The Evolution of Magnetotaxis in Bacteria

Magnetotactic bacteria (MTB) produce organelles known as magnetosomes which are magnetic magnetite or greigite nanocrystals encased in a membrane. These organelles allow them to align along magnetic field lines, a behaviour known as magnetotaxis. This trait is thought to aid the bacteria in locating and maintaining an optimal position for growth, at an oxic-anoxic interface. MTB have a wide distribution, occurring in most habitats including extreme environments, such as, hot springs and hypersaline waters. These bacteria are also very important in the cycling of key elements including sulphur, nitrogen, carbon and especially iron.

Biodiversity
MTB are a very diverse group of Gram-negative bacteria but only occur in minor populations. Currently all known MTB are found within the domain Bacteria, connected with three phyla. Alphaproteobacteria class represent the most dominant proportion of uncultured MTB, which have incorporated sulphur globules into their cells. Therefore, using reduced sulphur as an electron source is thought to be a common feature. Also the ability of nitrogen fixation was found in all MTB tested although one strain was found not to encode the gene.

Genetics determinants
In the majority of MTB, magnetosomes are aligned in varying lengths of chains, the most efficient orientation in terms of magnetic potential. This structure is thought to be under specific genetic control. Genes for bio-mineralization of crystals are organised in clusters within the genomes of MTB and are common in all, some keep them as a genomic island. However, it is unclear how environmental factors affect the production of these minerals. The presence and location of the genes within MTB genomes can contribute to the understanding of the origin and evolution of magnetosomes.

Evolution
The origin of this organelle seems to be monophyletic, vertical gene transfer seems to mainly responsible for the distribution of this trait whereas distribution among closely related groups is attributed to horizontal gene transfer.  If this was true and the common ancestor to all MTB then this suggests that the common ancestor of all Proteobacteria was magnetotatic or carried similar genes for this trait. The evidence shown suggests that when Proteobacteria originated approximately 2.5-3 billion years ago, there was low levels of oxygen. Therefore magnetotaxis would have been important in locating reactive oxygen species but when oxygen levels increased, this behaviour was no longer needed and the magnetosome genes were lost in some bacteria.

They are still many questions unanswered: This trait is also found in the domain eukarya, used for orientation, navigation and location of other organisms but how did they develop this ability? Is this trait found in the domain Archaea? Why do some bacteria still have this trait if they use sulphur compounds instead of reactive oxygen species? Do these organelles have another function and if not, why waste energy on its production?

Lefevre C.T. and Wu L. (2013) Evolution of the bacterial organelle responsible for magnetotaxis. Trends in Microbiology 21: 534-543

Are corals cooling the seas?

While searching for papers relating to symbiosis following Colins lecture yesterday I came across a really interesting paper linking symbiosis, the marine sulphur cycle and DMSP production that has strong evidence to suggest that it is not only algae that are responsible for DMSP production.

Dimethylsulphoniopropionate (DMSP) in an integral part of the sulphur cycle within the marine environment, which once metabolized to dimethylsulphide (DMS) is though to induce cloud formation and in turn may help reduce temperatures and also phytoplankton growth as part of a feedback mechanism (although this is still under contention).

Raina et al. have studied the Acropora corals of the indo-Pacific region as they acquire their symbiotic algae (Symbiodinium) from the environment following their larval stage.  Juveniles of two coral species, Acropora millepore and Acropora tenuis were cultured in an algae free environment and the absence of Symbiodinium was confirmed using five DNA markers.  DMSP levels were monitored and despite the total lack of symbionts, the larval corals still produces high levels of DMSP.  The levels detected were in fact greater than those reported for benthic algae found in the same region.  Once the larvae had settled the DMSP levels within the coral continued to increase (by 44% in A. millepore and 54% in A. tennis).

When subjected to thermal stress (32°C) over 6 days both species again showed a marked increase in DMSP levels.  They also exhibited a decrease in levels of the antioxidants acrylate and DMS; presumably this is attributed to their use in ROS detoxification.

To support the findings in the juveniles, adult corals were subjected to thermal stress until 84% of their Symbiodinium had been expelled and the remaining algal cells were all displaying signs of advanced necrosis.  Under these conditions the adult corals contained 68% more DMSP and 36% less acrylate than the control, confirming that the DMSP production in these corals is not just at one particular stage in its life.

