Bacterial Genes in Rice: A Cautionary Tale

Something very strange happened the other day.

I was fooling around looking for flagellum genes in various organisms, hoping to find homology between bacterial flagellum proteins and eukaryotic cilia proteins. All of a sudden, a search came back positive for a bacterial gene in rice, of all things.

On a lark, I decided to check further. ("If one gene transferred, maybe there are more," I reasoned.) It was late at night. Before going to bed, I downloaded the DNA sequence data for all 3,725 genes of Enterobacter cloacae subsp. cloacae strain NCTC 9394 and set up a brute-force BLAST search of the 3,725 bacterial genes against all 49,710 genes of Oryza sativa L. ssp. indica. I set the E-value threshold to the most stringent value allowed by the CoGeBlast interface, namely 1e-30, meaning: reject anything that has more than a one-in-10³⁰ chance of having matched by chance. I went to bed expecting the search to turn up nothing more than the one flagellum protein-match I'd found earlier.

When I woke up the next morning, I was stupefied to find that my brute force blast-n (DNA sequence) search had brought back more than 150 high-quality hits in the rice genome.

I later found 400 more bacterial genes, from Acidovorax, a common rice pathogen. (Enterobacter is not a known pathogen of rice, although it has been isolated from rice.)

But before you get the impression that this is some kind of major scientific find, let me cut the suspense right now by telling you the bottom line, which is that after many days of checking and rechecking my data, I no longer think there are really hundreds of horizontally transferred bacterial genes lurking in the rice genome. Oh sure, the genes are there, in the data (you can check for yourself), but this is actually just a sad case of garbage in, rubbish out. The Oryza sativa indica genome, I'm now convinced, suffers from sample contamination. That is to say: Bacterial cells were present in the rice sample prior to sequencing. Some of the bacterial genes were amplified and got into the contigs, and the assembly software dutifully spliced the bacterial data in with the rice data.

My first tipoff to the possibility of contamination (aside from finding several hundred bacterial genes where there shouldn't be any bacterial genes) came when I re-ran my BLAST searches using the most up-to-date copy of the indica genome. Suddenly, many of the hits I'd been seeing vanished. The most recent genome consists of 12 chromosome-sized contigs. The earlier genome I had been using had had the 12 chromosomes plus scores of tiny orphan contgis. When the orphan contigs went away, so did most of my hits.

When I looked at NCBI's master record for the Oryza sativa Indica Group, I noticed a footnote near the bottom of the page: "Contig AAAA02029393 was suppressed in Feb. 2011 because it may be a contaminant." (In actuality, a great many other contigs have been removed as well.)

When I ran my tests against the other sequenced rice genome, the Oryza sativa Japonica Group genome, I found no bacterial genes.

Contamination continues to plague the Indica Group genome. The 12 "official" chromosomes of Oryza sativa indica have Acidovorax genes all over the place, to this day. I suppose technically, it is possible those genes represent instances of horizontal gene transfer. But if that's what it is, then it's easily the biggest such transfer across species lines ever recorded. And it happened only in the indica variety of rice, not japonica. (The two varieties diverged 60,000 to 220,000 years ago.)

The following table shows some of the Acidovorax genes that can be found in the Oryza satisva Indica Group genome. This is by no means a complete list. Note that the Identities number in the far-right column pertains to DNA-sequence similarity, not amino-acid-sequence similarity.

Acidovorax Genes Ocurring in the Published Oryza sativa indica Genome

Query gene	Function	Rice gene	Query coverage	E	Identities
Aave_0021	phospho-2-dehydro-3-deoxyheptonate aldolase	OsI_15236	100.0%	0.0	93.6%
Aave_0289	orotate phosphoribosyltransferase	OsI_36535	100.0%	0.0	96.8%
Aave_0363	lipoate-protein ligase B	OsI_15083	100.0%	0.0	94.6%
Aave_0368	F0F1 ATP synthase subunit B	OsI_15082	100.0%	0.0	98.9%
Aave_0372	F0F1 ATP synthase subunit beta	None	100.1%	0.0	98.2%
Aave_0373	F0F1 ATP synthase subunit epsilon	OsI_15081	100.0%	0.0	97.8%
Aave_0637	twitching motility protein	OsI_37113	100.1%	0.0	95.5%
Aave_0916	general secretory pathway protein E	OsI_17332	86.9%	0.0	96.6%
Aave_1272	NADH-ubiquinone/plastoquinone oxidoreductase, chain 6	OsI_28652	100.0%	0.0	97.3%
Aave_1273	NADH-ubiquinone oxidoreductase, chain 4L	OsI_28651	100.0%	3e-174	100%
Aave_1301	DedA protein (DSG-1 protein)	OsI_21534	97.3%	0.0	96.8%
Aave_1312	hypothetical protein	OsI_15703	99.8%	0.0	93.4%
Aave_1948	histidine kinase internal region	OsI_23297	100.0%	0.0	96.3%
Aave_1950	hypothetical protein	OsI_23296	100.0%	0.0	96.6%
Aave_1957	penicillin-binding protein 1C	OsI_15534	100.1%	0.0	92.8%
Aave_1958	hypothetical protein	OsI_15533	99.2%	0.0	92.2%
Aave_2274	major facilitator superfamily transporter	OsI_33140	95.1%	0.0	92.5%
Aave_2484	2,3,4,5-tetrahydropyridine-2-carboxylate N-succinyltransferase	OsI_19753	100.0%	0.0	97.3%
Aave_3000	ferrochelatase	OsI_33935	100.0%	0.0	96.2%

So let this be a lesson to DIY genome-hackers everywhere. If you find what you think are dozens of putative horizontally transferred genes in a large genome, stop and consider: Which is more likely to occur, a massive horizontal gene transfer event involving several dozen genes crossing over into another life form, or contamination of a lab sample with bacteria? I think we all know the answer.

