Biology subjects

Kilb, A.

Publications and source records attributed to Kilb, A..

3 recordsLinked to original sources

bioRxiv2025

Bacillus competence pili are efficient single - and double stranded DNA uptake machines

Natural competence describes the ability of diverse bacterial species to take up DNA from the environment for integration into the host chromosome, allowing the acquisition of novel genetic information. Uptake in many species operates via a dynamic pilus structure that pulls DNA through the cell wall into the periplasm by pilus retraction, followed by transport of one of the two DNA strands into the cytosol by an active membrane transporter. How double stranded DNA with a bending contour of hundreds of base pairs can fit through a narrow pore accommodating a pilus has been a puzzling question. We show that purified major and minor pilins from two Bacillus species, B. subtilis and Geobacillus thermodenitrificans, show efficient binding to double as well as to single stranded DNA with similar affinities. Consistent with uptake, ssDNA accumulates and moves within the periplasm before transport through the cell membrane, with similar dynamics as dsDNA. Thus, Bacillus competence pili are dsDNA as well as ssDNA uptake machines. The binding of pili to ssDNA regions within environmental DNA likely increases the efficiency of competent bacteria to pull extended DNA strands through the cell wall. Uptake of environmental ssDNA may also provide a beneficial source of nutrition for bacteria during stationary growth phase.

microbiology↗

bioRxiv2023

Uptake of environmental DNA in Bacillus subtilis occurs all over the cell surface through a dynamic pilus structure

At the transition to stationary phase, a subpopulation of Bacillus subtilis cells can enter the developmental state of competence, where DNA is taken up through the cell envelope, is processed to single stranded DNA, which is incorporated into the genome if sufficient homology between sequences exists. We show here that the initial step of transport across the cell wall occurs via a true pilus structure, with an average length of about 500 nm, which assembles at various places on the cell surface. Once assembled, the pilus remains at one position and can be retracted in a time frame of seconds. The major pilin, ComGC, was studied at a single molecule level in live cells. ComGC was found in two distinct populations, one that would correspond to ComGC freely diffusing throughout the cell membrane, and one that is relatively stationary, likely reflecting pilus-incorporated molecules. The ratio of 65% diffusing and 35% stationary ComGC changed towards more stationary molecules upon addition of external DNA, while the number of pili in the population did not strongly increase. These findings suggest that the pilus assembles stochastically, but engages more pilin monomers from the membrane fraction in the presence of transport substrate. Our data reveal that localized transport of DNA into the cytosol at a single cell pole is preceded by localization throughout the periplasm and efficient uptake through the entire cell surface by a dynamic pilus. ImportanceHorizontal gene transfer (HGT) is important for bacterial evolution and the adaption to new environments. Through HGT, genes can be transferred between bacteria, which can lead to antibiotic resistance development, especially critical with regards to pathogenic bacterial species. One mechanism of HGT is natural competence, through which bacteria can take up DNA under natural conditions and incorporate it into their chromosomes. For DNA uptake, a complex will be formed that is conserved among Gram-positive and Gram-negative bacteria, in spite of different envelope architectures. We show that in Bacillus subtilis, where the part of the uptake complex spanning the cell membrane localizes to a single cell pole, transport through the cell wall is mediated by a visible pilus structure that can extend and retract to pull DNA into the periplasm. Thus, DNA uptake from the environment is not limited to polar sites, but is channelled into the cytosol at the pole. Single molecule dynamics of ComGC revealed a static, likely pilus-bound, and a mobile fraction of ComGC within the cell membrane. Addition of environmental DNA increased the static fraction of molecules, showing that pilus dynamics respond to DNA binding.

microbiology↗

bioRxiv2020

Single molecule dynamics of DNA receptor ComEA, membrane permease ComEC and taken up DNA in competent Bacillus subtilis cells

In competent gram-negative and gram-positive bacteria, double stranded DNA is taken up through the outer cell membrane and/or the cell wall, and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single stranded DNA, and transported through the cell membrane via ComEC. We show that in Bacillus subtilis, the C-terminus of ComEC, thought to act as a nuclease, is not only important for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single molecule tracking, we show that only 13% of ComEC molecules are statically localised at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C-terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as nuclease. By tracking fluorescently labeled DNA, we show that taken up DNA has a similar mobility within the periplasm as ComEA, suggesting that most taken up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken up DNA, before its entry into the cytosol. ImportanceBacteria can take up DNA from the environment and incorporate it into their chromosome in case similarity to the genome exists. This process of "natural competence" can result in the uptake of novel genetic information leading to horizontal gene transfer. We show that fluorescently labelled DNA moves within the periplasm of competent Bacillus subtilis cells with similar dynamics as DNA receptor ComEA, and thus takes a detour to get stored before uptake across the cell membrane into the cytosol by DNA permease ComEC. The latter assembles at a single cell pole, likely by a diffusion-capture mechanism, and requires its large C-terminus, including a conserved residue thought to confer nuclease function, for proper localization, function and mobility within the membrane.

microbiology↗