Benjamin Thompson

The bacterium is not only nasty but also very crafty. I’ve been reading a paper from PLoS ONE that explains how it can evade being destroyed by macrophages, the ‘first line of defence’ for the body’s immune system. These immune cells engulf invading bacteria through a process known as phagocytosis. The bacteria are then broken down inside the macrophage using a series of enzymes and toxic molecules, and the broken fragments of microbe are passed on to specialised immune cells that attack any remaining bacteria.

Stopping the macrophages doing their job is an important step for an invading pathogen. Some bacteria, such as Mycobacterium tuberculosis, do this by preventing the macrophages breaking them down. They get engulfed, but they just stay dormant and hide within the immune cells until they’re ready to emerge and cause the tuberculosis disease. N. meningitidis has a very different tactic: it makes the macrophages commit suicide. The proper name for this is apoptosis, or ‘programmed cell death’, a very important cellular pathway that usually happens in a highly regulated manner (you don’t want your cells dying for no reason).

The key to this is a protein found on the outer surface of the bacteria, with the snappy title ‘Neisseria hia/hsf homologue A’, or NhhA for short. This protein is used as an anchor, sticking the bacteria to the body’s epithelial cells. Without NhhA, for example, N. meningitidis is unable to colonise mouse nasal cavities.

NhhA is also able to bind to macrophages, and this greatly increases their rate of apoptosis. The group tested different chemical pathways that can result in cell death and concluded that caspase activation is the root of the apoptosis. Caspases are proteases, and their job is to break down other proteins. In bacteria lacking NhhA, apoptosis of macrophages was seen at a much lower rate.

This work gives an insight into the way N. meningitidis evades the immune system – essentially, by killing macrophages before they can engulf and kill the N. meningitidis bacteria. Given the severity of the disease caused by these bacteria, this new information is very welcome.

Benjamin Thompson

Sjölinder M, Altenbacher G, Hagner M, Sun W, Schedin-Weiss S, & Sjölinder H (2012). Meningococcal Outer Membrane Protein NhhA Triggers Apoptosis in Macrophages. PloS one, 7 (1) PMID: 22238624

Image Credit: Sanofi Pasteur on Flickr

Streptomyces

Streptomyces bacteria probably make antibiotics as chemical weapons to kill off competitors for the scarce nutrients in the soil and to use as signalling molecules to communicate with their neighbours. We know they are very good at sharing genes among themselves and other bacterial species through horizontal gene transfer – many antibiotic resistance genes originate in Streptomyces bacteria because they have to be resistant to their own antibiotics to avoid suicide – but, until now, very little was known about the way Streptomyces bacteria coexist and interact within the soil.

Kishony and colleagues, from Harvard Medical School, examined the interactions between all the Streptomyces species they isolated from three single grains of soil. Specifically, they looked at whether antibiotics secreted by individual ‘sender’ strains promoted or inhibited the growth of ‘receiver’ strains isolated from the same soil grains.

The results showed that closely related species were just as likely to inhibit each other’s growth as distant relatives. Remarkably, if a sender strain promoted (or inhibited) the growth of a receiver, the relationship was likely to be reciprocated. What’s more, two strains isolated from the same grain of soil were more likely to show this reciprocal relationship than two strains isolated from different grains of soil, regardless of how closely they are related.

This study is important because it overturns previously held assumptions that Streptomyces species happily share their antibiotic resistance genes with each other while competing with non-streptomycetes. In fact, they’ll compete with anyone, including their closest relatives. The reciprocal interactions suggest they can form mutually beneficial relationships or go to all-out war.

This study is impressive in its technical achievements, but it also provides a vital first step towards addressing the role of antibiotic production and resistance in the soil. Longer term, it could enable scientists to discover new antibiotics that are only made when Streptomyces species (positively or negatively) interact with their neighbours.

