Monthly Archives: July 2012

What Are Fosmids?

I’m starting a new series of short tutorials.  In a selfish way, these posts are for me – a vehicle for me to clarify my own comprehension of a given topic.  I might also tell you about a solution to a problem I have been troubleshooting.

The first post in this series is about fosmids.  There seems to be some public confusion as to what they are or what they can be used for – basically, I have been confused.  Apparently I had forgotten my microbiology coursework along the way.  Many genome sequencing projects – such as the human genome project – have utilized fosmids to create libraries prior to sequencing, but it wasn’t until hearing about JGI’s fungal and metagenomic sequencing initiatives did I hear the term fosmid mentioned frequently.

Fosmids are used when preparing genomic libraries for genome sequencing.  Fosmids are circular DNA of bacterial origin – technically plasmids – but where typical plasmids exist in high copy number (up to 100 copies per cell) and possess small (3 to 6 kb) inserts, fosmids are present as a single copy in a cell and may possess inserts upwards of 40 kb.  Fosmids are advantageous because they produce stable libraries for genome sequencing.  They have a tendency to provide fairly uniform coverage, so they are optimal for closing gaps in whole genome alignments.  In addition to genome sequencing, they have also been used for metagenomics and expression studies.

Fosmids are derived from the fertility plasmid (or F-plasmid) and are responsible for the formation of the sex pilus during bacterial conjugation.  This plasmid contains both origin and partitioning genes derived from the F’-episome and as a result, the plasmid is kept as a single copy clone, which comes in handy during genomic DNA library construction.  Fosmid vectors are derived from random shearing – which yields more uniform coverage when comparing against other library cloning methods.

Cosmids may also be useful for genome sequencing projects, but unlike fosmids, they are multi-copy vectors that are generally present at anywhere from 20-70 copies per cell and this high copy number leads to instability and lost segments of genomic DNA.  This can be an issue for closing gaps in genome alignment, but if you’ve got high sequencing depth and a small genome to sequence, it may not be much of an issue.  Most importantly, with high copy number plasmids, such as cosmids, the chance of recombination increases which can disrupt and rearrange genomic DNA inserts prior to sequencing.

fosmid figure

Lastly, fosmids can be useful for chromosome specific sequencing and as cytological markers for chromosome identification.  The image above — which comes from this paper — shows the identification of chloroplast genome isolation and sequencing from fosmids; a similar technique can be used to isolate and sequence specific chromosomes.  Also, fosmids may be used as cytological markers with in situ hybridization on metaphase karyotypes and sorted using flow cytometric methods.

Upcoming Mycological & Mycorrhizal Meetings 2012

Here’s three upcoming mycological meetings which may be of interest to you:

european nitrogen fixation conference 2012

As part of the 10th European Conference on Nitrogen Fixation held in Munich a satellite meeting called the 1st Molecular Mycorrhiza Meeting will be held over two days: Thursday, September 6th to Friday, September 7th.  The list of speakers is pretty amazing if (like me), mycorrhizae is your thing.  See here for registration and see here for abstract submission.

above & below-ground interactions meeting

The British Mycological Society will be having a meeting for Fungal Interactions on September 3rd to 6th, 2012, in Alicante, Spain.  The sessions look to be diversely balanced and focused on fungal interactions with all types of organisms.  See here for the preliminary meeting program and to register see here.

Finally, if you live in the New England area of North America (or don’t mind the travel) the first regional meeting of mycologists from the state of Massachusetts and the surround area will be held on October, 27th 2012.  It will be aptly named MassMyco.  The meeting will be held at Clark University and hosted by the Hibbett Lab.  I love these small regional meetings, so perhaps I’ll try to make the trek for this one.  Registration is not open yet, but check back soon.

Book Review: Cryptococcus – From Human Pathogen To Model Yeast

I wrote the following book review for the Mycological Society of America‘s Inoculum newsletter and I think the book is a great resource if you study Cryptococcus — so I am reproducing my review here.  You can also find a copy of the review here.

cryptococcus book cover for web

Cryptococcus: From Human Pathogen To Model Yeast. 2010.  Joseph Heitman, Thomas R. Kozel, Kyung J. Kwon-Chung, John R Perfect, and Arturo Casadevall (Eds.).  ASM Press, Washington, DC.

