The lipase LipA (PA2862) but not LipC (PA4813) from Pseudomonas aeruginosa influences regulation of pyoverdine production and expression of the sigma factor PvdS

A key element in iron-dependent regulation of iron metabolism and virulence-related functions for Pseudomonas aeruginosa is the sigma factor PvdS. PvdS expression itself is also influenced by iron-independent stimuli. We show that pyoverdine production and pvdS expression depend on one of the two lipases of P. aeruginosa.     

Cofactor trapping, a new method to produce flavin mononucleotide

We have purified flavin mononucleotide (FMN) from a flavoprotein-overexpressing Escherichia coli strain by cofactor trapping. This approach uses an overexpressed flavoprotein to trap FMN, which is thus removed from the cascade regulating FMN production in E. coli. This, in turn, allows the isolation of highly pure FMN.     

The BapF protein from Pseudomonas aeruginosa is a β-peptidyl aminopeptidase

A gene encoding a so far uncharacterized β-peptidyl aminopeptidase from the opportunistic human pathogen Pseudomonas aeruginosa PAO1 was cloned and actively expressed in the heterologue host Escherichia coli. The gene was identified in the genome sequence by its homology to the S58 family of peptidases. The sequence revealed an open reading frame of 1,101 bp with a deduced amino acid sequence of 366 amino acids. The gene was amplified by PCR, ligated into pET22b(+) and was successfully expressed in E. coli BL21 (DE3). It was shown that the enzyme consists of two polypeptides (α- and β-subunit), which are processed from the precursor. The enzyme is specific for N-terminal β-alanyl dipeptides (β-Ala-Xaa). BapF hydrolyses efficiently β-alanine at the N-terminal position, including H-β³hAla-pNA, H–D-β³hAla-pNA and β-Ala-l-His (l-carnosine). d- and l-alaninamide were also hydrolysed by the enzyme.     

A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus

The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins. Consequently, alternative bacterial expression systems have to be developed to provide novel strategies for protein synthesis exceeding the repertoire of the standard expression host E. coli.Here, we report on the construction of a novel expression system that combines the high processivity of T7 RNA polymerase with the unique physiological properties of the facultative photosynthetic bacterium Rhodobacter capsulatus. This system basically consists of a recombinant R. capsulatus T7 expression strain (R. capsulatus B10S-T7) harboring the respective polymerase gene under control of a fructose inducible promoter. In addition, a set of different broad-host-range vectors (pRho) was constructed allowing T7 RNA polymerase dependent and independent target gene expression in R. capsulatus and other Gram-negative bacteria. The expression efficiency of the novel system was studied in R. capsulatus and E. coli using the yellow fluorescent protein (YFP) as model protein. Expression levels were comparable in both expression hosts and yielded up to 80 mg/l YFP in phototrophically grown R. capsulatus cultures. This result clearly indicates that the novel R. capsulatus-based expression system is well suited for the high-level expression of soluble proteins.     

The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.     

Structural basis of carbohydrate recognition by the lectin LecB from Pseudomonas aeruginosa

The crystal structure of Pseudomonas aeruginosa fucose-specific lectin LecB was determined in its metal-bound and metal-free state as well as in complex with fucose, mannose and fructopyranose. All three monosaccharides bind isosterically via direct interactions with two calcium ions as well as direct hydrogen bonds with several side-chains. The higher affinity for fucose is explained by the details of the binding site around C6 and O1 of fucose. In the mannose and fructose complexes, a carboxylate oxygen atom and one or two hydroxyl groups are partly shielded from solvent upon sugar binding, preventing them from completely fulfilling their hydrogen bonding potential. In the fucose complex, no such defects are observed. Instead, C6 makes favourable interactions with a small hydrophobic patch. Upon demetallization, the C terminus as well as the otherwise rigid metal-binding loop become more mobile and adopt multiple conformations.     

The effect of water column mixing on phytoplankton succession, diversity and similarity

The lake number was used to describe the mixing condition forthree consecutive years (1992–1994) in Lake Tegel, Berlin,and compared to the successions of diatoms, dinoflagellatesand cyanobacteria, the main phytoplankton groups in the lake,as well as to diversity and similarity indices. Using both diversityand similarity indices in juxtaposition provides an indicationof the growth type of prevailing species (r- or K-strategists)and the degree of competition in the aqua-ecological system.A genera] pattern of these indices can be recognized as threephases: (i) high diversity—during spring, summer and autumn,interrupted by (ii) phases of low diversity during the latespring clear-water phase as the number of spring species plummeted,and (iii) during the late summer, climax populations of K-strategists.On a smaller time scale, similarity and diversity proved toreact sensitively to disturbances at frequencies intermediatein relation to the generation times of the phytoplankton. Thissupports the ‘intermediate disturbance hypothesis’,as proposed for phytoplankton by Padisak et al. [(eds) IntermediateDisturbance Hypothesis in Phytoplankton Ecology. Kluwer Academic,1993]. Diversity may remain quite high even for extended periodsduring summer climax situations, in conjunction with a highdegree of similarity, if deeper mixing of the epilimnion occursat time intervals of 2–3 weeks, as during the summer of1993. This enables the prevalence of ‘ruderal’ species,together with some motile K-strategists who actively seek optimaldepths for photosynthesis. During such summer situations describedby frequent occurrences of lower lake numbers, the epilimnionof Lake Tegel is mixed deeply enough to support ruderals, butnot too vigorously to counteract competitive advantages of motilespecies. Thus, vertical niche separation enhances diversity.     

Alternative hosts for functional (meta)genome analysis

Microorganisms are ubiquitous on earth, often forming complex microbial communities in numerous different habitats. Most of these organisms cannot be readily cultivated in the laboratory using standard media and growth conditions. However, it is possible to gain access to the vast genetic, enzymatic, and metabolic diversity present in these microbial communities using cultivation-independent approaches such as sequence- or function-based metagenomics. Function-based analysis is dependent on heterologous expression of metagenomic libraries in a genetically amenable cloning and expression host. To date, Escherichia coli is used in most cases; however, this has the drawback that many genes from heterologous genomes and complex metagenomes are expressed in E. coli either at very low levels or not at all. This review emphasizes the importance of establishing alternative microbial expression systems consisting of different genera and species as well as customized strains and vectors optimized for heterologous expression of membrane proteins, multigene clusters encoding protein complexes or entire metabolic pathways. The use of alternative host-vector systems will complement current metagenomic screening efforts and expand the yield of novel biocatalysts, metabolic pathways, and useful metabolites to be identified from environmental samples.