Small Things Considered

What’s Old is New: Genome Wide Manipulation of the Bacterial Chromosome in Vivo

by Michael G. Schmidt

Every now and then you come across an article that makes you sit back and hear yourself say Wow! This is what happened to me recently while preparing to discuss a paper on This Week in Microbiology (TWiM), a bi-weekly podcast produced by Vincent Racaniello and ASM, or as I think of it, a journal club open to any who are curious about all aspects of microbiology. The paper by Church and colleagues, published in Science, describes the precise manipulation of bacterial chromosomal sequences on a genome-wide scale. Titles matter, and this one, Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement, spoke volumes. Before even reading the abstract, it caused me to ask, Have these authors found the Holy Grail that synthetic biologists have been searching for? I’ll let you be the judge as I take you through the world of Multiplex Automated Genome Engineering (MAGE) and Conjugative Assembly Genome Engineering (CAGE).

Here's what they did: they replaced all of the 314 TAG amber stop codons, scattered across the 4.6 megabase genome of Escherichia coli with the synonymous TAA ochre codon. This means that they would then have strains with a free codon that can be reprogrammed to do other things. You might be asking, What things? This really is a subject for another STC piece on cellular engineering written from the perspective of a synthetic biologist. But, in keeping with the theme of What’s Old Is New, one could envision the alteration of a tRNA gene, sneaking it into the chromosome via a limited version of MAGE, such that its product will now recognize the TAG codon and substitute a novel or unique amino acid into a protein resulting in the desired/engineered phenotype. Mother nature has already provided the proof of concept: extragenic suppression of existing amber mutations.

Fine Reading: Microbial Genomics and Infectious Diseases

by Elio

Would you like to read a concise and well-written review about how genomics has influenced our understanding of infectious diseases? Click here for a satisfying account by David Relman, one of the leading contributors to this field.

After summarizing the dizzying accelerating pace at which complete genome sequences are being acquiredeukaryotic, viral, bacterial, archaeal—Relman discusses how microbial genomic diversity impacts on health and disease, as illustrated by concepts such as the pangenome, genome reduction, genomic islands, CRISPR loci, and others. We read about the evolution of pathogens such as the plague bacillus, including its origin between 2600 and 28,000 years ago in China, and that the common ancestor of all the strains in the USA today probably arrived in San Francisco in 1899. We learn that comparing genomic sequences of Helicobacter pylori strains has been used to determine the timing and direction of human migrations, and that Vibrio cholerae traveled, with human help, from South Asia to Haiti.

Going to Great Lengths

by Merry

Source.

Given the streamlined genomes and the frugal nature of the Bacteria and Archaea, one might expect their proteins to be short and to the point. However, a survey of the 580 prokaryotic sequenced genomes available in 2008 found many genes apparently encoding large proteins. Specifically, 0.2% of the ORFs (3732 genes) were longer than 5 kb. Of those, 80 were truly giants—more than 20 kb! These mammoths were found scattered about in 47 taxa in 8 different phyla. The longest two, both from the green sulfur bacterium Chlorobium chlorochromatii CaD3, encode proteins containing 36,806 and 20,647 amino acids, respectively.

Does this set an all-domain record? Not quite. They are bested by one of our own, ttn, the gene for titin, an abundant protein of critical importance in vertebrate striated muscles. This single gene encodes 363 exons that, upon translation, yields a protein with 38,138 amino acid residues with 244 individually folded protein domains. Each titin molecule spans half the length of an entire sarcomere. Score one for the eukaryotes.

An Inactive Mine Provides Active Opportunities

Acid mine drainage in Spring Creek down-
stream from the Richmond Mine, part of
the Iron Mountain Mine Superfund Site
nine miles northwest of Redding, Calif.
Microbes inside the mine eat pyrite─fool's
gold─to produce sulfuric acid, creating the
most acidic groundwater ever measured.
Spring Creek flows eventually into the
Sacramento River. (UC─Berkeley) Source.

by Elio

“...a riddle, wrapped in a mystery,
inside an enigma...”
                                  Winston Churchill

Metagenomics is a fine tool indeed for surveying a microbial community in concert, treating both the cultured and uncultured equally. When the sample studied is rich in microbial variety, as often is the case, the pieces of genomes can be reluctant to reveal the genetic heritage of whole microbes. But there are a few particular environments that are dominated by a handful of species at most, and here this approach allows the reconstruction of complete genomes. That is the case with the acid mine drainage from mineral or coal mines. When mining ceases, all hell can break loose (anthropocentrically speaking). Microbes oxidize sulfides such as pyrite (iron sulfide) into sulfuric acid, which in turn solubilizes iron, copper, arsenic, silver, gold, and other heavy metals. Water no longer being pumped from the mine, this gemish of minerals emerges from seeps and other openings to become a highly toxic, low pH stream that eventually pollutes larger bodies of water. But for some bacteria and archaea, this is a juicy place to live and thrive.

