Biology subjects

Graumann, P. L.

Publications and source records attributed to Graumann, P. L..

6 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↗

bioRxiv2023

A dynamic bactofilin cytoskeleton recruits an M23 endopeptidase to control bacterial morphogenesis

Bactofilins have emerged as a widespread family of cytoskeletal proteins with important roles in bacterial morphogenesis, but their precise mode of action is still incompletely understood. In this study, we identify the bactofilin cytoskeleton as a key regulator of cell growth in the stalked budding alphaproteobacterium Hyphomonas neptunium. We show that, in this species, bactofilin polymers localize dynamically to the stalk base and the bud neck, with their absence leading to unconstrained growth of the stalk and bud compartments, indicating a central role in the spatial regulation of cell wall biosynthesis. Database searches reveal that in a range of different species bactofilin genes are clustered with genes for cell wall hydrolases of the M23 peptidase family, suggesting a functional connection between these two types of proteins. In support of this notion, we find that the H. neptunium M23 peptidase homolog LmdC interacts directly with bactofilin in vitro and is required for proper cell shape in vivo. Complementary studies in the spiral-shaped alphaproteobacterium Rhodospirillum rubrum again reveal a close association of its bactofilin and LmdC homologs, which co-localize at the inner curve of the cell, modulating the degree of cell curvature. Collectively, these findings demonstrate that bactofilins and M23 peptidases form a conserved functional module that promotes local changes in the mode of cell wall biosynthesis, thereby driving cell shape determination in morphologically complex bacteria.

microbiology↗

bioRxiv2022

DipM controls multiple autolysins and mediates two regulatory feedback loops promoting cell constriction in C. crescentus

Proteins containing a catalytically inactive LytM-type endopeptidase domain have emerged as important regulators of cell wall-degrading enzymes in bacteria. Although these so-called LytM factors are wide-spread among species, the range of functions they fulfill and their precise modes of action are still incompletely understood. In this work, we study the LytM factor DipM, a protein required for proper cell division in the model species C. crescentus. We show that the LytM domain of DipM interacts directly with multiple autolysins, including the lytic transglycosylases SdpA and SdpB, the amidase AmiC and the putative carboxypeptidase CrbA, and stimulates the activities of SdpA and AmiC. The crystal structure of the LytM domain of DipM reveals conserved features, including a distinctive groove. Modeling studies suggest that this groove could represent the docking site of AmiC. The architecture of the binding interface in the DipM-AmiC complex is very similar to that observed for the LytM domain of EnvC in complex with its autoinhibitory restraining arm, suggesting a conserved role of the groove in the interaction of LytM factors with their (auto-)regulatory targets. In line with this hypothesis, a mutation in the groove abolishes DipM function. Interestingly, single-molecule tracking studies reveal that the recruitment of DipM and its regulatory targets SdpA and SdpB to the division site is mutually interdependent, with DipM establishing a self-reinforcing cycle that gradually increases lytic transglycosylase activity at the cell center as division progresses. At the same time, the DipM-dependent activation of AmiC leads to the production of denuded peptidoglycan, generating a spatial cue that attracts FtsN to the division site and thus, in turn, again promotes the recruitment of DipM. Collectively, these findings show that DipM is a central regulator that acts at the intersection of different peptidoglycan remodeling pathways and coordinates the activities of various classes of autolysins to promote cell constriction and daughter cell separation.

microbiology↗

bioRxiv2022

Protein secretion zones within the Gram-positive cell wall

Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram positive bacteria, which are widely used for industrial applications. We have studied secretion of -amylase AmyE within two different Bacillus strains. We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches with very low dynamics that are visible at many places within the lateral cell wall, for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term "secretion zones". Zones corresponded to visualized AmyE activity on the surface of cells, and frequently overlapped with SecA signals, but did not necessarily co-localize with the secretion ATPase. Single particle tracking showed much higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA show changes in distinct diffusion states of SecA, but suggest that AmyE overexpression does not overwhelm the system. Because secretion zones were only found during the transition to and within stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside, releases AmyE and thus probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall in Gram positive bacteria.

microbiology↗

bioRxiv2021

A GGDEF domain serves as a spatial on-switch for a phosphodiesterase by direct interaction with a polar landmark protein

In bacteria, the monopolar localization of enzymes and protein complexes can result in a bi-modal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here we show how PdeB polar recruitment is mediated by direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the C-terminal FimV domain of the polar landmark protein HubP. We demonstrate that this interaction is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. We further show that PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations that is generated by differences in abundance and temporal polar appearance of PdeB as well as by bi-modal distribution after cell fission orchestrates the population behavior with respect to cell-surface interaction and environmental spreading. SignificancePhenotypic heterogeneity benefits the proliferation of microbial populations in changing environments. Such heterogeneity can be created by recruitment of enzymatic activity to specific cellular compartments, e.g., the cell pole. Here we show how a GGDEF domain of a multidomain phosphodiesterase has adopted the function as a spatial on-switch that is specifically activated upon direct interaction with a polar landmark protein.

microbiology↗