Mikrobiol. Z. 2022; 84(5):50-61.
doi: https://doi.org/10.15407/microbiolj84.06.050

Effect of Tryptophane on Synthesis of Certain Exometabolites by Bacteria of Genus Acinetobacter,
Nocardia, and Rhodococcus and Their Properties

T.P. Pirog1,2, D.V. Piatetska1, V.I. Zhdanyuk1, N.O. Leonova2, T.A. Shevchuk2

1National University of Food Technologies
68 Volodymyrska Str., Kyiv, 01601, Ukraine

2Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

The efficiency of integrated microbial biotechnologies for obtaining several practically valuable metabolites in one technological process is determined both by the maximum concentration of these substances and their properties. This is especially true for secondary metabolites, the composition and properties of which vary depending on the cultivation conditions of the producer. Aim. To research the effect of tryptophan (a precursor of auxin biosynthesis) in the culture media on the synthesis of certain exometabolites by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241, and Nocardia vaccinii IMV B-7405 as well as their properties. Methods. R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-724, and N. vaccinii IMV B-7405 were cultivated in a medium containing refined and waste sunflower oil, biodiesel waste, or ethanol as a carbon source. The concentration of tryptophan in the medium was 300 mg/L. Surfactants were extracted from the supernatant of the cultural liquid with a modified Folch mixture. Phytohormones were isolated from the supernatant by sequential extraction with organic solvents after surfactant extraction. Thin-layer chromatography was used for preliminary purification and concentration of phytohormones. Qualitative and quantitative determination of auxins was performed using high-performance liquid chromatography. The antimicrobial activity of surfactants was analysed by the minimum inhibitory concentration. The activity of enzymes of surface-active glycoand aminolipids biosynthesis (phosphoenolpyruvate synthetase, phosphoenolcarboxykinase, and NADP+-dependent glutamate dehydrogenase) was determined spectrophotometrically during the oxidation of NADH or NADP. Results. It was found that the presence of tryptophan in the culture medium of the strains under study did not affect the number of synthesized surfactants, which was 1.80−1.90, 1.55−1.75, and 1.50−1.65 g/L, respectively. At the same time, cultivation of R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-724, and N. vaccinii IMV B-7405 in the media with tryptophan increased the number of phytohormones: it was higher than the amount of phytohormones synthesized during cultivation without a precursor. The introduction of tryptophan into the culture medium of the strains was accompanied by the formation of surfactants. These compounds showed 2−4 times higher antimicrobial activity against the phytopathogenic bacteria (Agrobacterium tumefaciens UCM B-1000, Pseudomonas syringae UCM B-1027T, Xanthomonas vesicatoria UCM B-1106, Pectobacterium carotovorum UCM B-1075T, Clavibacter michiganensis IMV B-102 and Pseudomonas syringae pv. tomato IMV B-9167) than compounds synthesized on a medium without a precursor. The antimicrobial activity of surfactants synthesized by A. calcoaceticus IMV B-7241 in the presence of tryptophan either did not change compared to that for surfactants obtained without tryptophan, or increased slightly. Data on the activity of surfactant biosynthesis enzymes correlated with the indicators of their antimicrobial activity. In the presence of tryptophan in the culture medium of N. vaccinii IMV B-7405 and R. erythropolis IMV Ac-5017, NADP+-dependent glutamate dehydrogenase activity in the cells of these strains (a key enzyme for biosynthesis of aminolipids responsible for antimicrobial activity) increased almost by 1.4 times compared to that on a tryptophan-free medium. Conclusions. As a result of this work, it was found that the presence of tryptophan in the culture medium of researched strains did not affect the number of surfactants. The antimicrobial activity of surfactants against phytopathogenic bacteria either increased or remained unchanged compared to that established for surfactants synthesized without a precursor of auxin biosynthesis. The obtained data testify to the high efficiency of the potential use of surfactants complex preparations and phytohormones in crop production to stimulate the growth of plants and biocontrol of phytopathogenic bacteria.

Keywords: surfactants, phytohormones, biosynthesis precursor, enzyme activity, antimicrobial activity.