Comments on the “Synthetic Cell”

Creation of Light, by Gustave Doré (1832-1883).
Source.

by Elio & Merry

The now famous announcement by the Venter group is based on their paper in Science entitled Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. We applaud this work for its impressive technical achievement and we acknowledge its future potential. However, we find the term “creation” to be misleading because the "new" cell was assembled using mainly preexisting constituents. We also find the term worrisome because, somewhat perversely, it may bring up creationism. We reprint a comment submitted by Bernie Strauss in response to the timely article published in Nature: Life After the Synthetic Cell. His words accurately express our sentiments.

Bernard S. Strauss wrote:

The first time I recall reading that biochemists had succeeded in synthesizing life was in 1967 when Arthur Kornberg and his colleagues succeeded in replicating a øX174 DNA template with E. coli polymerase I and ligase. According to the National Library of Medicine, U.S. President Lyndon Johnson looked at the Stanford press release and to Kornberg's dismay announced: "Some geniuses at Stanford University have created life in the test tube!" There was much discussion at the time including arguments similar to those published in the May 27th issue of Nature. The accomplishments are reasonably similar making allowance for the increase in technology over the past 40 years. A sequence of DNA has been replicated and then introduced into a cell where it takes over the functional apparatus to produce materials specified by the introduced DNA. This is certainly an important technical achievement and one with potential practical application. Does it tell us much about the origin or nature of life?

I would argue that the result is of little theoretical importance. The critical part of Kornberg's work 40 years ago (and one which he acknowledged) and of Venter's work this year is the necessity of a preformed cell. It is only this preformed cell that can convert the coded information in the DNA into functional products. In addition, that cell is not only a passive translator of genetic information but seems to include inherited information of its own, apart from the nucleic acid. As an example, membrane structures require preexisting structures as a template. It may be that at some date it will be possible to mix component membrane parts to form a structure into which all of the other cytoplasmic components can be added along with a synthetic DNA. So far however, all that has been done is to insert programming instructions into a completed machine. The basic unit of biology, and of life, remains the cell. Recent work emphasizes just how malleable the cell is but still requires the preexistence of this elementary life unit.

Anyone who has done science knows just how much work it takes to make any new finding. It is perhaps inevitable that having done all that work, one wants to make the most of it.

This brings to mind two earlier contributions to this blog by Franklin Harold (click here and here) where he discusses the essentialness of pre-existing membranes. It also prompts us to muse that every virus does something similar, with less expense and fanfare.

As always, we welcome your comments.

Bernard Strauss is Professor Emeritus, Department of Molecular Genetics and Cell Biology, The University of Chicago.

And The Winner Is…

by Elio

The transposases! The contest, in case you wonder, was for the most abundant set of genes in the known universe, or at least in the genomic data banks available on Earth. Aziz, Breitbart, and Edwards examined some 10 million genes from 2137 sequenced genomes (47 archaeal, 725 bacterial, 29 eukaryotic and 1336 viral) plus 187 metagenomes, in search of the gene set that is both most abundant and most ubiquitous. (Ubiquitous refers to genes that carry essential functions and are thus needed by every genome. A gene that is highly abundant in some ecosystems but not ubiquitous is likely to play an important habitat-specific role.) The winners, by far, were the genes for transposases. One could squawk that the genomes selected for sequencing to date reflect a bias towards “germs” and other microbes of human interest, and that so far the source environments for metagenomes represent only a small sample of what’s out there. But given the off-scale preponderance of the transposase genes, there is little to make one question their dominance.

Abundance of different functional roles in 2137 genomes plotted against the ubiquity of these
functional roles (defined as the number of genomes in which the functional role is represented
at least once). Source.

Not everybody seems to have transposases; only 1/3 of the viruses in this study have them. But those organisms that do tend to possess multiple copies—close to 40 on average. If you take a set of 2000 randomly sampled genes (the number found in a typical bacterial genome), 22 will encode transposases. They are also more common in free-living bacteria than in obligate pathogens or endosymbionts. The champion transposase-carrier? Possibly the marine cyanobacterium Crocosphaera watsonii, which has more than 400 copies.

