When did nylon-eating proteins actually evolve the ability to eat nylon?

It has been widely advertised that nylon eating genes evolved after 1940. I have no problem with that claim in principle since new antibiotic and malaria resistances have evolved since 1940. Even though I can easily accept the possibility of post-1940 nylon-eating evolution in principle, where is the slam dunk evidence that this is actually the case? Did a significant portion of the ability for bacteria to digest nylon take place after 1940 (or 1935 when nylon was first created)?

In an NCSE article New Proteins Without God’s Help we read:

Since nylon first came into commercial production in 1940, we know that the new enzymes have formed since that time.

A similar line of thinking was argued at BioLogos in Biological Information and Intelligent Design, De Novo or Ex Nihilo.

Three nylon eating genes NylA, NylB, NylC were discovered on the Flavobacteria plasmid pOAD2 from 1977-1992, but the researchers concede none of the three have significant sequence homology. Worse, in papers published in 2007, they reported other bacteria contain those same genes in their chromosomes. Unless the researchers have access to pre-1935 bacteria sitting in lab refrigerators, the claim that the genes actually evolved new proteins via mutation is dubious since we have no pre-1935 bacterial samples to actually do a comparison with, especially in the case of NylC. The claim that NylB’s nylon eating ability evolved by gene duplication from a non-functional NylB-prime gene could just as well be interpreted that a functionless NylB-prime gene is a defective copy of a functioning NylB gene!

What’s the proof new nylon eating genes actually evolved after 1940, or is it just speculation? Slam dunk proof would entail having strains of pre-1935 bacteria and then comparing it with the strains after 1935 that supposedly evolved new genes. Is that the case? No. Just speculation which began in 1977 but got less defensible over the next 40 years as more bacteria and non-sequence-homologous genes were discovered to have nylon eating capability.

At least we can credit Richard Lenski who can back up his claims of evolution because he has samples of bacteria in his lab before and after his creatures evolved, whereas the guys promoting the claim the nylon-eaters are new don’t have pre-1935 physical samples of the bacteria. The only pre-1935 samples of the bacteria they have are samples from their imagination.

Incidentally, it is worth pointing out the bacteria that is the centerpiece of the controversy has been mis-classified or misidentified, and thus the name of the bacteria has itself gone through some sort of macro evolution. It was first called Acromobacter gattatus, then renamed as Flavobacterium sp., but then renamed Arthrobacter sp. after it was realized it had been put in the wrong taxonomic category all along.

But anyway, here is the 1977 speculation that became uncritically accepted and exaggerated as fact for the last 40 years:

6-Aminohexanoic Acid Cyclic Dimer Hydrolase. A New Cyclic Amide Hydrolase Produced by Acromobacter guttatus KI 72

There are two possible mechanisms for an enzyme to be active towards unnatural synthetic substance such as 6-aminohexanoic acid cyclic dimer; one is that an unnatural compound is hydrolyzed as an analogue of the physiological substrate, and the other is that the compound is hydrolyzed by an evolved enzyme which originally had an activity on a physiological substrate but lost it by the evolutionary mutation. The data obtained in this experiment indicate that this enzyme did not hydrolyze any physiological substrates tested including peptides, cyclic amides, and amides. In addition to the lack of activity on natural compounds, its low turnover number (8 SKcI) compared to other cyclic amide hydrolases (35 – 260 s ~ for penicillinase [12]) supports the possibility that the enzyme has evolved by adaptation to a new synthetic substance which is a waste product of nylon-6 production.

I have no problem if they say “the enzyme has evolved by adaptation to a new synthetic substance which is a waste product of nylon-6 production” when they mean an existing enzyme has changed its pre-existing function to a different one. That is to say, it acquires a new function while also losing its previous one. However, I would have serious issue with the NCSE’s insinuation that this was such a spectacular change that it illustrates how to solve the origin things on the order of the spliceosome or ribosome.

They don’t have strains sitting in a refrigerator from which to compare the “newly evolved” genes with do they? They just guessed, and then 30 years later one of the same team (Negoro) found nylon eating genes on other bacteria, plus reasonable nylon-eating homologues on other bacteria to boot.

Worse, the claim that these are post-1935 genes is weakened by some facts that require a little elaboration. First there is the basic nylon monomer that forms the components of nylon oligomers. NylA, NylB, NylC have different specialties of which nylon oligomer they can degrade. These three enzymes provide what looks like a digestive cascade where N-oligomer substrates are degraded by NylC to dimers which can be further degraded by NylB (and even NylC but to lesser extent). The cascade is described in: Biodegradation of nylon oligomers.

