RESULTS:
1 - 3 of 3 for "Claus Sternberg"
Quantification of biofilm structures by the novel computer program comstat
The structural organization of four microbial communities was analysed by a novel computer program COMSTAT which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria Pseudomonas putida P. aureofaciens P. fluorescens and P. aeruginosa tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure – mean thickness roughness substratum coverage and surface to volume ratio – showed that the four Pseudomonas strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum and formed flat uniform biofilms. P. aureofaciens resembled P. aeruginosa but had a stronger tendency to form micro-colonies. Finally the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0·03 mM 0·1 mM or 0·5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover biofilm roughness increased with lower citrate concentrations whereas surface to volume ratio increased with higher citrate concentrations.
Mucoid conversion of Pseudomonas aeruginos by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung
The leading cause of mortality in patients with cystic fibrosis (CF) is respiratoy failure due in large part to chronic lung infection with Pseudomonas aeruginosa strains that undergo mucoid conversion display a biofilm mode of growth in vivo and resist the infiltration of polymorphonuclear leukocytes (PMNs) which release free oxygen radicals such as H2O2. The mucoid phenotype among the strains infecting CF patients indicates overproduction of a linear polysaccharide called alginate. To mimic the inflammatory environment of the CF lung P. aeruginosa PAO1 a typical non-mucoid strain was grown in a biofilm. This was treated with low levels of H2O2 as if released by the PMNs and the formation of mucoid variants was observed. These mucoid variants had mutations in mucA which encodes an anti-σ factor; this leads to the deregulation of an alternative σ factor (σ22 AlgT or AlgU) required for expression of the alginate biosynthetic operon. All of the mucoid variants tested showed the same mutation the mucA22 allele a common allele seen in CF isolates. The mucoid mucA22 variants when compared to the smooth parent strain PA01 produced 2--6-fold higher levels of alginate|ii) exhibited no detectable differences in growth rate|iii) showed an unaltered LPS profile|iv) were ~72% reduced in the amount of inducible-β-lactamase and (v) secreted little no LasA protease and only showed 44% elastase activity. A characteristic ~54 kDa protein associated with alginate overproducing strains was identified as AlgE (Alg76) by N-terminal sequence analysis. Thus the common phenotype of the mucoid variants which included a genetically engineered mucA22 mutant suggested that the only mutation incurred as a result of H2O2 treatment was in mucA. When a P. aeruginosa biofilm was repeatedly expose to activated PMNs in vitro mucoid variants were also observed mimicking in vivo observations. Thus PMNs and their oxygen by-products may cause P. aeruginosa to undergo the typical adaptation to the intractable mu- coid form in the CF lung. These findings indicate that gene activation in bacteria by toxic oxygen radicals similar to that found in plants and mammalian cells may serve as a defence mechanism for the bacteria. This suggests that mucoid conversion is a response to oxygen radical exposure and that this response is mechanism of defence by the bacteria. This is the first report to show that PMNs and their oxygen radicals can cause this phenotypic and genotypic change which is so typical of the intractable form of P. aeruginosa in the CF lung. These findings may provide a basis for the development of anti-oxidant and anti-inflammatory therapy for the early stages of infection in CF patients
Physiological responses of Pseudomonas putida KT2442 to phosphate starvation
The physiological responses of Pseudomonas putida KT2442 to phosphate starvation were examined with respect to cell morphology qualitative demonstration of the accumulation of the intracellular storage component poly-3-hydroxyalkanoate (PHA) cellular ATP and ribosome content and the rate of total protein synthesis. Upon prolonged incubation under phosphate-limiting conditions the number of viable cells decreased by two to three orders of magnitude during the first 3 weeks. However after this decline viability of the cultures remained remarkably constant for many weeks. The cells remained rod-shaped under phosphate starvation conditions with a tendency to swell in parallel with the accumulation of PHA. Protein synthesis and ribosome concentration were gradually reduced and ATP levels dropped to very low values after the onset of starvation; later however there was a return to near-normal ATP concentrations. Evidence was obtained that the strong selective pressure imposed by phosphate deprivation forces the selection of mutants with a competitive advantage. These mutants are able to grow possibly utilizing nutrients derived from dead cells and eventually take over the cultures. One frequently encountered mutant formed smaller colonies on rich solidified medium and displayed an altered cell morphology. This mutant was isolated and further characterized. By employing a bioluminescence-based marker system we demonstrated that this mutant is able to replace wild-type cells in mixed culture experiments. Thus long-term phosphate-deprived cultures represent dynamic regimes that can undergo population shifts.