Proline confers acid stress tolerance to Bacillus megaterium G18

Abstract

Proline plays a multifunctional role in several organisms including bacteria in conferring protection under stress conditions. In this paper we report the role of proline in conferring acid tolerance to Bacillus megaterium G18. An acid susceptible mutant of B. megaterium G18 which required proline for its growth under acid stress condition was generated through Tn5 mutagenesis. Further, targeted inactivation of proC involved in osmo-adaptive proline synthesis in B. megaterium G18 resulted in the loss of ability of the bacterium to grow at low pH (pH 4.5). Exogenous supply of proline (1 mM) to the growth medium restored the ability of the mutant cells to grow at pH 4.5 which was not the same in case of other osmoprotectants tested. Proline was produced and secreted to extracellular medium by B. megaterium G18 when growing in low pH condition as evidenced by the use of Escherichia coli proline auxotrophs and HPLC analysis. Further, pHT01 vector based expression of full length proC gene in the ∆proC mutant cells restored the survival capacity of the mutant cells in acidic pH, suggesting that proline production is an important strategy employed by B. megaterium G18 to survive under acid stress induced osmotic stress.

Figure 1

Acid tolerance and proline requirement test of the Tn5 mutant (BA5). (a) The growth of the Tn5 mutant (BA5) in minimal media (MM) adjusted to pH 4.5 was tested in presence and absence of l-proline and compared with the growth of WT at the same stressed media (b) The growth of the Tn5 mutant (BA5) in Nutrient Broth (NB) adjusted to pH 4.5 was tested in presence and absence of l-proline and compared with the growth of WT at the same stressed media. The error bar represents standard error of mean (n = 3).

Figure 2

Acid and salt tolerance response of the proC mutant (BM13A). (a) Comparing the growth of the proC mutant (BM13A) in minimal media (MM) adjusted to pH 4.5 in presence and absence of l-proline and compared with its growth at pH 7.0 and also with the growth of the WT at pH 7.0 and pH 4.5 at the same media. (b) Comparing the growth of the proC mutant (BM13A) in Nutrient Broth (NB) adjusted to pH 4.5 in presence and absence of l-proline and compared with its growth at pH 7.0 and also with the growth of the WT at pH 7.0 and pH 4.5 at the same media. (c) Comparing the growth of the proC mutant (BM13A) in minimal media (MM) of pH 7.0 in presence and absence of 0.5 M and 1 M NaCl, comparing its growth in the same media with the WT as well as the effect of l-proline in alleviating the salt stress of BM13A. The error bar represents standard error of mean (n = 3).

Figure 3

(a) Effect of osmoprotectants on the growth of the proC mutant (BM13A) in acid stress condition (pH 4.5). The growth of the proC mutant (BM13A) was compared by growing it in Minimal Media (MM) with and without the addition of osmoprotectants separately viz., l-proline, l-glutamate and beataine and also compared its growth with the WT grown in MM adjusted to pH 4.5. The error bar represents standard error of mean (n = 3). (b) Effect of osmoprotectants on the growth of B. megaterium G18 in acid stress condition. The growth of the proC mutant (BM13A) was compared by growing it in Minimal Media (MM) with and without the addition of osmoprotectants separately viz., l-proline, l-glutamate and beataine.

Figure 4

Spectrophotometric estimation of extracellular proline production by B. megaterium G18 after growing in minimal media adjusted to pH 4.5 for 6 h. The error bar represents standard error of mean (n = 3).

Figure 5

Validation of proline released by B. megaterium G18 in the culture media under acid stress using E. coli proline auxotrophs. (a) Growth of E. coli ΔproA strain (b) Growth of E. coli ΔproB strain and (c) Growth of E. coli ΔproC strain in the filtered media supernatant in which B. megaterium G18 was grown for 6 h; growth of the each strain in MM supplemented with l-proline serves as control. The error bar represents standard error of mean (n = 3).

Figure 6

Detection of extracellular of proline secreted under acid stress by B. megaterium G18 using HPLC. The isolate was grown in minimal media (MM) adjusted to pH 7.0 and pH 4.5 as well as MM of pH 7.0 containing 0.5 M NaCl for 1 h, 6 h and 16 h. Determination of l-proline in the media supernatant of all the 3 conditions (a) after 1 h (b) after 6 h and (c) after 16 h, the numerical 1 and 2 in figure (a–c) represents the peak of l-glutamate and l-proline respectively. (d) Graphical representation of the concentration of proline produced in each time point.

Figure 7

Representative results of Live/Dead BacLight staining on B. megaterium G18 cells grown in Minimal Media of pH 7.0, pH 4.5 and Minimal Media of pH 7.0 containing 0.5 M NaCl for 1 h, 6 h and 16 h. The cells were incubated for 5 min in the presence of 1 μL of 1.67 mM propidium iodide (PI) and 1.67 mM SYTO 9. Image of bacterial cells at 500 nm (green) for SYTO9 signal and at 635 nm (red) for PI signal were superimposed using Image J tool. (a–c) Images of B. megaterium G18 cells grown in Minimal media of pH 7.0 for 1 h, 6 h, and 16 h respectively. (d–f) Images of B. megaterium G18 cells grown in Minimal media of pH 4.5 for 1 h, 6 h and 16 h respectively. (g–i) Images of B. megaterium G18 cells grown in Minimal media of pH 7.0 amended with 0.5 M NaCl for 1 h, 6 h and 16 h respectively (magnification: ×100 with oil immersion for all images).

Figure 8

Comparing the relative expression of proC in acid- (pH 4.5) and salt stressed B. megaterium G18 cells with the non-stressed B. megaterium G18 cells grown at 1 h and 5 h. The error bar represents standard error of mean (n = 3). Different letter(s) above the indicates significant difference (p ≤ 0.05).

Figure 9

Microscopic analyses to examine the effect acid (pH 4.5) and salt stress (NaCl 0.5 M) on B. megaterium G18 and BM13A morphology in presence of l-proline (Pro) and l-glutamate (Glu). The B. megaterium G18 and BM13A cells (6 logCFU/mL) were grown in minimal media in acid and salt stressed condition for 1 h, harvested by centrifugation at 6000 rpm for 5 min, gram stained and observed under light microscope.