But important to note: NylA, NylB, NylC are not sequence homologous. In A New Nylon Oligomer Degradation Gene (nylC) on Plasmid pOAD2 from a Flavobactenium sp we read:

Sequence alignment by the method of Wirbur and Lipman (21) showed no significant homology among the
nylA, nylB, and nylC genes. These results suggest that the three nylon oligomer-degradative enzymes evolved independently.

There is no pre-1935 bacteria from which to compare with to establish NylC’s true history, much less anything beyond speculation about the nature of NylC’s ancestor. So how can anyone claim it is new (as in post-1935-new)? Just guesses. They’d have to argue these the genes evolved independently since 1935, and in the case of NylC, the ancestor is almost totally without detailed description.

They however argue, a functional NylB arose from a non-functioning NylB-prime gene via gene duplication in a 1991 paper entitled: Evolutionary adaptation of plasmid-encoded enzymes for degrading nylon oligomers.

But for all we know NylB’ is a defective copy of a functioning NylB! Where is a Ken-Miller-pseudo-gene-like argument when you need it?

In fairness, another bacteria called Pseudomonas was shown in lab conditions to acquire enzymatic nylon-eating function after a few months. The gene however in Pseudomonas, according to BLASTN, has no sequence homology with Flavobacteria. However, the protein according to BLASTP can align 91% at 37% homology. But even the researchers were astonishingly vague about what actually changed in the bacteria that enabled nylon degredation. The two relevant papers are: Characterization of the 6-aminohexanoate-dimer hydrolase from Pseudomonas sp. NK87 and Emergence of Nylon Oligomer Degradation Enzymes in Pseudomonas aeruginosa PAO through Experimental Evolution.

Unless we have strains sitting in a refrigerator of pre-1935 bacteria, how do we know that the genes actually evolved to digest bacteria? For all we know, it had that capability already or very close to it. And unless we have genes from all existing bacteria in 1935 and can prove all the bacteria on the globe do not have horizontally-transferrable nylon eating genes which could end up on the plasmids of Flavobacteria, we can’t unequivocally argue the three nylon eating genes (NylA, NylB, NylC) in Flavobacteria didn’t already exist pre-1935.

Even if the capability of nylon eating evolved, it is also not necessarily because of a new gene, but new regulation. A mini-example of this possibility had to be raised because even in their lab, they had bacteria with identical nylon eating genes but which could not digest nylon.

it is conceivable that expression of the nylC gene is enhanced in these strains and the elevated enzyme activities made the cells Nom+. However, the following possibilities could not be ignored: (i) KI725R strains may possess an additional nylon oligomer-degrading enzyme which is active
toward a substrate included in Noml, but K1725 has no degradative ability toward the substrate; (ii) nylon oligomer transport proteins are activated in K1725R strains; or (iii) the EIII proteins were altered by mutations in the coding region of the nylC gene by which the specific activities and/or
substrate specificity of the enzyme were changed. The last possibility should be negligible,

So my question is, if we ignore the NCSE hype, does the scientific literature really even prove unequivocally a significant part of nylon eating genes evolved after 1935 and absolutely rule out these genes pre-existed? Maybe yes, maybe no, but the NCSE can’t now brag that any significant evolution (like say compared to evolution of antibiotic resistance) bacteria definitely evolved after 1940 without God’s help. We simply don’t know.

NOTES:

Here are some accession numbers:

NylB (protein) in Flavobacteria: WP_012476894.1

which generates a BLASTP hit on Agromyces accession number: BAE97621.1

NylC (protein) in Flavobacteria: BAA01528

which generates a BLASTP hit also on Agromyces accession number: BAE97629.1

in Pseudomonas, the nylon eating gene accession numbers are : BAA01524 (protein), D10678.1 (DNA)

which generates BLAST hits on a variety of organisms as well

55 thoughts on “When did nylon-eating proteins actually evolve the ability to eat nylon?”

  1. stcordova Post author

    John Harshman:

    Or perhaps NylB’ is the duplicate. You need to separate the duplication process from the evolution of nylon-digesting activity. Either copy of the gene could have evolved that activity. I would suggest that it’s more likely that NylB is the original copy

    I concur. I somewhat (not exactly) suggested the possibility in the OP:

    The claim that NylB’s nylon eating ability evolved by gene duplication from a non-functional NylB-prime gene could just as well be interpreted that a functionless NylB-prime gene is a defective copy of a functioning NylB gene!

