Regulation of extracellular proteases in Achlya ambisexualis

1997 ◽  
Vol 75 (3) ◽  
pp. 440-444 ◽  
Author(s):  
Terry W. Hill ◽  
Markus P. Pott
Keyword(s):  
Glutamic Acid ◽  
Molecular Mass ◽  
Protease Activity ◽  
Defined Medium ◽  
Extracellular Proteases ◽  
Nutrient Source ◽  
Achlya Ambisexualis ◽  
Extracellular Protease Activity ◽  
Protease Secretion ◽  
Secreted Enzymes

The use of proteins as a nutrient source by Achlya ambisexualis was investigated, using media containing defined and undefined sources of carbon, nitrogen, and (or) sulfur. Release of extracellular proteases occurred during growth on all proteins and protein hydrolysates tested, but not during growth on yeast extract or in defined medium. In gelatin-containing media, three major bands of extracellular protease activity were detected by electrophoresis, with estimated molecular mass of 26, 48, and 58 kDa. Growth on gelatin was stimulated to a much greater degree by the addition of glucose to the medium than by additions of glutamic acid or methionine. This and the release of ammonia during growth indicate that gelatin is less effective in meeting metabolic needs for carbon than it is in meeting the needs for nitrogen and sulfur. Protease secretion is only partially regulated by glucose, whereas glucose, methionine, and glutamic acid in combination cause almost complete repression. The pattern of regulation indicated by these results is most consistent with one of induction + derepression. Key words: oomycetes, proteinases, regulation, secreted enzymes.

scholarly journals New Spatially Explicit Method for Detecting Extracellular Protease Activity in Biofilms

2001 ◽  
Vol 67 (9) ◽  
pp. 4329-4334 ◽  
Author(s):  
Steven N. Francoeur ◽  
Robert G. Wetzel ◽  
Robert K. Neely
Keyword(s):  
Protease Activity ◽  
Experimental Manipulation ◽  
Extracellular Protease ◽  
Confocal Scanning Laser Microscopy ◽  
Extracellular Proteases ◽  
Confocal Scanning ◽  
Novel Method ◽  
Extracellular Protease Activity ◽  
Confocal Scanning Laser ◽  
Fluorescent Molecules

ABSTRACT A novel method of detecting extracellular protease activity at biofilm-substratum interfaces was developed. This method utilizes fluorescent molecules bound to cellulose substrata with a lectin. Extracellular proteases degrade the lectin and release the fluorochrome into solution. This new technique and a standard dissolved-substrate assay detected similar responses of biofilm extracellular protease activity to experimental manipulation of N supply. Combination of this technique with confocal scanning laser microscopy allowed direct visualization of microspatial patterns of bacterial distribution and extracellular protease activity at the biofilm-substratum interface.

scholarly journals Amylase and Protease Secretion by the Marine Bacterium Vibrio gazogenes

1982 ◽  
Vol 35 (4) ◽  
pp. 457 ◽  
Author(s):  
Catherine Ratcliffe ◽  
RL Sanders ◽  
C Ludmila Tittel ◽  
RW O’Brien
Keyword(s):  
Protease Activity ◽  
Metal Ion ◽  
Rna Synthesis ◽  
De Novo ◽  
Defined Medium ◽  
Logarithmic Phase ◽  
Chloromethyl Ketone ◽  
Late Logarithmic Phase ◽  
Casamino Acids ◽  
Protease Secretion

