Matthieu Jules

Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis

Outside In Acquisition and analysis of large data sets promises to move us toward a greater understanding of the mechanisms by which biological systems are dynamically regulated to respond to external cues. Now, two papers explore the responses of a bacterium to changing nutritional conditions (see the Perspective by Chalancon et al.). Nicolas et al. (p. 1103) measured transcriptional regulation for more than 100 different conditions. Greater amounts of antisense RNA were generated than expected and appeared to be produced by alternative RNA polymerase targeting subunits called sigma factors. One transition, from malate to glucose as the primary nutrient, was studied in more detail by Buescher et al. (p. 1099) who monitored RNA abundance, promoter activity in live cells, protein abundance, and absolute concentrations of intracellular and extracellular metabolites. In this case, the bacteria responded rapidly and largely without transcriptional changes to life on malate, but only slowly adapted to use glucose, a shift that required changes in nearly half the transcription network. These data offer an initial understanding of why certain regulatory strategies may be favored during evolution of dynamic control systems. A horizontal analysis reveals the breadth of genes turned on and off as nutrients change. 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.

Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila

Adaptation to the host environment and exploitation of host cell functions are critical to the success of intracellular pathogens. Here, insight to these virulence mechanisms was obtained for the first time from the transcriptional program of the human pathogen Legionella pneumophila during infection of its natural host, Acanthamoeba castellanii. The biphasic life cycle of L. pneumophila was reflected by a major shift in gene expression from replicative to transmissive phase, concerning nearly half of the genes predicted in the genome. However, three different L. pneumophila strains showed similar in vivo gene expression patterns, indicating that common regulatory mechanisms govern the Legionella life cycle, despite the plasticity of its genome. During the replicative phase, in addition to components of aerobic metabolism and amino acid catabolism, the Entner‐Doudoroff pathway, a NADPH producing mechanism used for sugar and/or gluconate assimilation, was expressed, suggesting for the first time that intracellular L. pneumophila may also scavenge host carbohydrates as nutrients and not only proteins. Identification of genes only upregulated in vivo but not in vitro, may explain higher virulence of in vivo grown L. pneumophila. Late in the life cycle, L. pneumophila upregulates genes predicted to promote transmission and manipulation of a new host cell, therewith priming it for the next attack. These including substrates of the Dot/Icm secretion system, other factors associated previously with invasion and virulence, the motility and the type IV pilus machineries, and > 90 proteins not characterized so far. Analysis of a fliA (σ28) deletion mutant identified genes coregulated with the flagellar regulon, including GGDEF/EAL regulators and factors that promote host cell entry and survival.

Global Network Reorganization During Dynamic Adaptations of Bacillus subtilis Metabolism

Outside In Acquisition and analysis of large data sets promises to move us toward a greater understanding of the mechanisms by which biological systems are dynamically regulated to respond to external cues. Now, two papers explore the responses of a bacterium to changing nutritional conditions (see the Perspective by Chalancon et al.). Nicolas et al. (p. 1103) measured transcriptional regulation for more than 100 different conditions. Greater amounts of antisense RNA were generated than expected and appeared to be produced by alternative RNA polymerase targeting subunits called sigma factors. One transition, from malate to glucose as the primary nutrient, was studied in more detail by Buescher et al. (p. 1099) who monitored RNA abundance, promoter activity in live cells, protein abundance, and absolute concentrations of intracellular and extracellular metabolites. In this case, the bacteria responded rapidly and largely without transcriptional changes to life on malate, but only slowly adapted to use glucose, a shift that required changes in nearly half the transcription network. These data offer an initial understanding of why certain regulatory strategies may be favored during evolution of dynamic control systems. A vertical analysis reveals that a simple switch of one food for another evokes changes at many levels. 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.

Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila

To transit from intra‐ to extracellular environments, Legionella pneumophila differentiates from a replicative/non‐virulent to a transmissive/virulent form using the two‐component system LetA/LetS and the global repressor protein CsrA. While investigating how both regulators act co‐ordinately we characterized two ncRNAs, RsmY and RsmZ, that link the LetA/LetS and CsrA regulatory networks. We demonstrate that LetA directly regulates their expression and show that RsmY and RsmZ are functional in Escherichia coli and are able to bind CsrA in vitro. Single mutants have no (ΔrsmY) or a little (ΔrsmZ) impact on virulence, but the ΔrsmYZ strain shows a drastic defect in intracellular growth in Acanthamoeba castellanii and THP‐1 monocyte‐derived macrophages. Analysis of the transcriptional programmes of the ΔletA, ΔletS and ΔrsmYZ strains revealed that the switch to the transmissive phase is partially blocked. One major difference between the ΔletA, ΔletS and ΔrsmYZ strains was that the latter synthesizes flagella. Taken together, LetA activates transcription of RsmY and RsmZ, which sequester CsrA and abolish its post‐transcriptional repressive activity. However, the RsmYZ‐CsrA pathway appears not to be the main or only regulatory circuit governing flagella synthesis. We suggest that rather RpoS and LetA, by influencing LetE and probably cyclic‐di‐GMP levels, regulate motility in L. pneumophila.

