Sweet Stuff: Trehalose — the Stress-busting Sugar

There are sugars whose job involves much more than being sweet or providing the organism with energy. Trehalose, known to anybody who has studied brewers’ yeast, is one such bonbon. It is a disaccharide, which means that one molecule is made up of two joined-up basic sugar molecules (in this case a pair of glucose molecules) and it is found throughout the microbial, plant and animal kingdoms. Its role in yeast is the most studied, although it is probably best known to the man and woman on the street (and cosmetics scientists!) as the sugar that allows the resurrection plant (Selaginella) come back to life after months or even years of drought.

And it is not far off the mark to think of trehalose as the resurrection sugar — or at least the sugar that keeps organisms from drying out or dying from stress. Trehalose’s functions in yeast include protecting cells from the stressors which they encounters as they beaver away at producing the beer, wine and cider that we love so: high alcohol concentrations; heat; dehydration; oxidation; low pH; and osmotic stress. In a nutshell, if it weren’t for each yeast cell having thousands of trehalose molecules, these budding babies would never get very far into a fermentation: the conditions in the initial wort and during the first few hours of fermentation would see off 99.999% of old Saccharomyces cerevisiae. Imagine how long your cells would survive in a 12% sugar solution? Or in beer or wine?

So, how exactly does trehalose work in de-stressing cells? It’s all down to chemistry and how the two glucose molecules that make up trehalose shape up to one another and the outside world. Because of the 1,1-glycosidic bond (a bond between carbon atom 1 of the first sugar and carbon atom 1 of the second) between each glucose, trehalose forms a closed ring, and thanks to this tends to self-associate when in solution. In such associations, trehalose demonstrates an avidity for water that few other molecules match. And because of its binding properties the sugar can enhance yeast cell membranes by forming a stable matrix and sticking to charged groups of lipids and proteins, thereby preventing the disruption of proteins and undesirable reactions between them. From this buttressing of the cell membrane comes its usefulness when levels of ethanol (a solvent, let us remember) are high in the environment.

The stress caused to cells by a too-concentrated solution is called osmotic stress. And by golly are yeast cells under osmotic stress at the beginning of fermentation, where they are thrown into a 12% sugar solution (or more, in the case of high-gravity brewing). Trehalose would seem to counteract osmotic stress by coating proteins and stopping them turning into mush (denaturing). Stable proteins mean solid, correctly folded and associated cell contents.

So important is trehalose to brewing yeast that during fermentation it composes up to 5% of cell dry weight. In high-gravity fermentations, where there can be as much as 18 g of sugar per 100 ml, much higher levels are formed — up to 25% of the dry weight. Similar levels are found in dried yeast preparations where its presence and concentration correlate with the ability to withstand the rigours of rehydration.

What about what is known of trehalose’s role in other organisms? In the insect world, trehalose is the rocket fuel used for flight, chosen by Mother Nature to power the pullulation of butterflies and the buzzing of bees because of its efficiency as a storage carbohydrate (twice that of starch). The sugar is not found in abundance though in the plants and animals that humans regularly eat. Even though we have the necessary enzyme — trehalase — to break it down, it is only very recently that trehalose has become a regular part of the diet of non-shiitake mushroom and -oyster eaters (both of these are high in trehalose). Since the invention of an efficient method of trehalose extraction from starch, the sugar has been making its way into our diet through its addition to processed foods, especially frozen foods, where its cryoprotectant properties are used to lower the freezing point of things like ice cream. There is a theory out there that the rise in cases of Clostridium difficile infections may be down to our newfound ingestion of trehalose. This sweet-toothed (sweet-flagella’d?) bacterium seems to have quite a penchant for trehalose!

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Low-Alcohol Beer

Even in this day of rampant regulation, it is unclear what a low-alcohol beer is. Some textbooks will define a low-alcohol beer as having less than 2.5% abv (alcohol by volume). The EU used to regard beer with less than 2.8% abv as being low-alcohol, but has recently upped the threshold to 3.5% in order to “provide an incentive for brewers to be innovative and create new products [and to] encourage consumers to choose low-strength alcoholic drinks over standard ones, reducing alcohol intake”. But then, the EU Regulation on the provision of food information to consumers (1169/2011) exempts beverages with more than 1.2% abv from having to declare nutritional data, implying that this percentage is their threshold (as it officially is in the UK). Furthermore, we have the alcohol-free beers. The textbook definition of alcohol-free beer is of one containing less than 0.05% abv. Various European countries have different thresholds for alcohol-free beers: Italy and France classify alcohol-free beer as having less than 1.2% abv, Germany and Belgium 0.5% abv (the EU threshold) while in the Netherlands the term “alcohol free” can only be applied to beers with less than 0.1% abv . The UK’s limit is 0.05% abv.

