 |
 |
 |
 |
 |
 |
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 LING1,4,*
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
Diserahkan: 29 April 2021/Diterima: 5 Julai 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
RUJUKAN
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 Microbiology39(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 frigidisoliPB4T, 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 irgensiiKOPRI 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. Nature179(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
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 Bacteriology135(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 Factories11: 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. Life8(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-C15:0 fatty acid required for low-temperature growth of Listeria monocytogenes. Applied and Environmental Microbiology 71(12): 8002-8007.
*Pengarang untuk surat-menyurat; email: michaelw@ums.edu.my
|
|||
|
