Endo-polygalacturonate lyase of Cytophaga johnsonii

Summary An extracellular endopolygalacturonate lyase of Cytophaga johnsonii was purified from the culture filtrate. It appeared to be homogeneous as judged by polyacrylamide gel electrophoresis at pH 8.6 as well as pH 4.3. The purified enzyme had a pH optimum around 9.0 and required Ca⁺⁺ ions for its maximum activity. The apparent K _mfor polygalacturonic acid was found to be 0.22%. Both paper and column chromatography indicated formation and accumulation of an unsaturated monomer along with unsaturated di-, tri-, tetra- and pentamers from polygalacturonic acid by the enzyme action, indicating that the enzyme cleaved the substrate randomly in a non-hydrolytic manner. The glycosidic linkage next to the non-reducing end of polygalacturonic acid was not resistant to attack by this enzyme unlike in other known polygalacturonate lyases.Abbreviations PG lyase Polygalacturonate lyase - Tris Tris (hydroxymethyl) aminomethane     

Pectinase Production by Bacteria Associated with Improved Preservative Permeability in Sitka Spruce: Synthesis and Secretion of Polygalacturonate Lyase by Cytophaga johnsonii

Cytophaga johnsonii synthesized a polygalacturonate lyase which produced random cleavage of galacturonic acid polymers. No pectin methyl-esterase or hydrolytic pectinase activities could be detected in cultures of the organism. Polygalacturonate lyase synthesis was inducible and also subject to repression by glucose and other compounds. Galacturonic acid was the most effective inducer; lower activities were obtained with citrus pectin, polygalacturonic and polypectic acids. Glucose repression of lyase synthesis was not alleviated by 5 mM-adenosine-3'.5'-cyclic-monophosphate. Enzyme production was growth-linked and ceased when batch cultures entered the stationary phase. In steady-state chemostat cultures lyase activity was maximal at a dilution rate ( D ) of 0.19 h^-1. Polygalacturonate lyase was both cell-bound and free in the supernatant medium. The proportion of free enzyme increased throughout the batch growth cycle and in chemostat cultures over 70% of the activity was cell free at dilution rates below 0.05 h^-1.     

Accumulation of zinc and cadmium by Cytophaga johnsonae

Passive and active accumulation of zinc and cadmium by a common soil and freshwater bacterium, Cytophaga johnsonae, was studied using a radio-tracer batch distribution technique. The effects of variation of pH (3–10), as well as of ionic strength (0.007 and 0.07 m) on passive accumulation of the metals were examined. For both zinc and cadmium, accumulation was mainly due to passive processes, such as surface adsorption and/or diffusion into the periplasm. However, at low zinc concentrations, accumulation increased when glucose was added, suggesting an active uptake; at higher zinc concentrations such uptake was not detected, probably because it was masked by the stronger sorption properties of the cell wall. Adsorption of the metals was pH dependent: at higher ionic strength, accumulation was enhanced at pH values above 7; at lower ionic strength, adsorption differed and was markedly higher, with increased accumulation between pH 5 and 8.     

Alkaloids of Strychnos johnsonii

Twenty-three alkaloids have been identified in the root bark and stem bark of Strychnos johnsonii from Zaire. They are demethoxycarbonyl-3,14-dihydro-gambirtannine, angustine, dihydro-cycloakagerine, O-ethylakagerine, O-ethylakagerine lactone, dihydro decussine, normalindine, oxojanussine, tetrahydroalstonial, ajmalicinial, norepimalindine, norharman, harman, akagerine, akagerine lactone, janussines A and B, tetrahydro akagerine, demethoxycarbonyl-3,14,15,16,17,18-hexahydro-gambirtannine, dihydrocorynantheol, anthirine lactone, anthirine and isoanthirine.     

Production of yeast-lytic enzymes by Cytophaga species in batch culture

The conditions for culture storage, inoculum preparation, and growth of a Cytophaga species, constitutive with respect to yeast-lytic enzymes, have been established in shake-flask studies and in 5 liter fermentor experiments. A low cost medium was adopted for 900 liter-scale fermentation and gave an enzyme activity in the fermentation broth somewhat greater than the comparable laboratory-scale one.     

The lysis of Gram-negative Alcaligenes eutrophus by enzymes from Cytophaga

Summary The use of Cytophaga lysing enzymes was investigated for the liberation of poly--hydroxybutyrate (PHB) granules from the Gram-negative bacterium Alcaligenes eutrophus. Complete cell lysis was approached within a 60 minute period. Contrary to previous findings for the lysis of Gram-negative bacteria, prior removal of the outer membrane was not essential for enzymic lysis. The destabilisation of the outer membrane by the removal of divalent cations resulted in no significant improvement in the disruption process.     

The hydrolysis of algal galactans by enzymes from a Cytophaga species

1. Two bacteria were isolated from sea water by the enrichment culture technique, both of which could utilize the galactan sulphate, porphyran, as sole source of carbon. 2. From the cells of one bacterium, classified as a Cytophaga sp., hydrolytic enzymes were isolated. 3. Partial purification of the enzymes is described and some of the properties of the principal enzymes have been studied. 4. The action of the enzymes on several galactan sulphates of red algae suggests that an agarase is present in the mixture.     

