Antibiotic sensitivity reveals that wall teichoic acids mediate DNA binding during competence in Bacillus subtilis

Despite decades of investigation of genetic transformation in the model Gram-positive bacterium Bacillus subtilis, the factors responsible for exogenous DNA binding at the surface of competent cells remain to be identified. Here, we report that wall teichoic acids (WTAs), cell wall-anchored anionic glycopolymers associated to numerous critical functions in Gram-positive bacteria, are involved in this initial step of transformation. Using a combination of cell wall-targeting antibiotics and fluorescence microscopy, we show that competence-specific WTAs are produced and specifically localized in the competent cells to mediate DNA binding at the proximity of the transformation apparatus. Furthermore, we propose that TuaH, a putative glycosyl transferase induced during competence, modifies competence-induced WTAs in order to promote (directly or indirectly) DNA binding. On the basis of our results and previous knowledge in the field, we propose a model for DNA binding and transport during genetic transformation in B. subtilis

Antibiotic sensitivity reveals that wall teichoic acids mediate DNA binding during competence in Bacillus subtilis

Natural genetic transformation in bacteria requires DNA binding at the surface of competent cells. Here, Mirouze et al. show that wall teichoic acids are specifically produced or modified during competence in Bacillus subtilis and promote (directly or indirectly) DNA binding at the cell surface

Contrasting mechanisms of growth in two model rod-shaped bacteria

How cells control their shape and size is a long-standing question in cell biology. Many rod-shaped bacteria elongate their sidewalls by the action of cell wall synthesizing machineries that are associated to actin-like MreB cortical patches. However, little is known about how elongation is regulated to enable varied growth rates and sizes. Here we use total internal reflection fluorescence microscopy and single-particle tracking to visualize MreB isoforms, as a proxy for cell wall synthesis, in Bacillus subtilis and Escherichia coli cells growing in different media and during nutrient upshift. We find that these two model organisms appear to use orthogonal strategies to adapt to growth regime variations: B. subtilis regulates MreB patch speed, while E. coli may mainly regulate the production capacity of MreB-associated cell wall machineries. We present numerical models that link MreB-mediated sidewall synthesis and cell elongation, and argue that the distinct regulatory mechanism employed might reflect the different cell wall integrity constraints in Gram-positive and Gram-negative bacteria

MreB Forms Subdiffraction Nanofilaments during Active Growth in Bacillus subtilis

The construction of the bacterial cell envelope is a fundamental topic, as it confers its integrity to bacteria and is consequently the target of numerous antibiotics. MreB is an essential protein suspected to regulate the cell wall synthetic machineries. Despite two decades of study, its localization remains the subject of controversies, its description ranging from helical filaments spanning the entire cell to small discrete entities. The true structure of these filaments is important because it impacts the model describing how the machineries building the cell wall are associated, how they are coordinated at the scale of the entire cell, and how MreB mediates this regulation. Our results shed light on this debate, revealing the size of native filaments in B. subtilis during growth. They argue against models where MreB filament size directly affects the speed of synthesis of the cell wall and where MreB would coordinate distant machineries along the side wall.The actin-like MreB protein is a key player of the machinery controlling the elongation and maintenance of the cell shape of most rod-shaped bacteria. This protein is known to be highly dynamic, moving along the short axis of cells, presumably reflecting the movement of cell wall synthetic machineries during the enzymatic assembly of the peptidoglycan mesh. The ability of MreB proteins to form polymers is not debated, but their structure, length, and conditions of establishment have remained unclear and the subject of conflicting reports. Here we analyze various strains of Bacillus subtilis, the model for Gram-positive bacteria, and we show that MreB forms subdiffraction-limited, less than 200 nm-long nanofilaments on average during active growth, while micron-long filaments are a consequence of artificial overaccumulation of the protein. Our results also show the absence of impact of the size of the filaments on their speed, orientation, and other dynamic properties conferring a large tolerance to B. subtilis toward the levels and consequently the lengths of MreB polymers. Our data indicate that the density of mobile filaments remains constant in various strains regardless of their MreB levels, suggesting that another factor determines this constant

PamR, a new MarR-like regulator affecting prophages and metabolic genes expression in <i>Bacillus subtilis</i>

<div><p><i>B</i>. <i>subtilis</i> adapts to changing environments by reprogramming its genetic expression through a variety of transcriptional regulators from the global transition state regulators that allow a complete resetting of the cell genetic expression, to stress specific regulators controlling only a limited number of key genes required for optimal adaptation. Among them, MarR-type transcriptional regulators are known to respond to a variety of stresses including antibiotics or oxidative stress, and to control catabolic or virulence gene expression. Here we report the characterization of the <i>ydcFGH</i> operon of <i>B</i>. <i>subtilis</i>, containing a putative MarR-type transcriptional regulator. Using a combination of molecular genetics and high-throughput approaches, we show that this regulator, renamed PamR, controls directly its own expression and influence the expression of large sets of prophage-related and metabolic genes. The extent of the regulon impacted by PamR suggests that this regulator reprograms the metabolic landscape of <i>B</i>. <i>subtilis</i> in response to a yet unknown signal.</p></div

The YdcH regulon.

