Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron.

BACKGROUND: Ordered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce and the construction methodology remains in early stages of development. RESULTS: Here, we describe the assembly of a genome-scale ordered collection of transposon-insertion mutants in the model gut anaerobe Bacteroides thetaiotaomicron VPI-5482 that we created as a resource for the research community. We used flow cytometry to sort single cells from a pooled library, located mutants within this initial progenitor collection by applying a pooling strategy with barcode sequencing, and re-arrayed specific mutants to create a condensed collection with single-insertion strains covering >2500 genes. To demonstrate the potential of the condensed collection for phenotypic screening, we analyzed growth dynamics and cell morphology. We identified both growth defects and altered cell shape in mutants disrupting sphingolipid synthesis and thiamine scavenging. Finally, we analyzed the process of assembling the B. theta condensed collection to identify inefficiencies that limited coverage. We demonstrate as part of this analysis that the process of assembling an ordered collection can be accurately modeled using barcode sequencing data. CONCLUSION: We expect that utilization of this ordered collection will accelerate research into B. theta physiology and that lessons learned while assembling the collection will inform future efforts to assemble ordered mutant collections for an increasing number of gut microbiota members.

Fatty Acid Synthesis Knockdown Promotes Biofilm Wrinkling and Inhibits Sporulation in Bacillus subtilis.

Many bacterial species typically live in complex three-dimensional biofilms, yet much remains unknown about differences in essential processes between nonbiofilm and biofilm lifestyles. Here, we created a CRISPR interference (CRISPRi) library of knockdown strains covering all known essential genes in the biofilm-forming Bacillus subtilis strain NCIB 3610 and investigated growth, biofilm colony wrinkling, and sporulation phenotypes of the knockdown library. First, we showed that gene essentiality is largely conserved between liquid and surface growth and between two media. Second, we quantified biofilm colony wrinkling using a custom image analysis algorithm and found that fatty acid synthesis and DNA gyrase knockdown strains exhibited increased wrinkling independent of biofilm matrix gene expression. Third, we designed a high-throughput screen to quantify sporulation efficiency after essential gene knockdown; we found that partial knockdowns of essential genes remained competent for sporulation in a sporulation-inducing medium, but knockdown of essential genes involved in fatty acid synthesis exhibited reduced sporulation efficiency in LB, a medium with generally lower levels of sporulation. We conclude that a subset of essential genes are particularly important for biofilm structure and sporulation/germination and suggest a previously unappreciated and multifaceted role for fatty acid synthesis in bacterial lifestyles and developmental processes. IMPORTANCE For many bacteria, life typically involves growth in dense, three-dimensional communities called biofilms that contain cells with differentiated roles held together by extracellular matrix. To examine how essential gene function varies between vegetative growth and the developmental states of biofilm formation and sporulation, we created and screened a comprehensive library of strains using CRISPRi to knockdown expression of each essential gene in the biofilm-capable Bacillus subtilis strain 3610. High-throughput assays and computational algorithms identified a subset of essential genes involved in biofilm wrinkling and sporulation and indicated that fatty acid synthesis plays important and multifaceted roles in bacterial development.

Three-dimensional biofilm colony growth supports a mutualism involving matrix and nutrient sharing.

Life in a three-dimensional biofilm is typical for many bacteria, yet little is known about how strains interact in this context. Here, we created essential gene CRISPR interference knockdown libraries in biofilm-forming Bacillus subtilis and measured competitive fitness during colony co-culture with wild type. Partial knockdown of some translation-related genes reduced growth rates and led to out-competition. Media composition led some knockdowns to compete differentially as biofilm versus non-biofilm colonies. Cells depleted for the alanine racemase AlrA died in monoculture but survived in a biofilm colony co-culture via nutrient sharing. Rescue was enhanced in biofilm colony co-culture with a matrix-deficient parent due to a mutualism involving nutrient and matrix sharing. We identified several examples of mutualism involving matrix sharing that occurred in three-dimensional biofilm colonies but not when cultured in two dimensions. Thus, growth in a three-dimensional colony can promote genetic diversity through sharing of secreted factors and may drive evolution of mutualistic behavior.

Environmental and Physiological Factors Affecting High-Throughput Measurements of Bacterial Growth.

Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth.IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

Biosurfactant-Mediated Membrane Depolarization Maintains Viability during Oxygen Depletion in Bacillus subtilis.

The presence or absence of oxygen in the environment is a strong effector of cellular metabolism and physiology. Like many eukaryotes and some bacteria, Bacillus subtilis primarily utilizes oxygen during respiration to generate ATP. Despite the importance of oxygen for B. subtilis survival, we know little about how populations adapt to shifts in oxygen availability. Here, we find that when oxygen was depleted from stationary phase B. subtilis cultures, ∼90% of cells died while the remaining cells maintained colony-forming ability. We discover that production of the antimicrobial surfactin confers two oxygen-related fitness benefits: it increases aerobic growth yield by increasing oxygen diffusion, and it maintains viability during oxygen depletion by depolarizing the membrane. Strains unable to produce surfactin exhibited an ∼50-fold reduction in viability after oxygen depletion. Surfactin treatment of these cells led to membrane depolarization and reduced ATP production. Chemical and genetic perturbations that alter oxygen consumption or redox state support a model in which surfactin-mediated membrane depolarization maintains viability through slower oxygen consumption and/or a shift to a more reduced metabolic profile. These findings highlight the importance of membrane potential in regulating cell physiology and growth, and demonstrate that antimicrobials that depolarize cell membranes can benefit cells when the terminal electron acceptor in respiration is limiting. This foundational knowledge has deep implications for environmental microbiology, clinical anti-bacterial therapy, and industrial biotechnology.

