scholarly journals Control of Bacillus subtilis Replication Initiation during Physiological Transitions and Perturbations

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
John T. Sauls ◽  
Sarah E. Cox ◽  
Quynh Do ◽  
Victoria Castillo ◽  
Zulfar Ghulam-Jelani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bacillus Subtilis ◽  
Steady State ◽  
Single Cell ◽  
Cell Size ◽  
Physiological Parameters ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
E Coli

ABSTRACT Bacillus subtilis and Escherichia coli are evolutionarily divergent model organisms whose analysis has enabled elucidation of fundamental differences between Gram-positive and Gram-negative bacteria, respectively. Despite their differences in cell cycle control at the molecular level, the two organisms follow the same phenomenological principle, known as the adder principle, for cell size homeostasis. We thus asked to what extent B. subtilis and E. coli share common physiological principles in coordinating growth and the cell cycle. We measured physiological parameters of B. subtilis under various steady-state growth conditions with and without translation inhibition at both the population and single-cell levels. These experiments revealed core physiological principles shared between B. subtilis and E. coli. Specifically, both organisms maintain an invariant cell size per replication origin at initiation, under all steady-state conditions, and even during nutrient shifts at the single-cell level. Furthermore, the two organisms also inherit the same “hierarchy” of physiological parameters. On the basis of these findings, we suggest that the basic principles of coordination between growth and the cell cycle in bacteria may have been established early in evolutionary history. IMPORTANCE High-throughput, quantitative approaches have enabled the discovery of fundamental principles describing bacterial physiology. These principles provide a foundation for predicting the behavior of biological systems, a widely held aspiration. However, these approaches are often exclusively applied to the best-known model organism, E. coli. In this report, we investigate to what extent quantitative principles discovered in Gram-negative E. coli are applicable to Gram-positive B. subtilis. We found that these two extremely divergent bacterial species employ deeply similar strategies in order to coordinate growth, cell size, and the cell cycle. These similarities mean that the quantitative physiological principles described here can likely provide a beachhead for others who wish to understand additional, less-studied prokaryotes.

scholarly journals Gram-positive and Gram-negative Bacteria Share Common Principles to Coordinate Growth and the Cell Cycle at the Single-cell Level

2019 ◽  
Author(s):  
John T. Sauls ◽  
Sarah E. Cox ◽  
Quynh Do ◽  
Victoria Castillo ◽  
Zulfar Ghulam-Jelani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Cell Size ◽  
Physiological Parameters ◽  
Gram Negative Bacteria ◽  
Single Cell Level ◽  
Cell Level ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli

Bacillus subtilis and Escherichia coli are evolutionarily divergent model organisms that have elucidated fundamental differences between Gram-positive and Gram-negative bacteria, respectively. Despite their differences in cell cycle control at the molecular level, both organisms follow the same phenomenological principle for cell size homeostasis known as the adder. We thus asked to what extent B. subtilis and E. coli share common physiological principles in coordinating growth and the cell cycle. To answer this question, we measured physiological parameters of B. subtilis under various steady-state growth conditions with and without translation inhibition at both population and single-cell level. These experiments revealed core shared physiological principles between B. subtilis and E. coli. Specifically, we show that both organisms maintain an invariant cell size per replication origin at initiation, with and without growth inhibition, and even during nutrient shifts at the single-cell level. Furthermore, both organisms also inherit the same “hierarchy” of physiological parameters ranked by their coefficient of variation. Based on these findings, we suggest that the basic coordination principles between growth and the cell cycle in bacteria may have been established in the very early stages of evolution.

scholarly journals Organization of the Flagellar Switch Complex ofBacillus subtilis

2018 ◽  
Vol 201 (8) ◽  
Author(s):  
Elizabeth Ward ◽  
Eun A Kim ◽  
Joseph Panushka ◽  
Tayson Botelho ◽  
Trevor Meyer ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interaction ◽  
Protein Complex ◽  
Cross Linking ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Middle Domain ◽  
Flagellar Rotation

