Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca²⁺. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca²⁺ is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca²⁺, the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca²⁺ and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca²⁺ protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca²⁺ addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope.     

Enzymo-Serological Comparison of Proteolytic Enzymes Produced by Salmonella Species and Other Enterobacteria

The proteolytic enzymes produced by 11 Salmonella species of the Sub-genera I, II and IV, have been compared by a so-called enzymo-serological procedure. In sub-genus I, the enzymes of S. schleissheim and S. abortus-bovis showed an identical, or closely related, serological picture, whereas S. texas was serologically distinct. All the 7 examined strains of sub-genus II produced proteolytic enzymes which were serologically very similar, or identical. No enzymo-serological cross-reactions were observed between these organisms and the members of sub-genus I. The only examined species of sub-genus IV, S. argentina, was enzymo-serologically distinct from all other species. Intergeneric cross-reactions occurred between the enzymes from Salmonella species, Enterobacter (Aerobacter) cloacae and Serratia marcescens. The significance of these cross-reactions is discussed.     

Extracellular Proteolytic Enzymes of Azospirillum brasilense Strain Sp7 and Regulation of Their Activity by a Homologous Lectin

It was found that Azospirillum brasilense strain Sp7 is able to produce extracellular proteolytic enzymes. The enzymes were active within a broad range of pH values, with two peaks of activity being located in the acid and alkaline pH areas; required calcium ions; and exhibited substrate specificity with respect to azogelatin. Zymography allowed at least four proteolytic enzymes with molecular weights of 32, 45, 52, and 174 kDa to be detected in A. brasilense Sp7 culture liquid. It was shown that the lectin from A. brasilense Sp7 can inhibit proteolytic enzymes.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 444–448.Original Russian Text Copyright © 2005 by Chernyshova, Alen’kina, Nikitina, Ignatov.     

Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes

Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (~74, ~55, ~54, and ~37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokházi, G. Kóczán, F. Hudecz, L. Gráf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.     

Involvement of Proteolytic Enzymes and Their Inhibitors in Plant Protection (Review)

The literature data on possible ways of involvement of proteolytic enzymes and their inhibitors in protection of plants from pests and disease are analyzed. Certain practical applications of natural protease inhibitors to plant protection are discussed.     

Enzymes Produced by a Pseudomonas Species Which Inactivate Inhibitors of Certain Viral Hemagglutinins. I. Identification and Purification of a Proteinase and Phospholipase C

Filtrates from cultures of a psychrophilic Pseudomonas species, which inactivate serum inhibitors of certain viral hemagglutinins, were shown to contain both lecithinase (phospholipase C) and a proteolytic enzyme with elastase activity. The bacterium was cultivated under conditions favoring production of the respective enzymes, and the enzymes were purified by ammonium sulfate precipitation followed by column chromatography or by gel filtration. The elastase was obtained in crystalline form and was recrystallized. It has properties similar to those of a number of other bacterial elastases but is more heat-labile than most. Although a high degree of purification was achieved for the lecithinase, as evidenced by an increase in specific activity, it was not obtained in crystalline form. Partially purified preparations of the lecithinase had extremely high activity compared to that of commercial preparations of phospholipase C from Clostridium welchii.     

Localization of Polyvinyl Alcohol Oxidase Produced by a Bacterial Symbiont, Pseudomonas sp. Strain VM15C

An axenic culture of a polyvinyl alcohol (PVA)-degrading symbiont, Pseudomonas sp. strain VM15C, was established on PVA with a crude preparation of the growth factor (factor A) produced by the symbiotic partner Pseudomonas putida VM15A. An increase of factor A in the culture medium enhanced the cell-associated PVA oxidase activity as well as the growth rate, but decreased production of extracellular PVA oxidase. PVA oxidase in cells grown on PVA was present in the periplasmic space at a higher ratio than in cells grown on peptone. PVA degradation occurred rapidly with washed cells. PVA was also degraded by immobilized cells entrapped in agar gels.     

Bacterial Alginate Produced by a Mutant of Azotobacter vinelandii

The optimum conditions in shaken flasks for production of bacterial alginate by mutant C-14 of Azotobacter vinelandii NCIB 9068 and a comparison of the properties of bacterial and algal alginates were investigated. The largest amount of bacterial alginate was obtained in about 110 h by a culture grown on optimum medium at 34°C and 170-rpm shaking speed. The viscosity of the culture broth was 18,400 cps and the alginate concentration reached 6.22 g/liter. The viscosity of the purified bacterial alginate was as high as 11,200 cps at a low concentration (0.6%). A greater than fivefold concentration of algal alginate was required to reach the same viscosity at a low shear rate. A solution of bacterial alginate was more pseudoplastic than that of algal alginate was. No significant differences were observed in other properties of bacterial and algal alginates such as gel formation with calcium ion, thermostability, and effect of temperature, pH, and sodium chloride on viscosity.     

