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Old 01/04/2016, 07:12 AM   #2508
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Operating under the assumption that CFB 302T-1 is actually a cytophaga species, not a bacteroides, because that looks like the best lead we have, the genera of bacteria associated with O. lenticularis are...


AEROMONAS: Gamma-proteobacteria. Ubiquitous gram-negative rods. Facultative anaerobes. Saprophytic, meaning they participate in the decomposition of organic matter. One of the most common genera of bacteria found in salt and fresh water and frequently associated with diseases in fish, amphibians, reptiles, and birds. Considered a potentially serious emerging pathogen in humans. Aeromonas hydrophila can be fatal, and the genus is generally regarded as toxic and pathogenic (if you Google them, you'll get a lot of information about drinking water).

ALTEROMONAS: Gamma-proteobacteria related to Pseudomonas. Species have been repeatedly shuffled in and out of this genus in recent decades as a result of improved analytic techniques, and apparently there are currently only 8 of these aerobic and facultatively anaerobic gram-negative curved rods with single flagella. Happily, it seems none of them are pathogenic. Found in marine sediments and one species lives in seafloor hot springs, but mostly planktonic. One species is reportedly very good at hoovering up labile (very low molecular weight) dissolved organic carbon and is often associated with phyto blooms in NSW. Others are saprophytic -- an alternomonas species is among the bioluminescent bacteria that colonize marine snow, making it glow in hopes that it will be seen sinking through the darkness and eaten, giving the bacteria access to the nutrient-rich environment in a fish's intestines.

coryneform bacteria: "Coryneform" literally means "club-shaped" and apparently refers to unidentified bacteria cultured in the experiment from the '80s.

CYTOPHAGA: Ubiquitous gram-negative rods. This genus was massively reorganized on the basis of genetic evidence about 15 years ago, tearing it down to just two species -- Cytophaga hutchinsonii and C. aurantiaca -- and reassigning the other cytophagas to new genera. Formerly part of the CFB group, which no longer really exists but you still see references to it in the literature, cytophaga actually groups in a clade with the genus flexibacter, which was also torn down to the type species a while back and rebuilt on the basis of genetic evidence... Up until about 2005 or '06, there are bunches of papers about the Cytophaga-Flavobacterium Bacteroidetes group, aka the Cytophaga-Flavobacterium-Bacteroides group, or the Cytophaga-Flavobacterium/Flexibacter-Bacteroides group, or the Flexibacter–Bacteroides–Cytophaga phylum, or simply the Flavobacteriaceae, and several other names before it all shook out and settled on the classes Cytophagia, Flavobacteria, Bacteroidia, and Sphingobacteria of the phylum Bacteroidetes, representing an estimated 7000 different species, over half of them Flavobacteria which are easily recognizable by the large clocks they wear as necklaces. It's hard to tell what information from older studies of cytophaga from the CFB days is applicable to modern marine cytophaga, and further complicating matters from our POV, cytophaga are generally considered soil bacteria as the type species of the genus, C. hutchinsonii, was isolated from soil. Marine cytophaga had a reputation in some quarters as algicidal bacteria because they could consume macroalgal cell wall and structural polysaccharides like xylan, mannan, and cellulose, but on the other hand, one of the new genera into which old cytophaga species were moved is cellulophaga ("cellulose eater") -- yet C. hutch can grow using cellulose as its only source of organic carbon... Since O. ovata is epiphytic and can kill macroalgae, cytophaga would be plausible food bacteria for them if they were associated with disease and mortality in macro, and indeed I found some recent studies comfirming that marine cytophaga are among the bacteria associated with decaying phyto blooms and may opportunistically attack macroalgae. The genus' reputation is intact. Interestingly, cytophaga can consume lipids, and lipids + polysaccharides = lipopolysaccharides = bacterial cell wall proteins. An obligate association between ostis and a species of cytophaga bacteria thus makes sense on some level, as cytophaga look like excellent candidates for the CUC on the dinos' bacteria farms, and since they constitute about half the bacteria present in vitro, cytophaga sp. is also the obvious candidate for being the ostis' preferred food bacteria.

