The homology of the archaeal and eukaryotic ribosome provides one of the key pieces of evidence that underpins the idea that eukaryotes acquired their core information processing machinery from archaea. Since this discovery, reverse genetics has been used to study the functions of many archaeal proteins with eukaryotic homologues. Yet, our general understanding of archaeal growth and division remains unclear, in part because of difficulties of carrying out unbiased genetic screens in archaea. Here, by overcoming several technical hurdles we have used a screen of temperature sensitive mutants in Sulfolobus acidocaldarius to identify core regulators of cell growth and division. First, flow cytometry was used to define DNA content, identifying a set of mutants defective in cell cycle progression at elevated growth temperatures. Using genome sequencing and plasmid rescue, we then identified a point mutation in the large ribosomal subunit that inhibits translation and prevents entry into division following a shift to the restrictive temperature. This study reveals a link between translation and cell cycle control, and opens up the future possibility of using forward genetic screens in archaea to further our understanding of the similarities and differences in the cell biology of archaea, bacteria and eukaryotes. ### Competing Interest Statement The authors have declared no competing interest.

The homology of the archaeal and eukaryotic ribosome along with similarities in their apparatus involved in DNA replication and transcription provide key pieces of evidence underpinning the idea that eukaryotes acquired their core information processing machinery from archaea. Building on this discovery, reverse genetics in archaea has been used to study the functions of many archaeal proteins with eukaryotic homologues. Despite this progress, our general understanding of archaeal growth and division remains unclear, partly because of difficulties of carrying out unbiased genetic screens in archaeal relatives of eukaryotes. Here, we have used a screen of temperature-sensitive mutants in Sulfolobus acidocaldarius to identify regulators of cell growth and division. First, flow cytometry was used to define cellular DNA content - identifying a set of mutants defective in cell-cycle progression at elevated growth temperatures. Genome sequencing and plasmid rescuethen identified a point mutation in the large ribosomal subunit that inhibits translation and prevents growth and entry into division at the restrictive temperature. Taken together, these data reveal a link between translation and cell-cycle control in Sulfolobus, and show how forward genetic screens in archaea can be used to further explore similarities and differences in the cell biology of archaea, bacteria, and eukaryotes.

A small subset of acidic hot springs sampled in Yellowstone National Park yielded rod-shaped viruses which lysed liquid host cultures and formed clear plaques on lawns of host cells. Three isolates chosen for detailed analysis were found to be genetically related to previously described isolates of the Sulfolobus islandicus rod-shaped virus (SIRV), but distinct from them and from each other. Functional stability of the new isolates was assessed in a series of inactivation experiments. UV-C radiation inactivated one of the isolates somewhat faster than bacteriophage λ, suggesting that encapsidation in the SIRV-like virion did not confer unusual protection of the DNA from UV damage. With respect to high temperature, the new isolates were extremely, but not equally, stable. Several chemical treatments were found to inactivate the virions and, in some cases, to reveal apparent differences in virion stability among the isolates. Screening a larger set of isolates identified greater variation of these stability properties but found few correlations among the resulting profiles. The majority of host cells infected by the new isolates were killed, but survivors exhibited heritable resistance, which could not be attributed to CRISPR spacer acquisition or the loss of the pilus-related genes identified by earlier studies. Virus-resistant host variants arose at high frequency and most were resistant to multiple viral strains; conversely, resistant host clones generated virus-sensitive variants, also at high frequency. Virus-resistant cells lacked the ability of virus-sensitive cells to bind virions in liquid suspensions. Rapid interconversion of sensitive and resistant forms of a host strain suggests the operation of a yet-unidentified mechanism that acts to allow both the lytic virus and its host to propagate in highly localized natural populations, whereas variation of virion-stability phenotypes among the new viral isolates suggests that multiple molecular features contribute to the biological durability of these viruses.

Archaea and bacteria in geothermal environments are predicted to suffer DNA depurination in vivo at high rates, which raises questions regarding the biological roles of their abasic-site-repair enzymes. Gene deletion and enzymatic assay demonstrated that the saci_0015 gene of Sulfolobus acidocaldarius encodes an AP endonuclease (Apn) accounting for as much as 95% of the assayable activity in cell extracts and is not essential for viability. To identify genetic functions of this enzyme, deletion (ΔApn) strains were examined with respect to growth, spontaneous mutation, transformation by ssDNA containing an abasic site, and conjugation. Relative to its isogenic control, the ΔApn strain did not exhibit any change in growth rate or final cell density, rate or spectrum of spontaneous mutation, transformation by DNA containing an abasic site, or efficiency of DNA transfer and recombination. The apparent lack of genetic impact of removing the major AP endonuclease was unexpected and indicated that abasic sites are rarely bypassed directly by DNA polymerases in S. acidocaldarius . AP endonuclease deficiency had no obvious effect on survival of S. acidocaldarius under several test conditions, but it accelerated the death of cells at 4º C under illumination. Our results suggest that the normal level of AP endonuclease in S. acidocaldarius is well above the minimum required for growth and cell division but not for recovery from prolonged exposure to certain low-temperature conditions. This situation illustrates a biological challenge that has not been emphasized in experimental studies of extremophiles, i.e., the problem of long-term survival under “non-extreme” conditions.

