Evidence for Divisome Localization Mechanisms Independent of the Min System and SlmA in Escherichia coli

Cell division in Escherichia coli starts with assembly of FtsZ protofilaments into a ring-like structure, the Z-ring. Positioning of the Z-ring at midcell is thought to be coordinated by two regulatory systems, nucleoid occlusion and the Min system. In E. coli, nucleoid occlusion is mediated by the SlmA proteins. Here, we address the question of whether there are additional positioning systems that are capable of localizing the E. coli divisome with respect to the cell center. Using quantitative fluorescence imaging we show that slow growing cells lacking functional Min and SlmA nucleoid occlusion systems continue to divide preferentially at midcell. We find that the initial Z-ring assembly occurs over the center of the nucleoid instead of nucleoid-free regions under these conditions. We determine that Z-ring formation begins shortly after the arrival of the Ter macrodomain at the nucleoid center. Removal of either the MatP, ZapB, or ZapA proteins significantly affects the accuracy and precision of Z-ring positioning relative to the nucleoid center in these cells in accordance with the idea that these proteins link the Ter macrodomain and the Z-ring. Interestingly, even in the absence of Min, SlmA, and the putative Ter macrodomain – Z-ring link, there remains a weak midcell positioning bias for the Z-ring. Our work demonstrates that additional Z-ring localization systems are present in E. colithan are known currently. In particular, we identify that the Ter macrodomain acts as a landmark for the Z-ring in the presence of MatP, ZapB and ZapA proteins

The essential YycFG two-component system controls cell wall metabolism in Bacillus subtilis

THESIS 8328The work presented in this thesis describes a comprehensive analysis of the YycFG two-com ponent system (TCS) in Bacillus subtilis. TCSs are widespread mechanisms used by bacteria to sense and respond to the prevailing environmental conditions. YycFG is the only essential TCS encoded in the B. subtilis genome, but the nature of its essentiality has not been established. In addition, its regulon is poorly defined

DegU-P Represses Expression of the Motility fla-che Operon in Bacillus subtilis

Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth. The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility. The degU32(Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella. The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag, and of the fla-che operon is strongly reduced in a degU32(Hy) genetic background. To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32(Hy) that completely suppressed the motility deficiency. The mutations were genetically mapped and characterized by PCR and sequencing. Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che, thus allowing transcriptional readthrough from the cod operon. Two additional mutations improved the σ(A)-dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the P(A) promoter region of the fla-che operon. The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella

Suite of novel vectors for ectopic insertion of GFP, CFP and IYFP transcriptional fusions in single copy at the amyE andbglSloci in Bacillus subtilis

We report the development of a suite of sixintegrative vectors for construction of single copy transcriptional fusions with the gfpmut3, cfp and iyfp reporter genes in B. subtilis. The promoter fusions are constructed using the highly efficient ligation-independent cloning (LIC) technique making them suitable for high-throughput applications. The plasmids insert into the chromosomeby a double cross-over event at the amyEorbglS loci and integration at each site can be verified by a plate-based screening assay. The vectors allow expression of two different promoters to be determined in the same strain using the cfp and iyfp reporter genes since CFP and iYFP are spectrally distinct and have comparable half-lives of approximately 2 hours in exponentially growing B. subtilis cells. We demonstrate the versatility of these vectors by measuring expression of the tuaAand phoA operons singularly and in combination, during growth in phosphate limiting conditions

A matter of life and death: cell wall homeostasis and the WalKR (YycGF) essential signal transduction pathway

International audienceThe WalK/WalR (aka YycG/YycF) two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and specific to low G+C Gram-positive bacteria, including a number of important pathogens. An unusual feature is that this system is essential for viability in most of these bacteria. Recent studies have revealed conserved functions for this system, defining this signal transduction pathway as a crucial regulatory system for cell wall metabolism, that we have accordingly renamed WalK/WalR. Here we review the cellular role of the WalK/WalR TCS in different bacterial species, focusing on the function of genes in its regulon, as well as variations in walRK operon structure and the composition of its regulon. We also discuss the nature of its essentiality and the potential type of signal being sensed. The WalK histidine kinase of B. subtilis has been shown to localize to the divisome and we suggest that the WalKR system acts as an information conduit between extracytoplasmic cellular structures and intracellular processes required for their synthesis, playing a vital role in effectively co-ordinating peptidoglycan plasticity with the cell division process

Pre-reproductive maternal enrichment influences rat maternal care and offspring developmental trajectories. Behavioral performances and neuroplasticity correlates

