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Old 01/04/2016, 08:01 AM   #2511
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But on the other hand, they're known associates of toxic dinos and apparently part of the benthic dino holobiont -- a real bummer for me, as the easy story here would be, "Ostis like vibrio, and rosies don't like vibrio; therefore, rosies don't like ostis." So I tried to figure out if there was a common element to the association of Roseobacter Clade bacteria with dinos and corals, which of course have dinoflagellate endosymbionts...

Originally Posted by Diversity and dynamics of bacterial communities in early life stages of the Caribbean coral Porites astreoides
In this study, we examine microbial communities of early developmental stages of the coral Porites astreoides... Bacteria are associated with the ectoderm layer in newly released planula larvae, in 4-day-old planulae, and on the newly forming mesenteries surrounding developing septa in juvenile polyps after settlement. Roseobacter clade-associated (RCA) bacteria and Marinobacter sp. are consistently detected in specimens of P. astreoides spanning three early developmental stages, two locations in the Caribbean and 3 years of collection. ... The results are the first evidence of vertical transmission (from parent to offspring) of bacteria in corals. The results also show that at least two groups of bacterial taxa, the RCA bacteria and Marinobacter, are consistently associated with juvenile P. astreoides against a complex background of microbial associations, indicating that some components of the microbial community are long-term associates of the corals and may impact host health and survival. ...

The Rhodobacterales sequences in this study primarily represent the RCA group of bacteria. ... Roseobacter is an abundant and diverse genus in seawater communities, notably in coral reef habitats and coral mucus, and Roseobacter are among the first bacterial associates acquired from seawater by larvae of the Pacific spawning coral Pocillopora meandrina. Other RCA bacteria have been shown to exhibit antibacterial activity against known marine pathogens. Many RCA bacteria engage in associations with dinoflagellates in the marine environment and in laboratory culture. On the basis of the prevalence of chemotaxis toward dimethylsulfoniopropionate, a dinoflagellate metabolite, among RCA bacteria, it has been hypothesized that some RCA bacteria detected in corals are associated with Symbiodinium spp., though it is unknown whether this interpartner communication results in a fitness benefit to the Symbiodinium or to the coral host. Though RCA bacteria in some corals may provide potential benefits to the coral holobiont, other RCA strains have been found to be pathogenic, implicated in coral black band disease and juvenile oyster disease. Ribotypes identified in the clone libraries are affiliated (9899% sequence similiarity) with RCA sequences derived from disease bands in the coral Siderastrea siderea and Thalassomonas sp. and Rhodobacter sp. (9499% sequence similarity) associated with disease bands in other corals.
Emphasis mine.

Dimethylsulfoniopropionate, eh? Okay, obviously that needs to be looked into, but first and foremost, the link to BBD can't be ignored. What's the difference between rosies that protect corals and rosies that kill corals?

Originally Posted by Microbial community composition of black band disease on the coral host Siderastrea siderea from three regions of the wider Caribbean
Microbial communities associated with black band disease (BBD) on colonies of the reef building coral Siderastrea siderea from reefs in 3 regions of the wider Caribbean were studied... All of the clone libraries were dominated by Alphaproteobacteria and contained sequences associated with bacteria of the sulfur cycle... Additionally, all clone libraries had sequence types of bacteria associated with toxin producing dinoflagellates. These sequences were most abundant in a sewage impacted reef site in St. Croix, which also had the highest prevalence of BBD-infected colonies. ... We propose that with degrading water quality (i.e. increasing nutrients) certain proteobacteria thrive and increase BBD virulence. ...

BBD, first reported in the 1970s, is characterized by a dark, migrating band which moves across a living coral colony completely lysing coral tissue and often killing the entire colony in a matter of months. It is most accurately described as a horizontally migrating, cyanobacterial-dominated, sulfide-rich microbial mat but is unique in that it is pathogenic to corals and is motile. Thus far, in addition to the dominant cyanobacteria, sulfate-reducing bacteria (in particular Desulfovibrio spp.), sulfide-oxidizing bacteria, numerous heterotrophic bacteria, and fungi have been reported in the BBD community. Members of all of these groups have been proposed as primary pathogens; however, few have been isolated into culture and Kochs postulates have not been fulfilled for any of them. The fulfillment of Koch's postulates has classically been the 'gold standard' for verifying that a suspected microbial pathogen is responsible for a specific disease. ...

