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All cells must increase their volumes in response to biomass growth to maintain intracellular mass density within physiologically permissive bounds. Here, we investigate the regulation of volume growth in the Gram-positive bacterium Bacillus subtilis. To increase volume, bacteria enzymatically expand their cell envelopes and insert new envelope material. First, we demonstrate that cell-volume growth is determined indirectly, by expanding their envelopes in proportion to mass growth, similarly to the Gram-negative Escherichia coli, despite their fundamentally different envelope structures. Next, we studied, which pathways might be responsible for robust surface-to-mass coupling: We found that both peptidoglycan synthesis and membrane synthesis are required for proper surface-to-mass coupling. However, surprisingly, neither pathway is solely rate-limiting, contrary to wide-spread belief, since envelope growth continues at a reduced rate upon complete inhibition of either process. To arrest cell-envelope growth completely, the simultaneous inhibition of both envelope-synthesis processes is required. Thus, we suggest that multiple envelope-synthesis pathways collectively confer an important aspect of volume regulation, the coordination between surface growth, and biomass growth.

Significance Intracellular biomass density is an important variable for cellular physiology. It defines the crowded state of the cytoplasm and thus influences macromolecular interactions and transport. To control density during growth, bacteria must expand their cell volumes in synchrony with biomass. The regulation of volume growth and biomass density remain fundamentally not understood—in bacteria or any other organism. Using advanced microscopy, we demonstrate that cells control dry-mass density indirectly through two independent processes. First, cells expand surface area, rather than volume, in proportion with biomass growth. Second, cell width is controlled independently, with an important influence of turgor pressure. Our findings overturn a long-standing paradigm of mass-density constancy in bacteria and reveal fundamental determinants of dry-mass density and shape., During growth, cells must expand their cell volumes in coordination with biomass to control the level of cytoplasmic macromolecular crowding. Dry-mass density, the average ratio of dry mass to volume, is roughly constant between different nutrient conditions in bacteria, but it remains unknown whether cells maintain dry-mass density constant at the single-cell level and during nonsteady conditions. Furthermore, the regulation of dry-mass density is fundamentally not understood in any organism. Using quantitative phase microscopy and an advanced image-analysis pipeline, we measured absolute single-cell mass and shape of the model organisms Escherichia coli and Caulobacter crescentus with improved precision and accuracy. We found that cells control dry-mass density indirectly by expanding their surface, rather than volume, in direct proportion to biomass growth—according to an empirical surface growth law. At the same time, cell width is controlled independently. Therefore, cellular dry-mass density varies systematically with cell shape, both during the cell cycle or after nutrient shifts, while the surface-to-mass ratio remains nearly constant on the generation time scale. Transient deviations from constancy during nutrient shifts can be reconciled with turgor-pressure variations and the resulting elastic changes in surface area. Finally, we find that plastic changes of cell width after nutrient shifts are likely driven by turgor variations, demonstrating an important regulatory role of mechanical forces for width regulation. In conclusion, turgor-dependent cell width and a slowly varying surface-to-mass coupling constant are the independent variables that determine dry-mass density.

International audience; Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

Cells exhibit a high degree of intracellular crowding. To control the level of crowding during growth cells must increase their volumes in response to the accumulation of biomass. UsingEscherichia colias a model organism, we found that cells control cell volume indirectly, by increasing cell-surface area in proportion to biomass growth. Thus, dry-mass density, a readout of intracellular crowding, varies in proportion to the surface-to-volume ratio, both during the cell cycle and during perturbations such as nutrient shifts. On long time scales after shifts, initial dry-mass density is nearly restored by slow variations of the surface-to-mass ratio. Contrary to a long-standing paradigm, cell-envelope expansion is controlled independently of cell-wall synthesis but responds to the activity of cell-wall cleaving hydrolases. Finally, we observed rapid changes of Turgor pressure after nutrient shifts, which were likely responsible for initial changes of cell diameter and dry-mass-density. Together, our experiments reveal important regulatory relationships for cell volume and shape.

AbstracCell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shapedEscherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell, the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that class-A PBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level we then demonstrate that PBP1b, the major class-A PBP, contributes to cell-wall integrity by localizing and inserting peptidoglycan in direct response to local cell-wall defects.