Many thanks to professor Jonathan Eisen at U.C. Davis for providing valuable consultation.

Hydrogen Peroxide Powers Evolution

I'm about to offer a conjecture that is a bit preposterous-sounding but could well hold true. I actually think it does.

I propose that evolution, at the level of bacteria (though probably not at higher levels), is driven by hydrogen peroxide.

This theory rests on three assumptions: One is that the creation of new bacterial species happens almost entirely via lateral gene transfer, not heritable point-mutations. Secondly, bacteria (marine and terrestrial) are regularly exposed to challenges by hydrogen peroxide in the environment. Thirdly, those challenges drive lateral gene transfer.

Evidence for the first assumption is embarrassingly abundant. If you're not up to speed on the subject, I suggest you read the excellent paper, "Lateral Gene Transfer," by Olga Zhaxybayeva and W. Ford Doolittle in Current Biology, April 2011, 21:7, pp. R242-246 (unlocked copy here). It's now common to find that any given bacterial species can trace a good percentage of its protein base to "ancestors" that are too far removed horizontally to be ancestors in the conventional sense.

Consider E. coli. There are hundreds of strains of E. coli, with genes ranging in number from 4,100 to about 5,300 per strain. The problem is, the various strains of E. coli have only about 900 genes in common (and that's far too few genes to render a fully functional E. coli). The E. coli pan-genome actually takes in more than 15,000 gene families, total. Certainly, you can draw a family tree of E. coli based on 16S ribosomal polymorphisms, but that doesn't explain where the 15,000 pan-genome genes came from. The "family tree" metaphor quickly breaks down if you start drawing trees based on proteins. You get many conflicting trees—all of them correct.

Trees like this are fiction where bacteria are concerned.
The tree of life is more like a net of life or web
of life than a directed acyclic graph.

Where are all of the genes coming from? Other species, of course. They arrive by way of mechanisms like transformation, transduction, and conjugation. all of which allow direct entry of foreign DNA into a bacterial cell. At one time it was thought that conjugation could only occur between bacteria of the same species, but it is now known that cross-species conjugation also occurs (as, for example, between E. coli and Streptomyces or Mycobacterium).

Transduction, which is where viruses package up an infected host's genes in virus capsules that are then taken up by another cell, occurs naturally in bacterial populations in response to environmental factors like ultraviolet light and hydrogen peroxide. Exposure of a virus-carrying (lysogenic) cell to UV light or peroxide can induce runaway production of virus, and in fact this mechanism is used by Streptococcus to kill competitive Staphylococcus cells, in a clever bit of chemical warfare. It's been known for years that hydrogen peroxide can cause many types of bacteria to shed DNA. Now we know why: Hydrogen peroxide is a signalling molecule. It signals (among other things) lysogenic bacteria to go into a lytic cycle. It also signals cells to mount what's known as the SOS response, which is a global response to oxidative challenge. Years ago, Bruce Ames and his colleagues showed that exposing Salmonella to very dilute (60 micromolar) hydrogen peroxide caused the cells to differentially express 30 "SOS" proteins, including heat-shock proteins and low-fidelity DNA-repair systems. We know that hydrogen peroxide as dilute as 0.1 micromolar can induce phage (virus) production in up to 11% of marine bacteria. This is significant, because rainwater contains hydrogen peroxide in concentrations of 2 to 40 micromolar, and ocean water has been known to reach millimolar levels of H2O2 after a rain storm.

If you're wondering why rain contains hydrogen peroxide, the peroxide gets there in two ways. One is UV-frequency photochemistry (where water is cleaved to H and OH, then reforms as H2 and H2O2); the other is via ionization reactions caused by lightning. (Lightning is energetic enough to bring airborne oxygen and water to a plasma state. The resulting ionization and rearrangement of free atoms yields a certain amount of hydrogen peroxide.) The presence of H2O2 in rainwater has been confirmed many times, and in fact there's a well-preserved "fossil record" of it in polar icepacks, going back centuries. (Polar snowpacks contain from 10 to 900 ppb of H2O2; it varies seasonally, the max coming in summer.)

Bottom line, every rain event (over land, over sea) constitutes a hydrogen peroxide challenge for microbes. Which induces viral transduction (and a release of whole-cell DNA through lysis, some of which will be inevitably be used in transformation). It also induces low-fidelity DNA repair (which is guaranteed to help evolution along). Every rain event, in other words, is a chance for evolution to do its thing. For bacteria, that means gene-sharing within and across species lines.

Darwin's theory of a tree-like ancestor basis
for all living things is dead wrong, at
least for bacteria.

W. Ford Doolittle (who wrote a classic book chapter about lateral gene transfer called "If the Tree of Life Fell, Would We Recognize the Sound?") estimates that if a horizontal gene transfer occurs once every ten billion vertical replications, "it would be enough to ensure that no gene in any modern genome has an unbroken history of vertical descent back to some hypothetical last universal common ancestor." (See this article.)

It's obvious (to me, at least) that every rain event carries with it the potential to cause far more gene transfers than are necessary (according to Doolittle) to make vertical inheritance fade into insignificance as an evolutionary bringer of change. The hydrogen peroxide in rain has been driving lateral gene transfer in bacteria for eons. In fact, it is arguably the dominant driver of evolution in bacteria.

Sorry, Mr. Darwin. Point mutations handed down to sons and daughters just isn't cutting it.