Let’s ignore all the confusion concerning what constitutes a low- or no-alcohol beer for the minute and look at how this type of beer is produced. You make low-alcohol beer by applying either of the two Rs: restricting alcohol production during fermentation or removing excess alcohol from normal fermentations.

The most cost-effective approach for producing a low-alcohol beer would seem to be, applying first principles, restricting the quantity of alcohol produced by the yeast during fermentation. Because what’s the point in spending all that energy (sugars, heat, aeration, refrigeration for example) in producing alcohol if you’re only going to be removing it later? That’s like painting a whole room screaming red, when you only want a feature wall and then re-painting everything bar the said feature wall magnolia. And removing alcohol from beer in order to reduce the percentage from, say 3.5% to 1.2% abv, is energy intensive. You either evaporate the alcohol off via vacuum distillation (a type of snazzy boiling!) or physically remove the alcohol via reverse osmosis or dialysis. (Who would ever have thought that beer would one day be dialysed? It sounds tricky, highly technological and expensive — and, no surprise, it is.)

So, in an ideal, streamlined world you would restrict rather than remove. One approach to restricting the quantity of alcohol in the final product is to feed the yeast a smaller amount of sugars than they normally get. Less sugars mean less “fuel” for the yeasts, who then go on to metabolise less, grow less and spit out less alcohol. But if you feed yeast with a wort (the term for the sugary malt extract that yeast work on to produce beer) that’s as weak as water, you’ll get a low alcohol beer that tastes nothing like the nectar of the Gods and more like bubbly barley water. And this is the dilemma for brewers of low-alcohol beer: no matter how much R&D these boyos do they have found it near impossible to match the flavour, aroma and body of low- and non-alcoholic beers with standard beer. Even the snazziest of the snazzy removal systems extract not just the alcohol but also the flavour and aroma compounds associated with beer, which are chemically volatile (just like ethanol they boil off easily) and come from the ester, aldehyde, fusel alcohol and organic acid families.

But one method of producing low-alcohol beer is showing promise in terms of yielding a substance that is pleasing to the senses — the use of non-standard yeasts. Both ales and lager are the result of Saccharomyces fermentations; S. cerevisiae in the case of beer and S. pastorianus in the case of lager. The strains of Saccharomyces that have evolved over the centuries tend to yield beers with between 3% and 5% abv. It has proven very difficult to engineer these into become low alcohol producing strains. Even fancy biotechnological solutions such as immobilising Saccharomyces cells on special matrices and forcing them to rapidly ferment the wort at low temperatures has not produced satisfactory results. But non-Saccharomyces strains such as Mrakia gelida and Saccharomycodes ludwigii need no such tinkering in order to get them to make a low-alcoholic beer. Their fermentations naturally halt around 1.5% abv. Some reports claim that Mrakia beer has a flavour profile close enough to regular beer. Perhaps in the coming years we will be able to purchase a fine-tasting Mrakia beer and perhaps by then we will be able to say for certain what a low alcohol beer actually is.

 

The Most Famous Gram after Parsons

Gram_stain_01I love my bit of country rock, my wee daily dose of Americana. And ol’ Gram Parsons would be in the mix there, depending on the mood. But today I’m going to write about another Gram. A surname Gram, if you get my drift. And a man far removed from the world of the Byrds, Flying Burrito Brothers and cowboy boots.

Hans Christian Joachim Gram was a Danish microbiologist who could be described as the father of bacterial strain identification. For it was he who gave us the simple but effective staining method that has split the eubacterial kingdom right down the middle. After staining, a bacterium is either Gram-positive or -negative*.

In 1884, Gram was working in the morgue of a Berlin hospital and wished to devise a stain that would colour up the bugs from sections of lung tissue and allow them to be discriminated from the background human cells. After some trial and error and a few modifications, we got what is now called the Gram stain. The stain works on the basis of dyes that react with the bacterial cell wall, which is composed of peptidoglycan. One group of bacteria have a thick layer of peptidoglycan, while another has a thin layer that is surrounded on the outside by a thick (and sometimes gooey) lipid membrane. The first stain applied during Gram staining, crystal violet, stains both types of cells purple. The next additive, iodine, is a mordant#, which in the case of the thick-walled cells fixes the crystal violet’s purple colour, but is occluded from the second group of cells by the lipid layer. Alcohol is then applied to the cells. This washes out the loosely held dye from the thin-walled cells, decolourising them, while having little effect on the thick-walled cells, which remain purple. A second colour (either safranin or fuschin, both red) is then added to the cells. This addition does not affect the thick-walled cells, which remain purple, while the colourless, thin-walled cells are now stained red. And so we have in a simple staining method two broad groups of cells distinguished on the basis of cell envelope morphology. Combined with other morphological features that can be seen under a reasonably high-powered microscope, the Gram stain yields a deal of information about the mystery bugs under investigation.

