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Assembly-Driven Community Genomics of a Hypersaline Microbial Ecosystem  [PDF]
Sheila Podell, Juan A. Ugalde, Priya Narasingarao, Jillian F. Banfield, Karla B. Heidelberg, Eric E. Allen
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0061692
Abstract: Microbial populations inhabiting a natural hypersaline lake ecosystem in Lake Tyrrell, Victoria, Australia, have been characterized using deep metagenomic sampling, iterative de novo assembly, and multidimensional phylogenetic binning. Composite genomes representing habitat-specific microbial populations were reconstructed for eleven different archaea and one bacterium, comprising between 0.6 and 14.1% of the planktonic community. Eight of the eleven archaeal genomes were from microbial species without previously cultured representatives. These new genomes provide habitat-specific reference sequences enabling detailed, lineage-specific compartmentalization of predicted functional capabilities and cellular properties associated with both dominant and less abundant community members, including organisms previously known only by their 16S rRNA sequences. Together, these data provide a comprehensive, culture-independent genomic blueprint for ecosystem-wide analysis of protein functions, population structure, and lifestyles of co-existing, co-evolving microbial groups within the same natural habitat. The “assembly-driven” community genomic approach demonstrated in this study advances our ability to push beyond single gene investigations, and promotes genome-scale reconstructions as a tangible goal in the quest to define the metabolic, ecological, and evolutionary dynamics that underpin environmental microbial diversity.
CRISPR loci reveal networks of gene exchange in archaea
Avital Brodt, Mor N Lurie-Weinberger, Uri Gophna
Biology Direct , 2011, DOI: 10.1186/1745-6150-6-65
Abstract: Here we analyzed all currently known spacers present in archaeal genomes and identified their source by DNA similarity. While nearly 50% of archaeal spacers matched mobile genetic elements, such as plasmids or viruses, several others matched chromosomal genes of other organisms, primarily other archaea. Thus, networks of gene exchange between archaeal species were revealed by the spacer analysis, including many cases of inter-genus and inter-species gene transfer events. Spacers that recognize viral sequences tend to be located further away from the leader sequence, implying that there exists a selective pressure for their retention.CRISPR spacers provide direct evidence for extensive gene exchange in archaea, especially within genera, and support the current dogma where the primary role of the CRISPR/Cas system is anti-viral and anti-plasmid defense.This article was reviewed by: Profs. W. Ford Doolittle, John van der Oost, Christa Schleper (nominated by board member Prof. J Peter Gogarten)CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic Repeats)/Cas (CRISPR-associated) modules constitute acquired prokaryotic immune systems that protect prokaryotes against parasitic genetic elements, such as viruses [for recent reviews see [1-3]]. CRISPR/Cas systems contain repeated sequences that are interrupted by short non-repetitive DNA segments (20-50 bp long), termed spacers [4] (Figure 1). CRISPR arrays can be transcribed and processed into small crRNA molecules, which then lead to degradation of foreign nucleic acids by a mechanism based on complementary base-pairing [5,6]. CRISPR/Cas systems also have a mechanism that prevents targeting the locus encoding the CRISPR itself [7]. The systems are both adaptive, heritable and can be used to determine a history of past infections [7-9]CRISPR elements can be found in almost all archaeal genomes and in approximately 40% of sequenced bacterial genomes [10]. Archaeal CRISPR loci tend to be larger than those found in bac
Diversity of Halophilic Archaea in Hypersaline lakes of Inner Mongolia, China

PAN Hai-lian,ZHOU Cheng,WANG Hong-lei,XUE Yan-fen,MA Yan-he,

微生物学报 , 2006,
Abstract: The aims of this work were to explore the diversity of halophilic archaea in hypersaline lakes of Inner Mongolia, China and to collect novel halophilic archaea. One hundred and sixty-five halophilic archaea were isolated from the three different types of hypersaline lakes (Erliannor, shangmatala and Xilin soda lake) in Inner Mongolia. By analysis of the restriction patterns of amplified 16S rDNA (ARDRA) with the enzyme Afa I and Hae II, respectively, the isolates were clustered into 14 genotypes, and the representatives of each genotype were randomly chosen for the determination of 16S rDNA sequence. The phylogenetic analysis revealed that all of the isolates were clustered into 10 groups: Halorubrum, Natronococcus, Natronorubrum, Haloterrigena, Halorhabdus, Halobiforma, Haloarcula, Haloferax and other two unknown groups. Dominant isolates were related to Halorubrum spp. in all three lakes. Some of the isolates studied showed less affiliation with known taxa ( <98% sequence similarity) and may represent novel taxa. Two isolates HXH33 and HSH33 showed very less affiliation with known genus ( < 93% sequence similarity) and may represent two new genera. These results suggest that diverse archaea exist in and the unknown archaea thrive in the hypersaline lakes of Inner Mongolia.
