Multicellular Communities Research Articles

How bacteria block their own biofilms

Bacterial biofilms are surface-associated multicellular communities that are highly resistant to removal. Scheffler et al. discovered that Pseudomonas aeruginosa secretes a small molecule that inhibits other P. aeruginosa cells from adsorbing to surfaces by interfering with type IV pili dynamics. The inhibition of cell adsorption could present a method to prevent biofilm formation on sensitive surfaces in hospitals and industry.

Identification of signaling pathways, matrix-digestion enzymes, and motility components controlling Vibrio cholerae biofilm dispersal

Bacteria alternate between being free-swimming and existing as members of sessile multicellular communities called biofilms. The biofilm lifecycle occurs in three stages: cell attachment, biofilm maturation, and biofilm dispersal. Vibrio cholerae biofilms are hyperinfectious, and biofilm formation and dispersal are considered central to disease transmission. While biofilm formation is well studied, almost nothing is known about biofilm dispersal. Here, we conducted an imaging screen for V. cholerae mutants that fail to disperse, revealing three classes of dispersal components: signal transduction proteins, matrix-degradation enzymes, and motility factors. Signaling proteins dominated the screen and among them, we focused on an uncharacterized two-component sensory system that we term DbfS/DbfR for dispersal of biofilm sensor/regulator. Phospho-DbfR represses biofilm dispersal. DbfS dephosphorylates and thereby inactivates DbfR, which permits dispersal. Matrix degradation requires two enzymes: LapG, which cleaves adhesins, and RbmB, which digests matrix polysaccharides. Reorientation in swimming direction, mediated by CheY3, is necessary for cells to escape from the porous biofilm matrix. We suggest that these components act sequentially: signaling launches dispersal by terminating matrix production and triggering matrix digestion, and subsequent cell motility permits escape from biofilms. This study lays the groundwork for interventions aimed at modulating V. cholerae biofilm dispersal to ameliorate disease.

Emerging Roles of Functional Bacterial Amyloids in Gene Regulation, Toxicity, and Immunomodulation.

Bacteria often reside in multicellular communities, called biofilms, held together by an extracellular matrix. In many bacteria, the major proteinaceous component of the biofilm are amyloid fibers. Amyloids are highly stable and structured protein aggregates which were known mostly to be associated with neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases. In recent years, microbial amyloids were identified also in other species and shown to play major roles in microbial physiology and virulence. For example, amyloid fibers assemble on the bacterial cell surface as a part of the extracellular matrix and are extremely important to the scaffolding and structural integrity of biofilms, which contribute to microbial resilience and resistance. Furthermore, microbial amyloids play fundamental nonscaffold roles that contribute to the development of biofilms underlying numerous persistent infections. Here, we review several nonscaffold roles of bacterial amyloid proteins, including bridging cells during collective migration, acting as regulators of cell fate, as toxins against other bacteria or against host immune cells, and as modulators of the hosts' immune system. These overall points on the complexity of the amyloid fold in encoding numerous activities, which offer approaches for the development of a novel repertoire of antivirulence therapeutics.

Community effects allow bioelectrical reprogramming of cell membrane potentials in multicellular aggregates: Model simulations.

Bioelectrical patterns are established by spatiotemporal correlations of cell membrane potentials at the multicellular level, being crucial to development, regeneration, and tumorigenesis. We have conducted multicellular simulations on bioelectrical community effects and intercellular coupling in multicellular aggregates. The simulations aim at establishing under which conditions a local heterogeneity consisting of a small patch of cells can be stabilized against a large aggregate of surrounding identical cells which are in a different bioelectrical state. In this way, instructive bioelectrical information can be persistently encoded in spatiotemporal patterns of separated domains with different cell polarization states. The multicellular community effects obtained are regulated both at the single-cell and intercellular levels, and emerge from a delicate balance between the degrees of intercellular coupling in: (i) the small patch, (ii) the surrounding bulk, and (iii) the interface that separates these two regions. The model is experimentally motivated and consists of two generic voltage-gated ion channels that attempt to establish the depolarized and polarized cell states together with coupling conductances whose individual and intercellular different states permit a dynamic multicellular connectivity. The simulations suggest that community effects may allow the reprogramming of single-cell bioelectrical states, in agreement with recent experimental data. A better understanding of the resulting electrical regionalization can assist the electroceutical correction of abnormally depolarized regions initiated in the bulk of normal tissues as well as suggest new biophysical mechanisms for the establishment of target patterns in multicellular engineering.