Raina et al. then looked at the genetic level for evidence of homologous gene sequences that are found in other species.  These genes correspond to each of the enzymes in the four steps associated with the biosynthesis pathway for DMSP recently discovered within a diatom species.  They found orthologues for two of these genes, the first of which codes for an NADPH reductase enzyme that has many other orthologues in nature.  The second gene found however is thought to be specific to the DMSP pathway, and codes for methyltransferase which is responsible for regulating intracellular DMSP levels and until now has only been found in photosynthetic organisms and two eukaryotes.   The expression of this gene was initially high but decreased following settlement at a time when the coral would normally acquire its Symbiodinium. 

This research shows that the coral itself actually may contribute around half of the DMSP produced and I find it interesting that at a time of stress when the corals may have lost all symbionts they could still be capable of producing DMSP which has been linked to reducing water temperature as part of a feedback mechanism.  This could possibly give the corals time to recover and take on new symbionts that may be more tolerant to an overall increase in temperature. 

Raina, J. B., Tapiolas, D. M., Forêt, S., Lutz, A., Abrego, D., Ceh, J., ... & Motti, C. A. (2013). DMSP biosynthesis by an animal and its role in coral thermal stress response. Nature.

Activated ClpP kills persisters and eradicates a chronic biofilm infection.

A new addition to the antimicrobial arsenal.

As you will all know by now, antibiotics are becoming less and less effective with resistance increasing in natural populations. Methicillin resistant Staphylococcus aureus (MRSA) is probably the most infamous microbe in medicine as it targets immunocompromised patients and those who require invasive surgery. It’s not true to say that it is resistant to antibiotic action. In fact, in growing populations, antibiotics such as ciprofloxacin produce a biphasic pattern of killing but leave persister cells. These persisters primary function is to survive attack and remain viable until growth can resume.

So how do you kill a dormant persister, especially when they are usually buried deep inside a protective biofilm?

Ribosomal synthesis of proteins is not always perfect. Sometimes peptides are produced that are misfolded. The cell has a mechanism for the removal and recycling of these misfolded proteins, ClpP, a protease dependant on ATP. A known antibiotic for growing cells is Acyldepsipeptide (ADEP). This antibiotic activates the ClpP protease and keeps it’s catalytic chamber open, allowing for proteolysis to occur. It also removes the dependence on ATP. In this study ADEP4, a derivative from Streptomyces hawaiensis was chosen because of it’s previous success in eradicating Gram-positive bacteria.

Using proteomics on treated and untreated samples, ADEP4 was shown to activate the degradation of at least 417 proteins with FtsZ being a major target, hence the microbicidal action in growing populations. In an exponentially growing culture of S. aureus treated with antibiotics the population is reduced to persisters. With the addition of ADEP4 the population if further reduced to the limit of detection, implying that the ADEP4 causes self-digestion within the dormant cells. In a stationary phase culture of S. aureus most of the cells act as persisters and are extremely difficult to treat with antibiotics. ADEP4 here managed to reduce the population by 4 log₁₀ but a rebound in the population then occurred. This is because of the null clpP mutants. S. aureus do not require ClpP so null clpP wild type mutants arise readily. However when ADEP4 was coupled with rifampicin the stationary phase culture was eradicated. It was hypothesized that the null mutants have diminished fitness and are therefore susceptible to antibiotics. This may indicate that the null clpP mutants do not enter persister state, possibly due to their temperature sensitive phenotypic characteristics, which would make for inadequate dormant cells.

The combination of ADEP4 and rifampicin was tested on many known strains of MRSA (SA113, USA300, UAMS-1 & strain 37), none of which showed any colonies after incubation for 72h. One of the major problems with MRSA is its formation of antibiotic resistant biofilms. Using a biofilm produced by the osteomyelitis (infection of the bone) associated strain UAMS-1 the authors, again, showed the complete eradication of all living cells using ADEP4 and rifampicin. This has not been seen before with such low concentrations of antibiotics.

The final and most important question seems obvious. Can this work in an organism? For this they used a deep-seated mouse thigh infection model in which the mouse is made neutropenic (without neutrophils), infected with a large dose of pathogen and allowed 24h for incubation. This was used as a model for hard to treat human chronic infection. Treatment with ADEP4 and rifampicin, remarkably, completely cleared the infection although I doubt the mouse lived to tell the tale.

