N E W S L E T T E R O F F E M S F E D E R AT I O N O F E U RO P E A N M I C RO B I O L O G I C A L S O C I E T I E S

The limelight for this issue of FEMS Focus falls on a topic which currently generates enormous volumes of information and pushes science to immeasurable dimensions – genomics fuelling post-genomics such as RNomics. The rapid emergence of these new disciplines has provided researchers with a new looking-glass for scientists and non-scientists, microbiologists and non-microbiologists alike. Thereby, it is also providing a lot of new input, energizing the fields of life, earth and environmental sciences. With the boom of the genomics era, the decoding of complete human and microbial genome sequences is made pos-sible. Suddenly, man and microbes are spelled differently, with As, Ts, Gs and Cs. With that come endless discoveries – dis-eases illustrated in detail, probable solu-tions and new promise for the future. The race is on to sequence the genome of every living organism on the planet, to under-stand them better and provide answers to questions. This is impacting on microbiol-ogy, decoding its vast diversity. Indeed, we have entered the post-genomics era. In this new era, every minute counts.

Tone Tønjum, Editor & Chared Verschuur,

Communications Assistant

ased on her experience in the field and recent RNA-based work, Dr. Cossart and her co-workers ex-

plored the role of RNomics with focus on the bacterium Listeria monocytogenes. This bacterium has recently emerged as a multifaceted model in pathogenesis and is ubiquitous in the environment. It can lead to severe foodborne infections exceeding the fatality rates of Salmonel-la. How this bacterium switches from a saprophyte to a pathogen is largely unknown. By using tiling arrays and RNAs from wild-type and mutant bac-teria grown in vitro, ex vivo and in vivo in animal models, Cossart has analysed the entire Listeria transcriptome. What is your most important finding in Listeria?We have provided the complete Liste-ria operon map and have discovered far more diverse types of RNAs than ex-pected: in addition to 50 small RNAs, at least two of which are involved in vir-ulence in mice, we have identified anti-sense RNAs covering several open-read-ing frames and long overlapping 5’ and 3’ untranslated regions (UTRs). Long transcripts of 1-5 kb were discovered opposite of operons. These transcripts which had not been “annotated” in the Listeria genome may be remnants of genes or play other roles which should be analyzed.We have also discovered that riboswitch-es can act as terminators for upstream genes. Interestingly, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same

September 2009 No. 5

Genomics. Post- genomics. Hot topics in contemporary research contribut-ing vast amount of data and opening new avenues for science. In order to address a prime example of the role of genomics and post- genomics in contemporary science, FEMS Focus interviewed a major personality in the field – Professor Pascale Cossart. She is the head of the Bacteria-Cell Interac-tions Unit of the Pasteur Institute in Paris, France. Read on for her views on genomics and post-genomics with emphasis on RNomics/transcriptomics, on the connection between these fields and microbiology, and the challenges that these novel opportunities facilitate.

Genomics and RNomics: Taking the code further

From the Editorial Team

expression patterns as the virulence genes. Taken together, our data unravel successive and coordinated global tran-scriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

Prof. Pascale Cossart (with permission)

Professor Dr. Pascale Cossart heads the Bacterial-Cell Interactions Unit at the Institut Pasteur in Paris, France. Her research focuses on the food pathogen Listeria monocytogenes and employs molecular and cellular biology, RNomics, bioinformatics and animal models to sort out patho-genesis. Dr. Cossart received her Ph.D. in Biochemistry from the University of Paris in 1977. Her postdoctoral research was conducted at the Institut Pasteur. In 1998, she received the Richard Lounsberry Prize and the L’Oréal/UNESCO Award for Women in Science Leadership. In year 2000, the Swedish Society of Medicine awarded her the 2000 Louis Pasteur Gold Medal. Dr. Cossart is an “Officier de la Légion d’honneur” of the French Legion of Honor, and a member of the French Academie des Sciences and the German Leopoldina. She received an Advanced Investigator ERC grant in 2008, is a member of EMBO and just this year, became a member of the National Academy of Sciences in the US.

