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Bacillus cereus is a food-berne pathogen and a frequent contaminant of food production plants. The persistence of this pathogen in various environments results from the formation of spores and of biofilms. To investigate the role of the B. cereus flagellar apparatus in biofilm formation, we constructed a non-flagellated mutant and a flagellated but non-motile mutant. Unexpectedly, we found that the presence of flagella decreased the adhesion of the bacterium to g ass surfaces. We hypothesize that this decrease is a consequence of the flagella hindering a direct interaction between the bacterial cell wall and the surface. In contrast, in specific conditions, motility promotes biofilm formation. Our results suggest that motility could influence biofilm formation by three mechanisms. Motility is necessary for the bacteria to reach surfaces suitable for biofilm formation, In static conditions, reaching the air--liquid interface, where the biofilm forms, is a strong requirement, whereas in flow cells bacteria can have access to the bosom glass slide by sedimentation. Therefore, motility is important for biofilm formation in glass tubes and in microtitre plates, but not in flew cells. Motility also promotes recruitment of planktonic cells within the biofilm by allowing motile bacteria to invade the whole biofilm, Finally, motility is involved in the spreading of the biofilm on glass surfaces. DOI 10.1099/mic.0.034827-0

Bacillus subtilis is a widely used bacterial model to decipher biofilm formation, genetic determinants and their regulation. For several years, studies were conducted on colonies or pellicles formed at the interface with air, but more recent works showed that non-domesticated strains were able to form thick and structured biofilms on submerged surfaces. Taking advantage of time-lapse confocal laser scanning microscopy, we monitored bacterial colonization on the surface and observed an unexpected biphasic submerged biofilm development. Cells adhering to the surface firstly form elongated chains before being suddenly fragmented and released as free motile cells in the medium. This switching coincided with an oxygen depletion in the well which preceded the formation of the pellicle at the liquid-air interface. Residual bacteria still associated with the solid surface at the bottom of the well started to express matrix genes under anaerobic metabolism to build the typical biofilm protruding structures., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Interspecific interactions within biofilms determine relative species abundance, growth dynamics, community resilience, and success or failure of invasion by an extraneous organism. However, deciphering interspecific interactions and assessing their contribution to biofilm properties and function remain a challenge. Here, we describe the constitution of a model biofilm composed of four bacterial species belonging to four different genera (Rhodocyclus sp., Pseudomonas fluorescens, Kocuria varians, and Bacillus cereus), derived from a biofilm isolated from an industrial milk pasteurization unit. We demonstrate that the growth dynamics and equilibrium composition of this biofilm are highly reproducible. Based on its equilibrium composition, we show that the establishment of this four-species biofilm is highly robust against initial, transient perturbations but less so towards continuous perturbations. By comparing biofilms formed from different numbers and combinations of the constituent species and by fitting a growth model to the experimental data, we reveal a network of dynamic, positive, and negative interactions that determine the final composition of the biofilm. Furthermore, we reveal that the molecular determinant of one negative interaction is the thiocillin I synthesized by the B. cereus strain, and demonstrate its importance for species distribution and its impact on robustness by mutational analysis of the biofilm ecosystem., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

During biofilm growth, the coexistence of planktonic and sessile cells can lead to dynamic exchanges between the two populations. We have monitored the fate of these populations in glass tube assays, where the Bacillus thuringiensis 407 strain produces a floating pellicle. Time-lapse spectrophotometric measurement methods revealed that the planktonic population grew until the pellicle started to be produced. Thereafter, the planktonic population decreased rapidly down to a value close to zero while the biofilm was in continuous growth, showing no dispersal until 120 h of culture. We found that this decrease was induced by the presence of the pellicle, but did not occur when oxygen availability was limited, suggesting that it was independent of cell death or cell sedimentation and that the entire planktonic population has integrated the biofilm. To follow the distribution of recruited planktonic cells within the pellicle, we tagged planktonic cells with GFP and sessile cells with mCherry. Fluorescence binocular microscopy observations revealed that planktonic cells, injected through a 24-h-aged pellicle, were found only in specific areas of the biofilm, where the density of sessile cells was low, showing that spatial heterogeneity can occur between recruited cells and sessile cells in a monospecies biofilm.