The yeast-forming basidiomycete genus, Cryptococcus, has emerged as a significant model for both fungal genetics and pathogenicity.  A long history of research compounded with numerous laboratory resources, as well as two sequenced genomes, have yielded a great deal of information on this enigmatic fungus.  The new book Cryptococcus: From Human Pathogen To Model Yeast, edited by Heitman, Kozel, Kwon-Chung, Perfect, and Casadevall, features contributions from 123 authors and summarizes a vast amount of data as well as synthesizes disparate concepts on the biology of Cryptococcus.  If you consider Casadevall & Perfect’s 1998 tome Cryptococcus neoformans as the groundwork for this book, then these 646 pages are evidence for the explosive advance of knowledge on Cryptococcus that has accrued over the last 12 years.

Cryptococcus species, arguably the most important fungal pathogen of mammals, are common in immuno-compromised hosts; HIV-associated cryptococcosis alone infects more than 1 million people per year.  For example, Cryptococcus has been laboratory confirmed in Sub-Saharan African countries to be responsible for anywhere from 10 to 70% of fatal meningitis cases over the last two decades.  A well-publicized outbreak of a particularly virulent strain of C. gattii was determined to be the causative agent of more than 200 cases of human meningitis in non-immuno compromised individuals within the Pacific Northwest over the last decade.  A concerted global consortium of medical mycology researchers ­ the majority of whom are authors of chapters in this book ­have provided the foundation for establishing Cryptococcus as the model system for understanding fungal pathogenesis in both a medical and veterinary setting.

Species of Cryptococcus entered my personal radar when they kept turning up in plant-associated environmental samples.  Wanting to get up to speed with natural history, population genetics, and methods for typing Cryptococcal diversity, this book was an obvious entry point for me.  Chapters here are dedicated to identification from environmental niches – such as the description of avian- or plant-associated vectors – as well as population biology to phylogeography, and species complexes to hybridization.

Copiously illustrated throughout, notable figures include those documenting Cryptococcus morphology, cell and molecular biological networks, secondary metabolite chemistry, and gene and genome structure.  Chapters devoted to phylogeography and species complexes have detailed phylogenetic trees and distribution maps.  Additionally, this wouldn’t be a clinical textbook if it didn’t include a series of color and monochrome plates of human and animal infections that remind you why you have – or haven’t – studied medical mycology.

Mycologists aren’t the only ones who will find this resource useful.  Geared toward a wide array of specialists, this book is equally applicable to the interests of clinicians and physicians, microbiologists and immunologists, disease ecologists and epidemiologists, and, to a lesser extent, public health and policy administrators.  The book succeeds in connecting and interpreting basic research science and applying this knowledge in a clinical context.

The book consists of a whopping 44 chapters separated into seven sections.  These sections are devoted to general biology; genetics and genomics; virulence; environmental interactions and population biology; immune host responses; pathogenesis; and diagnosis, treatment, and prevention.  Each of the sections consist of five to eight chapters and each informative chapter stands on its own – concise enough to allow for discrete chunks of reading without overwhelming the reader.  In fact, I would argue that the book’s greatest strength is cohesive breadth blended with factual depth.  My only criticism ­ and this is an extremely minor one ­ is that the book as a whole is slightly overwhelming in scope.  This by no means indicates a lack of vision from the authors or editors, but reflects their desire to take into consideration the complete state of knowledge relating to Cryptococcus and its biology.  As a result, the contributors have not only provided a truly fascinating and utterly comprehensive collection of everything Cryptococcus, but have set the bar high for the best treatise on fungal biology at the genus level.  I would consider this book essential for anyone working directly with Cryptococcus ­ or wanting to get up to speed ­ and for mycologists looking for a framework to fully grasp the biology of an important model fungus.

ICOM7 – The 7th International Conference on Mycorrhiza, January 2013

ICOM7

Perhaps because I study mycorrhizae the ICOM meetings have a special place in my heart, so I’m excited to tell you that the next ICOM — the 7th International Conference on Mycorrhiza (ICOM7) — is open for registration.  The meeting will be held in New Dehli in January of 2013.  Here is the call for abstracts.  Here’s some more information from the meeting website:

The Organizing Committee cordially invites you to the 7th International Conference on Mycorrhiza (ICOM7) to be held from 6th to 11th January’ 2013 in New Delhi, the capital Republic of India. Organized by TERI under the auspices of the International Mycorrhiza Society and in collaboration with the Mycorrhiza Network, this 6 day gala event would bring the ICOM legacy to Asia for the first time.

The theme of this conference, “Mycorrhiza for all – An Under Earth Revolution” is wisely chosen so that it may prove to be the epicenter of a new revolution that our planet is in dire need of. A change that would help minimise the usage of chemical fertilizer on soil and hence leave the least environmental footprint.

