DNA replication and cell enlargement of Enterococcus faecalis protoplasts

Satoshi Kami; Rintaro Tsuchikado; Hiromi Nishida; Satoshi Kami; Rintaro Tsuchikado; Hiromi Nishida

doi:10.3934/microbiol.2019.4.347

AIMS Microbiology

2019, Volume 5, Issue 4: 347-357. doi: 10.3934/microbiol.2019.4.347

Previous Article Next Article

Research article Special Issues

DNA replication and cell enlargement of Enterococcus faecalis protoplasts

Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan

Received: 19 August 2019 Accepted: 23 October 2019 Published: 25 October 2019

Protoplasts of Enterococcus faecalis did not divide but enlarged in Difco Marine Broth containing penicillin. Our previous studies have demonstrated that transcription and translation were essential for bacterial cell enlargement. However, it was uncertain whether replication was also essential. In this study, we measured the amount of DNA in E. faecalis cells during the course of enlargement using quantitative polymerase chain reaction. The growth of normally divided cells (native forms) of E. faecalis exhibited a log phase before 6 h of incubation was reached. Although a difference in quantitation cycle (Cq) values between the replication initiation and termination regions was observed in the log phase, it was not present in the stationary growth phase. On the other hand, the amount of DNA in E. faecalis protoplasts increased during the cell enlargement incubation. The difference of Cq values between the protoplasts at 0 and 96 h of incubation was 8–9, indicating that the DNA amount at 96 h was 200–500 times higher than that at 0 h. The Cq values differed between the replication initiation and termination regions, indicating that the replication level was high. When novobiocin, a DNA replication inhibitor, was added to the medium at 24 h of incubation, DNA replication and cell enlargement were almost stopped. Thus, replication plays an important role in the enlargement of E. faecalis protoplasts.
- DNA replication,
- Enterococcus faecalis,
- novobiocin treatment,
- protoplast enlargement,
- replication initiation,
- replication termination
Citation: Satoshi Kami, Rintaro Tsuchikado, Hiromi Nishida. DNA replication and cell enlargement of Enterococcus faecalis protoplasts[J]. AIMS Microbiology, 2019, 5(4): 347-357. doi: 10.3934/microbiol.2019.4.347

Related Papers:

Abstract

Protoplasts of Enterococcus faecalis did not divide but enlarged in Difco Marine Broth containing penicillin. Our previous studies have demonstrated that transcription and translation were essential for bacterial cell enlargement. However, it was uncertain whether replication was also essential. In this study, we measured the amount of DNA in E. faecalis cells during the course of enlargement using quantitative polymerase chain reaction. The growth of normally divided cells (native forms) of E. faecalis exhibited a log phase before 6 h of incubation was reached. Although a difference in quantitation cycle (Cq) values between the replication initiation and termination regions was observed in the log phase, it was not present in the stationary growth phase. On the other hand, the amount of DNA in E. faecalis protoplasts increased during the cell enlargement incubation. The difference of Cq values between the protoplasts at 0 and 96 h of incubation was 8–9, indicating that the DNA amount at 96 h was 200–500 times higher than that at 0 h. The Cq values differed between the replication initiation and termination regions, indicating that the replication level was high. When novobiocin, a DNA replication inhibitor, was added to the medium at 24 h of incubation, DNA replication and cell enlargement were almost stopped. Thus, replication plays an important role in the enlargement of E. faecalis protoplasts.

Acknowledgments

We thank Sawako Takahashi for valuable comments. This work was funded by JSPS KAKENHI Grant Numbers 16K14891 (to HN).

