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The htx and ptx Operons of Pseudomonas stutzeri WM88 Are New Members of the Pho Regulon. Andrea K. White, 2004.The htx and ptx operons of Pseudomonas stutzeri WM88 allow for the use of the inorganic reduced phosphorus (P) compounds hypophosphite (P valence, +1) and phosphite (P valence, +3) as sole P sources . To support the proposed in vivo role for the htx and ptx operons, namely the use of phosphite and hypophosphite as alternative P sources, we used reporter gene fusions to examine their expression levels with respect to various P conditions . Expression of the htx and ptx operons was induced up to 17- and 22-fold, respectively, in cultures grown under phosphate starvation conditions relative to expression in medium with excess phosphate (Pi) . However, the presence of the reduced P substrate hypophosphite, phosphite, or methylphosphonate, in addition to excess Pi, did not result in an increase in the expression of either operon . To provide further support for a role of the htx and ptx operons in Pi acquisition, we identified P . stutzeri phoBR homologs and constructed deletion mutants . Induction of the htx and ptx reporter gene fusions in response to growth on limiting Pi was abolished in  phoB,
phoR,
and
phoBR
mutants, demonstrating that htx and ptx expression is
phoBR dependent . The putative LysR-type regulator encoded by
ptxE has no apparent role in the expression of the htx
and ptx operons, as no effect was observed on the level of
induction of either operon in a
ptxE
mutant .
Regulatory Elements of the Staphylococcus aureus Protein A (Spa) Promoter. Jinxin Gao, 2004.Staphylococcal protein A (Spa) is an important virulence factor of Staphylococcus aureus . Transcription of the spa determinant occurs during the exponential growth phase and is repressed when the cells enter the postexponential growth phase . Regulation of spa expression has been found to be complicated, with regulation involving multiple factors, including Agr, SarA, SarS, SarT, Rot, and MgrA . Our understanding of how these factors work on the spa promoter to regulate spa expression is incomplete . To identify regulatory sites within the spa promoter, analysis of deletion derivatives of the promoter in host strains deficient in one or more of the regulatory factors was undertaken, and several critical features of spa regulation were revealed . The transcriptional start sites of spa were determined by primer extension . The spa promoter sequences were subcloned in front of a promoterless chloramphenicol acetyltransferase reporter gene . Various lengths of spa truncations with the same 3' end were constructed, and the resultant plasmids were transduced into strains with different regulatory genetic backgrounds . Our results identified upstream promoter sequences necessary for Agr system regulation of spa expression . The cis elements for SarS activity, an activator of spa expression, and for SarA activity, a repressor of spa expression, were identified . The well-characterized SarA consensus sequence on the spa promoter was found to be insufficient for SarA repression of the spa promoter . Full repression required the presence of a second consensus site adjacent to the SarS binding site . Sequences directly upstream of the core promoter sequence were found to stimulate transcription . Anti-Aspergillus fumigatus Efficacy of Pentraxin 3 Alone and in Combination with Antifungals. Roberta Gaziano, 2004.The collectin pentraxin 3 (PTX3) is an essential component of host resistance to pulmonary aspergillosis . Here we examined the protective effects of administration of PTX3 alone or together with deoxycholate amphotericin B (Fungizone) or liposomal amphotericin B (AmBisome) against invasive aspergillosis in a murine model of allogeneic bone marrow transplantation . PTX3, alone or in combination with the polyenes, was given intranasally or parenterally either before, in concomitance with, or after the intranasal infection with Aspergillus fumigatus conidia . Mice were monitored for resistance to infection and parameters of innate and adaptive T-helper immunity . The results showed the following: (i) complete resistance to infection and reinfection was observed in mice treated with PTX3 alone; (ii) the protective effect of PTX3 was similar or superior to that observed with liposomal amphotericin B or deoxycholate amphotericin B, respectively; (iii) protection was associated with accelerated recovery of lung phagocytic cells and T-helper-1 lymphocytes and concomitant decrease of inflammatory pathology; and (iv) PTX3 potentiated the therapeutic efficacy of suboptimal doses of either antimycotic drug . Together, these data suggest the potential therapeutic use of PTX3 either alone or as an adjunctive therapy in A . fumigatus infections . Statistical Quantification of Detachment Rates and Size Distributions of Cell Clumps from Wild-Type (PAO1) and Cell Signaling Mutant (JP1) Pseudomonas aeruginosa Biofilms. Suzanne Wilson, 2004.The detachment of cells from bacterial biofilms is an important, yet poorly understood and largely unquantified phenomenon . Detached cell clumps from medical devices may form microemboli and lead to