (corresponding to Immersol 518F); accuracy computation = Best; wavelength = 516 nm (corresponding to Alexa Fluor 488 emission maximum); numerical aperture =1.4; pixelsize XY = 65 nm; Z-step = 250 nm; sizes XYZ—X = 256, Y = 256, Z = 65; display = Linear, 16-bits, Fire. The Z-stack composing the PSF was Z-projected to a single 256 × 256 pixel image by averaging the pixel intensities of each individual pixel in the 256 × 256 pixel array across the 65 Z-slices of the Z-stack, resulting in a 2D PSF. For Alexa Fluor 488 signal captured on the Zeiss Axio Imager.Z2 microscope configured as described above, the same procedure was performed to estimate the PSF, only changing Pixelsize XY to 110 nm. For Cy5 signal captured on the Zeiss Axio Imager.Z2 microscope configured as described above, all parameters were kept constant to estimate the PSF, only changing wavelength = 666 nm (corresponding to Cy5 emission maximum).
Epifluorescence microscopy images were deconvolved using the DeconvolutionLab2 plugin (EPFL Biomedical Imaging Group) for Fiji (NIH). Individual epifluorescence microscopy images were opened in Fiji, and the DeconvolutionLab2 plugin was run. The PSF generated as above, corresponding to a particular epifluorescence signal, was used to deconvolve that signal with DeconvolutionLab2 (algorithm = Richardson Lucy, 100 iterations).
Quantification of pili produced in comGC merodiploid strains
Images of Alexa Fluor 488-maleimide-labeled cells from each merodiploid strain, acquired as described previously, were opened in the Fiji image processing package (ImageJ2, NIH). The plugin ObjectJ was first used to identify 300 individual cells of each strain from phase-contrast images. Narrow filaments of length greater than 0.5 µM that were directly connected to the cell bodies of these 300 cells, which were surmised to be ComGC pili, were identified and counted in the green epifluorescence channel. The extracellular space immediately adjacent to the 300 cell bodies was scanned in the green epifluorescence channel for foci, which were likely sheared ComGC pili, and foci within 0.5 µM of the cell body were counted. These epifluorescence data were graphed using Microsoft Excel.
Measurement of ComGC pili lengths
Images of Alexa Fluor 488-maleimide-labeled cells of the comGCE56C and comGCS65C merodiploid strains, acquired as described previously, were opened in Fiji (NIH). The images were scaled up fivefold using bilinear interpolation, and the phase-contrast and green epifluorescence image channels were split for each pilus-producing cell. Each channel was converted into a binary image using Fiji’s default thresholding parameters, and then outlines of the signal present in each channel were produced. The outline view of the phase-contrast channel demarcated the cell boundary and extracellular space, while the green epifluorescence channel outline divided a pilus from the extracellular space. These outlines were merged together to simultaneously display both the cell boundary and pilus boundary. Pilus length was manually measured by placing the start of a segmented line on the cell boundary, approximately where the medial axis of the pilus would cross the cell boundary, and creating a line that roughly followed the pilus medial axis to the extreme tip of the pilus.
ComGC pilus retraction rate measurements
Time lapse microscopy images of Alexa Fluor 488-maleimide-labeled comGCS65C cells, acquired as described previously, were visually scanned for potential ComGC pilus retraction events using Fiji (NIH). Once identified, cells with retracting pili were isolated, and pilus length was measured for each time point of the time lapse as described previously. Frame-to-frame retraction rates were calculated by dividing the change in pilus length between frames by the time interval of the time lapse. These data were collected for 10 individual pilus retraction events, which included 44 instances of frame-to-frame retraction. Microsoft Excel was used to perform the t-test and ANOVA (using the Real Statistics Resource Pack release 8.6.3) referenced in the Results section.
Localization analysis of fluorescent proteins (ComGC, ComEA, ComFA) and PCR product
Images acquired as previously described for Alexa Fluor 488-maleimide-labeled comGCS65C
, and gfp-comFA
strains were opened in Fiji (NIH). For labeled ComGCS65C
pili, individual pilus-producing cells were isolated and identified in the phase-contrast channel, and a 4 × 4 pixel white square was added to the green epifluorescence channel where the pilus medial axis would cross the cell boundary to mark the base of pilus production. Because maleimide labeling also resulted in cell membrane staining, cell bodies were checked against the green fluorescence channel to identify cell septa not visible in phase-contrast images. If a septum was identified, a 1-pixel wide white line was added onto the phase-contrast image across the cell body to allow MicrobeJ to detect multiple cells in the image; this process was repeated for 232 individual pilus production events. MicrobeJ (version 5.11x) was used to define cell bodies and medial axes from phase-contrast images, and pili bases were identified from the green epifluorescence images by adjusting the plugin’s sensitivity parameters (tolerance and Z-score) until only the added white square was detected as a fluorescent focus (71
). The identified cells and pili boundaries were associated with one another, and a heatmap of pilus production localization on a size-normalized rod-shaped cell was made within the “Heatmap(s)” tab of the results window. This heatmap was scaled up tenfold using bilinear interpolation to create the final version included in Fig. 5
For localization of GFP-ComEA foci, cell bodies were identified by TMA-DPH epifluorescence signal, and the outline of each cell was filled in using a white rounded rectangle with a 6-pixel wide black border to enhance MicrobeJ detection of cells. GFP-ComEA foci were identified in the green epifluorescence channel and marked with a 4 × 4 pixel white square to enhance contrast. Microbe J (version 5.11x) was used as described above to generate a heatmap of GFP-ComEA localization. For localization of GFP-ComFA foci, cell bodies of isolated single cells were identified using phase-contrast images, and GFP-ComFA foci were identified in the green epifluorescence channel. A localization heatmap was generated as described previously. For localization of Cy5-labeled PCR product binding to Alexa Fluor 488-maleimide-labeled cells, time course images acquired as described previously were opened in Fiji (NIH). A binding event was defined as the co-localization of a Cy5 focus with either the cell body or a labeled ComGC filament for at least three consecutive frames (≥15 seconds of co-localization). Cell bodies were identified as described previously, and Cy5-labeled PCR product foci were identified in the red epifluorescence channel and contrast-enhanced with a 4 × 4 white square as described previously. A localization heatmap was then generated as described previously.