Regulation der respiratorischen Stoffwechselwege zur Energiegewinnung in D. shibae unter anaeroben Bedingungen mit Nitrat

Dinoroseobacter shibae is a marine bacterium and belongs to the ubiquitously found Roseobacter clade. Investigation of the genome sequence identified genes encoding enzymes of the anaerobic metabolism. 7 Crp/Fnr like regulators were annotated, which leads to the question for the gene regulatory network, proteomic and metabolomic networks controlling the transition between aerobic and anaerobic growth. To answer this question, an aerobic continuously cultivation of D. shibae in a chemostate in minimal media supplemented with was established. Time resolved shift experiments from oxic to anoxic growth conditions were performed to characterize the transcriptomic, proteomic and metabolomic adaptation of D. shibae. The transcriptome and proteome data revealed a strong expression of operons encoding proteins for the formation of the denitrification machinery. Furthermore, the expression of the genes encoding the Crp/Fnr like regulators dnrD, dnrE and dnrF was also found increased, which leads to the assumption of involvement in the regulatory network for the establishment of nitrate respiratory conditions. Moreover, the synthesis of PHB was observed under these conditions and verified by transmission electronic microscopy and HPLC analyses. Using a bioinformatics approach the phylogenetic affiliation of DnrE, DnrD and DnrF into the various subfamily of Crp/Fnr like regulators was achieved. Furthermore, comparative transcriptome analyses of wildtype and dnrF deletion mutant revealed the mainly repressing influence of DnrF on several genes encoding for enzymes of the energy generation machinery, transcription factors and iron-sulfur cluster proteins under oxic and anoxic conditions. This leads to the consumption of DnrF acts mainly as a oxygen independent repressor. To identify the essential genes of D. shibae for growth under nitrate respiratory conditions a transposon mutagenesis approach was established. Over 4500 mutants were screened for a growth failure under these conditions. Finally, 53 strains showed a growth defect or even no growth. Genes encoding the periplasmic nitrate reductase NapA and the molybdopterin biosynthesis protein MoeB were found to be essential. In comparison with the transcriptomic and proteomic profile of D. shibae only 3 genes (napA, phaA and the Na+/Pi antiporter gene Dshi_0543) showed an overlap of induced and essential. Furthermore, highly induced genes (nirS) are not essential for the growth under nitrate respiratory conditions.Dinoroseobacter shibae ist ein Meeresbakterium, das zu den ubiquitär zu findenden Roseobacter Cluster gehört. Die Untersuchung des sequenziertem Genoms identifizierte Gene, die für Enzyme des anaeroben Metabolismus codieren. 7 Crp/Fnr ähnliche Transkriptionsregulatoren annotiert, was die Frage nach dem genregulatorischem, proteomischem und metabolomischem Netzwerk aufwirft, das die Anpassung von aeroben zu anaeroben Wachstumsbedingungen kontrolliert. Daher wurde in dieser Arbeit zunächst eine kontinuierliche Kultivierung von Dinoroseobacter shibae im Chemostaten unter aeroben Bedingungen in Minimalmedium etabliert. Zeitaufgelöste Shift Experimente von oxischen zu nitratrespiratorischen Wachstumsbedingungen wurden zur Charakterisierung der Anpassung von D. shibae auf transkriptioneller, translationeller und metabolischer Ebene durchgeführt. Die Transkriptom- und Proteomdaten zeigten eine starke Expression der Operons, die für Proteine der Denitrifikation codieren. Weiterhin wurde die Expression von Genen, die für die Proteine der Crp/Fnr Familie dnrD, dnrE und dnrF codieren, auch induziert vorgefunden, was zu der Annahme führt, dass diese in das regulatorische Netzwerk der Adaption an Nitratrespiratorische Bedingungen beteiligt sind. Durch eine bioinformatische Stammbaumanalyse konnten DnrD, DnrE und DnrF in die Unterfamilie Dnr klassifiziert werden. Vergleichende DNA Microarray Experimente des Wildtyps und einer dnrF Deletionsmutante zeigten einen zumeist reprimierenden Einfluss von DnrF auf die Expression verschiedener Gene, codierend für Energiegewinnung, Transkriptionsfaktoren und Eisen-Schwefel Cluster Proteinen unter aeroben und anaeroben Bedingungen. Diese Ergebnisse weisen darauf hin, dass DnrF meist als Sauerstoff unabhängiger Repressor fungiert. Zur Identifizierung von essentiellen Genen, die in D. shibae für das Wachstum unter Nitratrespiratorischen Bedingungen benötigt werden, wurde eine Transposonmutagenese etabliert. Über 4500 Mutanten wurden nach Wachstumsversuche untersucht. Hier zeigten 53 Mutanten ein geringeres oder kein Wachstum. Essentiell für das Wachstum wurden z. B. die Gene, die für die periplasmatische Nitratreduktase NapA und das Molybdopterin Biosynthese Protein MoeB identifiziert. Im Vergleich mit dem Transkriptom und Proteom von D. shibae wurden nur 3 Gene (napA, phaA and the Na+/Pi antiporter gene Dshi_0543) identifiziert, die sowohl essentiell als auch induziert unter diesen Bedingungen sind

Characterization of the transcriptome of Haloferax volcanii, grown under four different conditions, with mixed RNA-Seq

Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did not contain any length information. Therefore, a mixed RNA-Seq approach was used to determine transcript length and to identify additional transcripts, which are not present under optimal conditions. In total, 50 million paired end reads of 150 nt length were obtained. 1861 protein-coding RNAs (cdRNAs) were detected, which encoded 3092 proteins. This nearly doubled the coverage of cdRNAs, compared to the previous dRNA-Seq study. About 2/3 of the cdRNAs were monocistronic, and 1/3 covered more than one gene. In addition, 1635 non-coding sRNAs were identified. The highest fraction of non-coding RNAs were cis antisense RNAs (asRNAs). Analysis of the length distribution revealed that sRNAs have a median length of about 150 nt. Based on the RNA-Seq and dRNA-Seq results, genes were chosen to exemplify characteristics of the H. volcanii transcriptome by Northern blot analyses, e.g. 1) the transcript patterns of gene clusters can be straightforward, but also very complex, 2) many transcripts differ in expression level under the four analyzed conditions, 3) some genes are transcribed into RNA isoforms of different length, which can be differentially regulated, 4) transcripts with very long 5’-UTRs and with very long 3’-UTRs exist, and 5) about 30% of all cdRNAs have overlapping 3’-ends, which indicates, together with the asRNAs, that H. volcanii makes ample use of sense-antisense interactions. Taken together, this RNA-Seq study, together with a previous dRNA-Seq study, enabled an unprecedented view on the H. volcanii transcriptome

FnrL and Three Dnr Regulators Are Used for the Metabolic Adaptation to Low Oxygen Tension in Dinoroseobacter shibae

The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated

Translational coupling via termination-reinitiation in archaea and bacteria

The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria