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Edited by John M. Denu Archaea are a distinct and deeply rooted lineage that harbor eukaryotic-like mechanisms, including several that manage chromosome function. In previous work, the thermoacidophilic crenarchaeon, Sulfolobus solfataricus, was subjected to adaptive laboratory evolution to produce three strains, called SARC, with a new heritable trait of super acid resistance. These strains acquired heritable conserved transcriptomes, yet one strain contained no mutations. Homologous recombination without allele replacement at SARC acid resistance genes caused changes in both phenotype and expression of the targeted gene. As recombination displaces chromatin proteins, their involvement was predicted in the SARC trait. Native chromatin proteins are basic and highly abundant and undergo post-translational modification through lysine monomethylation. In this work, their modification states were investigated. In all SARC lines, two chromatin proteins, Cren7 and Sso7d, were consistently undermethylated, whereas other chromatin proteins were unaltered. This pattern was heritable in the absence of selection and independent of transient exposure to acid stress. The bulk of Sso7d was undermethylated at three contiguous N-terminal lysine residues but not at central or C-terminal regions. The N-terminal region formed a solvent-exposed patch located on the opposite side of the binding domain associated with the DNA minor groove. By analogy to eukaryotic histones, this patch could interact with other chromosomal proteins and be modulated by differential post-translational modification. Previous work established an epigenetic-like mechanism of adaptation and inheritance in S. solfataricus. The identification of heritable epigenetic marks in this work further supports the occurrence of an epigenetic process in archaea.

lntroduction the cointegration of consEucts containing each of the o, 0, and yencoding sequences for hfib into a transcriptionally responsive chromosomal domain is needed. For the purposes

In vitro studies demonstrate that porcine relaxin may possess various therapeutic effects. In this study, we explore the possibility of expressing porcine relaxin in transgenic tobacco. Tobacco was selected because it is a non-food, non-feed crop, and recombinant protein production can readily be scaled up. The cDNA of porcine preprorelaxin was under the regulation of two different constitutive promoters. DNA analysis by polymerase chain reaction verified that all transgenic plants contained the correct size of gene insert. Preliminary studies showed the presence of putative prorelaxin bands in both silver-stained SDS-PAGE and western blot. The results also indicated that tobacco-produced prorelaxin may not be properly processed to yield the mature relaxin.

21Epigenetic variants of the archaeon Sulfolobus solfataricus called SARC have evolved heritable traits 22 including extreme acid resistance, enhanced genome integrity and a conserved "SARC" transcriptome 23 related to acid resistance. These traits appear to result from altered chromatin protein function related 24 to the heritable hypomethylation of chromatin proteins Cren7 and Sso7D. To clarify how this might 25 occur, ChIPseq and Affinity Purification Mass Spectrometry (AP-MS) were used to compare Cren7 and 26 Sso7D genome binding sites and protein networks between lineages (wild type and SARC) and culture 27 pH (pH 1 and 3). All SARC transcriptome loci were bound by these chromatin proteins but with invariant 28 patterns indicating binding alone was insufficient to mediate the SARC traits. In contrast, chromosome 29 association varied at other loci. Quantitative AP-MS was then used to identify protein interaction 30 networks and these included transcription and DNA repair proteins implicated in the evolved heritable 31 traits that varied in abundance between SARC and wild type strains. Protein networks included most of 32 the S-adenosylmethionine (SAM) synthesis pathway including serine hydroxymethyltransferase (SHMT), 33 whose abundance varied widely with culture pH. Because epigenetic marks are coupled to SAM pools 34 and oxidative stress in eukaryotes, occurrence of a similar process was investigated here. Archaeal SAM 35 pools were depleted by treatment with SAM pathway inhibitors, acid or oxidative stress and, like 36 eukaryotes, levels were raised by vitamin B12 and methionine supplementation. We propose that in 37 archaea, oxidation-induced SAM pool depletion acting through an SHMT sensor, drove chromatin 38 protein hypomethylation and thereby protein network changes that established the evolved SARC 39 epigenetic traits. 40 Significance Statement 41Archaea and eukaryotes share many molecular processes, including chromatin-mediated epigenetic 42 inheritance of traits. As with eukaryotes, archaeal protein complexes were formed between trait-related 43 proteins and chromatin proteins, subject to chromatin protein methylation state. Oxidation-induced 44 depletion of S-adenosylmethionine (SAM) pools likely resulted in chromatin protein hypomethylation. 45 Subsequent chromatin enrichment of serine hydroxymethyltransferase as a response to oxidative stress 46 could modulate methylation at specific genomic loci. The interplay between archaeal metabolism and 47 chromatin appear consistent with patterns observed in eukaryotes and indicate the existence of an 48 ancient oxidation signal transduction pathway controlling epigenetics. 49 50 Immunoprecipitation for ChIP-Seq. Triplicate crosslinked cell pellets were sonicated and subjected to 105 chromatin immunoprecipitation using polyclonal antibody serum for Cren7 or Sso7D as described 106 previously (30) with alterations described in and SI Appendix, Supplemental Methods. 107ChIP-Seq and data analysis. DNA samples were blunt ended, A-tailed, ligated to barcoded a...

Microbes belonging to the genus Metallosphaera oxidize sulfidic minerals. These organisms thrive at temperature extremes and are members of the archaeal phylum Crenarchaeota. Because they can employ a lithoautotrophic metabolism, energy availability likely limits their activity raising questions about how they conduct biogeochemical activity. Vesicles are membrane encapsulated structures produced by all biological lineages but using very different mechanisms. Across the Crenarchaeota, it has been proposed that a eukaryotic-like Endosomal Sorting Complex Required for Transport system promotes formation of these structures but in response to unknown signals and for undefined purposes. To address such questions, Metallosphaera sedula vesicle formation and function were studied under lithoautotrophic conditions. Energy deprivation was evaluated and found to stimulate vesicle synthesis while energy excess repressed vesicle formation. Purified vesicles adhered rapidly to the primary copper ore, chalcopyrite, and formed compact monolayers. These vesicle monolayers catalyzed iron oxidation and solubilization of mineralized copper in a time-dependent process. As these activities were membrane associated, their potential transfer by vesicle fusion to M. sedula cells was examined. Fluorophore-loaded vesicles rapidly transferred fluorescence under environmentally relevant conditions. Vesicles from a related archaeal species were also capable of fusion; however, this process was species-specific as vesicles from different species were incapable of fusion. In addition, vesicles produced by a copper-resistant M. sedula cell line transferred copper extrusion capacity along with improved viability over mutant M. sedula cells lacking copper transport proteins. Membrane vesicles may therefore play a role in modulating energy-related traits in geochemical environments by fusion-mediated protein delivery.

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