Bernd H. A. Rehm

A sensitive, viable colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds

Abstract The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of only 0.5 μg/ml, and growth of the cells occurred in the presence of the dyes. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The presence of Nile red or Nile blue A did not affect growth of the bacteria. This viable-colony staining method was in particular applicable to gram-negative bacteria such as Azotobacter vinelandii, Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. It was less suitable for discriminating between PHA-negative and PHA-positive strains of gram-positive bacteria such as Bacillus megaterium or Rhodococcus ruber, but it could also be used to discriminate between wax-ester- and triacylglycerol-negative and -positive strains of Acinetobacter calcoaceticus or Rhodococcus opacus. The potential of this new method and its application to further investigations of PHA synthases and PHA biosynthesis pathways are discussed.

Polyester synthases: natural catalysts for plastics

Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with a conserved cysteine residue as catalytic nucleophile. This review provides a survey of the known biochemical features of these unique enzymes and their proposed catalytic mechanism.

Bacterial polymers: biosynthesis, modifications and applications

Bacteria can synthesize a wide range of biopolymers that serve diverse biological functions and have material properties suitable for numerous industrial and medical applications. A better understanding of the fundamental processes involved in polymer biosynthesis and the regulation of these processes has created the foundation for metabolic- and protein-engineering approaches to improve economic-production efficiency and to produce tailor-made polymers with highly applicable material properties. Here, I summarize the key aspects of bacterial biopolymer production and highlight how a better understanding of polymer biosynthesis and material properties can lead to increased use of bacterial biopolymers as valuable renewable products.

Human Host Defense Peptide LL-37 Prevents Bacterial Biofilm Formation

ABSTRACT The ability to form biofilms is a critical factor in chronic infections by Pseudomonas aeruginosa and has made this bacterium a model organism with respect to biofilm formation. This study describes a new, previously unrecognized role for the human cationic host defense peptide LL-37. In addition to its key role in modulating the innate immune response and weak antimicrobial activity, LL-37 potently inhibited the formation of bacterial biofilms in vitro. This occurred at the very low and physiologically meaningful concentration of 0.5 μg/ml, far below that required to kill or inhibit growth (MIC = 64 μg/ml). LL-37 also affected existing, pregrown P. aeruginosa biofilms. Similar results were obtained using the bovine neutrophil peptide indolicidin, but no inhibitory effect on biofilm formation was detected using subinhibitory concentrations of the mouse peptide CRAMP, which shares 67% identity with LL-37, polymyxin B, or the bovine bactenecin homolog Bac2A. Using microarrays and follow-up studies, we were able to demonstrate that LL-37 affected biofilm formation by decreasing the attachment of bacterial cells, stimulating twitching motility, and influencing two major quorum sensing systems (Las and Rhl), leading to the downregulation of genes essential for biofilm development.

Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis

Polyhydroxyalkanoic acids (PHA) represent a complex class of storage polyesters that are synthesized by a wide range of different gram-positive and gram-negative bacteria as well as by some Archaea and that are deposited as insoluble cytoplasmic inclusions. PHA synthases, which are the key enzymes for PHA biosynthesis, have been characterized in much detail. At present 42 PHA synthase structural genes from 38 different bacteria have been cloned, and from 30 genes the nucleotide sequences were obtained. The strategies successfully employed to clone these genes and the current knowledge on the organization of the PHA synthase genes and other genes encoding proteins related to PHA metabolism will be compiled. In addition, the primary structures of the 30 PHA synthases were aligned and analyzed with respect to highly conserved amino acids and biochemical features. The direction, in which research should proceed, in order to increase our knowledge on biosynthesis of PHAs and to utilize this knowledge for the development of technically and economically feasible processes for the production of these polyesters will be outlined.

A New Metabolic Link between Fatty Acid de NovoSynthesis and Polyhydroxyalkanoic Acid Synthesis THE PHAG GENE FROM PSEUDOMONAS PUTIDAKT2440 ENCODES A 3-HYDROXYACYL-ACYL CARRIER PROTEIN-COENZYME A TRANSFERASE

To investigate the metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid (PHA) synthesis, we isolated mutants of Pseudomonas putida KT2440 deficient in this metabolic route. The gene phaG was cloned by phenotypic complementation of these mutants; it encoded a protein of 295 amino acids with a molecular mass of 33,876 Da, and the amino acid sequence exhibited 44% amino acid identity to the primary structure of the rhlA gene product, which is involved in the rhamnolipid biosynthesis in Pseudomonas aeruginosa PG201. S1 nuclease protection assay identified the transcriptional start site 239 base pairs upstream of the putative translational start codon. Transcriptional induction of phaG was observed when gluconate was provided, and PHA synthesis occurred from this carbon source. No complementation of the rhlA mutant P. aeruginosa UO299-harboring plasmid pBHR81, expressingphaG gene under lac promoter control, was obtained. Heterologous expression of phaG inPseudomonas oleovorans, which is not capable of PHA synthesis from gluconate, enabled PHA synthesis on gluconate as the carbon source. Native recombinant PhaG was purified by native polyacrylamide gel electrophoresis from P. oleovorans-harboring plasmid pBHR81. It catalyzes the transfer of the acyl moiety from in vitro synthesized 3-hydroxydecanoyl-CoA to acyl carrier protein, indicating that PhaG exhibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity.

