Isolation of <i>B. subtilis</i> mutants that have inactivated the <i>gudB</i> gene.

<p>(A) Lack of exogenous glutamate is the driving force allowing the selection of mutants with inactivated <i>gudB</i> alleles. (B) Prior to growth in the absence of glutamate, the <i>B. subtilis rocG^–</i> mutant strain GP801 (<i>ΔrocG gudB⁺</i>) expressing only the active GDH, GudB was grown in C minimal medium supplemented with glucose and glutamate as carbon and nitrogen sources (plus glutamate), respectively. During growth in minimal medium lacking glutamate (no glutamate) samples were taken at indicated time points. (C) 5 µl were plated from serial dilutions (from non-diluted till 10⁻⁶) of cell suspensions of the <i>gudB^CR</i> and <i>gudB⁺</i> control strains GP754 (<i>ΔrocG gudB^CR</i>) and GP801 (<i>ΔrocG gudB⁺</i>), respectively, and the isolated <i>gudB^–</i> mutants for phenotypic analyses. The dilutions were spotted on minimal medium agar plates supplemented either with glucose and ammonium or with glutamate and ammonium. The plates were incubated for 48 h at 37°C. (D) Western blot analysis to monitor synthesis of the GDH, GudB in the <i>gudB^–</i> isolates using GDH-specific antibodies. Results of the sequence analysis of the <i>gudB^–</i> alleles are summarized below (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066120#pone-0066120-t001" target="_blank">Table 1</a>).</p

Glutamate biosynthesis and degradation in <i>B. subtilis</i>.

<p>(A) The link between carbon and nitrogen metabolism. GS, glutamine synthetase; GOGAT, glutamate synthase; GDH, glutamate dehydrogenase. (B) In the presence of glucose GltC activates the <i>gltAB</i> operon and the synthesized GOGAT converts 2-oxoglutarate and glutamine to glutamate. In the presence of arginine the GDH RocG is synthesized and the catabolically active enzyme binds to GltC and inhibits its DNA-binding activity.</p

Selection-Driven Accumulation of Suppressor Mutants in <i>Bacillus subtilis</i>: The Apparent High Mutation Frequency of the Cryptic <i>gudB</i> Gene and the Rapid Clonal Expansion of <i>gudB⁺</i> Suppressors Are Due to Growth under Selection

<div><p>Soil bacteria like <i>Bacillus subtilis</i> can cope with many growth conditions by adjusting gene expression and metabolic pathways. Alternatively, bacteria can spontaneously accumulate beneficial mutations or shape their genomes in response to stress. Recently, it has been observed that a <i>B. subtilis</i> mutant lacking the catabolically active glutamate dehydrogenase (GDH), RocG, mutates the cryptic <i>gudB^CR</i> gene at a high frequency. The suppressor mutants express the active GDH GudB, which can fully replace the function of RocG. Interestingly, the cryptic <i>gudB^CR</i> allele is stably inherited as long as the bacteria synthesize the functional GDH RocG. Competition experiments revealed that the presence of the cryptic <i>gudB^CR</i> allele provides the bacteria with a selective growth advantage when glutamate is scarce. Moreover, the lack of exogenous glutamate is the driving force for the selection of mutants that have inactivated the active <i>gudB</i> gene. In contrast, two functional GDHs are beneficial for the cells when glutamate was available. Thus, the amount of GDH activity strongly affects fitness of the bacteria depending on the availability of exogenous glutamate. At a first glance the high mutation frequency of the cryptic <i>gudB^CR</i> allele might be attributed to stress-induced adaptive mutagenesis. However, other loci on the chromosome that could be potentially mutated during growth under the selective pressure that is exerted on a GDH-deficient mutant remained unaffected. Moreover, we show that a GDH-proficient <i>B. subtilis</i> strain has a strong selective growth advantage in a glutamate-dependent manner. Thus, the emergence and rapid clonal expansion of the active <i>gudB</i> allele can be in fact explained by spontaneous mutation and growth under selection without an increase of the mutation rate. Moreover, this study shows that the selective pressure that is exerted on a maladapted bacterium strongly affects the apparent mutation frequency of mutational hot spots.</p></div

Stabilities of DRs present in the native <i>gudB^CR</i> and in the <i>gudB^CR_Sac</i>_I<i>-gfp</i> alleles.

<p>(A) In addition to the native <i>gudB^CR</i> allele, a second <i>gudB^CR-gfp</i> fusion that could be potentially mutated during growth of a <i>B. subtilis ΔrocG</i> mutant under selective pressure was introduced into the <i>amyE</i> locus on the chromosome. (B) DNA species comprising the 9 bp DR were amplified by colony PCR using <i>gudB</i>-specific oligonucleotides (see Materials and Methods). To distinguish the DNA species derived from the two <i>gudB^CR</i> alleles, a <i>Sac</i>I site was introduced into the <i>gudB^CR-gfp</i> allele by exchanging G at position 402 by C. (C) Schematic illustration of the fragment pattern of DNA species obtained from cells collected prior to selective growth and after selection. The same samples were treated with <i>Sac</i>I. The emergence of a 147 bp DNA species shown by red letters would indicate the decryptification of the <i>gudB^CR-gfp</i> allele. (D) Fragment pattern of DNA species obtained from real samples.</p

Effect of glutamate supply on the clonal expansion of <i>gudB</i> mutants.