ERYTHROBACTER: Gram-negative non-motile aerobic alpha-proteobacteria. Erythrobacter are anoxygenic photoheterotrophs related to purple photosynthetic bacteria. Most commonly found in eutrophic coastal waters. Not all species make bacteriochlorophyll, and there was no mention of pigmented bacteria in either of the papers on the bacteria associated with O. lenticularis, so I would guess that whatever species of erythrobacter was present wasn't photoheterotrophic (...intuitively, it seems like benthic dinos shouldn't like photoheterotrophs because they're competing for the same real estate, but it makes sense that photoheterotrophs like dinos because they can swim and use this ability to stay in the light).

FLAVOBACTERIUM: Ubiquitous gram-negative rods of the phylum Bacteroidetes. Type genus of the family Flavobacteriaceae of the class Flavobacteria. Saprophytes that can consume carbohydrates, amino acids, proteins, and polysaccharides, but cannot consume alcohols, organic acids, hydrocarbons, and aromatics. Some species are pathogenic in FW but not in SW, and the genus likely evolved in FW and adapted to the marine environment. Marine pathogenic flavos afflict macroalgae as well as fish. Flavos are widely associated with phyto blooms, especially green algaes, in both FW and marine ecosystems, and they're often found in the benthic bacterial community though generally are not present in large numbers. Officially, flavos are aerobic, but poking around on Google Scholar suggests that at least some of them are facultative anaerobes -- even F. columnare, the cause of cotton mouth in FW fish, is described as aerobic but can reduce NO3 and release sulfide, both of which suggest the ability to use terminal electron receptors other than oxygen (...nitrate and sulfur are the classic fallbacks that you see over and over in facultative anaerobes: they go with NO3 because nitrate reduction works in marginally anoxic/micro-oxic conditions and NO3 yields the most energy of all the anaerobic terminal electron receptors, and they stick with sulfur because sulfur reduction is viable in full-on anoxia, and it's "Old Reliable" -- their aerobic metabolism was bolted onto biochemical machinery inherited from anaerobic ancestors, and the old system still works).

MESORHIZOBUM: Alpha-proteobacteria. Gram-negative facultative anaerobes generally considered soil bacteria, where they form symbiotic relationships with plants from the legume family and trade fixed nitrogen for organic carbon. Mesorhizobum species have been found in association with both terrestrial and marine worms. Can break down and consume cellulose and lignin, but none of the species in this genus is known to cause disease or even be an opportunistic pathogen in plants or algae.

NOCARDIA: Gram-positive rods of the class Actinobacteria. Saprophytic facultative anaerobes found in water and soil rich in organic matter (...the word for this in Science is "copiotrophic" -- copiotrophy is to heterotrophs as eutrophy is to autotrophs -- I think the root is the Latin "copi" meaning abundance, not the Greek "copros" meaning feces, but amusingly, for hobbyists it would make sense either way). Known to be very common in FW aquariums and a normal part of the intestinal microflora of tropical FW fish. Considered pathogenic in humans but with low virulence. Marine species can infect fish and mammals. Nocardia infections thought to be introduced through fish meal in high-protein feed pellets are a common problem for aquaculture operations, which I mention because this could be a vector that affects hobbyists, as well.

PSEUDOMONAS: Gamma-proteobacteria. Gram-negative flagellated rods found in soil and all aquatic environments. Formerly thought to be aerobic but now considered "metabolically diverse". Saprophytic, and terrestrial species are often found in association with plant roots, trading nutrients for carbohydrates and amino acids. Aquatic species are generally regarded as non-pathogenic or opportunistic pathogens that infect organisms with compromised immune systems. Psuedomonas fluorescens has been used as a probiotic treatment by aquaculturists to protect fish from bacteria and fungi with mixed success -- it seems to prevent vibriosis, for example, but not infection by aeromonas.

