A comprehensive analysis of the importance of translation initiation factors for Haloferax volcanii applying deletion and conditional depletion mutants

Translation is an important step in gene expression. The initiation of translation is phylogenetically diverse, since currently five different initiation mechanisms are known. For bacteria the three initiation factors IF1 – IF3 are described in contrast to archaea and eukaryotes, which contain a considerably higher number of initiation factor genes. As eukaryotes and archaea use a non-overlapping set of initiation mechanisms, orthologous proteins of both domains do not necessarily fulfill the same function. The genome of Haloferax volcanii contains 14 annotated genes that encode (subunits of) initiation factors. To gain a comprehensive overview of the importance of these genes, it was attempted to construct single gene deletion mutants of all genes. In 9 cases single deletion mutants were successfully constructed, showing that the respective genes are not essential. In contrast, the genes encoding initiation factors aIF1, aIF2γ, aIF5A, aIF5B, and aIF6 were found to be essential. Factors aIF1A and aIF2β are encoded by two orthologous genes in H. volcanii. Attempts to generate double mutants failed in both cases, indicating that also these factors are essential. A translatome analysis of one of the single aIF2β deletion mutants revealed that the translational efficiency of the second ortholog was enhanced tenfold and thus the two proteins can replace one another. The phenotypes of the single deletion mutants also revealed that the two aIF1As and aIF2βs have redundant but not identical functions. Remarkably, the gene encoding aIF2α, a subunit of aIF2 involved in initiator tRNA binding, could be deleted. However, the mutant had a severe growth defect under all tested conditions. Conditional depletion mutants were generated for the five essential genes. The phenotypes of deletion mutants and conditional depletion mutants were compared to that of the wild-type under various conditions, and growth characteristics are discussed

Untersuchung der Flagellen von Pyrococcus furiosus

Bei Pyrococcus furiosus, der rasenden Feuerkugel, handelt es sich um ein motiles Archaeum. Die bis zu 50 Flagellen, die pro Zelle vorliegen können, wurden mit der Motilität in Verbindung gebracht. Im Rahmen der vorliegenden Arbeit konnte gezeigt werden, dass die Flagellen von P. furiosus als ein multifunktionelles Organell dienen können, welche nicht nur die Fähigkeit zum Schwimmen verleihen. Die Flagellen sind für eine bislang einzigartige Vernetzung einzelner Zellen untereinander, aber auch in größeren Ansammlungen von Zellen verantwortlich. Dies führt letztendlich zur Ausbildung eines Biofilms. Die neuartige Verbindung zwischen den Zellen wird dabei von dem Flagellenbüschel einer Zelle hergestellt, wobei das gesamte Büschel an einer zweiten Zelle inseriert und die Zellen somit verbunden werden. Die Flagellen weisen zudem die Fähigkeit zur Anheftung an verschiedensten Oberflächen auf. Die Anheftung geschieht dabei selektiv, wobei es zur Bevorzugung oder Diskriminierung von Oberflächen kommen kann. Die getesteten Materialien (=Oberflächen) wurden dabei breit gefächert ausgewählt, auch im Hinblick auf eine mögliche industrielle Nutzung der Flagellinproteine innerhalb der Nano(bio)technologie als neuartigen, hitzestabilen �Biokleber�. Des Weiteren wurde eine systematische und detaillierte Studie zum Verlauf der Transkription des fla-Genclusters von P. furiosus erstellt. Die archaeelle Flagelle wird vermutlich ähnlich dem Mechanismus der bakteriellen Typ IV Pili von der Basis her assembliert, was einen Gegensatz zur Assemblierung der bakteriellen Geißel darstellt. Diese bereits lange bestehende Hypothese wird durch die durchgeführten Untersuchungen der (Ko)Transkriptionsmuster der Gene innerhalb dieses fla-Genlcusters unterstützt. Es konnte gezeigt werden, dass es im Verlauf der Wachstumskurve zu unterschiedlichen Transkriptionsmustern kommt. Es findet eine zeitnahe Kotranskription der wohl jeweils benötigten Komponenten zum Aufbau der Flagelle statt. Zunächst kommt es am Beginn der Wachstumskurve vorwiegend zur Expression des Hauptflagellins, welches für die Assemblierung des Hauptanteils der Flagelle benötigt wird. Weiterhin werden Proteine gefunden, die als membranständige bzw. membranassoziierte Proteine vermutlich als Assemblierungsbasis des Filaments und für den Transportmechanismus über die Membran dienen könnten. Im weiteren Verlauf der Wachstumskurve werden alle weiteren Komponenten transkribiert, die wohl für die Assemblierung der Nebenflagelline oder möglicherweise des Antriebs benötigt werden. Es konnte gezeigt werden, dass es nicht nur zur (Ko)Transkription von fla-Genen kommt. Einen weiteren neuen Aspekt stellt die Kotranskription der fla-Gene mit weiteren stromabwärts gelegenen Genen dar. Diese Proteine dienen vermutlich der Modifizierung einzelner Fla-Proteine bzw. der Regulation der Transkription

