A fluorescent assay for bacterial cell wall lytic enzymes

A sensitive fluorimetric assay is described for the measurement of N-acetylmuramic acid l-alanine amidase as well as lysozyme. The method uses Bacillus subtilis cell walls labeled with fluorescamine on the free amino group of diaminopimelic acid. The method can easily detect the lytic activity of 0.02 μg of pure N-acetylmuramic acid l-alanine amidase in 30 min and of 1 μg of hen egg-white lysozyme in the same period. The method is particularly suitable for measurement of competition between various cell wall preparations for the same enzyme.     

Cell wall mutants of Saccharomyces cerevisiae with increased digestibility by cell wall lytic enzymes and protein extractability

Yeast cell wall mutants were obtained by mutagenesis of Saccharomyces cerevisiae X2180-1A, a haplid strain, with N-methyl-N′-nitro-N-nitrosoguanidine. The two S. cerevisiae mutants showed considerable morphological changes and digestibilities by lytic enzymes. Sequential extractions of proteins and polysaccharides from the mutant and wild type cells indicate that the mutants have high protein extractability and lack some wall proteins as well as some polysaccharide fractions.     

Proteolytic activity of a yeast cell wall lytic Arthrobacter species

AIMS: To investigate properties of the proteolytic activity of a yeast cell wall lytic soil bacterium identified as an Arthrobacter species. METHODS AND RESULTS: The organism was grown at pH 7.5 and 30 degrees C in shake flasks on media with different complex subtrates. Highest proteolytic activity assayed with azocaseine was detected in media with wheat gluten. In addition, l-leucine, l-alanine exopeptidase activity and esterase activity were found. The proteolytic activity showed stability up to pH 12, with a maximum at pH 11. The temperature optimum was at 55 degrees C, but there was a loss in enzyme activity of 50% within 2 h. The proteolytic activity was inhibited by 3,4-dichloroisocumarin, whereas there was little or no effect with EDTA, pepstatin A or E64. CONCLUSIONS: The proteolytic activity is highly alkaline stable. The formation of the enzyme can be induced by media with high protein content.     

Chimeric pneumococcal cell wall lytic enzymes reveal important physiological and evolutionary traits.

Two novel chimeric pneumococcal cell wall lytic enzymes, named LC7 and CL7, have been constructed by in vitro recombination of the lytA gene encoding the major autolysin (LYTA amidase) of Streptococcus pneumoniae, a choline-dependent enzyme, and the cpl7 gene encoding the CPL7 lysozyme of phage Cp-7, a choline-independent enzyme. In remarkable contrast with previous chimeric constructions, we fused here two genes that lack nucleotide homology. The CL7 enzyme, which contains the N-terminal domain of CPL7 and C-terminal domain of LYTA, exhibited a choline-dependent lysozyme activity. This experimental rearrangement of domains might mimic the process that have generated the choline-dependent CPL1 lysozyme of phage Cp-1 during evolution, providing additional support to the modular theory of protein evolution. The LC7 enzyme, built up by fusion of the N-terminal domain of LYTA and the C-terminal domain of CPL7, exhibited an amidase activity capable of degrading ethanolamine-containing cell walls. The chimeric amidase behaved as an autolytic enzyme when it was cloned and expressed in S. pneumoniae. The chimeric enzymes provided new insights on the mechanisms involved in regulation of the host pneumococcal autolysins and on the participation of these enzymes in the process of cell separation. Furthermore, our experimental approach confirmed the basic role of the C-terminal domains in substrate recognition and revealed the influence of these domains on the optimal pH for catalytic activity.     

Barley pathogenesis-related proteins with fungal cell wall lytic activity inhibit the growth of yeasts.

Proteins from intercellular fluid extracts of chemically stressed barley (Hordeum vulgare L.) leaves were separated by native polyacrylamide gel electrophoresis at alkaline or acid pH. Polyacrylamide gels contained Saccharomyces cerevisiae (bakers' yeast) or Schizosaccharomyces pombe (fission yeast) crude cell walls for assaying yeast wall lysis. In parallel, gels were overlaid with a suspension of yeasts for assaying growth inhibition by pathogenesis-related proteins. The same assays were also performed with proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. In alkaline native polyacrylamide gels, only one band corresponding to yeast cell wall lytic activity was found to be inhibitory to bakers' yeast growth, whereas in acidic native polyacrylamide gels one band inhibited the growth of both yeasts. Under denaturing nonreducing conditions, one band of 19 kD inhibited the growth of both fungi. The 19-kD band corresponded to a basic protein after two-dimensional gel analysis. The 19-kD protein with yeast cell wall lytic activity and inhibitory to both yeasts was found to be different from previously reported barley chitosanases that were lytic to fungal spores. It could be different from other previously reported lytic antifungal activities related to pathogenesis-related proteins.     

