The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: Highlights from a multi-level omics approach

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas putida </it>KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by <it>P. putida </it>strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.</p> <p>Results</p> <p>We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)^-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other <it>P. putida </it>strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system.</p> <p>Conclusion</p> <p>This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.</p

Genotypic and phenotypic analyses of a Pseudomonas aeruginosa chronic bronchiectasis isolate reveal differences from cystic fibrosis and laboratory strains

Background Pseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized. Results This strain displays phenotypes that have been associated with chronic respiratory infections in CF including alginate over-production, rough lipopolysaccharide, quorum-sensing deficiency, loss of motility, decreased protease secretion, and hypermutation. Hypermutation is a key adaptation of this bacterium during the course of chronic respiratory infections and analysis indicates that PAHM4 encodes a mutated mutS gene responsible for a ~1,000-fold increase in mutation rate compared to wild-type laboratory strain P. aeruginosa PAO1. Antibiotic resistance profiles and sequence data indicate that this strain acquired numerous mutations associated with increased resistance levels to β-lactams, aminoglycosides, and fluoroquinolones when compared to PAO1. Sequencing of PAHM4 revealed a 6.38 Mbp genome, 5.9 % of which were unrecognized in previously reported P. aeruginosa genome sequences. Transcriptome analysis suggests a general down-regulation of virulence factors, while metabolism of amino acids and lipids is up-regulated when compared to PAO1 and metabolic modeling identified further potential differences between PAO1 and PAHM4. Conclusions This work provides insights into the potential differential adaptation of this bacterium to the lung of patients with bronchiectasis compared to other clinical settings such as cystic fibrosis, findings that should aid the development of disease-appropriate treatment strategies for P. aeruginosa infections

Modeling and analysis of flux distributions in the two branches of the phosphotransferase system in Pseudomonas putida

Abstract Background Signal transduction plays a fundamental role in the understanding of cellular physiology. The bacterial phosphotransferase system (PTS) together with the PEP/pyruvate node in central metabolism represents a signaling unit that acts as a sensory element and measures the activity of the central metabolism. Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru) and a second branch (PTSNtr), which communicate with each other by phosphate exchange. Recent experimental results showed a cross talk between the two branches. However, the functional role of the crosstalk remains open. Results A mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one of the PTS proteins, for different environmental conditions and different strain variants. Additionally, data from flux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based on the calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructose could be determined. Conclusion Our calculations show that during growth of the wild type strain on the PTS substrate fructose, the major part of the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a new role of the second branch of the PTS and will serve as a basis for further experimental studies.AK was funded in part by the FORSYS initiative from the German Federal Ministry of Education and Research (BMBF).Peer Reviewe

Phage-mediated Dispersal of Biofilm and Distribution of Bacterial Virulence Genes Is Induced by Quorum Sensing

The microbiome and the phage meta-genome within the human gut are influenced by antibiotic treatments. Identifying a novel mechanism, here we demonstrate that bacteria use the universal communication molecule AI-2 to induce virulence genes and transfer them via phage release. High concentrations (i.e. 100 mu M) of AI-2 promote dispersal of bacteria from already established biofilms, and is associated with release of phages capable of infecting other bacteria. Enterococcus faecalis V583 Delta ABC harbours 7 prophages in its genome, and a mutant deficient in one of these prophages (i.e. prophage 5) showed a greatly reduced dispersal of biofilm. Infection of a probiotic E. faecalis strain without lytic prophages with prophage 5 resulted in increased biofilm formation and also in biofilm dispersal upon induction with AI-2. Infection of the probiotic E. faecalis strain with phage-containing supernatants released through AI-2 from E. faecalis V583 Delta ABC resulted in a strong increase in pathogenicity of this strain. The polylysogenic probiotic strain was also more virulent in a mouse sepsis model and a rat endocarditis model. Both AI-2 and ciprofloxacin lead to phage release, indicating that conditions in the gastrointestinal tract of hospitalized patients treated with antibiotics might lead to distribution of virulence genes to apathogenic enterococci and possibly also to other commensals or even to beneficial probiotic strains

PCR results validating the presence or absence of phage sequences in the original <i>E. faecalis</i> Symbioflor 1, polylysogenic Symbioflor 1, and bacterial isolates from animals infected with polylysogenic Symbioflor 1.

<p>Pp1 to 7 represent the seven prophages from <i>E. faecalis</i> V583 ΔABC, + indicates that sequences of that prophage were found in the respective isolate by PCR. The results confirm that the inoculum strain <i>E. faecalis</i> Symbioflor 1 was stably transduced with prophages pp1, pp5 and pp7, while bacteria isolated from animals contained pp1, pp5, and pp7, only pp5, or only pp1 and pp7. All Symbioflor 1 strains contain pp2 (part of the core genome). Primers used are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004653#ppat.1004653.s002" target="_blank">S2 Table</a>.</p><p>PCR results validating the presence or absence of phage sequences in the original <i>E. faecalis</i> Symbioflor 1, polylysogenic Symbioflor 1, and bacterial isolates from animals infected with polylysogenic Symbioflor 1.</p

Time course of AI-2 production during growth of <i>E. faecalis</i> V583ΔABC (A) and biofilm formation of <i>E. faecalis</i> 12030, V583 ΔABC and two clinical isolates of <i>E. faecalis</i> (B).

<p>(<b>A)</b> AI-2 concentration in the supernatants was determined by a FRET-based assay for the growing culture (grey rectangle), values are shown on the left Y axis. The X-axis shows the time in hours. <b>(B)</b> A microtiter plate biofilm assay was done with staining of the attached bacteria by crystal violet. Biofilm formation was measured in absence or presence of 100 μM AI-2. Absorbance was measured at 600 nm. Statistical analysis was done by ANOVA (p<0.001) with Dunnett post test. * indicates p<0.05, ** p<0.01. Error bars represent standard error of the mean.</p