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1.
PLoS Genet ; 12(5): e1006025, 2016 05.
Article in English | MEDLINE | ID: mdl-27171414

ABSTRACT

Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.


Subject(s)
Chromosomal Proteins, Non-Histone/genetics , DNA Replication/genetics , DNA Topoisomerase IV/genetics , Escherichia coli Proteins/genetics , Integrases/genetics , Binding Sites/genetics , Catalytic Domain/genetics , Cell Cycle/genetics , Cell Division/genetics , Chromatids/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation/genetics , Chromosomes, Bacterial/genetics , DNA Topoisomerase IV/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/metabolism , Integrases/metabolism , Sister Chromatid Exchange/genetics
2.
Mol Syst Biol ; 9: 709, 2013 Nov 26.
Article in English | MEDLINE | ID: mdl-24281055

ABSTRACT

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.


Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Gene Expression Regulation, Bacterial , Metabolic Networks and Pathways , RNA, Messenger/genetics , Carbon Isotopes , Kinetics , RNA, Messenger/metabolism , Transcription, Genetic
3.
Mol Microbiol ; 86(6): 1285-90, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23078263

ABSTRACT

The mechanisms driving bacterial chromosome segregation remain poorly characterized. While a number of factors influencing chromosome segregation have been described in recent years, none of them appeared to play an essential role in the process comparable to the eukaryotic centromere/spindle complex. The research community involved in bacterial chromosome was becoming familiar with the fact that bacteria have selected multiple redundant systems to ensure correct chromosome segregation. Over the past few years a new perspective came out that entropic forces generated by the confinement of the chromosome in the crowded nucleoid shell could be sufficient to segregate the chromosome. The segregating factors would only be required to create adequate conditions for entropy to do its job. In the article by Yazdi et al. (2012) in this issue of Molecular Microbiology, this model was challenged experimentally in live Escherichia coli cells. A Fis-GFP fusion was used to follow nucleoid choreography and analyse it from a polymer physics perspective. Their results suggest strongly that E. coli nucleoids behave as self-adherent polymers. Such a structuring and the specific segregation patterns observed do not support an entropic like segregation model. Are we back to the pre-entropic era?


Subject(s)
Chromosomes, Bacterial/chemistry , Chromosomes, Bacterial/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/growth & development , Escherichia coli/metabolism , Nucleoproteins/metabolism
4.
Bioinformatics ; 28(20): 2705-6, 2012 Oct 15.
Article in English | MEDLINE | ID: mdl-22764159

ABSTRACT

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.


Subject(s)
Bacteria/genetics , Software , Transcription, Genetic , Bacteria/metabolism , Fluorescent Dyes , Genes, Reporter , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Promoter Regions, Genetic
5.
Science ; 335(6072): 1099-103, 2012 Mar 02.
Article in English | MEDLINE | ID: mdl-22383848

ABSTRACT

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.


Subject(s)
Adaptation, Physiological , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Gene Regulatory Networks , Glucose/metabolism , Malates/metabolism , Metabolic Networks and Pathways/genetics , Algorithms , Bacterial Proteins/metabolism , Computer Simulation , Data Interpretation, Statistical , Gene Expression Regulation, Bacterial , Genome, Bacterial , Metabolome , Metabolomics , Models, Biological , Operon , Promoter Regions, Genetic , Transcription Factors/metabolism , Transcription, Genetic
6.
Science ; 335(6072): 1103-6, 2012 Mar 02.
Article in English | MEDLINE | ID: mdl-22383849

ABSTRACT

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.


Subject(s)
Bacillus subtilis/genetics , Bacillus subtilis/physiology , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Transcription, Genetic , Transcriptome , Adaptation, Physiological , Algorithms , Binding Sites , Gene Expression Profiling , Gene Regulatory Networks , Oligonucleotide Array Sequence Analysis , RNA, Antisense/genetics , RNA, Antisense/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regulon , Sigma Factor/metabolism , Terminator Regions, Genetic
7.
Microbiology (Reading) ; 156(Pt 6): 1600-1608, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20150235

ABSTRACT

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.


Subject(s)
Bacillus subtilis/genetics , Gene Expression , Plasmids , Bacillus subtilis/metabolism , Base Sequence , Carbon/metabolism , Gene Expression Regulation, Bacterial , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Molecular Sequence Data , Phosphates/metabolism , Promoter Regions, Genetic , Transcription, Genetic
8.
J Biol Chem ; 285(3): 1587-96, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-19917605

ABSTRACT

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 (13)C 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 (13)C-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.


Subject(s)
Bacillus subtilis/drug effects , Bacillus subtilis/metabolism , Carbon/metabolism , Malates/pharmacology , Bacillus subtilis/genetics , Bacillus subtilis/growth & development , Carbon Isotopes , Genes, Bacterial/genetics , Glucose/metabolism , Glucose/pharmacology , Intracellular Space/drug effects , Intracellular Space/metabolism , Isotope Labeling , Malates/metabolism , Substrate Specificity , Thermodynamics , Transcription, Genetic/drug effects
9.
J Bacteriol ; 191(9): 3168-71, 2009 May.
Article in English | MEDLINE | ID: mdl-19270101

ABSTRACT

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.


Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Fructose-Bisphosphatase/genetics , Fructose-Bisphosphatase/metabolism , Bacillus subtilis/growth & development , Bacillus subtilis/metabolism , Genes, Bacterial , Glycolysis , Operon , Transcription, Genetic
10.
Ecol Lett ; 11(1): 44-51, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18021245

ABSTRACT

Virulence is generally considered to benefit parasites by enhancing resource-transfer from host to pathogen. Here, we offer an alternative framework where virulent immune-provoking behaviours and enhanced immune resistance are joint tactics of invading pathogens to eliminate resident competitors (transferring resources from resident to invading pathogen). The pathogen wins by creating a novel immunological challenge to which it is already adapted. We analyse a general ecological model of 'proactive invasion' where invaders not adapted to a local environment can succeed by changing it to one where they are better adapted than residents. However, the two-trait nature of the 'proactive' strategy (provocation of, and adaptation to environmental change) presents an evolutionary conundrum, as neither trait alone is favoured in a homogenous host population. We show that this conundrum can be resolved by allowing for host heterogeneity. We relate our model to emerging empirical findings on immunological mediation of parasite competition.


Subject(s)
Biological Evolution , Host-Parasite Interactions/immunology , Inflammation/parasitology , Models, Biological , Parasites/pathogenicity , Animals , Parasites/immunology , Virulence
11.
Mol Biochem Parasitol ; 153(1): 41-7, 2007 May.
Article in English | MEDLINE | ID: mdl-17335919

ABSTRACT

The malaria parasite encodes a wide range of proteases necessary to facilitate its many developmental transitions in vertebrate and insect hosts. Amongst these is a predicted cysteine protease structurally related to caspases, named Plasmodium metacaspase 1 (PxMC1). We have generated Plasmodium berghei parasites in which the PbMC1coding sequence is removed and replaced with a green fluorescent reporter gene to investigate the expression of PbMC1, its contribution to parasite development, and its involvement in previously reported apoptosis-like cell death of P. berghei ookinetes. Our results show that the pbmc1 gene is expressed in female gametocytes and all downstream mosquito stages including sporozoites, but not in asexual blood stages. We failed to detect an apparent loss-of-function phenotype, suggesting that PbMC1 constitutes a functionally redundant gene. We discuss these findings in the context of two other putative Plasmodium metacaspases that we describe here.


Subject(s)
Caspases/metabolism , Plasmodium berghei/enzymology , Plasmodium berghei/growth & development , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Apoptosis , Base Sequence , Caspases/genetics , DNA, Protozoan/genetics , Female , Gene Deletion , Genes, Protozoan , Genes, Reporter , Green Fluorescent Proteins/genetics , Molecular Sequence Data , Phenotype , Plasmodium berghei/cytology , Plasmodium berghei/genetics , Protozoan Proteins/genetics , Recombinant Proteins/genetics , Sequence Homology, Amino Acid
12.
Curr Biol ; 16(20): 2048-52, 2006 Oct 24.
Article in English | MEDLINE | ID: mdl-17055985

ABSTRACT

Locally adapted residents present a formidable barrier to invasion . One solution for invaders is to kill residents . Here, we explore the comparative ecological dynamics of two distinct microbial mechanisms of killing competitors, via the release of chemicals (e.g., bacteriocins ) and via the release of parasites (e.g., temperate phage ). We compared the short-term population dynamics of susceptible E. coli K12 and isogenic carriers of phage varphi80 in experimental cultures to that anticipated by mathematical models using independently derived experimental parameters. Whereas phages are a direct burden to their carriers because of probabilistic host lysis, by killing competitor bacteria they can indirectly benefit bacterial kin made immune by carrying isogenic phage. This is similar to previously described bacteriocin-mediated effects. However, unlike chemical killing, viable phage trigger an epidemic among susceptible competitors, which become factories producing more phage. Amplification makes phage carriers able to invade well-mixed susceptibles even faster when rare, whereas chemical killers can only win in a well-mixed environment when sufficiently abundant. We demonstrate that for plausible parameters, the release of chemical toxins is superior as a resident strategy to repel invasions, whereas the release of temperate phage is superior as a strategy of invasion.


Subject(s)
Bacteriophages/physiology , Competitive Behavior/physiology , Escherichia coli K12/physiology , Escherichia coli K12/virology , Models, Biological , Bacteriocins/metabolism , Ecology , Population Dynamics
13.
Microbiology (Reading) ; 152(Pt 1): 67-73, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16385116

ABSTRACT

In silico, it has been shown that mutator alleles that increase mutation rate can be selected for by generating adaptive mutations. In vitro and in vivo, competition between wild-type bacteria and isogenic mutator mutants is consistent with this view. However, in vivo, the gain of the mutator seems to be reduced when migration is allowed. In vitro, the advantage of mutators has been described as frequency-dependent, leading to mutator advantage only when they are sufficiently frequent. Using an in vitro system, it is demonstrated that (i) the selection of mutators is frequency-independent, yet depends on at least one mutator bacterium bearing an adaptive mutation (its presence depends on chance, mutation rates and population size of mutator bacteria); (ii) on average, the mutator gain is always equal to the ratio of the adaptive mutation frequency of the mutator versus wild-type; (iii) when migration into an empty niche is allowed, the mutator benefit is reduced if migration occurs after fixation of the adaptive mutation into the wild-type population. It is concluded that in all cases, mutator gain depends directly on the ratio of bacteria carrying a beneficial mutation in mutator versus wild-type lineages.


Subject(s)
Escherichia coli/genetics , Mutation , Selection, Genetic , Adaptation, Physiological/genetics , Alleles , Escherichia coli Proteins/genetics , Evolution, Molecular
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