Vindified (along with short histories of Microbiology and Earth's past)

This is a word coined by my nephew during a high-stakes Scrabble match against his wife. Jed’s one of the most competent people I know, but spelling’s never been a strong suit, and his whip-smart wife had challenged one of his words.  Much to both their surprise, his spelling was correct, Jed  yelled out ‘Vindified’. The newly coined word combining vindicated and justified is now part of our family vocabulary.

I bring this up in regards to two paper’s published in the past year relevant to my own career. The first is about Leptothrix ochracea, a bacterium that oxidizes iron and produces a rust-coated tubular sheath. This fascinating microbe was first described in 1797 making it one of the first bacteria to actually be given an official name, and was the subject of a number of papers through the 1800’s. This is not a coincidence, in the 1800’s microbiology was as much a field science as a lab science with the field melded into the lab through samples brought into the lab from nearly any environment you can think of, and peered at through the light microscope to see what tiny life might abound. Light microscopes were being perfected in the 1800’s, and considered the leading research instruments of the day. Unlike protozoa and larger single-celled organisms, most bacteria are very hard to see with a conventional (1800’s) microscope, and can only be visualized using a stain that gives contrast to the cell. Developing stains for microscopic analysis of bacteria was cutting edge science in the 1800s. Leptothrix, however, and some of it’s iron-oxidizing cousins, made its own stain by virtue of the rusty coated rind it produced. This made them easy to pick out with any basic light microscope with good magnification. As a result, when microscopists picked up rusty looking material from a stream bank and looked at in the microscope it was easy to see it teeming  with life and most prolific was Leptothrix, thus it was a quite a well known microbe well before Escherichia coli was discovered in 1885.

I became interested in Leptothrix ochracea in the 1980’s as a graduate student. Not much new had been learned about it since the turn of the 20th century, largely because it couldn’t be grown in the laboratory, and it was during the 20th century that microbiology changed from a field and lab science to almost strictly a lab science based around microbes that could be grown successfully on a petri plate, e.g. E. coli. Wild microbes were largely forgotten. Despite my fascination with Leptothrix, I also focused on wild microbes, but ones I could grow in the lab, and although I tried, I never had any success with growing L. ochracea. Fortunately, the field part of microbiology has undergone a renaissance, or a revolution, depending on your point of view, since the turn of the 21st century. We now have powerful, DNA, RNA, and protein techniques to study cells and their activities in nature without cultivating them in the lab. In fact, we now know that vast majority of microbes that live in nature have never — and most will never be — grown on the surface of a petri plate.

A small rusty water channel. I happened on this one a path next to the George Washington Parkway, near Washington D.C, where it flows into the Potomac River. It’s easy to imagine an 18th or 19th century naturalist coming upon this and scooping up at sample to look at under a microscope.

In the 2010’s I had some funding to study something related to iron and microbes. One of the things you learn as a scientist, is to figure out how to creatively use the money to study something that seems relevant to a funding agency to study a problem you are actually really interested in. One of my postdocs at the time, Emily Fleming, became fascinated with L. ochracea, and turned her attention to it. Emily’s tenacity and creativity were enough to actually get L. ochracea to grow just a little bit in the lab.   We were fortunate to be able to take advantage of state-of-the-art technologies for studying single cells from the natural environment. Using these tools Emily was able to partially sequence the genome of a single L. ochracea cell (I’ve actually come to the conclusion that L. ochracea is a poster microbe for not doing single cell genomics, but that’s another story). She also collaborated with colleagues in Germany to use Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) to study the metabolism of the few cells she had cultivated in the lab. NanoSIMS is as sophisticated as it sounds, so this took some time. In the end, she had part of a genome and some activity measurements on L. ochracea’s metabolism. So, what did this tell us? 

A closer view of an ‘iron mat’ from the channel above., The ocherous color is what gives L. ochracea its name. A number of earlier civilizations harvested this material to make ochre paints used in cave and wall paintings.

For that we need to jump into some basic life physiology about how cells acquire carbon to make more cells, and the energy source they use to power the process. There are heterotrophs that get their carbon and energy from organic molecules, for example glucose. Humans and most multicellular animals are heterotrophs. There are phototrophs that fix carbon dioxide out of the air and convert it to biomass using energy from the sun, e.g. photosynthesis by plants. Then there are lithotrophs that can also fix carbon dioxide but gain energy from oxidizing inorganic elements like iron, sulfur, nitrogen, or hydrogen. We knew that L. ochracea oxidized iron and it also had a key gene for fixing carbon dioxide, so maybe it was a lithotroph. But why could we never grow it the way I’ve successfully grown many other iron-oxidizing lithotrophs in my lab? 

Emily’s data seemed to indicate L. ochracea might be a mixotroph. Mixotrophy is exactly as the name implies a mixture of different growth physiologies. Let’s just say the complexities mixotrophy presents makes NanoSIMS seem simple. So, we published a paper with Emily’s results and concluded that L. ochracea was probably a mixotroph. However, I’ve always been a bit nervous about this claim being based on the analysis of a few single cells amongst literally millions or billions. It felt like we had caught an instantaneous glimpse of something very interesting, but at the extreme limit of our vision.  

