Markus Pötter

Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16

The H2-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H2 and CO2 as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organisms ability to produce and store large amounts of poly[R-(–)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.

Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16

Regulation of expression of the phasin PhaP, which is the major protein at the surface of polyhydroxyalkanoate (PHA) granules in Ralstonia eutropha H16, was studied and analysed at the molecular level. The regulation of PhaP expression is achieved by an autoregulated repressor, which is encoded by phaR in R. eutropha. The occurrence of PhaR homologues and the organization of phaR genes was analysed in detail in 29 different bacteria. Three kinds of molecule to which PhaR binds were identified in cells of R. eutropha, as revealed by gel-mobility-shift assays, DNaseI footprinting, cell fractionation, immunoelectron microscopy studies employing anti-PhaR antibodies raised against purified N-terminal hexahistidine-tagged PhaR and in vitro binding studies employing artificial PHA granules. PhaR binds upstream of phaP at two sites comprising the transcriptional start site plus the -10 region and a region immediately upstream of the -35 region of the sigma(70) promoter of phaP, where two imperfect 12 bp repeat sequences (GCAMMAAWTMMD) were identified on the sense and anti-sense strands. PhaR also binds 86 bp upstream of the phaR translational start codon, where the sigma(54)-dependent promoter was identified. PhaR also binds to the surface of PHA granules. In the cytoplasm of a phaROmegaKm mutant of R. eutropha H16, increased quantities of PhaP were detected and the cells formed by this strain were much smaller and had many more PHA granules present than the wild-type. These data support the following model for the regulation of phaP expression. Under cultivation conditions not permissive for PHA biosynthesis or in mutants defective in PHA biosynthesis, PhaR binds to the phaP promoter region and represses transcription of this gene. After the onset of PHA biosynthesis, under conditions that are permissive for the formation of nascent granules, PhaR binds to PHA granules and phaP is transcribed. At the later stages of PHA accumulation, PhaR no longer binds to the granules and the transcription of phaP is again repressed. In addition to this, phaR expression is subject to autoregulation. Excess PhaR that has not bound to the phaP upstream region or to PHA granules binds to the phaR upstream region, thereby repressing its own transcription.

Pseudomonas extremaustralis sp. nov., a Poly(3-hydroxybutyrate) Producer Isolated from an Antarctic Environment

A Gram-negative, mobile, rod-shaped, non-spore-forming bacterium (strain 14-3T) was isolated from a temporary pond in Antarctica. On the basis of 16S rRNA gene sequence similarity, strain 14-3T was shown to belong to the genus Pseudomonas sensu stricto. Physiological and biochemical tests supported the phylogenetic affiliation. Strain 14-3T is closely related to Pseudomonas veronii DSM 11331T, sharing 99.7% sequence similarity. DNA–DNA hybridization experiments between the two strains showed only moderate reassociation similarity (35.1%). Tests for arginine dihydrolase and nitrate reduction were positive, while those for denitrification, indol production, glucose acidification, urease, ß-galactosidase, esculin, caseine and gelatin hydrolysis were negative. Growth of this bacterium occurred in a range from 4 to 37°C but not at 42°C. It accumulated poly(3-hydroxybutyrate) when grown on sodium octanoate medium. Strain 14-3T therefore represents the type strain of a new species, for which the name Pseudomonas extremaustralis sp. nov. is proposed. The type strain 14-3T has been deposited as DSM 17835T and as CIP 109839T.

Binding of the Major Phasin, PhaP1, from Ralstonia eutropha H16 to Poly(3-Hydroxybutyrate) Granules

The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1(Reu) is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of PhaP1(Reu) which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1(Reu) or various regions of PhaP1(Reu) were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of PhaP1(Reu) contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified PhaP1(Reu), which revealed that PhaP1(Reu) is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnological applications of phasin fusion proteins and PHB granules in nanobiotechnology.

Biogenesis and Structure of Polyhydroxyalkanoate Granules

A large variety of prokaryotes are capable of accumulating polyhydroxyalkanoates (PHAs) as water-insoluble inclusions in the cytoplasm, and are referred to as PHA granules. Generally, PHAs represent storage compounds for carbon and energy, and they are synthesized under unbalanced growth conditions, i.e., when the carbon source is available in excess and when another nutrient is limited at the same time. In this case, further microbial growth is prevented, and PHAs are accumulated in the cytoplasm of the cells. These PHAs may possess molecular masses of up to several million daltons, and the polyester might represent the major cell constituent, contributing up to 90% or even more of the cellular dry weight. At the beginning of this chapter a brief overview about the PHA synthase and the different metabolic pathways occurring in prokaryotes will be given. The main topic focuses on the biogenesis of PHA granules and the chemical and physical properties of PHA granules produced by bacteria. The function of granule-associated proteins during the biogenesis and mobilization of PHA granules will also be discussed in detail. The chapter will be completed with an overview about applications of PHA granules as surface coatings and as nanoparticles.