ROSEOBACTER: Alpha-proteobacteria. Gram-negative flagellated heterotrophs. Named for the color of bacteriochlorophyll-a made by photoheterotrophic species, which were the first to be discovered. Not all roseobacters are photoheterotrophs, but some that don't make bacteriochlorophyll carry the genes for it and can be induced to do so by exposing them to lower salinity levels, which tracks with the general observation that photoheterotrophs tend to be most common in eutrophic, turbid coastal waters such as bays and estuaries where salinity levels are often lower than NSW.

THALASSOMONAS: Gamma-proteobacteria. Aerobic gram-negative halophilic flagellated rods found only in SW. Can break down gelatin, casein, starch and lecithin, but not alginate or agar. The type species of the genus, Thalassomonas viridans, was isolated from oysters and first described in 2001.

VIBRIO: Gamma-proteobacteria. Facultative anaerobes and aggressive practitioners of chemical warfare that typically dominate copiotrophic environments. Vibrio are common in marine sediments and also in the water column as bacterioplankton, where many species are adapted to an oligotrophic environment in the open ocean. Vibrio are part of the normal intestinal population of healthy fish, people, and other animals, and a few of the same species (and also others) are lethal pathogens. Of those that cause disease in humans, the best known is V. cholerae, the cause of cholera. Pathogenic vibrio are a major problem in the marine environment; fewer pathogenic species are known in FW. At least two pathogenic species (Vibrio harveyi and V. splendidus) luminesce upon reaching some critical population density, causing sick shrimp to glow so predators can easily find them and the bacteria will gain access to the copiotrophic environment inside a fish. Some vibrio bacteria like to live on pods, perhaps to take advantage of the organic carbon released by feeding (pods are messy eaters) and other biological processes, or perhaps because some species of vibrio can grow using chitin as their only source of organic carbon. Vibrio spp. were the second-most-common bacteria associated with O. lenticularis and are thus also candidates for being their food bacteria (amphidinium carterae as well as two species of prorocentrum dinos have been reported preying on vibrio parahaemolyticus, a human pathogen -- but on the other hand, these same bacteria are algicidal towards another dino, cochlodinium polykrikoides...).

ULVIBACTER: Gram-negative aerobic non-motile rods of the phylum Bacteroidetes and the family Flavobacteriaceae. These bacteria are marine and require Na+ sodium ions for growth. Only six species are known from this genus, which groups in a clade with genera populated by ex-cytophagas, including the genus cellulophaga. The type species, U. littoralis, was first described in 2004 after being isolated from the epiphytic community of the frondose green macroalgae Ulva fenestrata. Ulvibacters tend to bloom during the early stages of decay in phyto blooms, so their association with O. lenticularis is probably opportunistic.


So not only are proteobacteria and Bacteroidetes present, but they're pretty much all that's present... The only break from the pattern is nocardia, an Actinobacteria reported in 1989 but not 2006. While several of the gamma-pros are facultative anaerobes that might do well in standing water as bacterial respiration draws down oxygen levels, a protein skimmer looks tailor-made to create heaven for Bacteroidetes. It may be that anaerobic bacteroides, which as marine Bacteroidetes are adapted to metabolize recalcitrant proteins, are the main beneficiaries as the O2 level drops in stored skimmate.

Saprophytic bacteria are logical partners for benthic dinos, as they feed on detritus, which of course is in plentiful supply in most DT sand beds, and are exactly the sort of P-rich bacteria that dinos would need to eat to get all the phosphorous they require and still have some left over with which to recruit diazotrophic cyano. Additionally, there's a metabolic pathway that allows some heterotrophic bacteria to break down large, recalcitrant organic carbon molecules that are normally too large for their enzymes to work on, but they need labile organic carbon to make it work. In other words, the small DOC molecules the dinos exude may allow some of their associated bacteria to break up very large molecules and get at organic carbon (and other nutrients, as well, but for heterotrophic bacteria it's mostly about the carbon) they wouldn't ordinarily have access to. That's why carbon dosing is good for water clarity, incidentally.