Vetsigian K, Jajoo R, & Kishony R (2011). Structure and evolution of streptomyces interaction networks in soil and in silico. PLoS biology, 9 (10) PMID: 22039352

Image Credit: born1945 on Flickr

We all love living things that glow in the dark, and scientists are no exception. Roger Tsien won a Nobel Prize in 2008 for discovering and developing green fluorescent protein and – perhaps even more excitingly – evil scientists turned mice fluorescent in a recent episode of the BBC television series Sherlock.

For animals that live in the blackness of the deep ocean, a little bit of bioluminescence goes a long way. For the squid Euprymna scolopes, this bioluminescence is generated by Vibrio fischeri bacteria that live within its light organ. The light organ is incredible, and it helps to hide the squid’s silhouette. This symbiosis is a win–win situation: the bacteria get housed and fed, and the squid gets a built-in cloaking device. Free-living bacteria also generate bioluminescence – but if they’re not in a symbiotic relationship, why do they bother?

Researchers have shown for the first time that the bacteria produce light to attract predators. Although this might seem crazy, it actually provides them with a great method of dispersing themselves throughout the vast oceans, and they might even get some food on the way through their predators’ insides. How clever is that?

In a simple experiment, scientists put some zooplankton (tiny animals that eat bacteria) in an aquarium, along with a bag of glowing Photobacterium leiognathi bacteria placed at one end of the tank and a bag of non-glowing P. leiognathi at the other. They found that the zooplankton had moved during the experiment and were found exclusively on the surface of the glowing bag.

The scientists also showed that glowing zooplankton are much more likely to be eaten by fish, perhaps because they’re easier to see. Presumably, food is rather scarce in the deep ocean, so eating glowing bacteria – even if it means something bigger might eat you – is worth the risk.

Finally, when the researchers analysed poo from both the fish and the zooplankton, they discovered that it contained high numbers of living, glowing bacteria. It turns out that being bioluminescent isn’t just a good way to move around: fish guts might offer a better source of food than seawater. If you’re a bacterium living free in the ocean, getting eaten might not be such a bad thing.

Matt Hutchings

Zarubin, M., Belkin, S., Ionescu, M., & Genin, A. (2011). From the Cover: Bacterial bioluminescence as a lure for marine zooplankton and fish Proceedings of the National Academy of Sciences, 109 (3), 853-857 DOI: 10.1073/pnas.1116683109

Image Credit: AJC1 on Flickr

Puffer fish, in case you didn’t know, is quite the delicacy in many parts of Asia. In Japan, it’s known as fugu. I didn’t eat any, though. Why not? Well, the fish is one of the most poisonous animals in the world. It must be skilfully prepared, or it’s potentially lethal: a slight tingling of the lips, and it’s goodnight. Deaths are rare nowadays, but I didn’t want to take the risk. I try to avoid eating anything that might kill me (although I did once – and only once – eat a kebab).

The root of the problem is tetrodotoxin (TTX), a lethal neurotoxin found in many animals. It currently has no known antidote. The animals themselves are not poisonous; the TTX is probably produced by symbiotic bacteria that live within them. Indeed, if you grow a puffer fish in an enclosed water system, it contains no TTX. Release it into open water, or feed it toxic puffer fish liver, and it becomes poisonous. Numerous different TTX-producing species have been identified, including those from the Pseudomonas, Vibrio and Actinomyces.

I found a paper last week in Marine Drugs in which a group of researchers have isolated a species of bacteria not previously known to produce TTX from within the intestines of the Hong Kong marine puffer fish Takifugu niphobles. This species was identified as Raoultella terrigena, a Gram-negative, rod-shaped bacterium.

Five bacterial strains were successfully isolated from the fish’s intestines, but only one was able to produce any toxic effects. The presence of TTX was confirmed by mass spectrometry. The identity of the strain was suggested initially by comparing its membrane fatty acid profile to other species on record and confirmed by ribosomal DNA sequence comparison.

