If you can’t grow it, sequence it

Bacteria live almost everywhere, and use a staggering variety of strategies to get the energy they need to grow.  In the process, they make and recycle all kinds of globally important materials; and we often don’t understand how, biochemically, they do this.  One reason — apart from the sheer overwhelming number of different types of bacteria — is that many bacterial species are hard to culture in the laboratory.  Estimates of the proportion of bacteria that are “unculturable” (or, not cultured yet) range as high as 99%, based on sequencing of 16S rRNAs.   If the microorganism you want to study happens to be among the unlucky 99%, what are you supposed to do?  These days, you have a new option: sequence its genome.

In a paper published this month in PNAS (Lücker et al. 2010 A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria. Proc Natl Acad Sci U S A. 107, 13479-13484 PMID: 20624973), Lücker et al. do just that.  Frustrated by their inability to grow a nitrite-oxidizing bacterium — one that grows happily in sewage treatment facilities, what’s more, and therefore has no right to be fussy — they made an enriched preparation of it from sludge, and sequenced it.

You will realize that, while the genome sequencing is impressive, it wouldn’t have been possible without the much less glamorous activity of growing bacteria from sludge.  This was the work of Spieck et al. (Spieck et al. 2006. Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge Environmental Microbiology, 8 405-415 PMID: 16478447).   They incubated sludge from a wastewater treatment plant in Hamburg, Germany in a mineral-only medium, in the dark, with NaNO(₂) as the sole energy source.  Although they didn’t claim to achieve a pure culture, they got floc-like aggregates of something that eats NaNO(₂).  Electron microscopy and some other broad-brush characterization confirmed that this organism looked a lot like a bacterium.

Okay, step back for a second.  In 2006 we had something that grew out of sludge that was probably a bacterium.  In 2010, we have its genome sequence.  Let’s take a moment to marvel at the accelerating pace of science.

Done marveling?  OK, now let’s go on to what has been learned from this.  It’s a lot.  What’s at stake here is our understanding of the global nitrogen cycle, which (in addition, of course, to carbon) is another aspect of the ecology of planet Earth that humans have managed to change profoundly.  Nitrogen in the atmosphere is brought into the nitrogen cycle (“fixed”) by soil bacteria, which convert it into ammonia.  This is later oxidized to nitrite, and then to nitrate.  By far the most abundant and ubiquitous of the bacteria that perform the second oxidation, i.e. nitrite to nitrate, are the bacteria of the Nitrospira phylum.  After the nitrogen has been converted to nitrate, denitrifying bacteria get to work and return the nitrogen to the atmosphere.

Fixed nitrogen is an essential nutrient, but it is possible to have too much of a good thing.  As part of feeding ourselves, we grow enormous amounts of beans and other legumes, which carry nitrogen-fixing bacteria as symbionts, and we make artificial fertilizer by fixing nitrogen chemically.  As a result, there is now much more ammonia and oxides of nitrogen in the atmosphere and in the oceans, leading to unpredictable changes to ecosystems.  [The omnivore’s dilemma just got worse.  I used to think that beans were the most virtuous food available.  It’s undoubtedly better for the planet to eat beans than to eat methane-producing cows, or soon-to-be-extinct fish, but the clear blue light of virtue has been dimmed.  On the other hand, if it’s really true that phytoplankton are disappearing from the oceans — horrifyingly fast — and that phytoplankton growth is often limited by the availability of nitrogen, perhaps too much nitrogen in the oceans is a good thing, not a bad thing.  Life is so complicated.]

So understanding how Nitrospirae do what they do takes on an added interest.  The genome sequence is quite enlightening in several ways, though many questions remain open.  For example, the likely reason that Nitrospirae are more efficient than other nitrite-oxidizing bacteria, such as those of the Nitrobacter genus, is that the key enzyme, nitrite oxidoreductase (NXR) faces the periplasm in Nitrospira, and faces the cytoplasm in Nitrobacter.  The NXR function probably evolved independently in these lineages.  Having the NXR facing the periplasm means that nitrite and nitrate don’t have to be transported into/out of the cytoplasm in Nitrospira, which is a potentially significant advantage as the efficiency of the transporters can limit the activity of the NXR.  There are also energetic advantages to this set-up.  The result is that Nitrospira can grow at lower levels of nitrite than Nitrobacter, probably accounting for its dominance. Lücker et al. were able to make a fairly detailed model of the likely electron-transport chain, based only on the genome sequence and homology-gazing; but since 30% of the predicted coding sequences have no known homolog, there may be surprises to come.

Several lines of evidence suggest that Nitrospira evolved from an anaerobic ancestor.  For example, it doesn’t seem to have any of the common genes that protect bacteria from reactive oxygen species.  A practical consequence of this lack is that it would seem to be a good idea to keep an eye on oxygen levels in your wastewater treatment plant, if you want denitrification to be complete before your waste ends up in the groundwater.  The particular Nitrospira they sequenced (for which they suggest the name Nitrospira defluvii, from the Latin for outflow or sewage) does, however, have a very nice collection of multidrug efflux systems and transporters for heavy metals.  This would seem like a good idea if you’re going to hang out in sludge that came from who knows where.

And finally, quite a significant surprise.  There is another route back to nitrogen gas from ammonium and nitrite: it’s called anaerobic ammonium oxidation, abbreviated as anammox.  Not all that long ago, anammox was believed to be biochemically impossible: then bacteria were found that could do the job, and now we think that they’re responsible for half of the nitrogen-removing activity in the oceans.  Naturally, they turn out to be hard to culture; they also grow extremely slowly, dividing at most once a fortnight.

About four years ago, a large collaborative group (including some of the authors from Lücker et al.) reconstructed the genome of an anammox bacterium, K. stuttgartiensis, from sequences obtained from a bioreactor that was dominated by a single species — just as was done here for N. defluvii.  And it turns out that significant chunks of these two genomes look highly related.  Specifically, it looks as if the nitrite-oxidizing pathway of both bacteria came from the same place, via horizontal gene transfer, early in the evolution of the two lineages.  So the two major processes for getting nitrogen back into the atmosphere are actually, on some level, the same process; invented once — perhaps 800 million years ago — and used in two different, profoundly important ways.

Lücker S, Wagner M, Maixner F, Pelletier E, Koch H, Vacherie B, Rattei T, Damsté JS, Spieck E, Le Paslier D, & Daims H (2010). A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria. Proceedings of the National Academy of Sciences of the United States of America 107, 13479-13484 PMID: 20624973

Spieck, E., Hartwig, C., McCormack, I., Maixner, F., Wagner, M., Lipski, A., & Daims, H. (2006). Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge Environmental Microbiology, 8 (3), 405-415 DOI: 10.1111/j.1462-2920.2005.00905.x