A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium.

Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.

Crystal structure of staphylococcal LukF delineates conformational changes accompanying formation of a transmembrane channel.

Staphylococcal LukF, LukS, HgammaII, and alpha-hemolysin are self-assembling, channel-forming proteins related in sequence and function. In the alpha-hemolysin heptamer, the channel-forming beta-strands and the amino latch make long excursions from the protomer core. Here we report the crystal structure of the water soluble form of LukF. In the LukF structure the channel-forming region folds into an amphipathic, three-strand beta-sheet and the amino latch forms a beta-strand extending a central beta-sheet. The LukF structure illustrates how a channel-forming toxin masks protein-protein and protein-membrane interfaces prior to cell binding and assembly, and together with the alpha-hemolysin heptamer structure, they define the end points on the pathway of toxin assembly.

Further study on the two pivotal parts of Hlg2 for the full hemolytic activity of staphylococcal gamma-hemolysin.

Staphylococcal gamma-hemolysin consists of LukF of 34 kDa and Hlg2 (or H gamma II) of 32 kDa, which cooperatively lyse human and rabbit erythrocytes. Our previous data showed that the 5-residue segment K23R24L25A26I27 of Hlg2 is pivotal for the hemolytic activity [Nariya, H. and Kamio, Y., Biosci. Biotechnol. Biochem., 59, 1603-1604 (1997)]. Here, we identify an additional amino acid residue in Hlg2 necessary for the full gamma-hemolysin activity by measuring the toxin activity of Hlg2 mutants in the presence of LukF. The data obtained showed that Arg217 of Hlg2 is an additional pivotal amino acid residue besides the KRLAI segment for the full Hlg2-specific function in gamma-hemolysin. We also report evidence that the Hlg2 mutants showing a low or null hemolytic activity in the presence of LukF towards human erythrocytes had low or no binding activity to the cells, resulting in failure of formation of the ring-shaped pore-forming complex on the erythrocytes.

Phosphorylation of LukS by protein kinase A is crucial for the LukS-specific function of the staphylococcal leukocidin on human polymorphonuclear leukocytes.

Staphylococcal leukocidin (Luk) consists of two protein components, LukF and LukS, which cooperatively lyse human and rabbit polymorphonuclear leukocytes. Here, we demonstrate that the phosphorylation of LukS by protein kinase A is crucial for the LukS-specific leukocytolytic function of Luk on HPMNLs by using N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), which is a potent and selective inhibitor of protein kinase A. At 0.5 microM H-89 completely prevented the Luk-induced cell lysis accompanied by blocking of the incorporation of exogenous 32P-H3PO4 into LukS on HPMNLs. However, with LukS and LukF together, 0.5 microM H-89 did not inhibit the cell swelling which takes place before the cell lysis. HPMNLs also became swollen upon treating with both LukF and LukS mutants which could not be phosphorylated.

Identification of the minimum segment in which the threonine246 residue is a potential phosphorylated site by protein kinase A for the LukS-specific function of staphylococcal leukocidin.

Staphylococcal leukocidin and gamma-hemolysin consist of LukF and LukS for leukocidin and LukF and Hlg2 for gamma-hemolysin. In this report, we identify the minimum segment responsible for the LukS-specific function of leukocidin. After chemical analysis and homology study of the amino acid sequence of the C-terminal region between LukS and Hlg2, we found a unique 5-residue sequence I242K243R244S245T246 in LukS in which the 4-residue KRST is identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. To elucidate whether the 5-residue segment is essential for the LukS function, we created plasmids containing a series of mutant genes corresponding to the 5-residue sequence and expressed them in Escherichia coli. The mutant proteins were purified and assayed for their leukocytolytic activity with LukF. The mutant MLS-TS, in which the T246 in the 5-residue sequence was replaced by S, showed leukocidin activity 10 times higher than that of the intact LukS. However, neither mutant MLS-TY nor MLS-TA, in which T246 was replaced by Y or A, respectively, showed leukocidin activity. The 5-residue segment was found to be deleted in Hlg2. The mutant of Hlg2, in which the 5-residue segment was inserted at the position that the segment is deleted, showed leukocidin activity. The boiled LukS, MLS-TS, and MHS-Z were strongly phosphorylated with [gamma-32P]ATP in the presence of protein kinase A in a cell-free system. Thus, we conclude that the 5-residue segment 1242K243R244S245T246 is the pivotal segment of LukS responsible for the LukS function of staphylococcal leukocidin.

