Refuting Creationism - More Evidence of Endosybiosis in Progress

A group of diatoms with their fluorescently-labeled symbionts.

© Max Planck Institute for Marine Microbiology Bremen/Mertcan Esti

The rhizobial nitrogen fixing symbionts (fluorescently-labeled in orange and green using genetic probes) residing inside diatoms collected from the tropical North Atlantic. The nucleus of the diatom is shown in bright blue.

© Max Planck Institute for Marine Microbiology Bremen/Mertcan Esti

Long-standing marine mystery solved: How algae get nitrogen to grow

Readers my remeber my article about how a team of scientists have discovered a new cell organelle in the process of transforming from a free-living nitrogen-fixing bacterium to becoming an endosymbiont of a marine alga, in much the same way that cyanobacteria became the chloroplasts of plant cells and rickettsia bacteria became the mitochondria of all eukaryote cells.

Now a different team, from the Max Planck Institute for Marine Microbiology, the Alfred Wegener Institute and the University of Vienna, have reported on a similar phenomenon in the form of a bacterium closely related to the Rhizobia that form a symbiotic association with leguminous plants such as peas and bean, which has teamed up with a marine diatom. This symbiotic relationship involves the bacterium living within the single cell of the diatom, unlike the relationship between Rhizobia and legumes in which the bacteria live in special nodes on the roots of the plants, but not inside the plant cells as such.

What is the chemical pathway by which nitrogen-fixing bacteria convert atmospheric nitrogen into ammonia and nitrates? Nitrogen-fixing bacteria play a crucial role in the nitrogen cycle by converting atmospheric nitrogen (N₂) into ammonia (NH₃), which can be further converted into nitrates (NO₃⁻) by other microorganisms. The chemical pathway for nitrogen fixation primarily involves the enzyme nitrogenase, which catalyzes the reduction of atmospheric nitrogen to ammonia. Here is an overview of the pathway:

1. Nitrogen Fixation
   Nitrogenase Enzyme Complex:
   • Components: The nitrogenase complex consists of two main proteins: the iron (Fe) protein and the molybdenum-iron (MoFe) protein.
   • Reaction:
     \[ \begin{equation*} \begin{aligned} \text{N}_2 + 8H^+ + 8e^- + 16ATP & \\ \rightarrow 2NH_3 + H_2 + 16ADP + 16Pi & \end{aligned} \end{equation*} \]
   • Steps:
     1. Electron Donation: Electrons are donated from ferredoxin or flavodoxin to the Fe protein.
     2. ATP Hydrolysis: ATP binds to the Fe protein and is hydrolyzed, providing energy for the transfer of electrons.
     3. Electron Transfer: Electrons are transferred from the Fe protein to the MoFe protein.
     4. Nitrogen Reduction: The MoFe protein reduces atmospheric nitrogen (N₂) to ammonia (NH₃) in a series of steps involving the binding and reduction of N₂.
2. Ammonia Assimilation
   Once ammonia is produced, it can be assimilated into organic compounds or further processed into other nitrogenous compounds.
   
   Conversion to Nitrates
   The conversion of ammonia to nitrates occurs in two steps involving nitrifying bacteria:
   
   
   Step 1: Ammonia to Nitrite
   Bacteria Involved: Ammonia-oxidizing bacteria (AOB), such as Nitrosomonas. \[ \text{NH}_3 + \text{O}_2 \rightarrow \text{NO}_2^- + 3H^+ + 2e^- \] Step 2: Nitrite to Nitrate
   Bacteria Involved: Nitrite-oxidizing bacteria (NOB), such as Nitrobacter. \[ \text{NO}_2^- + \frac{1}{2}\text{O}_2 \rightarrow \text{NO}_3^- \]

Summary of Pathway

1. Nitrogen Fixation: Atmospheric nitrogen (N₂) is converted to ammonia (NH₃) by nitrogenase enzyme complex in nitrogen-fixing bacteria.
2. Ammonia Assimilation: Ammonia can be incorporated into organic molecules or further processed.
3. Nitrification: Ammonia is first oxidized to nitrite (NO₂⁻) by AOB and then nitrite is oxidized to nitrate (NO₃⁻) by NOB.