EDITORIAL article Front. Microbiol., 29 July 2021Sec. Biology of Archaea https://doi.org/10.3389/fmicb.2021.722509

Hyperthermophilic archaea are expected to have effective (and perhaps atypical) mechanisms to limit the genetic consequences of DNA damage, but few gene products have been demonstrated to have genome-preserving functions in vivo . This study confirmed by genetic criteria that the S. acidocaldarius Ogg protein avoids the characteristic mutagenesis of G oxidation. This enzyme and the bypass polymerase Dbh have similar impacts on genome stability but work independently and may comprise most of the DNA oxidation defense of S. acidocaldarius . The critical dependence of accurate oxoG bypass on the accessory DNA polymerase Dbh further argues that some form of polymerase exchange is important for accurate genome replication in Sulfolobus , and perhaps in related hyperthermophilic archaea.

The thermophilic crenarchaeon Sulfolobus acidocaldarius has four DNA polymerases (DNAPs): PolB1, PolB2, PolB3, and Dbh (PolY). Previous in vitro studies suggested that PolB1 is the main replicative DNAP of Sulfolobales whereas PolB2 and Y-family polymerases Dpo4 ( Saccharolobus solfataricus ) or Dbh are involved in DNA repair and translesion DNA synthesis. On the other hand, there are various opinions about the role of PolB3, which remains to be clearly resolved. In order to examine the roles of the DNAPs of S. acidocaldarius through in vivo experiments, we constructed polB2 , polB3 , and dbh deletion strains and characterized their phenotypes. Efforts to construct a polB1 deletion strain were not successful; in contrast, it was possible to isolate triple gene-deletion strains lacking polB2 , polB3 , and dbh . The growth of these strains was nearly the same as that of the parent strains under normal growth conditions. The polB2 , polB3 , and dbh single-deletion strains were sensitive to some types of DNA-damaging treatments, but exhibited normal sensitivity to UV irradiation and several other damaging treatments. Overall, the genotype which exhibited the greatest sensitivity to the DNA-damaging treatments we tested was the Δ polB2 Δ polB3 combination, providing the first evidence of overlapping function for these two DNAPs in vivo . The results of our study strongly suggest that PolB1 is responsible for the DNA replication of both the leading and lagging strands and is sufficient to complete the repair of most DNA damage under normal growth conditions in S. acidocaldarius .

ABSTRACT In order to analyze in molecular terms how Sulfolobus genomes diverge, damage-induced mutations and natural polymorphisms (PMs) were identified in laboratory constructs and wild-type isolates, respectively, of Sulfolobus acidocaldarius . Among wild-type isolates drawn from one local population, pairwise nucleotide divergence averaged 4 × 10 −6 , which is about 0.15% of the corresponding divergence reported for Sulfolobus islandicus . The most variable features of wild-type S. acidocaldarius genomes were homopolymer (mononucleotide) tracts and longer tandem repeats, consistent with the spontaneous mutations that occur under laboratory conditions. Natural isolates, however, also revealed large insertions/deletions and inversions, which did not occur in any of the laboratory-manipulated strains. Several of the large insertions/deletions could be attributed to the integration or excision of mobile genetic elements (MGEs), and each MGE represented a distinct system of site-specific recombination. The mode of recombination associated with one MGE, a provirus related to Sulfolobus turreted icosahedral virus , was also seen in certain chromosomal inversions. Artificially induced mutations, non-MGE insertions/deletions, and small PMs exhibited different distributions over the genome, suggesting that large-scale patterning of Sulfolobus genomes begins early in the divergence process. Unlike induced mutations, natural base pair substitutions occurred in clusters, and one cluster exhibited properties expected of nonreciprocal recombination (gene conversion) between dispersed imperfect repeats. Taken together, the results identify simple replication errors, slipped-strand events promoted by tandem repeats, homologous recombination, and rearrangements promoted by MGEs as the primary sources of genetic variation for this extremely acidophilic archaeon in its geothermal environment. IMPORTANCE The optimal growth temperatures of hyperthermophilic archaea accelerate DNA decomposition, which is expected to make DNA repair especially important for their genetic stability, yet these archaea lack certain broadly conserved types of DNA repair proteins. In this study, the genome of the extreme thermoacidophile Sulfolobus acidocaldarius was found to be remarkably stable, accumulating few mutations in many (though not all) laboratory manipulations and in natural populations. Furthermore, all the genetic processes that were inferred to diversify these genomes also operate in mesophilic bacteria and eukaryotes. This suggests that a common set of mechanisms produces most of the genetic variation in all microorganisms, despite the fundamental differences in physiology, DNA repair systems, and genome structure represented in the three domains of life.

