Project description:Transcriptome analysis based on total RNA-seq was performed on different Haloferax volcanii strains including mutants strains iincluding deletion strains for two small proteins. Differential expression analysis showed that a subset of genes were found to be regulated in the absence of the small proteins.
Project description:DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life1 and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whilst most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We have used deep sequencing to study replication in Haloferax volcanii. Four chromosomal origins of differing activity were identified. Deletion of individual origins resulted in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild-type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved. Measurement of replication dynamics (marker frequency analysis; MFA) for Haloferax volcanii strains, including wild-type, the laboratory strain, individual and combinations of replication origin deletions.
Project description:Purpose: The Haloferax volcanii ∆tfeB strain provided a unique opportunity to study a putative role of TFEβ in the regulation of gene expression. Results: The deletion of the tfeB gene in H. volcanii results in the aberrant expression of approximately one third of all genes, consistent with its function as a basal transcription initiation factor. Interestingly, tfeB deletion particularly affects foreign genes including a prophage region. Conclusions: Our results confirm the dual function of TFE as basal factor and regulator of transcription
Project description:The function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeon H. volcanii a plethora of sRNAs have been identified, however, in-depth functional analysis is missing for most of them. We selected a small RNA (s479) from H. volcanii for detailed characterization. The sRNA gene is encoded between a CRISPR RNA locus and the Cas protein gene cluster, the s479 deletion strain is viable and was characterized in detail. Transcriptome studies of wild type Haloferax cells and the deletion mutant revealed up-regulation of six genes in the deletion strain, showing that the sRNA has a distinct cellular function. Proteome comparison of wild type and deletion strains further expanded the regulon of s479 deeply rooting this sRNA within the metabolism of H. volcanii especially the regulation of transporter abundance.
2021-08-10 | PXD022750 | Pride
Project description:Haloferax volcanii oxsR deletion and oxsRHA strain sequencing
Project description:A potential origin that appears to stay dormant in its native host Haloferax volcanii lacking the main active origins becomes activated and competent for replication of the entire chromosome when integrated into the chromosome of the origin-deleted H. mediterranei. Measurement of replication dynamics (marker frequency analysis; MFA) for Haloferax mediterranei H13
Project description:Rhomboids are conserved intramembrane serine proteases involved in cell signaling processes. Their role in prokaryotes is scarcely known and remains to be investigated in Archaea. We previously constructed a rhomboid homologue deletion mutant (ΔrhoII) in Haloferax volcanii, which showed reduced motility, increased novobiocin sensitivity, and an Nglycosylation defect. To address the impact of rhoII deletion on H. volcanii physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. A total of 1847 proteins were identified (45.8% of H. volcanii predicted proteome), from which 103 differed in amount. Additionally, the mutant strain evidenced 99 proteins with altered electrophoretic migration, which suggested differential post-translational processing/modification. Integral membrane proteins that evidenced variations in concentration, electrophoretic migration, or semitryptic cleavage in the mutant were considered as potential RhoII targets. These included a PrsW protease homologue (which was less stable in the mutant strain), a predicted halocyanin, and six integral membrane proteins potentially related to the mutant glycosylation (S-layer glycoprotein, Agl15) and cell adhesion/motility (flagellin1, HVO_1153, PilA1, and PibD) defects. This study investigated for the first time the impact of a rhomboid protease on the whole proteome of an organism.
Project description:µ-proteins (≤ 70 amino acids) have important and often essential roles in all kingdoms of life, including cell motility, regulation of membrane transport and as transcription factors. In the halophilic archaeon and model system Haloferax volcanii a significant number of µ-proteins were predicted to be zinc finger proteins. Here we used mass spectrometry-based proteomics to systematically investigate the impact of single gene deletions of 19, previously uncharacterized, zinc finger µ-proteins on H. volcanii grown in glycerol media. We employed a state-of-the-art dia-PASEF acquisition strategy, detecting over 3400 proteins across the 19 deletion strains and the wild-type. The comprehensive proteome coverage enabled a systematic analysis of proteome remodeling. We found that in 11 out of the 19 mutants the proteome remodeling involved proteins annotated to play a role in cell motility, matching swarming and cell growth phenotypes we observed for these strains. Taken together, our data provide the most comprehensive proteome coverage of H. volcanii to date, and the effect of 19 different zinc-finger µ-proteins deletion strains on the proteome of this organism. The combined data provide a valuable resource for future research in the field.