For the first time, researchers are able to look at the need for every gene in a bacterial cell in a single experiment. The new method will transform the study of gene activity and the search for weaknesses in bacterial armouries.
Using a newly developed, next-gen sequencing method, a team established which genes Salmonella Typhi needs to survive and which are more of a luxury. The results and the method will be a boon to scientists tackling bacterial disease, allowing them to capitalize on the abundance of genomic sequence data from next-generation sequencing technologies.
Every year 22 million people are infected and 220,000 die from infection with S. Typhi. It is a special threat in the developing world, in areas with poor sanitation or a lack of clean drinking water.
The team were able to look at almost all the genes in S. Typhi and showed that it needs only 356 genes for survival: 4162 genes were not essential. Knowing which genes are essential to the survival of pathogens, researchers can seek treatments to target those genes.
"We developed a new method that is ten times more powerful than any previous technique," says Sanger Institute graduate student Gemma Langridge, one of the first authors on the paper. "By combining transposon-induced mutagenesis – a method whereby small chunks of cut-and-paste DNA sequence are inserted into the genome effectively disabling individual genes – and high-throughput sequencing, we have been able to determine which genes are essential for the survival of S. Typhi and which are non-essential."
"Crucially, our new method allows us to achieve all this in just a single experiment."
Using the novel method, which the team have named TraDIS (Transposon Directed Insertion site Sequencing), they inserted transposons into the S. Typhi genome to generate more than one million mutants. They then grew the bacteria and used next-generation sequencing to directly identify 370,000 insertion sites in the S. Typhi genome – an average of more than 80 insertion sites per gene. Previous methods produce only a few mutations per gene.
If a transposon inserts into an essential gene, the gene is silenced and that mutant cell will not grow and it – and the transposon insert – will be absent from the mutant pool. By sequencing DNA from the entire pool – approximately 1 million mutants in total – the team were able to identify genes in which no transposon insertions had been detected.
In a single experiment using the TraDIS method, the team were able to determine whether or not 99.6% of the S. Typhi genes are essential to its survival.
"Sequencing centres such as ours can produce vast amounts of genomic data at a pace unimaginable just a few years ago," explains Professor Julian Parkhill, Director of Sequencing and head of Pathogen Genomics at the Sanger Institute. "One of our aims is to develop high-throughput research methods that can exploit this explosion of genetic data, to ensure these resources can be used effectively. We can now discover which of all the genes in an organism are essential to its survival or required for growth under special conditions, such as infection. Our new TraDIS method will make a dramatic difference to the ability to carry out such genome-wide research."
Importantly, the team applied the method to a clinical problem by looking at how S. Typhi might survive in humans. Typhoid can be spread by carriers who, without showing symptoms, act as reservoirs, storing the bacterium in the gallbladder and passing it to others. The most famous such carrier was Typhoid Mary, who worked in the food industry in the US and spread typhoid fever without exhibiting any symptoms herself.
But, bacteria cannot survive in the fairly hostile environment of the gall bladder unless they are tolerant to bile – the fatty fluid secreted by the gall bladder. Looking at genes involved in bile resistance, allows us to see which genes are essential for helping S. Typhi persist in a carrier.
"We grew the bacteria in ox bile to pick out genes required for bile tolerance," says Keith Turner, Sanger Institute investigator and a senior author on the paper. "We found 169 genes involved in bile tolerance – many of these had not been suspected before and more than 30 are genes not characterized at all.
"Using TraDIS, we have highlighted several possible new targets for treatment that would pick on S. Typhi's need to survive in the gall bladder."
For the first time, it is possible to paint a comprehensive picture of essential, advantageous or burdensome genes in many phases of the bacterial life cycle, to determine functions necessary to support them throughout their entire disease cycle. Such a picture is important for discovery of new targets for treatment.
This elegant new method exemplifies how high-throughput research allows scientists to determine systematically the function of or requirement for individual genes in a single experiment, opening the door for similar analyses of other pathogenic genomes in the future.Publication Details
Published online before print at doi: 10.1101/gr.097097.109
This work was supported by the Wellcome Trust.
Participating CentresThe Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK
Laboratory of Gastrointestinal Pathogens, Centre for Infections, Health Protection Agency, Colindale, London, UK
The Wellcome Trust Sanger Institute, which receives the majority of its funding from the Wellcome Trust, was founded in 1992. The Institute is responsible for the completion of the sequence of approximately one-third of the human genome as well as genomes of model organisms and more than 90 pathogen genomes. In October 2006, new funding was awarded by the Wellcome Trust to exploit the wealth of genome data now available to answer important questions about health and disease. http://www.sanger.ac.uk
The Wellcome Trust is the largest charity in the UK. It funds innovative biomedical research, in the UK and internationally, spending over £600 million each year to support the brightest scientists with the best ideas. The Wellcome Trust supports public debate about biomedical research and its impact on health and wellbeing. http://www.wellcome.ac.uk
Don Powell | EurekAlert!
A Map of the Cell’s Power Station
18.08.2017 | Albert-Ludwigs-Universität Freiburg im Breisgau
On the way to developing a new active ingredient against chronic infections
18.08.2017 | Deutsches Zentrum für Infektionsforschung
Whether you call it effervescent, fizzy, or sparkling, carbonated water is making a comeback as a beverage. Aside from quenching thirst, researchers at the University of Illinois at Urbana-Champaign have discovered a new use for these "bubbly" concoctions that will have major impact on the manufacturer of the world's thinnest, flattest, and one most useful materials -- graphene.
As graphene's popularity grows as an advanced "wonder" material, the speed and quality at which it can be manufactured will be paramount. With that in mind,...
Physicists at the University of Bonn have managed to create optical hollows and more complex patterns into which the light of a Bose-Einstein condensate flows. The creation of such highly low-loss structures for light is a prerequisite for complex light circuits, such as for quantum information processing for a new generation of computers. The researchers are now presenting their results in the journal Nature Photonics.
Light particles (photons) occur as tiny, indivisible portions. Many thousands of these light portions can be merged to form a single super-photon if they are...
For the first time, scientists have shown that circular RNA is linked to brain function. When a RNA molecule called Cdr1as was deleted from the genome of mice, the animals had problems filtering out unnecessary information – like patients suffering from neuropsychiatric disorders.
While hundreds of circular RNAs (circRNAs) are abundant in mammalian brains, one big question has remained unanswered: What are they actually good for? In the...
An experimental small satellite has successfully collected and delivered data on a key measurement for predicting changes in Earth's climate.
The Radiometer Assessment using Vertically Aligned Nanotubes (RAVAN) CubeSat was launched into low-Earth orbit on Nov. 11, 2016, in order to test new...
A study led by scientists of the Max Planck Institute for the Structure and Dynamics of Matter (MPSD) at the Center for Free-Electron Laser Science in Hamburg presents evidence of the coexistence of superconductivity and “charge-density-waves” in compounds of the poorly-studied family of bismuthates. This observation opens up new perspectives for a deeper understanding of the phenomenon of high-temperature superconductivity, a topic which is at the core of condensed matter research since more than 30 years. The paper by Nicoletti et al has been published in the PNAS.
Since the beginning of the 20th century, superconductivity had been observed in some metals at temperatures only a few degrees above the absolute zero (minus...
16.08.2017 | Event News
04.08.2017 | Event News
26.07.2017 | Event News
18.08.2017 | Life Sciences
18.08.2017 | Physics and Astronomy
18.08.2017 | Materials Sciences