Metabolic Microenvironments Drive Microbial Differentiation and Antibiotic Resistance

For bacteria, the transition from unicellular entities to multicellular biofilm communities generates distinct metabolic microenvironments. Dynamic and programmed metabolic responses allow the biofilms to react to local changes in nutrient levels. Moreover, metabolic adaptations contribute to phenotypic antibiotic resistance of the community, suggesting novel therapeutic approaches to target biofilms.

Persistent Bacteriuria and Antibodies Recognizing Curli/eDNA Complexes From Escherichia coli Are Linked to Flares in Systemic Lupus Erythematosus.

Infections contribute to morbidity and mortality in systemic lupus erythematosus (SLE). Uropathogenic Escherichia coli (UPEC) are known to trigger urinary tract infections (UTIs) and form biofilms, which are multicellular communities of bacteria that are strengthened by amyloids such as curli. We previously reported that curli naturally form complexes with bacterial extracellular DNA (eDNA), and these curli/eDNA complexes induce hallmark features of lupus in mouse models. The present study was undertaken to investigate whether anti-curli/eDNA complex antibodies play a role in the pathogenesis of SLE or development of flares in SLE. In total, 96 SLE patients who met at least 4 Systemic Lupus International Collaborating Clinics disease criteria were investigated. Anti-curli/eDNA complex antibodies in the plasma were tested for both IgG and IgA subclasses. Results were compared to that in 54 age-, sex-, and race/ethnicity-matched healthy controls. Correlations of the levels of anti-curli/eDNA antibodies with clinical parameters, lupus disease status, and frequency of bacteriuria were assessed. Anti-curli/eDNA antibodies were detected in the plasma of SLE patients and healthy controls, and their levels correlated with the presence of asymptomatic persistent bacteriuria and occurrence of disease flares in lupus patients. Persistent bacteriuria contained curli-producing UPEC, and this was associated with an inflammatory phenotype. Finally, curli/eDNA complexes cross-reacted with lupus autoantigens, such as double-stranded DNA, in binding autoantibodies. These results suggest that UTIs and persistent bacteriuria are environmental triggers of lupus and its flares. Antibodies against curli/eDNA could serve as a sign of systemic exposure to bacterial products in SLE.

Clarithromycin Exerts an Antibiofilm Effect against Salmonella enterica Serovar Typhimurium rdar Biofilm Formation and Transforms the Physiology towards an Apparent Oxygen-Depleted Energy and Carbon Metabolism.

Upon biofilm formation, production of extracellular matrix components and alteration in physiology and metabolism allows bacteria to build up multicellular communities which can facilitate nutrient acquisition during unfavorable conditions and provide protection toward various forms of environmental stresses to individual cells. Thus, bacterial cells within biofilms become tolerant against antimicrobials and the immune system. In the present study, we evaluated the antibiofilm activity of the macrolides clarithromycin and azithromycin. Clarithromycin showed antibiofilm activity against rdar (red, dry, and rough) biofilm formation of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium ATCC 14028 (Nalr) at a 1.56 μM subinhibitory concentration in standing culture and dissolved cell aggregates at 15 μM in a microaerophilic environment, suggesting that the oxygen level affects the activity of the drug. Treatment with clarithromycin significantly decreased transcription and production of the rdar biofilm activator CsgD, with biofilm genes such as csgB and adrA to be concomitantly downregulated. Although fliA and other flagellar regulon genes were upregulated, apparent motility was downregulated. RNA sequencing showed a holistic cell response upon clarithromycin exposure, whereby not only genes involved in the biofilm-related regulatory pathways but also genes that likely contribute to intrinsic antimicrobial resistance, and the heat shock stress response were differentially regulated. Most significantly, clarithromycin exposure shifted the cells toward an apparent oxygen- and energy-depleted status, whereby the metabolism that channels into oxidative phosphorylation was downregulated, and energy gain by degradation of propane 1,2-diol, ethanolamine and l-arginine catabolism, potentially also to prevent cytosolic acidification, was upregulated. This analysis will allow the subsequent identification of novel intrinsic antimicrobial resistance determinants.