The authors of the paper stress that although a TTX-producing species of bacteria was found within a puffer fish, there is no direct proof that it’s the bacteria making the fish poisonous, although it seems likely. Given some of the odd symbioses we’ve reported on this blog, it wouldn’t surprise me. I think I’ll stick to puffer fish grown in sterile water for the time being…

Benjamin Thompson

Yu VC, Yu PH, Ho KC, & Lee FW (2011). Isolation and Identification of a New Tetrodotoxin-Producing Bacterial Species, Raoultella terrigena, from Hong Kong Marine Puffer Fish Takifugu niphobles. Marine drugs, 9 (11), 2384-96 PMID: 22163191

Image Credit: Joi on Flickr

I found a paper in PLoS One that looks at this very subject. Canadian scientists looked at how the communities of bacteria, archaea and tiny eukaryotes changed between 2003 and 2010 in the Beaufort Strait. This timeframe is significant because September 2007 saw Arctic ice shrink to a record low. This was due to the melting of ‘multiyear sea ice’ – ice that stays frozen through the summer months and contains less brine (and, therefore, is less salty) than other ice in the Arctic. In particular, the scientists looked at a layer of the sea known as the subsurface chlorophyll maxima, or SCM, which contains a high number of photosynthetic plankton.

As a result of the multiyear sea ice melting, the area the researchers tested is becoming more layered, with a clear divide between the fresh and salty water. This increasing division might reduce the cycling of nutrients between the layers.

The results showed that levels of nitrate found in the waters had decreased significantly between 2002 and 2010 as a result of the increased ice melt, which is important because nitrates are the limiting factor for photosynthesis in the seas. Large differences were also seen in the community of sea microorganisms before and after the 2007 record ice low. These differences were seen in the Bacteria, Archaea and Eukarya. Bacterial diversity fell, with a large decrease seen in the phylum Bacteroidetes. These bacteria prefer to grow on complex organic matter, and the authors suggest that their depletion is a marker for the changing levels of organic matter in Arctic waters.

Although the research only looked at microscopic eukaryotes between 0.2 and 3.0 microns in diameter, the work showed that ciliates became more common after 2007 and marine stramenophiles (which feed on bacteria) became harder to find, possibly because of the lower level of Bacteroidetes bacteria for them to eat.

It was among members of the Archaea that some of the most striking changes were seen. Marine Group I Phylum Thaumarchaeota (Thaum-MG-I), which had previously accounted for approximately 60 per cent of all Archaeal DNA sequences identified before 2007, had declined to less than 10 per cent by 2010. These microbes use ammonia as an energy source, which is one of the early steps in its conversion into nitrate. The authors of the paper speculate that this is contributing to the drop in nitrate levels. This, in turn, could promote the growth of organisms better at competing for the available nitrates, further upsetting the ecological niche.

This work shows that our seas, and the microbes that live within them, are changing in response to the warming planet. The increasing divisions between fresh and salty water and changes within microbial communities could have a substantial impact, not only on the complex and delicate Arctic food chain but also in the cycling and sequestering of carbon dioxide. Whether these initial results represent the tip of the iceberg remains to be seen.

Next time you see a polar bear struggling at the top of the Arctic food pyramid, spare a thought for the microorganisms that make up its base. If that crumbles, the whole thing could collapse.

Image Credit: Nasa Goddard Photo and Video on Flickr

You can read the it here.

A very happy New Year from Matt and I, thanks for taking the time to read our posts and we’ll be back in 2012 with lots of new stories!

Benjamin Thompson

A new paper by Djamei et al. has revealed the nature of one of the virulence factors of Ustilago maydis, the fungal agent that causes maize smut. U. maydis requires live plants to survive, secreting many protein effectors that suppress the plant’s defence response and alter its metabolic pathways to suit the fungus. Most of these proteins are of unknown function.

The researchers have identified an enzyme called chorismate mutase, Cmu1, which is involved in the conversion of chorismate into the amino acids tyrosine and phenylalanine. They demonstrate that the production of this enzyme is upregulated during plant colonisation and is secreted into plant cells. Mutants unable to produce the protein showed reduced virulence.