Produced by a Strain of Unidentified Actinomycetes sp.

The possibility of using two kinds of sorghum as raw materials in consolidated bioprocessing bioethanol production using Flammulina velutipes was investigated. Enzymatic saccharification of sweet sorghum was not as high as in brown mid-rib (bmr) mutated sorghum, but the amount of ethanol production was higher. Ethanol production from bmr mutated sorghum significantly increased when saccharification enzymes were added to the culture.     

Constituents of a Peptidal Antibiotic P168 Produced by Paecilomyces lilacinus (Thom) Samson

Two glycerol dehydrogenases, GD-I and GD-II, were purified from Geotrichum candidum by precipitation of ammonium sulfate, sequential column chromatography on Blue-Sepharose CL-4B and DEAE-Sepharose CL-6B and gel filtration on Cellulofine GC-700. The purified enzyme preparations were homogeneous upon disc gel electrophoresis. The molecular weights were estimated to be 135,000 for GD-I and 130,000 for GD-II, and their isoelectric points were 5.9 and 6.2, respectively. The specific activity of GD-I was twice that of GD-II. However, their other properties were very similar: Their optimum pHs for the oxidation of glycerol were 10.5, and those for the reduction of dihydroxyacetone were 5.5. The enzyme activities were highly activated by Cl-, but inhibited by PO₄^3?, BO₃^3? and 2-mercaptoethanol. The enzymes showed highest activity for glycerol, but also acted on several analogs of glycerol.     

Structure of a Peptidal Antibiotic P168 Produced by Paecilomyces lilacinus (Thom) Samson

( + )-α-Kainic acid (1) was synthesized by starting from a building block, N-Boc-3-acetoxyallylglycine ethyl ester (2). The key intermediate, a methyl 4-[(tert-butoxycarbonyl)prenylamino]-5-hydroxy-2-pentenoate derivative (9), was prepared from 2 in eight synthetic steps. After converting 10 into a methyl ester (11), intramolecular ene-carbocyclization of 11 gave a pyrrolidine derivative (12), which was converted to 1 in a moderate yield.     

新城疫病毒中国标准强毒株F48E9株F基因的酶切分析

含新城疫病毒Ｆ４８Ｅ９株Ｆ基因的重组质粒ｐＢＦＦ经单酶切,双酶切分析,结果表明,中国的标准强毒株Ｆ４８Ｅ５株的Ｆ基因与国外的一些强毒株相比有较大的差异,多出一个ＢａｍＨＩ酶切位点,一个ＳｅａＩ酶切位点,三个ｐｓｔＩ酶切位点,少了一个ｐｓｔＩ酶切位点。     

野生Bc菌株产黑色素的研究

Bc58是一株野生蜡状芽抱杆菌菌株,经L-酪氨酸诱导后可产生红棕色色素。通过红外光谱及各种化学测定证明该色素与Sigma公司标准黑色素（Melanin）的性质相似。生测结果显示添加Bc58黑色素的Bt制剂经紫外照射5h后的LC50为16．1μg／mL,与未经紫外照射的Bt制剂的LC50 15．2μg／mL基本相同,而比未添加黑色素的Bt制剂经紫外照射后的杀虫毒力高出近1倍。经SDS—PAGE检测表明该黑色素可保护苏云金杆菌晶体蛋白在紫外光下基本不降解,表明Bc58黑色素是一种优良的紫外保护剂。     

Characterization of a Bacteriocin-Like Substance Produced by a Vaginal Lactobacillus salivarius Strain

A novel bacteriocin-like substance produced by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328 with activity against Enterococcus faecalis, Enterococcus faecium, and Neisseria gonorrhoeae was characterized. The highest level of production of this heat-resistant peptide or protein occurred during the late exponential phase. Its mode of action was shown to be bactericidal. L. salivarius subsp. salivarius CRL 1328 could be used for the design of a probiotic to prevent urogenital infections.     

Technique for Estimating Low Numbers of a Bacterial Strain(s) in Soil

A technique is described for obtaining most probable number estimates of the number of cells of a bacterial strain(s) when it is present in low numbers in the soil. The technique is based on the bacteriophage response that is elicited when a known number of bacteriophage for the bacterium of interest is incubated with soil dilutions in a nutrient broth. The technique was evaluated for use with gram-negative bacteria.