Frank Rosenau

Complete genome sequence of the myxobacterium Sorangium cellulosum.

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strains complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase–like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.

Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1

The Sm-like protein Hfq has been implicated in the regulation of sigmaS-dependent and sigmaS-independent genes in E. coli and in the regulation of virulence factors in both, Yersinia enterocolitica and Brucella abortus. Here, we have studied the effect of Hfq on virulence and stress response of Pseudomonas aeruginosa (PAO1). We have constructed a PAO1hfq- mutant and a PAO1hfq-rpoS- double mutant to permit distinction between direct and indirect effects of Hfq. When compared to the wild-type and the rpoS- strains, the hfq knock out strain showed a reduced growth rate and was unable to utilize glucose as a sole carbon source. Elastase activity was 80% reduced in the hfq- mutant when compared to the wild-type or the rpoS- strain, whereas alginate production seemed to be solely affected by sigmaS. The production of catalase and pyocyanin was shown to be affected in an additive manner by both, Hfq and sigmaS. Moreover, twitching and swarming mediated by typeIV pili was shown to be impaired in the hfq- mutant. When compared to PAO1 wild-type and the rpoS- mutant, the hfq- mutant decreased virulence in Galleria mellonella by a factor of 1 x 10(4) and 5 x 10(3), respectively. Likewise, when compared to wild-type, the PAO1hfq- mutant was significantly attenuated in virulence when administered intraperitoneally in mice. These results strongly suggest that Hfq is a global regulator of PAO1 virulence and stress response which is not exclusively due to its role in stimulating the synthesis of sigmaS.

Inhibition and Dispersion of Pseudomonas aeruginosa Biofilms by Glycopeptide Dendrimers Targeting the Fucose-Specific Lectin LecB

The human pathogenic bacterium Pseudomonas aeruginosa produces a fucose-specific lectin, LecB, implicated in tissue attachment and the formation of biofilms. To investigate if LecB inhibition disrupts these processes, high-affinity ligands were obtained by screening two 15,536-member combinatorial libraries of multivalent fucosyl-peptide dendrimers. The most potent LecB-ligands identified were dendrimers FD2 (C-Fuc-LysProLeu)(4)(LysPheLysIle)(2)LysHisIleNH(2) (IC(50) = 0.14 microM by ELLA) and PA8 (OFuc-LysAlaAsp)(4)(LysSerGlyAla)(2)LysHisIleNH(2) (IC(50) = 0.11 microM by ELLA). Dendrimer FD2 led to complete inhibition of P. aeruginosa biofilm formation (IC(50) approximately 10 microM) and induced complete dispersion of established biofilms in the wild-type strain and in several clinical P. aeruginosa isolates. These experiments suggest that LecB inhibition by high-affinity multivalent ligands could represent a therapeutic approach against P. aeruginosa infections by inhibition of biofilm formation and dispersion of established biofilms.

Bacterial lipases from Pseudomonas: regulation of gene expression and mechanisms of secretion.

Lipases from Pseudomonas bacteria are widely used for a variety of biotechnological applications. Overexpression in heterologous hosts like Escherichia coli failed to produce enzymatically active lipase prompting to study the molecular mechanisms underlying the regulation of lipase gene expression and secretion. The prototype lipase from P. aeruginosa is encoded in a bicistronic operon which is transcribed from two different promotors, one of which depends on the alternative sigma factor RpoN (sigma(54)). Recently, a two-component regulatory system was identified as an element controlling transcription of the lipase operon. P. aeruginosa lipase is secreted via a type II pathway. The cytoplasmic prelipase contains a 26 amino acid N-terminal signal sequence mediating secretion across the inner membrane via the Sec-machinery. In the periplasm, lipase folds into an enzymatically active conformation assisted by its specific intermolecular chaperone Lif and by unspecific accessory folding catalysts including Dsb-proteins which catalyze the formation of a disulfide bond. Enzymatically active and secretion-competent lipase is finally transported through a complex secretion machinery consisting of 12 different Xcp-proteins of which XcpQ forms a pore-like structure in the outer membrane allowing the release of lipase into the extracellular medium. Biotechnologically important lipases from Burkholderia glumae and P. alcaligenes also use such a type II secretion pathway whereas lipases from P. fluorescens and Serratia marcescens, which lack a typical signal sequence are secreted via a type I pathway. Future challenges to produce Pseudomonas lipases may include artificial up-regulation of lipase gene transcription and construction of more efficient expression strains in which both folding and secretion of lipase are optimized.

Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440

BackgroundRhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.ResultsHere, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C10:C10). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/gglucose corresponding to 32% of the theoretical optimum. Whats more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation.ConclusionsA functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.

