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L-Proline Synthesis Mutants of Bacillus subtilis Overcome Osmotic Sensitivity by Genetically Adapting L-Arginine Metabolism.

ABSTRACT: The accumulation of the compatible solute L-proline by Bacillus subtilis via synthesis is a cornerstone in the cell's defense against high salinity as the genetic disruption of this biosynthetic process causes osmotic sensitivity. To understand how B. subtilis could potentially cope with high osmolarity surroundings without the functioning of its natural osmostress adaptive L-proline biosynthetic route (ProJ-ProA-ProH), we isolated suppressor strains of proA mutants under high-salinity growth conditions. These osmostress-tolerant strains carried mutations affecting either the AhrC transcriptional regulator or its operator positioned in front of the argCJBD-carAB-argF L-ornithine/L-citrulline/L-arginine biosynthetic operon. Osmostress protection assays, molecular analysis and targeted metabolomics showed that these mutations, in conjunction with regulatory mutations affecting rocR-rocDEF expression, connect and re-purpose three different physiological processes: (i) the biosynthetic pathway for L-arginine, (ii) the RocD-dependent degradation route for L-ornithine, and (iii) the last step in L-proline biosynthesis. Hence, osmostress adaptation without a functional ProJ-ProA-ProH route is made possible through a naturally existing, but inefficient, metabolic shunt that allows to substitute the enzyme activity of ProA by feeding the RocD-formed metabolite γ-glutamate-semialdehyde/Δ¹-pyrroline-5-carboxylate into the biosynthetic route for the compatible solute L-proline. Notably, in one class of mutants, not only substantial L-proline pools but also large pools of L-citrulline were accumulated, a rather uncommon compatible solute in microorganisms. Collectively, our data provide an example of the considerable genetic plasticity and metabolic resourcefulness of B. subtilis to cope with everchanging environmental conditions.

SUBMITTER: Stecker D

PROVIDER: S-EPMC9245794 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Genetic manipulation of bacterial spores of the genus Bacillus has shown potential for vaccination and for delivery of drugs or enzymes. Remarkably, proteins displayed on the spore surface retain activity and generally are not degraded. The heat stability of spores, coupled with their desiccation resistance, makes them suitable for delivery to humans or to animals by the oral route. Despite these attributes, one regulatory obstacle has remained regarding the fate of recombinant spores shed into the environment as viable spores. We have addressed the biological containment of GMO spores by utilizing the concept of a thymineless death, a phenomenon first reported 6 decades ago. Using Bacillus subtilis, we have inserted chimeric genes in the two thymidylate synthase genes, thyA and thyB, using a two-step process. Insertion is made first at thyA and then at thyB whereby resistance to trimethoprim enables selection of recombinants. Importantly, this method requires introduction of no new antibiotic resistance genes. Recombinant spores have a strict dependence on thymine (or thymidine), and in its absence cells lyse and die. Insertions are stable with no evidence for suppression or reversion. Using this system, we have successfully created a number of spore vaccines as well as spores displaying active enzymes.IMPORTANCE Genetic manipulation of bacterial spores offers a number of exciting possibilities for public and animal health, including their use as heat-stable vehicles for delivering vaccines or enzymes. Despite this, one remaining problem is the fate of recombinant spores released into the environment where they could survive in a dormant form indefinitely. We describe a solution whereby, following genetic manipulation, the bacterium is rendered dependent on thymine. As a consequence, spores if released would produce bacteria unable to survive, and they would exhibit a thymineless death due to rapid cessation of metabolism. The method we describe has been validated using a number of exemplars and solves a critical problem for containing spores of GMOs in the environment.

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L-Proline Synthesis Mutants of Bacillus subtilis Overcome Osmotic Sensitivity by Genetically Adapting L-Arginine Metabolism.

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