metastasis, especially if they are resistant to host defenses and antibiotics . In manufacturing plants detached clumps entering a process stream decrease product quality . Two strains of Pseudomonas aeruginosa, a wild type (PAO1) and a cell signaling mutant (JP1), were studied to (i) quantify and model detachment patterns and (ii) determine the influence of cell signaling on detachment . We collected effluent from a biofilm flowthrough reactor and determined the size distribution for cell detachment events by microscopic examination and image analysis . The two strains were similar in terms of both biofilm structure and detachment patterns . Most of the detachment events were single-cell events; however, multiple-cell detachment events contributed a large fraction of the total detached cells . The rates at which events containing multiple cells detached from the biofilm were estimated by fitting a statistical model to the size distribution data . For events consisting of at least 1,000 cells, the estimated rates were 4.5 events mm–2 min–1 for PAO1 and 4.3 events mm–2 min–1 for JP1 . These rates may be significant when they are scaled up to the total area of a real biofilm-contaminated medical device surface and to the hours or days of patient exposure . Acid Resistance Systems Required for Survival of Escherichia coli O157:H7 in the Bovine Gastrointestinal Tract and in Apple Cider Are Different. Stuart B. Price, 2004.Escherichia coli O157:H7 is a highly acid-resistant food-borne pathogen that survives in the bovine and human gastrointestinal tracts and in acidic foods such as apple cider . This property is thought to contribute to the low infectious dose of the organism . Three acid resistance (AR) systems are expressed in stationary-phase cells . AR system 1 is  S dependent, while AR systems 2 and 3 are glutamate and arginine dependent, respectively . In this study, we sought to determine which AR systems are important for survival in acidic foods and which are required for survival in the bovine intestinal tract . Wild-type and mutant E . coli O157:H7 strains deficient in AR system 1, 2, or 3 were challenged with apple cider and inoculated into calves . Wild-type cells, adapted at pH 5.5 in the absence of glucose (AR system 1 induced), survived well in apple cider . Conversely, the mutant deficient in AR system 1, shown previously to survive poorly in calves, was susceptible to apple cider (pH 3.5), and this sensitivity was shown to be caused by low pH . Interestingly, the AR system 2-deficient mutant survived in apple cider at high levels, but its shedding from calves was significantly decreased compared to that of wild-type cells . AR system 3-deficient cells survived well in both apple cider and calves . Taken together, these results indicate that E . coli O157:H7 utilizes different acid resistance systems based on the type of acidic environment encountered .
Transcriptional Expression of Escherichia coli Glutamate-Dependent Acid Resistance Genes gadA and gadBC in an hns rpoS Mutant. Scott R. Waterman, 2003.Resistance to being killed by acidic environments with pH values lower than 3 is an important feature of both pathogenic and nonpathogenic Escherichia coli . The most potent E . coli acid resistance system utilizes two isoforms of glutamate decarboxylase encoded by gadA and gadB and a putative glutamate:  -aminobutyric acid antiporter encoded by gadC . The gad system is controlled by two repressors (H-NS and CRP), one activator (GadX), one repressor-activator (GadW), and two sigma factors ( S and
70) . In contrast to results of previous reports, we demonstrate that gad transcription can be detected in an hns rpoS mutant strain of E . coli K-12, indicating that gad promoters can be initiated by
70 in the absence of H-NS .
Characteristics of a Novel Type of Bovine Cryptosporidium andersoni. Masaaki Satoh, 2003.We isolated oocysts that resemble Cryptosporidium andersoni from cattle grazing on a farm in Japan . The partial sequences of genes from the isolate were coincident with published sequences of genes of C . andersoni . Since the isolate was able to infect SCID mice, the isolate appears to be a novel type of C . andersoni . Assessment of Photodynamic Destruction of Escherichia coli O157:H7 and Listeria monocytogenes by Using ATP Bioluminescence. N. A. Romanova, 2003.Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses . It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP . Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB) . A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes . To evaluate the effects of photodestruction on E . coli and L . monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used . All tested photosensitizers were effective for photodynamic destruction of both bacteria . The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms . The MICs were two- to fourfold higher for E . coli O157:H7 than for L . monocytogenes . The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample . The time courses of photodestruction and intracellular ATP leakage were different for E . coli and L . monocytogenes . These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction .
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