After isolating a bug, Gram staining is the first port of call for a bug hunter. A bug being Gram-positive or -negative allows us microbiologists to rule our mystery organism out of belonging to about 50% of the eubacterial kingdom. Whatsmore, in taking a gawk at our isolate under the microscope to check for Gram status we also get to cop a look at cell morphology. Is the critter a rod? Or a coccus (round)? Is it boat-shaped? Sausage-shaped? Are there spores? Do the cells occur in bunches of grapes? Or in chains? Do the cells exist as tetrads? Et cetera. Us bug hunters then follow a protocol to narrow down the possibilities of what our isolate might be. We test for the presence of enzymes such as catalase and oxidase. We test for the bug’s ability to metabolise a host of carbon sources. And finally, if we want to be really precise, we sequence the blighter’s DNA and compare it to a database of previously identified isolates.

Thus, the modern microbiologist is using a mixture of techniques, some of which date from the nineteenth century, and some which were invented last year. And even in this day and age of next-generation sequencing and CRISPR and aptamers all that, it’s still not time for Gram to sling his guitar!

 

 

*There’s a couple of “buts” and “howevers”, however. Some strains are Gram-variable. Depending on culture age or physiological state, they can be positive or negative. But these are outliers in a very successful and venerable classification system.

#A mordant is a substance that combines with a dye or stain and thereby fixes it in a material.

Atten-shun! Legionnaires’ Disease

In a previous blog I wrote about bugs that hide out inside our cells to escape attack from our immune systems. I have another, fascinating creepy crawly to add to this list of undercover undesirables: Legionella. While the “ella” part of this particular nasty’s name may have the effect of making it sound all cute and cuddly (or even something akin to a lesser-known but no less delicious member of the family of hazelnut- and cocoa-based spreads) there is nothing warm or huggy about Legionella. She is insidious. And lethal.

She also has a great (albeit tragic) story-behind-the-name story. If you get me.

As described in this report in the New England Journal of Medicine, no such bug as Legionella had been isolated and characterised up until one fateful day in July 1976, when, at a convention of the American Legion at the Bellevue-Stratford Hotel, Philadelphia, 182 attendees contracted a form of pneumonia which ended up killing 29 of them. The outbreak is described as “explosive”, the agent “airborne” and the epicentre of the organism’s spread attributed to the hotel lobby or the area in its immediate vicinity. Perhaps the bug should have been called Lobby-ella!

Although the paper doesn’t engage in speculation as to the exact pinpoint of Legionella‘s debut outbreak’s ground zero, it was most likely the hotel’s air conditioning system or a water feature. Because, as we all know now, over forty years after the name was coined, Legionella is a veritable floozy in the jacuzzi: this femme fatale lurves nothing more than to schmooze (and reproduce) in the toasty waters of cooling towers, hot water pipes, fountains, mist sprayers, hot tubs, shower heads and the like. And it is really only in the last forty years that exposure to these Legionella breeding grounds has become a feature of everyday life. Hence the bacterium’s sudden appearance on the list of medical microbiologists’ most wanted. Like sin, Legionella has always been among us — it is only very recently in Homo sapiens’ history that the lass has gotten the opportunity to become a mass murderer of us. There were no cooling towers in the caves of Lascaux or the hanging gardens of Babylon!

Legionella is spread via aerosols. If breathed into the lungs the bug wiggles its way into our cells and proceeds to make itself very much at home*. It nests, forming structures called “Legionella-containg Vacuoles” (LCVs) which provide the perfect environment for replication, as well as protecting it from cellular components such as peroxisomes which might otherwise obliterate her. They aren’t just any old cells that the bacterium targets, by the way, but those charged with protecting the lungs — cells called alveolar macrophages. As such, a Legionella infection becomes a kind of ever decreasing circle of infection: it knocks out immune cells, which gives it a further chance of infecting even more cells leading to a further reduction of immune cells, which . . . Lethal cases of Legionnaires’ disease result from complications such as respiratory failure, septic shock or acute kidney failure. If caught in time, however, there are a number of effective treatments against the infection. It is important that the antibiotics used have high cellular penetration in order to bunker-bust Legionella in her LCV lairs.