<i>Pyrococcus furiosus</i> flagella: biochemical and transcriptional analyses identify the newly detected <i>flaB0</i> gene to encode the major flagellin

We have described previously that the flagella of the Euryarchaeon Pyrococcus furiosus are multifunctional cell appendages used for swimming, adhesion to surfaces and formation of cell-cell connections. Here, we characterize these organelles with respect to their biochemistry and transcription. Flagella were purified by shearing from cells followed by CsCl-gradient centrifugation and were found to consist mainly of a ca. 30 kDa glycoprotein. Polymerization studies of denatured flagella resulted in an ATP-independent formation of flagella-like filaments. N-terminal sequencing of the main flagellin revealed an unexpected N-terminus. Therefore, we resequenced the respective region of the genome, thereby discovering that the published genome sequence is not correct. A total of 771 bp are missing in the data base, resulting in the fact that a total of three flagellin genes are present. To keep in line with the earlier nomenclature we call these flaB0, flaB1, and flaB2. Very interestingly, the previously not identified flaB0 codes for the major flagellin. Transcriptional analyses of the newly defined flagellar operon identified various different transcripts depending on the growth phase

Flagella of Pyrococcus furiosus: multifunctional organelles, made for swimming, adhesion to various surfaces, and cell-cell contacts

Pyrococcus furiosus ("rushing fireball") was named for the ability of this archaeal coccus to rapidly swim at its optimal growth temperature, around 100 degrees C. Early electron microscopic studies identified up to 50 cell surface appendages originating from one pole of the coccus, which have been called flagella. We have analyzed these putative motility organelles and found them to be composed primarily (>95%) of a glycoprotein that is homologous to flagellins from other archaea. Using various electron microscopic techniques, we found that these flagella can aggregate into cable-like structures, forming cell-cell connections between ca. 5% of all cells during stationary growth phase. P. furiosus cells could adhere via their flagella to carbon-coated gold grids used for electron microscopic analyses, to sand grains collected from the original habitat (Porto di Levante, Vulcano, Italy), and to various other surfaces. P. furiosus grew on surfaces in biofilm-like structures, forming microcolonies with cells interconnected by flagella and adhering to the solid supports. Therefore, we concluded that P. furiosus probably uses flagella for swimming but that the cell surface appendages also enable this archaeon to form cable-like cell-cell connections and to adhere to solid surfaces

Membrane fatty acid composition and cell surface hydrophobicity of marine hydrocarbonoclastic alcanivorax borkumensis SK2 grown on diesel, biodiesel and rapeseed oil as carbon sources

The marine hydrocarbonoclastic bacterium Alcanivorax borkumensis is well known for its ability to successfully degrade various mixtures of n-alkanes occurring in marine oil spills. For effective growth on these compounds, the bacteria possess the unique capability not only to incorporate but also to modify fatty intermediates derived from the alkane degradation pathway. High efficiency of both these processes provides better competitiveness for a single bacteria species among hydrocarbon degraders. To examine the efficiency of A. borkumensis to cope with different sources of fatty acid intermediates, we studied the growth rates and membrane fatty acid patterns of this bacterium cultivated on diesel, biodiesel and rapeseed oil as carbon and energy source. Obtained results revealed significant differences in both parameters depending on growth substrate. Highest growth rates were observed with biodiesel, while growth rates on rapeseed oil and diesel were lower than on the standard reference compound (hexadecane). The most remarkable observation is that cells grown on rapeseed oil, biodiesel, and diesel showed significant amounts of the two polyunsaturated fatty acids linoleic acid and linolenic acid in their membrane. By direct incorporation of these external fatty acids, the bacteria save energy allowing them to degrade those pollutants in a more efficient way. Such fast adaptation may increase resilience of A. borkumensis and allow them to strive and maintain populations in more complex hydrocarbon degrading microbial communities