This pathway is essential for making atmospheric nitrogen available to plants and other organisms in a usable form, thereby sustaining the nitrogen cycle in ecosystems.

This association in the oceans accounts for the supply of fixed nitrogen in the seas which is then available for plant and ultimately animal life.

Of course, any intelligent designer would have given the diatoms the necessary genes to fix nitrogen themselves, just as it would have given legumes the same ability, but evolution, in its haphazard, unplanned and unintelligent way often produces sub-optimal, overly complex solutions to problems simply because that solution was better than what went before; there is no intelligence to think about using processes designed earlier or of the elegance of the solution.

The discovery is the subject of an open access paper in Nature and a press release from Vienna University (Universität Wien):

Long-standing marine mystery solved: How algae get nitrogen to grow

Newly discovered symbiosis between Rhizobia and diatoms could also open new avenues for agriculture

In a new study, scientists from the Max Planck Institute for Marine Microbiology, the Alfred Wegener Institute and the University of Vienna shed light on an unexpected partnership: A marine diatom and a bacterium that can account for a large share of nitrogen fixation in vast regions of the ocean. This symbiosis likely plays a key role for global marine nitrogen fixation and productivity, and thus uptake of carbon dioxide. The newly-discovered bacterial symbiont is closely related to the nitrogen-fixing Rhizobia which live in partnership with many crop plants and may also open up new avenues for engineering nitrogen-fixing plants. The results were published in the current print edition of the renowned journal Nature.

Nitrogen is an essential component of all living organisms. It is also the key element controlling the growth of crops on land, as well as the microscopic oceanic plants that produce half the oxygen on our planet. Atmospheric nitrogen gas is by far the largest pool of nitrogen, but plants cannot transform it into a usable form. Instead, some crop plants like soybeans, peas and alfalfa (collectively known as legumes) have acquired Rhizobial bacterial partners that "fix" atmospheric nitrogen into ammonium, which can be used by plants. This partnership makes legumes one of the most important sources of proteins in food production.

Yet, how marine plants obtain the nitrogen they need to grow has not yet been fully clarified. Scientists from the Max Planck Institute for Marine Microbiology, the Alfred Wegener Institute and the University of Vienna now report that Rhizobia can also form similar partnerships with tiny marine plants called diatoms – a discovery that solves a long-standing marine mystery and which has potentially far-reaching agricultural applications.

An enigmatic marine nitrogen fixer hiding within a diatom

For many years it was assumed that most nitrogen fixation in the oceans was carried out by photosynthetic organisms called cyanobacteria. However, in vast regions of the ocean there are not enough cyanobacteria to account for measured nitrogen fixation. Thus, many scientists hypothesized that non-cyanobacterial microorganisms must be responsible for the "missing" nitrogen fixation.

For years, we have been finding gene fragments encoding the nitrogen-fixing nitrogenase enzyme, which appeared to belong to one particular non-cyanobacterial nitrogen fixer but, we couldn’t work out precisely who the enigmatic organism was and therefore had no idea whether it was important for nitrogen fixation.

Marcel M. M. Kuypers, lead author
Max Planck Institute for Marine Microbiology, Bremen, Germany

In 2020, the scientists travelled from Bremen to the tropical North Atlantic to join an expedition involving two German research vessels. They collected hundreds of liters of seawater from the region, in which a large part of global marine nitrogen fixation takes place, hoping to both identify and quantify the importance of the mysterious nitrogen fixer. It took them the next three years to finally puzzle together its genome.

It was a long and painstaking piece of detective work but ultimately, the genome solved many mysteries.

Bernhard Tschitschko, first author
Max Planck Institute for Marine Microbiology, Bremen, Germany Now: Department of Microbiology
University of Innsbruck, Innsbruck, Austria.

Based on the nitrogenase gene fragment we had seen in many marine samples before, one would have expected to find this gene in a Vibrio-related organism, but by carefully piecing together the genetic information it turned out that instead, it belonged to a genome closely related to known Rhizobia, which typically live in symbiosis with legume plants.