Post infection, a tagged version of Cmu1 could be seen in the fungal hyphae, the interface between the plant and fungus and the cytoplasm of the maize cells. The researchers showed evidence that Cmu1 is able to move between plant cells, most likely through plasmodesmata – narrow tubes that connect the cytoplasm of neighbouring plant cells.

This work also showed that Cmu1 is capable of forming a 1:1 complex (also known as a dimer) with ZmCm2, a chorismate mutase enzyme made by the maize cells. It appears that this dimer prevents the maize producing salicylic acid (SA) – a compound involved in mediating plant defences against pathogen attack.

Indeed, maize plants infected by U. maydis mutants unable to produce Cmu1 showed a ten times higher level of SA than those infected by the wild-type fungus. Chorismate is a substrate for SA biosynthesis and the authors of the paper propose that Cmu1, in conjunction with ZmCm2, alters the maize’s metabolism to lower the levels of chorismate available for SA production. However, given the large number of secreted proteins, it is likely that many more effectors are involved in altering the plant’s metabolic pathways.

Secreted chorismate mutases are encoded in the genomes of many fungal pathogens of plants and these virulence factors may prove to be a key part of the fungal armoury.

Benjamin Thompson

Djamei A, Schipper K, Rabe F, Ghosh A, Vincon V, Kahnt J, Osorio S, Tohge T, Fernie AR, Feussner I, Feussner K, Meinicke P, Stierhof YD, Schwarz H, Macek B, Mann M, & Kahmann R (2011). Metabolic priming by a secreted fungal effector. Nature, 478 (7369), 395-8 PMID: 21976020

Image Credit: das_butzele on Flickr

Mining databases of microbial genomes has rapidly become a routine part of experimental work for microbiologists the world over. It must be impossible for this year’s new intake of PhD students to imagine a world without them. But let’s reminisce for a moment, just to see how far we’ve come.

If you needed to know the sequence of a piece of DNA in the 1970s, you had two choices: you could either use Maxam and Gilbert’s chemical sequencing or Sanger and Coulson’s chain termination methods. These produced read lengths of around 100 base pairs (bp), so you can imagine how long it took to complete the first sequenced genome, that of bacteriophage Φ174, which has 5386 bp (and was published ten months before I was born).

Fluorescence-based sequencing, developed from the Sanger method, has been the mainstay of DNA sequencing for over 30 years. It was used to produce the first genome sequence of a free-living organism, Haemophilus influenzae, in 1997 and the first draft of the human genome in 2001. DNA sequencing on this scale is expensive and labour intensive, so these projects were generally restricted to a few centres dotted around the world, such as the Wellcome Trust Sanger Institute in Cambridgeshire.

As a result of these large projects, everything began to change, as these graphs show. In 2001, the cost of 1 Mbp (1 million bp) of DNA sequence was approximately $5000 USD; now, it’s less than ten cents. The driver for this cost reduction has been competition between companies, each with their own sequencing methods, such as Illumina, Roche 454, PacBio, IonTorrent and Solid.

These can generate millions of DNA reads between 50 and 1000 bp in length in just a few hours. It is now possible to generate data for ~100 draft bacterial genome assemblies on an Illumina GA2 sequencer in a single run. However, given the high cost of these machines, the old model of having a few large, well-funded sequencing hubs still applies. BBSRC clearly agrees with this, as demonstrated by their investment in The Genome Analysis Centre (TGAC). Having said that, many smaller university departments are also investing in these technologies, as can be seen in this excellent map, maintained by Nick Loman at the University of Birmingham.

So where do we go from here? The answer to this appears to be the advent of scaled-down benchtop sequencers. There are at least three offerings in labs around the world; Illumina’s MiSeq, Life Technologies’ Ion Torrent and Roche’s GS Junior instruments. These instruments are much cheaper to buy and run than their bigger brothers and have the ability to put the genomics revolution directly into the hands of individual research groups: sequencing for the microbiological masses.

