I wrote this email back in Oct 25, 2020 to Daniel Zeigler, the Director of the Bacillus Genetic Stock Center. I thought putting it down somewhere on the internet to be referenced would be useful (I did get permission to post)! Many thanks to him for his impressive knowledge of Bacillus subtilis and being willing to share it all.
Hello,
I was curious about some procedures in Bacillus subtilis sporulation. I saw your posts on 2xSG media - is there a reason to use that over just raw LB? How many more spores can you get from 2xSG than from just letting cells sit in LB for a week? (I've used this previously with success)
In addition, I was thinking of trying to induce competence in conjunction with sporulation (perhaps with SCK6 or REG19). Would something like that work, or should I try to induce competence immediately after germination? The goal would be to go from spored Bacillus subtilis -> transformed Bacillus subtilis.
Thanks so much for your help,
Keoni
Hi Keoni,
Good questions as always! You do get a significantly better yield of spores using 2xSG as opposed to LB. There are a few reasons for this. One is simply that 2xSG is a richer medium and supports a higher growth density. But the efficiency of sporulation is also higher in the specialized medium. Sporulation is induced in most cells that enter stationary phase, but it can be repressed by excess carbon, nitrogen, or phosphorus sources. So it's possible to have a culture run of a nutrient and go into stationary phase, but repressing levels of some different nutrient still be present. Specialized media have the nutrients better balanced. One other issue is the inclusion of trace elements in 2xSG. Sporulation is especially dependent on sufficient Mn2+. The main reason is that in B. subtilis, the glycolytic enzyme phosphoglycerate phosphomutase has a strict manganese requirement. If you are using highly purified water, your LB may not have enough of the ion, and the culture will reach an early stationary phase and never sporulate. It's been 35 years since I did the experiments, but if memory serves me correctly, if I added trace amounts of MnCl2 to LB, I got an efficiency of about 30%, as opposed to closer to 80"% with 2xSG. But don't hold me to those exact numbers. Bottom line is that LB is ok if you don't care much about maximizing the yield. It's convenient. But for high yields, 2xSG is better.
Competence and sporulation are two mutually exclusive developmental pathways during stationary phase. Competence is part of the "K state," controlled by the master regulator SigK. During the K state, growth ceases, septum formation is suppressed, and the competence apparatus is assembled on the cell surface. It persists for a couple of hours in lab cultures. Most cells skip the K state entirely, and they don't get competent. Once cells pass through the K state, they can then enter sporulation. The pattern of gene expression is very different. The competence machinery is dismantled, an asymmetric septum is laid down, and away the cell goes with forming an endospore. So bottom line is that you can't package a competent cell in a spore. A further twist is that the supercompetent cells are induced with carbohydrates at levels that would suppress sporulation--but since they are locked in the K state while the inducer is present, it doesn't matter anyway.
You would have to germinate the spores and let them grow out and resume normal metabolism. At that point you could hit the REG19 or SCK6 with inducer and they would go immediately into competence. If you are using 1A1 and natural competence, you would have to inoculate the outgrown cells into the sporulation growth medium and start the protocol.
I can try to clarify any of these points. It's starting to get late Eastern Time, so it may not be as clear as I intended.
Daniel