Cell division cycle fluctuation of Pal concentration in Escherichia coli
This article has been Reviewed by the following groups
Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
The Tol-Pal proteins stabilize the outer membrane during cell division in many Gram-negative bacteria, including Escherichia coli . Pal is an outer membrane lipoprotein that can bind peptidoglycan. It accumulates at the septum during division by a mobilization-and-capture mechanism. This work further substantiates and extends knowledge of Pal’s localization in E. coli using immunolabelling; this method enables the detection of endogenous proteins. The midcell localization of Pal and TolB, as seen with fluorescent protein fusions, during cell division, was confirmed. The retention of Pal in newly formed cell poles seemed to persist longer than observed with fluorescent Pal fusions. The concentration of endogenous Pal during the cell division cycle fluctuated: it decreased initially (to half the fluorescence concentration (32.1 au µm −3 ) of the maximum (64.1 au µm −3 ) reached during the cell cycle) and then increased during the second half of the cell division cycle. We probed for possible regulators and proposed two new putative regulators of Pal. By deleting the periplasmic protease, Prc decreased the total Pal abundance (to ~65% of the fluorescence concentration in WT cells) and affected its concentration fluctuation during the cell cycle. This suggests that Prc controls a cell division stage-specific regulator of Pal. Immunolabelling also supported the prediction that the small RNA MicA suppresses Pal expression (the fluorescence concentration of Pal in cells without MicA is double that of Pal in WT cells). However, the regulation by MicA occurred in a cell cycle-independent manner. All these findings urge further research on the tight regulation of the dividing cell envelope stability.
Article activity feed
-
-
Thank you very much for submitting your revised manuscript to Access Microbiology and providing clarifications on the reviewer comments. The work presented, with a very refined methodology, is highly valuable and provides important insights to continue research on the regulation of cell division. I am pleased to let you know that it has now been accepted for publication. Congratulations to all authors!
-
-
The manuscript is interesting and well-written. It focuses on the Cell division cycle fluctuation of Pal concentration in Escherichia coli and is important for the field of cell biology. The literature review contains many relevant recent papers of good quality and the methodology is quite sound. Few minor corrections: - In the Abstract, when you talk about increase/decrease, provide values, fold-change and stats. - The second part of your Results section is much shorter than the first. - Fig.3 Why no error bars for the yellow line? - Fig.5 Are your error bars SEM or STDEV? Ensure that these details are provided. - In Discussion, you need more examples of similar studies. Highlight wgy your findings are novel. - Add a concluding paragraph.
-
Comments to Author
The ms "Cell division cycle fluctuation of Pal concentration in Escherichia coli" presented by Mertens et al. describes, as correctly indicated in the title, the spatiotemporal distribution and protein levels of the endogenous outer membrane lipoprotein Pal protein throughout the division cycle. As mentioned by the authors, it is very important for physiological studies in bacteria, but also for eukaryotes, to carefully corroborate published data obtained through mild or moderate overproduction of the studied item, whether being a wild-type or fluorescent variant. Therefore, it is no surprise to me that the contributing research groups, known to continuously question models and mechanism of protein-protein interactions within the cell division field, present this rigorously and detailed experimental …
Comments to Author
The ms "Cell division cycle fluctuation of Pal concentration in Escherichia coli" presented by Mertens et al. describes, as correctly indicated in the title, the spatiotemporal distribution and protein levels of the endogenous outer membrane lipoprotein Pal protein throughout the division cycle. As mentioned by the authors, it is very important for physiological studies in bacteria, but also for eukaryotes, to carefully corroborate published data obtained through mild or moderate overproduction of the studied item, whether being a wild-type or fluorescent variant. Therefore, it is no surprise to me that the contributing research groups, known to continuously question models and mechanism of protein-protein interactions within the cell division field, present this rigorously and detailed experimental strategy to shed some light on the behavior of Pal throughout the division cycle and its possible implication during the simultaneous invagination of the outer and cytoplasmic membrane during the cell division in E. coli. Experiments are performed very carefully and double checked for consistency. To detect Pal (and other cell division related proteins), authors rely on the time-consuming immunofluorescence microscopy, that has nowadays been replaced in many cases by real-time fluorescence imaging techniques. This replacement is because sample preparation and handling throughout immunofluorescence microscopy is time consuming and, in some cases, might lead to additional artefacts sometimes very hard to separate from the subject of study. Nonetheless, real-time fluorescence imaging techniques very often are based on the overproduction of fluorescent fusion protein of interest that when not directly expressed form the bacterial chromosome (after a clean substitution of the wild-type copy of the gene of interest) very often alter the physiological behavior of the cell. This trade-off is very complicated and should be balanced. Having said this, despite the effort undertaken by authors I have the feeling that the interesting part of the experiment is still waiting to be done. In the first part, authors show that Pal seems to reside, according to the current working model, in a mobile and a stationary form that is modulated through its direct physical temporary interaction with the periplasmic TolB protein. Throughout the early cell division stage TolB is tethered away from Pal by the cytoplasmic membrane protein complex TolA-Q-R complex. This allows Pal to interact with the newly synthesized septal PG until it is recaptured by TolB once cell division is completed. Pal resides at the new cell pole until the endogenous cellular concentration of TolB is sufficient to detach Pal from the PG layer and redistribute it along the cell until TolB is again highjacked by the midcell localized TolA-Q-R complex. Throughout this process authors not only see that the Pal protein is moving from midcell to the cell pole and back to midcell, but they also measure that the total cellular protein concentration sinuously changes throughout the cell cycle. First it drops during early divisiome assembly and then rises to almost double its initial cellular protein concentration during late divisome assembly and division. This observation prompted authors to look for potential candidates that regulate Pal throughout cell division cycle. In the second part they test whether strains lacking the genes for prc, nlpI and micA alter Pal protein levels in the cell. Prc is an endopeptidase, NlpI an endopeptidase adapter protein and MicA a sRNA that is predicted to bind anti-sense fashion twice within the pal gene and on other 18 locations within the E. coli chromosome (https://biocyc.org/gene?orgid=ECOLI&id=G0-8866#REGULON). The interesting result is that the cellular Pal levels did not significantly decrease and only slightly increase within the delta prc strain. For the micA deletion strain, they observe the cellular Pal protein concentration to fluctuate as seen for the wildtype with the difference that overall this mutant displayed a higher (up to 2 times) endogenous cellular protein concentration of Pal when compared to wt strain. I think this result is very interesting and that authors should have followed this lead to understand this result. (The fact that it is not in the ms does not say that authors did not do it). Without diminishing the presented work, I think that it would have a greater impact on the cell division field than verifying and corroborating already existing data and working models. The second part is very interesting, but very short and partly speculative. More general and minor comments: * I am puzzled by the use of LMC500 and BW25113 strain as E. coli wildtype controls. I understand that BW25113 is the parental strain of the KEIO collection and the parent of the used mutants and LMC500 is the generally used E. coli wit strain used for the physiological studies grown at 28ºC. Why di authors not P1 transduct mutants LMC500 strain? * All phase contrast microscopy images seem to be at a slightly smaller scale than the corresponding fluorescence microscopy image. Maybe they are not focused in the same plane? That might me an optical effect on paper. Overall, I like the ms. It is sound and consistent, but results are on one side not novel (first part) or too speculative (in the second part). More experiments are needed to understand what is happening.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: No concerns
-