Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of <i>Escherichia coli</i>

Abstract

None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work.cillin (an inhibitor of peptidoglycan synthesis) was shown to generate giant protoplasts (Kuroda et al., 1998;Kusaka, 1967;Nakamura et al., 2011).Generally, DNA duplicates before cell division, and it is distributed to each cell during cell division. Most bacteria undergo symmetric binary fission. During the growth of spheroplasts, although cell division was not observed, DNA replication was, indeed, observed (Kuroda et al., 1998;Nakamura et al., 2011). However, it is uncertain whether plasmid DNA replicates during spheroplast growth. Several factors influence the synchronous regulation between chromosome and plasmid (Nordström and Dasgupta, 2006).In this study, we measured the chromosomal and plasmid DNA of E. coli spheroplasts by using real-time quantitative PCR, in order to elucidate the change in copy numbers of chromosomal and plasmid DNA during the growth of spheroplasts. Materials and MethodsPreparation of giant spheroplasts from E. coli. Giant spheroplasts were prepared using a modified version of the method previously described by Kusaka (1967) and the spheroplast incubation method reported by Kuroda et al. (1998). E. coli SCS1 (Stratagene) cells, which was transformed with the plasmid pHRP311 (14 kbp) with an RSF1010 replicon, were grown on marine broth agar (Difco) containing streptomycin under aerobic conditions. The harvested cells (0.003 g) were suspended in a buffer (1 mL) consisting of 0.1 M Tris-HCl (pH 7.6) and 0.3 M sucrose. Lysozyme (Wako) (200 µg/mL) was added to the cell suspension, and the suspension was incubated at 37°C for 15 min. After the suspension was divided into 2 aliquots (500 µL each), the cells were harvested (centrifugation Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of Escherichia coli (Received August 8, 2015; Accepted September 9, 2015) Sawako Takahashi and Hiromi Nishida* Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Japan We generated spheroplasts from Escherichia coli carrying a broad-host-range plasmid. In the presence of penicillin, the spheroplasts did not divide but grew and enlarged in marine broth, whereas, in the absence of penicillin, they elongated. We quantified cellular DNA at different time points by using real-time quantitative PCR. Both chromosomal and plasmid DNA had replicated during spheroplast growth not only in the absence but also in the presence of penicillin. Thus, plasmid DNA and chromosomal DNA replication might be regulated synchronously during the growth of spheroplasts. Biotechnology

Requirement of dark culture condition for enlargement of spheroplasts of the aerobic anoxygenic photosynthetic marine bacterium <i>Erythrobacter litoralis</i>

“…3). This is consistent with our previous findings using Escherichia coli (Takahashi and Nishida, 2015). Therefore, the speed of DNA replication during enlargement is slower than during cell division.…”

“…Although DNA replication is generally related to cell division, spheroplasts have been shown to replicate their DNA without undergoing cell division under incubation conditions (Takahashi and Nishida, 2015). Furthermore, spheroplasts from Escherichia coli can recover to their native cellular morphology via cell wall resynthesis (Ranjit and Young, 2013).…”

Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts

“…To recover to their native forms, cell wall resynthesis is necessary [ 70 , 71 ]. Although bacterial protoplasts/spheroplasts cannot divide, DNA replication occurs during cell enlargement [ 19 , 21 , 49 , 72 ]. In the presence of the peptidoglycan biosynthetic inhibitor penicillin, E. coli forms a bulge at the site where the new cell wall is normally formed, which requires DNA replication [ 73 ].…”

Cell wall damage increases macromolecular crowding effects in the Escherichia coli cytoplasm

“…One study showed that these cells have the same biopolymer volume fraction as exponentially growing cells 1 . In contrast, other groups measured an increase in biomacromolecule content (DNA, RNA, and protein) 25,26,[28][29][30][31][32] : Spheroplasts continue to synthesize molecules and can remain viable and regain their usual rod-like shape after spheroplasting 25,[33][34][35] . Cell wall disruption can lead to a host of downstream effects.…”