Comparative analysis of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) loci in the genomes of halophilic archaea

Fan Zhang,Bin Zhang,Hua Xiang,Songnian Hu,

微生物学报 , 2009,
Abstract: Abstract: Objective] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a widespread system that provides acquired resistance against phages in bacteria and archaea. Here we aim to genome-widely analyze the CRISPR in extreme halophilic archaea, of which the whole genome sequences are available at present time. Methods] We used bioinformatics methods including alignment, conservation analysis, GC content and RNA structure prediction to analyze the CRISPR structures of 7 haloarchaeal genomes. Results] We identified the CRISPR structures in 5 halophilic archaea and revealed a conserved palindromic motif in the flanking regions of these CRISPR structures. In addition, we found that the repeat sequences of large CRISPR structures in halophilic archaea were greatly conserved, and two types of predicted RNA secondary structures derived from the repeat sequences were likely determined by the fourth base of the repeat sequence. Conclusion] Our results support the proposal that the leader sequence may function as recognition site by having palindromic structures in flanking regions, and the stem-loop secondary structure formed by repeat sequences may function in mediating the interaction between foreign genetic elements and CAS-encoded proteins.
Exploiting CRISPR/Cas: Interference Mechanisms and Applications  [PDF]
Hagen Richter,Lennart Randau,André Plagens
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms140714518
Abstract: The discovery of biological concepts can often provide a framework for the development of novel molecular tools, which can help us to further understand and manipulate life. One recent example is the elucidation of the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) that protects bacteria and archaea against viruses or conjugative plasmids. The immunity is based on small RNA molecules that are incorporated into versatile multi-domain proteins or protein complexes and specifically target viral nucleic acids via base complementarity. CRISPR/Cas interference machines are utilized to develop novel genome editing tools for different organisms. Here, we will review the latest progress in the elucidation and application of prokaryotic CRISPR/Cas systems and discuss possible future approaches to exploit the potential of these interference machineries.
T. Tsimpouki on I. Tyrrell’s Transnational Nation  [cached]
European Journal of American Studies , 2009, DOI: 10.4000/ejas.7516
Abstract: Ian Tyrrell. Transnational Nation. United States History in Global Perspective since 1789. Palgrave Macmillan, 2007. pp. 289. ISBN 978-1-4039-9368-7.When asked to choose among the most influential scholarly studies of history or theory from the last twenty years, Russ Castronovo picked Kaplan and Pease’s Cultures of United States Imperialism (1993) as the book that marks “a paradigmatic shift” in American Studies scholarship because it assesses “the emergence of academic interest in empire, b...
Dealing with the Evolutionary Downside of CRISPR Immunity: Bacteria and Beneficial Plasmids  [PDF]
Wenyan Jiang,Inbal Maniv,Fawaz Arain,Yaying Wang,Bruce R. Levin,Luciano A. Marraffini
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1003844
Abstract: The immune systems that protect organisms from infectious agents invariably have a cost for the host. In bacteria and archaea CRISPR-Cas loci can serve as adaptive immune systems that protect these microbes from infectiously transmitted DNAs. When those DNAs are borne by lytic viruses (phages), this protection can provide a considerable advantage. CRISPR-Cas immunity can also prevent cells from acquiring plasmids and free DNA bearing genes that increase their fitness. Here, we use a combination of experiments and mathematical-computer simulation models to explore this downside of CRISPR-Cas immunity and its implications for the maintenance of CRISPR-Cas loci in microbial populations. We analyzed the conjugational transfer of the staphylococcal plasmid pG0400 into Staphylococcus epidermidis RP62a recipients that bear a CRISPR-Cas locus targeting this plasmid. Contrary to what is anticipated for lytic phages, which evade CRISPR by mutations in the target region, the evasion of CRISPR immunity by plasmids occurs at the level of the host through loss of functional CRISPR-Cas immunity. The results of our experiments and models indicate that more than 10?4 of the cells in CRISPR-Cas positive populations are defective or deleted for the CRISPR-Cas region and thereby able to receive and carry the plasmid. Most intriguingly, the loss of CRISPR function even by large deletions can have little or no fitness cost in vitro. These theoretical and experimental results can account for the considerable variation in the existence, number and function of CRISPR-Cas loci within and between bacterial species. We postulate that as a consequence of the opposing positive and negative selection for immunity, CRISPR-Cas systems are in a continuous state of flux. They are lost when they bear immunity to laterally transferred beneficial genes, re-acquired by horizontal gene transfer, and ascend in environments where phage are a major source of mortality.
Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems
Kira S Makarova, L Aravind, Yuri I Wolf, Eugene V Koonin
Biology Direct , 2011, DOI: 10.1186/1745-6150-6-38
Abstract: A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs) containing a distinct form of the RNA Recognition Motif (RRM) domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes.Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure comparison continue to reveal unexpected homologous relationship among Cas proteins. Unification of Cas protein families previously considered unrelated provides for improvement in the classification of CRISPR-Cas systems and a reconstruction of their evolution.This article was reviewed by Malcolm White (nominated by Purficacion Lopez-Garcia), Frank Eisenhaber and Igor Zhulin. For the full reviews, see the Reviewers' Comments section.The CRISPR-Cas is an adaptive immunity system that is present in most archaea and many bacteria, and functions on a "Lamarckian inheritance" principle. The CRISPR-Cas loci in prokaryote genomes consist of an array of direct, typically palindromic repeats known as CRISPR (Clustered Regularly Interspaced Short Palindrom
Evolutionary conservation of sequence and secondary structures in CRISPR repeats
Victor Kunin, Rotem Sorek, Philip Hugenholtz
Genome Biology , 2007, DOI: 10.1186/gb-2007-8-4-r61
Abstract: Here we analyze CRISPR repeats identified in 195 microbial genomes and show that they can be organized into multiple clusters based on sequence similarity. Some of the clusters present stable, highly conserved RNA secondary structures, while others lack detectable structures. Stable secondary structures exhibit multiple compensatory base changes in the stem region, indicating evolutionary and functional conservation.We show that the repeat-based classification corresponds to, and expands upon, a previously reported CAS gene-based classification, including specific relationships between CRISPR and CAS subtypes.Clustered regularly interspaced short palindromic repeats (CRISPRs) are repetitive structures in Bacteria and Archaea composed of exact repeat sequences 24 to 48 bases long (herein called repeats) separated by unique spacers of similar length (herein called spacers) [1,2]. The CRISPR sequences appear to be among the most rapidly evolving elements in the genome, to the point that closely related species and strains, sometimes more than 99% identical at the DNA level, differ in their CRISPR composition [3,4].Up to 45 gene families, called CRISPR-associated sequences (CASs), appear in conjunction with these repeats and are hypothesized to be responsible for CRISPR propagation and functioning [2,5,6]. It has been proposed that CASs can be divided into seven or eight subtypes, according to their operon organization and gene phylogeny [5,6]. Phylogenetic analysis additionally indicates that CASs have undergone extensive horizontal gene transfer, as very similar CAS genes are found in distantly related organisms [6,7]. CRISPRs and CASs have been found on mobile genetic elements, such as plasmids, skin mobile elements, and even prophages, suggesting a possible distribution mechanism for the system [7-9].CRISPRs have been suggested to play roles in replicon partitioning [1], DNA repair [10], regulation [5] and chromosomal rearrangement [11]. It was recently reported tha
Persisting Viral Sequences Shape Microbial CRISPR-based Immunity  [PDF]
Ariel D. Weinberger ? ,Christine L. Sun ?,Mateusz M. Pluciński,Vincent J. Denef,Brian C. Thomas,Philippe Horvath,Rodolphe Barrangou,Michael S. Gilmore,Wayne M. Getz,Jillian F. Banfield
PLOS Computational Biology , 2012, DOI: 10.1371/journal.pcbi.1002475
Abstract: Well-studied innate immune systems exist throughout bacteria and archaea, but a more recently discovered genomic locus may offer prokaryotes surprising immunological adaptability. Mediated by a cassette-like genomic locus termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), the microbial adaptive immune system differs from its eukaryotic immune analogues by incorporating new immunities unidirectionally. CRISPR thus stores genomically recoverable timelines of virus-host coevolution in natural organisms refractory to laboratory cultivation. Here we combined a population genetic mathematical model of CRISPR-virus coevolution with six years of metagenomic sequencing to link the recoverable genomic dynamics of CRISPR loci to the unknown population dynamics of virus and host in natural communities. Metagenomic reconstructions in an acid-mine drainage system document CRISPR loci conserving ancestral immune elements to the base-pair across thousands of microbial generations. This ‘trailer-end conservation’ occurs despite rapid viral mutation and despite rapid prokaryotic genomic deletion. The trailer-ends of many reconstructed CRISPR loci are also largely identical across a population. ‘Trailer-end clonality’ occurs despite predictions of host immunological diversity due to negative frequency dependent selection (kill the winner dynamics). Statistical clustering and model simulations explain this lack of diversity by capturing rapid selective sweeps by highly immune CRISPR lineages. Potentially explaining ‘trailer-end conservation,’ we record the first example of a viral bloom overwhelming a CRISPR system. The polyclonal viruses bloom even though they share sequences previously targeted by host CRISPR loci. Simulations show how increasing random genomic deletions in CRISPR loci purges immunological controls on long-lived viral sequences, allowing polyclonal viruses to bloom and depressing host fitness. Our results thus link documented patterns of genomic conservation in CRISPR loci to an evolutionary advantage against persistent viruses. By maintaining old immunities, selection may be tuning CRISPR-mediated immunity against viruses reemerging from lysogeny or migration.
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