The Tails For Metamorphosis

ResearchBlogging.orgMarine tubeworms are sessile organisms found in variety of ocean ecosystems. Among the many different species of tubeworms some are real record breakers. If you ever heard of a tubeworm then you most likely have heard about Riftia pachyptila, usually called the Giant tubeworm, which can reach more than 2m in length and lives near deep-sea volcanoes and hydrothermal vents. The Giant tubeworms can experience temperature changes from the near boiling waters coming off the hydrothermal vents to just above freezing temperatures of the deep-sea waters. Tubeworms don’t have digestive system and rely on microbes to provide ready-made nutrients. In a special organ, called trophosome, tubeworms harbour thousands of symbiotic bacteria which provide them food. In the case of the Giant tubeworm these bacteria are adapted to metabolise sulphur-rich compounds (coming from the vent fluid), which ultimately leads to production of organic molecules that are taken up by the tubeworm.

Riftia pachyptila
Riftia pachyptila

Many marine invertebrates that live close to or at the bottom of a body of water (known as benthic environment) release swimming larvae during their life cycle. These larvae then have to find a place to attach and settle, whether that’s a patch of sand or a corral. After settlement the larvae metamorphose to become an adult. While this may sound a straightforward thing to do- just find some nice and empty place- there’s actually a whole symbiotic tail (yes, tail, not tale, though both nouns would work here) that goes into picking the right spot.

A marine tubeworm Hydroides elegans has been used for many years to answer the question: what environmental cues are used by tubeworms for finding the right settlement place. In early studies it was noted that a homogenate of adult tubeworms could induce settlement and metamorphosis of the H. elegans larvae. This observation has led to suggest that perhaps adults are releasing some chemical clues which act as attractants for young larvae. No such chemicals were ever found, however, it was noted that addition of antibiotics to the adult homogenate would lead to substantial reduction in larval metamorphosis. This and further investigations have later showed that bacterial biofilms present on benthic surfaces are the key to metamorphic induction in tubeworm larvae.

Bacterial biofilms are essentially close-knit layers of bacteria that are associated with surfaces. Biofilms are found in all sorts of places: on our teeth, in our lungs, on various household surfaces and, as you perhaps have deduced from the passage above, also on the various surfaces in the sea. But what is it about the biofilms that attracts tubeworms such as H. elegans?

A marine bacterium called Pseudoalteromonas luteoviolacea (HI1) can form biofilms and induce H. elegans metamorphosis in laboratory environment. In a study published by Huang et al. in 2011 scientists took HI1 and created a library of HI1 mutants using a transposon mutagenesis technique. In this library each HI1 has a transposon inserted into a random site of their genome. Consequently, if the transposon has been inserted into a site of a coding gene or a regulatory sequence the function of either one is lost. After screening the ability of individual mutants in HI1 library to induce H. elegans metamorphosis the scientists found two mutants that were no longer able the do so. In both mutants transposon insertion was revealed to be in an operon consisting of 7 different genes. When individually mutated 4 of the genes in this operon were found to be essential for H. elegans metamorphosis. Notably, deletion of only one of these four genes affected the structure of biofilm (making it denser and increasing its mass), whereas deletion mutants of all other genes retained wild type biofilm phenotype.

Mutant strains of HI1 (Plm9 and Plm45 ) can no longer induce H. elegans metamorphosis as compare to wild type strain (P.l.) or natural biofilm (NB) and fresh water (FSW) as negative control.
Mutant strains of HI1 (Plm9 and Plm45 ) can no longer induce H. elegans metamorphosis as compared to wild type strain (P.l.) or natural biofilm (NB). Autoclaved filtered seawater (FSW) was used as negative control.

So what do these genes do and why are they needed? Well, the 2011 study did not address this question directly apart from looking at the sequences of these genes and searching for the most similar sequences present in other organisms using bioinformatics. Their analysis suggested that one of the genes potentially codes for an adhesin-like protein, another gene did not have any recognisable sequence and the two remaining genes had domains which looked similar to phage tail-like proteins.