    You’ve refined, corrected, and improve my original speculation. You’re improvement however seems to suggest NylB’ came from NylB, but then NylB evolved, whereas I suggested NylB was already functional before it made a copy of NylB’, and then NylB’ de-evolved to something functionless. I can live with your alternate version, however.

    Thanks.

  2. stcordova Post author

    True, but Rubisco has two active sites. I think I read that NylB doesn’t (seem to?) in one of the papers. Don’t take my word for it, I have to go back and read it again.

    I believe Rubisco has one active site that can enable Carboxylase and Oxygenase activity. I got this from my Biochem textbook, Lehningher Principles of Biochem:

    Rubisco is not absolutely specific for CO2 as a substrate. Molecular oxygen (O2) competes with CO2 at the active site, and about once in every three or four turnovers, rubisco catalyzes the condensation of O2 with ribulose 1,5-bisphosphate to form 3-phosphoglycerate and 2-phosphoglycolate

    Nelson, David L.; Cox, Michael M.. Lehninger Principles of Biochemistry (Page 812). W.H. Freeman. Kindle Edition.

    Not that Rubisco has any necessary relevance, but it’s the only enzyme I can think of that can catalyze two fairly different reactions from the same active site. If you have a better enzyme to get the point across that nylonase activity possibly can coexist with other enzymatic functions, then I’ll happily use that example over Rubisco.

    If a NylB-like gene had been around millions of years, I have to imagine it was doing something before it got co-opted to eat nylon. How much it had to change before getting co-opted, we don’t know. There is a chance it didn’t change at all post-1935 but was used to catalyze a different reaction than nylon degredation pre-1935.

    Just for completeness:

    The enzyme that catalyzes incorporation of CO2 into an organic form is ribulose 1,5-bisphosphate carboxylase/oxygenase, a name mercifully shortened to rubisco. As a carboxylase, rubisco catalyzes the covalent attachment of CO2 to the five-carbon sugar ribulose 1,5-bisphosphate and cleavage of the unstable six-carbon intermediate to form two molecules of 3-phosphoglycerate, one of which bears the carbon introduced as CO2 in its carboxyl group (Fig. 20–4). The enzyme’s oxygenase activity is discussed in Section 20.2.

    Nelson, David L.; Cox, Michael M.. Lehninger Principles of Biochemistry (Page 802). W.H. Freeman. Kindle Edition.

  3. John HarshmanJohn Harshman

    stcordova,

    Sal, let me clarify. I don’t think that NylB’ is nonfunctional or that the precursor of NylB (the gene before it gained the nylon-eating adaptations) was nonfunctional. I suspect they were both functional, just not as nylon-eaters. At least there is no particular reason to hypothesize non-functionality for either of the genes, now or in the past.

  4. stcordova Post author

    John,

    Thanks for the clarification and thanks so much for taking the time to participate. The hypothesis that NylB came from NylB’ was put on the table before the discovery of NylC on the same plasmid. It may be that the technology wasn’t as good back then as it is now, so that’s probably why NylC was missed for almost a decade.

    The next thing on my list of things to do is to look more carefully at Ohno’s frameshift hypothesis and then the NylA gene. It may be a few days before I get back on this thread since there is so much to read and read carefully.

    I didn’t intend this thread to be contentious, just a review and discussion of what was actually published over the last 40 years and trying to understand the claims and looking at which claims may now be obsolete.

    Thanks for participating. I hope to drop in again after I’ve read some more.

  5. stcordova Post author

    From the paper on Agromyces,

    NylA has not been found in Agromyces sp. KY5R by either enzyme assays of cell extracts or by Western blot analysis (9) using antiserum for p2-NylA (pOAD2-encoded NylA). Moreover, Southern blot analysis (9) using the p2-nylA probe exhibited no hybridization signal against that of the total DNA fragments from KY5R (data not shown). These results suggest that the absence of NylA activity in KY5R cells is not the result of the repression of gene expression, but rather the result of the absence of the responsible genes. Owing to the lack of NylA activity, strain KY5R does not degrade the Ahx-cyclic dimer, but degrades Ahx-linear oligomers and Ahx-cyclic oligomers (degree of polymerization >3) through the cooperative action of NylB and NylC (8). Therefore, we focused further analysis on the nylB/nylC regions

    So, I take that as evidence the pOAD2 plasmid did not originate from Agromyces. It would appear, the genes in Agromyces were integrated from some other source, possibly a plasmid like pOAD2.

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