scholarly journals A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion.

1996 ◽  
Vol 178 (22) ◽  
pp. 6608-6617 ◽  
Author(s):  
J K Gustin ◽  
E Kessler ◽  
D E Ohman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzymatic Activity ◽  
Extracellular Secretion ◽  
Lasa Protease

scholarly journals Identification of Critical Residues in the Propeptide of LasA Protease of Pseudomonas aeruginosa Involved in the Formation of a Stable Mature Protease

2007 ◽  
Vol 189 (11) ◽  
pp. 3960-3968 ◽  
Author(s):  
Kerian K. Grande ◽  
Jean K. Gustin ◽  
Efrat Kessler ◽  
Dennis E. Ohman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Insertional Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Amino Terminal ◽  
Amino Acid Region ◽  
Lasa Protease ◽  
Mature Domain ◽  
Acid Region

ABSTRACT LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.

scholarly journals Study of Some Physical and Chemical Properties of Staphylolysin Enzyme purified from Pseudomonas aeruginosa

2014 ◽  
Vol 11 (2) ◽  
pp. 819-828
Author(s):  
Baghdad Science Journal
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sodium Chloride ◽  
Enzymatic Activity ◽  
Potassium Chloride ◽  
Ferric Chloride ◽  
Zinc Chloride ◽  
Enzyme Stability ◽  
Mercury Chloride ◽  
Initial Activity ◽  
Full Activity

Some of the characters of the Staphylolysin A and D enzymes purified from Pseudomonas aeruginosa P16 and P5 respectively were studied, the molecular weights of Staphylolysin A and D were 20.417 kilo dalton and 23.988 kilo Dalton respectively by SDS- polyacryl amide gel electrophoresis. The optimum pH for staphylolysin A activity was found to be 8 which gives higher activity reaches 150 unit/ml, and for enzyme stability was 7.5-8.5 in which the enzyme nearly retained its full activity, while it was 9.5 for staphylolysin D that gives higher activity of 16 unit/ml,and 8.5-9.5 for enzyme stability in which the enzyme nearly retained its full activity, Maximum activity of two enzymes was obtained at 40C in which the specific activity for staphylolysin A and D were 140 and 16.4 unit/ml, and the two enzymes remained approximately without change at 25-40C for one hour. When the effects of some materials on Staphylolysin A&D activity were studied, the results showed that both sodium chloride & potassium chloride at 1 & 5 mM had the activator effect on enzymatic activity compared with its control where the staphylolysin A and D retained 105% ,108% and 102%, 104% of their activity respectively when treated with sodium chloride, while they retained 110%, 114% and 133%, 118% of their activity respectively when treated with potassium chloride. The enzymatic activity for both enzymes were inhibited when treated with ferric , mercury and zinc chloride at variable ratios, Staphylolysin A kept 73% and 7% of its initial activity respectively when treated with 5mM of ferric chloride and mercury chloride respectively and it kept only 9% of its initial activity when treated with 0.1mM Zinc chloride . Staphylolysin D kept 45% and 13% of it is initial activity respectively when treated with 5mM of ferric chloride and mercury chloride respectively and it kept only 23% of its initial activity when treated with 0.1mM Zinc chloride while enzymatic activity for both enzymes were not affected when treated with EDTA at l0mM and phenyl methyl sulphonyl fluoride (PMSF) at 0.4mM.These results referred to that Staphylolysin A and D are Zn -metallo endopeptidase .

scholarly journals Effect of pH on the Electrochemical Behavior of Hydrogen Peroxide in the Presence of Pseudomonas aeruginosa

2021 ◽  
Vol 9 ◽  
Author(s):  
Javier Espinoza-Vergara ◽  
Paulo Molina ◽  
Mariana Walter ◽  
Miguel Gulppi ◽  
Nelson Vejar ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Pseudomonas Aeruginosa ◽  
Enzymatic Activity ◽  
Electrochemical Behavior ◽  
Electrochemical Techniques ◽  
Stable State ◽  
Reduction Reaction ◽  
Maximum Activity ◽  
Open Circuit ◽  
Mueller Hinton