So, why transposases? The authors point to their general usefulness to both viruses and cellular organisms in promoting the spread of mobile elements, thus leading to diversification. “Selfish genes” indeed! As the authors write, By securing their own replication and dissemination, transposases guarantee to thrive so long as nucleic acid-based life forms exist. This rather implies that they might imagine non-nucleic acid based life forms exist somewhere. Who says that bioinformaticists are just computational nerds?

Aziz RK, Breitbart M, & Edwards RA (2010). Transposases are the most abundant, most ubiquitous genes in nature. Nucleic acids research PMID: 20215432

Prophage Masquerade

by Merry

Source.

Roseovarius nubinhibens recently joined the exclusive club of about a thousand bacteria whose genomes have been sequenced. Why this honor? It’s a member of one of the most ubiquitous and most intensely studied clades of α-Proteobacteria, the marine roseobacters. This populous group participates in important jobs, including the global cycling of sulfur, climate regulation, and even modulation of our day-to-day weather. (For the latter, see our earlier post.) To appreciate the sulfuric importance of R. nubinhibens in particular, we need to begin with the major quantities of dimethylsulfoniopropionate (DMSP) made by marine algae for whom it serves as an osmolyte. Roseobacters then enter the picture, their work being to break down the DMSP by either of two pathways. One route converts DMSP into volatile DMS that can give rise to sulfate aerosols that act as cloud seeds. The other pathway leads to methanethiol, a primary sulfur source for marine bacteria. The relative amount of sulfur that flows each direction matters. R. nubinhibens was one of the first bacteria found to be able to carry out both conversions, thus is a potentially important switch point. The researchers reporting its isolation (José González and colleagues in Mary Ann Moran's lab) dubbed their new species Roseovarius nubinhibens (from the Latin nubes, for clouds, and inhibens, for inhibiting).

Virus-like particles generated by mitomycin C induction of R. nubinhibens
ISM. Siphovirus particles are commonly seen in the induced lysate, and
many of them appear to be “empty.” Bars = 50 nm. Source.

The R. nubinhibens genome has been sequenced. Any prophages on board? A routine search using Prophage Finder (software that looks for clusters of phage-related genes) turned up several candidates. Let's refine the question and ask whether there are any inducible prophages on board. Add mitomycin C (a potent DNA cross-linker) to a growing culture and wait 24 hours. Voilà! You get ~10¹⁰ VLPs per ml compared to ~10⁵ for control cultures. (What is a VLP? It's a Virus-Like Particle, i.e., something that looks like a virus but hasn't been shown to infect like a virus.) The induced VLPs have the long, flexible tails and polyhedral heads characteristic of siphoviruses, but most of these VLPs are empty-headed and/or have broken tails, suggesting that many are defective.

Was one of the predicted prophages the source of the VLPs? The DNA strands inside the VLPs were shown by pulsed-field gel electrophoresis (PFGE) to be about 30 kb, suggesting that the prophage might be about the same. None of the predicted prophages were near this size. So the researchers then searched the genome for any phage-related genes. They were lucky. They found an integrase gene along with a few other recognizable phage genes in a 27 kb region that Prophage Finder had overlooked. They concluded that they had found the prophage associated with the VLPs because they identified a gene in this stretch of DNA that encodes a major capsid protein and showed it to be identical to the capsid gene in the VLP DNA.

So what else does that prophage DNA encode? A few genes required for the prophage life cycle could be tentatively identified. Of the 28 genes that had significant hits in the GenBank database, 25 are most similar to genes of unknown function previously found in Rhodobacterales bacteria. Might those 25 "bacterial" genes actually be phage genes that are part of the unsequenced majority and that are residing in prophages? No wonder Prophage Finder—looking as it does for known phage-related genes—didn’t find the real prophage! Since more than 99.9% of phage diversity has not been captured in any database, hunting for prophages with Prophage Finder is like going birding with a field guide that includes less than one species out of a thousand.