ABSTRACTWhile the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied inEscherichia coliandSalmonella, less is known about the switch complex inBacillus subtilisor other Gram-positive species. Two component proteins (FliG and FliM) are shared betweenE. coliandB. subtilis, but in place of the protein FliN found inE. coli, theB. subtiliscomplex contains the larger protein FliY. Notably, inB. subtilisthe signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action inE. coli. Here, we have examined the architecture and function of the switch complex inB. subtilisusing targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, theB. subtilisswitch complex appears to be organized similarly to that inE. coli. The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that ofE. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences fromE. coliinvolve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCEFlagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex inBacillus subtilisor other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas inE. coliorSalmonellaCheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of theB. subtilisswitch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control inB. subtilisand other Gram-positive species.

scholarly journals A Novel Mobilizing Tool Based on the Conjugative Transfer System of the IncM Plasmid pCTX-M3

2020 ◽  
Vol 86 (17) ◽  
Author(s):  
Michał Dmowski ◽  
Izabela Kern-Zdanowicz
Keyword(s):  
Bacillus Subtilis ◽  
Lactococcus Lactis ◽  
Conjugative Transfer ◽  
Conjugation System ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Mobilizable Plasmid

ABSTRACT Conjugative plasmids are the main players in horizontal gene transfer in Gram-negative bacteria. DNA transfer tools constructed on the basis of such plasmids enable gene manipulation even in strains of clinical or environmental origin, which are often difficult to work with. The conjugation system of the IncM plasmid pCTX-M3 isolated from a clinical strain of Citrobacter freundii has been shown to enable efficient mobilization of oriTpCTX-M3-bearing plasmids into a broad range of hosts comprising Alpha-, Beta-, and Gammaproteobacteria. We constructed a helper plasmid, pMOBS, mediating such mobilization with an efficiency up to 1,000-fold higher than that achieved with native pCTX-M3. We also constructed Escherichia coli donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator (orf35) of the pCTX-M3 tra genes, and S25 and S26, devoid of two putative regulators (orf35 and orf36) of the pCTX-M3 tra genes. Strains S14 and S15 and strains S25 and S26 are, respectively, up to 100 and 1,000 times more efficient in mobilization than pCTX-M3. Moreover, they also enable plasmid mobilization into the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis. Additionally, the constructed E. coli strains carried no antibiotic resistance genes that are present in pCTX-M3 to facilitate manipulations with antibiotic-resistant recipient strains, such as those of clinical origin. To demonstrate possible application of the constructed tool, an antibacterial conjugation-based system was designed. Strain S26 was used for introduction of a mobilizable plasmid coding for a toxin, resulting in the elimination of over 90% of recipient E. coli cells. IMPORTANCE The conjugation of donor and recipient bacterial cells resulting in conjugative transfer of mobilizable plasmids is the preferred method enabling the introduction of DNA into strains for which other transfer methods are difficult to establish (e.g., clinical strains). We have constructed E. coli strains carrying the conjugation system of the IncM plasmid pCTX-M3 integrated into the chromosome. To increase the mobilization efficiency up to 1,000-fold, two putative regulators of this system, orf35 and orf36, were disabled. The constructed strains broaden the repertoire of tools for the introduction of DNA into the Gram-negative Alpha-, Beta-, and Gammaproteobacteria, as well as into Gram-positive bacteria such as Bacillus subtilis and Lactococcus lactis. The antibacterial procedure based on conjugation with the use of the orf35- and orf36-deficient strain lowered the recipient cell number by over 90% owing to the mobilizable plasmid-encoded toxin.

scholarly journals Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Robert S. Brzozowski ◽  
Brooke R. Tomlinson ◽  
Michael D. Sacco ◽  
Judy J. Chen ◽  
Anika N. Ali ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Cell Size ◽  
Unknown Function ◽  
Suppressor Mutation ◽  
Gram Positive ◽  
Content Type ◽  
Suppressor Mutations ◽  
Extragenic Suppressor ◽  
Cell Division Inhibition