The influence of pH on the electrochemical behavior of hydrogen peroxide in the presence of Pseudomonas aeruginosa was investigated using electrochemical techniques. Cyclic and square wave voltammetry were used to monitor the enzymatic activity. A modified cobalt phthalocyanine (CoPc) carbon electrode (OPG), a known catalyst for reducing O2 to H2O2, was used to detect species resulting from the enzyme activity. The electrolyte was a sterilized aqueous medium containing Mueller-Hinton (MH) broth. The open-circuit potential (OCP) of the Pseudomonas aeruginosa culture in MH decreased rapidly with time, reaching a stable state after 4 h. Peculiarities in the E / I response were observed in voltammograms conducted in less than 4 h of exposure to the culture medium. Such particular E/I responses are due to the catalase’s enzymatic action related to the conversion of hydrogen peroxide to oxygen, confirming the authors’ previous findings related to the behavior of other catalase-positive microorganisms. The enzymatic activity exhibits maximum activity at pH 7.5, assessed by the potential at which oxygen is reduced to hydrogen peroxide. At higher or lower pHs, the oxygen reduction reaction (ORR) occurs at higher overpotentials, i.e., at more negative potentials. In addition, and to assess the influence of bacterial adhesion on the electrochemical behavior, measurements of the bacterial-substrate metal interaction were performed at different pH using atomic force microscopy.

scholarly journals GidA Posttranscriptionally Regulates rhl Quorum Sensing in Pseudomonas aeruginosa

2009 ◽  
Vol 191 (18) ◽  
pp. 5785-5792 ◽  
Author(s):  
Rashmi Gupta ◽  
Timothy R. Gobble ◽  
Martin Schuster
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Flavin Adenine Dinucleotide ◽  
Skim Milk ◽  
Opportunistic Pathogen ◽  
Homoserine Lactone ◽  
Trna Modification ◽  
Loss Of Function ◽  
Novel Genes ◽  
Lasa Protease

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa utilizes two interconnected acyl-homoserine lactone quorum-sensing (acyl-HSL QS) systems, LasRI and RhlRI, to regulate the expression of hundreds of genes. The QS circuitry itself is integrated into a complex network of regulation by other factors. However, our understanding of this network is still unlikely to be complete, as a comprehensive, saturating approach to identifying regulatory components has never been attempted. Here, we utilized a nonredundant P. aeruginosa PA14 transposon library to identify additional genes that regulate QS at the level of LasRI/RhlRI. We initially screened all 5,459 mutants for loss of function in one QS-controlled trait (skim milk proteolysis) and then rescreened attenuated candidates for defects in other QS phenotypes (LasA protease, rhamnolipid, and pyocyanin production) to exclude mutants defective in functions other than QS. We identified several known and novel genes, but only two novel genes, gidA and pcnB, affected all of the traits assayed. We characterized gidA, which exhibited the most striking QS phenotypes, further. This gene is predicted to encode a conserved flavin adenine dinucleotide-binding protein involved in tRNA modification. Inactivation of the gene primarily affected rhlR-dependent QS phenotypes such as LasA, pyocyanin, and rhamnolipid production. GidA affected RhlR protein but not transcript levels and also had no impact on LasR and acyl-HSL production. Overexpression of rhlR in a gidA mutant partially restored QS-dependent phenotypes. Taken together, these results indicate that GidA selectively controls QS gene expression posttranscriptionally via RhlR-dependent and -independent pathways.

L-SERINE DEHYDRATASE (DEAMINASE) OF PSYCHROPHILES AND MESOPHILES FROM POLAR AND TEMPERATE HABITATS

1967 ◽  
Vol 13 (10) ◽  
pp. 1333-1342 ◽  
Author(s):  
James T. Staley ◽  
William L. Boyd
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Enzymatic Activity ◽  
Growth Temperature ◽  
Enzymic Activity ◽  
Direct Role ◽  
Natural Plant ◽  
Serine Dehydratase ◽  
The Relationship ◽  
Dehydratase Activity

Escherichia coli and Pseudomonas aeruginosa were grown at several temperatures ranging from 0° to 37 °C and assayed at each temperature for L-serine dehydratase activity. The relationship between enzymatic activity and growth temperature is quite different for the mesophile and psychrophile. In addition, the stimulatory effect of natural plant lecithin upon serine deamination was different for each organism, suggesting that there is either a difference in permeability or that this substance has a more direct role in enzymic activity. Also included are data confirming the results of other workers.