This discovery, to me, seems like a game changer, opening up more potential avenues of investigation involving self-digestive proteases and other potential activators. Although it does seem naïve to think that microbes will not respond with new strategies to combat this type of microbicidal action, we can still take comfort in the fact that there are always going to be new innovations on both sides.

Conlon, B. P., Nakayasu, E. S., Fleck, L. E., LaFleur, M. D., Isabella, V. M., Coleman, K., … Lewis, K. (2013). Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature, advance on.

In situ interactions between photosynthetic picoeukaryotes and bacterioplankton in the Atlantic Ocean: evidence for mixotrophy

Mixotrophy is classically the trophic strategy of specialized aplastidic protists in acquatic environments. In this article is presented evidence of mixotrophy carried out by phototrophic picoeukaryotes belonging Prymnesiophyceae, Chrysophyceae and Pelagophyceae groups of microalgae (<5 μm size).

Authors using flow cytometric cell sorting and dual tyramide signal amplification fluorescence in situ hybridization, present the interaction of individual picoeukaryotes cell and bacterioplankton. They show graphically the presence of picocyanobacteria cell inside phototrophic picoeukaryotes. Prochlorococcus and SAR11 clade are two major groups of bacterioplankton studied in this work, and is known that they both can’t establish symbiotic relationship with eukariotes.

Interesting results are that different groups of picoeukaryotes (predators) have likely favorite prey (e.g. Prymnesiophyceae incorporate mainly Prochlorococcus), opening a key role in the microbial food web of these important and abundand groups. Authors also suppose that global distribution of picosized Primnesiophyceae might be explained by their mixotrophic behaviour and the nutritional flexibility potentially gives a significant competitive advantage under different light and nutrient regimes in open ocean waters.

Another interesting result is the ingestion/assimilation ratio of prey biomass by predator that is equal about 50% meaning phototrophic picoeukaryotes might be as hightly efficient in prey assimilation as specialized protistan predators, such as microflagellates and planktonic ciliates. Is also supposed a selective predation on Prochlorococcus cell respect SAR11 clade cells because the last one are significantly more abundant in the environments studied here but are also smaller and hence less nutritious.

During the seminar we examined other two recent scientific articles about interactions between microalgae and cyanobacteria in the upper holigotrophic ocean waters. We talked  about other cases of symbiotic relationship between procariotic simbiont and eucariotic host, highlighting that phytoplankton organisms (one "simple" green cell… (not so simple!)) are not only able to express great metabolic plasticity in response to changing environmental conditions, but also can adapt their trophic strategy based on the most convenient available resources (e.g. light and nutrients or, for exemple, preys like delicious take-away Prochlorococcus cells) and partners for exemple exchanging fixed N and gaining back organic C with different group of eukariotic microalgae.

Hartmann, M., Zubkov, M. V., Scanlan, D. J., & Lepère, C. (2013). In situ interactions between photosynthetic picoeukaryotes and bacterioplankton in the Atlantic Ocean: evidence for mixotrophy. Environmental Microbiology Reports. Online Early.

Eating whale bone requires some serious symbiote diversity

Since whales do not cremate their dead, there exists a surprising community of extreme specialists who depend on sunken cetacean carcasses for sustenance and habitation. Within this group is the bone-eating polychaete worm, Osedax, an especially specific specialist that permeates whale bones with root-like structures. These structures are brimming with bacterial symbionts hypothesized to break bone down into nutrients which their host can use. Osedax depends entirely on these symbionts for nutrition, as it lacks a mouth and gut, like its fellow Siboglinidae member, the hydrothermal vent worm Riftia pachptila. Due the rarity of whalefall events, only 5 Osedax species have had their symbionts identified and so far all fall within the Oceanospirillales of the Gammaproteobacteria. This study aimed to fill in the knowledge gap regarding Osedax endosymbiont diversity and distribution within individuals, using O. mucofloris, comparative 16S rRNA sequencing and fluorescence in situ hybridization (FISH).

16S rRNA gene analysis found a paraphylectic group of eight phylogenetically distinct clusters (labelled A-H) with 99.5-99.7 % similarity; most O. mucofloris individuals were dominated by a single Oceanospirillales cluster. Endosymbiont microdiversity was impressive, with 61 of 76 full rRNA phylotype sequences being unique (differing by at least one nucleotide from all other endosymbiont sequences) and the majority of individual hosts had phylotypes unique to them, sometimes as many as nine. Geographic patterns of endosymbionts distribution showed no clear pattern, contrasting with clear depth patterns, as statistical and 16S rRNA analyses demonstrated strong phylogenetic distinctions between Osedax species found in shallow and deep water (<500-1000m<). Given the close relatedness of the host species studied, endosymbiote phylogeny is a more likely cause of depth differences, since endosymbionts likely have a free-living stage during which they infect a host.