A total of 26 clone sequences, from all 3 regions, were related (96-99% similarity by BLAST analysis) to bacteria associated with paralytic shellfish toxin (PST) producing dinoflagellates. [...among bacteria, the rule of thumb is that >=97% genetic similarity is probably the same species; 93-96% is probably another species from the same genus] ... Twelve sequences from all 3 regions were closely related (98-99% similarity by BLAST analysis) to an uncultured Alphaproteobacterium (AJ294357) associated with the toxic dinoflagellate Alexandrium. ... Five sequences, 3 from the Bahamas, one from the Florida Keys, and one from St. Croix were closely related (94-97% similarity) to the Alphaproteobacterium (AF260726) associated with the toxic dinoflagellate Alexandrium tamarense, with this identification confirmed by phylogenetic analysis. In Clone Libraries A and F, 2 sequences (DQ446084, and EF123311, respectively) were closely related (98% similarity) to an Alphaproteoacterium (AY701434) associated with the toxic dinoflagellate Gymnodinium catenatum. Phylogenetic analysis confirmed that the 2 sequences were closely related to the Hyphomonas group of bacteria, some members of which are associated with toxic dinoflagellates. In the clone library from the polluted St. Croix site (F), a sequence (DQ644019) was related (98% similarity) to a Bacteroidetes bacterium (AY701462) associated with a toxic dinoflagellate. ...

Any specific role in the BBD community of the dominant Alpha-proteobacteria is not yet known. Because they are not well represented in healthy coral mucus and coral tissue, it is worth noting the abundance of Roseobacter clade members in BBD samples. ... A recent study showed that many of the Roseobacter species produce antibacterial compounds that inhibit non-Roseobacter species, which may contribute to the dominance of members of this clade in diverse environments, including BBD. ...

It may be that degraded water quality is causing a shift within the population of Alpha-proteobacteria to the more toxic representatives of this group. ...

Two previous molecular studies of the BBD community detected sequences matching Roseovarius crassostreae, the bacterial pathogen associated with JOD [juvenile oyster disease -- interestingly, this is the only pathogenic rosie I've found mention of aside from the ones involved with BBD]. In the present study this sequence was again found in a large number (15) of clones, with sequence homologies of 94 to 97%, from the Bahamas and St. Croix sites. ...

Sequence types matching bacteria associated with toxic dinoflagellates were observed in the clone libraries from all 3 geographic regions and have been reported previously by our group from Bahamas samples. These sequences were not observed in the healthy SML [surface mucus layer] of samples of the same coral colonies. The present study showed their consistent presence in all the BBD samples examined, irrespective of the sampling location.
If life were like the movies, this would be the turning point in the narrative when I have a crucial flash of insight: "By Jove, if bacteria associated with toxic dinos can kill corals, then it just might be that bacteria associated with healthy corals can kill. Toxic. Dino. Flagellates. To the Sciencemobile!"

But that's not how it works in the real world (...nor, alas, do I have a Sciencemobile). In the real world, I bumbled around for weeks trying to puzzle through this, discarding several theories along the way that didn't pan out for one reason or another, and even when I thought I had the answer, it still took a couple of months to write this up because more than once I tugged on a loose thread during fact-checking and a chunk of the thing unraveled and had to be put back together a little bit differently. For example, sorting out what the heck was going on with the CFB group and in particular the genus cytophaga took like two weeks all by itself... So even though you already know where I'm going with all this, I'm taking you there the long way around because that's the scenic route.

I read another paper on BBD from 2006 and was able to identify a rosie in uninfected coral mucus that produces a well-known antibacterial and antifungal compound called tropodithietic acid (TDA), but this species was not present in the BBD biofilm, and two of the BBD rosies were species known not to produce TDA. And another paper I read indicated that most TDA synthesis in vitro appeared to occur at the water's surface in stagnant cultures -- TDA synthesis is known to be energy-intensive, so it's likely that producing significant amounts of it requires enough oxygen for the bacteria to run entirely in aerobic mode. If there really are beneficial rosies in skimmate, that could explain why Montireef's fresh skimmate was dino death but week-old skimmate is dino chow: as bacterial respiration draws down dissolved O2, the coral-friendly rosies don't have enough oxygen to make TDA and thus lose the battle for dominance in biofilms growing on colloidal detritus in the skimmer cup. Perhaps TDA-making rosies are one of the keystone species corals recruit to maintain a friendly bacteria population in their mucus...

At any rate, there's still this dimethylsulfoniopropionate that dinos make and which attracts rosies. That doesn't look like a clue so much as a big, flashing, polychromatic neon arrow.

Dimethylsulfoniopropionate, aka DMSP, is produced by a great many different species of algae and is the single most common organic sulfur compound in NSW. Dinos and coccolithophores, in particular, are known to make a lot of DMSP for some reason -- even a few heterotrophic dinos have been shown to make the stuff.

