None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work.Erythrobacter are rod shaped and approximately 0.4-0.5 × 1.0-5.0 µm in size (Yurkov and Beatty, 1998).Incubation of spheroplasts formed by lysozymes with penicillin, an inhibitor of peptidoglycan synthesis, was shown to generate giant protoplasts (Kuroda et al., 1998;Kusaka, 1967;Nakamura et al., 2011). 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).In this study, we evaluated DNA replication and cell morphology of spheroplasts from Erythrobacter litoralis during growth in the absence and presence of penicillin at different temperatures, in order to determine the optimal conditions for growth of Erythrobacter litoralis spheroplasts.
Materials and Methods
Cultivation of spheroplasts from Erythrobacter litoralis.Giant spheroplasts were prepared using a previously described method with modifications (Kusaka, 1967). Spheroplast incubation was performed according to the method reported by Kuroda et al. (1998). Cells of the Erythrobacter litoralis NBRC 102620 strain were grown in marine broth agar (Difco Co.) under aerobic conditions. The harvested cells (0.003 g) were suspended in buffer (1 mL) consisting of 0.1 M Tris-HCl (pH 7.4) and 0.3 M sucrose. Lysozyme (Wako Co.) (200 µg/mL) was added to the cell suspension, which was then incubated at 25°C for 15 min. Afterwards, the suspension was divided into 2 aliquots (500 µL each), followed by harvesting (centrifugation for 5 min at 3000 rpm) and resuspension in marine broth (500 µL) containing 600 µg/mL penicillin G (Serva). The susRequirement of dark culture condition for enlargement of spheroplasts of the aerobic anoxygenic photosynthetic marine bacterium Erythrobacter litoralis (Received September 15, 2015; Accepted October 23, 2015) Ayana Takayanagi, Sawako Takahashi, and Hiromi Nishida* Biotechnology, Toyama Prefectural University, Imizu, Japan In the present study, spheroplasts from the aerobic anoxygenic photosynthetic marine bacterium Erythrobacter litoralis were generated and cultivated. In the presence of penicillin, the spheroplasts grew and enlarged in marine broth without undergoing cell division. However, continuous light inhibited their enlargement, and they were therefore cultivated in the dark. Cellular DNA was quantified at various time points (0, 24, and 48 h) and temperatures (20°C, 25°C, and 30°C) using realtime quantitative PCR. The DNA content was highest at 30°C in the absence of penicillin, whereas there was no observable change with exposure to penicillin at all evaluated temperatures. During growth, larger spheroplasts were more frequently observed at 25°C in the presence o...