V. gazogenes secreted an amylase throughout the logarithmic phase of growth when starch or maltose was the carbon source for growth. The enzyme was apparently not constitutive and was repressed by glucose. The amylase was of the oc-type and had an optimum pH of 6·5. Protease secretion by V. gazogenes occurred when the organism was grown in a defined medium containing 0·005 % (w/v) yeast extract. The activity of the enzyme was increased 40-fold when the organism was grown in a medium containing peptone or Casamino Acids. Enzyme secretion started in the late logarithmic phase of growth and ceased when the cells entered the stationary phase of growth and was not repressed by glucose. The protease was neither induced nor repressed by glutamic acid, aspartic acid, alanine, serine, arginine, valine, threonine, lysine, leucine, proline or oc-ketoglutarate. Tryptophan delayed, but did not inhibit, protease secretion. Both the amylase and the protease were truly extracellular and were not released as a result of cell lysis. Phenylmethylsulfonyl fluoride and N-oc-p-tosyl-L-Iysine chloromethyl ketone hydrochloride, L-l-tosylamide-2-phenylethyl chloromethyl ketone, leupeptin, anti pain and chymostatin did not markedly inhibit the protease, indicating that it is not a serine protease nor similar to trypsin, chymotrypsin, papain or cathepsin B. Inhibitors of sulfhydryl enzymes were also without effect on protease activity, but 1,1O-phenanthroline, ethylenediaminetetraacetate and ethyleneglycol-bis-(p-aminoethyl ether) N,N'-tetraacetate almost completely inhibited the protease, indicating that it requires a divalent metal ion for activity. After dialysis against water, both amylase and protease lost over 90% of their activity. The amylase was almost completely reactivated by 15 mM Cl-, Be or 1-, but not by Ca 2+ or Mg2+. Protease was fully reactivated by about 30 mM Ca2+ or Mg2+ (at 70 ruM concentration these ions stimulated activity to about 140% of the rate of the undialysed enzyme). Mn2+ and Co2+ partially restored protease activity. Chloramphenicol, at concentrations that did not affect RNA synthesis, completely inhibited amylase and protease secretion, showing that secretion of both enzymes was a de novo process. When rifampin or actinomycin D, at concentrations that completely and rapidly inhibited cellular RNA synthesis, was added to cultures actively secreting amylase or protease, there was no inhibition of secretion for periods of 6-20 min, indicating the presence of a pool of mRNA specific for each enzyme. The protease was not affected by trypsin or oc-chymotrypsin during the secretion process, indicating that the enzyme had taken up its tertiary conformation before emergence from the cell membrane.

scholarly journals Proteases as Markers for Differentiation of Pathogenic and Nonpathogenic Species ofAcanthamoeba

2000 ◽  
Vol 38 (8) ◽  
pp. 2858-2861 ◽  
Author(s):  
Naveed A. Khan ◽  
Edward L. Jarroll ◽  
Noorjahan Panjwani ◽  
Zhiyi Cao ◽  
Timothy A. Paget
Keyword(s):  
Epithelial Cells ◽  
Conditioned Medium ◽  
Protease Activity ◽  
Extracellular Protease ◽  
Pathogenic Species ◽  
Extracellular Proteases ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Extracellular Protease Activity ◽  
Different Strains

Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genusAcanthamoeba. Although not allAcanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenicAcanthamoeba. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenicAcanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoebaisolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed withAcanthamoeba-conditioned medium as well as withAcanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenicAcanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5.

scholarly journals Plasmid-Encoded Regulator of Extracellular Proteases in Bacillus anthracis

2005 ◽  
Vol 187 (9) ◽  
pp. 3133-3138 ◽  
Author(s):  
Arthur I. Aronson ◽  
Chris Bell ◽  
Ben Fulroth
Keyword(s):  
Bacillus Subtilis ◽  
Bacillus Thuringiensis ◽  
Bacillus Anthracis ◽  
Related Species ◽  
Protease Activity ◽  
Extracellular Proteases ◽  
Closely Related Species ◽  
Strain Transformation ◽  
Pleiotropic Regulator ◽  
Secreted Enzymes

ABSTRACT Bacillus anthracis Sterne cured of the pXO1 plasmid had enhanced secreted protease activity during the postexponential phase but no change in hemolytic or lecithinase activities. A zymogen profile revealed at least six proteases, including serine, metal, and perhaps cysteine types. There were similar amounts of protease secreted by the closely related species Bacillus cereus and Bacillus thuringiensis, but the patterns differed. Among the pXO1 plasmid-encoded proteins, there is a tetratricopeptide protein designated Cot43 that is related to the Rap proteins of Bacillus subtilis and the PlcR pleiotropic regulator of secreted enzymes and toxins in B. thuringiensis. A disruption of the cot43 gene resulted in overproduction of several proteases to a somewhat greater extent than in the plasmid-cured strain. Transformation of either of these strains with a clone of the cot43 gene resulted in the inhibition of accumulation of some of the proteases and induction of at least one. On the basis of lacZ fusions, transcription of the cot43 gene increased in late exponential cells at the time of protease accumulation. The expression of lacZ fusions to the upstream regions of two B. anthracis extracellular protease genes was greater in the strain with the disruption of cot43 than in the Sterne strain, indicating regulation at the level of transcription. In B. anthracis, a pXO1 plasmid-encoded protein directly modulates or indirectly regulates the transcription of genes for several chromosomally encoded extracellular proteases.