ABSTRACT Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

scholarly journals Sinorhizobium meliloti, a Slow-Growing Bacterium, Exhibits Growth Rate Dependence of Cell Size under Nutrient Limitation

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Xiongfeng Dai ◽  
Zichu Shen ◽  
Yiheng Wang ◽  
Manlu Zhu
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Cell Size ◽  
Sinorhizobium Meliloti ◽  
Cell Cycle Progression ◽  
Bacterial Species ◽  
Progression Rate ◽  
Content Type ◽  
E Coli ◽  
Slow Growing

ABSTRACTBacterial cells need to coordinate the cell cycle with biomass growth to maintain cell size homeostasis. For fast-growing bacterial species likeEscherichia coliandBacillus subtilis, it is well-known that cell size exhibits a strong dependence on the growth rate under different nutrient conditions (known as the nutrient growth law). However, cell size changes little with slow growth (doubling time of >90 min) forE. coli, posing the interesting question of whether slow-growing bacteria species also observe the nutrient growth law. Here, we quantitatively characterize the cell size and cell cycle parameter of a slow-growing bacterium,Sinorhizobium meliloti, at different nutrient conditions. We find thatS. melilotiexhibits a threefold change in its cell size when its doubling time varies from 2 h to 6 h. Moreover, the progression rate of its cell cycle is much longer than that ofE. coli, suggesting a delicate coordination between the cell cycle progression rate and the biomass growth rate. Our study shows that the nutrient growth law holds robustly regardless of the growth capacity of the bacterial species, generalizing its applicability among the bacterial kingdom.IMPORTANCEThe dependence of cell size on growth rate is a fundamental principle in the field of bacterial cell size regulation. Previous studies of cell size regulation mainly focus on fast-growing bacterial species such asEscherichia coliandBacillussubtilis. We find here thatSinorhizobium meliloti, a slow-growing bacterium, exhibits a remarkable growth rate-dependent cell size pattern under nutrient limitation, generalizing the applicability of the empirical nutrient growth law of cell size. Moreover,S. melilotiexhibits a much slower speed of cell cycle progression thanE. colidoes, suggesting a delicate coordination between the cell cycle progression rate and the biomass growth rate.

Photocatalytic Inactivation of Bioaerosols by TiO2 Coated Membrane

2005 ◽  
Vol 3 (1) ◽  
Author(s):  
Amrita Pal ◽  
Xu Min ◽  
Liya E Yu ◽  
Simo O Pehkonen ◽  
Madhumita B. Ray
Keyword(s):  
Bacillus Subtilis ◽  
Large Scale ◽  
Indoor Air Pollution ◽  
Bacterial Species ◽  
Public Health Problem ◽  
Continuous Systems ◽  
Gram Positive ◽  
Gram Negative ◽  
Tropical Regions ◽  
E Coli

Indoor air pollution by microbial contaminants is increasingly receiving attention as a public health problem. Under a suitable environment, such as in heating, ventilation and air conditioning (HVAC) system, airborne bacteria are able to proliferate and grow causing various allergies and illnesses. This can be particularly serious in tropical regions due to high relative humidity and warm temperatures all round the year. Application of photocatalysis using UV-A and TiO2 to inactivate air-borne bacteria is relatively new and systematic parametric study is required for the engineering design of a process based on this technology. This study investigates the effects of TiO2 mediated inactivation of various bacterial species in batch and continuous systems using different TiO2 loadings and radiation intensities. Gram-negative bacteria, E. coli and two Gram-positive bacteria, Microbacterium sp. and Bacillus subtilis were used for the inactivation studies. In both systems, inactivation rates of Gram-negative E. coli are higher than the Gram-positive Bacillus subtilis and Microbacterium sp. and the inactivation rates increased in presence of TiO2 for all bacteria. Depending on the type of bacteria, TiO2 loading and light intensity, an increase of 1.3-5.8 times in the inactivation rates was obtained from those in the absence of TiO2. The inactivation rates in the batch and continuous systems were reasonably comparable. Inactivation rates in the continuous system are somewhat higher than those in the batch system due to the unaccounted loss of bacteria via adsorption and settling on the reactor walls in the flow system. The study demonstrates an approach that can be used for the designing of large scale systems for the treatment of bioaerosol.