FISH confirmed endosymbiont presence in O. mucofloris root tissues and revealed further diversity undetected by 16S rRNA sequencing, as general FISH probes for Osedax endosymbionts hybridized to novel endosymbionts which did not belong to any of the eight known phylogenetic clusters. FISH demonstrated dominance of cluster A across most hosts, closely associated with clusters B, C, D and E. FISH analysis of small sections of root tissue suggested low endosymbiont co-occurrence within bacteriocytes, with a single cluster dominating most and minor clusters occupying peripheral root tissue. Endosymbionts also occurred occasionally in epithelial cells and often in the root surface mucus layer, also dominated by a single cluster.

Apart from endosymbionts, other O. mucofloris associated bacteria included Bacteroidetes, Epsilonproteobacteria and Alphaproteobacteria, both of which were never found in worm tissue, had way less diverse sequence libraries than the Oceanospirillales and included whalefall-associated members of these groups. These bacteria are present as epibiota on all Osedax species and their role is unknown.

Endosymbiont and host genetic similarities did not correlate and bacteria were absent from eggs or sperm, strongly indicating horizontal transmission as the means of endosymbiont acquisition. 68% of endosymbiont variability was explained by the effect of host individuality, resulting from heterogeneity in the environment, the greater size and diversity of water column bacteria compared to that of the hosts and competition between endosymbionts.

Despite its unique and unusual nature, this symbiosis lends itself well to being a model for understanding symbiosis formation, symbiont transmission and the influence of genomic associations between host and symbiont on evolution. Particularly prominent is the compartmentalisation of distinct endosymbionts, suggesting a strong requirement for the separation of their respective metabolic roles. The mysterious role of the bacterial epibiota could well be one involving endosymbiont acquirement, depending on whether the skin is the route of uptake or not and also the relative timing of epibiota and endosymbiont acquirement during development. Much is unanswered regarding these bone-worms; how do their symbioses change between growing on whale bone and waiting for the next whalefall? Is all whale bone equally usable by all symbioses? Do shallow water Siboglinid worms also favour horizontal endosymbiont transmission, or is is vertical transmission just too conservative to succeed in the harsh, nutrient scarce deep ocean? How significant is the role of bone being broken down by these endosymbionts in terms of carbon, nitrogen and phosphorus cycling?

Verna, C., Ramette, A., Wiklund, H., Dahlgren, T. G., Glover, A. G., Gaill, F., & Dubilier, N. (2010). High symbiont diversity in the bone‐eating worm Osedax mucofloris from shallow whale‐falls in the North Atlantic. Environmental Microbiology, 12(8), 2355-2370.

Updating the human gut microbiome to degrade seaweed

The microbial community of the human gut possess “carbohydrate active enzymes”, or CAZymes, that are absent from the human genome. These CAZymes are used to digest and metabolise polysaccharides that our own enzymes are incapable of affecting.  Metagenomic analyses of the gut microbiomes of American, Spanish and Japanese populations has shown the presence of CAZymes from marine bacteria, obtained via horizontal gene transfer, that confer the ability to digest algal polysaccharides such as those found in sushi.

Before this study, it was already known that the two major phyla in the gut community are the Firmicutes and the Bacteroidetes, and that the majority of their enzyme systems are devoted to the breakdown of host glycans and the terrestrial plant material that makes up the majority of the human diet. What remained unknown, or at least required more evidence before an evaluation could be made with any degree of certainty, was how the microbial gut community evolves to process new sources of carbohydrate.

The authors have provided this evidence, by establishing how a horizontally acquired integrative and conjugative element (ICE) was able to confer Bacteroides plebeius with the ability to metabolise the red seaweed polysaccharide porphyran. They were able to show via X-ray crystallographic and biochemical analysis that the obtained ‘polysaccharide utilization locus’ (PUL) consisted of 40 genes coding for proteins that included β-porphyranases. Growth experiments showed that B. plebeius was able to utilise the PUL to grow on the porphyran, whilst it could not grow on similar carbohydrates such as agarose and carrageenan. Transcriptomics showed that the genes of the PUL were upregulated when the bacterium was grown on porphyran. They also identified two other gut Bacteroides, B. uniformis NP1 and B. thetaiotaomicron VPI-3731, that were able to grow on agar and carrageenan, which contrasted with previous studies suggesting that such metabolic pathways were absent from the gut microbes.