Bacteria can adjust the structure of colonies and biofilms to enhance their survival rate under external stress. Here, we explore the link between bacterial interaction forces and colony structure. We show that the activity of extracellular pilus motors enhances local ordering and accelerates fusion dynamics of bacterial colonies. The radial distribution function of mature colonies shows local fluidlike order. The degree and dynamics of ordering are dependent on motor activity. At a larger scale, the fusion dynamics of two colonies shows liquidlike behavior whereby motor activity strongly affects surface tension and viscosity.

International audience; Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which RNA polymerase "kicks out" dCas9 from the target and completes transcription. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. Alternatively, tuning repression by changing dCas9 concentration is noisy and promoter-strength dependent. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto-repressor. Second, we study the impact of amount variations of cell-wall synthesizing enzymes on cell morphology. Finally, we multiplex the system to obtain any combination of fractional repression of two genes.

Extracellular DNA is an important structural component of many bacterial biofilms. It is unknown, however, to which extent external DNA is used to transfer genes by means of transformation. Here, we quantified the acquisition of multidrug resistance and visualized its spread under selective and nonselective conditions in biofilms formed by Neisseria gonorrhoeae . The density and architecture of the biofilms were controlled by microstructuring the substratum for bacterial adhesion. Horizontal transfer of antibiotic resistance genes between cocultured strains, each carrying a single resistance, occurred efficiently in early biofilms. The efficiency of gene transfer was higher in early biofilms than between planktonic cells. It was strongly reduced after 24 h and independent of biofilm density. Pilin antigenic variation caused a high fraction of nonpiliated bacteria but was not responsible for the reduced gene transfer at later stages. When selective pressure was applied to dense biofilms using antibiotics at their MIC, the double-resistant bacteria did not show a significant growth advantage. In loosely connected biofilms, the spreading of double-resistant clones was prominent. We conclude that multidrug resistance readily develops in early gonococcal biofilms through horizontal gene transfer. However, selection and spreading of the multiresistant clones are heavily suppressed in dense biofilms. IMPORTANCE Biofilms are considered ideal reaction chambers for horizontal gene transfer and development of multidrug resistances. The rate at which genes are exchanged within biofilms is unknown. Here, we quantified the acquisition of double-drug resistance by gene transfer between gonococci with single resistances. At early biofilm stages, the transfer efficiency was higher than for planktonic cells but then decreased with biofilm age. The surface topography affected the architecture of the biofilm. While the efficiency of gene transfer was independent of the architecture, spreading of double-resistant bacteria under selective conditions was strongly enhanced in loose biofilms. We propose that while biofilms help generating multiresistant strains, selection takes place mostly after dispersal from the biofilm.

Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which dCas9 successfully blocks the RNA polymerase. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto-repressor. Second, we study the impact of copy-number variations of cell-wall synthesizing enzymes on cell morphology. Finally, we demonstrate that this system can be multiplexed to obtain any combination of fractional repression of two genes.

Cellular positioning towards the surface of bacterial colonies and biofilms can enhance dispersal, provide a selective advantage due to increased nutrient and space availability, or shield interior cells from external stresses. Little is known about the molecular mechanisms that govern bacterial positioning. Using the type IV pilus (T4P) of Neisseria gonorrhoeae, we tested the hypothesis that the processes of phase and antigenic variation govern positioning and thus enhance bacterial fitness in expanding gonococcal colonies. By independently tuning growth rate and T4P-mediated interaction forces, we show that the loss of T4P and the subsequent segregation to the front confers a strong selective advantage. Sequencing of the major pilin gene of the spatially segregated sub-populations and an investigation of the spatio-temporal population dynamics was carried out. Our findings indicate that pilin phase and antigenic variation generate a standing variation of pilin sequences within the inoculation zone, while variants associated with a non-piliated phenotype segregate to the front of the growing colony. We conclude that tuning of attractive forces by phase and antigenic variation is a powerful mechanism for governing the dynamics of bacterial colonies.