scholarly journals A Bacteriophage Capsid Protein Is an Inhibitor of a Conserved Transcription Terminator of Various Bacterial Pathogens

2017 ◽  
Vol 200 (1) ◽  
Author(s):  
Gairika Ghosh ◽  
Jayavardhana Reddy ◽  
Susmit Sambhare ◽  
Ranjan Sen
Keyword(s):  
Capsid Protein ◽  
Antibiotic Treatment ◽  
Rho Proteins ◽  
Gram Positive ◽  
Transcription Terminator ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Bacteriophage P4

ABSTRACTRho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibitsEscherichia coliRho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, includingMycobacterium smegmatis,Mycobacterium bovis,Mycobacterium tuberculosis,Xanthomonas campestris,Xanthomonas oryzae,Corynebacterium glutamicum,Vibrio cholerae,Salmonella enterica, andPseudomonas syringae. The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from theE. colitranscription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins.In vivopulldown assays revealed direct binding of Psu with many of these Rho proteins.In vivoexpression ofpsuinduced killing ofM. smegmatis,M. bovis,X. campestris, andE. coliexpressingS. entericaRho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the “universal” inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions.IMPORTANCEBacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of theE. colitranscription terminator Rho. Here we show that apart from antagonizingE. coliRho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psuin vivokills various pathogens, such asMycobacteriumandXanthomonasspecies. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens.

scholarly journals Cationized Magnetoferritin Enables Rapid Labeling and Concentration of Gram-Positive and Gram-Negative Bacteria in Magnetic Cell Separation Columns

2016 ◽  
Vol 82 (12) ◽  
pp. 3599-3604 ◽  
Author(s):  
S. Correia Carreira ◽  
J. Spencer ◽  
W. Schwarzacher ◽  
A. M. Seddon
Keyword(s):  
Magnetic Nanoparticles ◽  
Cell Separation ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Magnetic Cell Separation ◽  
Magnetic Labeling ◽  
Separation Columns

ABSTRACTIn order to identify pathogens rapidly and reliably, bacterial capture and concentration from large sample volumes into smaller ones are often required. Magnetic labeling and capture of bacteria using a magnetic field hold great promise for achieving this goal, but the current protocols have poor capture efficiency. Here, we present a rapid and highly efficient approach to magnetic labeling and capture of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria using cationized magnetoferritin (cat-MF). Magnetic labeling was achieved within a 1-min incubation period with cat-MF, and 99.97% of the labeled bacteria were immobilized in commercially available magnetic cell separation (MACS) columns. Longer incubation times led to more efficient capture, withS. aureusbeing immobilized to a greater extent thanE. coli. Finally, low numbers of magnetically labeledE. colibacteria (<100 CFU per ml) were immobilized with 100% efficiency and concentrated 7-fold within 15 min. Therefore, our study provides a novel protocol for rapid and highly efficient magnetic labeling, capture, and concentration of both Gram-positive and Gram-negative bacteria.IMPORTANCEAntimicrobial resistance (AMR) is a significant global challenge. Rapid identification of pathogens will retard the spread of AMR by enabling targeted treatment with suitable agents and by reducing inappropriate antimicrobial use. Rapid detection methods based on microfluidic devices require that bacteria are concentrated from large volumes into much smaller ones. Concentration of bacteria is also important to detect low numbers of pathogens with confidence. Here, we demonstrate that magnetic separation columns capture small amounts of bacteria with 100% efficiency. Rapid magnetization was achieved by exposing bacteria to cationic magnetic nanoparticles, and magnetized bacteria were concentrated 7-fold inside the column. Thus, bacterial capture and concentration were achieved within 15 min. This approach could be extended to encompass the capture and concentration of specific pathogens, for example, by functionalizing magnetic nanoparticles with antibodies or small molecule probes.