So now, ten years later my colleagues at the University of Delaware published a new paper on L. ochracea (I’m actually an author on it too, but played a pretty minor role). This work is based on lots of genomic evidence gathered through a process called metagenomics. Metagenomics allows you to sequence multiple genomes of closely related environmental microbes without growing the cells in the lab. Its widespread use wasn’t really available to us at the time of Emily’s work, but since then it’s become quite cheap. The metagenome analysis of L. ochracea (I actually think L. ochracea is a poster child for metagenomics, but that’s another story) confirmed our earlier findings, and due to there being way more data, this provided more confidence in the conclusion that it is a mixotroph. 

What makes it really interesting is that L. ochracea appears to require mixotrophy. Mixotrophy is turning out to be quite common in microbes, especially the more people look. Nonetheless, generally while a microbe might mix metabolisms, for example heterotrophy and phototrophy, it can grow nearly as well, or better using just one set, i.e. it doesn’t need both. L. ochracea might be one of the few microbes we know about that has to mix both heterotrophy and lithotrophy, it can’t grow with either alone, so it’s possible that it’s a true or obligate mixotroph. Why? Your guess might not be quite as good as mine, but it would be surprisingly close, and if you just shrugged, ‘how should I know?’ you might be even closer! Needless to say, there are still many mysteries to be resolved about L. ochracea, and about mixotrophy, but it was cool to see Emily’s faith in some hard-won data confirmed by today’s availability of metagenomics .

Leptothrix ochraca tubular sheaths coated in iron oxide as  viewed under a microscope from an ocherous iron mat like the one pictured above. Each filament is about one micrometer wide (1/100 the width of a human hair). Under the right conditions these sheaths can become micro-fossils.

The second paper has to do with Earth’s history and iron. Not surprisingly, from my point of view, iron has played a number of key roles in Earth history including in the development of human civilization. This latter point is due to the development of the steel industry, and the ubiquitous use of steel in modern society. A principal reason steel is abundant and cheap is because its main ingredient, iron oxide, is relatively easy to mine and refine. Good iron ore deposits can contain 60 to 70% iron oxides. Geologists have determined that there were periods in Earth history when large deposits of iron oxides accumulated rapidly, typically in shallow ocean or estuarine basins at a time when there was very little or at most moderate amounts of oxygen in the atmosphere.  

So, what caused these large-scale iron formations? Microbes are thought to have played a key role through photosynthesis. The clearest role belongs to photosynthetic cyanobacteria (classic phototrophs) that evolved to produce oxygen driven by photosynthesis somewhere around 3 billion years ago, a time when the atmosphere contained vanishingly small amounts of oxygen. In one sense oxygen is a waste product of their metabolism, lucky for us! These cyanobacteria gradually oxygenated the atmosphere, and since there was a lot of soluble iron in seawater at that time (but not today), the oxygen reacted with the iron to oxidize it and produce iron oxides or rust that sank to the seafloor and accumulated to form massive iron oxide deposits. Some of these are what we mine today. 

Now what’s always really interested me in this story is that if there were iron-oxidizing lithotrophs or mixotrophs, e.g. L. ochracea, present at this time, could they have played an important part in precipitating these iron oxide deposits? It would have been perfect for them, a little oxygen that they like, and a lot of ferrous iron for energy. Some of my colleagues and I have speculated on the role they might have played, but to this day, it’s most common for Earth scientists writing on the subject to just say the rise of oxygen led to formation of large iron deposits with the implication that this was all due to the chemical oxidation of the iron, no direct microbial reaction required, and in essence ignoring the role of iron-oxidizing bacteria. 

This is a little frustrating, on the other hand, my work is really focused on latter day microbes and processes, and I don’t directly study these deep time aspects, so I kind of ignore them right back! Now coming back to this new paper, these folks analyzed iron-rich rocks using several different methods and concluded that iron-oxidizing bacteria played an important role in their deposition. A key piece of evidence they present are micro-fossils that look nearly identical to modern iron-oxidizing bacteria like L. ochracea. Just like the iron rinds that iron oxidizing bacteria produce made them easily visible to 19th century microscopists, these same rinds, given the right conditions can produce excellent micro-fossils! Personally, I am a more visual scientist, so all the detailed elemental analysis, isotopic analysis and organic molecular tends to go over my head, but show me a well preserved micro-fossil and that’s a smoking gun! 

I really like the title of this paper: 'Living in their Heyday: Iron-oxidizing Bacterial Bloomed in Shallow-Marine, Subtidal Environments at ca. 1.88Ga'. I’d even go a little further out on a limb and speculate that there could easily have been a billion years worth of heydays for iron-oxidizing bacteria to abound in.

So it’s exciting and a bit gratifying after years of work to see strong evidence for mixotrophy in L. ochracea, and making a case for the importance of iron-oxidizers in Earth’s history. In neither case do I feel vindicated by this work, since that would imply that everyone else was wrong and I was correct, it’s much more subtle than that. So vindicated, no, but vindified, absolutely!