Cultivation of Bacteria Producing Polyamino Acids with Liquid Manure as Carbon and Nitrogen Source

ABSTRACT Poly(γ-d-glutamic acid) (PGA)-producing strains ofBacillus species were investigated to determine their ability to contribute to reducing the amount of ammonium nitrogen in liquid manures and their ability to convert some of the ammonium into this polyamino acid as a transient depot for nitrogen. Organisms that do these things should help solve the serious environmental problems which are caused by the use of large amounts of liquid manure resulting from intensified agriculture; these problems are mainly due to the high content of ammonium nitrogen. Bacillus licheniformis ATCC 9945 and Bacillus subtilis were able to grow in liquid manure and to produce PGA in the presence of sodium gluconate. On artificial liquid manure these two strains were able to produce 0.85 and 0.79 g of PGA per liter, respectively. Under conditions that are found in intensified farming situations the ammonia content was reduced within 48 h from 1.3 to 0.75 g/liter. One mutant of B. subtilis 1551 impaired in the catabolism of PGA was obtained after nitrosoguanidine mutagenesis. This mutant produced PGA at a final concentration of 4.8 g/liter, whereas the wild type produced only 3.7 g/liter.

Ralstonia eutropha H16 Flagellation Changes According to Nutrient Supply and State of Poly(3-Hydroxybutyrate) Accumulation

ABSTRACT Two-dimensional polyacrylamide gel electrophoresis (2D PAGE), in combination with matrix-assisted laser desorption ionization-time of flight analysis, and the recently revealed genome sequence of Ralstonia eutropha H16 were employed to detect and identify proteins that are differentially expressed during different phases of poly(3-hydroxybutyric acid) (PHB) metabolism. For this, a modified protein extraction protocol applicable to PHB-harboring cells was developed to enable 2D PAGE-based proteome analysis of such cells. Subsequently, samples from (i) the exponential growth phase, (ii) the stationary growth phase permissive for PHB biosynthesis, and (iii) a phase permissive for PHB mobilization were analyzed. Among several proteins exhibiting quantitative changes during the time course of a cultivation experiment, flagellin, which is the main protein of bacterial flagella, was identified. Initial investigations that report on changes of flagellation for R. eutropha were done, but 2D PAGE and electron microscopic examinations of cells revealed clear evidence that R. eutropha exhibited further significant changes in flagellation depending on the life cycle, nutritional supply, and, in particular, PHB metabolism. The results of our study suggest that R. eutropha is strongly flagellated in the exponential growth phase and loses a certain number of flagella in transition to the stationary phase. In the stationary phase under conditions permissive for PHB biosynthesis, flagellation of cells admittedly stagnated. However, under conditions permissive for intracellular PHB mobilization after a nitrogen source was added to cells that are carbon deprived but with full PHB accumulation, flagella are lost. This might be due to a degradation of flagella; at least, the cells stopped flagellin synthesis while normal degradation continued. In contrast, under nutrient limitation or the loss of phasins, cells retained their flagella.

Überblick über die Mikroorganismen

Zu Beginn des Mikrobiologischen Praktikums sollen einige einfuhrende Anmerkungen gemacht werden, welche die in jeglicher Hinsicht auserordentlich grose Diversitat der Mikroorganismen verdeutlichen und einen Uberblick uber die verschiedenen Gruppen der Mikroorganismen und ihre Lebensweisen geben. Die letzte offizielle Bilanzierung des taxonomischen Standardwerkes Bergey’s Manual of Systematic Bacteriology aus dem Jahre 2003 wies exakt 6466 validierte Spezies aus, beim Schreiben dieser Auflage ist die Zahl auf rund 9000 gestiegen (angegeben in der List of Prokaryotic Names with Standing in Nomenclature, http://www.bacterio.cict.fr ). Bakterien zeichnen sich durch eine ungewohnlich grose Vielfalt aus. Entgegen haufig getatigten Auserungen betrifft dies sogar die Zellmorphologie und die Zellgrose. Besonders vielseitig ist der Stoffwechsel der Bakterien. Hier gibt es zahlreiche Stoffwechselleistungen, die ausschlieslich von Prokaryonten katalysiert werden konnen (Monopole der Prokaryonten) und ohne die ein Leben auf unserem Planeten auf lange Sicht nicht moglich ware. Diese kurzen Ausfuhrungen konnen die Lekture von Lehrbuchern der Mikrobiologie nicht ersetzen; sie sollen jedoch auf das Mikrobiologischen Praktikums einstimmen und einige besondere Aspekte beim Umgang mit Mikroorganismen hervorheben.

Modulare Zusammenstellung von Versuchen für unterschiedliche Zielgruppen

In der nachfolgenden Tabelle (◉ Tab. 7.1) sind samtliche im Mikrobiologischen Praktikum beschriebenen Versuche, Exkursionen und Demonstrationen aufgefuhrt. Daneben sind acht Zielgruppen bzw. Ausbildungsrichtungen aufgefuhrt, denen Versuche zugeordnet sind, die uns fur die jeweilige Zielgruppe bzw. Ausbildungsrichtung besonders geeignet erscheinen (✓). Die Abschatzung der Durchfuhrbarkeit basiert auf Erfahrungswerten und berucksichtigt die fur die Versuche notwendigen Geratschaften sowie den Kenntnisstand der Teilnehmer.

Chemikalien, Nachweisreagenzien und Medien

Die unten aufgefuhrte Liste der Medien, die im Mikrobiologischen Praktikum Verwendung finden, verzichtet zugunsten der Ubersichtlichkeit auf das detaillierte Beschreiben der Zubereitungsweisen. Fur Ungeubte und Anfanger auf dem Gebiet sei auf die Methoden 1 und 2 verwiesen; dort sind allgemeingultige Ratschlage hierzu aufgefuhrt.