<p>Pie charts summarizing genome-wide transcriptional profiling by RNAseq comparing gene expressions in a WT (ABS2005) and a Δ<i>ydcH</i> strain (ASEC56). The 363 genes retained (left chart) were reproducibly and statistically induced (182, right up) or repressed (181, right down) in the mutant compared to the wt by at least a two-fold factor. Genes were sorted by functional categories (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189694#pone.0189694.s007" target="_blank">S5 Table</a> for complete results), then regrouped into families of functions: Metabolism (carbon sources, amino acids, lipids, nucleotides and other metabolic pathways; electron transport & ATP synthesis; transport of sugars and other metabolites), stress response, information processing (DNA replication, segmentation, modification, recombination and repair; RNA and protein synthesis, modification and degradation), cellular processes (cell division; cell envelope synthesis, modification and degradation; ion homeostasis), lifestyles (motility & chemotaxis; biofilms formation; competence; sporulation), prophages & mobile genetic elements, and unknown. Numbers indicate the number of gene for each category.</p

YdcH binds specifically to inverted repeats in the promoter region of <i>ydcFGH</i>.

<p>A. Sequence of the region upstream of the <i>ydcFGH</i> operon. The two identified IR are indicated as green arrows. The transcriptional upshift previously identified is indicated as “up”, putative -35, -10 and rbs sequences are underlined, and the <i>ydcF</i> orf is boxed. B. EMSAs (right panels) showing the specific binding of PamR to DNA fragments corresponding to the wild type (wt) and mutated (IR1*) <i>ydcF</i> promoter, and schematic representation of the corresponding area (left panel). IRs are drawn as facing triangles, plain for the wild types and hollowed for the mutated. The quantity of YdcH (in pmol) incubated with 0.1 pmol of labeled target DNA is indicated above each lane.</p

The expression of <i>ydcFGH</i> is driven by two promoters.

<p>A. Schematic representation of the DNA fragments of the <i>ydcFGH</i> locus used for generating <i>lacZ</i> reporter fusions. The two putative promoters are indicated by arrows and the names of the resulting transcriptional fusion to <i>lacZ</i> are indicated below. On the right is displayed a picture of an X-Gal-LB plate to visualize LacZ activity of colonies harboring <i>lacZ</i> transcriptional fusions to P_<i>ydc1</i>, P_<i>ydc2</i>, P_{<i>ydc1-2</i>} or P_<i>ydc0</i> placed in either WT (ABS1761; ABS1763; ABS1765; ABS1767, respectively), or Δ<i>ydcH</i> (ABS1820; ABS1821; ABS1822; ABS1823, respectively), and to P_<i>ydc1</i> or P_<i>ydc2</i> in Δ<i>ydcF</i> (ASEC297; ASEC333) or Δ<i>ydcG</i> (ASEC301; ASEC335) background. B. Expression of a P_<i>ydc1</i> <i>luxABCDE</i> transcriptional fusion in cells grown in LB medium, in a wild type (red; ABS2005) or mutant for <i>ydcF</i> (green; ASEC325), <i>ydcG</i> (purple; ASEC327) or <i>ydcH</i> (blue; ASEC329) background. Note that the Δ<i>ydcH</i> data are relative to the upper part of the ordinate axis (in blue). Growth curves are presented as dotted lines and correspond to the optical density at 600nm while luciferase activities (plain lines) are relative luminescence units normalized by the OD_600nm.</p

<i>ydcFGH</i>, an operon of unknown function induced in a Δ<i>mreB</i> strain.

<p>A. Schematic representation of the genetic organization of the <i>B</i>. <i>subtilis ydcFGH</i> locus. Gene size of the orfs and putative functions are indicated above each gene. “P_<i>ydc</i> <i>lacZ</i>” shows the approximate size and localization on the locus of the DNA fragment amplified to construct the transcriptional reporter fusion to <i>lacZ</i> (strain ABS1761). B. A P_<i>ydc</i> <i>lacZ</i> fusion is induced in a strain lacking <i>mreB</i> (3725 “Δ<i>mreB”</i>; ABS1762) but not <i>mbl</i> (Δ<i>mbl</i>; ABS1769) nor in its wild type parent (Wt; ABS1761). C. Transformation of chromosomal DNA from strain ABS1761 (<i>amyE</i>::P_<i>ydc</i> <i>lacZ-spc</i>) into the recipient 3725 (neo- Δ<i>mreB</i>) leads to 100% of the spectinomycin/kanamycin resistant colonies expressing the <i>lacZ</i> reporter fusion (left) while the reverse transformation (right) leads to a limited number of blue colonies, indicating the absence of genetic link between Δ<i>mreB</i> and the factor inducing the reporter.</p

Sequence variations detected in strain 3725.

<p>Sequence variations detected in strain 3725.</p