Selection of a Streptomyces strain able to produce cell wall degrading enzymes and active against Sclerotinia sclerotiorum

Control of plant pathogen Sclerotinia sclerotiorum is an ongoing challenge because of its wide host range and the persistence of its sclerotia in soil. Fungicides are the most commonly used method to control this fungus but these can have ecotoxicity impacts. Chitinolytic Streptomyces strains isolated from Brazilian tropical soils were capable of inhibiting S. sclerotiorum growth in vitro, offering new possibilities for integrated pest management and biocontrol, with a new approach to dealing with an old problem. Strain Streptomyces sp. 80 was capable of irreversibly inhibiting fungal growth. Compared to other strains, its crude enzymes had the highest chitinolytic levels when measured at 25°C and strongly inhibited sclerotia from S. sclerotiorum. It produced four hydrolytic enzymes involved in fungal cell wall degradation when cultured in presence of the fungal mycelium. The best production, obtained after three days, was 0.75 U/ml for exochitinase, 0.9 U/ml for endochitinase, 0.16 U/ml for glucanase, and 1.78 U/ml for peptidase. Zymogram analysis confirmed two hydrolytic bands of chitinolytic activity with apparent molecular masses of 45.8 and 206.8 kDa. One glucanase activity with an apparent molecular mass of 55 kDa was also recorded, as well as seven bands of peptidase activity with apparent molecular masses ranging from 15.5 to 108.4 kDa. Differential interference contrast microscopy also showed alterations of hyphal morphology after co-culture. Streptomyces sp. 80 seems to be promising as a biocontrol agent against S. sclerotiorum, contributing to the development of new methods for controlling plant diseases and reducing the negative impact of using fungicides.     

Species-specificity of the lytic activity of the cell wall in the genus Chlorella

Cell wall lytic activity was compared among strains IAM C-27, C-87, SAG 211-1c, -1d, -9a, -8b, -8c, -8l, -11f, -8k, -11g, and -11h/9 of the genus Chlorella . The optimal pH was alkaline in strains with glucosamine as the characteristic group of the rigid wall, and acidic in strains characterised by glucan groups. The lytic enzymes of strains in the former type of algae lyzed the cell wall mainly to soluble high molecular oligosaccharides. The lytic activity of the Chlorella cell wall thus appears species- and strain-speicific.     

Enzyme formation by a yeast cell wall lytic Arthrobacter species: chitinolytic activity

The kinetics of the release of chitinolytic activity (endochitinase EC 3.2.1.14, \-N-acetyglucosaminidase EC 3.2.1.30) by a yeast cell wall lytic Arthrobacter species was studied. The organism was cultivated on yeast cell wall, mycelium of Trichoderma reesei, colloidal chitin, N-acetylglucosamine, glucosamine and mixtures with acetate. With the exception of yeast cell wall, these substrates were used as the sole source of carbon and nitrogen. The growth on colloidal chitin (0.5%) proceeded at a maximum specific growth rate (_umax) of 0.23 h^–1 and yielded 2700 mU1^–1 chitinase. Yeast cell wall and mycelium of T. reesei supported more rapid growth (_max = 0.30 h^–1 and 0.25 h^–1 respectively) but yielded reduced chitinase activity (565 mUl^–1 and 700 mUl^–1). The growth rate on glucosamine (_max = 0.24 h^–1) was reduced when this was mixed with acetate (_max = 0.12 h^–1), whereas the enzyme yield was increased from 720 mUl^–1 to 960 mUl^–1. The same effect on growth rate was observed with glucose and equimolar mixtures of glucose and acetate, indicating a strong impact of the organic acid on carbohydrate transport or metabolism. The growth of adapted cells on N-acetylglucosamine was comparable to that observed on an equimolar mixture of glucosamine and acetate, indicating that N-acetylglucosamine is rapidly hydrolysed by adapted cells.     

