Phosphorylation controls the functioning of Staphylococcus aureus isocitrate dehydrogenase – favours biofilm formation

Figures & data

Table 1. Primers used for PCR amplification.

Gene name	Orientation	Primer sequence (5′ to 3′)	Reference	Amplicon size
PknB-F1	Forward	CATGATAGGTAAAATA	Present Study	2000
PknB-R1	Reverse	TTATACATCATCATA	Present Study
ALP-F1	Forward	TGCAGTTGCTAACCT	Present Study	1425
ALP-R1	Reverse	TGCGCTTTCTGCATT	Present Study
IDH-F1	Forward	CATGCTGATGTAGAAACT	Prasad et al.13	902
IDH-R1	Reverse	TCAAGGAACTGAAGGAT	Prasad et al.13

Figure 1. (A) Lane 1 PCR amplification of PknB gene (2.0 kb) from the chromosomal DNA of S. aureus ATCC12600, Lane M supermix DNA ladder obtained from Merck Bioscience Pvt. Ltd. (B) SDS-PAGE (10%) analysis of rPknB expression. Lane 1 cytosolic fraction of IPTG-induced PV1 clone, Lane 2 Nickel metal agarose column purified PknB and Lane 3 cytosolic fraction of uninduced PV1 clone and Lane M molecular size marker obtained from Merck Bioscience Pvt. Ltd. (C) Separation of pure phosphorylated PknB identified by reagent-A. L1: Pure phosphorylated PknB. L2 & L3: phosphorylated stpks. (D) Lanes 2 and 3 PCR amplification of alp gene (1.425 kb) from the chromosomal DNA of S. aureus ATCC12600, L1 supermix DNA ladder obtained from Merck Bioscience Pvt. Ltd. (E) SDS-PAGE (10%) analysis of rALP-III expression. Lane 1 uninduced cell lysate of UVPALP-3 clone, L2 IPTG-induced cell lysate of UVP-ALP3 clone and L3 pure rUVP-ALP3 eluted from nickel metal chelate agarose chromatographic column, and Lane M molecular size marker obtained from Merck Bioscience Pvt. Ltd. (F) Separation of pure phospharylated IDH identified by reagent-A. L1: Pure IDH. Lane M: Marker. L2 & L3: phosphorylated IDH.

Figure 2. (A) Multiple sequence alignment of ALP sequence with other bacterial ALP. (B) Multiple sequence alignment of PknB sequence with other bacterial PknB.

Table 2. Enzyme kinetics of rPknB and rALP III.

Source of enzyme	Protein concentration (µg/µl)	Enzyme activity (µM/ml/min)	Km (mM)	Vmax (µM/mg/min)
rPknB (Substrate level)	4	0.162 ± 0.08	0.720 ± 0.08	1.98 ± 0.2
rPknB (Auto phosphorylation)	4	0.316 ± 0.090	0.380 ± 0.07	8.0 ± 0.43
rALP III	3.89	0.203 ± 0.013	4.703 ± 0.619	7.1 ± 0.96

Values are the mean ± SD from three determinations.

Table 3. Enzyme kinetics of S. aureus IDH grown in LB and BHI media.

	Aerobic media (LB)				Anaerobic media (BHI)
Source of enzyme	Vmax (µM/mg/min)	Km (µm)	Kcat	BU	Vmax (µM/mg/min)	Km (µm)	Kcat	BU
IDH in the cytosolic Fraction  of S. aureus ATCC12600	751 ± 58.18	8.2 ± 0.28	3.75 ± 0.06	0.075 ± 0.045	550 ± 38.18	8.2 ± 0.28	2.75 ± 0.07	0.091 ± 0.06

Values are the mean ± SD from three determinations.

Figure 3. Effect of phosphorylation and dephosphorylation in S. aureus biofilm formation.

Table 4. IDH regulation by phosphorylation and dephosphorylation.

IDH regulation	Enzyme activity units
Pure rIDH	23.5 ± 0.28
IDH-P by PknB	Inactive
Dephosphorylation by ALP	20.4 ± 0.24

Values are the mean ± SD from three determinations.

Prasad UV, Vasu D, Kumar YN, et al. Cloning, expression and characterization of NADP-dependent isocitrate dehydrogenase from Staphylococcus aureus. Appl Biochem Biotechnol 2013;169:862–9

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