Stress Responses of the Industrial Workhorse <i>Bacillus licheniformis</i> to Osmotic Challenges

<div><p>The Gram-positive endospore-forming bacterium <i>Bacillus licheniformis</i> can be found widely in nature and it is exploited in industrial processes for the manufacturing of antibiotics, specialty chemicals, and enzymes. Both in its varied natural habitats and in industrial settings, <i>B. licheniformis</i> cells will be exposed to increases in the external osmolarity, conditions that trigger water efflux, impair turgor, cause the cessation of growth, and negatively affect the productivity of cell factories in biotechnological processes. We have taken here both systems-wide and targeted physiological approaches to unravel the core of the osmostress responses of <i>B. licheniformis</i>. Cells were suddenly subjected to an osmotic upshift of considerable magnitude (with 1 M NaCl), and their transcriptional profile was then recorded in a time-resolved fashion on a genome-wide scale. A bioinformatics cluster analysis was used to group the osmotically up-regulated genes into categories that are functionally associated with the synthesis and import of osmostress-relieving compounds (compatible solutes), the SigB-controlled general stress response, and genes whose functional annotation suggests that salt stress triggers secondary oxidative stress responses in <i>B. licheniformis</i>. The data set focusing on the transcriptional profile of <i>B. licheniformis</i> was enriched by proteomics aimed at identifying those proteins that were accumulated by the cells through increased biosynthesis in response to osmotic stress. Furthermore, these global approaches were augmented by a set of experiments that addressed the synthesis of the compatible solutes proline and glycine betaine and assessed the growth-enhancing effects of various osmoprotectants. Combined, our data provide a blueprint of the cellular adjustment processes of <i>B. licheniformis</i> to both sudden and sustained osmotic stress. </p> </div

Induction of <i>proHJAA</i> transcription in <i>B. licheniformis</i> DSM 13^T in response to salt stress.

<p>(A) Genetic organization of the <i>B. licheniformis</i> DSM 13^T<i>proHJAA</i> locus with its indicated promoter and transcriptional terminator regions. The localization of the single-stranded anti-sense RNA used as probes in the Northern blot analysis of the <i>proHJAA</i> gene cluster are indicated as black bars below the individual gene symbols. (B) Northern blot analysis of the <i>proHJAA</i> transcript. Total RNA was isolated from cultures of <i>B. licheniformis</i> DSM 13^T that were grown in SMM either in the absence (-) or the presence (+) of 0.8 M NaCl. Gene-specific RNA transcripts were identified by hybridization of total RNA to DIG-labeled single-stranded anti-sense RNA probes. The arrow indicates the position of an approximately 3,400 nucleotide mRNA species that corresponds to the full-length mRNA of the <i>proHJAA</i> operon. (C) DNA sequence of the <i>proH</i> promoter regions of the <i>B. subtilis</i> and <i>B. licheniformis</i> DSM 13^T chromosomes. The start site (indicated by an arrow) mapped for the <i>B. subtilis </i><i>proHJ</i> mRNA transcript via primer extension analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B54" target="_blank">54</a>] revealed a SigA–type promoter (shown in red, with boxed -10, -16 and -35 sequences [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B79" target="_blank">79</a>]) and a putative ribosome-binding site (RBS) located upstream of the predicted ATG start codon of the <i>proH</i> coding region. DNA sequences resembling those of the <i>B. subtilis </i><i>proHJ</i> promoter [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B54" target="_blank">54</a>] can be found in the <i>B. licheniformis</i> DSM 13^T<i>proHJAA</i> promoter region.</p

Use of L-proline as a nutrient by <i>B. licheniformis</i> DSM 13^T.

<p>The proline catabolic system has been studied in <i>B. subtilis</i> where a high-affinity proline transporter (PutP), two proline catabolic enzymes (PutB-PutC) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B82" target="_blank">82</a>], and a proline-responsive activator protein (PutR) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B83" target="_blank">83</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B84" target="_blank">84</a>] have been functionally studied. (A) This proline catabolic pathway is predicted from the genome sequence to be present in <i>B. licheniformis</i> DSM 13^T as well [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B13" target="_blank">13</a>]. (B) Genetic organization of the <i>put</i> locus of <i>B. licheniformis</i> DSM 13^T. (C) <i>B. licheniformis</i> DSM 13^T cells were grown in SMM with glucose as the carbon source without the addition of a nitrogen (N) source (-) or in the presence of 15 mM (NH₄)₂SO₄ [NH₄⁺, 30 mM L-proline [Pro], or 30 mM glycine betaine [GB], respectively. Growth yields of the cultures were measured after 16 h of incubation at 37 °C in a shaking water bath. (D) <i>B. licheniformis</i> DSM 13^T cells were grown in SMM with (NH₄)₂SO₄ [15 mM] in the absence (-) of a carbon source or in the presence of 27 mM glucose [Gluc], 32.4 mM L-proline [Pro] or 32.4 mM glycine betaine [GB]. Growth yields of the cultures were measured after 16 h of incubation at 37 °C in a shaking water bath. The error bars give the standard deviation of three independently grown cultures. </p

Growth yields, proline production and osmoprotection of <i>B. licheniformis</i> DSM 13^T by compatible solutes.