Daan R. Speth, co-author
Max Planck Institute for Marine Microbiology, Bremen, Germany Now: Centre for Microbiology and Environmental Systems Science
Division of Microbial Ecology
University of Vienna, Vienna, Austria,

Together with its surprisingly small genome, this raised the possibility that the marine Rhizobia might be a symbiont.

The first known symbiosis of this kind

Spurred on by these discoveries, the authors developed a genetic probe which could be used to fluorescently label the Rhizobia.

This allowed us to visualize the Rhizobia directly in their native habitat - the complex environmental samples collected in the Atlantic.

Katharina Kitzinger, co-author
Max Planck Institute for Marine Microbiology, Bremen, Germany
Now: Centre for Microbiology and Environmental Systems Science
Division of Microbial Ecology
University of Vienna, Vienna, Austria.

Indeed, their suspicions about it being a symbiont were quickly confirmed.

We were finding sets of four Rhizobia, always sitting in the same spot inside the diatoms. It was very exciting as this is the first known symbiosis between a diatom and a non-cyanobacterial nitrogen fixer.

Marcel M. M. Kuypers.

The scientists named the newly discovered symbiont Candidatus Tectiglobus diatomicola. Having finally worked out the identity of the missing nitrogen fixer, they focused their attention on working out how the bacteria and diatom live in partnership. Using a technology called nanoSIMS, they could show that the Rhizobia exchanges fixed nitrogen with the diatom in return for carbon. And it puts a lot of effort into it:

In order to support the diatom’s growth, the bacterium fixes 100-fold more nitrogen than it needs for itself.

Wiebke Mohr, co-author
Max Planck Institute for Marine Microbiology, Bremen, Germany

A crucial role in sustaining marine productivity

Next the team turned back to the oceans to discover how widespread the new symbiosis might be in the environment. It quickly turned out that the newly discovered partnership is found throughout the world’s oceans, especially in regions where cyanobacterial nitrogen fixers are rare. Thus, these tiny organisms are likely major players in total oceanic nitrogen fixation, and therefore play a crucial role in sustaining marine productivity and the global oceanic uptake of carbon dioxide.

A key candidate for agricultural engineering?

Aside from its importance to nitrogen fixation in the oceans, the discovery of the symbiosis hints at other exciting opportunities in the future. Kuypers is particularly excited about what the discovery means from an evolutionary perspective.

The evolutionary adaptations of Ca. T. diatomicola are very similar to the endosymbiotic cyanobacterium UCYN-A, which functions as an early-stage nitrogen-fixing organelle. Therefore, it’s really tempting to speculate that Ca. T. diatomicola and its diatom host might also be in the early stages of becoming a single organism.

Marcel M. M. Kuypers.

Tschitschko agrees that the identity and organelle like nature of the symbiont is particularly intriguing.

So far, such organelles have only been shown to originate from the cyanobacteria, but the implications of finding them amongst the Rhizobiales are very exciting, considering that these bacteria are incredibly important for agriculture. The small size and organelle-like nature of the marine Rhizobiales means that it might be a key candidate to engineer nitrogen-fixing plants someday.

Bernhard Tschitschko

The scientists will now continue to study the newly discovered symbiosis and see if more like it also exist in the oceans.

Original publication:
Bernhard Tschitschko, Mertcan Esti, Miriam Philippi, Abiel T. Kidane, Sten Littmann, Katharina Kitzinger, Daan R. Speth, Shengjie Li, Alexandra Kraberg, Daniela Tienken, Hannah K. Marchant, Boran Kartal, Jana Milucka, Wiebke Mohr, Marcel M. M. Kuypers (2024): Rhizobia-diatom symbiosis fixes missing nitrogen in the ocean. Nature (2024) DOI: 10.1038/s41586-024-07495-w

Participating institutions:

• Max Planck Institute for Marine Microbiology, Bremen, Germany
• Alfred Wegener Institute - Helmholtz-Centre for Polar and Marine Research, Bremerhaven, Germany
• University of Vienna, Vienna, Austria

