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Hüseyin Kahraman, Cennet Canan Karaderi; Proline Formation (Produce) in the Bacteria: under Kcl Stress, Trends Journal of Sciences Research, Volume 3, Issue 2, September 25, 2018, Pages 90-95, 10.31586/Biology.0302.05


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Proline Formation (Produce) in the Bacteria: under Kcl Stress

Trends Journal of Sciences Research, Volume 3, Issue 2, 2018, Pages 90–95.

https://doi.org/10.31586/Biology.0302.05

Received August 19, 2018; Revised September 20, 2018; Accepted September 23, 2018;
Published September 25, 2018

Abstract

Salinity is one of the important abiotic stress affecting microorganism growth and productivity. To survive these stresses most organisms have to stress-adaptation mechanisms. It's one of these things proline. We did not find any similar studies with P. aeruginosa and E. faecalis in our studies. The highest value for proline production at 30 °C 100 rpm was in E. faecalis 11,074 U/ml and in E. coli 6,833 U/ml. The highest proline production in LB medium containing 37 °C 100 rpm KCl was found to be in E. faecalis 14,604 U/ml and in E. coli 6,557 U/ml. However, there are studies with E. coli. This experiment revealed that the extracellular proline concentration is proportionally linked to the KCl stress. We should first mention that studies similar to those we were less common in the literature. We did not find any similar studies with P. aeruginosa and E. faecalis in our studies.

Introduction

Salinity is one of the important abiotic stress affecting microorganism growth and productivity 1 and under severe stress conditions, lead to growth inhibition or cell disruptions 2. No microorganism can actively pump water in or out of the cell to compensate for water fluxes caused by changes in the external osmotic condition 3. To survive these stresses most organisms have to stress-adaptation mechanisms 2. It's one of these things proline 4.

L-Proline is synthesized from L-glutamic acid via three enzymatic reactions in microorganisms 5. The proline is not only involved in protecting the intercellular structure and regulating the cytosolic pH, but also in promoting protein integrity and activating enzyme activities. Proline is particularly important in prokaryotic and eukaryotic cells have been reported to be as an osmolyte 4, 6. The amount of proline is an amino acid that increases in stress conditions, participates in the detoxification of free oxygen radicals and has protective properties that play an important role in resistance to stress conditions 7. In addition, many organisms proline has been shown to have multiple functions in vitro it enhances the stability of proteins and membranes during freezing, dehydration, or high temperatures on cell physiology 3.

Pseudomonas aeruginosa is a Gram-negative, rod-shaped, motile organism (polar flagella) which characteristically produces water-soluble pigments. It is commonly found free living in moist environments. P. aeruginosa is also a pathogen of plants, animals, and humans. It is not an active fermented of carbohydrates and produces acid, but no gas, in glucose and is lactose-negative. Although an aerobic atmosphere is necessary for optimal growth, most strains will multiply slowly in an anaerobic environment if nitrate is present as hydrogen acceptors. It is able to grow at 42 °C 8, 9. Escherichia coli is a gram-negative, (is capable of switching to or if oxygen is absent), rod-shaped, and coli form bacterium. Strains that possess are motile. The flagella have a arrangement. Most E. coli are harmless. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and preventing colonization of the intestine with pathogenic bacteria. Cells are typically rod-shaped, and are about 2.0 long and 0.25-1.0 μm in diameter, with a cell volume of 0.6-0.7 μm3. E. coli is not damaged by penicillin. E. coli can live on a wide variety of substrates and uses mixed-acid fermentation in anaerobic conditions 10. Enterococcus faecalis is a non-motile, gram-positive, spherical bacterium. It can be observed singly, in pairs, or in short chains, and is most often found in the large intestine of humans. It is a facultative anaerobe with a fermentative metabolism 11.

As a result of these changes in the osmolarity and salinity of the habitats, the P. aeruginosa and E. coli cells has to cope with osmotically instigated water fluxes across the cytoplasmic membrane. This paper deals with the different conditions of bacteria producing much proline.

Materials and Methods

Solvents and Chemicals. Bactopepton, Yeast Extract (Mast Diagnostics); NaCl, Glucose (Meck); Tris-HCl, NaOH, Acetic acid (Sigma); EDTA (Carlo-Erba); SDS (ADR Group-Lab Chem); Agar (Fluka), Phosphoric acid, Ninhidrine, Benzene.

Bacterial Strains. E. coli ATCC 20715, P. aeruginosa ATCC 27853 and E. faecalis ATCC 25539 were used in this study.