But what really caught my eye when I was first looking into this was the association between O. lenticularis and vibrio bacteria. What if O. ovata is buddies with vibrio, too? That would be an Axis of Evil -- I mean, there are a lot of toxic dinos in the sea, and there are plenty of pathogenic bacteria out there, too, but O. ovata teaming up with vibrio would be like Lex Luthor teaming up with Sauron. It's pretty much a worst case scenario.

And it also totally makes sense. As hobbyists, our basic understanding of how dinos do business is that they kill everything and hoover up the nutrients that are released as stuff decays, and the bacteria associated with Ostreopsis lenticularis are consistent with this model. Ostis, perhaps in combination with vibrio and other potentially pathogenic bacteria, can kill benthic fauna and algae; saprophytic bacteria then feed on the deceased; and the ostis feed on the bacteria (...note, however, that this is an idealized model, and it's surely not so tidy IRL -- check out this paper to get some idea of the potential complexities of dino-centric food webs, and it also bears mentioning that ostis probably have a "long tail" of biodiversity in the bacteria associated with them, but the laboratory techniques for identifying these bacteria, which by definition are only present in small numbers, are still pretty new and apparently haven't been used on dinos yet). The same dynamic would work for O. ovata, and the similarities the two species share, in habitat preferences and external characteristics and the fact that they're toxic dinoflagellates from the same genus, suggest they have essentially the same lifestyle and perhaps even similar bacterial partners. But like I said, I can only get away with this kind of thinking to the extent that I can reason by inference, so I went looking for evidence to provide some theoretical support for such intuitive leaps...

Originally Posted by Phylogenetic and functional diversity of the cultivable bacterial community associated with the paralytic shellfish poisoning dinoflagellate Gymnodinium catenatum
A total of 61 distinct bacteria spanning three phyla were cultured from the seven strains of G. catenatum. Thirty (49%) of the bacterial strains were affiliated with the Alphaproteobacteria... Thirteen (21%) isolates were affiliated with the Gammaproteobacteria... The remaining isolates came from two phyla, the Bacteroidetes (26%) and the high G+C% Gram-positive Actinobacteria (3%) [...note that the nocardia bacteria reported with O. lenticularis in 1989 are gram-positive actinobacteria]. ... The abundance of Gammaproteobacteria and Bacteroidetes in G. catenatum cultures averaged 5 and 4% respectively. ...

A number of the bacterial strains isolated were phylogenetically closely related to one another, while having originated from G. catenatum cultures from different parts of the world. ... Another distinct group was a specific clade of Roseobacter/Roseovarius-like strains that originated from G. catenatum cultures isolated from the sea areas as separate as Australia, Korea, Japan and Spain. In addition, many strains were also closely related to bacteria identified in association with other dinoflagellates such as the Paralytic Shellfish Toxin producing Alexandrium tamarense, Alexandrium lusitanicum and Alexandrium affine, the diarrhetic shellfish poisoning (DSP) Prorocentrum lima, and the non-toxic dinoflagellate Scrippsiella trochoidea. These similarities were especially evident among the dinoflagellate-derived strains belonging to the Rhodobacteraceae and Alteromonadaceae families. ...

In summary, the Alphaproteobacteria dominated the strains isolated, and an individual Alphaproteobacteria (Rhodobacteraceae) was always the most numerically abundant bacterium present in each culture. Half of all of the Alphaproteobacteria isolated were capable of a mode of photosynthetic growth, termed AAP [aerobic anoxygenic photosynthesis]. The second trend was for there to be cultivable oligotrophic and/or hydrocarbon-degrading Gammaproteobacteria present in almost all of the cultures. And thirdly, one or more cultivable isolates belonging within the Flexibacteraceae or Flavobacteriaceae families of the Bacteroidetes were always present in each culture.