Bacterial alginates : biosynthesis and applications

Abstract Alginate is a copolymer of β-d-mannuronic acid and α-l-guluronic acid (GulA), linked together by 1–4 linkages. The polymer is a well-established industrial product obtained commercially by harvesting brown seaweeds. Some bacteria, mostly derived from the genus Pseudomonas and belonging to the RNA superfamily I, are also capable of producing copious amounts of this polymer as an exopolysaccharide. The molecular genetics, regulation and biochemistry of alginate biosynthesis have been particularly well characterized in the opportunistic human pathogen Pseudomonas aeruginosa, although the biochemistry of the polymerization process is still poorly understood. In the last 3 years major aspects of the molecular genetics of alginate biosynthesis in Azotobacter vinelandii have also been reported. In both organisms the immediate precursor of polymerization is GDP-mannuronic acid, and the sugar residues in this compound are polymerized into mannuronan. This uniform polymer is then further modified by acetylation at positions O-2 and/or O-3 and by epimerization of some of the residues, leading to a variable content of acetyl groups and GulA residues. In contrast, seaweed alginates are not acetylated. The nature of the epimerization steps are more complex in A. vinelandii than in P. aeruginosa, while other aspects of the biochemistry and genetics of alginate biosynthesis appear to be similar. The GulA residue content and distribution strongly affect the physicochemical properties of alginates, and the epimerization process is therefore of great interest from an applied point of view. This article presents a survey of our current knowledge of the molecular genetics and biochemistry of bacterial alginate biosynthesis, as well as of the biotechnological potential of such polymers.

Bacterial Polyhydroxyalkanoate Granules: Biogenesis, Structure, and Potential Use as Nano-/Micro-Beads in Biotechnological and Biomedical Applications

Polyhydroxyalkanoates (PHAs) are naturally occurring organic polyesters that are of interest for industrial and biomedical applications. These polymers are synthesized by most bacteria in times of unbalanced nutrient availability from a variety of substrates and they are deposited intracellularly as insoluble spherical inclusions or PHA granules. The granules consist of a polyester core, surrounded by a boundary layer with embedded or attached proteins that include the PHA synthase, phasins, depolymerizing enzymes, and regulatory proteins. Apart from ongoing industrial interest in the material PHA, more recently there has also been increasing interest in applications of the PHA granules as nano-/micro-beads after it was conceived that fusions to the granule associated proteins (GAPs) provide a way to immobilize target proteins at the granule surface. This review gives an overview of PHA granules in general, including biogenesis and GAPs, and focuses on their potential use as nano-/micro-beads in biotechnological and biomedical applications.

Role of Exopolysaccharides in Pseudomonas aeruginosa Biofilm Formation and Architecture

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslA Δalg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.

Polymer production by two newly isolated extremely halophilic archaea: application of a novel corrosion-resistant bioreactor

Abstract A novel corrosion-resistant bioreactor composed of polyetherether ketone (PEEK), tech glass and silicium nitrite ceramics was constructed and applied for the cultivation of two newly isolated, extremely halophilic archaea producing poly(γ-glutamic acid) (PGA), or poly(β-hydroxy butyric acid) (PHB), respectively. These bacteria were isolated from hypersaline soil close to Aswan (Egypt). The isolate strain 40, which is related to the genus Natrialba, produced large amounts of PGA when cultivated on solid medium. Culture conditions were optimised applying the corrosion-resistant bioreactor. PGA production was dependent on NaCl concentration and occurred about at 20% (w/v) NaCl in the medium. A maximum cell density of about 1.6 g cell dry matter/l was obtained when the bioreactor was stirred and aerated in a batch fermentation process using proteose-peptone medium. The supernatant was monitored with respect to PGA formation, and after 90 h a maximum of 470 mg/l culture volume was detected by HPLC analysis. Culture conditions were optimized for the isolate 56, which accumulated PHB as intracellular granules. Batch fermentations in the stirred and aerated bioreactor applying acetate and n-butyric acid as carbon sources led to cell density of 2.28 g cell dry matter/l and a maximum PHB accumulation contributing to about 53% of cellular dry weight. About 4.6 g PHB were isolated from 10.6 g dried cells of strain 56, which exhibited a weight average molar mass of 2.3 × 105 g mol−1 and a polydispersity of about 1.4.