scholarly journals Functional Implementation of the Posttranslational SecB-SecA Protein-Targeting Pathway in Bacillus subtilis

2011 ◽  
Vol 78 (3) ◽  
pp. 651-659 ◽  
Author(s):  
Liuyang Diao ◽  
Qilei Dong ◽  
Zhaohui Xu ◽  
Sheng Yang ◽  
Jiahai Zhou ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Targeting ◽  
Heterologous Proteins ◽  
Gram Positive ◽  
Content Type ◽  
E Coli ◽  
New Strategy ◽  
Secretory Production ◽  
Close Relatives

ABSTRACTBacillus subtilisand its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria,B. subtilisdoes not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway intoB. subtilisby coexpressing SecB fromEscherichia colitogether with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of theB. subtilisSecA were replaced by the corresponding part of SecA fromE. coli.In vitropulldown experiments showed that, in contrast toB. subtilisSecA, the hybrid SecA protein gained the ability to efficiently bind toE. coliSecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins byB. subtiliswas significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.

scholarly journals Single-Cell Microscopy Reveals That Levels of Cyclic di-GMP Vary amongBacillus subtilisSubpopulations

2019 ◽  
Vol 201 (16) ◽  
Author(s):  
Cordelia A. Weiss ◽  
Jakob A. Hoberg ◽  
Kuanqing Liu ◽  
Benjamin P. Tu ◽  
Wade C. Winkler
Keyword(s):  
Bacillus Subtilis ◽  
Single Cell ◽  
Relative Abundance ◽  
Biofilm Formation ◽  
Single Cells ◽  
Cell Types ◽  
Population Heterogeneity ◽  
Competent Cell ◽  
Gram Positive ◽  
Content Type

ABSTRACTThe synthesis of signaling molecules is one strategy bacteria employ to sense alterations in their environment and rapidly adjust to those changes. In Gram-negative bacteria, bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile state. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. To that end, we constructed a fluorescentyfpreporter based on a c-di-GMP-responsive riboswitch to visualize the relative abundance of c-di-GMP for single cells of the Gram-positive model organismBacillus subtilis. Coupled with cell-type-specific fluorescent reporters, this riboswitch reporter revealed that c-di-GMP levels are markedly different amongB. subtiliscellular subpopulations. For example, cells that have made the decision to become matrix producers maintain higher intracellular c-di-GMP concentrations than motile cells. Similarly, we find that c-di-GMP levels differ between sporulating and competent cell types. These results suggest that biochemical measurements of c-di-GMP abundance are likely to be inaccurate for a bulk ensemble ofB. subtiliscells, as such measurements will average c-di-GMP levels across the population. Moreover, the significant variation in c-di-GMP levels between cell types hints that c-di-GMP might play an important role duringB. subtilisbiofilm formation. This study therefore emphasizes the importance of using single-cell approaches for analyzing metabolic trends within ensemble bacterial populations.IMPORTANCEMany bacteria have been shown to differentiate into genetically identical yet morphologically distinct cell types. Such population heterogeneity is especially prevalent among biofilms, where multicellular communities are primed for unexpected environmental conditions and can efficiently distribute metabolic responsibilities.Bacillus subtilisis a model system for studying population heterogeneity; however, a role for c-di-GMP in these processes has not been thoroughly investigated. Herein, we introduce a fluorescent reporter, based on a c-di-GMP-responsive riboswitch, to visualize the relative abundance of c-di-GMP for singleB. subtiliscells. Our analysis shows that c-di-GMP levels are conspicuously different amongB. subtiliscellular subtypes, suggesting a role for c-di-GMP during biofilm formation. These data highlight the utility of riboswitches as tools for imaging metabolic changes within individual bacterial cells. Analyses such as these offer new insight into c-di-GMP-regulated phenotypes, especially given that other biofilms also consist of multicellular communities.

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