Fumbling for the switch in Streptomyces

Posted on September 18, 2023   by Dr. Valentin Waschulin

Dr. Valentin Waschulin takes us behind the scenes of their latest publication, 'Design and validation of a PCR screen for γ-butyrolactone-like regulatory systems in Streptomyces' published in Access Microbiology.

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Prof. Christophe Corre

Back in the autumn of 2018, just after starting my PhD, I would often wander down to the lab to inspect the bacteria I was cultivating. As I opened the incubator doors, a waft of warm air would hit me, and the sweet, earthy smell of soil after rain would make me think I was in the forest rather than a university laboratory. Taking out a Petri dish, the bacterium I was growing was a sprinkling of fuzzy white dots that slowly swelled, getting wrinkly as they aged, and then became dotted with the tiny droplets of deep blue that the bacterium owes its name to: Streptomyces coelicolor (i.e. sky blue). Both the colour and the smell are caused by compounds produced by this wondrous creature - actinorhodin for the blue colour and geosmin for the smell. These are just two of the thousands of compounds found in the genus Streptomyces so far. And in contrast to geosmin and actinorhodin, which merely look pretty and smell pleasant, some of these compounds have important medicinal uses as antibiotics, antiparasitic agents and immunosuppressants. It’s safe to say that modern medicine wouldn’t be the same without the humble Streptomyces.

However, it’s not easy to coax Streptomyces into producing these magical compounds. Imagine yourself as a Streptomyces growing in a Petri dish. Like every form of life, your biological goal is to produce the most offspring you possibly can. Since you’re the only one growing in this warm, cosy and nutrient-rich Petri dish, why would you expend unnecessary energy producing large amounts of complicated compounds like antibiotics, when you could invest this energy into more copies of yourself? So your internal regulation systems just shut production down, to be activated only when the compounds are needed. These systems work on many levels and are affected by a lot of factors, so compound production can seem frustratingly random to researchers.

Questionable anthropomorphisms aside, it is a recognised problem that Streptomyces don’t live up to their full potential when grown in the lab. Genome sequencing of a strain often reveals the presence of genes for the production of dozens of compounds, while we can only actually observe a handful of them in the lab. One way we can approach this problem is to manipulate the regulatory systems with genetic engineering. My co-supervisor Christophe Corre’s research on the production of a compound called methylenomycin has shown that a specific combination of regulatory genes that “switches on” methylenomycin production can be found in virtually the same layout across many different Streptomyces strains, doing exactly the same thing - just with different compounds. Imagine a child figuring out that by flicking a switch, they can not only turn on the lights, but also the TV, the fan or the toaster - what a revelation!

Now the problem is that these switches, aka regulatory gene cassettes, are hidden in the genomes, and to find them, you have to sequence the whole genome. This can nowadays be done for the price of a nice bottle of Scotch, which most labs can afford. However, while it’s fine to treat yourself to a nice dram or a hybrid genome assembly once in a while, it can quickly add up the more you drink and/or sequence. In your glass, you can replace your Lagavulin 16 with Aldi’s Highland Earl (even though I wouldn’t recommend that), but you can’t really replace genome sequencing. Or at least until now you couldn’t. In my recent paper in Access Microbiology, I present a cheap and easy screen to see if a Streptomyces strain contains a regulatory gene cassette that could potentially be engineered to turn on the production of a compound.

The technique used for this test is PCR, polymerase chain reaction. In essence, PCR is a photocopier that makes millions of copies of a single, little piece of DNA - so many that you can see them with the naked eye if you add a dye and some UV light. To target the DNA sequence you want to copy, all you need is two short pieces of DNA that match the left and right end of the bit of DNA you want to copy - they’re called primers and you can mail-order them for a couple of pounds. You then add a DNA-copying enzyme and some other bits, put it all in a machine, and an hour later you will know if the target gene is present in the sample. With a successful PCR test, we could test any Streptomyces strain to see if there is a potential “switch” to turn on the production of some unknown compound - at the cost of mere pennies. Basically, Highland Earl for Streptomyces researchers.

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© iStock/vkovalcik The PCR reaction containing primers, target DNA and enzymes is inserted into the PCR machine where the target stretch of DNA will be amplified. Machines like this can be found in biology laboratories around the globe.

A challenge I had to consider was that the same gene in two different Streptomyces strains will not have the exact same sequence in both strains. In fact, it might only be 30% or so identical! Therefore, if I used the DNA sequence of only one strain to design my primers to target the genes I want to copy, there would be a big chance that these primers would work only in this one strain. I, however, wanted to design primers that would work in as many strains as possible. I addressed this by downloading all 2000 or so Streptomyces genome sequences available in public databases and comparing them with each other. Through this, I was able to identify stretches of the DNA that were almost identical across all Streptomyces species - little islands of sequence identity in an ever-changing sea of mutations accumulated over time. To design my primers, I chose two of these islands - one in the first gene of my cassette, one in the second gene, to ensure that I would only detect co-occurring instances of these genes. According to my calculations based on the distributions of different gene variants, I would be able to detect 69% of the regulatory gene cassettes. This means that if there is a regulatory gene cassette present in a Streptomyces strain, this assay would detect it in 69% of cases. Testing the primers in the laboratory confirmed that they worked as expected - which made me very happy, as this was the first real result of my PhD research!

Soon after this, my research got dragged into a whole other direction and then Covid hit, so I didn’t look at these primers again until I was long out of the lab, writing up my thesis. I chose to publish this paper because I think that this little dram of research from the first months of my PhD could be useful for the Streptomyces research community. It’s great that journals like Access Microbiology encourage the publication of relatively small pieces of research, as well as preliminary data and negative results. They are useful for the community and would otherwise often rot in a drawer because they do not meet the criteria often required by many other journals. I hope that other researchers will pick up this primer set, swirl it in their glass, sniff it and use it to discover some novel compounds - whether they be fragrant, beautiful or maybe even medicinally useful.

Thumbnail image credit: Prof. Christophe Corre. A Petri dish fully grown over with Streptomyces coelicolor. Blue droplets full of actinorhodin have formed on its surface.