Streptococcus pneumoniae is a type of bacteria that causes respiratory infections which can lead to meningitis and other serious medical conditions. It is a Gram-positive bacteria, which means that it has a singular inner membrane with a thick, impermeable outer cell wall made of a substance called peptidoglycan. It is able to acquire DNA from non-competent cells—neighboring cells that lack certain enzymes that make them immune to lysis by other cells. However, there is a distinct difference in the machinery that allows bacteria to uptake DNA between Gram-positive and Gram-negative bacteria. Gram-negative bacteria, which have an inner and outer membrane with a much thinner layer of peptidoglycan in-between, have a complex system of genes that are translated into type IV pili. These pili are miniature anchors that adhere to surfaces or host cells and are essential in twitching movement. They also control the shape of the colony as well as surface sensing—all of which require retraction. Unlike Gram-negative bacteria that need to transfer DNA through the outer membrane, Gram-positive bacteria use something called Com pili to retract and bring DNA through the peptidoglycan cell wall for processing. The Com pilus is the equivalent of type IV pili in Gram-negative bacteria, but there is something special about this type of pilus. S. pneumoniae is the only Gram-positive bacteria where the Com pilus has been directly seen as an external organ, which makes it ideal to study.
A diagram showing the complex cellular machinery that allows Gram-positive bacteria to uptake DNA from non-competent cells.
In Gram-positive bacteria such as S. pneumoniae, this process of genetic uptake (natural transformation) and its cellular machinery, coded by what is called competence genes, are governed by two linked genes, comG and pilD. ComG codes for a pilus, or a hair-like structure on the surface of the cell that anchors to the non-competent cell’s DNA and brings it into the competent cell’s cytoplasm. The Com pilus is comprised of eight genes: comGC (the main protein subunit, called the major pilin), downstream genes comGD, comGE, comGF, and comGG that form minor protein subunits, comGA (an enzyme that is the driving force behind pilus extension), and comGB, which encodes a membrane protein. The pilD genes encode an enzyme that is not linked to the comG genes but is coregulated throughout the induction of competence. In Gram-negative bacteria, the machinery behind this process includes two separate proteins for extension and retraction. However, in Gram-positive bacteria such as S. pneumoniae, the pili lack a protein for retraction. Lam et al. (2021) set out to investigate how exactly natural transformation can occur in S. pneumoniae without this second protein.
Figure 5 from Lam et al. (2021). Competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake. Molecular Microbiology, 116: 381- 396.
In this study, Lam et al. first had to show that pilus retraction was necessary for natural transformation. To do this, they fluorescently labeled the pilus with a compound called biotin and added a bulky compound called neutravidin, which has a high binding affinity to biotin. The fluorescence of these treatments is shown in Figure 5b. The time-lapse of these experiments is shown in Figures 5a1-a4. In the experiments with neutravidin (5a3 and 5a4), the white arrows indicate static pili, which were unable to retract. Figure 5c shows the percent of cells within the treatments that were able to produce motile vs. non-motile (static and tangle) pili. Cells that were treated with neutravidin had a lower percentage of transformation than the control group (Figure 5d), showing that the retraction of the pili is necessary for natural transformation. Thus, Lam et al. were able to determine that even though the pili lack a protein for retraction, there is retraction included in natural transformation.
In terms of the aforementioned missing protein for retraction, there is a small possibility that ComFA (another enzyme) might be the missing piece of the puzzle. However, it is unlikely as ComFA in pneumococcal bacteria has been identified as a single-stranded DNA-binding protein that only interacts with itself, another small protein, and ComFC, but not ComGA. It would also be unexpected for such a small protein to be able to have a secondary function (independent of its other function) that was involved in pilus retraction. An alternative to this is that there is a singular enzyme that is associated with the comG operon that acts as a reversible enzyme. This is similar to the Gram-negative bacteria Caulobacter crescentus, where a single enzyme controls the movement (both extension and retraction) of type IV pili. Pneumococcal Com pili may be more flexible than those of V. cholerae, the Gram-negative bacteria used as a model organism in this paper. In Gram-positive bacteria, there is no outer membrane pore to limit the size of the DNA complex, which indicates that there may be different roles for Com pili in Gram-positive bacteria vs Gram-negative bacteria; it is also possible that the route of Com pili through the peptidoglycan cell wall may better adjust to the DNA molecule.
Even with these breakthroughs in the role and nature of the Com pili in S. pneumoniae, there is still much to be explored. The roles of minor pilins are unknown—all four are needed for assembly, but their function remains unclear. The nature of pilus-dependent transport, as well as trans-membrane transport, remains elusive: are these events coordinated in any way? Much is still unknown about the nature of gene transfer from competent to non-competent cells. The world of natural transformation in S. pneumoniae remains as ever, com-plex.
About the Author:
Mars Jordan ‘22 is a Biology major from Orange County, CA with a passion for plants. After Mount Holyoke, they hope to pursue a career in public horticulture. Outside of academics, they enjoy journaling, tatting, and reading.
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