New genes, old function

Ken mused a while back here on MT about the improbability of finding a DNA sequence that had no similarity to sequence from any known organism.  And this is as we'd expect, if all life on Earth shares a common ancestor, and nothing that has been discovered since Darwin first proposed this in 1859 suggests otherwise.

So, why are researchers reporting DNA enzyme sequences that don't appear to be homologous to any known sequences for similar enzymes?  François Delavat et al. have sequenced genes from organisms found in an acid mine drainage, organisms that are resistant to being cultured (i.e., that can't be readily grown up in the lab), looking for novel genes or function.  They report their findings in the open-access Nature journal, Scientific Reports ("Amylases without known homologues discovered in an acid mine drainage: significance and impact").

Amylases are enzymes that catalyze the breakdown of carbohydrates, or in the case of bacteria described here, degrade polymers found in their acidic, metal-heavy surroundings.  Amylases from bacteria that grow in culture have been well-studied, and have been classified into several families based on their structure and other characteristics.  Some have been found in extreme environments, but no one had reported the sequencing of DNA from Acid Mine Drainages before this paper.  These are very low pH, very high metal environments. 

The authors

...decided to perform a function-based screening for the well-known amylases, using standard techniques. This strategy allowed the isolation of 28 positive clones, 2 of them being subcloned, the proteins purified and characterized in vitro. In silico analyses based on the nucleotidic sequence and both the primary and the predicted tertiary structures revealed that they are completely different from other known hydrolases as both genes encode a « protein of unknown function » and display no known conserved amylolytic domain. Nevertheless, in vitro tests confirmed the amylolytic activity of these 2 enzymes.

That is, these genes did degrade polysaccharide, but neither of the subclones matched any known amylase sequences in the databases. 

As Delavat et al. point out, much is known about lab-friendly bacteria, and a whole lot less about organisms that can't be grown in the lab.  Thus, if these results are confirmed, the fact that these genes, from organisms that were found in an extreme previously unexplored environment, don't look like other known amylase sequences doesn't at all suggest that these bacteria are unique, or that they don't share the same common origin the rest of us share.  Rather, it suggests that the lab-centric biology of the last century has given us a lab-centric view of the world.  It's no surprise that bacteria that live in the low pH, high metal extremes of Acid Mine Drainages would have evolved particular enzymes appropriate for that environment.  But it's also not a surprise that these enzymes have a function that is common to bacteria in every environment.

That the genes that code for these enzymes are unlike any of the subset of amylases yet described is another example of phenogenetic drift, the conservation of a biological trait or function even when its underlying genetic basis has changed.  These genes may look novel now, but as more bacteria are characterized from non-lab environments it's likely that more will be found that share some of the characteristics of these amylase genes. 

Note, also, that these sequences are genes -- they have protein coding structures and are identifiable from sequences as such.  They are not 'random' sequences with no known relation to the usual characteristics of genes.

Metagenomics in action

'Metagenomics', the direct sequencing of all the DNA found in environmental samples.  From one perspective, this is yet another 'omics', a way to keep all those expensive sequencing machines going.  And a way to avoid having to have hypotheses about what's going on in nature before you start your project--a somewhat strange twist in modern biology.  Still, 'omics' is done because we have the tools to do it and because it does promise to find something, on the single underlying assumption, namely, that something is there, even if we don't know what it might be.

In fact an interesting use of this methodology is reported in Nature this week, in a paper about the sequencing of all the microbes in Alaskan permafrost soil samples pre- and post-thaw.  The paper reports rapid changes in the abundance of many phylogenetic and functional genes and pathways, suggesting a rapid response to changing environment.  In this case, there need not have been any kind of a priori hypothesis about what one would specifically find for this to be a valuable kind of work, which this study has proven to be.

Mackelprang et al. collected 3 frozen soil cores from an area in Alaska that they had previously characterized.  They removed samples from these and let them thaw over 7 days.  They monitored the carbon dioxide and methane concentrations in the headspace of the helium filled tubes in which the samples were incubated, and extracted DNA for 16S ribosomal RNA and metagenome sequencing.

The authors were particularly interested in what happened to the methane and carbon dioxide because, of course, these are greenhouse gases.  They document changing levels of these gases as the soil samples thawed, and corresponding increases in genes in their metagenome from microbes that produce these gases as metabolic byproducts.  

Specifically:

The metagenome data revealed core-specific shifts in some community members, including the orders Proteobacteria, Bacteriodetes and Firmicutes. We found that Actinobacteria increased in both cores during thaw. Actinobacteria have previously been found at high abundance in permafrost, which is thought to be caused by their maintenance of metabolic activity and DNA repair mechanisms at low temperatures. Most archaeal sequences identified in the metagenomic data were methanogens in the phylum Euryarchaeota (62–95%), including the Methanomicrobia that was represented in our draft genome. In total, four orders of methanogens (Methanosarcinales, Methanomicrobiales, Methanomicrobia and Methanobacterales) were detected. As the permafrost thawed, the methanogens (including Methanomicrobia) increased in relative abundance. These orders are known to be metabolically versatile and can use a variety of substrates.

They also found that methane was consumed post-thaw.  But, to us what is most interesting about this, not being climatologists or microbiologists, is what they found about the differences between samples post-thaw, as they describe here.