Other sequenced members of the Rhodobacterales harbor related prophages, suggesting that this type of prophage is common and may be active in horizontal gene transfer (HGT) among these bacteria. Tantalizingly, there may be a subplot to this story, with even more HGT going on. The PFGE analysis of the VLP DNA also found a few short segments of 3, 4, and 12 kb. The authors suspect that these may be random bits of host DNA packaged inside capsids, i.e. gene transfer agents (GTAs). (For our earlier post about GTAs, click here.) GTAs transfer genes between members of the same species. Their production is controlled by the bacteria, and neither viral infection or cell lysis is involved. GTAs have been found only in α-Proteobacteria, with a particularly high incidence among the roseobacters. Previously, 21 of the 22 sequenced members of the marine Roseobacter clade had been found to carry the genes for GTA production; now R. nubinhibens makes it 22 out of 23.

What do GTAs have to do with prophages? Did one evolve from the other? If so, in which direction? In the words of two researchers in this field: The overall pattern of relationships between these various GTA, phage and prophage elements supports the notion of a continuum of sequence relationships resulting from transfers throughout a global phage gene pool. And we have barely dipped our toes into that global pool.

Zhao Y, Wang K, Ackermann HW, Halden RU, Jiao N, & Chen F (2010). Searching for a "hidden" prophage in a marine bacterium. Applied and environmental microbiology, 76 (2), 589-95 PMID: 19948862

Genomic Secrets of P. infestans, the Master of Potato Blight

This is the third and final post for our week of the oomycetes. In our first post, Elio provided the foundation by answering five key questions about this often neglected and misunderstood group. This was followed by a reminder from Mercè Piqueras of the role one member, the potato blight pathogen, has played in our history. Here in this final offering, Merry explores what we now—more than 150 years after The Great Famine—know about this pathogen's tactics from its genome sequence.

by Merry

No fungicide has ever been found to which P. infestans could not ultimately adjust…Indeed, no potato has ever been developed with defenses that Phytophthora could not ultimately breach. (Glenn Garelik. Source)

More than 150 years after the historic Irish potato famine, the deadly pathogen responsible for potato blight, Phytophthora infestans, is now destroying more than $3 billion worth of potatoes each year. What is the secret of it's pathogenic success? Hoping to find some answers in its genome, a team of 96 researchers have sequenced virtually its entire 240 Mb genome. This is by far the largest and most complex genome sequenced among the chromalveolates, a diverse eukaryote supergroup that includes not only the stramenopiles (the oomycetes, diatoms, and some algae) but also the dinoflagellates and the apicomplexans.

Why so much DNA? That's more than double that of Caenorhabditis elegans.

The Genes, The Whole Genes, & Nothing But The Genes

by Merry

SEM of Oxytricha in the process of reproducing.
Source.

We have come to expect the unexpected of ciliates, and Oxytricha trifallax, with its genomic capers, does not disappoint. Like many of its more famous ciliate relatives (e.g., paramecium, tetrahymena, stentor), Oxytricha is a complex unicellular organism with many specialized cellular structures. Of course, they have the requisite cilia for locomotion, feeding, and sensing their environment. But they also have a "mouth," food vacuoles where digestion takes place, kidney-like contractile vacuoles for osmoregulation, and a pseudo-anus—the cytoproct. Not surprisingly, ciliates tend to be large cells, some measured in millimeters. Combined with a fast rate of growth, this may require more gene transcription than a diploid genome can support. To cope with this demand, ciliates employ a convoluted strategy, one unique so far to this group.

Of Terms in Biology: Obligate Parasite

by Elio

Drawing a line in the sand? Source.

For bacteria, the term "obligate parasite" can have at least two distinct meanings.

1. A parasite that naturally only reproduces within a host and cannot be artificially cultured on cell-free media. Included are all the viruses, most bacterial endosymbionts, and bacteria such as Treponema pallidum, Mycobacterium leprae, the rickettsiae, and the chlamydiae.
2. A parasite that naturally only reproduces within a host, whether or not it can be artificially cultured on cell-free media. A large number of bacterial parasites fall into this category, e.g., Pneumococci, Group B strep, gonococci, H. influenzae, mycoplasmas, etc.

In my experience, the prevailing definition for obligate parasite is # 1. A number of people whom I asked said that they call pathogenic bacteria that can grow on artificial media “facultative parasites” rather than "obligate parasites."

What brings this up? A recent paper purports that obligate parasites have genomes smaller than 1Mb. Mycoplasmas and others that can be cultured in cell-free media fall below this arbitrary threshold; thus, they are not obligate parasites by the first definition and are by the second one. But the second definition includes many organisms with genomes much larger than 1 Mb. Obligate parasites and free living organisms can therefore not be divided on the basis of genome size.