Aboveground-Belowground Interactions Meeting, London, October 2012

This meeting, “Aboveground-belowground interactions: technologies and new approaches” looks to be an interesting one.  The meeting is co-sponsored by three British organizations — the British Ecological Society, the Biochemical Society, and the Society for Experimental biology — and will be held at Charles Darwin House, London, from October 8th to 10th, 2012.

Here’s more information from the meeting website:

The conference aims to identify the specific skills that biochemists, molecular biologists, physiologists and ecologists can bring to cross-disciplinary research in this area, thus creating new opportunities for collaboration in above-belowground research. The conference will introduce examples where cross-discipline and across-scale research has successfully examined the mechanistic basis of multi-trophic interactions, and will aim to identify the potential for applying such approaches to above–belowground systems, which are traditionally more intractable.  The following three key questions will be addressed:

• What are the major challenges in above–belowground research?
• What tools and approaches can be adopted from different disciplines to address these challenges?
• How can high-throughput tools aid integration across disciplines and scales to facilitate a unified approach to studies of above–belowground systems?

Genetics Of Fagaceae & Nothofagaceae Meeting, October 2012

The IUFRO (International Union of Forest Research Organizations) working group has organized the “Genetics of Fagaceae & Nothofagaceae” meeting to be held in Bordeaux, France from October 9th to 12th, 2012.

This meeting will build upon a successful “Genomics of Forest and Ecosystem Health in the Fagaceae (Beech Family)” meeting held in North Carolina Research Triangle Park in 2009.

The aim of this international conference is to present new scientific findings in the area of genetics and genomics of species within the Fagaceae and Nothofagaceae.

Registration is open.

Carbohydrate binding gene family expansion in the amphibian pathogen Batrachochytrium dendrobatidis

You’d have to be living under a rock – as some amphibians do – to not be aware of the massive extinction facing our vertebrate friends living within aquatic habitats.  Researchers still don’t fully understand what is causing the amphibian mass-extinction – stress from habitat loss, increased chemical concentrations in the environment, and an auto-immune degrading infection have all been proposed.  What is known is that the chytrid fungus Batrachochytrium dendrobatidis – opportunistic or not – is infecting and killing a large number of amphibians.

What is not fully understood about B. dendrobatidis is its pathogenicity and what mechanisms it employs to cause infection.  A recent paper, “Species-Specific Chitin-Binding Module 18 Expansion in the Amphibian Pathogen Batrachochyrium dendrobatidis”, published in the mBio journal by John Abramyam & Jason Stajich at UC Riverside, begins to address this pathogenicity.  As the authors point out – more than 100,000 species of fungi have been described to date and very few of them are pathogenic.  This means that the ability to be pathogenic is derived from somewhere: genome expansion events, gene family duplication and diversification events – and we’re only starting to understand horizontal gene transfer events in fungi. This paper addresses the expansion of a gene family across two B. dendrobatidis genomes that are associated with pathogenicity.

When comparing the genomes of B. dendrobatidis with the genomes from other chytrid fungi there has been an expansion of genes within the family Carbohydrate-Binding Module Family 18 (CBM18).  The CBM18 family is a large group of proteins that have been implicated in other fungal pathogenic infections on both plants and animals.  The authors here question whether this interesting lineage specific expansion of CBM18 in B. dendrobatidis could be associated with the virulence of its pathogenicity on amphibians.

The authors used the CBM18 protein family domain HMM to search across the B. dendrobatidis genomes and found an increase in the number of domains when comparing it to genome of its closest relative.  When constructing phylogenetic trees of the CBM18 gene family, three monophyletic and strongly supported clades emerged.  When focusing on divergence of the protein domains, the authors determined that individual domain groups were monophyletic and showed a general pattern with regards to their genome locations.

More specifically, clades of the CBM18 family appears to possess different gene functions, some of which appear to be similar to lectins (LL), tyrosinase/catechol oxidases (TL), and chitin deacetylases (DL).  The function of these genes has yet to be experimentally determined, but the authors make some deductions based on DNA sequences.  The lectin-like genes may be involved in the sequestering of chitin, which could then be disrupting the amphibian immune response.  The tyrosinase/catechol oxidase gene family is associated with melanin synthesis, which could be disrupting the electron transport of the infected amphibians.  Lastly, chitin deacetylases may be involved in suppressing defense mechanisms in place to suppress the fungal infection of the host.  The authors plan to continue to elucidate the pathogenicity of B. dendrobatidis in an attempt to understand the ecology and evolution of its infection on amphibians.