Extracellular Elastolytic Activity in Human Lung Lavage: A Comparative study between Smokers and Non-Smokers

1985 ◽  
Vol 69 (1) ◽  
pp. 17-27 ◽  
Author(s):  
S. F. Smith ◽  
A. Guz ◽  
N. T. Cooke ◽  
G. H. Burton ◽  
T. D. Tetley
Keyword(s):  
Proteinase Inhibitor ◽  
Protease Activity ◽  
Human Lung ◽  
Lung Lavage ◽  
Low Molecular Weight ◽  
Elastase Activity ◽  
Significant Difference ◽  
Elastolytic Activity ◽  
Bronchial Mucus ◽  
Extracellular Protease Activity

1. Unrestrained proteolysis in the lung is believed to initiate emphysema, a disease common among tobacco smokers. However, few studies have found extracellular protease activity in human lung lavage. 2. In this investigation, elastase and serine protease activities were measured in broncho-alveolar lavage supernatants (BAL) from patients undergoing routine investigations. Significantly more elastolytic activity (against insoluble [3H]-elastin) was recovered in the lavage of smokers than that of non-smokers. However, no significant difference was found when the levels of serine proteolytic activity (against N-succinyl-L-trialanyl-p-nitroanilide) were compared. 3. The elastolytic component of the protease activity rose from 5% in non-smokers’ BAL to over 30% in that of smokers, suggesting that elastase activity is selectively enhanced by smoking. In lavages from most smokers, 80% or more of the elastase activity was serine-dependent, whereas lavages from non-smokers contained variable proportions of serine elastase. 4. Both α1-proteinase inhibitor (α1-PI) and a low molecular weight antiprotease, bronchial mucus proteinase inhibitor (BMPI) were detectable in the lavage samples, the latter contributing up to 76% of the total antiprotease quantified in the lavage. Functional antiprotease was detected in 85% of the lavages. 5. Since there were no differences in either antiprotease levels or functional inhibitory capacities between lavages from smokers and controls, it is concluded that the imbalance in the protease/antiprotease profile of the smokers’ lung results from an enhancement of proteases, specifically of elastolytic activity, rather than a reduction in inhibitory capacity.

Extracellular Heat-Resistant Proteases of Psychrotrophic Pseudomonads

1983 ◽  
Vol 46 (2) ◽  
pp. 90-94 ◽  
Author(s):  
THAKOR R. PATEL ◽  
FRANCIS M. BARTLETT ◽  
JAWED HAMID
Keyword(s):  
Protease Activity ◽  
Milk Powder ◽  
Milk Proteins ◽  
Raw Milk ◽  
Enzyme Synthesis ◽  
Maximum Activity ◽  
Protease Production ◽  
Divalent Metal Ions ◽  
Extracellular Proteases ◽  
Heat Resistant

Several bacterial isolates from raw milk produced proteases. Most of such 28 isolates were gram-negative rods which were oxidase- and catalase-positive. All the isolates grew at temperatures in the range of 0–35°C, but failed to grow at 37°C. Nineteen of these isolates were tentatively assigned to genus Pseudomonas, and were used in the present investigation. Extracellular proteases from these psychrotrophic pseudomonads were heat-resistant, being able to retain partial activity even after heat-treatment at 120°C for 10 min. Milk proteins were preferred substrates by these proteases although some also hydrolysed bovine serum albumin, hemoglobin and ovalbumin. The optimum pH for the maximum activity was between pH 7.2 and 7.4. Divalent metal ions like Cu2+, Co2+, Hg2+, and Zn2+ were inhibitory to protease activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect on the proteases. Induced levels of protease production were observed when cultures were grown in minimal media containing either casein or nonfat dried milk powder. Glucose, citrate and lactose repressed enzyme synthesis in a minmal salts medium containing either casein or nonfat dried milk powder. Protease activity was also detected in cultures grown in minimal medium containing glutamine. Proteases from different isolates varied in their molecular weights.