Imagine that you isolate a new strain of Streptomyces that produces a novel antibiotic compound – you can now sequence that strain in less than a week and start identifying the genes required to make this new compound (just like Microbelog’s Matt has been doing). It remains to be seen what impact these benchtop machines may have on clinical medicine – we’re a long way from having sequencers in clinical labs and surgeries. That said, the recent crowdsourcing effort to sequence and analyse the recent E. coli O104:H4 outbreak in Germany demonstrates how quickly this data becomes available to clinicians and researchers alike.

One of the big questions is this: is the traditional model of funding a few centralised genomics hubs still valid? In 1995, sequencing the H. influenzae genome was a large genomics project. It required many highly trained people, from microbiologists and molecular biologists to bioinformaticians. This is now a relatively small task that can be run by one or two people in less than a week using a benchtop sequencer. It should be noted, however, that much of the expertise (both lab and computational) required for this work has been discovered and disseminated by the large sequencing centres like the Sanger Institute.

At the recent SGM Autumn meeting in York, it became clear that many projects now contend with sequencing as many as 100 strains in a single study. This is clearly beyond the capability of benchtop instruments and individual labs. However, if you just want to sequence a couple of unusual strains, you could be in for a bit of a wait (and a lot of paperwork) to get these done at a large sequencing centre. There’s no hanging about with a benchtop instrument.

So, do I think the future of DNA sequencing is in benchtop machines? Well, we’re taking delivery of a GS Junior shortly, so ask me in a couple of months…

Nick Tucker is a lecturer in microbiology at the University of Strathclyde. His research focus is bacterial gene regulation and Pseudomonas aeruginosa genomics.

Image credit: Matt Hutchings

History dictates that Alexander Fleming will be best remembered for his discovery of penicillin, what with him winning the Nobel Prize and all. Perhaps what he should be remembered for, however, are the paintings he made on agar using different coloured bacteria. Quite why this culturally important art form never caught on is anybody’s guess.

It may have been Fleming’s strange hobby that inspired a group of scientists to go one stage further and develop an encrypted messaging system (obviously), which uses printed patterns, known as arrays, of different coloured bacteria. These bugs are coloured by the expression of genetically engineered fluorescent proteins, and the scientists in question have called the system ‘steganography by printed arrays of microbes’, or (ahem) SPAM for short…

Once I got past my initial scepticism and read the paper, I realised this is actually pretty cool stuff. The researchers use Escherichia coli cells that only produce the coloured proteins following treatment with a chemical called IPTG.

So how does it work? You grow your bacterial strains, each capable of expressing a different fluorescent protein, in liquid media in 96-well plates, then print the bacterial cultures onto agar plates – a bit like doing potato printing. Once the bacteria have grown on the plates, they can be pressed onto velvet (an old microbiology trick) or onto a nitrocellulose membrane and put in the post. All the recipient has to do is add IPTG to the material and wait for the bacteria to express their fluorescent proteins.

Of course, a series of fluorescent dots doesn’t really mean anything on its own, so you’d need some kind of cipher to work out what the pattern actually said. Ingeniously, just knowing the cipher isn’t enough – you need to know the chemical inducer needed to make the bugs glow. Add the right one, and the correct fluorescence shows; add the wrong one, and you could get a completely different set of genes expressed, giving you a very different text. Another great aspect is that the message fades when the inducer runs out – a Mission Impossible-style self-destruct.

The future of covert communication’s bright, the future’s fluorescent orange bacteria.

Matt Hutchings

Palacios MA, Benito-Peña E, Manesse M, Mazzeo AD, Lafratta CN, Whitesides GM, & Walt DR (2011). InfoBiology by printed arrays of microorganism colonies for timed and on-demand release of messages. Proceedings of the National Academy of Sciences of the United States of America, 108 (40), 16510-4 PMID: 21949364

Image Credit: kat m research on Flickr