No abstract

The Krebs cycle dictates oxidative and reductive conditions in Staphylococcus aureus and is mainly regulated by isocitrate dehydrogenase (IDH) which plays pivotal role in the growth and pathogenesis of the bacteria. In the present study, IDH gene from S. aureus ATCC12600 was cloned in the Sma I site of pQE 30 vector; the resultant clone was named as UVIDH1. The insert in the clone was sequenced (accession number HM067707), and the sequence showed complete homology with IDH sequence of other S. aureus strains reported in the database indicating presence of single enzyme in S. aureus, and considerable sequence homology with other bacteria was observed; however, only 24% homology was found with NADP-dependent human IDH. Phylogenetically, the S. aureus IDH showed close identity with Bacillus subtilis and high degree of variability with other bacteria and human IDH. The expression of IDH in the clone UVIDH1 was induced with 1 mM IPTG, and the recombinant IDH was purified by passing through nickel metal chelate column; the purified recombinant IDH showed a single band in SDS-PAGE with a molecular weight of 40 kDa; K(m) and V(max) for isocitrate are 8.2 ± 0.28 and 525 ± 25 μM NADPH/mg/min, respectively, and for cofactor NADP 67.5 ± 2.82 μM and V(max) 50.5 ± 2.12 μM NADPH/mg/min.

Isocitrate dehydrogenase (IDH) gene from Staphylococcus aureus ATCC12600 was cloned, sequenced and characterized (HM067707). PknB site was observed in the active site of IDH; thus, it was predicted as IDH may be regulated by phosphorylation. Therefore, in this study, PknB, alkaline phosphatase III (SAOV 2675) and IDH genes (JN695616, JN645811 and HM067707) of S. aureus ATCC12600 were over expressed from clones PV 1, UVPALP-3 and UVIDH 1. On passing the cytosloic fractions through nickel metal chelate column, pure enzymes were obtained. Phosphorylation of pure IDH by PknB resulted in the complete loss of activity and was restored upon dephosphorylation with SAOV 2675 which indicated that phosphorylation and dephosphorylation regulate IDH activity in S. aureus. Further, when S. aureus ATCC12600 was grown in BHI broth, decreased IDH activity and increased biofilm units were observed; therefore, this regulation of IDH alters redox status in this pathogen favouring 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 KM of 0.69 ± 0.02 µM, while the KM of 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-014-0248-3) contains supplementary material, which is available to authorized users.

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48 h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.