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Zakikhany K.,Karolinska Institutet | Harrington C.R.,UK Institute of Food Research | Nimtz M.,Helmholtz Center for Infection Biology | Hinton J.C.D.,UK Institute of Food Research | And 2 more authors.
Molecular Microbiology | Year: 2010

The transcriptional regulator CsgD of Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major regulator of biofilm formation required for the expression of csgBA, which encodes curli fimbriae, and adrA, coding for a diguanylate cyclase. CsgD is a response regulator with an N-terminal receiver domain with a conserved aspartate (D59) as a putative target site for phosphorylation and a C-terminal LuxR-like helix-turn-helix DNA binding motif, but the mechanisms of target gene activation remained unclear. To study the DNA-binding properties of CsgD we used electrophoretic mobility shift assays and DNase I footprint analysis to show that unphosphorylated CsgD-His6 binds specifically to the csgBA and adrA promoter regions. In vitro transcription analysis revealed that CsgD-His6 is crucial for the expression of csgBA and adrA. CsgD-His6 is phosphorylated by acetyl phosphate in vitro, which decreases its DNA-binding properties. The functional impact of D59 in vivo was demonstrated as S. Typhimurium strains expressing modified CsgD protein (D59E and D59N) were dramatically reduced in biofilm formation due to decreased protein stability and DNA-binding properties in the case of D59E. In summary, our findings suggest that the response regulator CsgD functions in its unphosphorylated form under the conditions of biofilm formation investigated in this study. © 2010 Blackwell Publishing Ltd.

Poblete-Castro I.,Andrés Bello University | Poblete-Castro I.,Helmholtz Center for Infection Biology | Binger D.,Helmholtz Center for Infection Biology | Oehlert R.,Helmholtz Center for Infection Biology | Rohde M.,Helmholtz Center for Infection Biology
BMC Biotechnology | Year: 2014

Background: Achieving a sustainable society requires, among other things, the use of renewable feedstocks to replace chemicals obtained from petroleum-derived compounds. Crude glycerol synthesized inexpensively as a byproduct of biodiesel production is currently considered a waste product, which can potentially be converted into value-added compounds by bacterial fermentation. This study aimed at evaluating several characterized P. putida strains to produce medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA) using raw glycerol as the only carbon/energy source. Results: Among all tested strains, P. putida KT2440 most efficiently synthesized mcl-PHA under nitrogen-limiting conditions, amassing more than 34% of its cell dry weight as PHA. Disruption of the PHA depolymerase gene (phaZ) in P. putida KT2440 enhanced the biopolymer titer up to 47% PHA (%wt/wt). The low biomass and PHA titer found in the mutant strain and the wild-type strain KT2440 seems to be triggered by the high production of the side-product citrate during the fermentation process which shows a high yield of 0.6 g/g. Conclusions: Overall, this work demonstrates the importance of choosing an appropriate microbe for the synthesis of mcl-PHA from waste materials, and a close inspection of the cell metabolism in order to identify undesired compounds that diminish the availability of precursors in the synthesis of biopolymers such as polyhydroxyalkanoates. Future metabolic engineering works should focus on reducing the production of citrate in order to modulate resource allocation in the cell's metabolism of P. putida, and finally increase the biopolymer production. © 2014 Poblete-Castro et al.; licensee BioMed Central.

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