Haemophilus influenzae persists in biofilm communities in a smoke-exposed ferret model of COPD.

RationaleNon-typeable Haemophilus influenzae (NTHi) is a common inhabitant of the human nasopharynx and upper airways that can cause opportunistic infections of the airway mucosa including bronchopulmonary infections in patients with chronic obstructive pulmonary disease (COPD). It is clear that opportunistic infections contribute significantly to inflammatory exacerbations of COPD; however, there remains much to be learned regarding specific host and microbial determinants of persistence and/or clearance in this context.MethodsIn this study, we used a recently described ferret model for COPD, in which animals undergo chronic long-term exposure to cigarette smoke, to define host–pathogen interactions during COPD-related NTHi infections.ResultsNTHi bacteria colonised the lungs of smoke-exposed animals to a greater extent than controls, and elicited acute host inflammation and neutrophilic influx and activation, along with a significant increase in airway resistance and a decrease in inspiratory capacity consistent with inflammatory exacerbation; notably, these findings were not observed in air-exposed control animals. NTHi bacteria persisted within multicellular biofilm communities within the airway lumen, as evidenced by immunofluorescent detection of bacterial aggregates encased within a sialylated matrix as is typical of NTHi biofilms and differential bacterial gene expression consistent with the biofilm mode of growth.ConclusionsBased on these results, we conclude that acute infection with NTHi initiates inflammatory exacerbation of COPD disease. The data also support the widely held hypothesis that NTHi bacteria persist within multicellular biofilm communities in the lungs of patients with COPD.

Indole signaling decreases biofilm formation and related virulence of Listeria monocytogenes

Bacterial communication system known as quorum sensing (QS) is a pivotal system for bacterial survival, adaptation and pathogenesis. Members in the multicellular community may synthesize or acquire a signaling molecule in order to elicit downstream cellular processes. Roles of indole and derivatives, a new class of quorum-sensing signal molecules, in various bacterial physiologies and virulence have been reported recently. Indole is normally found in mammal gastrointestinal tract as a metabolite of tryptophan metabolism by microbiota. Therefore, interspecies connection via indole signaling among commensal bacteria and enteric pathogens could be anticipated. Effects of indole exposure on the virulence of Listeria monocytogenes were investigated by phenotypic and molecular approaches. Results demonstrated that synthetic indole and indole-rich conditioned medium significantly diminished biofilm formation and related virulence of L. monocytogenes including motility, cell aggregation and exopolysaccharide production. Transcript levels of virulence-associated (pssE, dltA, flaA, fliI, motB, agrA and hly) and regulatory genes (codY, sigB, prfA and gmaR) were substantially downregulated in indole-treated cells. Only mogR gene encoding for a repressor of motility genes was upregulated after indole exposure. Our findings raise the possibility that L. monocytogenes may acquire indole signaling from gut microbiota for resource-effective adaptation upon transition to new environment.

Endodontic Biofilm - An Enigma to the dentist : A review article

Biofilm can be defined as a sessile multi-cellular microbial community characterized by cells that are firmly attached to a surface and enmeshed in a self produced matrix of extracellular polymeric substances.1,2 These are very prevalent in the apical root canals of teeth with primary and post-treatment apical periodontitis.3 It can be categorised as intracanal biofilms, extra radicular biofilms, periapical biofilms and biomaterial centered infections.4 The infected root canal harbors a polymicrobial population of aerobic, anaerobic, Gram-positive, and Gram-negative bacteria in a biofilm mode of growth. Bacterias involved are E. faecalis, Coagulase–negative Staphylococcus, S. aureus, Streptococci, P.aeruginosa, fungi, F. nucleatum, P. gingivalis, T. forsythensis, Actinomyces species and P. Propionicum. These are commonly isolated by culture, microscopy, immunological methods and molecular biology methods.3 The microbial communities grown in biofilm are remarkably difficult to eradicate with antimicrobial agents. Therefore, different antimicrobials ranging from antimicrobial irrigants to advanced antimicrobial methods such as lasers, photoactivated disinfection, and nanoparticles are employed in the management of infected root canal systems.4

Bacillus Responses to Plant-Associated Fungal and Bacterial Communities.