Abstract
Nitrogen (N₂) fixation in oligotrophic surface waters is the main source of new nitrogen to the ocean¹ and has a key role in fuelling the biological carbon pump². Oceanic N₂ fixation has been attributed almost exclusively to cyanobacteria, even though genes encoding nitrogenase, the enzyme that fixes N₂ into ammonia, are widespread among marine bacteria and archaea^3,4,5. Little is known about these non-cyanobacterial N₂ fixers, and direct proof that they can fix nitrogen in the ocean has so far been lacking. Here we report the discovery of a non-cyanobacterial N₂-fixing symbiont, ‘Candidatus Tectiglobus diatomicola’, which provides its diatom host with fixed nitrogen in return for photosynthetic carbon. The N₂-fixing symbiont belongs to the order Rhizobiales and its association with a unicellular diatom expands the known hosts for this order beyond the well-known N₂-fixing rhizobia–legume symbioses on land⁶. Our results show that the rhizobia–diatom symbioses can contribute as much fixed nitrogen as can cyanobacterial N₂ fixers in the tropical North Atlantic, and that they might be responsible for N₂ fixation in the vast regions of the ocean in which cyanobacteria are too rare to account for the measured rates.

Main
Nitrogen is an essential component of all living organisms and limits life in the ocean. Atmospheric N₂ gas is the largest reservoir of freely accessible nitrogen, but it is biologically available only to microorganisms that carry the nitrogenase metalloenzyme and thus can fix N₂ into ammonia⁷. Even though a wide diversity of marine bacteria and archaea encode nitrogenase, the bulk of nitrogen fixation in the ocean has been attributed to cyanobacteria (ref. ⁴ and references therein). These phototrophs are capable of both free-living and symbiotic lifestyles, and can directly or indirectly contribute to carbon fixation and export production in the regions where they are abundant, such as oligotrophic coastal waters and margins of subtropical gyres⁸. Notably, in vast regions of the ocean, such as the centres of subtropical gyres, cyanobacterial N₂ fixers are too rare to account for the measured rates of N₂ fixation. Instead, a role of non-cyanobacterial N₂ fixers has been invoked, on the basis of the abundance of nitrogenase-encoding gene sequences (nifH), most of which belong to uncultured proteobacteria (for example, refs. ^3,5,9,10,11). So far, the most frequently detected non-cyanobacterial N₂ fixer is the so-called gamma-A, named after its nifH gene phylogeny that clusters within the Gammaproteobacteria¹². This enigmatic microorganism has been shown to be distributed in most world oceans, and its potential activity has been inferred from in situ nifH transcription^13,14. To date, however, there is no proof that gamma-A fixes N₂ in situ, and essentially all aspects of its physiology remain unknown.

An N₂-fixing rhizobial diatom endophyte We investigated the role of non-cyanobacterial N₂ fixation in the tropical North Atlantic during an expedition in January–February 2020. This region is responsible for around 20% of oceanic N₂ fixation⁸, and cyanobacteria can only explain approximately half of the rates measured in the region¹⁰. We detected high N₂ fixation rates of up to 40 nmol N l⁻¹ d⁻¹ in the surface waters (Extended Data Table 1), and the presence of both cyanobacterial and heterotrophic N₂ fixers—specifically, gamma-A—was confirmed by metagenomic sequencing (Extended Data Fig. 1a). Gamma-A nifH sequences were retrieved only from the large size fraction (greater than 3 µm) suggesting particle attachment or an association with a host organism (Extended Data Fig. 1a). We recovered a near-complete metagenome-assembled genome (MAG; 1.7 Mb, 37.8% GC, 98% completion with 0% redundancy) containing the gamma-A nifH gene, as well as a complete cluster of rRNA genes (Supplementary Table 1). Although the retrieved nifH sequence clustered within the Gammaproteobacteria as previously reported^3,14,15 (Extended Data Fig. 2), both 16S-rRNA-gene-based and whole-genome-based taxonomy¹⁶ firmly placed this MAG within the alphaproteobacterial family Hyphomicrobiaceae (Fig. 1a). This family belongs to the order Rhizobiales, which comprises the prominent rhizobial symbionts of nodule-forming terrestrial legumes^6,17,18. In addition to nifH, most other genes of the nif regulon are of gammaproteobacterial origin, including nifD and nifK, which encode the catalytic component of the nitrogenase; nifE, nifN and nifB, which encode the iron-molybdenum cofactor assembly proteins; and nifS, which is involved in metallocluster biosynthesis (Extended Data Fig. 2a). Almost all other genes in the gamma-A MAG are of alphaproteobacterial origin (Supplementary Table 1). On the basis of these results, we conclude that the gamma-A N₂ fixer is, in fact, an alphaproteobacterium that has acquired its nitrogenase genes through horizontal gene transfer from a gammaproteobacterial donor. Besides gamma-A, several other bacteria, including members of the order Rhizobiales, obtained their nitrogenase genes through horizontal gene transfer from a gammaproteobacterial donor (Extended Data Fig. 2b). Such horizontal gene transfer across classes, resulting in the acquisition of nitrogenase genes, has been reported previously for other N₂ fixers^19,20.