The Autotransporter Esterase EstA of Pseudomonas aeruginosa Is Required for Rhamnolipid Production, Cell Motility, and Biofilm Formation

Pseudomonas aeruginosa PAO1 produces the biodetergent rhamnolipid and secretes it into the extracellular environment. The role of rhamnolipids in the life cycle and pathogenicity of P. aeruginosa has not been completely understood, but they are known to affect outer membrane composition, cell motility, and biofilm formation. This report is focused on the influence of the outer membrane-bound esterase EstA of P. aeruginosa PAO1 on rhamnolipid production. EstA is an autotransporter protein which exposes its catalytically active esterase domain on the cell surface. Here we report that the overexpression of EstA in the wild-type background of P. aeruginosa PAO1 results in an increased production of rhamnolipids whereas an estA deletion mutant produced only marginal amounts of rhamnolipids. Also the known rhamnolipid-dependent cellular motility and biofilm formation were affected. Although only a dependence of swarming motility on rhamnolipids has been known so far, the other kinds of motility displayed by P. aeruginosa PAO1, swimming and twitching, were also affected by an estA mutation. In order to demonstrate that EstA enzyme activity is responsible for these effects, inactive variant EstA* was constructed by replacement of the active serine by alanine. None of the mutant phenotypes could be complemented by expression of EstA*, demonstrating that the phenotypes affected by the estA mutation depend on the enzymatically active protein.

Bacterial lipases for biotechnological applications

Abstract Lipase genes originating from the Gram-negative bacteria Serratia marcescens and Pseudomonas aeruginosa were cloned. S. marcescens lipase was overexpressed in Escherichia coli yielding inclusion bodies which were purified and finally refolded to give enzymatically active lipase. The lipase operon of P. aeruginosa consisting of genes lipA and lipH was cloned behind the T7 φ10 promoter and overexpressed in a lipase-negative P. aeruginosa strain carrying a chromosomal insertion of the gene encoding T7 RNA polymerase. A 3D structural model was built for P. aeruginosa lipase using the coordinates of the Burkholderia cepacia lipase structure which has recently been solved in its open conformation by X-ray crystallography. Both lipases have been purified to homogeneity and were tested for their potential to catalyze biotechnologically important reactions. S. marcescens lipase stereoselectively hydrolyzed racemic isopropylideneglycerol acetate which is a basic building block in a variety of organic synthesis reactions. P. aeruginosa lipase was successfully used for kinetic resolution of chiral alcohols and amines giving enantiomeric excess values of ≥ 95% at reaction rates of 40–50%. Our results demonstrate that both lipases can be produced at levels of 100 mg/l for S. marcescens and 150 mg/l for P. aeruginosa . The recombinant lipase proteins are promising candidates for biotechnological applications.

Extracellular enzymes affect biofilm formation of mucoid Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes a variety of hydrolases, many of which contribute to virulence or are thought to play a role in the nutrition of the bacterium. As most studies concerning extracellular enzymes have been performed on planktonic cultures of non-mucoid P. aeruginosa strains, knowledge of the potential role of these enzymes in biofilm formation in mucoid (alginate-producing) P. aeruginosa remains limited. Here we show that mucoid P. aeruginosa produces extracellular hydrolases during biofilm growth. Overexpression of the extracellular lipases LipA and LipC, the esterase EstA and the proteolytic elastase LasB from plasmids revealed that some of these hydrolases affected the composition and physicochemical properties of the extracellular polymeric substances (EPS). While no influence of LipA was observed, the overexpression of estA and lasB led to increased concentrations of extracellular rhamnolipids with enhanced levels of mono-rhamnolipids, elevated amounts of total carbohydrates and decreased alginate concentrations, resulting in increased EPS hydrophobicity and viscosity. Moreover, we observed an influence of the enzymes on cellular motility. Overexpression of estA resulted in a loss of twitching motility, although it enhanced the ability to swim and swarm. The lasB-overexpression strain showed an overall enhanced motility compared with the parent strain. Moreover, the EstA- and LasB-overproduction strains completely lost the ability to form 3D biofilms, whereas the overproduction of LipC increased cell aggregation and the heterogeneity of the biofilms formed. Overall, these findings indicate that directly or indirectly, the secreted enzymes EstA, LasB and LipC can influence the formation and architecture of mucoid P. aeruginosa biofilms as a result of changes in EPS composition and properties, as well as the motility of the cells.

Lipase-Specific Foldases

Lipases represent the most important class of enzymes used in biotechnology. Many bacteria produce and secrete lipases but the enzymes originating from Pseudomonas and Burkholderia species seem to be particularly useful for a wide variety of different biocatalytic applications. These enzymes are usually encoded in an operon together with a second gene which codes for a lipase‐specific foldase, Lif, which is necessary to obtain enzymatically active lipase. A detailed analysis based on amino acid homology has suggested the classification of Lif proteins into four different families and also revealed the presence of a conserved motif, Rx1x2FDY(F/C)L(S/T)A. Recent experimental evidence suggests that Lifs are so‐called steric chaperones, which exert their physiological function by lowering energetic barriers during the folding of their cognate lipases, thereby providing essential steric information needed to fold lipases into their enzymatically active conformation.

Combinatorial variation of branching length and multivalency in a large (390 625 member) glycopeptide dendrimer library: ligands for fucose-specific lectins

A new approach to dendrimer property tuning is demonstrated by combinatorial variation of dendrimer branch length and multivalency level, using a large (390625-member) self-encoded glycopeptide dendrimer library. Ligands for the fucose lectins of Ulex europaeus were identified by screening a combinatorial library of dendrimers with variable arm length (0–4 amino acids) and multivalency (2–5 end groups). The ligands identified by this approach also bind to the fucose lectin LecB (PA-IIL) of Pseudomonas aeruginosa, and might provide a possible starting point to develop anti-infective agents against this antibiotic resistant pathogen which causes lethal respiratory tract infections in cystic fibrosis patients.