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

References

[1]	Marvin DA (1968) Control of DNA replication by membrane. Nature 219: 485–486. doi: 10.1038/219485a0
[2]	Sueoka N, Quinn WG (1968) Membrane attachment of the chromosome replication origin in Bacillus subtilis. Cold Spring Harb Symp Quant Biol 33: 695–705. doi: 10.1101/SQB.1968.033.01.078
[3]	Earhart CF, Tremblay GY, Daniels MJ, et al. (1968) DNA replication studied by a new method for the isolation of cell membrane-DNA complexes. Cold Spring Harb Symp Quant Biol 33: 707–710. doi: 10.1101/SQB.1968.033.01.079
[4]	Leibowitz PJ, Schaechter M (1975) The attachment of the bacterial chromosome to the cell membrane. Int Rev Cytol 41: 1–28. doi: 10.1016/S0074-7696(08)60964-X
[5]	Garner J, Crooke E (1996) Membrane regulation of the chromosomal replication activity of E. coli DnaA requires a discrete site on the protein. EMBO J 15: 3477–3485.
[6]	Boeneman K, Crooke E (2005) Chromosomal replication and the cell membrane. Cuur Opin Microbiol 8: 143–148. doi: 10.1016/j.mib.2005.02.006
[7]	Hill NS, Kadoya R, Chattoraj DK, et al. (2012) Cell size and the initiation of DNA replication in bacteria. PLOS Genet 8: e1002549. doi: 10.1371/journal.pgen.1002549
[8]	Wallden M, Fange D, Lundius EG, et al. (2016) The synchronization of replication and division cycles in individual E. coli cells. Cell 166: 729–739.
[9]	Kusaka I (1967) Growth and division of protoplasts of Bacillus megaterium and inhibition of division by penicillin. J Bacteriol 94: 884–888.
[10]	Ranjit DK, Young KD (2013) The Rcs stress response and accessory envelope proteins are required for de novo generation of cell shape in Escherichia coli. J Bacteriol 195: 2452–2462. doi: 10.1128/JB.00160-13
[11]	Takahashi S, Nishida H (2015) Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of Escherichia coli. J Gen Appl Microbiol 61: 262–265. doi: 10.2323/jgam.61.262
[12]	Takayanagi A, Takahashi S, Nishida H (2016) Requirement of dark condition for enlargement of the aerobic anoxygenic photosynthetic marine bacterium Erythrobacter litoralis. J Gen Appl Microbiol 62: 14–17. doi: 10.2323/jgam.62.14
[13]	Takahashi S, Takayanagi A, Takahashi Y, et al. (2016) Comparison of transcriptomes of enlarged spheroplasts of Erythrobacter litoralis and Lelliottia amnigena. AIMS Microbiol 2: 152–189. doi: 10.3934/microbiol.2016.2.152
[14]	Nakazawa M, Nishida H (2017) Effects of light and oxygen on the enlargement of spheroplasts of the facultative anaerobic anoxygenic photosynthetic bacterium Rhodospirillum rubrum. Jacobs J Biotechnol Bioeng 3: 014.
[15]	Nishino K, Morita Y, Takahashi S, et al. (2018) Enlargement of Deinococcus grandis spheroplasts requires Mg²⁺ or Ca²⁺. Microbiology 164: 1361–1371. doi: 10.1099/mic.0.000716
[16]	Kuroda T, Okuda N, Saitoh N, et al. (1998) Patch clamp studies on ion pumps of the cytoplasmic membrane of Escherichia coli. Formation, preparation, and utilization of giant vacuole-like structures consisting of everted cytoplasmic membrane. J Biol Chem 273: 16897–16904.
[17]	Nakamura K, Ikeda S, Matsuo T, et al. (2011) Patch clamp analysis of the respiratory chain in Bacillus subtilis. Biochim Biophys Acta 1808: 1103–1107. doi: 10.1016/j.bbamem.2011.01.006
[18]	Fisher K, Phillips C (2009) The ecology, epidemiology and virulence of Enterococcus. Microbiology 155: 1749–1757. doi: 10.1099/mic.0.026385-0
[19]	McBride SM, Fischetti VA, LeBlanc DJ, et al. (2007) Genetic diversity among Enterococcus faecalis. PLoS One 2: e582. doi: 10.1371/journal.pone.0000582
[20]	Palmer KL, Godfrey P, Griggs A, et al. (2012) Comparative genomics of Enterococci: variation in Enterococcus faecalis, clade structure in E. faecium, and defining characteristics of E. gallinarum and E. casseliflavus. mBio 3: e00318–11.
[21]	Paulsen IT, Banerjei L, Myers GSA, et al. (2003) Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science 299: 2071–2074. doi: 10.1126/science.1080613
[22]	Bourgogne A, Garsin DA, Qin X, et al. (2008) Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF. Genome Biol 9: R110. doi: 10.1186/gb-2008-9-7-r110
[23]	Brede DA, Snipen LG, Ussery DW, et al. (2011) Complete genome sequence of the commensal Enterococcus faecalis 62, isolated from a healthy Norwegian infant. J Bacteriol 193: 2377–2378. doi: 10.1128/JB.00183-11
[24]	Zischka M, Kuenne C, Blom J, et al. (2012) Complete genome sequence of the porcine isolate Enterococcus faecalis D32. J Bacteriol 194: 5490–5491. doi: 10.1128/JB.01298-12
[25]	Fritzenwanker M, Kuenne C, Billion A, et al. (2013) Complete genome sequence of the probiotic Enterococcus faecalis symbioflor 1 clone DSM 16431. Genome Announc 1: e00165–12.
[26]	Yu Z, Chen Z, Cheng H, et al. (2014) Complete genome sequencing and comparative analysis of the linezolid-resistant Enterococcus faecalis strain DENG1. Arch Microbiol 196: 513–516. doi: 10.1007/s00203-014-0986-y
[27]	Minogue TD, Daligault HE, Davenport KW, et al. (2014) Complete genome assembly of Enterococcus faecalis 29212, a laboratory reference strain. Genome Announc 2: e00968–14.
[28]	Jiao Y, Zhang L, Liu F, et al. (2016) Complete genome sequence of Enterococcus faecalis LD33, a bacteriocin-producing strain. J Biotechnol 227: 79–80. doi: 10.1016/j.jbiotec.2016.04.030
[29]	Uchimura Y, Wyss M, Brugiroux S, et al. (2016) Complete genome sequences of 12 species of stable defined moderately diverse mouse microbiota 2. Genome Announc 4: e00951–16.
[30]	Watanabe S, Ohbayashi R, Shiwa Y, et al. (2012) Light-dependent and asynchronous replication of cyanobacterial multi-copy chromosomes. Mol Microbiol 83: 856–865. doi: 10.1111/j.1365-2958.2012.07971.x
[31]	Gellert M, O'Dea MH, Itoh T, et al. (1976) Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc Natl Acad Sci USA 73: 4474–4478. doi: 10.1073/pnas.73.12.4474
[32]	Sugino A, Higgins NP, Brown PO, et al. (1978) Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc Natl Acad Sci USA 75: 4838–4842. doi: 10.1073/pnas.75.10.4838
[33]	Khodursky AB, Peter BJ, Schmid MB, et al. (2000) Analysis of topoisomerase function in bacterial replication fork movement: use of DNA microarrays. Proc Natl Acad Sci USA 97: 9419–9424. doi: 10.1073/pnas.97.17.9419

microbiol-05-04-347-s001.pdf

Reader Comments

Your name:*

Email:*
© 2019 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)