Identification of the minimum segment essential for the H gamma II-specific function of staphylococcal gamma-hemolysin.

Staphylococcal gamma-hemolysin consists of H gamma I (or LukF) of 34 kDa and H gamma II of 32 kDa, which cooperatively lyse human erythrocytes. Our previous data showed that the N-terminal 57-residue segment of H gamma II is the essential region for the H gamma II function [H. Nariya and Y. Kamio, Biosci. Biotech. Biochem., 59, 1603-1604 (1995)]. To identify the minimum amino acid residues in the 57-residue segment responsible for the specific hemolytic activity, a series of mutant genes were constructed and expressed in Escherichia coli. The mutant proteins were purified and assayed for their hemolytic activity. The results indicate that the 5-residue segment (K23R24L25A26I27) of H gamma II is the minimum region essential for the H gamma II function.

Identification of the essential amino acid residues in lukS for the hemolytic activity of staphylococcal leukocidin towards rabbit erythrocytes.

Staphylococcus aureus V8 (ATCC 49775) produces Panton-Valentine leukocidin (PVL) and leukocidin (Luk). PVL and Luk consist of LukF-PV and LukS-PV, and LukF and LukS, respectively. LukF and LukS cooperatively and strongly lyse rabbit erythrocytes besides human and rabbit polymorphonuclear leukocytes. LukF and LukS-PV also cooperatively lysed rabbit erythrocytes, but its activity was only 4% of that in combination [LukF-LukS] after 15 min of incubation. To identify the pivotal region responsible for the hemolytic function of LukS towards rabbit erythrocytes, we created a series of chimeric genes (lukS-PV/lukS) and mutant genes of lukS-PV and had them expressed in Escherichia coli. The chimeric and mutant proteins purified from the sonicated extract from the cells of E. coli were assayed for their hemolytic activities towards rabbit erythrocytes in combination with LukF or LukF-PV. The results indicate that a 2-residue segment (D12I13) of lukS is the minimum region essential for the hemolytic function of LukS towards rabbit erythrocytes.

Identification of the essential regions for LukS- and H gamma II-specific functions of staphylococcal leukocidin and gamma-hemolysin.

Staphylococcal leukocidin and gamma-hemolysin consist of LukF and LukS for leudocidin and LukF and H gamma II for gamma-hemolysin. To identify the pivotal domain(s) of LukS and H gamma II responsible for their specific cytolytic activities, a series of chimeric genes (lukS/hlg2) were constructed and expressed in Escherichia coli. The results indicate that the C-terminal 24-residue and the N-terminal 57-residue segments of LukS and H gamma II, respectively, are the essential regions for the LukS and H gamma II functions.

Inactivation of gamma-hemolysin H gamma II component by addition of monosialoganglioside GM1 to human erythrocyte.