PCR remains one of the most versatile and widely used techniques in molecular biology, yet the capabilities of PCR seem rudimentary when compared to genome replication in living cells. Modifying Taq polymerase, or supplementing or replacing it with other proteins of thermophilic archaea or bacteria, has enhanced the functional properties of PCR and created other techniques with extended or complementary capabilities. The enhancements to PCR include increased fidelity, the ability to amplify damaged template, and tolerance of otherwise inhibitory substances in analytical samples, whereas the complementary techniques include whole-genome amplification, high-resolution genotyping, and improved performance of massively parallel sequencing. A tremendous functional diversity of thermostable DNA-replication enzymes is provided by (i) the native proteins of thermophiles currently in culture, (ii) those cloned from uncultivated thermophiles, and (iii) molecular engineering of clones obtained from both these sources. The extent and depth of this molecular diversity argues that the pace of technological innovation involving DNA synthesis at elevated temperatures will continue to accelerate.

Codon-tRNA coevolution to maximize protein production has been, until recently, the dominant hypothesis to explain codon-usage bias in highly expressed bacterial genes. Two predictions of this hypothesis are 1) selection is weak; and 2) similar silent replacements at different codons should have similar fitness consequence. We used an allele-replacement strategy to change five specific 3rd-codon-position (silent) sites in the highly expressed Escherichia coli ribosomal protein gene rplQ from the wild type to a less-preferred alternative. We introduced the five mutations within a 10-codon region. Four of the silent sites were chosen to test the second prediction, with a CTG to CTA mutation being introduced at two closely linked leucine codons and an AAA to AAG mutation being introduced at two closely linked lysine codons. We also introduced a fifth silent mutation, a GTG to GTA mutation at a valine codon in the same genic region. We measured the fitness effect of the individual mutations by competing each single-mutant strain against the parental wild-type strain, using a disrupted form of the araA gene as a selectively neutral phenotypic marker to distinguish between strains in direct competition experiments. Three of the silent mutations had a fitness effect of |s| > 0.02, which is contradictory to the prediction that selection will be weak. The two leucine mutations had significantly different fitness effects, as did the two lysine mutations, contradictory to the prediction that similar mutations at different codons should have similar fitness effects. We also constructed a strain carrying all five silent mutations in combination. Its fitness effect was greater than that predicted from the individual fitness values, suggesting that negative synergistic epistasis acts on the combination allele.

Although hyperthermophilic archaea arguably have a great need for efficient DNA repair, they lack members of several DNA repair protein families broadly conserved among bacteria and eukaryotes. Conversely, the putative DNA repair genes that do occur in these archaea often do not generate the expected phenotype when deleted. The prospect that hyperthermophilic archaea have some unique strategies for coping with DNA damage and replication errors has intellectual and technological appeal, but resolving this question will require alternative coping mechanisms to be proposed and tested experimentally. This review evaluates a combination of four enigmatic properties that distinguishes the hyperthermophilic archaea from all other organisms: DNA polymerase stalling at dU, apparent lack of conventional NER, lack of MutSL homologs, and apparent essentiality of homologous recombination proteins. Hypothetical damage-coping strategies that could explain this set of properties may provide new starting points for efforts to define how archaea differ from conventional models of DNA repair and replication fidelity.