Some of the enzyme-coding genes on the PUL were shown to be expressed even in the absence of their polysaccharide substrate, in what the authors dub ‘surveillance’ levels.  This is common in Bacteroides, the production of small amounts of an enzyme to initially recognise and breakdown a polysaccharide which then triggers upregulation of that enzyme. A similar process occurs in the lac operon of other enteric bacteria.

The findings of this study are likely to have health implications, as red algae galactans (a class of polysaccharides including carrageenans, agars and porphyrans) are known to have a range of pharmacological effects. Health concerns have been associated with the products of carrageenan catabolism, as they have been shown to cause ulcerative colitis in animal trials. B. thetaiotaomicron VPI-3731 showed strong growth on carrageenan, inferring it must carrageenases and be capable of producing these harmful products. Further research would be required to discern if their presence in the gut microbiome has detrimental effects on the host. However, red algae galactans are also known to have antiviral, anticancer, anti-inflammatory and anti-oxidative effects, so many of the microbes catabolising them in the gut are likely to have a beneficial effect on their host.

This is purely speculative however. The main finding from this paper supports the hypothesis that the gut microbiome coevolves with host diet through horizontal gene transfer from extrinsic microbes enabling the catabolism of new carbohydrates.  

Hehemann, J. H., Kelly, A. G., Pudlo, N. A., Martens, E. C., & Boraston, A. B. (2012). Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes. Proceedings of the National Academy of Sciences, 109(48), 19786-19791.

Viral lysis of Micromonas pusilla: Impacts on dissolved organic matter production and composition

Viral lysis of Micromonas pusilla: Impacts on dissolved organic matter production and composition

Dissolved Organic Carbon (DOC) is a fundamental source of nutrients in the open ocean. There are several suggested methods of release or production of DOC (mentioned in Nianzhi Jiao’s paper [Colin’s lecture], Microbial production of recalcitrant dissolved organic matter). DOC has 3 categories, labile (<1%, minutes-days to break down), semi-labile (~50%, days to years) and refractory (~50%, years to 1000’s). This paper uses Micromonas pusilla to assess gaps in knowledge of how viral lysis of phytoplankton affects composition, lability and cycling of DOM.

The authors tested the organic matter dynamics and came up with 3 main conclusions:

Viral lysis enhances production of both labile and refractory DOC, impacting microbial web and perhaps decreasing the efficiency of the biological pump. Tests showed that in viral infected cultures, DOC concentration increased 4.5x faster and has an end point 2.6x higher than in uninfected cultures. Measuring labile amino acids and refractory humic-like substances, it was shown that viral infection increases labile DOC by 4.1x and refractory DOC by 2.8x.

Viral lysis changes the optical signature (composition) of DOM. Viral infection by itself was shown to change the cell composition of the infected phytoplankton host, shaping the signature of the released/lysed DOM. The viral presence influences host fatty acid and pigment concentration, DNA content and cell levels of DMS and DMSP (among other things).

Viral lysis increases the production of TEP, which is critical in the formation of biofilms, and possibly influences particle aggregation, affecting Marine Snow formation. Tests showed that in Viral infected cultures, Transparent Exopolymer Particles were produced 1.8x faster and had an end point 1.5x higher than uninfected cultures.

Separately, viral infection and then subsequent lysis are believed to more than double the pool of labile DOC. It was shown that 98% of decrease in POC was down to viral lysis, suggesting an efficient transformation pathway of cells into DOC. Lysis did not only increase DOC, but other important factors, such as organic nutrients and trace metals. In comparison to literature derived estimates, viral production of DOC was 1.2-1.9x higher in the observed experiment.

In summation, this paper provides an answer to the lack of knowledge highlighted by Jiao in terms of the production of DOC by viral lysis. This is a very important process, as 10²⁹ infections are believed to occur daily, with a daily total of 10⁹ tonnes of organic carbon being released to DOM, changing everything from composition to lability and affecting the cycling of the biological pump and microbial web.

Christian Lonborg, Mathias Middelboe and Corina P.D. Brussaard. (2013). Viral lysis of Micromonas pusilla: Impacts on dissolved organic matter production and composition. Biogeochemistry.