Structural analysis and biological significance of the cell wall lytic enzymes of Streptococcus pneumoniae and its bacteriophage

The development of an appropriate technique for the identification of autolysin-defective mutants of pneumococcus has been a fundamental step to carry out studies on the molecular characteristics of the lytic enzymes of Streptococcus pneumoniae and its bacteriophage. Our results show that the principal pneumococcal autolysin (an amidase) is responsible for the separation of the daughter cells at the end of the cell division. On the other hand, this system provides a reliable experimental model to support the extended idea concerning the modular organization of most proteins. The comparative analyses of the deduced amino acid sequences of these enzymes, as well as the construction of functional chimeric phage-bacterial enzymes, demonstrate that the C-terminal domain, which contains a large number of repeated amino acid motifs, is the substrate-binding domain, whereas the N-terminal domain provides enzymatic specificity. We propose that the pneumococcal lytic enzymes have evolved by modular exchange providing examples of the types of novel genes that the bacteria or the phage might create to allow them to become adapted to new environmental situations.     

Studies on a trace cell lytic activity associated with alpha-lactalbumin

alpha-Lactalbumin (alpha-LA) has been examined with a new and sensitive method for determination of lysozyme activity. Samples of bovine, human, equine, and rat alpha-LA exhibited cell lytic activity, from 2 X 10(-6) to 45 X 10(-6) of the specific activity of hen eggwhite lysozyme. The activity was chromatographically inseparable from bovine and human alpha-LA. Bovine serum albumin and purified beta-lactoglobulin were inactive. The pH profiles and reaction kinetics of bovine and human alpha-LA showed differences from those of the corresponding milk lysozymes, indicating that their lytic activities were not likely to have resulted from trace lysozyme content. Thus, it appears that a weak cell lytic activity is inherent to alpha-LA.     

Cementing the wall: cell wall polysaccharide synthesising enzymes

The year under review has seen the first molecular characterisation, with proof of functionality, of Golgi membrane-bound glycosyltransferase enzymes catalysing the synthesis of non-cellulosic plant cell-wall polysaccharides.     

Candida albicans cell wall lytic enzyme produced by Streptomyces thermodiastaticus

The production of lytic enzyme by Streptomyces thermodiastaticus was found to be affected by some growth conditions and nutritional factors. The highest enzyme production was obtained after 18 h of incubation at pH 5.5 and at 50 degrees C. The carbon source influenced the lytic enzyme production. A higher enzyme yield was obtained when Candida albicans cell wall (1 g/100 ml) was used as the sole carbon source. NaNO3 at 0.1 g/100 ml was the best nitrogen source for enzyme production. From all phosphorous sources, microelements, and growth factors tested, KH2PO4 (1 g/l), ZnSO4 (1 mg/I) and Tween 80 (0.1%), respectively, were found to favour the highest production of lytic enzymes by S. thermodiastaticus. The lytic enzymes mainly produced chitinolytic and proteolytic activities.     

Substrate specificity of thePenicillium lilacinum enzyme lytic to the cell wall ofRhodotorula glutinis and the structure of theRhodotorula cell wall glucomannan

The lytic enzyme active on cell walls ofRhodotorula, which was produced byPenicillium lilacinum ATCC 36010, decomposed an extracellular mannan fromRhodotorula glutinis IFO 0695 and was confined to the β-1,3-mannoside bond on the reducing side of the β-1,4-linkedd-mannopyranose. Based on these results, the structure of the glucomannan, which was a major component ofRhodotorula cell walls, was proposed.     

A conserved Hpa2 protein has lytic activity against the bacterial cell wall in phytopathogenic Xanthomonas oryzae

The type III secretion system (TTSS) proteins form a needle-like structure injecting effector proteins into eukaryotic target cells. Although the TTSS forms an important pathway for bacterium-host interaction, its assembly process in vivo is poorly understood. The process is thought to include the opening of a pore before TTSS proteins are inserted into the bacterial cell wall. The proteins that break the bacterial cell wall have not yet been identified. We hypothesize that a hypersensitive response and pathogenicity (hrp) gene functions to digest the bacterial cell wall because it contains a conserved protein sequence similar to lytic transglycosylase. In this study, we cloned hrp-associated 2 (hpa2) genes from the bacteria Xanthomonas oryzae pathovars. We show in vitro that expressed Hpa2 protein has a lytic activity against bacterial cell walls. The analysis of a loss-of-function mutant of the hpa2 gene suggests that the hpa2 affects bacterial proliferation in host plants and a hypersensitive response in nonhost plants. As this is the first of such enzyme activity identified in the Hrp protein family, we speculate that the Hpa2 contributes to the assembly of the TTSS by enlarging gaps in the peptidoglycan meshwork of bacterial cell walls.