Direct comparison of spatial transcriptional heterogeneity across diverse Bacillus subtilis biofilm communities.

Bacillus subtilis can form various types of spatially organised communities on surfaces, such as colonies, pellicles and submerged biofilms. These communities share similarities and differences, and phenotypic heterogeneity has been reported for each type of community. Here, we studied spatial transcriptional heterogeneity across the three types of surface-associated communities. Using RNA-seq analysis of different regions or populations for each community type, we identified genes that are specifically expressed within each selected population. We constructed fluorescent transcriptional fusions for 17 of these genes, and observed their expression in submerged biofilms using time-lapse confocal laser scanning microscopy (CLSM). We found mosaic expression patterns for some genes; in particular, we observed spatially segregated cells displaying opposite regulation of carbon metabolism genes (gapA and gapB), indicative of distinct glycolytic or gluconeogenic regimes coexisting in the same biofilm region. Overall, our study provides a direct comparison of spatial transcriptional heterogeneity, at different scales, for the three main models of B. subtilis surface-associated communities.

Bacillus subtilis NDmed, a model strain for biofilm genetic studies.

The Bacillus subtilis strain NDmed was isolated from an endoscope washer-disinfector in a medical environment. NDmed can form complex macrocolonies with highly wrinkled architectural structures on solid medium. In static liquid culture, it produces thick pellicles at the interface with air as well as remarkable highly protruding ''beanstalk-like'' submerged biofilm structures at the solid surface. Since these mucoid submerged structures are hyper-resistant to biocides, NDmed has the ability to protect pathogens embedded in mixed-species biofilms by sheltering them from the action of these agents. Additionally, this non-domesticated and highly biofilm forming strain has the propensity of being genetically manipulated. Due to all these properties, the NDmed strain becomes a valuable model for the study of B. subtilis biofilms. This review focuses on several studies performed with NDmed that have highlighted the sophisticated genetic dynamics at play during B. subtilis biofilm formation. Further studies in project using modern molecular tools of advanced technologies with this strain, will allow to deepen our knowledge on the emerging properties of multicellular bacterial life.

The coordinated population redistribution between Bacillus subtilis submerged biofilm and liquid-air pellicle.

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.

Comparison of the Genetic Features Involved in Bacillus subtilis Biofilm Formation Using Multi-Culturing Approaches.

Surface-associated multicellular assemblage is an important bacterial trait to withstand harsh environmental conditions. Bacillus subtilis is one of the most studied Gram-positive bacteria, serving as a model for the study of genetic pathways involved in the different steps of 3D biofilm formation. B. subtilis biofilm studies have mainly focused on pellicle formation at the air-liquid interface or complex macrocolonies formed on nutritive agar. However, only few studies focus on the genetic features of B. subtilis submerged biofilm formation and their link with other multicellular models at the air interface. NDmed, an undomesticated B. subtilis strain isolated from a hospital, has demonstrated the ability to produce highly structured immersed biofilms when compared to strains classically used for studying B. subtilis biofilms. In this contribution, we have conducted a multi-culturing comparison (between macrocolony, swarming, pellicle, and submerged biofilm) of B. subtilis multicellular communities using the NDmed strain and mutated derivatives for genes shown to be required for motility and biofilm formation in pellicle and macrocolony models. For the 15 mutated NDmed strains studied, all showed an altered phenotype for at least one of the different culture laboratory assays. Mutation of genes involved in matrix production (i.e., tasA, epsA-O, cap, ypqP) caused a negative impact on all biofilm phenotypes but favored swarming motility on semi-solid surfaces. Mutation of bslA, a gene coding for an amphiphilic protein, affected the stability of the pellicle at the air-liquid interface with no impact on the submerged biofilm model. Moreover, mutation of lytF, an autolysin gene required for cell separation, had a greater effect on the submerged biofilm model than that formed at aerial level, opposite to the observation for lytABC mutant. In addition, B. subtilis NDmed with sinR mutation formed wrinkled macrocolony, less than that formed by the wild type, but was unable to form neither thick pellicle nor structured submerged biofilm. The results are discussed in terms of the relevancy to determine whether genes involved in colony and pellicle formation also govern submerged biofilm formation, by regarding the specificities in each model.

Biofilm Formation and Synthesis of Antimicrobial Compounds by the Biocontrol Agent Bacillus velezensis QST713 in an Agaricus bisporus Compost Micromodel.

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.

Complete genome sequence of Bacillus velezensis QST713: A biocontrol agent that protects Agaricus bisporus crops against the green mould disease.

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.

Molecular and Physiological Logics of the Pyruvate-Induced Response of a Novel Transporter in Bacillus subtilis.

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. subtilisIMPORTANCE 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.

Should the biofilm mode of life be taken into consideration for microbial biocontrol agents?

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.

Pathogens protection against the action of disinfectants in multispecies biofilms.

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.

Identification of ypqP as a New Bacillus subtilis biofilm determinant that mediates the protection of Staphylococcus aureus against antimicrobial agents in mixed-species communities.

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 SPß 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 SPß 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.

Genome Sequences of Two Nondomesticated Bacillus subtilis Strains Able To Form Thick Biofilms on Submerged Surfaces.

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.

Biofilms of a Bacillus subtilis hospital isolate protect Staphylococcus aureus from biocide action.

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.

13C-flux analysis reveals NADPH-balancing transhydrogenation cycles in stationary phase of nitrogen-starving Bacillus subtilis.

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.

Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states.

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.

Malate-mediated carbon catabolite repression in Bacillus subtilis involves the HPrK/CcpA pathway.

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.

Absolute quantification of gene expression in individual bacterial cells using two-photon fluctuation microscopy.

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.

Prevention of cross-talk in conserved regulatory systems: identification of specificity determinants in RNA-binding anti-termination proteins of the BglG family.

Each family of signal transduction systems requires specificity determinants that link individual signals to the correct regulatory output. In Bacillus subtilis, a family of four anti-terminator proteins controls the expression of genes for the utilisation of alternative sugars. These regulatory systems contain the anti-terminator proteins and a RNA structure, the RNA anti-terminator (RAT) that is bound by the anti-terminator proteins. We have studied three of these proteins (SacT, SacY, and LicT) to understand how they can transmit a specific signal in spite of their strong structural homology. A screen for random mutations that render SacT capable to bind a RNA structure recognized by LicT only revealed a substitution (P26S) at one of the few non-conserved residues that are in contact with the RNA. We have randomly modified this position in SacT together with another non-conserved RNA-contacting residue (Q31). Surprisingly, the mutant proteins could bind all RAT structures that are present in B. subtilis. In a complementary approach, reciprocal amino acid exchanges have been introduced in LicT and SacY at non-conserved positions of the RNA-binding site. This analysis revealed the key role of an arginine side-chain for both the high affinity and specificity of LicT for its cognate RAT. Introduction of this Arg at the equivalent position of SacY (A26) increased the RNA binding in vitro but also resulted in a relaxed specificity. Altogether our results suggest that this family of anti-termination proteins has evolved to reach a compromise between RNA binding efficacy and specific interaction with individual target sequences.

The spatial architecture of Bacillus subtilis biofilms deciphered using a surface-associated model and in situ imaging.

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.

The Bacillus subtilis ywjI (glpX) gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the class III Fbp enzyme.

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions.

CcpN controls central carbon fluxes in Bacillus subtilis.

The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes.