Student projects – University of Copenhagen

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Bachelor/Master Projects

Master Project: Archaeal thermophilic proteases and esterases: Physiological roles and applications

Microorganisms are rich resource of proteases and esterases, enzymes that catalyze protein and ester degradation. While cellular proteases are essential to cellular life since they degrade dysfunctional proteins in the cell, extracellular proteases often function in converting proteinaceous substance into nitrogen source to the cell. Proteases encoded in thermophiles are featured with an extraordinarily large number and diversity and this is probably due to that proteinaceous substrates not only provide nitrogen source, but also serve as primary carbon and energy sources to their growth. As a result, thermophilic proteases exhibit regulated expression in these organisms as exemplified by tryptone-inducible expression of several extracellular proteases in Sulfolobus solfataricus (1). Furthermore, archaea encode small archaeal ubiquitin-like modifier proteins (SAMPs), which modify archaeal proteins in a ubiquitination-like mechanism, although the physiological function of the SAMP modification remains to be investigated. To date, only a few of archaeal proteases have been studied and this is primarily because they are expressed only to a low level in thermophilic organisms and their recombinant enzymes are almost insoluble in mesophilic hosts.

Recently, we have developed a robust protein expression system for Sulfolobus islandicus (2) for which versatile genetic tools are available for genetic study of gene function (3,4). These methodologies allow thermophilic proteases/esterasses to be expressed and purified from a homologous host and characterized whereas their physiological roles to be investigated. The expressed proteases/esterases shall be tested for biocatalysis in making sugar esters.

A highly motivated student will be hired as soon as possible to over-express important thermophilic proteases/esterases in S. islandicus and purified them for biochemical characterization. Their functions in nitrogen, carbon and energy metabolisms of S. islandicus as well as their regulation will also be studied and the results to be obtained will guide strain construction to strongly elevate protease production in this archaeon. The project will be in collaboration with industry and Danish university other than KU and the student will have study stay outside the KU campus with extra cost living covered.

References

  1. Cannio R. Catara G, Fiume I, Balestrieri M, Rossi M, Palmieri G. (2010) Protein Pept Lett. 17(1):78-85.
  2. Peng N, Deng L, Mei Y, Jiang D, Hu Y, Awayez M, Liang Y, She Q. (2012) Appl Environ Microbiol. 78(16):5630-7.
  3. Zheng T, Huang Q, Zhang C, Ni J, She Q, Shen Y. (2012) Appl Environ Microbiol. 78(2):568-74. 4. Zhang C, Tian B, Li S, Ao X, Dalgaard K, Gökce S, Liang Y, She Q. (2013) Biochem Soc Trans. 41(1):405-10.

Contact: Qunxin She (qunxin@bio.ku.dk), tel. 3532 2013

Global regulation of gene expression in Sulfolobus islandicus, a crenarchaeal model organism

Many interesting viruses have been isolated and characterized from diverse archaeal species. Sulfolobus SSV2 and pSSVx, the first characterized archaeal helper and satellite viruses, belong to a very interesting group. Our recent work on characterizing interactions between viruses SSV2 and pSSVx and their hosts has revealed that the virus replication is strongly induced at a late growth stage in the natural host. As a consequence, virus induction completely inhibits host growth (Contursi et al. 2006). Recently, a Sulfolobus genome microarray was constructed for Sulfolobus solfataricus (She et al. 2001) and several genetic elements (including SSV2 and pSSVx - Stedman et al. 2003) by an international consortium and this provided a useful tool for studying the molecular mechanisms behind this interesting phenomenon. We have conducted a pilot experiment with this array and observed major changes in the expression of many genes at the stages of before and after virus induction (Figure).

The genome array contains ca. 4000 probes of 55-70 long oligonucleotides that were spotted onto a glass slide in duplicates. Each contains a specific sequence derived from a host or viral/plasmidic gene. Only a portion of the image obtained from the microarray analysis is shown above where each dot in the picture represents a gene. Total RNAs were prepared from cell samples before, and after, SSV2 virus induction, and labeled with Cy3 (red) and Cy5 (green), respectively. Green spots indicate high levels of gene expression after the induction; red spots, very low levels/no gene expression after the induction (strong inhibition), yellow spots indicate similar levels of expression at both stages (no changes), whereas other colors record induced/inhibited gene expression during the induction. Quantification of the red and the green components in each spot yields induction or inhibition fold for each gene.


