Sains Malaysiana 51(11)(2022): 3621-3633

http://doi.org/10.17576/jsm-2022-5111-09

Expression Analysis using Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) Suggests Different Strategies in Parageobacillus caldoxylosilyticus ER4B under Exposure to Cold Shock

(Analisis Pengekspresan menggunakan Transkripsi Berbalik Kuantitatif Masa-Nyata PCR (RT-qPCR) Mencadangkan Strategi Berbeza untuk Parageobacillus caldoxylosilyticus ER4B di bawah Pendedahan kepada Renjatan Sejuk)

CHING XIN JIE¹, NOOR HYDAYATY MD YUSUF¹, NAZALAN NAJIMUDIN ², CHEAH YOKE KQUEEN³ & CLEMENTE MICHAEL WONG VUI LING^1,4,*

¹Biotechnology Research Institute, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia

²School of Biological Science, Universiti Sains Malaysia, Persiaran Bukit Jambul, 11900 Bayan Lepas, Penang, Malaysia

³Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

⁴National Antarctic Research Centre, Universiti Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

Received: 29 April 2021/Accepted: 5 July 2022

Abstract

Microorganisms have acquired both common and unique abilities to withstand cold stress on Earth. Many studies on bacterial cold shock have been conducted, however, the majority of the studies were focused on mesophiles and psychrophiles. To date, limited information is available on the response of thermophilic bacteria to cold stress and therefore, it is not known how thermophilic bacteria would respond to different cold shocks. To address this question, the cold shock responses of a thermophilic Parageobacillus caldoxylosilyticus ER4B which has an optimal growth temperature at 64 °C were determined using Real-Time PCR (RT-qPCR). When the bacterium was exposed to mild cold shock at 54 °C, the expressions of gene encoding for pyruvate kinase and acetolactate synthase were significantly upregulated, suggesting that more pyruvate molecules were produced to synthesize branched-chain amino acids that could alter the fatty acid profile on the cell membrane. Accumulation of pyruvate in the bacterium could also help to scavenge cold-induced reactive oxygen species (ROS). Meanwhile, exposing the bacterium to extreme cold shock at 10 °C resulted in significant upregulation of genes encoding for γ-glutamylcyclotransferase, cold shock protein B and competence protein ComEA. An increase in these enzymes expression indicated more extreme measures including apoptosis and transformation were adopted during extreme cold shock.

Keywords: Cold shock; cold stress response; Parageobacillus caldoxylosilyticus; reverse transcription quantitative real-time PCR (RT-qPCR); thermophilic bacterium

Abstrak

Mikroorganisma telah memperoleh ciri umum dan unik untuk bertahan daripada tekanan suhu sejuk di bumi. Banyak kajian mengenai kejutan sejuk bakteria telah dijalankan, namun kebanyakannya memfokuskan kepada bakteria mesofil dan psikofil. Sehingga kini, terdapat maklumat yang terhad mengenai gerak balas bakteria termofilik terhadap tekanan suhu rendah dan sehubungan itu, bagaimana bakteria termofilik bergerak balas terhadap kejutan sejuk yang berbeza masih belum diketahui. Untuk menjawab persoalan ini, gerak balas kejutan sejuk daripada termofilik Parageobacillus caldoxylosilyticus ER4B yang mempunyai suhu pertumbuhan optimum pada suhu 64 °C telah ditentukan melalui PCR Masa-Nyata (RT-qPCR). Apabila bakteria terdedah kepada kejutan sejuk 54 °C, pengekspresan gen yang mengekodkan piruvat kinase dan asetolaktat sintase menunjukkan peningkatan transkrip, mencadangkan bahawa lebih banyak molekul piruvat dihasilkan untuk mensintesis asid amino rantai bercabang yang dapat mengubah profil asid lemak pada membran sel. Pengumpulan piruvat pada bakteria juga dapat membantu menghapuskan spesies oksigen reaktif (ROS) yang disebabkan oleh suhu rendah. Sementara itu, pendedahan bakteria kepada kejutan sejuk yang melampau pada 10 °C mengakibatkan peningkatan pengawalaturan pengekodan gen untuk γ-glutamil siklotransferase, protein kejutan sejuk B dan protein kompetensi ComEA. Peningkatan pengekspresan enzim tersebut menunjukkan pendekatan yang lebih ekstrem termasuk apoptosis dan transformasi dilakukan semasa kejutan sejuk yang melampau.