Fig. 1: Phylogeny and visualization of Candidatus Tectiglobus diatomicola and its diatom host.

a, Maximum likelihood phylogenetic tree of concatenated bacterial marker genes from the order Rhizobiales, showing the placement of Ca. T. diatomicola within the Hyphomicrobiaceae family (see Methods). The novel genus Ca. Tectiglobus, comprising Ca. T. diatomicola and its closest relative Ca. T. profundi, is highlighted in pink. Families within the Rhizobiales that contain known N₂-fixing legume symbionts and their exemplary host plants are shown. The order Parvibaculales was used as an outgroup. Black dots indicate more than 95% bootstrap support. Scale bar indicates amino acid substitutions per site. Plant icons were designed by Freepik (Neptunia oleracea) or created with BioRender.com. b,c, False coloured scanning electron microscopy (SEM) image (b) and confocal laser scanning microscopy image (c) of a Haslea diatom. Four Ca. T. diatomicola cells (pink, overlay of Hypho1147 and Hypho734 fluorescence in situ hybridization (FISH) probes; Extended Data Table 2) were detected next to the host nucleus (white; stained with DAPI). Scale bars, 5 µm.

We name the newly discovered species ‘Candidatus Tectiglobus diatomicola’ within a novel genus ‘Candidatus Tectiglobus’ (see Methods for etymology). One other marine MAG from the North Pacific, which we now name ‘Candidatus Tectiglobus profundi’, is affiliated with this novel genus, with 72% average amino acid identity with Ca. T. diatomicola (Supplementary Methods). Compared with their closest relative, a MAG from the Mediterranean Sea, both Ca. Tectiglobus species have a substantially reduced genome size (around 1.7 Mb versus around 5 Mb) and a strongly decreased GC content (around 38% versus around 54%) (Extended Data Fig. 3), which are features typical of endosymbionts²¹. Notably, a similar reduction in genome size and GC content is observed for the N₂-fixing cyanobacterial endosymbiont Candidatus Atelocyanobacterium thalassa, or UCYN-A, which lives in symbiosis with a haptophyte alga^22,23. Thus, the genome properties of Ca. T. diatomicola, together with its presence in the large size fraction, strongly indicate a host-associated lifestyle.

A couple of points here for creationists to ignore or lie about:

• The authors show no doubts that this is an evolutionary process and show no evidence of adopting creationist superstition as a better explanation.
• The endosymbionts have a much reduced genome compared to their free-living relatives, showing that their evolution has involved a loss of genetic information typical of both endosymbionts and endo-parasites, despite the creationist assertion that a loss of genetic information is either fatal or 'devolutionary' [sic]
• This process illustrates the processes involved in the earlier evolution of biodiversity which produced eukaryote cells from associations of prokaryotes, which itself illustrates how 'selfish' genes form co-operative alliances with other 'selfish' genes, despite creationists assertions that selfish genes can only produce selfish individuals.

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