<p>(A) Cultures of <i>B. licheniformis</i> DSM13^T were grown at 37° C in SMM with glucose as the carbon source in the presence of the indicated NaCl concentrations. Growth yields of the cultures (as assessed by measuring the OD₅₇₈) were determined after 14 h of incubation. (B) Proline content of osmotically stressed <i>B. licheniformis</i> DSM 13^T cells. Cultures were grown in SMM with the indicated salinities either in the absence (red symbol) or in the presence (black symbol) of 1 mM of the osmoprotectant glycine betaine to an optical density (OD₅₇₈) of approximately 2. The proline content of the cells was determined by HPLC analysis. The data shown represent one typical experiment. (C) Salt-stress protection of <i>B. licheniformis</i> DSM 13^T by exogenously provided compatible solutes. Cultures of <i>B. licheniformis</i> DSM 13^T were grown in SMM either in the absence (hatched bar) or in the presence of 1.3 M NaCl (black bars) in the absence (-) or in the presence of various compatible solutes. GB: glycine betaine; Cho: choline; Pro: proline; PB: proline betaine; Car: carnitine; COS: choline-O-sulfate; DMSP: dimethylsulfoniopropionate; Ect: ectoine; OHEct: hydroxyectoine. The compatible solutes were added to the growth medium at a final concentration of 1 mM. Growth yields of the cultures were measured after 14 h of incubation at 37 °C in a shaking water bath. The data shown were derived from two independently grown cultures. </p

Physiological complementation of a <i>B. subtilis</i><i>proHJ</i> mutant strain by the heterologous <i>proHJAA</i> operon of <i>B. licheniformis</i> DSM 13^T.

<p>The <i>proHJAA</i> operon of <i>B. licheniformis</i> DSM 13^T was cloned into plasmid pX, yielding plasmid pTMB20. pTMB20 and the empty cloning vector pX (used as a control) were recombined in a single copy into the chromosomal <i>amyE</i> sites of the <i>B. subtilis</i> wild-type strain JH642 and its [Δ(<i>proHJ</i>::<i>tet</i>)1] mutant derivative JSB8 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B72" target="_blank">72</a>]. This resulted in the construction of the following <i>B. subtilis</i> strains: TMB134 [<i>proHJ</i> wild type and <i>amyE</i>::pX], TMB135 [<i>proHJ</i> wild type and <i>amyE::proHJAA</i>], TMB136 [Δ(<i>proHJ</i>::<i>tet</i>)1 and <i>amyE</i>::pX], and TMB137 [Δ(<i>proHJ</i>::<i>tet</i>)1 and <i>amyE::proHJAA</i>]. (A) Cultures of these strains were grown in SMM without (black bars) or with 0.8 M NaCl (hatched bars). Their growth yields (OD₅₇₈) were measured after 16 h of incubation at 37 °C. (B) Proline content of recombinant <i>B. subtilis</i> strains grown in SMM without (black bars) or with 0.8 M NaCl (hatched bars). When the cultures had reached mid-exponential growth phase (OD₅₇₈ of about 2), the cells were harvested, their total solute pool was extracted and the intracellular proline concentrations were determined by HPLC analysis. The error bars represent the standard deviations of the proline pools found in three independently grown cultures. The same set of strains as that shown in panel (A) was used for this experiment.</p

Predicted secondary structures of the <i>B. licheniformis</i> DSM 13^T<i>proI</i> and <i>proBA</i> mRNA leader transcripts.

<p>(A) Overview of the genetic organization of the structural genes in the genome of <i>B. licheniformis</i> DSM 13^T [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B13" target="_blank">13</a>] for the ProB-ProA-ProI anabolic proline biosynthetic route. The predicted secondary structures of the non-coding 5’-regions of the <i>proI</i> (B) and <i>proBA</i> (C) mRNA leader sequences were generated with the Mfold algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B115" target="_blank">115</a>] and edited manually for their termination and anti-termination configurations. The suggested proline-specific specifier codons (CCU) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B72" target="_blank">72</a>] in the T-box element for the <i>proI</i> and <i>proBA</i> mRNA leader sequences are shown in green, and the T-box signature sequences are marked in red. Asterisks indicate other short sequences conserved in the T-box gene family [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B76" target="_blank">76</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B116" target="_blank">116</a>].</p

Synthesis of glycine betaine from the precursor choline by <i>B. licheniformis</i> DSM 13^T in response to high salinity.

