atl is a newly discovered autolysin gene in Staphylococcus aureus. The gene product, ATL, is a unique, bifunctional protein that has an amidase domain and a glucosaminidase domain. It undergoes proteolytic processing to generate two extracellular peptidoglycan hydrolases, a 51-kDa endo-␤-N-acetylglucosaminidase and a 62-kDa N-acetylmuramyl-L-alanine amidase. It has been suggested that these enzymes are involved in the separation of daughter cells after cell division. We recently demonstrated that atl gene products are cell associated (unpublished data). The cell surface localization of the atl gene products was investigated by immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-␤-Nacetylglucosaminidase immunoglobulin G. Protein A-gold particles reacting with the antigen-antibody complex were found to form a ring structure on the cell surface at the septal region for the next cell division site. Electron microscopic examination of an ultrathin section of the preembedded sample revealed preferential distribution of the gold particles at the presumptive sites for cell separation where the new septa had not been completed. The distribution of the gold particles on the surface of protoplast cells and the association of the gold particles with fibrous materials extending from the cells suggested that some atl gene products were associated with a cellular component extending from the cell membrane, such as lipoteichoic acid. The formation of a ring structure of atl gene products may be required for efficient partitioning of daughter cells after cell division.Peptidoglycan hydrolases are a group of enzymes which catalyze the turnover or degradation of peptidoglycan in bacteria. They include N-acetylglucosaminidases, N-acetylmuramidases, N-acetylmuramyl-L-alanine amidases (AMs), endopeptidases, and transglycosylases (for reviews, see references 1, 18, 22, 25, 26, and 34). These enzymes degrade peptidoglycan saccules, resulting in cell lysis. Therefore, the activities of these enzymes should be strictly regulated. Various physiological functions of these enzymes have been proposed, including their roles in cell separation, wall growth, wall turnover, muropeptide recycling, sporulation, formation of flagella, and transformation (for reviews, see references 22, 25, 26, and 34). Peptidoglycan hydrolases have also been implicated in autolysis induced by ␤-lactams or in bacterial pathogenesis (1, 2, 13-15, 19; for reviews, see references 22 and 26).Bacterial cell separation is a dynamic event in the cell cycle and requires cleavage of peptidoglycan connecting daughter cells. In many bacteria, correlation of a lack of peptidoglycan hydrolase(s) activity and a failure in cell separation has been reported (4,5,7,8,10,11,23,26,27,31,34,35). Recent studies have identified peptidoglycan hydrolases involved in cell separation in Listeria monocytogenes and in Lactococcus lactis (3, 36).We have previously described the identification of a 51-kDa endo-␤-N-acetylglucosaminidase (GL) and a 62...

Abstract:The

In methicillin-resistant Staphylococcus aureus (MRSA) strains, Triton X-100 reduced the oxacillin resistance level, although the degree of reduction varied from strain to strain. To study the responses of MRSA strains to Triton X-100, we isolated a Tn551 insertion mutant of the COL strain that became more susceptible to oxacillin in the presence of 0.02% Triton X-100. The Tn551 insertion of the mutant was transduced back to the parent strain, other MRSA strains (strains KSA8 and NCTC 10443), and methicillin-susceptible strain RN450. All transductants of MRSA strains had reduced levels of resistance to oxacillin in the presence of 0.02% Triton X-100, while those of RN450 did not. Tn551 mutants of KSA8 and NCTC 10443 also had reduced levels of resistance in the absence of 0.02% Triton X-100. The autolysis rates of the transductants in the presence of 0.02% Triton X-100 were significantly increased. Amino acid analysis of peptidoglycan and testing of heat-inactivated cells for their susceptibilities to several bacteriolytic enzymes showed that there were no significant differences between the parents and the respective Tn551 mutants. The Tn551 insertion site mapped at a location different from the previously identified fem and llm sites. Cloning and sequencing showed that Tn551 had inserted at the C-terminal region of a novel gene designated fmt. The putative Fmt protein showed a hydropathy pattern similar to that of S. aureus penicillin-binding proteins and contained two of the three conserved motifs shared by penicillin-binding proteins and beta-lactamases, suggesting that fmt may be involved in cell wall synthesis.

We investigated the cell surface localization of the atl gene products of Staphylococcus aureus exposed to a lytic concentration (4 MIC) of penicillin G (PCG) by means of immunoelectron microscopy using anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-␤-N-acetylglucosaminidase immunoglobulin G. Protein A-gold conjugates reacting with antigen-antibody complex localized at sites of defects of the cell wall at the nascent cross wall. Anti-62-kDa N-acetylmuramyl-L-alanine amidase or anti-51-kDa endo-␤-N-acetylglucosaminidase immunoglobulin G inhibited the decreased turbidity caused by PCG-induced lysis and the formation of defects in the wall. The autolysis-defective mutant, S. aureus RUSAL2 (atl::Tn551), exposed to 4 MIC of PCG resisted autolysis and formation of the wall defect. These results suggest that activation or deregulation of the atl gene products at localized sites where formation of new cross wall was disturbed by PCG causes small defects in the cell wall in situ, eventually leading to general autolysis.

1. The effect of dietary Angelica keiskei on lipid metabolism was examined in stroke-prone spontaneously hypertensive rats (SHRSP). 2. Six-week-old male SHRSP were fed diets containing 0.2% A. keiskei extract (ethyl acetate extract from the yellow liquid of stems) for 6 weeks with free access to the diet and water. 3. Elevation of systolic blood pressure tended to be suppressed on and after 2 weeks; however, this effect was not statistically significant. 4. Serum levels of cholesterol and phospholipid in SHRSP were significantly elevated after treatment with A. keiskei extract and this effect was accompanied by significant increases in serum apolipoprotein (Apo) A-I and ApoE concentrations. These changes in the serum were due to increases in high-density lipoprotein (HDL) containing ApoA-I and ApoE. 5. In the liver, significant decreases in relative weight and triglyceride content were observed in SHRSP after treatment with A. keiskei extract. An investigation of mRNA expression of enzymes involved in hepatic triglyceride metabolism indicated a decreased level of hepatic Acyl-coenzyme A synthetase mRNA expression. 6. In conclusion, dietary A. keiskei produces elevation of serum HDL levels and a reduction of liver triglyceride levels in SHRSP.