Some members of root-associated Bacillus species have been developed as biocontrol agents due to their contribution to plant protection by directly interfering with the growth of pathogens or by stimulating systemic resistance in their host. As rhizosphere-dwelling bacteria, these bacilli are surrounded and constantly interacting with other microbes via different types of communications. With this review, we provide an updated vision of the molecular and phenotypic responses of Bacillus upon sensing other rhizosphere microorganisms and/or their metabolites. We illustrate how Bacillus spp. may react by modulating the production of secondary metabolites, such as cyclic lipopeptides or polyketides. On the other hand, some developmental processes, such as biofilm formation, motility, and sporulation may also be modified upon interaction, reflecting the adaptation of Bacillus multicellular communities to microbial competitors for preserving their ecological persistence. This review also points out the limited data available and a global lack of knowledge indicating that more research is needed in order to, not only better understand the ecology of bacilli in their natural soil niche, but also to better assess and improve their promising biocontrol potential.

Strain variability in biofilm formation: A food safety and quality perspective

The inherent differences in microbial behavior among identically treated strains of the same microbial species, referred to as “strain variability”, are regarded as an important source of variability in microbiological studies. Biofilms are defined as the structured multicellular communities with complex architecture that enable microorganisms to grow adhered to abiotic or living surfaces and constitute a fundamental aspect of microbial ecology. The research studies assessing the strain variability in biofilm formation are relatively few compared to the ones evaluating other aspects of microbial behavior such as virulence, growth and stress resistance. Among the available research data on intra-species variability in biofilm formation, compiled and discussed in the present review, most of them refer to foodborne pathogens as compared to spoilage microorganisms. Molecular and physiological aspects of biofilm formation potentially related to strain-specific responses, as well as information on the characterization and quantitative description of this type of biological variability are presented and discussed. Despite the considerable amount of available information on the strain variability in biofilm formation, there are certain data gaps and still-existing challenges that future research should cover and address. Current and future advances in systems biology and omics technologies are expected to aid significantly in the explanation of phenotypic strain variability, including biofilm formation variability, allowing for its integration in microbiological risk assessment.

Enzymatic and Mutational Analysis of the PruA Pteridine Reductase Required for Pterin-Dependent Control of Biofilm Formation in Agrobacterium tumefaciens.

Pterins are ubiquitous biomolecules with diverse functions including roles as cofactors, pigments, and redox mediators. Recently, a novel pterin-dependent signaling pathway that controls biofilm formation was identified in the plant pathogen, Agrobacterium tumefaciens A key player in this pathway is a pteridine reductase termed PruA, where its enzymatic activity has been shown to control surface attachment and limit biofilm formation. Here, we biochemically characterize PruA to investigate the catalytic properties and substrate specificity of this pteridine reductase. PruA demonstrates maximal catalytic efficiency with dihydrobiopterin and comparable activities with the stereoisomers dihydromonapterin and dihydroneopterin. Since A. tumefaciens does not synthesize or utilize biopterins, the likely physiological substrate is dihydromonapterin or dihydroneopterin, or both. Notably, PruA does not exhibit pteridine reductase activity with dihydrofolate or fully oxidized pterins. Site-directed mutagenesis studies of a conserved tyrosine residue, the key component of a putative catalytic triad, indicate that this tyrosine is not directly involved in PruA catalysis but may be important for substrate or cofactor binding. Additionally, mutagenesis of the arginine residue in the N-terminal TGX3RXG motif significantly reduces the catalytic efficiency of PruA, supporting its proposed role in pterin binding and catalysis. Finally, we report the enzymatic characterization of PruA homologs from Pseudomonas aeruginosa and Brucella abortus, thus expanding the roles and potential significance of pteridine reductases in diverse bacteria.Importance Biofilms are complex multicellular communities that are formed by diverse bacteria. In the plant pathogen, Agrobacterium tumefaciens, the transition from a free-living motile state to a non-motile biofilm state is governed by a novel signaling pathway involving small molecules called pterins. The involvement of pterins in biofilm formation is unexpected and prompts many questions about the molecular details of this pathway. This work biochemically characterizes the PruA pteridine reductase involved in the signaling pathway to reveal its enzymatic properties and substrate preference, thus providing important insight into pterin biosynthesis and its role in A. tumefaciens biofilm control. Additionally, the enzymatic characteristics of related pteridine reductases from mammalian pathogens are examined to uncover potential roles of these enzymes in other bacteria.