Hyperthermophilic archaea offer certain advantages as models of genome replication, and Sulfolobus Y-family polymerases Dpo4 (S. solfataricus) and Dbh (S. acidocaldarius) have been studied intensively in vitro as biochemical and structural models of trans-lesion DNA synthesis (TLS). However, the genetic functions of these enzymes have not been determined in the native context of living cells. We developed the first quantitative genetic assays of replication past defined DNA lesions and error-prone motifs in Sulfolobus chromosomes and used them to measure the efficiency and accuracy of bypass in normal and dbh(-) strains of Sulfolobus acidocaldarius. Oligonucleotide-mediated transformation allowed low levels of abasic-site bypass to be observed in S. acidocaldarius and demonstrated that the local sequence context affected bypass specificity; in addition, most erroneous TLS did not require Dbh function. Applying the technique to another common lesion, 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), revealed an antimutagenic role of Dbh. The efficiency and accuracy of replication past 8-oxo-dG was higher in the presence of Dbh, and up to 90% of the Dbh-dependent events inserted dC. A third set of assays, based on phenotypic reversion, showed no effect of Dbh function on spontaneous -1 frameshifts in mononucleotide tracts in vivo, despite the extremely frequent slippage at these motifs documented in vitro. Taken together, the results indicate that a primary genetic role of Dbh is to avoid mutations at 8-oxo-dG that occur when other Sulfolobus enzymes replicate past this lesion. The genetic evidence that Dbh is recruited to 8-oxo-dG raises questions regarding the mechanism of recruitment, since Sulfolobus spp. have eukaryotic-like replisomes but no ubiquitin.

On 19 January 2014 Rolf ('Roffe') Bernander passed away unexpectedly. Rolf was a dedicated scientist; his research aimed at unravelling the cell biology of the archaeal domain of life, especially cell cycle-related questions, but he also made important contributions in other areas of microbiology. Rolf had a professor position in the Molecular Evolution programme at Uppsala University, Sweden for about 8 years, and in January 2013 he became chair professor at the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University in Sweden. Rolf was an exceptional colleague and will be deeply missed by his family and friends, and the colleagues and co-workers that he leaves behind in the scientific community. He will be remembered for his endless enthusiasm for science, his analytical mind, and his quirky sense of humour.

Molecular MicrobiologyVolume 93, Issue 3 p. 582-582 CorrigendumFree Access Rolf Bernander (1956–2014): pioneer of the archaeal cell cycle This article corrects the following: Rolf Bernander (1956–2014): pioneer of the archaeal cell cycle Thijs J. G. Ettema, Ann-Christin Lindås, Karin Hjort, Andrzej B. Poplawski, Henrik Kaessmann, Dennis W. Grogan, Zvi Kelman, Anders F. Andersson, Erik A. Pelve, Magnus Lundgren, Staffan G. Svärd, Volume 92Issue 5Molecular Microbiology pages: 903-909 First Published online: May 28, 2014 First published: 28 July 2014 https://doi.org/10.1111/mmi.12691AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Thijs J. G. Ettema,1* Ann-Christin Lindås,2 Karin Hjort,3 Andrzej B. Poplawski,4 Henrik Kaessmann,5,6 Dennis W. Grogan,7 Zvi Kelman,8,9 Anders F. Andersson,10 Erik Pelve,11 Magnus Lundgren1 and Staffan G. Svärd1 1Department of Cell- and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. 2Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden. 3Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden. 4Department of Genetics, University of Alabama at Birmingham, USA. 5Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland. 6Swiss Institute of Bioinformatics, Lausanne, Switzerland. 7Department of Biological Sciences, University of Cincinnati, Cincinnati, USA 8Institute for Bioscience and Biotechnology Research, Rockville, USA. 9National Institute of Standards and Technology, Rockville, USA. 10KTH Royal Institute of Technology, Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Stockholm, Sweden. 11Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, USA. It was brought to our attention that the obituary to the memory of Rolf Bernander (Ettema et al., 2) contained a factual error. We incorrectly reported that the discovery of the involvement of the ESCRT-III machinery in cell division was first made in archaea, and that this was only later shown for eukaryotes. Two independent studies, one published by the Martin-Serrano lab (Carlton and Martin-Serrano, 1) and one by the Sundquist lab (Morita et al., 4), revealed that the ESCRT system is involved in the final stages of abscission in mammalian cells. Those papers were both published in 2007, and hence predate the studies of the Bernander lab (Lindås et al., 3) and the Bell lab (Samson et al., 5), which were both published in 2008. We are grateful to Dr. Steve Bell (Indiana University, USA) for pointing out the mistake. References 1Carlton, J.G., and Martin-Serrano, J. (2007) Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery. Science 316: 1908– 1912, doi:10.1126/science.1143422CrossrefCASPubMedWeb of Science®Google Scholar 2Ettema, T.J., Lindås, A.C., Hjort, K., Poplawski, A.B., Kaessmann, H., Grogan, D.W. et al. (2014) Rolf Bernander (1956–2014): pioneer of the archaeal cell cycle. Mol Microbiol 92: 903– 909, doi:10.1111/mmi.12608Wiley Online LibraryCASWeb of Science®Google Scholar 3Lindås, A.-C., Karlsson, E.A., Lindgren, M.T., Ettema, T.J., and Bernander, R. (2008) A unique cell division machinery in the Archaea. Proc Natl Acad Sci USA 105: 18942– 18946, doi:10.1073/pnas.0809467105CrossrefCASPubMedWeb of Science®Google Scholar 4Morita, E., Sandrin, V., Chung, H.Y., Morham, S.G., Gygi, S.P., Rodesch, C.K., and Sundquist, W.I. (2007) Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis. EMBO J 26: 4215– 4227, doi:10.1038/sj.emboj.7601850Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 5Samson, R.Y., Obita, T., Freund, S.M., Williams, R.L., and Bell, S.D. (2008) A role for the ESCRT system in cell division in archaea. Science 322: 1710– 1713, doi:10.1126/science.1165322CrossrefCASPubMedWeb of Science®Google Scholar Volume93, Issue3August 2014Pages 582-582 ReferencesRelatedInformation

