Siblings Strike Again
Cain Killing Abel, Italian School. Source.
by S. Marvin Friedman
Bacteria capable of sporulation go out of their way to grow rather than sporulate. They will therefore try to obtain needed nutrients, even at the cost of killing their neighbors. When starved for nutrients, cells of Bacillus subtilis engage in cannibalism, that is they lyse their siblings and use the nutrients thus obtained to postpone their own sporulation. A similar phenomenon, termed fratricide, has been reported in Streptococcus pneumoniae where the killing of siblings is linked to the induction of competence and the release of DNA from lysed cells. The antimicrobial agents secreted within the same colony by either cannibalism or fratricide belong to the family of bacteriocins. Be’er and his colleagues now report a third unique situation where bacterial stress leads to sibling death.
Panebacillus dendritiformis (T-morphotype) forms branched colonies on low-nutrient, hard agar plates. When two neighboring colonies are produced by inoculation from the same culture, their growth is inhibited at the zone of closest contact. All the cells at the interface of the inhibited region are killed. Death occurs even at relatively high nutrient levels, thus ruling out cannibalism (killing to obtain nutrients) as the mechanism that operates here.
(A) Two sibling colonies of Panebacterium dendritiformis (T-
morphotype) on an agar plate. (B) Proteins extracted from agar
between colonies and analyzed by SDS-PAGE and identified by
peptide sequencing as flagellin (32 kDa), subtilisin (30 kDa)
and Slf (12 kDa). (C) A single colony of P. dendritiformis grown
on an agar plate. (D) SDS-PAGE of proteins surrounding a single
colony, without (-) or with (+) added subtilisin, as shown in
panels C and E. (E) A single colony with subtilisin (0.1mg)
added 1 day after inoculation (black dot). Source.
In order to further characterize the lethal factor, proteins extracted from the agar between the colonies were analyzed by SDS-polyacrylamide gel electrophoresis. The proteins detected were flagellin (32 kDa), the protease subtilisin (30 kDa), and an unknown 12 kDa protein. When spotted at a high concentration near a growing colony, the 12 kDa protein inhibited growth, whereas flagellin had no effect. For that reason, the 12 kDa protein was designated Slf (sibling lethal factor). However, its gene (dfsB) codes for a 20 kDa protein with 173 amino acids. When the 20 kDa DfsB protein was produced by cloning the dfsB gene, the protein was found to be inactive. This is where subtilisin comes in: treatment of the 20 kDa DfsB with this protease yielded a 12 kDa protein that comigrated with the 12 kDa protein isolated from the agar plates and that also inhibited growing colonies. In addition, when Slf was added to liquid cultures prior to inoculation, no growth ensued. Adding it to a growing culture resulted in cell lysis. Thus, Slf, the 12 kDa protein, was identified as the lethal factor.
The sequence of the 173 amino acids encoded by the dfsB
gene. Large letters indicate the segment (amino acids 5-169)
that is associated with a conserved Pfam family domain. Bold
letters indicate the segment that composes the isolated Slf
The authors propose that a negative feedback loop regulates the concentration of subtilisin at the front of a growing colony. When added up to a threshold concentration, subtilisin promotes colony growth and expansion. When this level is exceeded due to the secretion of additional subtilisin by a neighboring colony or by nutrient depletion in the inhibited area between colonies, subtilisin cleaves the innocuous DfsB to form the lytic Slf. The resulting reduction in the population is important for survival since the reproduction rate at the front of the colony would otherwise become faster than the ability of the colony to expand. To quote the authors: A colony’s expansion is limited by surface tension; it cannot expand fast enough to create new space for the reproducing bacteria. An alternative solution could have been for the cells to sporulate, but sporulating bacteria produce high levels of subtilisin, which would only aggravate the situation.
Homologs of Slf occur in many Gram-positive bacteria and also in the yeast Saccharomyces cerevisiae. It will be interesting to see if these Slf-like proteins play the same role in regulating competition between colonies.
S. Marvin Friedman is Professor Emeritus, Department of Biological Sciences, Hunter College of CUNY, New York City.
Be'er A, Ariel G, Kalisman O, Helman Y, Sirota-Madi A, Zhang HP, Florin EL, Payne SM, Ben-Jacob E, & Swinney HL (2010). Lethal protein produced in response to competition between sibling bacterial colonies. Proceedings of the National Academy of Sciences of the United States of America, 107 (14), 6258-63 PMID: 20308591