The next step is to characterize the induction process in detail. It is our hope that some motivated students will join us at this stage to conduct more microarray analyses to determine whether a cascade activation of the expression of transcription factors is involved in the induction process. Subsequently, the identified archaeal transcription factors will be analyzed using genetic and functional genomic tools, both of which have recently been developed and/or established in our laboratory.

Contact: Qunxin She

References

  1. P. Contursi, S. Jensen, T. Aucelli, M. Rossi, S. Bartolucci & Q. She. Characterization of the Sulfolobus Host-SSV2 Virus Interaction. Extremophiles 10, 615-627. 2006
  2. Q. She, R. K. Singh, F. Confalonieri, Y. Zivanovic, G. Allard, M. J. Awayez, C. C. Chan-Weiher, I. G, Clausen, B. A. Curtis, A. de Moors, G. Erauso, P. M. K. Gordon, I. Heikamp de Jong, A. C. Jeffries, C. J. Kozera, N. Medina, X. Peng, H. P. Thi-Ngoc, P. Redder, M. E. Schenk, C. Theriault, N. Tolstrup, R. L. Charlebois, W. F. Doolittle, M. Duguet, T. Gaasterland, R. A. Garrett, M. A. Ragan, C. W. Sensen & J. van der Oost. The complete genome of the crenarchaeote Sulfolobus solfataricus P2. Proc. Natl. Acad. Sci. USA 98, 7835-7840, 2001.
  3. K.M. Stedman, Q. She, H. Phan, H. P. Arnold, I. Holz, R.A. Garrett & W. Zillig. Biological and genetic relationships between fuselloviruses infecting the extremely thermophilic archaeon Sulfolobus: SSV1 and SSV2. Res. Microbiol. 154, 295-302, 2003

Isolation and characterization of small RNAs involved in virus-host developement in hyperthermophilic archaea

Archaeal viruses exhibit unusual morphologies (Fig. 1) and genomic contents, suggestive of novel mechanisms involved in the life cycles of the viruses and their host interactions. As part of a major project, this bachelor/Master student project is to isolate and characterize small non-protein-coding RNAs which are completely novel and are involved in interactions between the virus SIRV2 and its hyperthermophilic host Sulfolobus.

Electron micrographs of archaeal viruses with highly diverse and exceptional morphologies, all but the rod shape have never been observed in the other two domains of life, Bacteria and Eukarya.

Work plan

  1. Infect Sulfolobus cells with virus SIRV2 and grow it at 80oC, pH 3.
  2. Extract total RNA from SIRV2-infected Sulfolobus cultures.
  3. Isolate small RNAs from gels and reverse transcribe them into cDNA. Then clone them into a vector to obtain a cDNA library.
  4. Sequenicng the cDNA library and identify the RNAs using bioinformatic tools.
  5. Verify the cDNA sequencing results by RT-PCR and Northern hybridization.

Contact: Xu Peng

References

  1. Basta T, Smyth J, Forterre P, Prangishvili D. Peng X. 2008. A novel conjugative plasmid pAH1 and its interactions with lipothrixvirus AFV1. Mol Microbiol. In press
  2. Peng X, Kessler A, Phan H, Garrett RA, Prangishvili D. 2004. Multiple variants of the archaeal DNA rudivirus SIRV1 in a single host and a novel mechanism of genomic variation. Mol Microbiol. 54:366-75.
  3. Peng X, Basta T, Häring M, Garrett RA, Prangishvili D. 2007. Genome of the Acidianus bottle-shaped virus and insights into the replication and packaging mechanisms. Virology 364:237-43.

CRISPR– a newly discovered antivirus and/or virus-host regulating system in Bacteria and Archaea

Recently, a novel family of repeats, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), were discovered in almost all archaeal genomes including hyperthermophilic Sulfolobus and in about 40% of sequenced bacterial genomes. CRISPRs are composed of short direct repeats (24 – 47 bp) that are interspaced by non-repetitive spacer sequences (26 – 72 bp). The number of repeats per cluster varies from 2 to 250 and genomes can have 1 to 18 CRISPR loci. Since the spacer sequences were often found to match short genomic regions of viruses and plasmids, it was proposed that CRISPR spacers mediate immunity against infection by extrachromosomal elements. Experimental evidence in support of this was recently provided in two bacterial systems (1,2). However, in archaea the CRISPR system appears to be much more complex than in bacteria and it has been suggested that it may play a more regulatory role in archaea (3).

