Design

This sequence includes the promoter(forward) of gltAB (glutamate synthase) and the promoter (backward) of gltC (LysR family transcriptional regulator). GltC can bind specific DNA site on it. Thus the expression of downstream proteins is upregulated, which is repressed by high concentration glutamate.

Since P_gltAB can sense intracellular glutamate level, we combined it with proteins such as GFP and LacI, so that these proteins can respond to intracellular glutamate concentration and carry out their own function.

Proof of Function

Detection of gltC transcription level in LL3-P_gltAB-GFP under different glutamate concentrations

LL3-P_gltAB-GFP was cultured in M9 medium with different extracellular glutamate concentrations. From the 6th hour, we extracted the total RNA of LL3-P_gltAB-GFP every 3 hours and tested the transcription of gltC together with the respective intracellular glutamate concentrations. Transcription level of gltC in plateau phase is shown in Fig. 1. It could be indicated that the transcription of gltC was repressed with the increasing intracellular glutamate concentration. Primers used in the assay are listed in Table 1.

Fig. 1. The intracellular glutamate concentration and the relative expression level of gltC in LL3 with P_gltAB-GFP in plateau stage. a. The intracellular glutamate concentration of LL3 with P_gltAB-GFP in plateau stage. *Significantly different (P < 0.05) by Student's t-test.b. The relative expression level of gltC in plateau stage. The value illustrates the effect of glutamate concentration on the transcription of gltC. ***Very very significantly different (P < 0.005) by Student <s t-test. The strains were cultured at 37 °C in M9 medium with 5 µg/mL chloromycetin under different extracellular glutamate concentration (0 g/L, 2.5 g/L, 5.0 g/L) for 24 hours. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.

GFP fluorescent intensity (FI) reports the P_gltAB function

P_gltAB-GFP and P₄₃-GFP were converted into both B. amyloliquefaciens LL3 Δbam and B. amyloliquefaciens LL3 Δ bam -icd strain (with stronger promoter before icd gene), which were designated as LL3-P_gltAB-GFP and LL3-icd-P_gltAB-GFP respectively. The two mutants were cultured in M9 culture medium for 24 hours. If needed the medium was supplemented with antibiotics or glutamate at the following concentrations: 5 µg/mL chloramphenicol, 0 g/L, 0.5 g/L, 1.0 g/L, 2.5 g/L or 5.0 g/L glutamate. During the fermentation, 1.5mL bacteria culture was taken out every 3 hours, of which 600µL was for GFP FI measurement (395nm\509nm) by microplate reader, and 900µL for OD₆₀₀ measurement.

Fig.2 Principle for detecting the P_gltAB function. Under high glutamate concentration, GltC level goes down, reducing the level of GFP.

Fig.3 FI of GFP in LL3-P_gltAB-GFP and LL3–icd-P_gltAB-GFP under different extracellular glutamate concentrations in plateau stage. a. The intracellular glutamate concentration under different extracellular glutamate concentrations in plateau stage. The value illustrates the relationship between glutamate concentration in medium and intracellular glutamate concentration. *Significantly different (P < 0.05) by Student's t-test. b. FI of GFP in LL3-P_gltAB-GFP under different extracellular glutamate concentrations in plateau stage. **Very significantly different (P < 0.01) by Student's t-test. c. FI of GFP in LL3-icd-P_gltAB-GFP under different extracellular glutamate concentrations in plateau stage. *** Very very significantly different (P < 0.005) by Student's t-test. The strains were cultured at 37 °C in M9 medium with 5 µg/mL chloromycetin for 24 hours under different extracellular glutamate concentration (0 g/L, 0.5 g/L, 1.0 g/L, 2.5 g/L, 5.0 g/L). Intracellular glutamate concentration, fluorescence intensity of GFP and the OD₆₀₀ were measured. FI of GFP was normalized against OD₆₀₀. Data indicate mean values ± standard deviations from three independent experiments performed in triplicates.

Reference

Weitao G, Mingfeng C, Cunjiang S et al. Complete Genome Sequence of Bacillus amyloliquefaciens LL3, Which Exhibits Glutamic Acid-Independent Production of Poly-γ-Glutamic Acid. J Bacteriol. 2011 Jul; 193(13): 3393–3394.

Picossi S, Belitsky B R, Sonenshein A L. Molecular mechanism of the regulation of Bacillus subtilis gltAB expression by GltC[J]. J Mol Biol., 2007, 365(5):1298-1313.

Commichau FM, Herzberg C, Tripal P et al. A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC. Mol Microbiol. 2007 Aug;65(3):642-654.

Bohannon D E and Sonenshein A L. Positive regulation of glutamate biosynthesis in Bacillus subtilis. J Bacteriol. 1989 Sep; 171(9): 4718–4727.