Cloning and Expression of the Gene for a Novel Protein from Mycobacterium smegmatis with Functional Similarity to Eukaryotic Calmodulin

Abstract

A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame. M. smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids (16%) in M. smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M. smegmatis CAMLP. Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M. smegmatis CAMLP.Calmodulin, a highly conserved and ubiquitous protein in eukaryotic cells (16,17), is a small, heat-and acid-stable protein composed of about 155 amino acids; it undergoes a conformational change upon binding calcium and modulates the function of a number of target proteins (20,21,40). While calmodulin was initially considered to be absent in bacteria (3, 6), calmodulin-like proteins (CAMLPs) exhibiting either calcium-binding properties or bovine phosphodiesterase (PDE)-stimulating properties were found in some bacteria (8,10,12,13,15,24,34,36) A917, 1993]. These results support the view that mycobacteria contain a protein functionally similar to eukaryotic calmodulin. As part of our studies on a possible regulatory role of CAMLP in the growth and metabolism of mycobacteria, we report here the purification of CAMLP from M. smegmatis, the nucleotide sequence of the gene encoding CAMLP, and the expression and partial characterization of the protein.Purification of M. smegmatis CAMLP. The activity of M. smegmatis CAMLP was assayed by the method of Fry et al. (11) based on the stimulation of bovine heart PDE activity by eukaryotic calmodulin. M. smegmatis ATCC 14468 was grown from a stab in Brodie and Gray's medium (1.3% BactoCasamino Acids, 0.1% potassium fumarate, 0.2% Tween 80, 0.1% K 2 HPO 4 , 0.03% MgSO 4 , 0.002% FeSO 4 [pH 7.0], with KOH) (2) at 37°C in a rotary shaker for 2 to 3 days. With this used as a fresh inoculum, 12 1-liter cultures were grown overnight. The cells were sedimented at 10,000 ϫ g for 10 min at 4°C and washed with 10 mM Tris-HCl (pH 7.5). About 70 g (wet weight) of cells was obtained.All operations were carried out at 4°C except for the high-

A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis

“…Bacteria are known to possess calmodulin-like and EF-hand proteins, which are speculated to play a role in Ca 2+ storage and signaling. M. smegmatis has a calmodulin-like protein (CAMLP) with functional similarity to eukaryotic calmodulin ( 28 ). This protein was implicated to have a role in phospholipid metabolism ( 29 ), and CAMLP from Mycobacterium tuberculosis has recently been shown to be important during infection ( 30 ).…”

cDNA cloning and characterization of a novel calmodulin‐like protein from pearl oyster Pinctada fucata

“…In addition, recent lines of evidence also demonstrate that CaM plays an important role in regulating the function of mature osteoclasts and osteoclastogenesis, a bone biomineralization related process [15]. CaM‐like protein as a multifunctional calcium sensor belongs to a new member of CaM superfamily, which has been found in bacteria [16], nematode [17], Drosophila [18], plant [19], chicken [20,21], rat [22] and human [23–25]. In human beings, CaLP proteins are involved in epithelial cell differentiation [25,26].…”

Role for malonyl coenzyme A:acyl carrier protein transacylase (MCAT) in the growth-inhibitory effect of the calmodulin antagonist trifluoperazine in Mycobacterium bovis BCG

“…Some evidence points to the existence of calmodulin-like activities in Mycobacterium. 12 Recently, Murthy and co-workers 13 identified the first calmodulin-like protein from Mycobacterium smegmatis and demonstrated trifluoperazinedependent inhibition of its activity. Does trifluoperazine exert its antibacterial activity via inhibition of calmodulin-like proteins in mycobacteria?…”