As an aside, as well as seeing off 29 Legionnaires, the outbreak also finished off the Bellevue, at the time one of North America’s most iconic hotels. After its appearance in the news headlines, the hotel’s occupancy rate fell to 4%, forcing it to close for business. The Bellevue was thereafter thoroughly restored and has been back in business for over two decades, offering the city of Philadelphia, among other things, its highest dining experience.

 

*The infectious dose can be as low as one organism, especially if one’s lungs are in a sufficiently receptive state (i.e. damaged) to “welcome” the bug,  — smokers have a far higher chance of contracting Legionnaires’ disease than non-smokers

How Bugs Turn Your Food Waste into Fuel

They are far from appetising, and out-of-date meat and dairy products may even induce the gagging reflex, but the food trimmings, leftovers and beyond-best-befores that go into your garbage are still high in energy. Even though they might be in the process of breaking down, your mouldy oranges, crusts of bread and rancid walnuts are still chock full of calorie-giving molecules such as sugars, starches, fats, proteins and organic acids. In the context of global warming we all want to be carbon neutral (or even good little carbon sinks), so the responsible thing to do is to capture and re-use all that trapped energy.

In the old days when we all lived on farms and when even urban dwellers had pigs and chickens to deal with slops, very little food waste was sent to landfill. At the very least, back in our grandparents’ time potato skins, gristle and the like was put on the compost heap to eventually be recycled back into the soil. At present we are throwing out worrying amounts of food, to the extent that even swivel-eyed Brexit politicians and the Pope are expressing concern. Most of the food waste we dump goes to landfill, meaning that the carbon that cost so much in terms of energy, water and other resources to incorporate into our munchables returns slowly to the atmosphere with nobody benefiting except the dumpsite decomposers (beetles, worms, fungi and bacteria) whose job it is to gobble up macromolecules and spit out water, carbon dioxide and a bit of methane and hydrogen sulphide.

What can be done to recapture the energy we are losing through discarding so much food? One excellent option is to convert it to biogas. And guess who are the stars in the story of the conversion of old soggy spinach and crusty peanut butter to methane? You guessed it, our old friends, bacteria. And not just one bacterium or one group of bacteria, but a whole bunch of them working in co-operative layers (called “trophic” layers). Here’s a helicopter view of the process, which takes place in a huge tank known as an anaerobic digester. Your green waste management company collects you and thousands of others’ food waste and transports it to its site where after some pre-processing it is packed into the temperature-controlled digester. The bugs get to work on it immediately. The first group of bugs, hydrolysers, chop up the big molecules in the waste (e.g. starches) into smaller ones (e.g. sugars). These simpler molecules then become the foodstuff for acidogenic bacteria, who produce acidy compounds like those found in pickles. A third group of bugs work on the acids to form primarily acetic acid (found in vinegar), carbon dioxide and hydrogen. Finally, methanogenic bacteria mop up the waste products of the other three groups and produce — you guessed it — methane. This gas is our biofuel and can be burned to produce heat or drive turbines that generate electricity. It’s not just waste disposal companies who are getting into anaerobic digestion: many factories now have their own on-site anaerobic digestors to convert their own waste into usable energy.

You may be wondering why “anaerobic” digestion? Anaerobic means “in the absence of air. More correctly, we are talking about the absence of oxygen. Aerobic digestion carried out by aerobic bacteria is basically like slow-burning your waste: you oxidise (burn) your macromolecules and generate heat, carbon dioxide and water, but no usable high-energy compounds such as methane are left behind by the process.

Finally, a special form of recycling food waste, is the conversion of cooking oil to biodiesel. Companies such as Olleco collect used cooking oil from chippers, Chineses and caffs and, using a chemical process (no bacteria involved, unfortunately), turn what would be a potent building block for sewer fatbergs into fuel.

Waste not want not!

Some Like it Hot

Milk nowadays is a heck of a lot safer than it used to be. Before pasteurisation became widespread in the period following World War Two, you could have been taking your life into your hands by drinking a glass of raw milk. As well as a whole list of nasties including many that cause food poisoning (E. coli, Staphylococcus aureus, Salmonella and Campylobacter), raw milk also harboured the bacteria behind deadly diseases such as tuberculosis, brucellosis, diphtheria and scarlet fever. As the son of a couple both of whom were affected by tuberculosis as children, I have little time for the irresponsible quackery spouted by those who advocate the benefits of drinking raw milk (see this pseudoscientific article by Darina Allen, for example). Pasteurisation has saved and continues to save lives. Full stop. End of argument.