The regulation of agarase production by resting cells of Cytophaga flevensis

The regulation of the synthesis of extracellular agarase by Cytophaga flevensis was studied in resting-cell suspensions. Enzyme synthesis was strictly dependent on the presence of a suitable inducer. Enzyme production was maximal at 20 C in phosphate buffer pH 6.9 in the presence of 1.3mm calcium chloride, 0.03% casamino acids and inducer. Enzyme production was virtually the same at 15 and 20 C, reduced to 50% at 25 C and was not detectable at 30 C. It was highly stimulated by the presence of 0.03% of casamino acids in the incubation mixture and was also favoured by the presence of 1.3mm calcium ions. Of a variety of compounds tested, only melibiose or neoagaro-oligosaccharides were effective inducers. Among the neoagaro-oligosaccharides, neoagarotetraose was the best inducer. At higher concentrations of inducer compounds catabolite repression of enzyme synthesis was apparent. This was also found when glucose was added to the incubation mixture. This repression was not relieved by the addition of cyclic AMP. Indications were found that the excretion process was limiting the rate of production of extracellular enzyme.     

Inhibition of Cytophaga U67 gliding motility by inhibitors of polypeptide synthesis

Gliding motility of Cytophaga U67 and several other cytophagas was inhibited by a growth-permissive concentration of chloramphenicol (50 g/ml). Several other inhibitors of polypeptide synthesis also demonstrated this effect. Short-term exposure to several of these inhibitors resulted in reversible inhibition of gliding by growing cells. In wet mounts chloramphenicol-grown cells demonstrated non-translocational tumbling. Electrophoretic patterns of polypeptides released by ethylenediaminetetraacetate treatment of control and chloramphenicol-grown cells were distinct. Gliding of a spontaneous mutant was resistant to chloramphenicol at 50 g/ml; its motility was inhibited at the growth-permissive concentration of 400 g/ml.     

Cell-associated amylase activity in Cytophaga johnsonae

Eighty-eight per cent of the amylase activity of cultures of Cytophaga johnsonae is cell-associated. The cell-associated amylase has optimal activity at pH 7 and 45°C and is completely inhibited by 50 mmol/1 EDTA. Starch in the growth medium induces amylase activity and the respiration rate of starch-grown cells of C. johnsonae is stimulated by starch, glycogen, maltose or glucose.     

Structural insight into chitin perception by chitin elicitor receptor kinase 1 of Oryza sativa

Plants have developed innate immune systems to fight against pathogenic fungi by monitoring pathogenic signals known as pathogen-associated molecular patterns (PAMP) and have established endo symbiosis with arbuscular mycorrhizal (AM) fungi through recognition of mycorrhizal (Myc) factors. Chitin elicitor receptor kinase 1 of Oryza sativa subsp. Japonica (OsCERK1) plays a bifunctional role in mediating both chitin-triggered immunity and symbiotic relationships with AM fungi. However, it remains unclear whether OsCERK1 can directly recognize chitin molecules. In this study, we show that OsCERK1 binds to the chitin hexamer ((NAG)₆) and tetramer ((NAG)₄) directly and determine the crystal structure of the OsCERK1-(NAG)₆ complex at 2 Å. The structure shows that one OsCERK1 is associated with one (NAG)₆. Upon recognition, chitin hexamer binds OsCERK1 by interacting with the shallow groove on the surface of LysM2. These structural findings, complemented by mutational analyses, demonstrate that LysM2 is crucial for recognition of both (NAG)₆ and (NAG)₄. Altogether, these findings provide structural insights into the ability of OsCERK1 in chitin perception, which will lead to a better understanding of the role of OsCERK1 in mediating both immunity and symbiosis in rice.     

Structure and Biosynthesis of Two Exopolysaccharides Produced by Lactobacillus johnsonii FI9785

Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1–3)-β-Glcp-(1–5)-β-Galf-(1–6)-α-Glcp-(1–4)-β-Galp-(1–4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.     

Identification of three proteins up-regulated by raw starch in Cytophaga sp.

Raw starch-digesting amylases (RSDAs) in many microorganisms convert starch granules into maltodextrins and simple sugars. We cloned and sequenced from Cytophaga sp. an RSDA with an excellent raw starch digestion activity. This RSDA was highly inducible by raw starch, but not by other sugars, suggesting that an unknown signal transduction mechanism is involved in the degradation of raw starch. We used a proteomic approach to investigate the effect of raw starch on protein expression in Cytophaga sp. Using MALDI–TOF MS protein analysis, we have identified three proteins up-regulated by raw starch, i.e., a 60-kDa chaperonin (cpn60), glutaminase, and pyruvate phosphate dikinase (PPDK). Subsequent time-course studies detected an increased expression of RSDA as well as the highest expression of PPDK occurring 6 h post-incubation with raw corn starch, implying that the latter enzyme may work along with RSDA on the digestion of raw starch. Finding these proteins up-regulated by raw starch may provide an insight into how Cytophaga sp. cells respond to raw starch stimulation.     

Hydrolysis of glycol chitin by chitinolytic enzymes

• 1.1. The hydrolysis of glycol chitin preparations by several β-N-acetylglucosaminidases was monitored colorimetrically with the potassium ferriferrocyanide reagent.
• 2.2. Glycol chitin samples from crab and insect sources varied considerably in chemical composition and susceptibility to enzymatic hydrolysis.
• 3.3. Insect endochitinase preferred crab glycol chitin as substrate while hen's egg white lysozyme preferred commercial glycol chitin.
• 4.4. Insect glycol chitin was well hydrolyzed by both enzymes.
• 5.5. Insect exochitinase did not digest glycol chitin.