<p>(A) Externally provided choline is predicted to be taken up via the ABC-transporter OpuC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B64" target="_blank">64</a>] and then converted into the compatible solute glycine betaine in a two-step oxidation reaction that involves the GbsB (choline dehydogenase) and GbsA (glycine betaine aldehyde dehydrogenase) enzymes in <i>B. subtilis</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B61" target="_blank">61</a>] and their counterparts in <i>B. licheniformis</i> DSM 13^T. (B) Genetic organization of the osmotically inducible <i>opuC</i> [<i>opuCA-opuCB-opuCC-opuCD</i>] cluster encoding the OpuC transporter, the <i>gbsAB</i> biosynthetic operon, and the <i>gbsR</i> regulatory gene [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B59" target="_blank">59</a>] in the genome of <i>B. licheniformis</i> DSM 13^T [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B13" target="_blank">13</a>]. (C) The intracellular choline and (D) glycine betaine content of the cells were analyzed by ¹H-NMR spectroscopy from unstressed and NaCl-stressed cells (the final NaCl concentration in the growth medium was 1 M). For this experiment, the cells were grown to mid-exponential growth phase in BMM or BMM with 1 M NaCl either in the absence (-) or in the presence of 1 mM choline (+ Cho). Intracellular choline and glycine betaine concentrations were absolutely quantified and normalized to cell dry weight (CDW) [nmol/mg CDW]. The error bars give the standard deviation of three independently grown cultures. </p

Cluster analysis of transcriptional changes in response to a sudden salt challenge.

<p>Cells of <i>B. licheniformis</i> DSM 13^T were cultivated in BMM with glucose as the carbon source until they reached early exponential growth phase (OD_500nm of about 0.4) when they were exposed to a sudden salt shock (the final NaCl concentration in the growth medium was 1.0 M). Immediately before and at the indicated time intervals subsequent to the imposed increase in the external salinity, cells were withdrawn and used for the isolation of total RNA for a genome-wide transcriptional analysis. The derived data were then subjected to a cluster analysis and grouped according to known salt stress response clusters from <i>B. subtilis</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B39" target="_blank">39</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#B65" target="_blank">65</a>]: (A) Synthesis and transport of compatible solutes, (B) general stress responses, (C) ECF-sigma factor genes, and (D) secondary oxidative stress response. The correlation of the transcription patterns of the different clustered genes is represented on the X-axis (cosine correlation). Detailed values for the transcriptional profile of individual genes are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080956#pone.0080956.s001" target="_blank">Table S1</a>. Genes marked in red represents those whose transcription is up-regulated in response to osmotic stress. </p

The cytosolic proteome of salt-stressed <i>B. licheniformis</i> DSM 13^T cells 30 min after the imposition of a NaCl shock.

<p>Cells of <i>B. licheniformis</i> DSM 13^T were cultivated in BMM with glucose as the carbon source until they reached early exponential growth phase (OD_500nm of about 0.4) when they were exposed to a sudden salt shock (the final NaCl concentration in the growth medium was 1.0 M). 30 min after the exposure to the salt stress, samples of cells were harvested and processed for 2D-PAGE analysis; proteins were separated in a pH gradient 4‑7. The control sample of the cells was harvested just prior to the imposed salt shock. Cell samples were labeled with L-[³⁵S]-methionine during the exponential growth phase (control, OD_500nm 0.4) and 30 min after the addition of NaCl. Dual channel images were created from the 2D-gels with the Delta 2D software (Decodon GmbH, Greifswald, Germany). </p

Novel Class of Potent and Cellularly Active Inhibitors Devalidates MTH1 as Broad-Spectrum Cancer Target

MTH1 is a hydrolase responsible for sanitization of oxidized purine nucleoside triphosphates to prevent their incorporation into replicating DNA. Early tool compounds published in the literature inhibited the enzymatic activity of MTH1 and subsequently induced cancer cell death; however recent studies have questioned the reported link between these two events. Therefore, it is important to validate MTH1 as a cancer dependency with high quality chemical probes. Here, we present BAY-707, a substrate-competitive, highly potent and selective inhibitor of MTH1, chemically distinct compared to those previously published. Despite superior cellular target engagement and pharmacokinetic properties, inhibition of MTH1 with BAY-707 resulted in a clear lack of <i>in vitro</i> or <i>in vivo</i> anticancer efficacy either in mono- or in combination therapies. Therefore, we conclude that MTH1 is dispensable for cancer cell survival