Making DMSP on an industrial scale is a serious problem for a dino bloom because DMSP and/or its breakdown product DMS serve as chemotactic cues for a vast variety of species, ranging from many different species of bacteria (including vibrios and other gamma-pros as well as rosies) and protists all the way up the food chain to fish, penguins, and seals. It seems like freakin' everything in the ocean can detect and home in on DMSP, DMS, or both -- including pods, though DMSP may also deter pods from feeding... AFAIK the jury's still out on whether producing lots of DMSP is a net plus or minus for dinos on the getting-eaten-by-pods problem, but I'm guessing the latter, as DMSP production by dinos must be an obligate physiological process or they'd stop making so darn much of it, so if DMSP is a problem for pods, it's a problem they've had millions of years to solve. Pods can evolve resistance to dino toxins (some pods have been shown to concentrate dino toxins from red tides and pass them up the food chain) so why not DMSP? And BTW, not only symbiodinium dinos but coral polyps themselves make DMSP, so coral mucus is full of the stuff.

In other words, corals are pod magnets. Neat.

And corals are also vibrio magnets. In fact, there's N-acetyl-D-glucosamine (the monomers from which chitin is assembled) in coral mucus, which is not so neat.

So now the big, flashing, polychromatic neon arrow is surrounded by chase lights, flanked by dancing girls, and skylined by fireworks.

Nobody really knows why dinos make so much DMSP. It has several potential uses, including osmoregulation, deterring grazers, helping to protect photosynthesis machinery from being damaged by the oxygen it produces, and DMSP has been shown to inhibit the attachment of some bacteria to macroalgae, notably cytophaga which specialize in consuming high molecular weight organic carbon and thus are likely opportunistic pathogens that can exploit any existing damage or infection. You'd think DMSP would do the same for dinos, so perhaps DMSP is one of the biochemical tricks ostis use to keep their food from eating them.

The most convincing theory I found about DMSP is that synthesizing it consumes organic carbon and two sulfide ions, the fully reduced form of sulfur. So basically, DMSP is where primary producers dump excess photosynthate and sulfide when they're nutrient-limited and/or their biological processes are being impeded by the accumulation of sulfide, which is necessary for the synthesis of the amino acids cysteine and methionine but is also toxic and highly reactive, so you can't just leave it sitting around. This would explain why dinos make a lot of DMSP, as mixotrophic organisms that get most of their nutrients by eating bacteria would end up with surplus sulfur (...and it may also explain why heterotrophic dinos have been observed to steal plastids from their prey but only keep them a short time, sometimes less than an hour, before digesting them). And fun science fact: coccolithophores, which also produce relatively high levels of DMSP, are also mixotrophic. That DMSP is biologically useful in a variety of ways is just how evolution works -- if you need to make an organic sulfur compound as a sink for excess sulfide and fixed carbon, picking a molecule that's good for something would give you a competitive advantage.

The garbage can hypothesis for the synthesis of DMSP finds indirect support from another paper on BBD that I read...

Originally Posted by Microbial Communities in the Surface Mucopolysaccharide Layer and the Black Band Microbial Mat of Black Band-Diseased Siderastrea siderea
We found the BBD-associated microbial communities to be highly diverse compared to the SML [surface mucus layer] communities. High bacterial diversity associated with BBD has been reported previously for four different species of corals (M. annularis, M. cavernosa, D. strigosa, and C. natans) in studies that used molecular methods. The present study, which investigated a fifth BBD host coral (S. siderea), also revealed much variability in the species composition in BBD, even between two black band-diseased colonies of the same species on the same reef. A large percentage of members of Rhodobacterales (alpha-proteobacteria) were observed in our BBD clone libraries, in particular, members of the Roseobacter spp. The Roseobacter clade is one of the major marine groups and comprises more than 20% of the coastal bacterioplankton community. ... A reason for the numerical abundance of these bacteria in association with black band-diseased corals has yet to be elucidated.

Members of the alpha-proteobacteria that are associated with toxin-producing dinoflagellates and the Juvenile Oyster Disease-causing bacterium Roseovarius crassostreae (Rhodobacterales, alpha-proteobacteria) were observed in our BBD clone libraries. Other important members of the alpha-proteobacteria, Sulfitobacter sp. strain ARCTIC-P49 and Sulfitobacter pontiacus, were also observed in our BBD clone libraries. The presence of S. pontiacus in BBD-associated corals has been reported previously. Members of the Sulfitobacter genus have been reported to be involved in oxidation of sulfite, which would be important in the active sulfuretum present in BBD. As in the previous studies, we detected sulfate-reducing bacteria in (one of) our BBD clone libraries. ... In contrast to the earlier studies, we did not find any sulfate-reducing bacteria in the SML of apparently healthy tissue (although the other studies analyzed samples of the tissue and not the SML).
This study suggests that coral mucus selects against sulfate-reducing bacteria while acknowledging that other studies have detected such bacteria in coral tissue, meaning that the coral polyps themselves contain or live alongside sulfate reducers that release sulfide at night when oxygen is in short supply. In other words, it looks like corals make DMSP in part to detoxify the sulfide released by their symbiotic and commensal bacteria when the polyps go hypoxic at night and the bacteria are forced to use sulfur as a terminal electron receptor (...changing from aerobic to anaerobic isn't a strict either/or thing with facultative anaerobes -- they'll use whatever oxygen they can get and rely on their anaerobic metabolism to whatever extent they have to). To avoid making this problem any worse than it already is, the bacteria in surface mucus layer select against bacteria that will release still more sulfide, potentially smothering the polyps.