Who's Your DadA? d-Alanine Levels Regulate Bacterial Stiffness.

A central question in mechanobiology is how cellular-scale structures are established and regulated. In bacteria, the cell envelope is essential for mechanical integrity, protecting against environmental stresses and bearing the load from high turgor pressures. Trivedi et al. (mBio 9:e01340-18, 2018, https://doi.org/10.1128/mBio.01340-18) screened a Pseudomonas aeruginosa transposon library and identified genes that influence cell stiffness by measuring cell growth while cells were embedded in an agarose gel. Their findings provide a broad knowledge base for how biochemical pathways regulate cellular mechanical properties in this pathogen. Dozens of genes across diverse functional categories were implicated, suggesting that cellular mechanics is a systems-level emergent property. Furthermore, changes in d-alanine levels in a dadA (d-alanine dehydrogenase) mutant resulted in decreases in the expression of cell wall enzymes, cross-linking density, and cell stiffness. These insights into the biochemical and mechanical roles of dadA highlight the importance of systems-level investigations into the physical properties of cells.

Species-Independent Attraction to Biofilms through Electrical Signaling.

Bacteria residing within biofilm communities can coordinate their behavior through cell-to-cell signaling. However, it remains unclear if these signals can also influence the behavior of distant cells that are not part of the community. Using a microfluidic approach, we find that potassium ion channel-mediated electrical signaling generated by a Bacillus subtilis biofilm can attract distant cells. Integration of experiments and mathematical modeling indicates that extracellular potassium emitted from the biofilm alters the membrane potential of distant cells, thereby directing their motility. This electrically mediated attraction appears to be a generic mechanism that enables cross-species interactions, as Pseudomonas aeruginosa cells also become attracted to the electrical signal released by the B. subtilis biofilm. Cells within a biofilm community can thus not only coordinate their own behavior but also influence the behavior of diverse bacteria at a distance through long-range electrical signaling. PAPERCLIP.

Mutations in the bacterial cell division protein FtsZ highlight the role of GTP binding and longitudinal subunit interactions in assembly and function.

BACKGROUND: Assembly of the tubulin-like GTPase, FtsZ, at the future division site initiates the process of bacterial cytokinesis. The FtsZ ring serves as a platform for assembly of the division machinery and constricts at the leading edge of the invaginating septum during cytokinesis. In vitro, FtsZ assembles in a GTP-dependent manner, forming straight filaments that curve upon GTP hydrolysis. FtsZ binds but cannot hydrolyze GTP as a monomer. Instead, the active site for GTP hydrolysis is formed at the monomer-monomer interface upon dimerization. While the dynamics of GTP hydrolysis and assembly have been extensively studied in vitro, significantly less is known about the role of GTP binding and hydrolysis in vivo. ftsZ84, a GTPase defective allele of Escherichia coli ftsZ, provides a striking example of the disconnect between in vivo and in vitro FtsZ assembly. RESULTS: Although ftsZ84 mutants are defective for FtsZ ring formation and division under nonpermissive conditions, they are near wild type for ring formation and division under permissive conditions. In vitro, however, purified FtsZ84 is defective in GTP binding, hydrolysis and assembly under standard reaction conditions. To clarify the nature of the FtsZ84 assembly defect, we isolated and characterized three intragenic suppressors of ftsZ84. All three suppressor mutations increased the apparent affinity of FtsZ84 for GTP, consistent with improved subunit-subunit interactions along the longitudinal interface. Although kinetic analysis indicates that the suppressor mutations increase the affinity of FtsZ84 for GTP, all three exhibit reduced rates of GTP hydrolysis and fail to support assembly in vitro. CONCLUSION: Together, our data suggest that FtsZ, and potentially other enzymes whose assembly is similarly regulated, can compensate for defects in catalysis through increases in substrate binding and subunit-subunit interactions. In addition, these results highlight the dichotomy between commonly used in vitro assembly conditions and FtsZ ring formation in the complex intracellular milieu.

Failsafe mechanisms couple division and DNA replication in bacteria.

The past 20 years have seen tremendous advances in our understanding of the mechanisms underlying bacterial cytokinesis, particularly the composition of the division machinery and the factors controlling its assembly [1]. At the same time, we understand very little about the relationship between cell division and other cell-cycle events in bacteria. Here we report that inhibiting division in Bacillus subtilis and Staphylococcus aureus quickly leads to an arrest in the initiation of new rounds of DNA replication, followed by a complete arrest in cell growth. Arrested cells are metabolically active but are unable to initiate new rounds of either DNA replication or division when shifted to permissive conditions. Inhibiting DNA replication results in entry into a similar quiescent state in which cells are unable to resume growth or division when returned to permissive conditions. Our data suggest the presence of two failsafe mechanisms: one linking division to the initiation of DNA replication and another linking the initiation of DNA replication to division. These findings contradict the prevailing view of the bacterial cell cycle as a series of coordinated but uncoupled events. Importantly, the terminal nature of the cell-cycle arrest validates the bacterial cell-cycle machinery as an effective target for antimicrobial development.