The Staphylococcal toxins leukocidin and gamma-hemolysin consist of two protein components: F and S in leukocidin and H gamma I and H gamma II in gamma-hemolysin. The two toxins share one component (F = H gamma I). We found that the H gamma II component was completely inactivated by the addition of monosialoganglioside GM1 at the molar ratio of 1:1. Disialogangliosides GD1a and GD1b had little effect on the inactivation of H gamma II. The molar ratios of GD1a and GD1b to H gamma II needed for maximum inactivation were 30:1 and 100:1, respectively. Related glycolipids caused little if any inactivation. H gamma II bound to GM1 to form H gamma II-GM1 complexes. Analysis of the intrinsic aromatic amino acid fluorescence in H gamma II and H gamma II-GM1 with 280 nm as the excitation wavelength showed that GM1 in the complex reduced the fluorescence intensity of H gamma II by 12% without changing the wavelength of maximum emission (325 nm). We concluded that GM1 is a receptor of the H gamma II component on human erythrocytes and that H gamma II takes on a different conformation when it binds to GM1.

The C-terminal region of the S component of staphylococcal leukocidin is essential for the biological activity of the toxin.

The Staphylococcal toxin leukocidin consists of two protein components, F and S. From a culture medium of Staphylococcus aureus RIMD 310925, we isolated a truncated form of S (LS2), of which the C-terminal 17-residue segment is missing. Unlike intact S, LS2, showed neither leukocytolytic activity in the presence of F nor affinity for monosialoganglioside GM1 (GM1). When excited at 280 nm, both S and LS2 exhibited intrinsic tryptophan fluorescence with an emission maximum at 318 nm. Upon binding to GM1, the emission maximum of S underwent a blue shift to 310 nm, whereas no change in fluorescence took place on mixing GM1 with LS2. We conclude that the C-terminal region of S is essential for its biological activity as well as for its binding to GM1 and that this binding is accompanied by a conformational change of the S protein.

The two Staphylococcal bi-component toxins, leukocidin and gamma-hemolysin, share one component in common.

Staphylococcal bi-component toxins, leukocidin and gamma-hemolysin, consist of two protein components, i.e. F and S for leukocidin and H gamma I and H gamma II for gamma-hemolysin. In this study we purified H gamma I and H gamma II to homogeneity from the culture medium of Staphylococcus aureus RIMD 310925 and compared their properties with those of F and S purified from the same source. The N-terminal 59- and C-terminal 2-residue amino acid sequences, apparent molecular mass, and isoelectric point of purified H gamma I were the same as those of F. In an Ouchterlony double diffusion test a fused line without spur was formed between F and H gamma I using either anti-F or anti-H gamma I antibodies. A synergistic action of F and H gamma II caused hemolysis of human red blood cells, and H gamma I acted synergistically with S to exhibit leukocidin activity. We conclude that the two toxins share one protein component (F = H gamma I) in common and leukocidin- and gamma-hemolysin-specific activities are determined by S and H gamma II, respectively. It is also reported that the N-terminal 58-residue sequence of H gamma II is 72% similar to the corresponding sequence of S.

Molecular cloning and nucleotide sequence of leukocidin F-component gene (lukF) from methicillin resistant Staphylococcus aureus.

A lukF gene encoding F-component of Staphylococcal leukocidin from methicillin resistant Staphylococcus aureus (MRSA) was cloned. The nucleotide sequence of lukF gene was determined. The sequence data have revealed an open reading frame, which encodes a polypeptide with 323 amino acid residues. Inspection of the amino acid sequence deduced from nucleotide sequence of lukF and that from F-component of leukocidin from S. aureus V8 clarified that pre-matured F-component contains a typical signal peptide at the NH2 terminus and ATG starting codon for pre-matured F-component was present one base downstream to the TGA which is translation termination codon for S-component of leukocidin [A. Rahman et al. (1991) Biochem. Biophys. Res. Commun. 181, 138-144]. The nucleotide sequence of 5'-flanking region of lukF showed the presence of the consensus sequence of ribosome binding site in the internal region of the structural gene of S-component. The lukF was transcribed in the same direction as that of lukS. No Pribnow box can be discerned in the intercistronic region between the lukS and lukF genes. The amino acid sequence homology between S- and F-components was 31%. F-component was expressed in Escherichia coli DH5 alpha harboring plasmid pFRK92 which contained lukF gene.