How does pasteurisation work? Simple: you apply enough heat to a liquid food product to bring about a reduction in the number of bugs it harbours. Pasteurisation is carried out for two reasons: to make a product safe; and to give it a longer shelf life. Beer is pasteurised, not to make it safe (beer is intrinsically safe because no food-poisoning organism studied to date has been able to grow in its high-alcohol, low pH and anaerobic environment) but to kill bacteria that would otherwise shorten its shelf life through the production of off-flavours, “wrong” mouthfeels, gushing or cloudiness. Milk is pasteurised mainly to render the product safe: none of the bugs referred to above make it through the pasteurisation process. But by no means does pasteurisation kill all the bacteria in milk. Milk is not a sterile product. That’s why it has a best-before date and why that distended carton you find in your fridge after you come back from your two weeks in Marbella reeks to high heaven when you gingerly take a whiff.

The microbial lads and lassies who survive the standard pasteurisation applied to milk are referred to as “thermodurics”, from the Greek thermo (heat) and Latin durare (to last). There are six main genera of thermodurics found in milk: Bacillus; Micrococcus; Enterococcus; Lactobacillus; Corynebacterium; Streptococcus. The vast majority of the bugs belonging to these genera are not harmful to your health when present in low numbers. It is generally only when they multiply to such levels that their toxins reach threatening concentrations or the amount of cells themselves cross that threshold referred to as “infectious dose” that milk becomes dangerous from a food safety outlook. But nine hundred and ninety-nine thousand nine-hundred and nine-nine times out of a million your milk will smell so bad by then and look so strange (ropey, clotted, yellow, orange) that even one of The Walking Dead‘s finest would turn their nose up at it. Each of the organisms produces a distinctive off quality in milk as a function of its metabolism and what enzymes it secretes. Bacillus species cause “bitty” or “broken” cream and “sweet curdling” while Lactobacillus will acidify your milk (these are, after all the gals behind the fermentation [of milk] that produces yoghurt).

While it is very, very rare these days for in-date pasteurised milk to be the cause of serious food poisoning, cases have been documented. Rigorous and all as dairies’ quality systems are, there is the odd one-in-a-million batch that escapes all the testing performed on milk. But nothing like the level of disease raw milk would be causing were it still widely consumed. To finish up with a quote from Wendie L.Claey et al. whose paper “Raw or heated cow milk consumption: Review of risks and benefits” makes for sobering reading:

raw milk poses a realistic health threat due to a possible contamination with human pathogens. It is therefore strongly recommended that milk should be heated before consumption. With the exception of an altered organoleptic profile, heating (in particularly ultra high temperature and similar treatments) will not substantially change the nutritional value of raw milk or other benefits associated with raw milk consumption.

Microbiological Miasmata

A miasma as defined by the Oxford English Dictionary is an “an unpleasant or unhealthy smell or vapour”. The word is of Greek origin and refers to a widely held belief that originated in the Middle Ages that noxious vapours or “night airs” were responsible for spreading disease. Miasmata (the plural of miasma) rose from rotting vegetation, soil and brackish water, causing every sort of disease from the common cold to bunions to bubonic plague. The zymotic theory behind miasmata reflected the best medieval medical thinking could offer to explain contagious disease in an era prior to the discovery of bacteria and viruses. Even a century and half after Pasteur and his fellow pioneering microbiologists put miasmata to bed with germ theory there are still vestiges of belief in disease-causing vapours and airs. If I’d a penny for every time my mother complained of catching a cold from a draught or heard an aul’ one wittering on about catching their death from the few raindrops landing on their sleeves I’d be able to afford a Gibson Chet Atkins guitar signed by Robert Smith himself!

While it is scientific heresy of the order of deserving a visit from a modern-day lab-coat-wearing Torquemada to suggest that air is a disease-causing agent, a recent review by Govindaraj Dev Kumar and co-workers gave me pause for thought. In this excellent review the authors offer evidence for air as means of carriage of disease-causing bacteria and viruses — or rather the dust particles carried by the air act as agents of dispersal of microbes.