What is characteristic of the interface between RNomics and microbiology?The field of RNomics, or transcriptomics, is exploding, demanding a lot of bioin-formatics power in order to handle all the data. Actually, it’s time for transcriptomics in microbiology. The diversity of the field and the unique traits of all the microbial entities to be unraveled make this a most timely challenge. A significant opportu-nity in this context is to combine RNom-ics/transcriptomics with deep sequencing in order to sort out the nature 3’/5’ UTR markings. What are the future impacts of RNomics on microbiology and beyond?RNomics will clearly open new avenues in microbiology. In this context, it is im-portant to realize that the RNAs/sDNAs we currently put on the tiling arrays is an average of the population under investiga-tion. Each bacterium might have a differ-ent transcriptome than its neighbour, and only the differential expression profile might sort out what is really significant. We need to get to the level of single cell analysis in order to sort out the gene ex-pression levels of the diversity of a popu-lation. In medicine, RNomics has identified and characterized disease-associated non-protein coding RNAs for applications as diagnostic markers and therapeutic tar-gets, and experimental and bioinformat-ics methods have been developed for this task. Meta-transcriptomics analyses show that these data sets can reveal new infor-mation about the diversity, taxonomic distribution and abundance of sRNAs in naturally occurring microbial com-munities, and indicate their involvement in environmentally relevant processes in-cluding carbon metabolism and nutrient acquisition.

arge-scale sequencing aims at sequenc-ing very long DNA pieces, such as whole chromosomes. Common approaches

consist of cutting or shearing large DNA fragments into shorter fragments. The frag-mented DNA is cloned into a DNA vector, and amplified in Escherichia coli. Short DNA fragments purified from individual bacte-rial colonies are individually sequenced and assembled electronically into one long, con-tiguous sequence. This method does not re-quire any pre-existing information about the sequence of the DNA and is referred to as de novo sequencing. Gaps in the assembled se-quence may be filled by primer walking. The different strategies have different tradeoffs in speed and accuracy; shotgun methods are of-ten used for sequencing large genomes, but its assembly is complex and difficult, particu-larly with sequence repeats often causing gaps in genome assembly.

Next generation sequencing methods

High-throughput sequencingThe high demand for low-cost sequencing has driven the development of high-through-put sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at once. All the new systems aim to avoid cloning of DNA fragments by employing different forms of parallel amplification and various forms of solid-phase approaches for signal detection. High-throughput (HTP) sequencing tech-nologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods. In vitro clonal amplificationMolecular detection methods are not yet sen-sitive enough for single molecule sequencing, so most approaches use an in vitro cloning step to amplify individual DNA molecules. Emulsion PCR isolates individual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase. Polymerase chain reaction (PCR) then coats each bead with clonal copies of the DNA molecule followed by immobilization for later sequencing. Another method for in vitro clonal amplification is bridge PCR, where fragments are amplified upon primers

attached to a solid surface. The single-mol-ecule method developed by Stephen Quake’s laboratory (later commercialized by Helicos) skips this amplification step, directly fixing DNA molecules to a surface.The revolutionizing aspect of the next gen-eration technologies compared to the original Sanger method for sequencing, is that ampli-fication of the single-stranded DNA gener-ated does not occur by cloning. This is ad-vantageous, since not all DNA fragments do not all have equal cloning efficiency. Instead, amplification is based on emulsion-PCR. The DNA molecules to be sequenced are bound to monodisperse and hydrophilic beads and amplified in a water- and oil-mixture, where the beads are located in the water drops.

Genomics facilitating post-genomicsIn recent years, major technological developments have enabled large-scale sequencing of complete genomes and other large-scale sequencing projects to be performed quickly outside sequencing factories and to a reasonable price. However, current methods can directly sequence only relatively short DNA fragments in a single reaction. The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucle-otide. As of July 2009, 934 microbial genome sequences are completed, while 1870 are in progress (source: http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi).

Source: wikipedia

DNA Translocation, the complexity of post-genomics (image by Tremani)

The amounts of raw sequence data that HTP Genome Sequencers generate are vast, with over 100 million bases per instrument per run.

In addition, the reads are short, represent-ing new challenges in bioinformatics. For HTP genomics studies, this presents a very

large data set with several challenges that need to be addressed. In fact, bioinformatics analysis represents

the new bottle-neck to unleash the optimal output of next-genera-tion technologies.