scholarly journals The Virulence Function and Regulation of the Metalloprotease Gene prtA in the Plant-Pathogenic Bacterium Burkholderia glumae

2019 ◽  
Vol 32 (7) ◽  
pp. 841-852 ◽  
Author(s):  
Tiago Lelis ◽  
Jingyu Peng ◽  
Inderjit Barphagha ◽  
Ruoxi Chen ◽  
Jong Hyun Ham
Keyword(s):  
Protease Activity ◽  
Virulent Strain ◽  
Regulatory Gene ◽  
Extracellular Protease ◽  
Bacterial Disease ◽  
Rna Seq ◽  
Burkholderia Glumae ◽  
Responsible Gene ◽  
Extracellular Protease Activity ◽  
Serine Metalloprotease

Bacterial panicle blight caused by Burkholderia glumae is a major bacterial disease of rice. Our preliminary RNA-seq study showed that a serine metalloprotease gene, prtA, is regulated in a similar manner to the genes for the biosynthesis and transport of toxoflavin, which is a known major virulence factor of B. glumae. prtA null mutants of the virulent strain B. glumae 336gr-1 did not show a detectable extracellular protease activity, indicating that prtA is the solely responsible gene for the extracellular protease activity detected from this bacterium. In addition, inoculation of rice panicles with the prtA mutants resulted in a significant reduction of disease severity compared with the wild-type parent strain, suggesting the requirement of prtA for the full virulence of B. glumae. A double mutant deficient in both serine metalloprotease and toxoflavin (ΔtoxA/prtA−) exhibited a further numeric but not statistically significant decrease of disease development compared with the ΔtoxA strain. Both the prtA-driven extracellular protease activity and the toxoflavin production were dependent on both the tofI/tofR quorum-sensing and the global regulatory gene qsmR, indicating the important roles of the two global regulatory factors for the bacterial pathogenesis by this pathogen.

Nutritional requirements for cell wall synthesis in Micrococcus lysodeikticus

1969 ◽  
Vol 15 (4) ◽  
pp. 327-334
Author(s):  
M. P. Hatton
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Glutamic Acid ◽  
Dry Weight ◽  
Defined Medium ◽  
Complete Medium ◽  
Magnesium Ions ◽  
Cell Wall Synthesis ◽  
Micrococcus Lysodeikticus ◽  
Total Dry Weight

Preferential cell wall synthesis in Micrococcus lysodeikticus, as determined by an increase in the dry weight of the cell wall, took place in a medium containing DL-glutamic acid, DL-alanine, L-lysine, glycine, magnesium ions, glucose and phosphate buffer, pH 7.0. Cell wall synthesis could not be completely dissociated from protein synthesis in the 'cell wall' medium. The cell wall synthesized in the defined medium accounted for 40–56% of the total dry weight increase of the cells. Chloramphenicol had no effect on cell wall synthesis. Incorporation of uracil and guanine in the medium did not result in any increase in the amount of cell wall synthesized. DL-Glutamic acid alone, or a mixture of the three amino acids DL-alanine, L-lysine, and glycine, were capable of replacing the four amino acids present in the complete medium, but under these conditions the total dry weight of cell wall synthesized was only 75% of that produced in the complete medium. There was no reduction in cell wall synthesis when L-glutamic acid replaced DL-glutamic acid, L-alanine replaced DL-alanine, or sucrose replaced glucose in the cell wall medium. Deprivation of magnesium ions produced the greatest decrease in wall synthesis; this was the most important single factor involved in cell wall synthesis which was studied in the present investigation. There was no observable change in the chemical composition of the cell wall synthesized in the 'wall' medium when compared to that synthesized by cells grown in a complex medium.

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