The bifunctional role of LiuE from Pseudomonas aeruginosa, displays additionally HIHG-CoA lyase enzymatic activity

2009 ◽  
Vol 37 (4) ◽  
pp. 1787-1791 ◽  
Author(s):  
Mauricio Chávez-Avilés ◽  
Alma Laura Díaz-Pérez ◽  
Jesús Campos-García
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzymatic Activity

scholarly journals Pseudomonas aeruginosa LasA Protease in Treatment of Experimental Staphylococcal Keratitis

2004 ◽  
Vol 48 (5) ◽  
pp. 1681-1687 ◽  
Author(s):  
Irina S. Barequet ◽  
Guy J. Ben Simon ◽  
Mary Safrin ◽  
Dennis E. Ohman ◽  
Efrat Kessler
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Rabbit Model ◽  
Treatment Protocols ◽  
Methicillin Resistant ◽  
Clinical Scores ◽  
Lasa Protease ◽  
Late Treatment ◽  
Intrastromal Injection ◽  
Staphylococcus Simulans

ABSTRACT LasA protease is a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa. We have examined the effectiveness of LasA protease in the treatment of staphylococcal keratitis caused by methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates in a rabbit model. Keratitis was induced by intrastromal injection of the bacteria. The eyes were treated topically, and the efficacy of LasA protease was compared to those of lysostaphin (a staphylolytic protease secreted by Staphylococcus simulans) and vancomycin. When treatment was initiated early (4 h) after infection, practically all of the MSSA- and MRSA-infected corneas were sterilized by LasA protease, and its efficacy in eradicating the bacteria was comparable to those of lysostaphin and vancomycin. By contrast, most of the control corneas were heavily infected, with median values of 4.5 × 106 (MSSA) and 5 × 105 (MRSA) CFU/cornea (P < 0.001). When treatment was initiated late (10 h) after infection, LasA protease reduced the numbers of CFU in both MSSA- and MRSA-infected corneas by 3 to 4 orders of magnitude compared to the numbers of CFU for the controls (median values, 1,380 and 30 CFU/cornea, respectively, for the treated animals compared to 1.2 × 106 and 5 × 105 CFU/cornea for the respective controls [P = 0.001]), and it was more effective than vancomycin in eradicating MRSA cells (P = 0.02). In both the early- and the late-treatment protocols, the clinical scores for eyes treated with LasA protease were significantly lower than those for the eyes of the corresponding controls and comparable to those for the lysostaphin- and vancomycin-treated eyes. We conclude that LasA protease is effective in the treatment of experimental S. aureus keratitis in rabbits and may have potential for the treatment of disease in humans.

scholarly journals Pseudomonas aeruginosa AmpR Is a Global Transcriptional Factor That Regulates Expression of AmpC and PoxB β-Lactamases, Proteases, Quorum Sensing, and Other Virulence Factors

2005 ◽  
Vol 49 (11) ◽  
pp. 4567-4575 ◽  
Author(s):  
Kok-Fai Kong ◽  
Suriya Ravi Jayawardena ◽  
Shalaka Dayaram Indulkar ◽  
Aimee del Puerto ◽  
Chong-Lek Koh ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Promoter Activity ◽  
Wild Type Strain ◽  
Transcriptional Factor ◽  
Wild Type ◽  
Global Regulator ◽  
Concomitant Increase ◽  
Expression Levels ◽  
Lasa Protease ◽  
The Family

ABSTRACT In members of the family Enterobacteriaceae, ampC, which encodes a β-lactamase, is regulated by an upstream, divergently transcribed gene, ampR. However, in Pseudomonas aeruginosa, the regulation of ampC is not understood. In this study, we compared the characteristics of a P. aeruginosa ampR mutant, PAOampR, with that of an isogenic ampR + parent. The ampR mutation greatly altered AmpC production. In the absence of antibiotic, PAOampR expressed increased basal β-lactamase levels. However, this increase was not followed by a concomitant increase in the P ampC promoter activity. The discrepancy in protein and transcription analyses led us to discover the presence of another chromosomal AmpR-regulated β-lactamase, PoxB. We found that the expression of P. aeruginosa ampR greatly altered the β-lactamase production from ampC and poxB in Escherichia coli: it up-regulated AmpC but down-regulated PoxB activities. In addition, the constitutive P ampR promoter activity in PAOampR indicated that AmpR did not autoregulate in the absence or presence of inducers. We further demonstrated that AmpR is a global regulator because the strain carrying the ampR mutation produced higher levels of pyocyanin and LasA protease and lower levels of LasB elastase than the wild-type strain. The increase in LasA levels was positively correlated with the P lasA , P lasI , and P lasR expression. The reduction in the LasB activity was positively correlated with the P rhlR expression. Thus, AmpR plays a dual role, positively regulating the ampC, lasB, and rhlR expression levels and negatively regulating the poxB, lasA, lasI, and lasR expression levels.

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