Indole is an important biological signalling molecule produced by many Gram positive and Gram negative bacterial species, including Escherichia coli. Here we study the effect of indole on the electrical properties of lipid membranes. Using electrophysiology, we show that two indole molecules act cooperatively to transport charge across the hydrophobic core of the lipid membrane. To enhance charge transport, induced by indole across the lipid membrane, we use an indole derivative, 4 fluoro-indole. We demonstrate parallels between charge transport through artificial lipid membranes and the function of complex eukaryotic membrane systems by showing that physiological indole concentrations increase the rate of mitochondrial oxygen consumption. Our data provide a biophysical explanation for how indole may link the metabolism of bacterial and eukaryotic cells.

Bacterial biofilms can generate micro-heterogeneity in terms of surface structures. However, little is known about the associated changes in the physics of cell–cell interaction and its impact on the architecture of biofilms. In this study, we used the type IV pilus of Neisseria gonorrhoeae to test whether variation of surface structures induces cell-sorting. We show that the rupture forces between pili are fine-tuned by post-translational modification. Bacterial sorting was dependent on pilus post-translational modification and pilus density. Active force generation was necessary for defined morphologies of mixed microcolonies. The observed morphotypes were in remarkable agreement with the differential strength of adhesion hypothesis proposing that a tug-of-war among surface structures of different cells governs cell sorting. We conclude that in early biofilms the density and rupture force of bacterial surface structures can trigger cell sorting based on similar physical principles as in developing embryos. DOI: http://dx.doi.org/10.7554/eLife.10811.001, eLife digest Communities of bacterial cells can live together embedded within a slime-like molecular matrix as a biofilm. This allows the bacteria to hide from external stresses. A single bacterium can replicate itself and develop into a biofilm, and over time the bacterial cells in specific regions of the biofilm will start to interact with their neighbors in different ways. These interactions occur via structures on the surface of the bacterial cells, and the differences in these interactions resemble those that occur as cells specialize during the development of animal embryos. Previous research into embryonic development has shown how differences in the physical interactions between embryonic cells are essential for sorting the cells into their correct locations and shaping the embryo. However, little is known about which processes govern the development of biofilms. Now, Oldewurtel et al. have asked whether differences in the physical interactions between bacteria trigger cell sorting during the early stages of biofilm development. The experiments involved measuring the force required to break the cell–cell connections (called the ‘rupture force’) in biofilms of a bacterium called Neisseria gonorrhoeae. Oldewurtel et al. found that, in agreement with previous predictions, physical interactions were important for sorting bacterial cells into clusters based on the structures on their surfaces. Bacterial cells actively pull on the surface structures of their neighbors, which allows the cells to sort themselves in a tug-of-war fashion. This means that a cell will move in the direction where it can pull the strongest (i.e., in the direction where the rupture force is highest). While bacteria and embryos use different molecules to generate these pulling forces, these findings indicate that the basic physical principles are similar in both systems. One of the next challenges will be to evaluate how biofilms might benefit from the structures that develop due to cell sorting. DOI: http://dx.doi.org/10.7554/eLife.10811.002

Competence for transformation is widespread among bacterial species. In the case of Gram-negative systems, a key step to transformation is the import of DNA across the outer membrane. Although multiple factors are known to affect DNA transport, little is known about the dynamics of DNA import. Here, we characterized the spatio-temporal dynamics of DNA import into the periplasm of Neisseria gonorrhoeae. DNA was imported into the periplasm at random locations around the cell contour. Subsequently, it was recruited at the septum of diplococci at a time scale that increased with DNA length. We found using fluorescent DNA that the periplasm was saturable within minutes with ∼40 kbp DNA. The DNA-binding protein ComE quantitatively governed the carrying capacity of the periplasm in a gene-dosage-dependent fashion. As seen using a fluorescent-tagged derivative protein, ComE was homogeneously distributed in the periplasm in the absence of external DNA. Upon addition of external DNA, ComE was relocalized to form discrete foci colocalized with imported DNA. We conclude that the periplasm can act as a considerable reservoir for imported DNA with ComE governing the amount of DNA stored potentially for transport through the inner membrane., Author Summary Bacterial transformation is the import and inheritable integration of external DNA. As such, it is believed to be a major evolutionary force. A key step is the import of DNA through the outer membrane. Here, we have characterized the spatio-temporal dynamics of DNA during import and residence in the periplasm of the Gram-negative pathogen Neisseria gonorrhoeae. We found that the periplasm can serve as a reservoir for imported DNA that can fill within five minutes by importing DNA from the environment. The amount of imported DNA roughly corresponds to the size of a phage genome. The periplasmic DNA-binding protein ComE is homogeneously distributed in the periplasm in the absence of extracellular DNA. It relocates rapidly to imported DNA when external DNA is added to competent gonococci. As ComE governs the carrying capacity of the periplasm, we propose that it might condense DNA, thus linking DNA uptake to its compaction. Although the import through the outer membrane was localized all around the cell contour, the major part of the imported DNA relocated to the septum at the center of diplococci. Our findings strongly support the idea that the periplasm masses DNA independently of transport through the inner membrane.

