BasyLiCA: a tool for automatic processing of a bacterial live cell array

ABSTRACT Summary: 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 standardised 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 a

Stratégies adaptatives dans la colonisation de nouveaux environnements

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

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

Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome

International audienceCatenation links between sister chromatids are formed progressively during DNA replica-tion 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 chro-matin 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

Ecology of microbial invasions: amplification allows virus carriers to invade more rapidly when rare.

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

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

Role of the <i>dif</i> site for the management of circular chromosomes.

<p>A) Southern blot analysis of Topo IV cleavage at the <i>dif</i> and 2.56 Mb sites in the <i>mukB</i> mutant grown in minimal medium at 22°C. B) Southern blot analysis of Topo IV cleavage at the <i>dif</i> and 2.56 Mb sites in the <i>seqA</i> mutant grown in minimal medium at 37°C. C) Southern blot analysis of Topo IV cleavage at the <i>dif</i> and 1.9 Mb sites in the <i>matP</i> mutant grown in LB at 37°C. D) Colony Forming Unit (CFU) analysis of the WT and <i>nalR</i> strains deleted for the <i>dif</i> site, the <i>xerC</i> and <i>matP</i> genes in the presence of ciprofloxacin. E) Colony Forming Unit (CFU) analysis of the WT, <i>parEts</i> and <i>gyrBts</i> strains deleted for the <i>matP</i> at a semi permissive temperature (38°C). F) Southern blot analysis of the Topo IV cleavage at the <i>dif</i> and 1.9Mb sites in cells with a circular or linearized chromosome. G) Phenotypes observed during exponential growth in LB in the <i>matP</i> mutant strains with circular or linear chromosome (DNA is labeled with DAPI, green). Scale bar is 5μm.</p

Topo IV binding pattern of replicating chromosome.

<p>A) Circos plot of the ChIP-seq experiments for ParC-flag and ParE-flag. The IP / input ratio over the entire <i>E</i>. <i>coli</i> genome is presented for three independent experiments, one IP on the <i>parC-flag</i> strain and two IPs on the <i>parE-flag</i> strain. From the center to the outside, circles represent: genomic coordinates, macrodomain map, position of tRNA genes and ribosomal operons, ParE-Flag 1 ChIP-seq (untreated data, orange), ParE-Flag 1 ChIP-seq (filtered data, red), ParE-Flag 2 ChIP-seq (untreated data, orange), ParE-Flag 2 ChIP-seq (filtered data, red), ParC-Flag ChIP-seq (untreated data, orange), ParC-Flag ChIP-seq (filtered data, red), position of the 19 validated Topo IV binding sites. The right panels represent magnifications for four specific Topo IV binding sites, position 1.25 Mb, position 1.58 Mb (<i>dif</i>), position 1.85 Mb and position 2.56Mb. The three first rows correspond to filtered IP/Input ratio for ParC-Flag, ParE-Flag1 and ParE-Flag2 IPs, the fourth and fifth rows correspond respectively to the forward and reverse raw read numbers of the <i>parC-flag</i> experiment. The position and orientation of genes are illustrated at the bottom of each panel. B) Sliding averages of the IP (blue, left Y axis), Input (red, left Y axis) and IP/input (green, right Y axis) data for the <i>parC-flag</i> experiment over 60 kb regions along the genome. To facilitate the reading, <i>oriC</i> is positioned at 0 and 4.639 Mb. C) Analysis of Topo IV binding during the bacterial cell cycle. Marker frequency analysis was used to demonstrate the synchrony of the population at each time point. Stars represent the position of the selected Topo IV sites. D) IP/input ratio for 7 regions presenting specific Topo IV enrichment during S and G2 phases. For each genomic position the maximum scale is set to the maximum IP/Input ratio observed.</p