The extracellular matrix protein TasA is a developmental cue that maintains a motile subpopulation within Bacillus subtilis biofilms.

In nature, bacteria form biofilms-differentiated multicellular communities attached to surfaces. Within these generally sessile biofilms, a subset of cells continues to express motility genes. We found that this subpopulation enabled Bacillus subtilis biofilms to expand on high-friction surfaces. The extracellular matrix (ECM) protein TasA was required for the expression of flagellar genes. In addition to its structural role as an adhesive fiber for cell attachment, TasA acted as a developmental signal stimulating a subset of biofilm cells to revert to a motile phenotype. Transcriptomic analysis revealed that TasA stimulated the expression of a specific subset of genes whose products promote motility and repress ECM production. Spontaneous suppressor mutations that restored motility in the absence of TasA revealed that activation of the biofilm-motility switch by the two-component system CssR/CssS antagonized the TasA-mediated reversion to motility in biofilm cells. Our results suggest that although mostly sessile, biofilms retain a degree of motility by actively maintaining a motile subpopulation.

Dual functionality of the amyloid protein TasA in Bacillus physiology and fitness on the phylloplane

Bacteria can form biofilms that consist of multicellular communities embedded in an extracellular matrix (ECM). In Bacillus subtilis, the main protein component of the ECM is the functional amyloid TasA. Here, we study further the roles played by TasA in B. subtilis physiology and biofilm formation on plant leaves and in vitro. We show that ΔtasA cells exhibit a range of cytological symptoms indicative of excessive cellular stress leading to increased cell death. TasA associates to the detergent-resistant fraction of the cell membrane, and the distribution of the flotillin-like protein FloT is altered in ΔtasA cells. We propose that, in addition to a structural function during ECM assembly and interactions with plants, TasA contributes to the stabilization of membrane dynamics as cells enter stationary phase.

Blue-Light-Switchable Bacterial Cell–Cell Adhesions Enable the Control of Multicellular Bacterial Communities

Although the fundamental importance and biotechnological potential of multibacterial communities, also called biofilms, are well-known, our ability to control them is limited. We present a new way of dynamically controlling bacteria-bacteria adhesions by using blue light and how these photoswitchable adhesions can be used to regulate multicellularity and associated bacterial behavior. To achieve this, the photoswitchable proteins nMagHigh and pMagHigh were expressed on bacterial surfaces as adhesins to allow multicellular clusters to assemble under blue light and reversibly disassemble in the dark. Regulation of the bacterial cell-cell adhesions with visible light provides unique advantages including high spatiotemporal control, tunability, and noninvasive remote regulation. Moreover, these photoswitchable adhesions make it possible to regulate collective bacterial functions including aggregation, quorum sensing, biofilm formation, and metabolic cross-feeding between auxotrophic bacteria with light. Overall, the photoregulation of bacteria-bacteria adhesions provides a new way of studying bacterial cell biology and will enable the design of biofilms for biotechnological applications.