Okay, so now we've got the dinos' side of what's going on with DMSP, but there's still the question of why rosies are attracted to the stuff...

The Roseobacter Bacteria Clade are a sprawling, highly diverse subset of the alpha-proteobacteria that are found in virtually every marine environment from sea ice to black smokers. According to the most recent information I was able to access, the RBC currently includes more than 50 genera and thousands of species, most of which are known only by genetic fragments from shotgun sequencing of bacterial DNA in seawater. They're most common in coastal environments and cooler waters and seem to be least common in the Sargasso Sea, probably because it's too ULNS -- they like a bit of eutrophy -- and the seaweed apparently doesn't like them very much. Rosies tend to dominate algal bacterial communities in the marine environment, and they're typically among the first bacteria to colonize exposed surfaces in NSW and start forming biofilms (...perhaps because of TDA-making species). The name is an historical artifact describing the color of bacteriochlorophyll-a, as the first roseobacter species to be discovered was among the earliest known aerobic anoxygenic phototrophic bacteria. This turns out to be an important niche: it's now believed that anoxygenic phototrophy accounts for as much as 10% of the solar energy captured by marine plankton. Some rosies are photoautotrophs but others lack the genes for fixing carbon and are photoheterotrophic. One study of phototrophy in the RBC found that not all the rosies that carried the genes for bacteriochlorophyll-a were actually making the stuff. Interestingly, some of those that weren't could be induced to do so by reducing salinity, which tracks with photoheterotrophic bacteria in general being common in turbid estuaries where the salinity is often lower than NSW.

The defining feature of the RBC is that rosies store an unusually large amount of DNA in distinct structures called plasmids that are separate from their chromosomes and can be readily passed from one bacterium to another. Bacteria use plasmids to store and circulate particularly useful bits of DNA, allowing them to add new, special-purpose DNA to the genes that they start out with so they can do something they weren't previously able to do, and rosies are particularly keen on sharing genes with each other. All bacteria do this -- a classic example is vibrio cholerae, which normally isn't pathogenic but becomes lethally so if upgraded with the right DNA. It's called "lateral gene transfer" or "horizontal gene transfer" and seriously messes with the heads of paleomicrobiologists trying to work out the origins and evolution of bacteria and archaea. And it's also why there's a rule of thumb that a genetic similarity of 97% indicates that two bacteria are probably members of the same species, when human and chimpanzee genomes are >98% identical (...though on the other hand, the whole concept of a "species" kind of breaks down in a world were genes can be passed around like baseball cards). Bacteria can move DNA across species boundaries in at least three different ways that I'm aware of, but they share genes most readily with other bacteria that live alongside them in the same environment, especially if they're from the same species or genus. Though on the other hand, just because two species are in the same genus doesn't mean they get along -- there's a lot of cutthroat competition among vibrio bacteria, for example, some of which make antibiotics that can kill other vibrios.

In essence, bacteria use distributed storage to address the problem of the limited amount of DNA any one bacterium can contain. Rosies, however, take this to the next level: their winning evolutionary strategy is to continue to share genes even as they evolve away from each other, and the bigger and more diverse the clade becomes, pushing into new environments and bumping up against new competitors, the more interesting bits of DNA they're able to collect, and the more effective their strategy becomes. Each of them has a small part of a vast library of genetic shareware that any of them can use to solve a problem or exploit an opportunity.

There are so many species in the RBC occupying so many different ecological niches that there are bound to be counterexamples to refute any generalization made about them, but broadly speaking, rosies are organoheterotrophic, sulfur-oxidizing facultative anaerobes that can use NO3 and inorganic sulfur as terminal electron receptors. A few are full-on denitrifiers that release N2, and some others have the genes for denitrification but don't seem to use them. Rosies can survive in permanently anoxic sediments, even deep in the heart of the anerobic community several centimeters below the surface, though in substantially reduced numbers compared to the saprophytic zone (the top 1-2 cm or so of eutrophic coastal sediments) where they can comprise as much as 10-15% of the population under favorable conditions.


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