Mechanism of action of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-positive bacteria: role of the cell wall anionic polymers of the target bacteria

The study of the extracellular bacteriolytic enzymes of Lysobacter sp. showed that they can efficiently hydrolyze the peptidoglycan of gram-positive bacteria provided that there is an electrostatic interaction of these enzymes with the cell wall anionic polymers, teichoic and teichuronic acids in particular. The hydrolytic action of bacteriolytic enzymes on the cell wall largely depends on the negative charge of teichoic and teichuronic acids, rather than on their chemical composition.     

The effect of metal ions on the synthesis of extracellular enzymes by sporulating bacteria]

The action of metal ions which are present in nutritious medium on the synthesis of extracellular enzymes by sporulating bacteria is analysed. An important role of these ions in post-secretory modification of protein molecules and formation of functionally active molecules of enzyme is shown. The effect of metal ions on some cell envelope properties and extracellular enzyme secretion is under discussion.     

Secretion of bacteriolytic endopeptidase L5 of Lysobacter sp. XL1 into the medium by means of outer membrane vesicles

The Gram-negative bacterium Lysobacter sp. XL1 secretes various proteins, including bacteriolytic enzymes (L1-L5), into the culture medium. These proteins are able to degrade Gram-positive bacteria. The mechanism of secretion of extracellular proteins by Lysobacter sp. XL1 has not been studied hitherto. Electron microscopic investigations revealed the phenomenon of the formation of extracellular vesicles by Lysobacter sp. XL1. These vesicles contained components of the Lysobacter sp. XL1 outer membrane, and demonstrated bacteriolytic activity against Gram-positive and Gram-negative bacteria: Staphylococcus aureus 209-P and Erwinia marcescens EC1, respectively. Western blotting analysis with antibodies to homologous bacteriolytic endopeptidases L1 and L5 showed that endopeptidase L5 was secreted into the culture medium by means of vesicles, unlike its homolog, endopeptidase L1. When inside the vesicles, endopeptidase L5 actively lysed the Gram-negative bacterium Erwinia marcescens; outside the vesicles, it lost this ability. The secretion of bacteriolytic endopeptidase L5 through the outer membrane vesicles is of great biological significance: because of this ability, Lysobacter sp. XL1 can compete in nature with both Gram-positive and Gram-negative bacteria.     

Comparison of the D-glutamate-adding enzymes from selected gram-positive and gram-negative bacteria.

The biochemical properties of the D-glutamate-adding enzymes (MurD) from Escherichia coli, Haemophilus influenzae, Enterococcus faecalis, and Staphylococcus aureus were investigated to detect any differences in the activity of this enzyme between gram-positive and gram-negative bacteria. The genes (murD) that encode these enzymes were cloned into pMAL-c2 fusion vector and overexpressed as maltose-binding protein-MurD fusion proteins. Each fusion protein was purified to homogeneity by affinity to amylose resin. Proteolytic treatments of the fusion proteins with factor Xa regenerated the individual MurD proteins. It was found that these fusion proteins retain D-glutamate-adding activity and have Km and Vmax values similar to those of the regenerated MurDs, except for the H. influenzae enzyme. Substrate inhibition by UDP-N-acetylmuramyl-L-alanine, the acceptor substrate, was observed at concentrations greater than 15 and 30 microM for E. coli and H. influenzae MurD, respectively. Such substrate inhibition was not observed with the E. faecalis and S. aureus enzymes, up to a substrate concentration of 1 to 2 mM. In addition, the two MurDs of gram-negative origin were shown to require monocations such as NH4+ and/or K+, but not Na+, for optimal activity, while anions such as Cl- and SO4(2-) had no effect on the enzyme activities. The activities of the two MurDs of gram-positive origin, on the other hand, were not affected by any of the ions tested. All four enzymes required Mg2+ for the ligase activity and exhibited optimal activities around pH 8. These differences observed between the gram-positive and gram-negative MurDs indicated that the two gram-negative bacteria may apply a more stringent regulation of cell wall biosynthesis at the early stage of peptidoglycan biosynthesis pathway than do the two gram-positive bacteria. Therefore, the MurD-catalyzed reaction may constitute a fine-tuning step necessary for the gram-negative bacteria to optimally maintain its relatively thin yet essential cell wall structure during all stages of growth.     