Kata kunci: Bakteria termofilik; gerak balas tekanan sejuk; kejutan sejuk; Parageobacillus caldoxylosilyticus; PCR masa nyata-kuantitatif transkripsi berbalik

REFERENCES

Abramson, J., Riistama, S., Larsson, G., Jasitis, A., Svensson-Ek, M., Laakkonen, L., Puustinen, A., Iwata, S. & Wikström, M. 2000. The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site. Nature Structural and Molecular Biology 7: 910-917.

Aliyu, H., Lebre, P., Blom, J., Cowan, D. & De Maayer, P. 2016. Phylogenomic re-assessment of the thermophilic genus Geobacillus. Systematic and Applied Microbiology 39(8): 527-533.

Alles, J., Karaiskos, N., Praktiknjo, S.D., Grosswendt, S., Wahle, P., Ruffault, P., Ayoub, S., Schreyer, L., Boltengagen, A., Birchmeier, C., Zinzen, R., Kocks, C. & Rajewsky, N. 2017. Cell fixation and preservation for droplet-based single-cell transcriptomics. BMC Biology 15(1): 44.

Allocati, N., Masulli, M., Ilio, C.D. & Laurenzi, V.D. 2015. Die for the community: An overview of programmed cell death in bacteria. Cell Death & Disease 6: e1609.

Angel, R. 2012. Total nucleic acid extraction from soil. Protocol Exchange. doi.org/10.1038/protex.2012.046

Bajerski, F., Wagner, D. & Mangelsdorf, K. 2017. Cell membrane fatty acid composition of Chryseobacterium frigidisoli PB4^T, isolated from Antarctic glacier forefield soils, in response to changing temperature and pH conditions. Frontiers in Microbiology 8: 677.

Beck, H. 2005. Branched-chain fatty acid biosynthesis in a branched-chain amino acid aminotransferase mutant of Staphylococcus carnosus. FEMS Microbiology Letters 243(1): 37-44.

Berendsen, E.M., Wells-Bennik, M.H.J., Krawczyk, A.O., de Jong, A., van Heel, A., Holsappel, S., Eijlander, R.T. & Kuipers, O.P. 2016. Draft genome sequences of seven thermophilic spore-forming bacteria isolated from foods that produce highly heat-resistant spores, comprising Geobacillus spp., Caldibacillus debilis, and Anoxybacillus flavithermus. Genome Announcement 4(3): e00105-e00106.

Bersolin, G., Neuhaus, K., Scherer, S. & Fuchs, T.M. 2006. Transciptional analysis of long-term adaptation of Yersinia enterocolitica to low-temperature growth. Journal of Bacteriology 188(8): 2945-2958.

Brock, T.D. 1986. Thermophiles: General, Molecular, and Applied Biology. New Jersey: John Wiley and Sons Ltd.

Cellini, L., Robuffo, I., Maraldi, N.M. & Donelli, G. 2001. Searching the point of no return in Helicobacter pylori life: Necrosis and/or programmed death? Journal of Applied Microbiology 90(5): 727-732.

Chattopadhyay, M.K., Raghu, G., Sharma, Y.V.R.K., Biju, A.R., Rajasekharan, M.V. & Shivaji, S. 2011. Increase in oxidative stress at low temperature in an Antarctic bacterium. Current Microbiology62(2): 544-546.

Ching, X.J., Najumudin, N., Cheah, Y.K. & Wong, C.M.V.L. 2021. Complete genome sequence data of tropical thermophilic bacterium Parageobacillus caldoxylosilyticus ER4B. Data In Brief 40: 107764.

Circu, M.L. & Aw, T.Y. 2008. Glutathione and apoptosis. Free Radicals Research 42(8): 689-706.

Craig, J.E., Boyle, D., Francis, K.P. & Gallagher, M.P. 1998. Expression of the cold-shock gene cspB in Salmonella typhimurium occurs below a threshold temperature. Microbiology 144(Pt 3): 697-704.

Dezfulian, M.H., Foreman, C., Jalili, E., Pal, M., Dhaliwal, R.K., Roberto, D.K.A., Imre, K.M., Kohalmi, S.E. & Crosby, W.L. 2017. Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development. BMC Plant Biology 17: 71.

Etchegaray, J. & Inouye, M. 1999. CspA, CspB, and CspG, Major cold shock proteins of Escherichia coli, are induced at low temperature under conditions that completely block protein synthesis. Journal of Bacteriology 181(6): 1827-1830.

Fedurayev, P.V., Mironov, K.S., Gabrielyam, D.A., Bedbenov, V.S., Zorina, A.A., Shumskaya, M. & Los, D.A. 2018. Hydrogen peroxide participates in perception and transduction of cold stress signal in Synechocystis.  Plant and Cell Physiology 59(6): 1255-1264.

