scholarly journals DsbA and DsbC Affect Extracellular Enzyme Formation in Pseudomonas aeruginosa

2001 ◽  
Vol 183 (2) ◽  
pp. 587-596 ◽  
Author(s):  
Andreas Urban ◽  
Martina Leipelt ◽  
Thorsten Eggert ◽  
Karl-Erich Jaeger
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipase Activity ◽  
Disulfide Bonds ◽  
Extracellular Enzymes ◽  
Extracellular Enzyme ◽  
Lipase Production ◽  
Lipase Gene ◽  
Cell Extracts ◽  
A Cell

ABSTRACT DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in Pseudomonas aeruginosa were identified and shown to play an important role for the formation of extracellular enzymes. Mutants deficient in either dsbA ordsbC or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. The dsbA mutant no longer produced these enzymes, whereas the lipase activity was doubled in the dsbC mutant. Also, extracellar lipase production was severely reduced in a P. aeruginosa dsbA mutant in which an inactive DsbA variant carrying the mutation C34S was expressed. Even when the lipase genelipA was constitutively expressed in trans in alipA dsbA double mutant, lipase activity in cell extracts and culture supernatants was still reduced to about 25%. Interestingly, the presence of dithiothreitol in the growth medium completely inhibited the formation of extracellular lipase whereas the addition of dithiothreitol to a cell-free culture supernatant did not affect lipase activity. We conclude that the correct formation of the disulfide bond catalyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type II pathway.

Lipoprotein lipase-suppressing mediator in serum of endotoxin-treated rats

1986 ◽  
Vol 251 (4) ◽  
pp. E470-E476 ◽  
Author(s):  
G. J. Bagby ◽  
C. B. Corll ◽  
J. J. Thompson ◽  
L. A. Wilson
Keyword(s):  
Lipoprotein Lipase ◽  
Exposure Time ◽  
Lipase Activity ◽  
Significant Suppression ◽  
Lipoprotein Lipase Activity ◽  
Activity Maximum ◽  
A Cell

The conditions under which lipoprotein lipase-suppressing mediator is present in serum of endotoxin-treated rats was determined in this study. The suppression of lipoprotein lipase activity in 3T3-L1 cells was used as a bioassay for mediator in serum. Endotoxin (0.1-10 micrograms/ml) and serum from control rats did not suppress lipoprotein lipase activity. Maximum suppression of cell lipoprotein lipase activity (70%) by serum from endotoxic rats required a cell exposure time of 5 h. At the highest dose of endotoxin used (1 mg/100 g), significant suppression was achieved when cells were incubated with 0.5% serum from endotoxic rats (P less than 0.05). Serum obtained 2-3 h after endotoxin injection possessed the maximal ability to suppress lipase activity, but suppressing activity was not present in serum collected 8 h after endotoxin. Rats rendered tolerant to endotoxin by 5 daily injections (0.1 mg/100 g) did not contain detectable levels of mediator in serum after endotoxin injection. The results demonstrate that the presence of lipoprotein lipase activity-suppressing mediator is transitory after in vivo exposure of naive rats to endotoxin, but does not appear in serum of endotoxin tolerant rats.

scholarly journals Rhamnolipid and lipase production by Pseudomonas aeruginosa san-ai: the process comparison analysis by statistical approach

2013 ◽  
Vol 67 (4) ◽  
pp. 677-685 ◽  
Author(s):  
Sonja Jakovetic ◽  
Zorica Knezevic-Jugovic ◽  
Sanja Grbavcic ◽  
Dejan Bezbradica ◽  
Natasa Avramovic ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipase Activity ◽  
Sunflower Oil ◽  
Hydrolytic Enzymes ◽  
Tween 80 ◽  
Lipase Production ◽  
Fermentation Time ◽  
Process Comparison ◽  
Central Composite Experimental Design ◽  
Oil Concentration

Pseudomonas aeruginosa was repeatedly reported as powerful producer of rhamnolipid biosurfactants as well as producer of hydrolytic enzymes. In this study effects of four fermentation factors were evaluated using response surface methodology and experiments were performed in accordance with a four-factor and five-level central composite experimental design. Investigated factors were: fermentation temperature, time of fermentation, concentration of sunflower oil and concentration of Tween? 80. The most important finding was that regression coefficients of the highest values were those that describe interactions between factors and that they differ for lipase and rhamnolipid production, which were both investigated in this study. Production of both metabolites was optimized and response equations were obtained, making it possible to predict rhamnolipid concentration or lipase activity from known values of the four factors. The highest achieved rhamnolipid concentration and lipase activity were 138 mg dm-3 (sunflower oil concentration 0.8 %, Tween? 80 concentration 0.05 %, temperature 30?C, and fermentation time 72 h) and 11111 IU dm-3(sunflower concentration of 0.4 %, Tween? 80 concentration of 0.05 %, temperature of 30?C, and fermentation time of 120 h), respectively.