The extracellular biofilm matrix often contains a network of amyloid fibers which, in the human opportunistic pathogen Bacillus cereus, includes the two homologous proteins TasA and CalY. We show here, in the closely related entomopathogenic species Bacillus thuringiensis, that CalY also displays a second function. In the early stationary phase of planktonic cultures, CalY was located at the bacterial cell-surface, as shown by immunodetection. Deletion of calY revealed that this protein plays a major role in adhesion to HeLa epithelial cells, to the insect Galleria mellonella hemocytes and in the bacterial virulence against larvae of this insect, suggesting that CalY is a cell-surface adhesin. In mid-stationary phase and in biofilms, the location of CalY shifted from the cell surface to the extracellular medium, where it was found as fibers. The transcription study and the deletion of sipW suggested that CalY change of location is due to a delayed activity of the SipW signal peptidase. Using purified CalY, we found that the protein polymerization occurred only in the presence of cell-surface components. CalY is, therefore, a bifunctional protein, which switches from a cell-surface adhesin activity in early stationary phase, to the production of fibers in mid-stationary phase and in biofilms., (© 2018 John Wiley & Sons Ltd.)

Bacillus velezensis QST713 is widely used as a biological control agent for crop protection and disease suppression. This strain is used industrially in France for the protection of Agaricus bisporus against Trichoderma aggressivum f. europaeum , which causes green mold disease. The efficacy of this biocontrol process was evaluated in a previous study, yet the mode of its action has not been explored under production conditions. In order to decipher the underlying biocontrol mechanisms for effective biofilm formation by strain QST713 in the compost and for the involvement of antimicrobial compounds, we developed a simplified micromodel for the culture of A. bisporus during its early culture cycle. By using this micromodel system, we studied the transcriptional response of strain QST713 in the presence or absence of A. bisporus and/or T. aggressivum in axenic industrial compost. We report the overexpression of several genes of the biocontrol agent involved in biofilm formation in the compost compared to their expression during growth in broth compost extract either in the exponential growth phase (the epsC , blsA , and tapA genes) or in the stationary growth phase (the tapA gene), while a gene encoding a flagellar protein ( hag ) was underexpressed. We also report the overexpression of Bacillus velezensis QST713 genes related to surfactin ( srfAA ) and fengycin ( fenA ) production in the presence of the fungal pathogen in the compost. IMPORTANCE Biocontrol agents are increasingly used to replace chemical pesticides to prevent crop diseases. In the button mushroom field in France, the use of Bacillus velezensis QST713 as a biocontrol agent against the green mold Trichoderma aggressivum has been shown to be efficient. However, the biocontrol mechanisms effective in the Agaricus bisporus / Trichoderma aggressivum / Bacillus velezensis QST713 pathosystem are still unknown. Our paper focuses on the exploration of the bioprotection mechanisms of the biocontrol agent Bacillus velezensis QST713 during culture of the button mushroom ( Agaricus bisporus ) in a micromodel culture system to study the specific response of strain QST713 in the presence of T. aggressivum and/or A. bisporus ., (Copyright © 2019 American Society for Microbiology.)

Bacillus subtilis QST713 is extensively used as a biological control agent in agricultural fields including in the button mushroom culture, Agaricus bisporus. This last use exploits its inhibitory activity against microbial pathogens such as Trichoderma aggressivum f. europaeum, the main button mushroom green mould competitor. Here, we report the complete genome sequence of this bacterium with a genome size of 4 233 757 bp, 4263 predicted genes and an average GC content of 45.9%. Based on phylogenomic analyses, strain QST713 is finally designated as Bacillus velezensis. Genomic analyses revealed two clusters encoding potential new antimicrobials with NRPS and TransATPKS synthetase. B. velezensis QST713 genome also harbours several genes previously described as being involved in surface colonization and biofilm formation. This strain shows a strong ability to form in vitro spatially organized biofilm and to antagonize T. aggressivum. The availability of this genome sequence could bring new elements to understand the interactions with micro or/and macroorganisms in crops., (Copyright © 2018 Elsevier B.V. All rights reserved.)

