Effects of Light and Oxygen on the Enlargement of Spheroplasts of the Facultative

Short Communication

Effects of Light and Oxygen on the Enlargement of Spheroplasts of the Facultative

Corresponding author: Hiromi Nishida, Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan, Email: hnishida@pu-toyama.ac.jp

Abstract

TRhodospirillum rubrum spheroplasts were enlarged under anaerobic dark conditions. In addition, they were under anaerobic continuous light conditions. These results suggest that they may be able to grow using energy generated from fermentation and anaerobic respiration. Thus, growth of R. rubrum spheroplasts differs from that of Erythrobacter litoralis spheroplasts under anaerobic conditions.

Keywords: Rhodospirillum Rubrum; Spheroplasts Enlargement; Light; Oxygen; Fermentation

Introduction

In marine broth containing penicillin, an inhibitor of peptidoglycan synthesis, bacterial spheroplasts do not divide, but instead enlarge [1-3]. We previously showed that the growth of normally dividing bacterial cells differs from that of spheroplasts [1-5]. Thus, although number of cells increases during the growth of normal cells, each cell enlarges during the growth of spheroplasts.

Photosynthetic purple bacteria, belonging to the Alphaproteobacteria, contain both inner and outer membranes. Within the inner membrane, there are photosynthesis-related proteins [6, 7]. The anoxygenic photosynthetic apparatus of purple bacteria, which is synthesized in the dark [8, 9], transforms light energy into an electrochemical gradient of protons across the inner membrane [9]. This photosynthetic system may share components with the respiration system [8, 10-12]. The modeling the electron transport chain of purple bacteria was reported [13].

Erythrobacter litoralis, an aerobic anoxygenic photosynthetic bacterium, does not perform aerobic photosynthesis [14]. As far as we know, E. litoralis cannot grow anaerobically in the dark. Generally, aerobic anoxygenic photosynthetic bacteria photosynthesize under aerobic condition but do not under anaerobic condition [9, 15]. Interestingly, E. litoralis spheroplasts grow under anaerobic light-dark conditions [5]. Our findings showed that E. litoralis spheroplasts can photosynthesize and grow [3, 5, 16, 17]. Thus, although normal cells of E. litoralis do not photosynthesize under anaerobic light-dark conditions, the spheroplasts photosynthesize [5].

Rhodospirillum rubrum, a facultative anaerobic anoxygenic photosynthetic bacterium, is capable of growth either anaerobically in the light (mainly by photosynthesis) or aerobically in the dark (mainly by respiration) [18-20]. In addition, R. rubrum can grow anaerobically in the dark by fermentation and anaerobic respiration [21]. Thus, the R. rubrum photosynthetic system differs from that of E. litoralis in normally divided cells. In this study, we elucidated whether the photosynthetic system also differs or not in the spheroplasts between these two purple bacteria.

In this study, we evaluated the morphology of spheroplasts of R. rubrum during growth in marine broth containing 12, 30, 60, or 300 μg/mL of penicillin at 25°C. R. rubrum spheroplasts were incubated at 25°C in the dark, in the light, and in the light and dark (12-h light-dark cycle). A fluorescent lamp was used to maintain the light conditions. Spheroplasts isolated from different cultures at various time points were evaluated morphologically. BIONIX2 (Sugiyama-Gen, Tokyo, Japan) was used to culture spheroplasts under oxygen-free conditions. Culture dishes were placed in a plastic bag, and a capsule containing a deoxidizing agent was opened and placed in the plastic bag along with an oxygen meter.

biotech fig 14.1

Figure 1. Phase-contrast micrographs of Rhodospirillum rubrum cells in marine broth containing 12 μg/mL of penicillin.

Cells under anaerobic (oxygen-free) conditions. (B) Cells under aerobic (20.5% oxygen) conditions. Phase-contrast microscopy images were obtained using an Olympus CKK41 microscope. Scale bar = 5 μm. Arrows show filamentous cells.

We observed some filamentous cells after 48 h and 144 h of growth in cultures containing 12 μg/mL of penicillin, but we did not observe filamentous cells in cultures containing 30 μg/ mL of penicillin (Figure. 1 and 2). The filamentous cells wereobserved in marine broth in the absence or low concentration of penicillin [1, 2]. We showed that the gene expression in theenlarged spheroplasts is different from the filamentous cells in Lelliottia amnigena [22].

biotech fig 14.2

Figure 2. Phase-contrast micrographs of Rhodospirillum rubrum cells in marine broth containing 30 μg/mL of penicillin.

Cells under anaerobic (oxygen-free) conditions. (B) Cells under aerobic (20.5% oxygen) conditions. Phase-contract microscopy images were obtained using an Olympus CKK41 microscope. Scale bar = 5 μm.

The energy supplying system in the filamentous cells is unknown. The results in cultures containing 60 and 300 μg/mL of penicillin did not differ from those in cultures containing 30 μg/mL of penicillin (data not shown). R. rubrum spheroplastswere enlarged under anaerobic dark conditions (Figure. 1 and 2), indicating that R. rubrum spheroplasts did not require oxygen for enlargement in the dark. Thus, they may be able to grow using energy generated from fermentation and anaerobic respiration.

Enlargement of R. rubrum spheroplasts under anaerobic lightdark conditions (Figure. 1 and 2) suggested that they synthesized ATP through photosynthesis, although it is uncertain how many photosynthetic apparatus in the membrane of enlarged cell of R. rubrum. This indicates that the dark is essential for photosynthesis, likely because the photosynthetic apparatus of purple bacteria is synthesized in the dark [9]. Continuous light inhibited the enlargement of R. rubrum spheroplasts under aerobic conditions (Figure 1 and 2). This result is consistent with those of previous studies in E. litoralis [3, 5]. Thus, continuous light may damage not only the photosynthetic system but also the respiration system. Surprisingly, some spheroplasts were enlarged under anaerobic continuous light conditions (Figure 1 and 2), which suggests that continuous light may not damage the fermentation and anaerobic respiration systems in R. rubrum spheroplasts.

Acknowledgements

This work was supported by grant from The Cannon Foundation (to HN) and JSPS KAKENHI Grant Number 16K14891 (toHN).

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