Flu virus finally sequenced in its native form

The genome of the flu virus has been fully sequenced in its native RNA form for the first time. Previously, all influenza genomes — as well as those of other viruses that store their genetic material as RNA — had been determined by copying the molecule into DNA. The native flu genome was generated using ‘nanopore’ sequencing technology, which reads RNA strands as they stream through a tiny molecular channel.

Virus de la gripe

RNA is chemically similar to its better known cousin, DNA. In cellular organisms, it serves as an intermediary between DNA-encoded genes and proteins, and performs other tasks in the cell. But many viruses — including those behind polio, Ebola and the common cold — store their genetic information as RNA, rather than DNA. Barnes, head of the CDC’s influenza-genomics team, says that no one had sequenced the virus’s RNA genome before because it seemed nearly impossible. Previous methods for sequencing native RNA strands involved degrading one chemical base, or letter, at a time, and these techniques have changed little since their invention in the late 1970s². To compensate, nearly all ‘RNA sequencing’ instead uses a viral enzyme called reverse transcriptase, which copies RNA into sequencer-friendly DNA strands.

Tiny is mighty

Nanopores offer a simpler way of sequencing actual RNA molecules, such as viral genomes. The technology is based on applying electrical current across a nanoscale molecular pore, and then measuring tell-tale current fluctuations as genetic material snakes through.

Fast track: nanopore sequencing identifies individual bases as a strand of DNA is passed through a pore.

In January, researchers at a leading developer of the technology, UK-based Oxford Nanopore Technologies, directly sequenced RNA using a chocolate-bar-sized device called the MinION³. That effort looked at transcripts of messenger RNA, the family of RNA molecules that conveys information from DNA to build proteins.

Oxford Nanopore

Oxford Nanopore's MinION sequencer can read DNA fragments up to 10 kilobases long

Barnes’s team applied this method to the genome of influenza A, which is roughly 13,500 RNA letters long and composed of eight segments. Barnes says his team’s approach isn’t ready for prime time. It required a lot of flu virus, and to iron out inevitable sequencing errors, the raw data had to be processed many times over. But nanopore technology is advancing quickly, and Barnes hopes that with further improvements, direct sequencing of flu and other RNA viruses will become routine.

At the top of his and other scientists’ wish lists are methods for identifying chemical modifications to RNA. More than 100 have been identified so far, but researchers have little idea what most of them do, in large part because it has been impossible to study them systematically. The Oxford Nanopore team was able to detect two common RNA modifications, or tags. Birney, who is a paid consultant to the company, expects that the technology will be able to find many more once machine-learning algorithms are used to unpick the tags’ signatures.

Sequencing modified bases of RNA would be “a big deal” for the field, says Bryan Cullen, a virologist at Duke University in Durham, North Carolina. His team found last year⁴ that a tag called m⁶A seems to alter the expression of influenza genes during infection in mice, which promotes viral replication. Current methods for detecting such modifications are time-consuming and expensive, he adds. Stacy Horner, co-director of Duke’s Center for RNA Biology, says that nanopore sequencing could also reveal hidden diversity in RNA viruses, which is lost with other methods that cobble together much shorter stretches of genetic material to generate a genome.

Although the methods aren’t yet perfect, Birney says, biologists are still excited about the possibility of soon being able to sequence entire viral genomes and other RNA molecules in their natural forms. “Suddenly, we’ve got the technology to do this. It’s kind of amazing.”

Nature 556, 420 (2018)

doi: 10.1038/d41586-018-04908-5

Blending genomes: Distributive Conjugal Transfer in Mycobacteria, a sexier form of HGT

This review discusses a novel form of horizontal gene transfer (HGT) found in mycobacteria called Distributive Conjugal Transfer (DCT). While satisfying the criteria for conjugation, DCT occurs by a mechanism so distinct from oriT‐mediated conjugation that it could be considered a fourth category of HGT. DCT involves the transfer of chromosomal DNA between mycobacteria and, most significantly, generates transconjugants with mosaic genomes of the parental strains. Multiple segments of donor chromosomal DNA can be co‐transferred regardless of their location or the genetic selection and, as a result, the transconjugant genome contains many donor‐derived segments; hence the name DCT. This distinguishing feature of DCT separates it from the other known mechanisms of HGT, which generally result in the introduction of a single, defined segment of DNA into the recipient chromosome. Moreover, these mosaic progeny are generated from a single conjugal event, which provides enormous capacity for rapid adaptation and evolution, again distinguishing it from the three classical modes of HGT. Unsurprisingly, the unusual mosaic products of DCT are generated by a conjugal mechanism that is also unusual. Here, we will describe the unique features of DCT and contrast those to other mechanisms of HGT, both from a mechanistic and an evolutionary perspective. Our focus will be on transfer of chromosomal DNA, as opposed to plasmid mobilization, because DCT mediates transfer of chromosomal DNA and is a chromosomally encoded process.

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Culture-independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant Gram-positive pathogens

Despite the wide availability of antibiotics, infectious diseases remain a leading cause of death worldwide. In the absence of new therapies, mortality rates due to untreatable infections are predicted to rise more than tenfold by 2050. Natural products (NPs) made by cultured bacteria have been a major source of clinically useful antibiotics. In spite of decades of productivity, the use of bacteria in the search for new antibiotics was largely abandoned due to high rediscovery rates. As only a fraction of bacterial diversity is regularly cultivated in the laboratory and just a fraction of the chemistries encoded by cultured bacteria are detected in fermentation experiments, most bacterial NPs remain hidden in the global microbiome. In an effort to access these hidden NPs, we have developed a culture-independent NP discovery platform that involves sequencing, bioinformatic analysis and heterologous expression of biosynthetic gene clusters captured on DNA extracted from environmental samples. Here, we describe the application of this platform to the discovery of the malacidins, a distinctive class of antibiotics that are commonly encoded in soil microbiomes but have never been reported in culture-based NP discovery efforts. The malacidins are active against multidrug-resistant pathogens, sterilize methicillin-resistant Staphylococcus aureus skin infections in an animal wound model and did not select for resistance under our laboratory conditions.








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