At the heart of central carbon metabolism, pyruvate is a pivotal metabolite in all living cells. Bacillus subtilis is able to excrete pyruvate as well as to use it as the sole carbon source. We herein reveal that ysbAB (renamed pftAB ), the only operon specifically induced in pyruvate-grown B. subtilis cells, encodes a hetero-oligomeric membrane complex which operates as a facilitated transport system specific for pyruvate, thereby defining a novel class of transporter. We demonstrate that the LytST two-component system is responsible for the induction of pftAB in the presence of pyruvate by binding of the LytT response regulator to a palindromic region upstream of pftAB We show that both glucose and malate, the preferred carbon sources for B. subtilis , trigger the binding of CcpA upstream of pftAB , which results in its catabolite repression. However, an additional CcpA-independent mechanism represses pftAB in the presence of malate. Screening a genome-wide transposon mutant library, we find that an active malic enzyme replenishing the pyruvate pool is required for this repression. We next reveal that the higher the influx of pyruvate, the stronger the CcpA-independent repression of pftAB , which suggests that intracellular pyruvate retroinhibits pftAB induction via LytST. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry. Overall, we provide evidence for a complete system of sensors, feed-forward and feedback controllers that play a major role in environmental growth of B. subtilis IMPORTANCE Pyruvate is a small-molecule metabolite ubiquitous in living cells. Several species also use it as a carbon source as well as excrete it into the environment. The bacterial systems for pyruvate import/export have yet to be discovered. Here, we identified in the model bacterium Bacillus subtilis the first import/export system specific for pyruvate, PftAB, which defines a novel class of transporter. In this bacterium, extracellular pyruvate acts as the signal molecule for the LytST two-component system (TCS), which in turn induces expression of PftAB. However, when the pyruvate influx is high, LytST activity is drastically retroinhibited. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry., (Copyright © 2017 Charbonnier et al.)

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho-null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks.

Almost one-third of crop yields are lost every year due to microbial alterations and diseases. The main control strategy to limit these losses is the use of an array of chemicals active against spoilage and unwanted pathogenic microorganisms. Their massive use has led to extensive environmental pollution, human poisoning and a variety of diseases. An emerging alternative to this chemical approach is the use of microbial biocontrol agents. Biopesticides have been used with success in several fields, but a better understanding of their mode of action is necessary to better control their activity and increase their use. Very few studies have considered that biofilms are the preferred mode of life of microorganisms in the target agricultural biotopes. Increasing evidence shows that the spatial organization of microbial communities on crop surfaces may drive important bioprotection mechanisms. The aim of this review is to summarize the evidence of biofilm formation by biocontrol agents on crops and discuss how this surface-associated mode of life may influence their biology and interactions with other microorganisms and the host and, finally, their overall beneficial activity., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Complex regulatory programs control cell adaptation to environmental changes by setting condition-specific proteomes. In balanced growth, bacterial protein abundances depend on the dilution rate, transcript abundances and transcript-specific translation efficiencies. We revisited the current theory claiming the invariance of bacterial translation efficiency. By integrating genome-wide transcriptome datasets and datasets from a library of synthetic gfp-reporter fusions, we demonstrated that translation efficiencies in Bacillus subtilis decreased up to fourfold from slow to fast growth. The translation initiation regions elicited a growth rate-dependent, differential production of proteins without regulators, hence revealing a unique, hard-coded, growth rate-dependent mode of regulation. We combined model-based data analyses of transcript and protein abundances genome-wide and revealed that this global regulation is extensively used in B. subtilis We eventually developed a knowledge-based, three-step translation initiation model, experimentally challenged the model predictions and proposed that a growth rate-dependent drop in free ribosome abundance accounted for the differential protein production., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

Predicting resource allocation between cell processes is the primary step towards decoding the evolutionary constraints governing bacterial growth under various conditions. Quantitative prediction at genome-scale remains a computational challenge as current methods are limited by the tractability of the problem or by simplifying hypotheses. Here, we show that the constraint-based modeling method Resource Balance Analysis (RBA), calibrated using genome-wide absolute protein quantification data, accurately predicts resource allocation in the model bacterium Bacillus subtilis for a wide range of growth conditions. The regulation of most cellular processes is consistent with the objective of growth rate maximization except for a few suboptimal processes which likely integrate more complex objectives such as coping with stressful conditions and survival. As a proof of principle by using simulations, we illustrated how calibrated RBA could aid rational design of strains for maximizing protein production, offering new opportunities to investigate design principles in prokaryotes and to exploit them for biotechnological applications., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Biofilms constitute the prevalent way of life for microorganisms in both natural and man-made environments. Biofilm-dwelling cells display greater tolerance to antimicrobial agents than those that are free-living, and the mechanisms by which this occurs have been investigated extensively using single-strain axenic models. However, there is growing evidence that interspecies interactions may profoundly alter the response of the community to such toxic exposure. In this paper, we propose an overview of the studies dealing with multispecies biofilms resistance to biocides, with particular reference to the protection of pathogenic species by resident surface flora when subjected to disinfectants treatments. The mechanisms involved in such protection include interspecies signaling, interference between biocides molecules and public goods in the matrix, or the physiology and genetic plasticity associated with a structural spatial arrangement. After describing these different mechanisms, we will discuss the experimental methods available for their analysis in the context of complex multispecies biofilms.