Physical interactions and in particular mechanical forces are involved in sorting of different cell types. These requirements have been established in the field of embryonic development [3]. The development of bacterial biofilms resembles eukaryotic development in various aspects [4]. In comparison, the bacterial cell body in our model can be considered as a point-object and the forces of pilus retraction are always attractive.We have tested the hypothesis that differential physical interactions between bacteria cause cell sorting in early biofilms [2]. We generated a toolbox of gonococcal type IV pili varying in number per bacterium and pilus-pilus rupture forces. The morphotypes of mixed microcolonies were in remarkable agreement with the predictions of the differential strength of adhesion hypothesis proposing that cells sort based on differential net rupture forces. Our findings suggest that similar to embryonic development, mechanical forces govern cell sorting in early biofilm formation. Furthermore we demonstrated that oxygen is required for gonococcal microcolony formation by enhancing pilus-pilus interaction [1]. Mechanistically, the strength of pilus-pilus interaction decreases with oxygen depletion and influences the microcolony stability.We conclude that differential cell-cell interactions govern the formation of and sorting within early bacterial biofilms.1.Lena Dewenter∗, Thorsten E. Volkmann and Berenike Maier; (2015) Oxygen governs gonococcal microcolony stability by enhancing the interaction force between type IV pili. Integr. Biol., 10.1039/c5ib00018a2.Enno R Oldewurtel, Nadzeya Kouzel, Lena Dewenter∗, Katja Henseler, Berenike Maier; (2015) Differential interaction forces govern bacterial sorting in early biofilms. eLife, 10.7554/eLife.108113.Gonzalez-Rodriguez D, Guevorkian K, Douezan S,Brochard-Wyart F (2012) Soft matter models of developing tissues and tumors. Science 338: 910-917. 5694.Monds RD, O'Toole GA (2009) The developmental model of microbial biofilms: ten years of a paradigm up for review. Trends in microbiology 17: 73-87.

Twitching motility enables bacteria to move over surfaces using type IV pili as grappling hooks. Here it is shown that the motility of the round Neisseria gonorrhoeae as well as of rod-shaped Myxococcus xanthus is guided by elevations with dimension and depth corresponding to the size of the bacteria.

Various bacterial species carry mutation hotspots on their genomes that act at high frequencies to reversibly switch off the generation of surface molecules. This process of phase variation is believed to be a major adaptive strategy to escape the immune system. However, most new clones are likely to get lost within a population due to the stochastic fluctuations in the early fixation process.Here, we directly visualised the spatio-temporal dynamics of a new clone and its progeny within a growing microcolony of the human pathogen Neisseria gonorrhoeae. At a high rate N. gonorrhoeae switches off the production of a major virulence factor, the type IV pilus. Imitating this, we designed DNA to replace an essential pilus gene with the gene for an egfp reporter and plated it on a surface. Stochastically, individual P+ cells within an expanding colony growing on this surface integrated this DNA into their genome, lost their pili (P-) and became fluorescent.Hardly any clones arising more than six bacterial diameters behind the expanding front fixed. Closer to the front, we found the probability of fixation of P- clones to be strongly increased compared to the probability for neutral P+ clones to reach the front. P- bacteria were expelled from the P+ microcolony. Due to high surface tension of P+ colonies, P- bacteria accumulated at the front of expanding colonies. This could be confirmed by characterising spontaneous mutations from the P+ to the P- state in range expansion assays.We conclude that the reduced physical interaction between P- and P+ cells, leads to an increased probability of fixation for P- gonococci within a microcolony. It is tempting to speculate that gonococci can shield their major antigen, the type IV pilus, by surrounding themselves with P- cells.