Environmental enrichment (EE) is a widely used paradigm for investigating the influence of complex stimulations on brain and behavior. Here we examined whether pre-reproductive exposure to EE of female rats may influence their maternal care and offspring cognitive performances. To this aim, from weaning to breeding age enriched females (EF) were reared in enriched environments. Females reared in standard conditions were used as controls. At 2.5 months of age all females were mated and reared in standard conditions with their offspring. Maternal care behaviors and nesting activity were assessed in lactating dams. Their male pups were also behaviorally evaluated at different post-natal days (pnd). Brain BDNF, reelin and adult hippocampal neurogenesis levels were measured as biochemical correlates of neuroplasticity. EF showed more complex maternal care than controls due to their higher levels of licking, crouching and nest building activities. Moreover, their offspring showed higher discriminative (maternal odor preference T-maze, pnd 10) and spatial (Morris Water Maze, pnd 45; Open Field with objects, pnd 55) performances, with no differences in social abilities (Sociability test, pnd 35), in comparison to controls. BDNF levels were increased in EF frontal cortex at pups' weaning and in their offspring hippocampus at pnd 21 and 55. No differences in offspring reelin and adult hippocampal neurogenesis levels were found. In conclusion, our study indicates that pre-reproductive maternal enrichment positively influences female rats' maternal care and cognitive development of their offspring, demonstrating thus a transgenerational transmission of EE benefits linked to enhanced BDNF-induced neuroplasticity. BDNF; cognition; environmental enrichment; maternal behavior; neurogenesis; reeli

Evidence for Divisome Localization Mechanisms Independent of the Min System and SlmA in <i>Escherichia coli</i>

<div><p>Cell division in <i>Escherichia coli</i> starts with assembly of FtsZ protofilaments into a ring-like structure, the Z-ring. Positioning of the Z-ring at midcell is thought to be coordinated by two regulatory systems, nucleoid occlusion and the Min system. In <i>E. coli</i>, nucleoid occlusion is mediated by the SlmA proteins. Here, we address the question of whether there are additional positioning systems that are capable of localizing the <i>E. coli</i> divisome with respect to the cell center. Using quantitative fluorescence imaging we show that slow growing cells lacking functional Min and SlmA nucleoid occlusion systems continue to divide preferentially at midcell. We find that the initial Z-ring assembly occurs over the center of the nucleoid instead of nucleoid-free regions under these conditions. We determine that Z-ring formation begins shortly after the arrival of the Ter macrodomain at the nucleoid center. Removal of either the MatP, ZapB, or ZapA proteins significantly affects the accuracy and precision of Z-ring positioning relative to the nucleoid center in these cells in accordance with the idea that these proteins link the Ter macrodomain and the Z-ring. Interestingly, even in the absence of Min, SlmA, and the putative Ter macrodomain – Z-ring link, there remains a weak midcell positioning bias for the Z-ring. Our work demonstrates that additional Z-ring localization systems are present in <i>E. coli</i> than are known currently. In particular, we identify that the Ter macrodomain acts as a landmark for the Z-ring in the presence of MatP, ZapB and ZapA proteins.</p></div

Division frequency at the 1/4 and 1/2 cell positions with respect to mother cell length.

<p>(A) Data for the Δ<i>slmA</i> Δ<i>minC</i> double mutant strain (PB194); (B) Δ<i>minC</i> strain (JW1165). Cell lengths are binned at 0.25 µm intervals. Arrows point to transition regions from centrally occurring divisions to divisions at cell quarters. The lengths of the mother cells are measured just before cell division. Note that only a few cells from both strains are longer than 8 µm, limiting analysis for longer cells.</p

Positioning of the Z-rings relative to the cell and nucleoid centers in triple deletion strains.

<p>Composite of DAPI labelled nucleoid (red), ZipA-GFP (green) and phase contrast image in (A) Δ<i>slmA</i> Δ<i>min</i> Δ<i>matP</i>, (B) Δ<i>slmA</i> Δ<i>min</i> Δ<i>zapB</i>, and (C) Δ<i>slmA</i> Δ<i>min</i> Δ<i>zapA</i> cells. Scale bar is 2 µm. (D)–(F) Distribution of distances between the Z-ring center and nucleoid center for Δ<i>slmA</i> Δ<i>min</i> Δ<i>matP</i>, Δ<i>slmA</i> Δ<i>min</i> Δ<i>zapB</i>, and Δ<i>slmA</i> Δ<i>min</i> Δ<i>zapA</i> cells, respectively. (G)–(I) ΔX_z vs. ΔX_n in Δ<i>slmA</i> Δ<i>min</i> Δ<i>matP</i>, Δ<i>slmA</i> Δ<i>min</i> Δ<i>zapB</i>, and Δ<i>slmA</i> Δ<i>min</i> Δ<i>zapA</i> cells, respectively. Data are from cells with a single compact nucleoid and a central Z-ring. Straight lines correspond to .</p