T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis

Brill, Jeanette; Hoffmann, Tamara; Putzer, Harald; Bremer, Erhard

doi:10.1099/mic.0.047357-0

Cited by 31 publications

(74 citation statements)

References 39 publications

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“…7). It thus emerges from our data that the B. subtilis cell can somehow distinguish exogenously provided L-proline from an intracellular L-proline pool built up via de novo synthesis for either anabolic stress (pool size, about 10 to 16 mM) (12,67) or osmostress (11,67) protective purposes. The molecular mechanism(s) responsible for these different effects of external and internal L-proline on the induction of the catabolic putBCP operon is unexplored.…”

Section: Discussionmentioning

confidence: 91%

Proline Utilization by Bacillus subtilis: Uptake and Catabolism

et al. 2012

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L-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced L-proline utilization genetically to the putBCP (ycgMNO) locus. The putBCP gene cluster encodes a high-affinity proline transporter (PutP) and two enzymes, the proline dehydrogenase PutB and the ⌬ 1 -pyrroline-5-carboxylate dehydrogenase PutC, which jointly catabolize L-proline to L-glutamate. Northern blotting, primer extension, and putB-treA reporter gene fusion analysis showed that the putBCP locus is transcribed as an L-proline-inducible operon. Its expression was mediated by a SigA-type promoter and was dependent on the proline-responsive PutR activator protein. Induction of putBCP expression was triggered by the presence of submillimolar concentrations of L-proline in the growth medium. However, the very large quantities of L-proline (up to several hundred millimolar) synthesized by B. subtilis as a stress protectant against high osmolarity did not induce putBCP transcription. Induction of putBCP transcription by external L-proline was not dependent on L-proline uptake via the substrate-inducible PutP or the osmotically inducible OpuE transporter. It was also not dependent on the chemoreceptor protein McpC required for chemotaxis toward L-proline. Our findings imply that B. subtilis can distinguish externally supplied L-proline from internal L-proline pools generated through de novo synthesis. The molecular basis of this regulatory phenomenon is not understood. However, it provides the B. subtilis cell with a means to avoid a futile cycle of de novo L-proline synthesis and consumption by not triggering the expression of the putBCP L-proline catabolic genes in response to the osmoadaptive production of the compatible solute L-proline.

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Section: Discussionmentioning

confidence: 91%

Proline Utilization by Bacillus subtilis: Uptake and Catabolism

et al. 2012

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“…The expression of chromosomal opuAtreA reporter gene fusions was monitored by assaying TreA [phospho-␣-(1,1)-glucosidase] enzyme activity (46) by using the chromogenic substrate para-nitrophenyl-␣-D-glucopyranoside as detailed previously (19). One unit of TreA activity is defined as 1 mol of substrate converted per min per mg of protein.…”

Section: Chemicalsmentioning

confidence: 99%

“…1) (9,16,19). Predictably, the genetic disruption of this osmostress-adaptive proline biosynthetic route causes osmotic sensitivity (9).…”

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confidence: 99%

“…The synthesis of glycine betaine from the precursor choline (taken up via the OpuB and OpuC transporters) is catalyzed through a two-step oxidation process by the GbsB and GbsA enzymes, with glycine betaine aldehyde as the intermediate (24,25). The anabolic and osmoadaptive proline biosynthetic routes and their genetic control have been described elsewhere (9,19). In addition to the ⌬ 1 -pyrroline-5-carboxylase reductases ProI and ProH, shown here, a third protein (ProG) with ⌬ 1 -pyrroline-5-carboxylase reductase activity operates in B. subtilis (94); its physiological role is unclear.…”

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confidence: 99%

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Osmotic Control of opuA Expression in Bacillus subtilis and Its Modulation in Response to Intracellular Glycine Betaine and Proline Pools

Hoffmann¹,

Wensing²,

Brosius³

et al. 2012

Journal of Bacteriology

172

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bGlycine betaine is an effective osmoprotectant for Bacillus subtilis. Its import into osmotically stressed cells led to the buildup of large pools, whose size was sensitively determined by the degree of the osmotic stress imposed. The amassing of glycine betaine caused repression of the formation of an osmostress-adaptive pool of proline, the only osmoprotectant that B. subtilis can synthesize de novo. The ABC transporter OpuA is the main glycine betaine uptake system of B. subtilis. Expression of opuA was upregulated in response to both sudden and sustained increases in the external osmolarity. Nonionic osmolytes exerted a stronger inducing effect on transcription than ionic osmolytes, and this was reflected in the development of corresponding OpuA-mediated glycine betaine pools. Primer extension analysis and site-directed mutagenesis pinpointed the osmotically controlled opuA promoter. Deviations from the consensus sequence of SigA-type promoters serve to keep the transcriptional activity of the opuA promoter low in the absence of osmotic stress. opuA expression was downregulated in a finely tuned manner in response to increases in the intracellular glycine betaine pool, regardless of whether this osmoprotectant was imported or was newly synthesized from choline. Such an effect was also exerted by carnitine, an effective osmoprotectant for B. subtilis that is not a substrate for the OpuA transporter. opuA expression was upregulated in a B. subtilis mutant that was unable to synthesize proline in response to osmotic stress. Collectively, our data suggest that the intracellular solute pool is a key determinant for the osmotic control of opuA expression.

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“…To fuel protein synthesis, B. subtilis produces proline from the precursor glutamate (17,18), a pathway that is present in many bacterial species (19). The anabolic proline synthesis route (ProB-ProA-ProI [ProG]) is interconnected with the osmostress-relieving production route for proline (ProJ-ProA-ProH) via the ␥-glutamyl phosphate reductase (ProA) (14).…”

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The γ-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

et al. 2014

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bPutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-DL-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the ␥-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHPresistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates.

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T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis

Cited by 31 publications

References 39 publications

Proline Utilization by Bacillus subtilis: Uptake and Catabolism

Proline Utilization by Bacillus subtilis: Uptake and Catabolism

Osmotic Control of opuA Expression in Bacillus subtilis and Its Modulation in Response to Intracellular Glycine Betaine and Proline Pools

The γ-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

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