The Legionella pneumophila F‐box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication

The environmental pathogen Legionella pneumophila encodes three proteins containing F‐box domains and additional protein–protein interaction domains, reminiscent of eukaryotic SCF ubiquitin–protein ligases. Here we show that the F‐box proteins of L. pneumophila strain Paris are Dot/Icm effectors involved in the accumulation of ubiquitinated proteins associated with the Legionella‐containing vacuole. Single, double and triple mutants of the F‐box protein encoding genes were impaired in infection of Acanthamoeba castellanii, THP‐1 macrophages and human lung epithelial cells. Lpp2082/AnkB was essential for infection of the lungs of A/J mice in vivo, and bound Skp1, the interaction partner of the SCF complex in mammalian cells, similar to AnkB from strain AA100/130b. Using a yeast two‐hybrid screen and co‐immunoprecipitation analysis we identified ParvB a protein present in focal adhesions and in lamellipodia, as a target. Immunofluorescence analysis confirmed that ectopically expressed Lpp2082/AnkB colocalized with ParvB at the periphery of lamellipodia. Unexpectedly, ubiquitination tests revealed that Lpp2082/AnkB diminishes endogenous ubiquitination of ParvB. Based on these results we propose that L. pneumophila modulates ubiquitination of ParvB by competing with eukaryotic E3 ligases for the specific protein–protein interaction site of ParvB, thereby revealing a new mechanism by which L. pneumophila may employ translocated effector proteins to promote bacterial survival.

Transcriptional regulation is insufficient to explain substrate‐induced flux changes in Bacillus subtilis

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.

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Commonly glucose is considered to be the only preferred substrate in Bacillus subtilis whose presence represses utilization of other alternative substrates. Because recent data indicate that malate might be an exception, we quantify here the carbon source utilization hierarchy. Based on physiology and transcriptional data during co-utilization experiments with eight carbon substrates, we demonstrate that malate is a second preferred carbon source for B. subtilis, which is rapidly co-utilized with glucose and strongly represses the uptake of alternative substrates. From the different hierarchy and degree of catabolite repression exerted by glucose and malate, we conclude that both substrates might act through different molecular mechanisms. To obtain a quantitative and functional network view of how malate is (co)metabolized, we developed a novel approach to metabolic flux analysis that avoids error-prone, intuitive, and ad hoc decisions on 13C rearrangements. In particular, we developed a rigorous approach for deriving reaction reversibilities by combining in vivo intracellular metabolite concentrations with a thermodynamic feasibility analysis. The thus-obtained analytical model of metabolism was then used for network-wide isotopologue balancing to estimate the intracellular fluxes. These 13C-flux data revealed an extraordinarily high malate influx that is primarily catabolized via the gluconeogenic reactions and toward overflow metabolism. Furthermore, a considerable NADPH-producing malic enzyme flux is required to supply the biosynthetically required NADPH in the presence of malate. Co-utilization of glucose and malate resulted in a synergistic decrease of the respiratory tricarboxylic acid cycle flux.

Legionella pneumophila adaptation to intracellular life and the host response: clues from genomics and transcriptomics.

Legionella pneumophila is the causative agent of the pneumonia‐like Legionnaires’ disease. The bacteriums survival and spread depend on the ability to replicate inside eukaryotic phagocytic cells. A particular feature of Legionella is its dual host system allowing the intracellular growth in protozoa like Acanthamoeba castellanii, and during infection in human alveolar macrophages. Genome analysis and comparisons as well as expression profiling of the pathogen and the host helped to identify regulatory circuits mediating adaptation of the L. pneumophila transcriptome to the intracellular environment and gave clues for the metabolic needs of intracellular Legionella. This review will summarize what is currently known about intracellular gene expression of L. pneumophila, the transcriptional host response of the model host Dictyostelium discoideum and will present hypotheses drawn from these data with respect to subversion of host cell functions and virulence of L. pneumophila.

pBaSysBioII: an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

Plasmid pBaSysBioII was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type event and is non-mutagenic, placing the fusion at the homologous chromosomal locus. Using phoA, murAA, gapB, ptsG and cggR promoters that are responsive to phosphate availability, growth rate and carbon source, we show that detailed profiles of promoter activity can be established, with responses to changing conditions being measurable within 1 min of the stimulus. This makes pBaSysBioII a highly versatile tool for real-time gene expression analysis in growing cells of 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.