In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species ("public goods"), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SPβ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SPβ prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Genomes of two nondomesticated strains of Bacillus subtilis subspecies subtilis, NDmed and NDfood, have been sequenced. Both strains form very thick and spatially complex biofilms on submerged surfaces. Moreover, biofilms of the NDmed isolate were shown to be highly resistant to antimicrobials action., (Copyright © 2014 Sanchez-Vizuete et al.)

Background: Many organisms coordinate cell growth and division through size control mechanisms: cells must reach a critical size to trigger a cell cycle event. Bacterial division is often assumed to be controlled in this way, but experimental evidence to support this assumption is still lacking. Theoretical arguments show that size control is required to maintain size homeostasis in the case of exponential growth of individual cells. Nevertheless, if the growth law deviates slightly from exponential for very small cells, homeostasis can be maintained with a simple 'timer' triggering division. Therefore, deciding whether division control in bacteria relies on a 'timer' or 'sizer' mechanism requires quantitative comparisons between models and data., Results: The timer and sizer hypotheses find a natural expression in models based on partial differential equations. Here we test these models with recent data on single-cell growth of Escherichia coli. We demonstrate that a size-independent timer mechanism for division control, though theoretically possible, is quantitatively incompatible with the data and extremely sensitive to slight variations in the growth law. In contrast, a sizer model is robust and fits the data well. In addition, we tested the effect of variability in individual growth rates and noise in septum positioning and found that size control is robust to this phenotypic noise., Conclusions: Confrontations between cell cycle models and data usually suffer from a lack of high-quality data and suitable statistical estimation techniques. Here we overcome these limitations by using high precision measurements of tens of thousands of single bacterial cells combined with recent statistical inference methods to estimate the division rate within the models. We therefore provide the first precise quantitative assessment of different cell cycle models.

The entomopathogen Bacillus thuringiensis produces dense biofilms under various conditions. Here, we report that the transition phase regulators Spo0A, AbrB and SinR control biofilm formation and swimming motility in B. thuringiensis, just as they control biofilm formation and swarming motility in the closely related saprophyte species B. subtilis. However, microarray analysis indicated that in B. thuringiensis, in contrast to B. subtilis, SinR does not control an eps operon involved in exopolysaccharides production, but regulates genes involved in the biosynthesis of the lipopeptide kurstakin. This lipopeptide is required for biofilm formation and was previously shown to be important for survival in the host cadaver (necrotrophism). Microarray analysis also revealed that the SinR regulon contains genes coding for the Hbl enterotoxin. Transcriptional fusion assays, Western blots and hemolysis assays confirmed that SinR controls Hbl expression, together with PlcR, the main virulence regulator in B. thuringiensis. We show that Hbl is expressed in a sustained way in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses Hbl. The gene coding for SinI, an antagonist of SinR, is expressed in the same biofilm subpopulation as hbl, suggesting that hbl transcription heterogeneity is SinI-dependent. B. thuringiensis and B. cereus are enteric bacteria which possibly form biofilms lining the host intestinal epithelium. Toxins produced in biofilms could therefore be delivered directly to the target tissue.

One of the key ways in which microbes are thought to regulate their metabolism is by modulating the availability of enzymes through transcriptional regulation. However, the limited success of efforts to manipulate metabolic fluxes by rewiring the transcriptional network has cast doubt on the idea that transcript abundance controls metabolic fluxes. In this study, we investigate control of metabolic flux in the model bacterium Bacillus subtilis by quantifying fluxes, transcripts, and metabolites in eight metabolic states enforced by different environmental conditions. We find that most enzymes whose flux switches between on and off states, such as those involved in substrate uptake, exhibit large corresponding transcriptional changes. However, for the majority of enzymes in central metabolism, enzyme concentrations were insufficient to explain the observed fluxes--only for a number of reactions in the tricarboxylic acid cycle were enzyme changes approximately proportional to flux changes. Surprisingly, substrate changes revealed by metabolomics were also insufficient to explain observed fluxes, leaving a large role for allosteric regulation and enzyme modification in the control of metabolic fluxes.

