The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the ⑀-amino group of lysine 983 (Lys 983 ) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys 860 ), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additional acylation site by replacing Lys 860 of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of The adenylate cyclase toxin-hemolysin (ACT, 1 AC-Hly, or CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis and a promising protective antigen candidate for acellular pertussis vaccines (1-5). ACT belongs to the RTX (repeats in toxin) protein family (6) and has the capacity to form small cation-selective membrane channels, which account for its weak hemolytic activity (8 -11). The major cytotoxic activity of the 1706-residue-long protein, however, consists in its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane (12-14) and to deliver into cells a catalytic adenylate cyclase (AC) domain. This intoxicates cells by unregulated conversion of ATP to cAMP (15-18) and causes impairment of microbicidal functions of immune effector cells and apoptosis of lung macrophages (19).The capacity of ACT to penetrate into target cell membranes and to intoxicate cells depends on a posttranslational activation by the accessory protein, CyaC (20, 21). It was first established for the Escherichia coli ␣-hemolysin (HlyA) that the activation of RTX toxins consists in amide linked fatty-acylation (22), and Hackett et al. (23) have demonstrated by mass spectrometric analysis that native ACT produced by Bordetella (Bp-ACT) is mono-acylated by a palmitoyl residue at the ⑀-amino group of lysine 983. In contrast, the HlyA from E. coli was found to be acylated at two lysines, both in vitro and in vivo (24,25). Moreover, two highly conserved RTX acylation sites (25), corresponding to lysine 983 (Lys 983 ) and lysine 860 (Lys 860 ) are also found in ACT, and for an unknown reason, the CyaCactivated recombinant ACT produced in E. coli (r-Ec-ACT) is palmitoylated also at Lys 860 , in addition to acylation of Lys 983 (26). When compared with the native mono-acylated Bp-ACT, the doubly acylated r-Ec-ACT exhibits about four times lower specific hemolytic activity on sheep erythrocytes (20) and about 10 times lower specific channel-forming activity in artificial planar lipid bilayers (10). At the same time, however, the characteristics of the channels formed by both proteins are identical, and both proteins have identical capacity to insert ...

Monitoring the external environment and responding to its changes are essential for the survival of all living organisms. The transmission of extracellular signals in prokaryotes is mediated mainly by twocomponent systems. In addition, genomic analyses have revealed that many bacteria contain eukaryotictype Ser/Thr protein kinases. The human pathogen Streptococcus pneumoniae encodes 13 two-component systems and has a single copy of a eukaryotic-like Ser/Thr protein kinase gene designated stkP. Previous studies demonstrated the pleiotropic role of the transmembrane protein kinase StkP in pneumococcal physiology. StkP regulates virulence, competence, and stress resistance and plays a role in the regulation of gene expression. To determine the intracellular signaling pathways controlled by StkP, we used a proteomic approach for identification of its substrates. We detected six proteins phosphorylated on threonine by StkP continuously during growth. We identified three new substrates of StkP: the Mndependent inorganic pyrophosphatase PpaC, the hypothetical protein spr0334, and the cell division protein DivIVA. Contrary to the results of a previous study, we did not confirm that the ␣-subunit of RNA polymerase is a target of StkP. We showed that StkP activation and substrate recognition depend on the presence of a peptidoglycan-binding domain comprising four extracellular penicillin-binding protein-and Ser/Thr kinase-associated domain (PASTA domain) repeats. We found that StkP is regulated in a growth-dependent manner and likely senses intracellular peptidoglycan subunits present in the cell division septa. In addition, stkP inactivation results in cell division defects. Thus, the data presented here suggest that StkP plays an important role in the regulation of cell division in pneumococcus.Protein phosphorylation is considered the universal language for inter-and intracellular communication in all living organisms. This process, catalyzed by protein kinases, enables translation of extracellular signals into cellular responses and adaptation to a constantly changing environment. Although previous work indicated that histidine kinases of two-component regulatory systems are the most prominent kinases in prokaryotes, recent data provide evidence that eukaryotic-type Ser/Thr protein kinases (ESTPKs) play an important role in prokaryotic cell signaling. The distribution of these enzymes among bacteria is broad but not universal. ESTPKs have been found in nearly two-thirds of the sequenced bacterial strains. Most of these enzymes are encoded by genes in strains belonging to the phyla Proteobacteria (Myxococcales), Actinobacteria, Cyanobacteria, Chloroflexi, Acidobacteria, and Planctomycetes (38). We surveyed sequenced bacterial genomes and found ESTPK-encoding genes also in the bacterial taxa Aquificae,

Silent information regulators are NAD(+)-dependent enzymes that display differential specificity toward acetylated substrates. This report provides first evidence for deacetylation activity of CobB1 in Streptomyces coelicolor. The protein is highly conserved in streptomycetes. The CobB1 protein catalytically removes the acetyl group from acetylated bovine serum albumin. In the absence of NAD+ or when NAD+ was substituted with nicotinamide, deacetylation was stopped. We isolated gene encoding AcetylCoA synthetaseA. The recombinant enzyme produces Acetyl-CoA from acetate. The highest acsA-mRNA level was detected in cells from the exponential phase of growth, and then decreased in transition and stationary phases of growth. Acetylated acsA loses the ability to transfer acetate to CoA. Deacetylation of the enzyme required CobB1, ATP-Mg2, and NAD+. Using specific antibodies against acetylated lys, CobB1, and acsA, we found relationship between level of CobB1 and acetylation of acsA, indicating that CobB1 is involved in regulating the acetylation level of acsA and consequently its activity. It was found that 1-acetyl-tetrahydroxy and 1-acetyl pentahydroxy antraquinone inhibit the deacetylation activity of CobB1.