Imagine this scenario: you run a small cheese-making operation. Your production facility is state-of-the-art, with food-grade stainless steel surfaces and process equipment. Your staff are highly trained, possess all the necessary safe handling certificates. Their hygiene is impeccable. Your HACCP (Hazard Analysis and Critical Control Points) is excellent. Your raw material, milk, is of the highest standard — low total bacterial counts, low somatic cell counts. Your pasteurisers yield milk that meets the most stringent regulatory standards. But, inexplicably, from time to time you produce batches of cheese that fail your quality control. The cheese looks off, tastes off and it has the wrong texture. It cannot be sold. You look for reasons why these batches fail. You find a wild Lactococcus strain has outgrown and displaced your starter strains and you scratch your head and wonder where the “bad” Lactococcus comes from. How has it entered your gleaming, space-age factory? Could it have come in on a stray draught? You examine the environment surrounding your factory. Et violà — five hundred yards from your facility is a barn in which silage is stored. On certain mornings an easterly wind carries the aroma of ripening silage right to the factory door. As well as the smell, you deduce that the breeze is also brining dust particles laden with bacteria. After a bit of classic investigative microbiology you find your wild Lactococcus growing in the silage and you prove that the dust that arrives to your factory also harbours the little mite. Your problem is solved by HEPPA-filtering your factory’s air.

Govindaraj Dev Kumar et al‘s review stresses the importance of wind-driven (technically called “aeolian”) dust-mediated contamination of foodstuffs. While currently there is much focus on water quality for the irrigation and washing of fruits and vegetables, and on the microbiological quality of the soil in which they are grown, as well as on the water used to wash down animal carcasses the authors highlight the risks of dust-borne contamination on foods of plant and animal origin. They cite cases of tree-grown fruit being contaminated with enteric bacteria associated with ruminant faeces. How does cow doo-doo make its way on to trees? Cows can’t fly! Faeces dry out, break down into dust and are blown up into the branches. It’s probably not a good idea to have a cherry orchard beside a cattle ranch!

The authors break down the sources of dust-borne contamination into natural (soil, decaying vegetation, feral animal droppings) and man-made (manure-amended soils, silage, municipal sewage-based biosolids, composting and animal production facilities). The most prominent species found to be involved in dust-borne contamination of food to date include: Escherichia coli O157:H7, Salmonella, Listeria monocytogenes, Campylobacter and methicillin-resistant Staphylococcus aureus (MRSA). The speed and distance of wind-driven carriage of microbes is impressive: the authors cite one instance of Xanthomonas malvacearum being scattered across 40 hectares in 20 minutes by a single whirlwind. The most alarming phenomenon described in the review is the internal contamination of fruits by dust-borne bacteria. Bacteria made their way on to the fruit through dust landing on blossoms and were internalised as the fertilised ovum developed into a fruit. No washing will eliminate bacteria harboured on the inside of a fruit and since much fruit goes uncooked the ingestion of a single contaminated orange or apple could introduce millions of pathogens into an unfortunate consumer’s gastrointestinal system.

In conclusion, there is cause for us microbiologists to start taking an interest in miasmata!

FISH-ing for Bacteria

PMC3061963_1472-6831-11-8-1Just as there are many ways to skin a cat, there are dozens of methods for looking for bacteria. Methods can be divided into a number of categories. There are slow methods, the foremost of which is the traditional “plate and wait” Petri dish-based plate count. There are rapid methods such as PCR or flow cytometry. There are bulk methods, which detect the metabolism or metabolic by-products of large (and undefined) numbers of bugs, the ATP bioluminescence assay being one of the best known. There are methods with allow you to count the numbers of individual cells present, including any number of microscopic methods. Then there are molecular methods, involving the detection of sequences of DNA or RNA. Most DNA-based methods would be also classified as rapid as they do not necessitate the growth of the bugs. If you are looking for a fella who likes to take his time in dividing (notorious strains include Mycobacterium tuberculosis, which causes tuberculosis, and Helicobacter pylori, which causes stomach ulcers) rapid methods for their detection are the holy grail.

One such rapid, molecular method is fluorescent in-situ hybridisation (FISH). Like all DNA or RNA detection methods, FISH is based on the chemistry of these macromolecules; the complementarity of pairs of the nucleotides that make up DNA or RNA. In the case of DNA, A (adenine) sticks to T (thymine), and G (guanine) sticks to C (cytosine). In RNA U (uracil) replaces T. During the great molecular biology revolution of the late 1970s and early 1980s scientists figured out that if they could synthesise short strands of DNA which were complimentary to sequences in the chromosomes of cells, if they stuck a fluorescent tag onto the synthetic molecules and if they could get these tagged sequences into cells, then they could light up very specific spots on chromosomes. So, you have a sequence A-T-T-A-G-G-G-C on chromosome 22 that you want to detect. Perhaps it’s a mutation, perhaps a risk factor for a disease. You design a probe which runs T-A-A-T-C-C-G, stick a fluorescent molecule such as fluorescein isothyocyanate onto it, et violà, if you can get your probe into the cell and focus on it using a fluorescent microscope, you are detecting your sequence of interest using FISH. You’re directly detecting a mutation or risk factor. Initially, FISH was used to detect mutations in human chromosomes and proved a great advance for the diagnosis of genetic conditions and certain types of cancers. It wasn’t long, though before microbiologists got in on the act. They saw the benefits of a rapid, non-growth-based method. But microbiologists weren’t primarily interested in looking for mutations in their bugs — they were just looking for them.