Pyrococcus furiosus flagella: biochemical and transcriptional analyses identify the newly detected flaB0 gene to encode the major flagellin

We have described previously that the flagella of the Euryarchaeon Pyrococcus furiosus are multifunctional cell appendages used for swimming, adhesion to surfaces and formation of cell-cell connections. Here, we characterize these organelles with respect to their biochemistry and transcription. Flagella were purified by shearing from cells followed by CsCl-gradient centrifugation and were found to consist mainly of a ca. 30 kDa glycoprotein. Polymerization studies of denatured flagella resulted in an ATP-independent formation of flagella-like filaments. N-terminal sequencing of the main flagellin revealed an unexpected N-terminus. Therefore, we resequenced the respective region of the genome, thereby discovering that the published genome sequence is not correct. A total of 771 bp are missing in the data base, resulting in the fact that a total of three flagellin genes are present. To keep in line with the earlier nomenclature we call these flaB0, flaB1, and flaB2. Very interestingly, the previously not identified flaB0 codes for the major flagellin. Transcriptional analyses of the newly defined flagellar operon identified various different transcripts depending on the growth phase

DNA as a phosphate storage polymer and the alternative advantages of polyploidy for growth or survival

Haloferax volcanii uses extracellular DNA as a source for carbon, nitrogen, and phosphorous. However, it can also grow to a limited extend in the absence of added phosphorous, indicating that it contains an intracellular phosphate storage molecule. As Hfx. volcanii is polyploid, it was investigated whether DNA might be used as storage polymer, in addition to its role as genetic material. It could be verified that during phosphate starvation cells multiply by distributing as well as by degrading their chromosomes. In contrast, the number of ribosomes stayed constant, revealing that ribosomes are distributed to descendant cells, but not degraded. These results suggest that the phosphate of phosphate-containing biomolecules (other than DNA and RNA) originates from that stored in DNA, not in rRNA. Adding phosphate to chromosome depleted cells rapidly restores polyploidy. Quantification of desiccation survival of cells with different ploidy levels showed that under phosphate starvation Hfx. volcanii diminishes genetic advantages of polyploidy in favor of cell multiplication. The consequences of the usage of genomic DNA as phosphate storage polymer are discussed as well as the hypothesis that DNA might have initially evolved in evolution as a storage polymer, and the various genetic benefits evolved later

Phenotypical characterization of an <i>aIF2B</i> double deletion mutant.

<p>The <i>aIF2B</i> double deletion mutant (<i>HVO_1934, HVO_2706</i>) was grown in medium with six different carbon sources and growth was compared to that of the wild-type (dotted black lines). Growth conditions with phenotypic differences between double mutant and wild-type are shown in (A), conditions under which double mutant and wild-type grew indistinguishably are shown in (B). Average results from triplicate cultures and their standard deviations are shown. The color code is defined with red squares for complex media, green triangles for sucrose media, and blue diamonds for xylose in Figure A. In B glucose (violet squares), CAS (light green triangles) and acetate (yellow diamonds) are shown.</p

Dispensable and redundantly encoded genes for translation initiation factors of <i>H. volcanii</i> and the phenotypes of single gene deletion mutants.

<p>td = doubling time; g.y. = growth yield; s.d. = standard deviation; n.a. = not available (no growth of the mutant); bold print = phenotypes; equal = equal to the wild-type.</p

Essential translation initiation factors of <i>H. volcanii</i> and the phenotypes of conditional depletion mutants with (d) or without (nd) depletion.

<p>d = depleted; nd = non-depleted; t_d = doubling time; g.y. = growth yield; s.d. = standard deviation, bold print = phenotypes; equal = equal to the wild-type;</p>*<p> = these values are not related to the wild-type shown in this table but to a separate experiment.</p