In Staphylococcus aureus the regulation of pyruvate kinase activity by serine/threonine protein kinase favors biofilm formation.
Staphylococcus aureus, a natural inhabitant of nasopharyngeal tract, survives mainly as biofilms. Previously we have observed that S. aureus ATCC 12600 grown under anaerobic conditions exhibited high rate of biofilm formation and L-lactate dehydrogenase activity. Thus, the concentration of pyruvate plays a critical role in S. aureus, which is primarily catalyzed by pyruvate kinase (PK). Analyses of the PK gene sequence (JN645815) revealed presence of PknB site in PK gene indicating that phosphorylation may be influencing the functioning of PK. To establish this hypothesis the pure enzymes of S. aureus ATCC 12600 were obtained by expressing these genes in PK 1 and PV 1 (JN695616) clones and passing the cytosolic fractions through nickel metal chelate column. The molecular weights of pure recombinant PK and PknB are 63 and 73 kDa, respectively. The enzyme kinetics of pure PK showed KMof 0.69 ± 0.02 µM, while the KMof PknB for stpks (stpks = NLCNIPCSALLSSDITASVNCAK) substrate was 0.720 ± 0.08 mM and 0.380 ± 0.07 mM for autophosphorylation. The phosphorylated PK exhibited 40 % reduced activity (PK = 0.2 ± 0.015 μM NADH/min/ml to P-PK = 0.12 ± 0.01 μM NADH/min/ml). Elevated synthesis of pyruvate kinase was observed in S. aureus ATCC 12600 grown in anaerobic conditions suggesting that the formed pyruvate is more utilized in the synthesis phase, supporting increased rate of biofilm formation.
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India.
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D
Vasu
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India.
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MM
Sunitha
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India.
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Srikanth
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India.
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Swarupa
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India.
U Venkateswara
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Prasad
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India.
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Sireesha
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India.
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Yeswanth
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India.
P Santhosh
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Kumar
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India.
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Venkatesh
Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India.
Abhijit
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Chaudhary
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, 517507, India. sarmasvims@gmail.com.
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Sarma
Biofilm; K M; PK; PknB; Stpks