As the DNA code of more and more bacteria came to be known, microbiologists began using this data to design FISH probes specific to their species (or group of species) of interest. A whole new world was opened up to us. Because FISH does not require the cultivation of bacteria, bugs who we had never succeeded in getting to grow on plates before lit up our microscopes and flow cytometers like the fourth of July. Where no evidence had previously existed of the presence of certain strains in water, soil, desert sands, marine sludges, biofilms in food-processing environments, et cetera, FISH was showing the little critters in all their fluorescence-tagged glory. Bacteria were found in the most unexpected niches — weird and wonderful bacteria, for which we didn’t even have names. It is fair to say that FISH turned microbiology upside down, especially environmental microbiology. It is because of FISH and other molecular methods that we know that us microbiologists have only managed to grow up and study in the lab far less than 1% of all the species that are out there.

FISH is not the panacea, however, for slow and tedious growth-based microbiology. It is a difficult technique. Probe design must be done thoughtfully and meticulously. If your probe is too specific it may only stick to its target under very favourable conditions. If it’s too loose, you’ll get lots of false positives. Probes must enter the cell to stick to their DNA or RNA target. This means permeabilising the bacteria’s not inconsiderable outer layers. Some Gram-positive bugs are a dream to permeabilise. Other lads, with mucous layers and thick walls and extra membranes are a nightmare. You also have to fix your cells (not in the sense of fixing your car) but in the sense of using a chemical such as formaldehyde to dehydrate them and cross-link their proteins so they maintain their contents and structure. Fixing kills cells, which means you have no idea if the FISH-ed cell you are seeing on your microscope or cytometer is was alive or dead at the time of sampling. Not good for food or water quality analysis. There are ways around this, but it adds an extra level of complexity to your analysis.

FISH, while not being a routine method for looking for bugs, has found in niche in environmental microbiology. It is a technique which has shown us that there were many more species in our surroundings than the lassies we were able to grow in the lab. In combination with other techniques such as cell sorting and single cell sequencing, FISH will allow the capture, growth and genetic sequencing of specific bugs from any environment one might wish. The way things are going FISH may be used some day to show that there is bacterial life on Mars!

Some Good Sporeformers

Sporeforming bacteria have a bad rep. The food industry hates them; there are entire conferences dedicated to the elimination of Bacillus and Clostridium from milk powders and infant formula. Doctors and hospital managers quake at reports of high levels of C. difficile in their hospitals. There’s nothing to instill more fear in the general public than talk of an anthrax outbreak, be it related to farm animals or bioterrorism. It is spores’ extreme resistance to all the usual treatments that we use to eliminate bacteria, combined with their ability to survive for years in a dormant state that causes our fear and loathing of them. But we must not forget that, in the case of certain sporeformers, these properties can be harnessed by biotechnologists. Not all spores are villains.

The first sporeforming good ‘un that I came across in my study of microbiology all those years ago was Bacillus thuringiensis. Many readers will have encountered the terms “Bt” or “Bt proteins” or “Bt crystals”, and perhaps some of you will have applied a product containing these terms in its list of ingredients to a cabbage patch or a flower bed. B. thuringiensis spores contain protein crystals called δ-endotoxins which have insecticidal action and have been used for a hundred years as biocontrol agents for the caterpillars of moths and butterflies. You spray your field with a preparation of Bt spores. When the caterpillars get a-munching they ingest the spores whose protein crystals then proceed to turn the poor wigglers’ insides into mush. The great thing about applying Bt spores to your land is that they persist in the soil for years. They don’t wash into the groundwater like conventional chemical pesticides, and they do not cause any damage to the environment or the man or woman charged with applying them. Bt preparations are the insecticide of choice for organic farmers. Genetically modified crops containing the gene sequence for a Bt protein, and which therefore produced endogenous Bt protein, were a cause celebre in the GM wars in the 1990s. I’m not going there!