Future developments / Demands and incentives

driving technologyurrent demands in HTP sequencing are whipping technology forward. The four major HTP sequence technology plat-

forms available today are 454, Solexa/Illumina, ABI (SOLiD) and Helicos. Among these, 454 yields the longer sequence reads. On the other hand, the latter platforms generate a greater abundance of fragments read, making them ideal for re-sequencing/deep-sequencing, how-ever, less useful for de novo sequencing. In addi-tion, stakeholder are offering incentives to fuel technology development. By the next few years, a next-next generation platform is expected to be invented, based on single-molecule-reads (without amplification of DNA). This is the way to go to achieve the ultimate goal: to be able to sequence the human genome in a couple of days for 1000 Euro. If the “1000 Euro”-ge-nome will be reality, this will represent a major change in genome-based medicine, for instance, the mutation rate in various somatic tissues and bacteria can be monitored any time.

Parallelized sequencingDNA molecules are physically bound to a surface, and sequenced in parallel. Sequenc-ing by synthesis, like dye-termination electro-phoretic sequencing, uses a DNA polymerase to determine the base sequence. Reversible terminator methods (used by Illumina and Helicos) use reversible versions of dye-termi-nators, adding one nucleotide at a time, de-

Some typical professional concepts and terms in the wake of HTP genomics are:

Transcriptomics: whole cell or tissue gene expression measurements by DNA microarrays or serial analysis of gene expression

RNomics: defining the complete transcriptome (all gene transcripts) of a cell or cell population

Proteomics: complete identification of proteins and protein expression patterns of a cell or tissue through two-dimensional gel electrophoresis and mass spectrometry or multi-dimen-sional protein identification techniques

Metabolomics: identification and measurement of all small-molecules metabolites within a cell or tissue

Glycomics: identification of the entirety of all carbohydrates in a cell or tissue.

The “-omics” of post-genomics

454 Life Sciences, a Roche biotechnology company, is a

company specializing in high-throughput DNA sequencing

using a novel massively parallel sequencing-by-synthesis

approach. 454 has experienced rapid growth since its acquisi-

tion by Roche Diagnostics and release of the GS20 sequencing

machine in 2005, the first next-generation DNA sequencer on

the market. The Genome Sequencer FLX instrument was re-

leased in 2007. In 2008, 454 Sequencing launched the GS FLX

Titanium series reagents for use on the current instrument,

with the ability to sequence 400-600 million base pairs with

400-500 base pair read lengths. With its high accuracy, low

cost, and long reads, many researchers have migrated away

from traditional Sanger capillary sequencing instruments and

toward the 454 Sequencing platform for a variety of genome

projects.

454 Life sciences was founded by Jonathan Rothberg, and the

underlying technology is based on pyrosequencing and was

conceived while he was on paternity leave and wanted a way

to sequence the genome of his new born son who had been

placed in new born intensive care.

Genomics in industry – the example of pyrosequencing

1953 Discovery of the structure of the DNA double helix by Watson and Crick1972 Development of recombinant DNA technology, which permits isolation of defined fragments of DNA; prior to this, the only accessible samples for sequencing were from bacteriophage or virus DNA1975 The first complete DNA genome to be sequenced is that of bacteriophage φX1741977 Allan Maxam and Walter Gilbert publish “DNA sequencing by chemical degradation”. Frederick Sanger, independently, publishes “DNA sequencing by enzymatic synthesis”1980 Frederick Sanger and Walter Gilbert receive the Nobel Prize in Chemistry1984 Medical Research Council scientists decipher the complete DNA sequence of the Epstein-Barr virus, 170 kb1986 Leroy E. Hood’s laboratory at the California Institute of Technology and Smith announce the first semi-automated DNA sequencing machine1987 Applied Biosystems markets first automated sequencing machine, the model ABI 3701990 The U.S. National Institutes of Health (NIH) begins large-scale sequencing trials on Mycoplasma capricolum, Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae1995 Richard Mathies et al. publish dye-based sequencing1995 The first complete bacterial genome sequence on Haemophilus influenzae is published by Fleischmann, et al.2004 Large-scale pyrosequencing is introducedJuly 2009: 2800 bacterial complete genome sequences are available on-line, more or less completely annotated

