Synthesis of the compatible solute proline by Bacillus subtilis: point mutations rendering the osmotically controlled proHJ promoter hyperactive

Abstract

The ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ-ProA-ProH), osmostress-adaptive synthesis of the compatible solute proline. The proA-encoded gamma-glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB-ProA-ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress-responsive proline production routes. A key player for enhanced osmostress-adaptive proline production is the osmotically regulated proHJ promoter. We used site-directed mutagenesis to study the salient features of this stress-responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA-type promoters serve to keep transcription low under non-inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA-type promoters of either 16-bp (the natural proHJ promoter), or 18-bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress-inducibility of transcription.

Role of the Extremolytes Ectoine and Hydroxyectoine as Stress Protectants and Nutrients: Genetics, Phylogenomics, Biochemistry, and Structural Analysis

“…Hence, despite the fact that E. coli does not synthesize ectoines naturally [ 187 ], the P. stutzeri ect promoter retained its exquisitely sensitive osmotic control in the heterologous host bacterium, indicating that osmoregulation of this promoter is an inherent feature of the rather small regulatory region per se. It is not yet clear yet how this can be accomplished mechanistically, but Czech et al [ 116 ] speculated that RNA polymerase alone, perhaps in response to changes in osmotically triggered changes in DNA supercoiling [ 210 ], and in combination with changes in the intracellular ion pool (in particular the pair of K + and l -glutamate) [ 207 , 208 , 211 ] and the size of the compatible solute pool [ 212 , 213 ], might afford osmoregulation of ect expression. It is currently difficult to grasp intuitively that the exquisitely sensitive osmotic control of the ect promoter and the tuning of its strength via incremental increases in sustained osmotic stress can be explained by this molecular mechanism alone.…”

“…The dampening effect of imported compatible solutes on ect transcription is not unique to this particular type of promoter(s), as the activity of many osmostress responsive promoters is down-regulated when externally provided compatible solutes are accumulated [ 84 , 212 , 213 ]. Hence, it seems plausible that newly synthesized ectoines will influence ect promoter activity when the cellular pools of these compatible solutes rise in response to increased osmotic stress.…”

Two MarR-Type Repressors Balance Precursor Uptake and Glycine Betaine Synthesis in Bacillus subtilis to Provide Cytoprotection Against Sustained Osmotic Stress

“…It is thus able to sustain growth under osmotically unfavorable conditions ( Hoffmann and Bremer, 2016 , 2017 ). However, the ability of B. subtilis to synthesize compatible solutes is limited because only L-proline can be produced de novo ( Whatmore et al, 1990 ; Brill et al, 2011 ; Hoffmann et al, 2017 ). In contrast, the synthesis of glycine betaine requires the prior import of the precursor choline ( Boch et al, 1994 , 1996 ; Kappes et al, 1999 ).…”

OpuF, a New Bacillus Compatible Solute ABC Transporter with a Substrate-Binding Protein Fused to the Transmembrane Domain

“…When confronted by high osmolarity, B. subtilis synthesizes large amounts of L-proline (14,58,59), a compatible solute widely used by both bacteria and plants (2,60,61), and an amino acid that serves also as a functionpreserving chemical chaperone (62,63). The massive osmostress-responsive synthesis of L-proline by B. subtilis (14,58,64) is an energy-demanding process because the production of just a single molecule of L-proline requires the expenditure of 20 ATP equivalents (65). To preserve precious energetic and biosynthetic resources (18), B. subtilis generally prefers the import of preformed osmostress protectants that can be found in its varied habitats (46,53,(66)(67)(68).…”

“…To preserve precious energetic and biosynthetic resources (18), B. subtilis generally prefers the import of preformed osmostress protectants that can be found in its varied habitats (46,53,(66)(67)(68). In turn, their accumulation suppresses the transcription of the osmostress-adaptive L-proline biosynthesis genes (14,58,64). B. subtilis can also synthesize the compatible solute glycine betaine (69,70), but this process requires the prior import of the precursor choline for its subsequent two-step oxidation to glycine betaine (46,53,69,70).…”