We tracked simultaneous shifts in the total gene complement from the metagenome data to obtain a global view of functional response to thaw. The active layer samples were relatively similar before and after thaw. By contrast, the two frozen permafrost metagenomes differed dramatically before thaw. In addition, functional genes in frozen active layer and permafrost samples were distinct from each other, including differences in several key metabolic pathways such as energy metabolism, nitrogen fixation, amino-acid transport, oxidative phosphorylation and anaerobic respiration. During thaw, the permafrost metagenomes rapidly converged and neared those in the active layer samples. The convergence of function was not matched by a convergence of phylogenetic composition during this short-term incubation, suggesting that disparate community responses to thaw can have similar functional consequences.

As we said yesterday in our post about ostrich penises, however the job can get done, evolution can support it.  Whether or not the job being done here is good or bad for humans vis-à-vis climate change is another story.  But, convergence of function in the communities of microbes analyzed by these researchers happened in communities with very different compositions of microbes.

Organisms have responsive genomes. Indeed, the 'job' of cells is to sequester their special ingredients within, but to monitor the external environment to determine how to behave most successfully.  They are changeable, within their genomic repertoire.  Mutation followed by natural selection can lead to specific genetically committed responses, but that isn't always necessary, because due to whatever earlier processes, even humble microbes have evolved to be able to respond to the conditions they find themselves in.  And, whether or not warming temperatures are beneficial to specific microbes, the community adapts. 

Based on comparative morphology and modern-day science, roughly 4 billion-year-old aggregates of bacteria (fossil biofilms calleld  'stromatolites') look strikingly like their modern descendants.  Today, biofilms are known to be bacterial responses to changes in conditions--even different species can aggregate in the same biofilm.  This means that not that long after the origin of life (and, indeed, of the earth itself), fundamental facultative adaptability had already been built by evolution into the genomes of the earliest cells--and that means it is a basic property of cells.  This is a point we stress in our book, The Mermaid's Tale, and we're always gratified to see it confirmed.

Gutting it out

Only a small fraction of bacteria can be grown in laboratories; apparently nobody understands what they need in their environment well enough. This can be a problem for microbiologists trying to identify the bacterium causing a new infectious disease, but it also means that it has not been possible to know all of the little bugs to which we our bodies are willing or unwilling hosts. The same would be true for other animals, wild ones as well as our pets and farm species. We say 'willing or unwilling' because, of these pathogens, some are presumed to be harmless commensals, and others are necessary to our survival (such as the E. coli in our intestine, that we depend on for digestion, and similarly for other mammals such as grazers like cows and goats who need bacteria in their rumens to digest cellulose).

One of the characteristics of bacteria is that they can exchange structures (e.g., plasmids) that contain some actively used genes. This is where many if not all of the genes are that lead the bacteria to resist nasty things in their environments such as antibiotics that bacterial targets have evolved to protect themselves. A vulnerable strain of bacteria can acquire a gene that makes them antibiotic resistant. This is of course a very important current problem in farm animals and humans, driven by the amount of antibiotics we ingest. And farm animals are reservoirs of pathogens that can affect humans, so humans and the animals they live with are part of large bacterial-host ecosystems.

Since we can't culture most bacteria, our knowledge of who's where, and the characteristics of many bacterial species, has been quite limited. But DNA sequencing technology has opened the way to identifying our visitors. By extracting all the DNA from a sample of some tissue, fragmenting the DNA and sequencing the fragments, their owners can be identified, and their genetic makeup and function characterized. This is done by comparing the sequence fragments against all sequences currently known (in Genbank). Even if we don't identify an exact match, we can find a known species that is close enough to our tissue-sampled sequence to identify its place in the bacterial tree of life. Antibiotic resistance genes can be identified in the same way, too, since many are already known.

A new paper in Science (Functional Characterization of the Antibiotic Resistance Reservoir in the Human Microflora, Sommer et al., Aug 28, 2009, 1128-1131) reports on a detailed analysis of the antibiotic resistance genes found in the microbiome, the resident bacteria, of healthy individuals. The study was done in an effort to learn more about how antibiotic resistance genes are acquired by pathogens that infect humans.

This study found that, indeed, many of the resistance genes in multidrug resistant bacteria were acquired by lateral gene transfer--that is, the gene hopped into the pathogen on a plasmid from a different bacterium. Bacteria in the wild usually live in large communities of many different species, where they can promiscuously exchange plasmids, thus antibiotic resistance can spread rapidly.

The authors wondered if the extensive history of antibiotic use in humans might mean that microflora in the human gut might be a reservoir of resistance genes readily transferable to pathogens. They isolated DNA from saliva and fecal samples from two healthy individuals who had not taken antibiotics for at least a year, and sequenced fragments of bacterial DNA that were resistant to all the antibiotics they tested.

They found that there was some, but by no means complete overlap between the two people who were sampled, and that nearly half the resistance genes they identified in this way were identical to resistance genes in human pathogens. This doesn't tell them whether gene transfer went from the commensal microflora to the pathogen or vice versa, but Sommer et al. suggest that it's quite plausible that our gut microflora are a reservoir of resistance genes just waiting to jump into pathogens which are now controllable with drugs. The remaining genes, although not yet found in pathogens, were functional when transferred to E. coli, suggesting that if they do eventually find their way into pathogens, they will be active, although there seems to currently be a barrier to lateral gene transfer which isn't yet understood.

The authors conclude:

Many commensal bacterial species, which were once considered relatively harmless residents of the human microbiome, have recently emerged as multidrug-resistant disease-causing organisms. In the absence of in-depth characterization of the resistance reservoir of the human microbiome, the process by which antibiotic resistance emerges in human pathogens will remain unclear.

This study provides interesting ecological information about bacterial dynamics, and of course warns us that antibiotic resistance may be more complex, challenging, and difficult to predict than we have thought. It's evolution in action.