Full text

  1. Arumugam A, Furhana Shereen M. Bioconversion of Calophyllum inophyllum oilcake for intensification of rhamnolipid and polyhydroxyalkanoates co-production by Enterobacter aerogenes. Bioresour Technol. 2020; 296:122321. https://doi.org/10.1016/j.biortech.2019.122321
  2. Romero Soto L, Thabet H, Maghembe R, Gameiro D, Van-Thuoc D, Dishisha T, et al. Metabolic potential of the moderate halophile Yangia sp. ND199 for co-production of polyhydroxyalkanoates and exopolysaccharides. Microbiologyopen. 2021;10(1):e1160. https://doi.org/10.1002/mbo3.1160
  3. Yadav B, Talan A, Tyagi RD, Drogui P. Concomitant production of value-added products with polyhydroxyalkanoate (PHA) synthesis: A review. Bioresour Technol. 2021; 337:125419. https://doi.org/10.1016/j.biortech.2021.125419
  4. Raajaraam L, Raman K. A Computational framework to identify metabolic engineering strategies for the  coproduction of metabolites. Front Bioeng Biotechnol. 2022; 9:779405. https://doi.org/10.3389/fbioe.2021.779405
  5. Dvořák P, Kováč J, de Lorenzo V. Biotransformation of d-xylose to d-xylonate coupled to medium-chainlength polyhydroxyalkanoate production in cellobiose-grown Pseudomonas putida EM42. Microb Biotechnol. 2020;13(4):1273-83. https://doi.org/10.1111/1751-7915.13574
  6. Pirog TP, Kliuchka LV, Klymenko NO, Shevchuk TA, Iutynska GO. [Integrated technologies of microbial synthesis of several final products]. Mikrobiol Z. 2019; 81(6):110‒30. Ukrainian. https://doi.org/10.15407/microbiolj81.06.110
  7. Pirog T, Leonova N, Piatetska D, Klymenko N, Shevchuk T. Influence of tryptophan on auxin-synthesizing ability of surfactant producer Acinetobacter calcoaceticus IMV B-7241. Ukrainian Food Journal. 2020; 9(1): 175‒84. https://doi.org/10.24263/2304-974X-2020-9-1-15
  8. Pirog TP, Leonova NO, Piatetska DV, Klymenko NO, Zhdanyuk VI, Shevchuk TA. Induction of auxins synthesis by Rhodococcus erythropolis IMV Ac-5017 with the addition of tryptophan to the cultivation medium. Mikrobiol Z. 2020; 82(6):3‒12. https://doi.org/10.15407/microbiolj82.06.003
  9. Pirog TP, Shevchuk TA, Voloshina IN, Gregirchak NN. Use of claydite-immobilized oil-oxidizing microbial cells for purification of water from oil. Appl Biochem Microbiol. 2005; 41(1):51—5. https://doi.org/10.1007/s10438-005-0010-z
  10. Bligh EG, Dyer WJ. A rapid method for total lipid extraction and purification. Can J Biochem Physiol. 1959; 37(8): 911−7. https://doi.org/10.1139/o59-099
  11. Pirog TP, Lutsai DA, Shevchuk TA, Iutynska GO. Synthesis and biological activity of Acinetobacter calcoaceticus IMV B-7241 surfactants depending on monovalent cations content in cultivation medium. Mikrobiol Z. 2021; 83(2):36‒47. https://doi.org/10.15407/microbiolj83.02.020
  12. Sha R, Meng Q. Antifungal activity of rhamnolipids against dimorphic fungi. J Gen Appl Microbiol. 2016; 62(5):233‒9. https://doi.org/10.2323/jgam.2016.04.004
  13. Rodrigues AI, Gudiña EJ, Teixeira JA, Rodrigues LR. Sodium chloride effect on the aggregation behaviour of rhamnolipids and their antifungal activity. Sci Rep. 2017; 7(1):12907. https://doi.org/10.1038/s41598-017-13424-x
  14. Monnier N, Furlan A, Botcazon C, Dahi A, Mongelard G, Cordelier S, et al. Rhamnolipids from Pseudomonas aeruginosa are elicitors triggering Brassica napus protection against Botrytis cinerea without physiological disorders. Front Plant Sci. 2018; 9:1170. https://doi.org/10.3389/fpls.2018.01170
  15. Adeniji AA, Aremu OS, Babalola OO. Selecting lipopeptide-producing, Fusarium-suppressing Bacillus spp.: Metabolomic and genomic probing of Bacillus velezensis NWUMFkBS10.5. Microbiologyopen. 2019; 8(6):e00742. https://doi.org/10.1002/mbo3.742
  16. Hazarika DJ, Goswami G, Gautom T, Parveen A, Das P, Barooah M, et al. Lipopeptide mediated biocontrol activity of endophytic Bacillus subtilis against fungal phytopathogens. BMC Microbiol. 2019; 19(1):71. https://doi.org/10.1186/s12866-019-1440-8
  17. Kim J, Le KD, Yu NH, Kim JI, Kim JC, Lee CW. Structure and antifungal activity of pelgipeptins from Paenibacillus elgii against phytopathogenic fungi. Pestic Biochem Physiol. 2020; 163:154‒63. https://doi.org/10.1016/j.pestbp.2019.11.009
  18. Abdallah DB, Tounsi S, Gharsallah H, Hammami A, Frikha-Gargouri O. Lipopeptides from Bacillus amyloliquefaciens strain 32a as promising biocontrol compounds against the plant pathogen Agrobacterium tumefaciens. Environ Sci Pollut Res Int. 2018; 25(36):36518‒29. https://doi.org/10.1007/s11356-018-3570-1
