Sweet surprise for scientists

What happens to bacteria when their favourite sugar source runs out? They stop growing for a while to adapt their enzyme and uptake systems to a new carbon source. This is what the microbiology textbooks have said for over seventy years, but it turns out they are not quite right. University of Groningen microbiologists topple the dogma in a paper published in Proceedings of the National Academy of Sciences on 5 May.

University of Groningen microbiologist Oscar Kuipers usually wears a broad smile, and today it is broader than usual. His PhD student Ana Solopova used modern microscopic techniques to shed new light on the ‘lag phase’ that occurs when bacteria switch to a new energy source.

‘This was described over seventy years ago by Jacques Monod ’, says Kuipers. When bacteria are grown in the presence of two different sugars, they will first use the preferred sugar source (often glucose) to fuel their growth. When this runs out, Monod observed, they will stop growing for some time (the lag phase) before growth resumes. His explanation was that the cells need to turn on protein systems before they can import and digest the new sugar source.

‘That is indeed what you find when you sample a flask of growing bacteria. You see that the presence of the enzyme systems needed to use the new sugar source increases during the lag phase’, Kuipers explains. The reason why the enzyme systems are not yet turned on was also obvious: the energy required to produce all these enzymes and transporters. And so Monod’s explanation of ‘enzymatic adaptation’ stood the test of time for decades.

Until now. Ana Solopova studied the lag phase under a microscope that allows her to image growing cells. The time-lapse microscopy setup is in a special lab. On top of a microscope is a climate chamber, with inside a glass slide with a tiny growth chamber for the Lactococcus lactis bacteria. Solopova used fluorescent labels to make the cells that use the new sugar source (in her case cellobiose) stand out.

‘The camera automatically records an image every few minutes’, Solopova explains. The result is a time-lapse movie of how the individual bacteria grow. She used this setup to investigate how cells respond to the switch from glucose to cellobiose. What she saw makes a major modification of Monod’s explanation necessary.

‘What happens is not that the bacteria switch from one system to another, but that a small group of cells which are prepared to use cellobiose begin to dominate the population’, Kuipers explains. The rest of the cells become dormant – they turn black in the time-lapse films. ‘They probably don’t have enough energy to make the switch to using cellobiose’, says Kuipers.

Another observation that Solopova made is that the response of cells can be manipulated. If they are grown on cellobiose before the experiment in which they must switch from glucose to cellobiose, the lag phase is shorter. Kuipers: ‘In this case, there are still some cellobiose transporters present in the membranes of the cell.’ In each division, the two daughter cells will inherit part of the cell membrane and proteins from the ‘mother’ cell, and these will contain cellobiose transporters if the cells were grown on this sugar source.

‘This could be of interest to the dairy industry’, says Kuipers. ‘Lactococcus lactis is used in cheese-making. Special companies provide starter cultures to make the milk curdle. What we have shown is that you can adapt the bacteria in this starter culture to a specific growth condition, thereby influencing their future behaviour.’ This is why four different dairy companies supported the project in collaboration with technology foundation STW, which funds joint projects that realize the transfer of knowledge between the technical sciences and users.

Why are these bacteria not equal, seeing as bacteria grow by cell division and therefore produce identical daughters? ‘That is the third observation we make in our study’, says Kuipers. ‘Cells in a culture are not all the same: there is heterogeneity of phenotypes.’ To explain this, collaboration was sought with the Faculty’s theoretical biology group.

Kuipers: ‘What their work shows is that it pays for bacterial cultures growing in a changing environment to spread the risk, something we know as “bet hedging”. This reduces the time needed to adapt to new circumstances.’ But this ability comes at a price. Solopova tested how well cells that are adapted to cellobiose respond to a subsequent switch to galactose. ‘It turns out that they fare very poorly on this new substrate, while glucose-adapted cells fare extremely well on galactose.’

Why did it take seventy years to topple Monod’s dogma of ‘enzymatic adaptation’? Kuipers gives two reasons: ‘The topic of heterogeneity in bacterial populations only became popular in the last ten years, and you need the right equipment to study individual cells. If you analyze samples from a flask of bacteria, you will find millions of cells on average.’ The microscopes his group is using are a recent development, and they carry a six-figure price tag.

The investment in a number of these microscopes would seem a safe bet: the field is booming. ‘There are all sorts of interesting phenomena in which heterogeneity is important, not just the response to different sugars but also when cells become resistant to antibiotics.’ There is plenty of work to do on individual cells.

Reference: Bet-hedging during bacterial diauxic shift ( doi: 10.1073/pnas.1320063111 ) Ana Solopova, Jordi van Gestel, Franz J. Weissing, Herwig Bachmann, Bas Teusink, Jan Kok, and Oscar P. Kuipers