Design

This part includes the promoter P_gltAB and green fluorescent protein, the expression of GFP is repressed by high level cellular glutamate because P_gltAB is upregulated by GltC, which is repressed by high intracellular concentration of glutamate. A restriction enzyme KpnI cutting site is added between P_gltAB and GFP.

The part was applied to test the repression function of GltC to P_gltAB, for which we fermented under different glutamate concentration and tested the fluorescence level changes in LL3.

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 Δ bam 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.

With the extracellular glutamate concentration increasing, the FI of GFP was decreasing, which means higher glutamate concentration can indeed repress the promoter P_gltAB's effect. The FI first rose and then fell, which may due to the extra glutamate adding that can promote cell growth. (Fig. 2 and Fig. 3.)

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, 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, 65(3):642-654.

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