Dynamics of Acetyl Homeostasis in Bacillus subtilis

Bacillus subtilis biofilms are effective models for studying prokaryotic multicellular systems. These biofilms, multicellular bacterial communities encased in a protective polysaccharide and protein matrix, are associated with several bacterial and chronic infections but can also serve beneficial purposes in agriculture and wastewater treatment. Recent investigation into post‐translational protein modification in bacteria has elucidated important regulatory mechanisms for growth and biofilm formation. Protein acetylation has been observed to regulate bacterial multicellularity, and it is closely linked to cellular metabolism by using acetyl metabolites as donors of acetyl groups. The goal of this study is to examine the role of protein lysine acetylation as a regulatory mechanism for metabolism B. subtilis. We hypothesize that bacteria use acetylation as a buffering mechanism for acetyl metabolites to balance levels and prevent metabolite toxicity by increasing acetylation during times of high acetyl metabolite levels and deacetylating proteins to restore metabolites during times of low levels. Wild type bacteria as well as knockout mutants affecting protein acetylation and the carbon overflow pathway are grown in biofilm‐inducing minimal media, and cell and supernatant samples are collected at various ODs. Acetyl‐CoA, acetyl‐phosphate, and acetate levels over the course of growth are measured using fluorometric and colorimetric metabolite test level kits. Dynamics of global lysine acetylation levels are also measured by an anti‐acetyl lysine Western blot. Work so far has focused primarily on optimizing and troubleshooting metabolite assay kits by modifying growth and sample preparation conditions. Acetate dynamics have been measured for the wild type bacteria, showing a continuous secretion of acetate into the supernatant throughout exponential growth. This result is expected, as rapid growth should coincide with continuous utilization of the carbon overflow pathway. Initial Western blots show increased acetylation in the ΔackAΔacuCΔsrtN triple knockout mutant, which is expected due to a buildup of acetyl phosphate and inhibition of deacetylase activity. Future research will also focus on analyzing mutated genes in Δpta suppressor mutants to uncover genes related to acetyl‐CoA‐mediated cell death. Findings from this study will promote the understanding of the regulatory mechanisms behind biofilm formation and potentially uncover novel information about bacterial survival strategies and programmed cell death.Support or Funding InformationThis project was funded by grants from the National Science Foundation, the Northeastern University Honors Program, and the Northeastern University Office of Undergraduate Research and Fellowships.

Acinetobacter baumannii virulence assay in Caenorhabditis elegans

Acinetobacter baumannii is a pathogen that infects hospitalized patients and evades antibiotics by acquiring resistances. It is unclear how A. baumannii infects its host. We used two different strains of A. baumannii, one is the wild type (wt) and the other is a ΔRecA, which we have shown form prominent biofilms. These are multicellular microbial communities encased in protective bacterial produced substances that protects them from antibiotics. We hypothesized that ΔRecA is more virulent than the wt since biofilms increase virulence. To test our hypothesis and better understand the toxicity of A. baumannii, we are developing an assay to test the virulence of the bacteria on the survival of the model organism Caenorhabditis elegans. The assay tests the virulence of A. baumannii using adult C. elegans that have exhausted a lawn of E. coli, picked and placed in a liquid culture containing ~1×108A. baumannii cells and a food dye. The C. elegans are left in this mix for a set amount of time and then deposited onto empty agar plates to allow for easier worm counting. After the liquid from the C. elegans mix has dried out, the survival rates for C. elegans is found. Through these tests, the assay has shown that there is approximately an 80% survival rate for C. elegans picked into an E. coli liquid mix. This result was expected as E. coli does not kill C. elegans. The assay showed there was a 28% survival rate for C. elegans picked into an A. baumannii liquid mix, and a 4% survival rate for C. elegans picked into an A. baumannii ΔRecA liquid mix. These data are in agreement with our hypothesis. To probe on the C. elegans side for what would be the genes responsible for detecting A. baumannii, or other harmful bacteria, we used a daf 7 C. elegans mutant. This strain has a loss of function in a neuron based environment sensing signaling pathway. It has been shown that daf 7 are continuously in a state similar to that of starved wt worms, where feeding rate is slowed down and fat stores are grown. Daf 7 are shown to grow to smaller sizes and to live longer than wt worms. This assay has been repeated with the clean knockout of daf 7 C. elegans, which remarkably and unexpectedly has increased survival rates, by approximately 16‐fold for ΔRecA, when compared to the parental worm. By further understanding the mechanisms underlining A. baumanii virulence, we hope to provide innovative strategies to eliminate this robust bacterium from clinical wards.Support or Funding InformationFunding Provided By Northeastern URFThese photos show C. elegans after the blue dye assay. The blue strips in the C. elegans are their intestines. A: This C. elegans was from a well containing E. coli. B: This C. elegans was from a well containing Ab Wt. C: This C. elegans was from a well containing Ab delta recA.Figure 1The survival rates of C. elegans in the blue dye assay with wt C. elegans in the darker shade and Δdaf 7 C. elegans in the lighter shade. Each bar represents the survival rates for approximately 100 C. elegans. Each survival rate for the wt C. elegans is statistically significant from the other survival rates at a p‐value of 0.05. In Δdaf 7, E. coli and Ab ΔrecA are statistically significant from Ab wt, but not from each other.Figure 2