Major landmarks in DNA sequencing

tect fluorescence at each position in real time, by repeated removal of the blocking group to allow polymerization of another nucleotide. Pyrosequencing (used by 454) also uses DNA polymerization, adding one nucleotide spe-cies at a time and detecting and quantifying the number of nucleotides added to a given location through the light emitted by the re-lease of attached pyrophosphates. Sequencing by ligationThis enzymatic sequencing method uses a DNA ligase to determine the target sequence. Used in the polony method and in the SOLiD technology, it uses a pool of all possible oligo-nucleotides of a fixed length, labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential liga-tion by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Other se-quencing technologies are currently being developed.

Bioinformatics for HTP genomics

The 50S subunit of a bacterial ribosome with 90000 atoms (image: www.ballview.org)

The FEMS Focus is published by the FEMS Central Office. Whom to contact? Prof. Dr. Tone Tønjum (tone.tonjum@medisin.uio.no).Design & production: ilumina@ilumina.siFEMS is a registered charity (no. 1072117) and also a company limited by guarantee (no. 3565643). © 2009 Federation of European Microbiological Societies

FEMS Central OfficeKeverling Buismanweg 4 2628 CL Delft The NetherlandsTel: +31-15-269 3920 Fax: +31-15-269 3921E-mail: fems@fems-microbiology.org

www.fems-microbiology.org

Post-genomics events

Post-genomics funding opportunitiesDistinguished Visiting Scientist Stipend programme, Netherlands Genomics InitiativeClosing date: 1 April 2010Budget: 0,5 million euro

Frontiers of Functional Genomics, European Science Foundation Calls for ProposalsCall for Science Meeting proposals - deadline 25th September 2009 17:00 CETCall for Short Visit and Exchange Grant applications - deadline 25th September 2009 17:00 CET

Post-genomics links and resources

http://www.genomics.nl/http://genomicsnetwork.ac.uk/http://www.genome.gov/http://www.cdc.gov/genomics/http://www.nerc.ac.uk/research/pro-grammes/proteomics/http://www.genomenewsnetwork.org/

FEMS grants deadlines

Meeting Attendance Grants

September 1, 2009

and April 1, 2010

FEMS Advanced Fellowship

October 1, 2009

FEMS Research Fellowship

December 15, 2009

Meeting Grants

March 1 of every year preceding that

in which the meeting takes place.

The FEMS-Jensen Award

is open for applications until

November 30, 2009.

Online subscription to the full set of FEMS journals for only 175 Euro

5th International DNA Sampling Conference16 – 18 September 2009, Alberta, Canada

The Genomics of Common Diseases 2009September 23-26, 2009, Cambridge, UK

Mapping the Genomic EraMeasurements and Meanings, 07 – 09 October 2009, Cardiff, UK

Life Sciences Momentum 200920 October 2009, World Forum, The Hague, The Netherlands

Genome InformaticsOctober 27 - 30, 2009, Cold Spring Harbor, New York

IPG (Integrative Post-Genomics) Lyon’s International Multidisciplinary Meeting on Post-Genomics, 18-20 November 2009, Lyon, France

Gene 20091-7 December 2009, Foshan, China

Register as a FEMS Affiliate!

JOIN US ATJOIN US AT

4th Congress of European Microbiologists

Geneva, SwitzerlandJune 26-30, 2011

Advancing Knowledge on Microbeswww.kenes.com/fems-microbiology

Camejo et al. In vivo transcriptional profiling of Listeria monocytogenes and mutagen-esis identify new virulence factors involved in infection. PLoS Pathogen 5(5):e1000449, 2009.Toledo-Arana et al. The Listeria transcriptional landscape from saprophytism to viru-lence. Nature 459:950-6, 2009.Shi et al. Metatranscriptomics reveals unique microbial small RNAs in the ocean’s water column. Nature 459(7244):266-9, 2009.Liu JM, Livny J, Lawrence MS, Kimball MD, Waldor MK, Camilli A. Experimental dis-covery of sRNAs in Vibrio cholerae by direct cloning, 5S/tRNA depletion and parallel sequencing. Nucleic Acids Res 37:e46, 2009.

Recent publication highlights on microbial RNomics

only 175 Euro

Thematic issue now available