Fogarty, C.E & Bergmann, A. 2015. The sound of silence: Signaling by apoptotic cells. Current Topics in Developmental Biology 114: 241-265.

Franco, T.M.A. & Blanchard, J.S. 2017. Bacterial branched-chain amino acid biosynthesis: Structures, mechanisms, and drugability. Biochemistry 56(44): 5849-5865.

Franco, R., Bortner, C., Schmitz, I. & CIidlowski, J.A. 2014. Glutathione depletion regulates both extrinsic and intrinsic apoptotic signaling cascades independent from multidrug resistance protein 1. Apoptosis 19(1): 117-134.

Gray, L.R., Tompkins, S.C. & Taylor, E.B. 2014. Regulation of pyruvate metabolism and human disease. Cellular and Molecular Life Sciences 71(14): 2577-2604.

Hassan, N., Anesio, A.M., Rafiq, M., Holtvoeth, J., Bull, I., Haleem, A., Shah, A.A. & Hasan, F. 2020. Temperature driven membrane lipid adaptation in glacial psychrophilic bacteria. Frontiers in Microbiology 11: 824. https://doi.org/10.3389/fmicb.2020.00824

Hecker, M. & Völker, U. 2001. General stress response of Bacillus subtilis and other bacteria. Advances in Microbial Physiology 44: 35-91.

Hong, Y., Zeng, J., Wang, X., Drlica, K. & Zhao, X. 2019. Post-stress bacterial cell death mediated by reactive oxygen species. PNAS 116(20): 10064-10071.

Janßen, H.J. & Steinbüchel, A. 2014. Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnology for Biofuels 7(1): 7.

Jin, B., Jeong, K. & Kim, Y. 2014. Structure and flexibility of thermophilic cold-shock protein of Thermus aquaticus. Biochemical and Biophysical Research Communications 451(3): 402-407.

Jung, Y.H., Lee, Y.K., Lee, H.K., Lee, K. & Im, H. 2018. CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freezing-tolerance. Brazilian Journal of Microbiology 49(1): 97-103.

Katsyv, A., Schoelmerich, M.C., Basen, M. & Muller, V. 2021. The pyruvate: Ferredoxin oxidoreductase of the thermophilic acetogen, Thermoanaerobacter kivui. FEBS Open Bio 11(5): 1332-1342.

Kaur, A., Gautam, R., Srivastava, R., Chanded, A., Kumar, A., Karthikeyan, S. & Bachhawat, A.K. 2017. ChaC2: An enzyme for slow turnover of cytosolic glutathione. Journal of Biological Chemistry292(2): 638-651.

Keto-Timonen, R., Hietala, N., Palonen, E., Hakakorpi, A., Lindström, M. & Korkeala, H. 2016. Cold shock proteins: A minireview with special emphasis on Csp-family of enteropathogenic Yersinia. Frontiers in Microbiology 7: 1151.

Kiesel, V.A., Sheeley, M.P., Coleman, M.F., Cotul, E.K., Donkin, S.S., Hursting, S.D., Wendt, M.K. & Teegarden, D. 2021. Pyruvate caxylase and cancer progression. Cancer & Metabolism 9(1): 20.

Kimball, S.R. & Jefferson, L.S. 2001. Regulation of protein synthesis by branched-chain amino acids. Current Opinion in Clinical Nutrition and Metabolic Care 4(1): 39-43.

Klein, W., Weber, M.H.W. & Marahiel, M.A. 1999. Cold shock response of Bacillus subtilis: Isoleucine-dependent switch in the fatty acid branching pattern for membrane adaptation to low temperatures. Journal of Bacteriology 181(17): 5341-5349.

Kloska, A., Cech, G.M., Sadowska, M., Krause, K., Szalewska-Pałasz, A. & Olszewski, P. 2020. Adaptation of the marine bacterium Shewanella baltica to low temperature stress. International Journal of Molecular Sciences 21(12): 4338.

Kornberg, H.L. & Krebs, H.A. 1957. Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle. Nature 179(4568): 988-991.

Krivoruchko, A., Zhang, Y., Siewers, V., Chen, Y. & Nielsen, J. 2014. Microbial acetyl-CoA metabolism and metabolic engineering. Metabolic Engineering 28: 28-42.

Kulkarni, Y.S., Liao, Q., Petrović, D., Krüger, D.M., Strodel, B., Amyes, T.L., Richard, J.P. & Kamerlin, S.C.L. 2017. Enzyme architecture: Modelling the operation of a hydrophobic clamp in catalysis by triosephosphate isomerase. Journal of the American Chemical Society 139(30): 10514-10525.