Unlabelled: Live Cell Array (LCA) technology allows the acquisition of high-resolution time-course profiles of bacterial gene expression by the systematic assessment of fluorescence in living cells carrying either transcriptional or translational fluorescent protein fusion. However, the direct estimation of promoter activities by time-dependent derivation of the fluorescence datasets generates high levels of noise. Here, we present BasyLiCA, a user-friendly open-source interface and database dedicated to the automatic storage and standardized treatment of LCA data. Data quality reports are generated automatically. Growth rates and promoter activities are calculated by tunable discrete Kalman filters that can be set to incorporate data from biological replicates, significantly reducing the impact of noise measurement in activity estimations., Availability: The BasyLiCA software and the related documentation are available at http://genome.jouy.inra.fr/basylica.

In their natural habitat, microorganisms are typically confronted with nutritional limitations that restrict growth and force them to persevere in a stationary phase. Despite the importance of this phase, little is known about the metabolic state(s) that sustains it. Here, we investigate metabolically active but non-growing Bacillus subtilis during nitrogen starvation. In the absence of biomass formation as the major NADPH sink, the intracellular flux distribution in these resting B. subtilis reveals a large apparent catabolic NADPH overproduction of 5.0 ± 0.6 mmol g(-1)h(-1) that was partly caused by high pentose phosphate pathway fluxes. Combining transcriptome analysis, stationary (13)C-flux analysis in metabolic deletion mutants, (2)H-labeling experiments, and kinetic flux profiling, we demonstrate that about half of the catabolic excess NADPH is oxidized by two transhydrogenation cycles, i.e. isoenzyme pairs of dehydrogenases with different cofactor specificities that operate in reverse directions. These transhydrogenation cycles were constituted by the combined activities of the glyceraldehyde 3-phosphate dehydrogenases GapA/GapB and the malic enzymes MalS/YtsJ. At least an additional 6% of the overproduced NADPH is reoxidized by continuous cycling between ana- and catabolism of glutamate. Furthermore, in vitro enzyme data show that a not yet identified transhydrogenase could potentially reoxidize ∼20% of the overproduced NADPH. Overall, we demonstrate the interplay between several metabolic mechanisms that concertedly enable network-wide NADPH homeostasis under conditions of high catabolic NADPH production in the absence of cell growth in B. subtilis.

Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations.

Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs.

Assessing gene expression noise in order to obtain mechanistic insights requires accurate quantification of gene expression on many individual cells over a large dynamic range. We used a unique method based on 2-photon fluorescence fluctuation microscopy to measure directly, at the single cell level and with single-molecule sensitivity, the absolute concentration of fluorescent proteins produced from the two Bacillus subtilis promoters that control the switch between glycolysis and gluconeogenesis. We quantified cell-to-cell variations in GFP concentrations in reporter strains grown on glucose or malate, including very weakly transcribed genes under strong catabolite repression. Results revealed strong transcriptional bursting, particularly for the glycolytic promoter. Noise pattern parameters of the two antagonistic promoters controlling the nutrient switch were differentially affected on glycolytic and gluconeogenic carbon sources, discriminating between the different mechanisms that control their activity. Our stochastic model for the transcription events reproduced the observed noise patterns and identified the critical parameters responsible for the differences in expression profiles of the promoters. The model also resolved apparent contradictions between in vitro operator affinity and in vivo repressor activity at these promoters. Finally, our results demonstrate that negative feedback is not noise-reducing in the case of strong transcriptional bursting.