A more recent study by Shikuma et al. from 2014 has looked more closely at the genes identified by Huang et al. as well as at the region in the genome of HI1 that these genes are located. It turns out that the previously identified genes are adjacent to a set of genes that code for bacteriocins. Bacteriocins are made up of phage tail-like proteins and are used by bacteria to puncture membranes of other bacteria. Mechanistically bacteriocin works like a tube with a needle inside, the outer tube attaches to the surface of a target bacterium and then the needle inside it is pushed through and punctures the bacterial membrane. The final outcome is essentially a flat tyre left from the target bacterium. We say bacteriocin is made of ‘phage tail-like proteins’ because the proteins it is composed of highly resembles the structure of bacteriophage without its head domain and it is thought that the two have a common evolutionary origin. Based on their predicted roles Shikuma et al. have called the bacteriocin genes that they identified the metamorphosis-associated contractile structure (mac) genes.

This is what bacteriophage looks like on the left and how bacteriocin looks like on the right
This shows how bacteriophage looks like on the left and how bacteriocin looks like on the right.

To investigate if mac genes are required for H. elegans metamorphosis deletion mutants of HI1 were made for each individual member of mac cluster. It was observed that deletion of mac members led to inhibition of H. elegans metamorphosis.

 

H. elegans larva (A) and juvenile adult (B) 12 hours after exposure to a P. luteoviolacea biofilm. Growth of HI1 is not affected by mac deletion mutants (C). Metamorphosis of H. elegans is inhibited if HI1 lacks mac genes (D)
H. elegans larva (A) and juvenile adult (B) 12 hours after exposure to a HI1 biofilm. Growth of HI1 is not affected by mac deletion mutants (C). Metamorphosis of H. elegans is inhibited if HI1 lacks mac genes (D)

Using electron microscopy Shikuma et al. could also observe phage tail–like structures in the extracellular space and tagging one of the mac members with a green fluorescence protein showed that it could also be secreted. Notably, some HI1 cells appeared to be potentially close to lysis caused by extensive aggregation of tail-like structures (MAC proteins) in their cytoplasm.

(A) MAC proteins packed inside HI1 cell (inner membrane; O, outer membrane; S, storage granule; white arrow, filamentous connections). (H) Reconstruction of MAC arrays coalescing in an amorphous core.
(A) MAC proteins packed inside HI1 cell (inner membrane; O, outer membrane; S, storage granule; white arrow, filamentous connections). (H) Reconstruction of MAC arrays coalescing in an amorphous core.

As an ultimate indicator that these MACs can independently induce H. elegans morphogenesis a careful cell-free preparation of purified MACs was incubated together with H. elegans larvae. As predicted the larvae underwent metamorphosis in the presence of purified MACs but not if the inoculum was filtered through a filter that prevented proteins of the predicted MAC member size passing through. Interestingly, same MAC extracts did not have bactericidal effect when incubated with a bacterium related to HI1 suggesting that either they have lost their bactericidal activity or can only work under different conditions.

It is still not clear exactly how MACs interact with H. elegans to induce metamorphosis. Mac like genes are found in other marine bacteria that can induce H. elegans metamorphosis as well and could potentially have similar functions. Then again, as the authors note, we don’t yet know what benefit, if any, association with tubeworms gives to HI1 and similar species. It may well be that MACs have some other function in HI1 altogether that is independent of association with tubeworms and were somehow only secondarily co-opted as ‘metamorphosis factors’. It seems that this tale of tails still waits stories to be told.

Shikuma NJ, Pilhofer M, Weiss GL, Hadfield MG, Jensen GJ, & Newman DK (2014). Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science (New York, N.Y.), 343 (6170), 529-33 PMID: 24407482

Huang, Y., Callahan, S., & Hadfield, M. (2012). Recruitment in the sea: bacterial genes required for inducing larval settlement in a polychaete worm Scientific Reports, 2 DOI: 10.1038/srep00228

Hadfield, Michael. (2001-06-13) Natural Chemical Cues for Settlement and Metamorphosis of Marine-Invertebrate Larvae. , 431-461. DOI: 10.1201/9781420036602.ch13

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