The bacterial flora of G. catenatum generally mirrors that found associated with other dinoflagellates, being dominated by the Alphaproteobacteria (principally the Rhodobacteraceae -- frequently referred to as Roseobacter clade). For example, 50% of all phylotypes identified in four Pfiesteria sp. cultures were affiliated with the Alphaproteobacteria, with the Rhodobacteraceae Rg. algicola and Hyphomonas jannaschiana-like bacteria among the most numerous of these phylotypes. Rhodobacteraceae were also a dominant feature of the bacterial flora associated with the DSP-producing dinoflagellate P. lima, and from which the association Rg. algicola was originally described. The bacterial flora of Alexandrium spp. and S. trochoidea cultures were also dominated by Alphaproteobacteria, with the Roseobacter clade dominating both the cultivable species and ribotype clones identified. Like G. catenatum, members of the Alteromonadaceae (Marinobacter and Alteromonas) were consistently identified in other dinoflagellate cultures.

The high incidence of Alphaproteobacteria associated with algae does not appear to be restricted to the dinoflagellates, as Alphaproteobacteria, primarily Rhodobacteraceae, were always identified in association with each of six different species of diatom culture. Bacterial culture from the domoic acid-producing pennate diatoms, Pseudo-nitzschia multiseries, Pseudo-nitzschia seriata and non-toxic Pseudo-nitzschia delicatissima, consistently identified one or more Alphaproteobacteria associated with each of these cultures.

A striking feature of the bacterial flora of G. catenatum was the high degree of genetic similarity of members of the Alpha- and Gammaproteobacteria (Rhodobacteraceae and the Alteromonadaceae, respectively) compared to other dinoflagellates, particularly the PST-producing genus Alexandrium. ...

The similarities of bacterial flora across different dinoflagellates have two potential explanations. Firstly, there are selective mechanisms operating in laboratory cultures that favour genera from within the Rhodobacteraceae and Gammaproteobacteria...

The second explanation for the similarities in the bacterial community across G. catenatum cultures and with other dinoflagellates is that the bacteria from these groups may be of specific importance to the growth and physiology of dinoflagellate cells. Bacterial mineralisation of the algal extracellular products and phytodetritus is recognised as being an important part of the 'microbial loop', re-supplying algal cells with readily utilisable forms of C, N and P. The supply of vitamins, chelated iron by bacterially produced siderophores, or the production of cytokinins are examples where bacterially produced factors have been shown to stimulate algal growth. It may also be that the aerobic photoheterotrophs (AAP) identified in this study, which dominated the cultivable bacterial flora of G. catenatum cultures, may have a role in contributing energy to G. catenatum growth. ...

Three reports have identified specific bacteria as key components of the bacterial flora associated with the stimulation of dinoflagellate growth. ... Importantly, these bacteria belong to the two bacterial families consistently encountered in G. catenatum and other dinoflagellate bacterial communities.
Translated from the Science, that means there's a dinoflagellate holobiont (...actually, this paper is about bacteria and thus establishes that there's a dinoflagellate microbiome, but as noted above, IRL there's a fairly complex micro-ecosystem associated with dinos, and since I'm not a scientist and can freely point out the obvious in public, I'm just gonna go ahead and call it: there's a dinoflagellate holobiont). These guys were talking about pelagic dinos, not benthic species, but if multiple species of dinoflagellates across different genera have similar bacterial communities associated with them, then there's probably a benthic dinoflagellate holobiont, as well.

And it's obviously significant that the bacteria associated with O. lenticularis are essentially the same as those associated with pelagic dinos: alpha-pros, gamma-pros, and Bacteroidetes; and the alpha-pros in O. lenticularis' microbiome include potential photoheterotrophs (erythrobacter and roseobacter); even the exception to the pattern is a gram-positive actinobacteria. That's a strong correlation. The primary difference is which among these groups is dominant, as alpha-pros are numerically dominant in the pelagic dino holobiont, while the bacteria reported with O. lenticularis were about 50% cytophaga (or bacteroides) which are Bacteroidetes, followed by gamma-pros, and alpha-pros were the least numerous group. This suggests that the benthic dinoflagellate microbiome is a version of the pelagic dino microbiome with the bacterial CUC adapted to a different environment, which is further circumstantial evidence supporting the notion that there's substantial similarity between the bacterial populations associated with O. lenticularis and O. ovata.


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