The crenarchaeote Sulfolobus solfataricus is an aerobic irregular coccus that grows optimally around 80ºC and pH 2-4. In the genome of S. solfataricus, there are multiple CRISPRs at different genomic loci comprising over 200 repeats with a 25 nt repeat unit and 36-39 nt spacer sequences. We are investigating the functions/mechanisms of CRISPRs in Archaea using Sulfolobus as a model organims.

CRISPR system for archaeal and bacterial viral defense. CRISPR RNA is transcribed from the conserved flanking sequence (a) and processed by a Dicer enzyme (pDicer) (b) to generate small RNAs corresponding in size and sequence.

Biochemical and molecular biology techniques are used to study the expression of CRISPRs, and genetic systems are employed to study the in vivo function (4, 5). We have a few PhD and Master’s students working on the complex system and a new student can easily be integrated into the group with an exciting project.

Contact: Xu Peng

References

  1. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 315:1709-12.
  2. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. 2008. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 321:960-4.
  3. Prangishvili D, Forterre P, Garrett RA. 2006. Viruses of the Archaea: a unifying view. Nat Rev Microbiol. 4:837-48.
  4. Lillestøl RK, Redder P, Garrett RA, Brügger K. 2006. A putative viral defence mechanism in archaeal cells. Archaea. 2(1):59-72.
  5. Peng X, Brugger K, Shen B, Chen L, She Q, Garrett RA. 2003 Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes. J Bacteriol. 185(8):2410-7

Novel protein-RNA functional systems of hyperthermophilic archaeal virus PSV

Parobaculum Spherical Virus (PSV) can propagate in the hyperthermophilic archaea Parobaculum and Thermoproteus growing at 85oC.

The genome of 28 kbp encodes 48 putative proteins, very few with known function. Recent X-ray crystallographic and bioinfomatic studies in collaboration between our laboratory, St Andrews University, Scotland and the Pasteur Institute, Paris have revealed 4 RNA-binding proteins and 2 putative RNAs (Figs. 1 and 2).

Putative secondary structures of RNAs 1 and 2.

Hypothesis

Our working hypothesis is that the 4 proteins and 2 RNAs represent two independent and novel functional systems involved in viral gene expression/replication and possibly in anti-host defence.

Experiments

  1. RT-PCR and Northern blot to study the in vivo expression of RNAs 1 and 2.
  2. Gel mobility shift assay to test RNA-protein interactions.
  3. Protein pull-down assays to reveal cellular/viral components interacting with the proteins.

Aim
The aim is to determine the structural and functional roles of the completely novel protein-RNA viral systems.

Contact: Xu Peng

Genome sequencing and analysis of novel archaeal viruses

Several novel viruses have been discovered over the past few years which have quite exceptional morphotypes and genomes and they have been classified into seven new viral families. Many of the viruses were characterised in collaboration with Dr. David Prangishvili's laboratory at the Pasteur Institute, Paris (Prangishvili & Garrett, 2004, 2005). The project involves isolating very small amounts of viral DNA from a new virus, amplifying the whole genomic DNA, clone library construction, sequencing the genome, analysing the sequence and annotating the genes.

Contact: Roger Garrett, Xu Peng

Archaeal virus replication and packaging

Genomic analyses of a few archaeal viruses have yielded limited insights into their different viral replication mechanisms and into possible mechanisms of packaging (Peng et al., 2001; Bettstetter et al., 2003; Häring et al., 2004; Häring et al., 2005; Vestergaard et al., 2005). These recent results, obtained in collaboration with Dr. David Prangishvili's laboratory at the Pasteur Institute, Paris, suggest a few student projects which would involve testing these putative mechanisms experimentally. Each project will involve learning basic nucleic acid and protein biochemical techniques, including cloning and expression, PCR methods, RNA expression, working with virus-host systems at high temperatures, as well as the bioinformatics of viral genomes.

Contact: Roger Garrett, Xu Peng

A special mechanism of genomic change in an Archaeal Rudivirus

The genome of one of the archaeal rudiviruses (SIRV1) undergoes rapid rearrangement in a foreign Sulfolobus host. One of the major mechanisms involves the insertion/deletion of 12 bp elements which appear, to an unquantified degree, to be mobile in the genome (Peng et al., 2004). The project involves determining the nature, and the mechanism of mobility, of these unusual elements which profoundly affect both the genome structure and the individual genes of the virus. The experiments will require the application of various biochemical methods and the student will learn to work with RNA and DNA techniques as well as methods for studying protein- nucleic acid interactions.

Contact: Xu Peng