Characterization of Biofilms Produced by Bacillus Wild Isolates

Many bacteria form complex multicellular communities known as biofilms. Biofilms are a common source of human infections and are associated with chronic infections and with infections resulting from biomedical implants. Bacillus subtilis has been used as a model organism to study biofilms, leading to important insights into how biofilms are regulated and form. However, most research has examined biofilm formation in a single laboratory strain of B. subtilis. We sought to examine differences in biofilm formation between twenty wild isolates that had been published by others to belong to the species B. subtilis. We characterized the biofilm phenotypes of the strains on several types of media and also sequenced the whole genome of each of the strains. We found a wide array of biofilm phenotypes with most being more intense than biofilms of the laboratory strain. Furthermore, biofilm phenotypes and intensity of the biofilm were media‐dependent. Our genetic results indicated that nine of the twenty strains are not actually Bacillus subtilis, but are likely to be a related species, Bacillus velezencis. Interestingly, biofilm strength did not correlate with species. Among the B. subtilis strains, several coding genetic variations were identified in genes important for biofilm formation. Together our study provides insight into variation in biofilm formation among related bacteria and creates a list of possible variations that can be tested for their impact on biofilm formation.Support or Funding InformationResearch was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM135861. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Lack of dormancy to protect diversity: Decrease in diversity of active zooplankton community observed in lake with depauperate egg bank

The study of zooplankton communities in freshwater resources under anthropogenic pressures rarely includes the simultaneous assessment of dormant embryos in bottom sediments and active life-stages in the water column. A coastal lake with a history of coal-ash contamination and disruption by hurricanes provided an ideal opportunity to demonstrate the power of examining both dormant and active zooplankton. The primary objective of this study was to evaluate changes in structure of a multicellular zooplankton community that is under simultaneous pressure from anthropogenic pollution and hurricane-induced flooding. To evaluate change in community structure, the active zooplankton community in 2015 was compared to that observed in 1985. Shannon-Wiener and Simpson indices demonstrate that diversity of the active zooplankton community decreased during this 30-year span. In total, 31% of zooplankton species were lost, and new colonization accounts for 27% of species richness. Dominant species of all major taxonomic groupings changed. Because most zooplankton in freshwater lakes depend on dormant embryos to reestablish active populations after major disruptions, dormant embryos in the sediment “egg bank” were also quantified. Dormant cladoceran ephippia are present in bottom sediments, but dormant copepods and rotifers are missing. The existence of a dormant egg bank that is less diverse than the active community in a freshwater lake is unprecedented, and a depauperate “egg bank” would certainly impair community recovery after severe flooding from hurricanes. It is argued that a paradigm shift is needed in the ecological assessment of inland lakes in order to account for the critical role that dormant embryos (egg banks) play in freshwater zooplankton communities. Two challenges to achieving this are that 1. long-term monitoring is expensive and 2. data on dormant zooplankton are rarely available. This study provides an example of how to conduct such studies by leveraging historic data when long-term monitoring is not possible.