Abstract Extreme thermophiles are microorganisms adapted to temperatures normally found only in hot springs, hydrothermal vents and similar sites of geothermal activity. These unicellular organisms include diverse archaea and bacteria, and they span a wide range of metabolic strategies. Various molecular features enable the cells of extreme thermophiles to function optimally at temperatures that kill other cells. These features include low‐molecular weight compounds that stabilise the conformations of proteins and nucleic acids, enzymes with intrinsically stable folding of the polypeptide and unusual lipids that form highly impermeable membranes. The intrinsically stable enzymes of extreme thermophiles offer advantages for industrial and diagnostic processes ranging from ore processing to molecular genotyping. Key Concepts: Bacteria and archaea consist of very simple (prokaryotic) cells but vary greatly with respect to metabolic capabilities, physiological limits and other fundamental properties. Diverse bacteria and archaea require the temperatures found in geothermal environments for optimal growth, and are classified as extreme thermophiles. Geothermal activity typically provides chemical energy and nutrients that microorganisms can utilise. Extreme thermophiles use two basic strategies to avoid thermal denaturation of their enzymes: extrinsic stabilisation, conferred by certain small molecules, and intrinsic stabilisation, conferred by the specific structure and conformation of the enzyme itself. Intrinsically thermostable enzymes of extreme thermophiles allow certain chemical reactions to be catalysed with high specificity at high temperatures or under other harsh conditions.

Although homologous recombination (HR) is known to influence the structure, stability, and evolution of microbial genomes, few of its functional properties have been measured in cells of hyperthermophilic archaea. The present study manipulated various properties of the parental DNAs in high-resolution assays of Sulfolobus acidocaldarius transformation, and measured the impact on the efficiency and pattern of marker transfer to the recipient chromosome. The relative orientation of homologous sequences, the type and position of chromosomal mutation being replaced, and the length of DNA flanking the marked region all affected the efficiency, linkage, tract continuity, and other parameters of marker transfer. Effects predicted specifically by the classical reciprocal-exchange model of HR were not observed. One analysis observed only 90 % linkage between markers defined by adjacent bases; in another series of experiments, sequence divergence up to 4 % had no detectable impact on overall efficiency of HR or on the co-transfer of a distal non-selected marker. The effects of introducing DNA via conjugation, rather than transformation, were more difficult to assess, but appeared to increase co-transfer (i.e. linkage) of relatively distant non-selected markers. The results indicate that HR events between gene-sized duplex DNAs and the S. acidocaldarius chromosome typically involve neither crossing over nor interference from a mismatch-activated anti-recombination system. Instead, the donor DNA may anneal to a transient chromosomal gap, as in the mechanism proposed for oligonucleotide-mediated transformation of Sulfolobus and other micro-organisms.

In a recent description of the rate and character of spontaneous mutation in the hyperthermophilic bacterium Thermus thermophilus, the mutation rate was observed to be substantially lower than seen in several mesophiles. Subsequently, a report appeared indicating that this bacterium maintains an average of about 4.5 genomes per cell. This number of genomes might result in a segregation lag for the expression of a recessive mutation and might therefore lead to an underestimate of the rate of mutation. Here we describe some kinds of problems that may arise when estimating mutation rates and outline ways to adjust the rates accordingly. The emphasis is mainly on differential rates of growth of mutants versus their parents and on various kinds of phenotypic lag. We then apply these methods to the T. thermophilus data and conclude that there is as yet no reliable impact on a previously described rate.

Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.