  19. Cao Y, Pi H, Chandrangsu P, Li Y, Wang Y, Zhou H, et al. Antagonism of two plant-growth promoting Bacillus velezensis isolates against Ralstonia solanacearum and Fusarium oxysporum. Sci Rep. 2018; 8(1):4360. https://doi.org/10.1038/s41598-018-22782-z
  20. Le KD, Kim J, Yu NH, Kim B, Lee CW, Kim JC. Biological control of tomato bacterial wilt, kimchi cabbage soft rot, and red pepper bacterial leaf spot using Paenibacillus elgii JCK-5075. Front Plant Sci. 2020; 11:775. https://doi.org/10.3389/fpls.2020.00775
  21. Chen M, Wang J, Liu B, Zhu Y, Xiao R, Yang W, et al. Biocontrol of tomato bacterial wilt by the new strain Bacillus velezensis FJAT-46737 and its lipopeptides. BMC Microbiol. 2020; 20(1):160. https://doi.org/10.1186/s12866-020-01851-2
  22. Kim BS, Lee JY, Hwang BK. In vivo control and in vitro antifungal activity of rhamnolipid B, a glycolipid antibiotic, against Phytophthora capsici and Colletotrichum orbiculare. Pest Manag Sci. 2000; 56(12):1029—35. https://doi.org/10.1002/1526-4998(200012)56:12<1029::AID-PS238>3.0.CO;2-Q
  23. Sanchez L, Courteaux B, Hubert J, Kauffmann S, Renault JH, Clément C, et al. Rhamnolipids elicit defense responses and induce disease resistance against biotrophic,  emibiotrophic, and necrotrophic pathogens that require different signaling pathways in Arabidopsis and highlight a central role for salicylic acid. Plant Physiol. 2012; 160(3):1630−41. https://doi.org/10.1104/pp.112.201913
  24. Azaiez S, Ben Slimene I, Karkouch I, Essid R, Jallouli S, Djebali N, et al. Biological control of the soft rot bacterium Pectobacterium carotovorum by Bacillus amyloliquefaciens strain Ar10 producing glycolipid-like compounds. Microbiol Res. 2018; 217:23‒3. https://doi.org/10.1016/j.micres.2018.08.013
  25. Chopra A, Bobate S, Rahi P, Banpurkar A, Mazumder PB, Satpute S. Pseudomonas aeruginosa RTE4: A tea rhizobacterium with potential for plant growth promotion and biosurfactant production. Front Bioeng Biotechnol. 2020; 8:861. https://doi.org/10.3389/fbioe.2020.00861
  26. Pirog TP, Piatetska DV, Yarova HA, Iutynska GO. Effect on phytopathogenic microorganisms of surfactants of microbial origin. Mikrobiol Z. 2021; 83(6):75‒94. https://doi.org/10.15407/microbiolj83.06.075
  27. [Phytopathogenic bacteria. Bacterial plant diseases]. Patyka VP, editor. Kyiv: OOO NVP Interservis, 2011. Ukrainian.
  28. Pirog T, Sofilkanych A, Konon A, Shevchuk T, Ivanov S. Intensification of surfactants’ synthesis by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii K-8 on fried oil and glycerol containing medium. Food Bioprod Proces. 2013; 91 (2):149−57. https://doi.org/10.1016/j.fbp.2013.01.001
  29. Ding L, Zhang S, Guo W, Chen X. Exogenous indole regulates lipopeptide biosynthesis in antarctic Bacillus amyloliquefaciens Pc3. J Microbiol Biotechnol. 2018; 28(5):784‒95.
  30. Sabaté DC, Brandan CP, Petroselli G, Erra-Balsells R, Audisio MC. Biocontrol of Sclerotinia sclerotiorum (Lib.) de Bary on common bean by native lipopeptide-producer Bacillus strains. Microbiol Res. 2018; 211:21‒30. https://doi.org/10.1016/j.micres.2018.04.003
  31. Chlebek D, Pinski A, Żur J, Michalska J, Hupert-Kocurek K. Genome mining and evaluation of the biocontrol potential of Pseudomonas fluorescens BRZ63, a new endophyte of oilseed rape (Brassica napus L.) against fungal pathogens. Int J Mol Sci. 2020; 21(22):8740. https://doi.org/10.3390/ijms21228740
  32. Bolivar-Anillo HJ, González-Rodríguez VE, Cantoral JM, García-Sánchez D, Collado IG, et al. Endophytic bacteria Bacillus subtilis, isolated from Zea mays, as potential biocontrol agent against Botrytis cinerea. Biology (Basel). 2021; 10(6):492. https://doi.org/10.3390/biology10060492
  33. Ghadamgahi F, Tarighi S, Taheri P, Saripella GV, Anzalone A, Kalyandurg PB, et al. Plant growth-promoting activity of Pseudomonas aeruginosa FG106 and its ability to act as a biocontrol agent against potato, tomato and taro pathogens. Biology (Basel). 2022; 11(1):140. https://doi.org/10.3390/biology11010140
  34. Chen L, Zhang H, Zhao S, Xiang B, Yao Z. Lipopeptide production by Bacillus atrophaeus strain B44 and its biocontrol efficacy against cotton rhizoctoniosis. Biotechnol Lett. 2021; 43(6):1183‒93. https://doi.org/10.1007/s10529-021-03114-0