Browsing by Subject "Halophiles"

Protein-based compaction of the genome is a feature found in species across the tree of life. In Archaea, the majority of species contain a histone fold domain-containing protein, and these have been shown to compact DNA through the formation of nucleosomes and extended structures called hypernucleosomes. However, the role of the histone-like proteins of halophilic archaea is unclear. Previous work in the model species Halobacterium salinarum indicated that its sole histone gene, hpyA, is dispensable for growth and is expressed at very low levels. I hypothesize that the unique high-salt environment of halophilic archaea has selected for an alternative histone function, and that they function instead as transcription factors.

This hypothesis was addressed with genetic approaches including the creation of knockout and complementation strains, traditional microbiology techniques including growth assays and microscopy, and high-throughput genomics approaches: ChIP-Seq to study genome-wide binding, and RNA-Seq to study differential expression in ΔhpyA strain. It was found that hpyA is required for optimal growth in hypo-osmotic conditions, and exhibits strong salt-dependent binding patterns and gene regulation. It directly regulates genes involved in iron uptake, and indirectly regulates genes in ion transport and nucleotide metabolism. These results validate the link between histone function and the high-salt environment of halophilic archaea.

Similar to hpyA, I found that the sole histone gene of another model halophile: hstA of Haloferax volcanii, could be deleted, and that knockout cells remained viable. The genome-wide binding of both halophilic histones was studied, and compared with publicly available data regarding the binding patterns from transcription factors (TFs), nucleoid-associated proteins (NAPs), and eukaryotic histones. Halophilic histones bind in narrow, discrete, and relatively rare peaks, just like TFs; however, this binding is not enriched at the promoter, and they instead bind evenly in both intergenic and coding regions (like some NAPs). Their occupancy profile across gene start sites do not resemble those of histones or TFs. In terms of sequence specificity, HpyA exhibits a histone-like preference for 10bp periodicity, while HstA exhibits a TF-like trait in preferentially binding a palindromic sequence motif. When considering all the data, I conclude that halophilic histones blur the line between TFs, NAPs, and histones.

A major technical challenge in generating this data was the removal of rRNA prior to carrying out RNA-Seq. Several approaches were tested across four model species of halophiles, and the reasons for differences in performance for these approaches were analyzed. Methods that deliver efficient rRNA removal targeted to a particular species, or to halophilic archaea in general, are highlighted.

Together these results shed light on the unusual function and binding modes of the histone-like proteins of halophilic archaea. In combination with other recent work, they suggest that histone function is linked with the physical environment of archaeal species.

Like most Archaea, the hypersaline-adapted organism Halobacterium salinarum exhibits characteristics from all three domains of life, including a eukaryotic histone protein, a universal propensity to genetic rearrangements, and homologs of bacterial cell division proteins. Here we investigate the ancestral function of histone protein in the Archaea. Transcriptomics, proteomics, and phenotypic assays of histone mutants determine that histone regulates gene expression and cell shape but not genome compaction in H. salinarum. We further explore the regulation of gene expression on a genome-wide scale through the study of genomic instability. Genomic deletions and duplications are detected through the meta-analysis of 1154 previously published gene expression arrays and 48 chromatin immunoprecipitation arrays. We discover that a 90 kb duplication event in the megaplasmid pNRC100 directly leads to increased gene expression, and find evidence that the chromosome is far more unstable than previously assumed. These events are all linked with the presence of mobile insertion elements. Finally, in response to questions generated by these experiments, we develop a novel time-lapse protocol for H. salinarum and ask basic questions about single cell dynamics during division. Fluorescent labeling of homologs to bacterial cell division proteins confirms their involvement in cell division but localization dynamics contradict the basic bacterial model. The discovery of unusual facets of morphology during cell division is consistent with these novel protein dynamics and opens up new avenues of inquiry into archaeal cell division.