Lambros, M., Pechuan-Jorge, X., Biro, D., Ye, K. & Bergman, A. 2021. Emerging adaptive strategies under temperature fluctuations in a laboratory evolution experiment of Escherichia coli. Frontiers in Microbiology 12: 724982.

Lebre, P.H., Aliyu, H., De Maayer, P. & Cowan, D.A. 2018. In silico characterization of the global Geobacillus and Parageobacillus secretome. Microbial Cell Factories 17(1): 156.

Livak, K.J. & Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the  method. Methods 25(4): 402-408.

Los, D.A. & Murata, N. 2004. Membrane fluidity and its roles in the perception of environmental signals. Biochimica et Biophysica Acta 1666(1-2): 142-157.

Luo, F., Li, Y., Yuan, F. & Zuo, J. 2019. Hexokinase II promotes the Warburg effect by phosphorylating alpha subunit of pyruvate dehydrogenase. Chinese Journal of Cancer Research 31(3): 521-532.

Mazzon, R.R., Lang, E.A.S., Silva, C.A.P.T. & Marques, M.V. 2012. Cold shock genes cspA and cspB from Caulobacter crescentusare posttranscriptionally regulated and important for cold adaptation. Journal of Bacteriology 194(23): 6507-6517.

Mitta, M., Fang, L. & Inouye, M. 1997. Deletion analysis of cspA of Escherichia coli: Requirement of the AT-rich UP element for cspA transcription and the downstream box in the coding region for its cold shock induction. Molecular Microbiology 26(2): 321-335.

Mocali, S., Chiellini, C., Fabiani, A., Decuzzi, S., de Pascale, D., Parrilli, E., Tutino, M.L., Perrin, E., Bosi, E., Fondi, M., Giudice, A.L. & Fani, R. 2017. Ecology of cold environments: New insights of bacterial metabolic adaptation through an integrated genomic-phenomic approach. Scientific Reports7: 839. https://doi.org/10.1038/s41598-017-00876-4

Monirujjaman, M. & Ferdouse, A. 2014. Metabolic and physiological roles of branched-chain amino acids. Advances in Molecular Biology 2014: 364976.

Morgan-Kiss, R.M., Priscu, J.C., Pocock, T., Gudynaite-Savitch, L. & Huner, N.P.A. 2006. Adaptation and acclimation of photosynthetic microorganisms to permanently cold environments. Microbiology and Molecular Biology Reviews 70(1): 222-252.

Morita, R.Y. 1975. Psychrophilic bacteria. Bacteriology Review 39(2): 144-167.

Mostofian, B., Zhuang, T., Cheng, X. & Nickels, J.D. 2019. Branched-chain fatty acid content modulates structure, fluidity, and phase in model microbial cell membranes. Journal of Physical Chemistry B 123(27): 5814-5821.

Nagamalleswari, E., Rao, S., Vasu, K. & Nagaraja, V. 2017. Restriction endonuclease triggered bacterial apoptosis as a mechanism for long time survival. Nucleic Acid Research 45(14): 8423-8434.

Neinast, M., Murashige, D. & Arany, Z. 2019. Branched chain amino acids. Annual Reviews of Physiology8: 139-164.

Niehaus, T.D., Elbadawi-Sidhu, M., de Crécy-Lagard, V., Fiehn, O. & Hanson, A.D. 2017. Discovery of a widespread prokaryotic 5-oxoprolinase that was hiding in plain sight. Journal of Biological Chemistry 292(39): 16360-16367.

Orlowski, M., Richman, P.G. & Meister, A. 1969. Isolation and properties of γ-L-glutamyl cyclotransferase from human brain. Biochemistry 8(3): 1048-1055.

Park, C.B., Ryu, D.D.Y. & Lee, S.B. 2003. Inhibitory effect of L-pyroglutamate on extremophiles: Correlation with growth temperature and pH. FEMS Microbiology Letters 221(2): 187-190.

Park, C.B., Lee, S.B. & Ryu, D.D. 2001. L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus. Applied and Environmental Microbiology 67: 3650-3654.

Paton, J.C., McMurchie, E.J., May, B.K. & Elliott, W.H. 1978. Effect of growth temperature on membrane fatty acid composition and susceptibility to cold shock of Bacillus amyloliquefaciens. Journal of Bacteriology 135(3): 754-759.

Pophaly, S.D., Singh, R., Pophaly, S.D., Kaushik, J.K. & Tomar, S.K. 2012. Current status and emerging role of glutathione in food grade lactic acid bacteria. Microbial Cell Factories 11: 114.

Provvedi, R. & Dubnau, D. 1999. ComEA is a DNA receptor for transformation of competent Bacillus subtilis. Molecular Microbiology31(1): 271-280.

Schormann, N., Hayden, K.L., Lee, P., Banerjee, S. & Chattopadhyay, D. 2019. An overview of structure, function, and regulation of pyruvate kinases. Protein Science 28: 1771-1784.

Shaeer, A., Aslam, M. & Rashid, N. 2019. A highly stable manganese catalase from Geobacillus thermopakistaniensis: Molecular cloning and characterization. Extremophiles 23(6): 707-718.

Sibon, O.C.M. & Strauss, E. 2016. Coenzyme A: To make it or uptake it? Nature Reviews Molecular Cell Biology 17(10): 605-606.

Smartt, A.B. 2014. A genomic and transcriptomic approach to understanding cold acclimation in Pseudomonas fluorescens HK44. Master Theses, University of Tennessee, Knoxville (Unpublished).

Smirnova, G.V. & Oktyabrsky, O.N. 2005. Glutathione in bacteria. Biochemistry 70(11): 1199-1211.

Smirnova, G.V., Zakirova, O.N. & Oktyabrskii, O.N. 2001. The role of antioxidant systems in the cold stress response of Escherichia coli. Microbiology 70: 45-50.

Sonenshein, A.L. 2005. CodY, a global regulator of stationary phase and virulence in Gram-positive bacteria. Current Opinion in Microbiology 8(2): 203-207.

Su, Y., Peng, B., Hui, L., Cheng, Z., Zhang, T., Zhu, J., Li, D., Li, M., Ye, J., Du, C., Zhang, S., Zhao, X., Yang, M. & Peng, X. 2018. Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria. PNAS 115(7): E1578-1587.

Sun, W., Wang, Z., Cao, J., Cui, H. & Ma, Z. 2016. Cold stress increases reactive oxygen species formation via TRPA1 activation in A549 cells. Cell Stress Chaperones 21(2): 367-372.

Suyal, D.C., Kumar, S., Yadav, A., Shouche, Y. & Goel, R. 2017. Cold stress and nitrogen deficiency affected protein expression of psychrotrophic Dyadabacter psychrophilusB2 and Pseudomonas jessenii MP1. Frontiers in Microbiology 2017(8): 430.

Suzuki, K., Maeda, S. & Morokuma, K. 2019. Roles of closed- and open-loop conformations in large scale structural transitions of L-lactate dehydrogenase. American Chemical Society Omega 4(1): 1178-1184.

Taton, A., Erikson, C., Yang, Y., Rubin, B.E., Rifkin, S.A., Golden, J.W. & Golden, S.S. 2020. The circadian clock and darkness control natural competence in cyanobacteria. Nature Communications11: 1688.

Tribelli, P.M. & López, N.I. 2018. Reporting key features in cold-adapted bacteria. Life 8(1): 8.

Tukey, J. 1949. Comparing individual means in the analysis of variance. Biometrics 5(2): 99-114.

Willey, J.M., Sherwood, L. & Woolverton, C.J. 2008. Prescott, Harley, and Klein’s Microbiology. New York: McGraw-Hill Higher Education.

Xie, T., Pang, R., Wu, Q., Zhang, J., Lei, T., Li, Y., Wang, J., Ding, Y., Chen, M. & Bai, J. 2019. Cold tolerance regulated by the pyruvate metabolism in Vibrio parahaemolyticus. Frontiers in Microbiology 10: 178.

Yeh, J.I., Chinte, U. & Du, S. 2008. Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism. PNAS 105(9): 3280-3285.

Zeigler, D.R. 2014. The Geobacillus paradox: Why is a thermophilic bacterial genus so prevalent on a mesophilic planet. Microbiology 160(Pt 1): 1-11.

Zhang, S. & Bryant, D.A. 2015. Biochemical validation of the glyoxylate cycle in the cyanobacterium Chlorogloeopsis fritschiistrain PCC 9212. Journal of Biological Chemistry 290(22): 14019-14030.

Zhang, X., St Leger, R.J. & Fang, W. 2017. Pyruvate accumulation is the first line of cell defense against heat stress in a fungus. mBio 8(5): e01284-e01317.

Zhu, K., Ding, X., Julotok, M. & Wilkinson, B.J. 2005. Exogenous isoleucine and fatty acid shortening ensure the high content of anteiso-C_15:0 fatty acid required for low-temperature growth of Listeria monocytogenes. Applied and Environmental Microbiology 71(12): 8002-8007.

^*Corresponding author; email: michaelw@ums.edu.my