scholarly journals The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study

Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 328-333 ◽  
Author(s):  
Elizabeta Nemeth ◽  
Gloria C. Preza ◽  
Chun-Ling Jung ◽  
Jerry Kaplan ◽  
Alan J. Waring ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Progressive Decrease ◽  
Cellular Iron ◽  
Gfp Fusion Protein ◽  
N Terminus ◽  
A Cell ◽  
Terminal Amino ◽  
Peptide Treatment

Abstract Hepcidin is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid (aa) peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in mice by the induction of hypoferremia. Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when 5 N-terminal aa's were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin and did not interfere with ferroportin internalization by native hepcidin. Deletion of 2 C-terminal aa's did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not affect peptide activity in vitro. However, these peptides were less active in vivo, likely because of their decreased stability in circulation. G71D and K83R, substitutions previously described in humans, did not affect hepcidin activity. Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters

1981 ◽  
Vol 1 (7) ◽  
pp. 635-651
Author(s):  
D C Lee ◽  
R G Roeder
Keyword(s):  
Adenovirus Type ◽  
Free System ◽  
Ribonucleic Acids ◽  
Cell Free System ◽  
Cell Extracts ◽  
A Cell ◽  
Adenovirus Type 2

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 373-383 ◽  
Author(s):  
Thomas Bjarnsholt ◽  
Peter Østrup Jensen ◽  
Mette Burmølle ◽  
Morten Hentzer ◽  
Janus A. J. Haagensen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Polymorphonuclear Leukocytes ◽  
Present Report ◽  
Cell Communication ◽  
A Cell ◽  
Opportunistic Human Pathogen ◽  
Conventional Antibiotics

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell–cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and H2O2, and are readily phagocytosed by PMNs, in contrast to bacteria with functional QS systems. In contrast to the wild-type, QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently, the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

scholarly journals Assembly of the MexAB-OprM Multidrug Pump of Pseudomonas aeruginosa: Component Interactions Defined by the Study of Pump Mutant Suppressors

2007 ◽  
Vol 189 (17) ◽  
pp. 6118-6127 ◽  
Author(s):  
Dominic Nehme ◽  
Keith Poole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Drug Efflux ◽  
Efflux System ◽  
Cell Extracts ◽  
Suppressor Mutations ◽  
Helical Hairpin ◽  
Pump Assembly ◽  
Extragenic Suppressors ◽  
Inactivating Mutations

ABSTRACT In an effort to identify key domains of the Pseudomonas aeruginosa MexAB-OprM drug efflux system involved in component interactions, extragenic suppressors of various inactivating mutations in individual pump constituents were isolated and studied. The multidrug hypersusceptibility of P. aeruginosa expressing MexB with a mutation in a region of the protein implicated in oligomerization (G220S) was suppressed by mutations in the α/β domain of MexA. MexB(G220S) showed a reduced ability to bind MexA in vivo while representative MexA suppressors (V66M and V259F) restored the MexA-MexB interaction. Interestingly, these suppressors also restored resistance in P. aeruginosa expressing OprM proteins with mutations at the proximal (periplasmic) tip of OprM that is predicted to interact with MexB, suggesting that these suppressors generally overcame defects in MexA-MexB and MexB-OprM interaction. The multidrug hypersusceptibility arising from a mutation in the helical hairpin of MexA implicated in OprM interaction (V129M) was suppressed by mutations (T198I and F439I) in the periplasmic α-helical barrel of OprM. Again, the MexA mutation compromised an in vivo interaction with OprM that was restored by the T198I and F439I substitutions in OprM, consistent with the hairpin domain mediating MexA binding to this region of OprM. Interestingly, these OprM suppressor mutations restored multidrug resistance in P. aeruginosa expressing MexB(G220S). Finally, the oprM(T198I) suppressor mutation enhanced the yields of all three constituents of a MexA-MexB-OprM(T198I) pump as detected in whole-cell extracts. These data highlight the importance of MexA and interactions with this adapter in promoting MexAB-OprM pump assembly and in stabilizing the pump complex.

scholarly journals Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters.

1981 ◽  
Vol 1 (7) ◽  
pp. 635-651 ◽  
Author(s):  
D C Lee ◽  
R G Roeder
Keyword(s):  
Adenovirus Type ◽  
Free System ◽  
Ribonucleic Acids ◽  
Cell Free System ◽  
Cell Extracts ◽  
A Cell ◽  
Adenovirus Type 2

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

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