The Effect of the Extracellular Bacteriolytic Enzymes of Lysobacter sp. on Gram-Negative Bacteria

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria was studied. These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria. The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells was altered by treating them with polymyxin B.     

Intracellular distribution of enzymes of phospholipid metabolism in several gram-negative bacteria.

Cell-free extracts of Salmonella typhimurium, Serratia marcescens, Enterobacter aerogenes, and Micrococcus cerificans contained the following enzymatic activities related to phospholipid metabolism: cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride):l-serine O-phosphatidyltransferase (phosphatidylserine synthase), phosphatidylserine decarboxylase, CDP-diglyceride:sn-glycero-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase), phosphatidylglycerophosphate phosphatase, and CDP-diglyceride hydrolase. The intracellular distribution of these enzymatic activities as determined by sucrose density gradient centrifugation of cell-free extracts was shown to be similar in each species investigated. The phosphatidylserine decarboxylase, phosphatidylglycerophosphate synthase, and CDP-diglyceride hydrolase activities were all associated with the cell envelope fraction, whereas the phosphatidylserine synthase activity was associated mainly with the ribosomal fraction. These enzymatic activities are comparable and have an intracellular distribution similar to those found in Escherichia coli cell-free extracts. Therefore, the pathways established for phospholipid biosynthesis in E. coli can also account for the synthesis of the major phospholipids (phosphatidylethanolamine and phosphatidylglycerol) in several other gram-negative organisms. In addition, the unusual ribosomal association of the phosphatidylserine synthase from E. coli (Raetz and Kennedy, J. Biol. Chem. 247:2008-2014, 1972) appears to be a general property for this activity in several other bacterial species.     

Reverse effect of gram-positive bacteria vs. gram-negative bacteria on adjuvant-induced arthritis in germfree rats

Germfree (GF) F344 rats developed severe adjuvant-induced arthritis with a 100% incidence after a single intradermal injection of heat-killed Mycobacterium bovis (BCG). Specific pathogene-free (SPF) rats developed less severe arthritis with a lower incidence. The rats colonized with Escherichia coli or Bacteroides developed mild disease comparable to that in SPF rats. The rats colonized with Bifidobacterium, Propionibacterium acnes, Lactobacillus casei, L. fermentum, L. murini, and L. acidophilus developed more severe disease than that in GF rats. Furthermore, the rats colonized with a mixture of E. coli and the above lactobacilli developed very mild disease similar to that in SPF rats. These results suggest that gram-negative bacteria, such as E. coli and Bacteroides, may suppress the disease, possibly through their lipopolysaccharides, and may be responsible for the lower susceptibility of SPF rats; gram-positive bacteria, such as Bifidobacterium, P. acnes, and lactobacilli, may enhance the disease, possibly through their peptidoglycans; and E. coli may play a dominant role in modulating the development of adjuvant-induced arthritis.     

链霉菌RX-17产溶细菌酶的研究

通过下磁针试验,确定了链霉菌（Streptomyces sp.)RX-17产溶细菌酶的最佳碳氮源比例即蔗糖3%,大豆蛋白胨1.25%。牛肉膏0.25%,最适培养条件的研究表明,通气量对该菌产酶十分关键,对酶的基本性质进行了研究,酶作用的最适温度和最适pH分别是60℃和6.0,在碱性范围内酶的稳定性较好,60℃1h残存酶活36.3%,溶菌活性对离子强度的变化高度敏感,溶菌谱测定显示,该酶对卵清溶菌酶不能作用的变链球菌（Streptococcus mutans),金黄色葡萄球菌（Staphylococcus aureus)有很强的溶解活性。