Culture-Media. E. coli, P. aeruginosa and E. faecalis strains were routinely maintained on Luria-Bertani (LB) agar plates. LB medium (g/L; Bactopepton 10, NaCl 10, Yeast extract 5, and for Petri dish 1,5 % Agar) was used in this study.

Growth Conditions. E. coli ATCC 20715, P. aeruginosa ATCC 27853 and E. faecalis ATCC 25539 were cultivated routinely at 30 or 37 °C on Luria-Bertani medium O/N and 0, 100 rpm or 200 rpm shaker, respectively. Growths of culture were determining the optical density at 600 nm using a UV/VIS spectrophotometer. The value (OD600) was increased from 0,5 and diluted to obtain an average of 0,3 - 0,4. A 100 µl culture were inoculums LB medium 10 ml/100 ml Erlenmeyer flasks. 30 or 37 °C, 0, 100 rpm or 200 rpm shaker and KCl, respectively.

Proline Assay. Bacteria were grown in a 10 ml flask containing 100 ml of LB medium. These cultures grew aerobically at 30 or 37 °C in a shaking set at static, 100 or 200 rpm rpm for 24 h. For L-proline assay, cells were harvested at 24 h growth phase. 1 ml culture was pipetted ependorf tube and centrifuged at 14.000 rpm for 20 min at room temperature, cellular debris was removed by centrifugation, and the supernatant was then used for prolin activity assays 12.

Proline Activity Assay. After centrifugation supernatant was discarded and then on pellet 100 µl GTE buffer ( glucose, Tris-HCl, EDTA) was added. Later room conditions were held for 1 min and then 200 µl lysis buffers was added and 5 min stay room temperature. Over 500 µl acidic ninhidrin (4 ml phosphoric acid, 6 ml glacial acetic acid, 0, 25 gr ninhidrin) was added. The tubes were kept in boiling water for 30 minutes. After tubes were then cooled. The mixture was pipettes into new set tubes, and 2 ml benzene was added. The mixed in tubes gently vortexed and left at room temperature for 1 h until separation of the different two phases. The aqueous phase (i.e., the lower phase) was discarded by dipping a pipette through the organic phase add new tubes (i.e., the upper phase). Each sample reads at 520 nm against using benzene a blank 12, 13. The proline concentration in samples was determined from a predetermined standard curve using proline. The reaction for each sample was performed in triplicate. For clarity, no error bars are given, but they are mostly less than 10 % of the respective data point.

Results

Proline production of bacteria at static 30 °C and 37 °C. The highest proline activity value in the 30 °C static environment was in E. faecalis; 3,257 U/ml and the lowest value in E. coli; it was found to be 0,437 U/ml. The highest proline production value in 37 °C static medium was found to be in E. faecalis 14,598 U/ml and in E. coli 5,071 U/ml (Figure 1, Figure 2).

Figure 1.
Proline levels of P. aeruginosa, E. coli and E. faecalis, grown in LB medium under static conditions at 30 °C normal (○) and KCl (●), respectively.
Figure 2.
Proline levels of P. aeruginosa, E. coli and E. faecalis, grown in LB medium under static conditions at 37 °C normal (○) and KCl (●), respectively.

Proline production of bacteria at 30 °C and 37 °C at 100 rpm. In this study; the highest value at 30 °C 100 rpm is in E. faecalis 13,480 U/ml and the lowest value is in E. coli 6,912 U/ml was found. At 37 °C 100 rpm, the highest proline production value was in E. faecalis 14,795 U/ml and the lowest value in E. coli; 6,222 U/ml was found (Figure 3, Figure 4).

Figure 3.
Proline levels of P. aeruginosa, E. coli and E. faecalis, grown in LB medium under 100 rpm agitation conditions at 30 °C normal (○) and KCl (●), respectively.

Proline production of bacteria at 30 °C and 37 °C at 200 rpm. The highest value at 30 °C 200 rpm was found to be in E. faecalis 1,705 U/ml and the lowest value were found to be in E. coli 0,410 U/ml. At 37 °C 200 rpm, proline production was the highest at E. faecalis 15,176 U/ml and in E. coli 7,254 U/ml (Figure 5, Figure 6).

Production of proline in bacterial KCl static at 30 °C and 37 °C. The highest proline production in LB medium containing 30 °C KCl was found to be in E. faecalis 16,925 U/ml and in E. coli 9,358 U/ml. The highest proline production in LB medium containing 37 °C KCl was found to be in E. faecalis 12,172 U/ml and in E. coli 2,395 U / ml (Figure 1, Figure 2).