The development of a biofilm constitutes a survival strategy by providing bacteria a protective environment safe from stresses such as microbicide action and can thus lead to important health-care problems. In this study, biofilm resistance of a Bacillus subtilis strain (called hereafter ND(medical)) recently isolated from endoscope washer-disinfectors to peracetic acid was investigated and its ability to protect the pathogen Staphylococcus aureus in mixed biofilms was evaluated. Biocide action within Bacillus subtilis biofilms was visualised in real time using a non-invasive 4D confocal imaging method. The resistance of single species and mixed biofilms to peracetic acid was quantified using standard plate counting methods and their architecture was explored using confocal imaging and electronic microscopy. The results showed that the ND(medical) strain demonstrates the ability to make very large amount of biofilm together with hyper-resistance to the concentration of PAA used in many formulations (3500 ppm). Evidences strongly suggest that the enhanced resistance of the ND(medical) strain was related to the specific three-dimensional structure of the biofilm and the large amount of the extracellular matrix produced which can hinder the penetration of peracetic acid. When grown in mixed biofilm with Staphylococcus aureus, the ND(medical) strain demonstrated the ability to protect the pathogen from PAA action, thus enabling its persistence in the environment. This work points out the ability of bacteria to adapt to an extremely hostile environment, and the necessity of considering multi-organism ecosystems instead of single species model to decipher the mechanisms of biofilm resistance to antimicrobials agents.

Quantification of promoter activity or protein expression in gene regulatory networks is generally achieved via measurement of fluorescent protein (FP) intensity, which is related to the true FP concentration by an unknown scaling factor, thereby limiting analysis and interpretation. Here, using approaches originally developed for eukaryotic cells, we show that two-photon (2p) fluorescence fluctuation microscopy, specifically scanning number and brightness (sN&B) analysis, can be applied to determine the absolute concentrations of diffusing FPs in live bacterial cells. First, we demonstrate the validity of the approach, despite the small size of the bacteria, using the central pixels and spatial averaging. We established the lower detection limit at or below 75 nM (~3 molecules of FP/vol(ex)) and the upper detection limit at approximately 10 μM, which can be extended using intensity measurements. We found that the uncertainty inherent in our measurements (<5%) was smaller than the high cell-cell variations observed for stochastic leakage from FP fusions of the lac promoter in the repressed state or the 10 to 25% variation observed on induction. This demonstrates that a reliable and absolute measure of transcriptional noise can be made using our approach, which should make it particularly appropriate for the investigation of stochasticity in gene expression networks., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate.

Genomic tiling array transcriptomics and RNA-seq are two powerful and rapidly developing approaches for unbiased transcriptome analysis. Providing comprehensive identification and quantification of transcripts with an unprecedented resolution, they are leading to major breakthroughs in systems biology. Here we review each step of the analysis from library preparation to the interpretation of the data, with particular attention paid to the possible sources of artifacts. Methodological requirements and statistical frameworks are often similar in both the approaches despite differences in the nature of the data. Tiling array analysis does not require rRNA depletion and benefits from a more mature computational workflow, whereas RNA-Seq has a clear lead in terms of background noise and dynamic range with a considerable potential for evolution with the improvements of sequencing technologies. Being independent of prior sequence knowledge, RNA-seq will boost metatranscriptomics and evolutionary transcriptomics applications., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The formation of multicellular communities known as biofilms is the part of bacterial life cycle in which bacteria display cooperative behaviour and differentiated phenotypes leading to specific functions. Bacillus subtilis is a Gram-positive bacterium that has served for a decade as a model to study the molecular pathways that control biofilm formation. Most of the data on B. subtilis biofilms have come from studies on the formation of pellicles at the air-liquid interface, or on the complex macrocolonies that develop on semi-solid nutritive agar. Here, using confocal laser scanning microcopy, we show that B. subtilis strains of different origins are capable of forming biofilms on immersed surfaces with dramatically protruding "beanstalk-like" structures with certain strains. Indeed, these structures can reach a height of more than 300 µm with one undomesticated strain from a medical environment. Using 14 GFP-labeled mutants previously described as affecting pellicle or complex colony formation, we have identified four genes whose inactivation significantly impeded immersed biofilm development, and one mutation triggering hyperbiofilm formation. We also identified mutations causing the three-dimensional architecture of the biofilm to be altered. Taken together, our results reveal that B. subtilis is able to form specific biofilm features on immersed surfaces, and that the development of these multicellular surface-associated communities involves regulation pathways that are common to those governing the formation of pellicle and/or complex colonies, and also some specific mechanisms. Finally, we propose the submerged surface-associated biofilm as another relevant model for the study of B. subtilis multicellular communities.

NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD(+) degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H(2)O(2) produced during the oxidative process or added exogenously., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)