Another triplet of sporeformers are being looked at as natural pesticides which would see use similar to Bt. Lysinibacillus sphaericus (formerly Bacillus sphaericus — these crazy taxonomists can’t leave any poor bug in peace!) is a mosquitocidal bacterium which harbours its insecticidal crystal in a mesh-like parasporal envelope and could be applied to swamplands close to towns and villages to reduce the impact of malaria-carrying mosquitoes. Imagine the lives that could be saved by such a simple and safe intervention. Pasteuria penetrans is a parasite of root-knot nematodes — pests to which are attributed 5% of annual global crop loss. The spores have been commercialized as a biological control agent. A cousin of P. penetrans, P. ramosa infects water fleas of the Daphnia genus. Infected hosts are completely sterilized and have a reduced life span.

Now, it’s time to describe a more esoteric and technical use for bacterial spores. Spores of the genus, Bacillus, all display a protein on their outer surface, the exosporium, called BclA. Genetic engineering of this protein — splicing a gene from another species onto the anchor region of BclA — allows scientists to stick any protein they want on the outside of these spores. What use is this, you may ask. Because of spores’ unique stability in the environment and inside living organisms, as well as their amenability to mass production processes such as freeze drying and inclusion in “wet” formulations, bioengineered spores offer huge possibilities. Recombinant protein-decorated spores can be purified and used as microparticles for industrial, bioremediation, or vaccine applications. Imagine a Bacillus spore genetically modified to present an antigen from the malaria parasite on their surface. You have a candidate for a malaria vaccine there. What’s more, an orally delivered vaccine (spores will survive passage through the low pH environment of the stomach). Because it has been shown that enzymes retain their activity when tethered to the exosporium, GM Bacillus are ideal for a range of biocatalytic processes; the clean-up of oil slicks, waste water treatment or the manufacture of bioethanol.

Anthrax and Argi(nase)-bargy

I’ve p2pha_humanarginasereviously referred to the evolutionary toing and froing between microbes and their hosts as an “arms race“. A microbe develops a sneaky new way to attack or evade its host. A couple of millennia later the host species evolves a way to overcome its foe. A new strain of the dastardly bug crops up, replete with a mutation that allows it slink past its host’s beefed-up and shiny new defences. The host species finds a way to block this latest ruse. Et cetera, et cetera. And so the deadly dance escalates.

The more you learn about disease-causing microbes (or, to be fancy, pathogens) the more one comes to appreciate the extent to which evolution has honed these suckers into becoming laser-guided killers. It would seem as if every single gene in their genome, every single structure from wall to flagellum — heck, every single molecule they possess — is purposed with doing damage to their host in order to win the ageless battle between pathogens and us decent folk.

Let’s take one example — that of Bacillus anthracis and one (just one of many!) of the weapons in its disease-causing arsenal (technically known as virulence factors): arginase. Everybody knows that B. anthracis is the causative agent of the deadly disease anthrax. It forms resistant spores that can persist in the environment for centuries and which can be freeze-dried and stored as a powder, making it the bug of choice for back room bio-terrorists. In a nutshell, B. anthracis infects the body by worming its way into cells, hiding out there, dividing, spreading to uninfected cells, and of course producing its lethal toxins. It (or evolution) has developed a myriad of ways to hide from or counteract the immune system. Arginase is just one of these.

One of the main cells charged with the elimination of B. anthracis and other invaders from the body is the macrophage. In Greek, macrophage means “big eater”, which pretty much encapsulates what these immune cells do — they travel around the body gobbling up bugs. But they don’t use teeth to masticate the microbial malfeasants — they use enzymes and chemicals to digest and obliterate them. One of the most effective chemicals employed by macrophages to scuttle invading bugs is nitric oxide, which acts on them in the same manner as a solar wind on a spaceship without deflector shields. Fizzle, fizzle, fizzle! Not very many bugs can withstand a blast of the old NO, and not very many bugs have developed a defence against it. Except, that is, B. anthracis.

Which is where arginase comes in. B. anthracis spores have significant amounts of the enzyme in their outer layer (the exosporium). The arginase competes with the macrophage’s nitric oxide synthase (the enzyme responsible for making NO) for its L-arginine substrate, snatching the cell’s raw materials before it gets the chance to stock up on the gas. No NO means the spores and the vegetative cells that germinate from them are spared a blast of solar wind and so have a higher chance of survival and reproduction. And killing us.

What better example of the immunological arms race could there be? One bug, one host, one defence mechanism (NO) and one way around it (arginase).