Proline production of bacterial KCl at 30 °C and 37 °C at 100 rpm. The highest value for proline production at 30 °C 100 rpm was in E. faecalis 11,074 U/ml and in E. coli 6,833 U/ml. The highest proline production in LB medium containing 100 rpm KCl was found to be in E. faecalis 14,604 U/ml and in E. coli 6,557 U/ml (Figure 3, Figure 4).

Figure 4.
Proline levels of P. aeruginosa, E. coli and E. faecalis, grown in LB medium under 100 rpm agitation conditions at normal (○) and KCl (●), respectively

Proline production of bacterial KCl at 30 °C and 37 °C at 200 rpm. The highest proline production at 30 °C 200 rpm in LB containing KCl was found to be in E. faecalis 12,067 U/ml and in E. coli 5,380 U/ml. The highest proline production in LB medium containing 37 °C 200 rpm KCl was found to be in E. faecalis 11,646 U/ml and in E. coli 4,493 U/ml (Figure 5, Figure 6).

At 0 rpm at 30 °C, 23-fold the activity of KCl in E. coli; P. aeruginosa 20; and E. faecalis 5-fold more proline production was observed, respectively. Similarly, at 37 °C, there was no difference in the presence of KCl in E. coli and E. faecalis; in P. aeruginosa 9-fold more proline production was observed. However, in the study with E. coli, P. aeruginosa and E. faecalis only at 30 °C and 200 rpm, 14, 7, 6-fold more proline production was observed in the presence of KCl, respectively (Figure 5).

Figure 5.
Proline levels of P. aeruginosa, E. coli and E. faecalis, grown in LB medium under 200 rpm agitation conditions at 30 °C normal (○) and KCl (●), respectively

In the study with P. aeruginosa and E. faecalis without KCl 8 and 7-fold more proline production was observed at 30 °C and 37 °C 200 rpm, respectively (Figure 5, Figure 6).

Figure 6.
Proline levels of P. aeruginosa, E. coli and E. faecalis, grown in LB medium under 200 rpm agitation conditions at 37 °C normal (○) and KCl (●), respectively

Discussion

Proline production has been studied in E. coli the first time. We should first mention that studies similar to those we were less common in the literature. We did not find any similar studies with P. aeruginosa and E. faecalis in our studies. However, there are studies with E. coli. This experiment revealed that the extracellular proline concentration is proportionally linked to the KCl stress. KCl is not a natural compound. It was chosen for this reason. As a result, there was a significant increase in the amount of proline, which is a struthen conservation task under KCl ventilated conditions. Proline production, which shows little variability and continuity in the presence of KCl, has been achieved. Both temperatures and presence of KCl decrease the amount of proline in all controls. The greatest decrease in proline production occurs in E. coli. However, the temperature increase increases the amount of proline. E. coli was produced proline at 400 rpm for 16 hours at 14. Bacteria can synthesize glutamate to proline. E. coli has been shown to produce 60 mg/ml proline at 30 °C for 120 hours and 600 rpm 15. Application of analog-resistance to the construction of proline overproducing strains was reported in Escherichia coli, although the productivity was low (Sugiura et al., 1985) 13. Increase in temperature increases proline production. The highest increase was in E. faecalis. An increase in proline is observed at 100 rpm according to 0 rpm. The amount of proline in the presence of KCl increases significantly compared to controls at 200 rpm, and the largest increase was observed in E. faecalis.

The proline amount increased independently of KCl with temperature increase. There was also an increase in proline in the presence of KCl at 30 °C. At 37 °C it was not possible to observe. The amount of proline in the presence of KCl decreases with the temperature increase in the static environment. At 100 rpm, the amount of proline is increased with temperature increase. Temperature increases at 200 rpm and the presence of KCl does not change the amount of proline. When all the results are examined, the highest proline mix is E. faecalis static 30 °C and in the presence of KCl. The lowest amount of proline was produced in E. coli at 30 °C with 0.4 g. Proline-overproducing mutants of Escherichia coli, Salmonella typhimurium, and Serratia marcescens clearly show enhanced osmotolerance. Overproduction of proline increased tolerance against drought and salinity stresses 2. The enteric organisms S. typhimuirium, E. coli and K. pneumoniae are entirely dependent on the presence of exogenous proline to be able to accumulate it to high concentrations during osmotic stress 16. The maximum value of L-proline produced was 16,9 µg/ml by E. faecalis in KCl. Temperature increase leads to increase production of proline. The results showed that proline production was best achieved by E. faecalis in comparison to other bactericides (P. aeruginosa and E. coli).

Acknowledgements

This work was supported by a Grant (APYB 2015/23) from Research Fund Unit of Inonu University.

Conflıct of ınterest

No conflict of interest was declared by the authors.

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