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The main point of this paper was that CidA, a bacterial holin protein, promotes cell death which releases genomic DNA into the environment which is required for biofilm formation. I want to hit on a couple of big picture points without going into to too much detail and then focus on one aspect of the paper for teaching purposes.
First, the field of biofilm formation is, in my opinion, much like a biofilm itself.....a diffuse poorly defined thing. OK, this is not really true, but many scientists use the term so loosely as to make it meaningless. A biofilm is commonly referred to as a collection of microbes encased within an extracellular polymer. Biofilms are important medically as microbes within a biofilm are often insensitive to antibiotics and microbial biofilms can grow on catheters and artificial valves and serve as sites of continual infection/dissemination. Because the cells within the biofilm are not all uniform like what would be observed during logarithmic planktonic grown, there is heterogeneity within this structure. In fact, a biofilm could be considered analogous to a microbial tissue.
Second, programmed cell death in unicellular microbes is an interesting phenomenon/idea. However, researchers in the area of microbial programmed cell death try too hard to make it directly comparable to multi-cellular organism programmed cell death. The idea of some organisms in a population sacrificing themselves for the rest of the population is well documented and makes some sense intellectually. The idea that bacteria do it the same way as the cells in your primordial hand do it is weak to wishful thinking in my opinion.
Alright, that said, the paper. First, the authors show that wild-type cells release a cytoplasmic enzyme into the environment once they enter stationary phase (basically when they stop growing). However, cells containing a null mutation in the cidA gene do not. This demonstrates a role for CidA in cell lysis, but says nothing about biofilm formation. The authors then show several assays that demonstrate that the cidA mutant does not form a robust biofilm like wild-type cells and the authors show that extracellular DNA (presumably from lysed cells) is required for proper biofilm formation in wild-type cells. (Since cidA mutants fail to lyse there is little DNA released to help form the biofilm.) Overall this is a nice analysis that provides some insights into the role of cell lysis and DNA in biofilm formation and this analysis explains the requirement for CidA in biofilm formation.
HOWEVER, this is a simple genetic analysis paper. These type of studies are done all the time by thousands of researchers working on thousands of genes in hundreds of organisms. The authors of this study break one of the
"Previously, we were unable to complement the antibiotic tolerance and murein hydrolase phenotypes of KB1050 (24), which was also the case in the present study, as the biofilm-defective phenotype was not complemented by supplying cidA on a plasmid (data not shown). However, this mutant phenotype is unlikely caused by a secondary site mutation because similar cidA mutations in different S. aureus genetic backgrounds also decreased their ability to form adherent biofilm (SI Fig. 7). This phenotype was also not caused by a polar effect on the downstream cid genes, because isogenic cidBC and cidC mutants grown under the same conditions produced biofilm comparable to that of UAMS-1 (data not shown)."
Let me break this down a little. When you make a genomic mutation, regardless of how you do it, there is a chance something else happened to the genomic DNA beyond what you were doing (life is a bitch). So you discover an outstanding phenotype in your mutant background...WOOHOO, contact Science, have them hold the presses.....Hold on cowboy, how do you know your mutation causes the phenotype and not some other horseshit that occurred when you made your mutant? In other words, how can you be sure the mutation and phenotype are linked? One quick and easy way, is to re-introduce a wild-type copy of the gene you mutated. The prediction (I love science) is that if the mutation causes the phenotype, putting a wild-type copy back in should rescue/restore/reverse the phenotype. If horseshit causes the phenotype, putting a wild-type copy back in will not rescue/restore/reverse the phenotype.
So, back to the authors and Im paraphrasing: our complementation tests failed for other phenotypes, and they failed for the phenotypes we present here (A, see below). We know it looks bad, but we are sure the phenotypes are not due to horseshit because when we make the mutant a bunch of times we see the same rescue (B) and other downstream genes are not required for the phenotype (C).
A. Not being able to complement one phenotype is bad, but not being able to complement a subsequent phenotype is not supportive evidence that things are working well.
B. Making the mutation a bunch of times independently does indeed tell you that the phenotype is not due to unlinked horseshit, but it could still be linked horseshit.
C. This is a little trickier. Generally bacterial genes required for a given process are found in operons, which are essentially chains of linked open reading frames (protein coding units). One or two transcriptional start sites exist to make mRNA which is then used to translate all the proteins in the operon. This allows for an efficient way to regulate all the components in a process simultaneously. So, the fact the cidB and cidC can be mutated without having an effect on biofilm formation tells us they aren't required for the phenotype. What the authors are considering is that the cidA mutation affects expression of cidB and/or cidC and that these are the true genes required for biofilm formation, not cidA. This would explain the lack of complementation because the authors add back cidA on a plasmid not in the chromosome (the authors did it right if you ask me), so if the cidA mutation disrupts cidB expression, putting the cidA gene back into the cell wouldn't help, because you still wouldn't get cidB expression.
OK, lets give the authors credit, they addressed some possible issues of horseshit. Here's one not addressed, what if the gene next to cidA (opposite cidB and cidC) is screwed up because of the cidA mutation. Let's say the mutation destroys the promoter of this hypothetical gene. You could make the cidA mutant over and over again and you would get the same phenotype that could not be complemented with cidA because this other gene would always be fucked. You could delete cidB and cidC with any affect on the phenotype. See my model fits all their data, actually mine is better because it fits all their data including the inability to complement the mutation. The authors never actually propose a model to explain this, they simply consider two possibilities and sweep the issue under the rug.
Now that being said, I still think the authors main conclusions are correct. There are many reasons to explain why you may not get complementation, although the authors failed to provide evidence for one of the reasons. However, and I think this is important, if you cannot complement your mutation, the bar is much higher to justify your conclusions. My problem is that the authors didn't consider a trivial explanation (the one I proposed) and based on my reading I expect the authors only mentioned anything regarding complementation and the lack therein in response to a reviewer. Again based on what the CidA protein does, the authors are likely correct. However, I think this paper allows us to highlight the importance of complementation testing in a rigorous molecular genetic study.
Edited for a couple of grammatical errors and to make a couple of points more clear.