<p>The <i>B. subtilis ΔrocG</i> mutant strain GP754 (<i>ΔrocG gudB^–</i>) was grown in C minimal medium supplemented with glucose and ammonium (no glutamate), glucose and ammonium/glutamate (plus glutamate), and in SP (rich) medium. The bars represent standard deviations for four independently repeated experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066120#pone.0066120.s010" target="_blank">Table S5</a>). The amount of <i>gudB^CR</i> and <i>gudB⁺</i> mutants at the indicated time points are shown by light brown and black bars, respectively.</p

Intraspecies competition experiment to identify the selective advantage for keeping the <i>gudB^CR</i> allele in the laboratory strain 168.

<p>(A) Mixed populations of strains BP40 (<i>rocG⁺ gudB^CR amyE::yfp</i>) and BP52 (<i>rocG⁺ gudB⁺ amyE::cfp</i>) or BP41 (<i>rocG⁺ gudB^CR amyE::cfp</i>) and BP156 (<i>rocG⁺ gudB⁺ amyE::yfp</i>) were grown for up to 24 h in C minimal medium supplemented with glucose and ammonium, and in minimal medium supplemented with glucose, ammonium and glutamate. (B) Prior to co-cultivation (0 h), and after 7 h and 24 h of growth dilutions of cells were plated on complex medium. The surviving cells that emerged after 12 h of incubation were identified by fluorescence microscopy and counted. Exposure time, 0.6 s; Scale bar, 1 mm. (C) Outcome of the competition experiment. The bars represent standard deviations for at least four independently repeated experiments.</p

Stabilities of DRs during growth under strong selective pressure that is exerted on a <i>B. subtilis rocG^– gudB^CR</i> mutant.

<p>The stabilities of DRs in a population of <i>gudB⁺</i> cells that originated from the <i>rocG^–</i> mutant GP747 were analyzed by colony PCR.</p

A GFP-based system to monitor the state of the <i>gudB</i> allele in <i>B. subtilis</i>.

<p>(A) The principle of the system is based on the stabilities of the inactive and active GudB^CR and GudB proteins, respectively. (B) Growth assay to confirm the enzymatic activity of the GFP-GudB fusion protein. C minimal medium supplemented with glucose and glutamate as the carbon and nitrogen sources, respectively, served as the positive control. Strains GP1165 (<i>ΔrocG gudB⁺</i>) and BP23 (<i>ΔrocG gfp-gudB⁺</i>) synthesizing the active GudB (dark green) and GFP-GudB (light green) alleles, respectively, were capable of catabolizing glutamate. The strains GP1163 (<i>ΔrocG gudB^CR</i>) and BP22 (<i>ΔrocG gfp-gudB^CR</i>) synthesizing the inactive <i>gudB^CR</i> (yellow) and <i>gfp-gudB^CR</i> (blue) alleles, respectively, did not grow with glutamate as the single source of carbon and nitrogen. (C) Western blot analysis to evaluate the stabilities of the inactive and active GudB^CR and GudB variants in strains GP754 (<i>ΔrocG gudB^CR</i>) and GP801 (<i>ΔrocG gudB⁺</i>), respectively, using polyclonal antibodies raised against GDH and GFP. The corresponding GFP-GudB^CR and GFP-GudB fusion proteins are synthesized in strains BP22 (<i>ΔrocG gfp-gudB^CR</i>) and BP23 (<i>ΔrocG gfp-gudB⁺</i>), respectively. (D) Fluorescence of microcolonies of strains BP22 and BP23 that express the cryptic <i>gfp-gudB^CR</i> and the active <i>gfp-gudB⁺</i> fusion genes, respectively. Exposure time, 1 s; scale bar, 5 µm.</p

Le Miroir des sports : publication hebdomadaire illustrée

17 août 19371937/08/17 (N964)-1937/08/17

Additional file 1: Figure S1. of Comparative genome and phenotypic analysis of three Clostridioides difficile strains isolated from a single patient provide insight into multiple infection of C. difficile

Growth curves in BHIS of DSM 27638, DSM 27639 and DSM 27640. The isolates were grown in brain heart infusion medium containing 0.5% (w/v) yeast extract und 0.03% (w/v) L-cysteine. All isolates have the same growth rate under laboratory conditions as shown as the means of three replicates with standard deviation. (DOCX 55 kb