జర్నల్ ఆఫ్ బయోక్వివలెన్స్ & బయోఎవైలబిలిటీ

నైరూప్య

Drastic Decline in Optical Density during Stationary Phase Meant that Bacillus subtilis NRS-762 is not Suitable as Model Organism in Microbial Survivability Studies

Wenfa NG

Survival of microbes on various surfaces and habitats is a question of importance to basic science, as well as health care, water treatment and distribution, ecology, and search for life in other planetary bodies. To this end, various model organisms known to be resilient against a variety of environmental stressors are used for understanding the mechanisms underlying survival in extreme environments, or conditions mimicking those of the investigated habitats. Observations of drastic decline in optical density of Bacillus subtilis NRS-762 (ATCC 8473) in LB Lennox and Tryptic Soy Broth (TSB) at temperatures of 25°C, 30°C and 37°C, after the aerobic shake flask culture reached maximal cell density at stationary phase, pointed to possible cell lysis as mechanism for cell death. Specifically, optical density of the bacterium declined from 5.4 at 22.5 hours post-inoculation in LB Lennox medium to 2.5 after 38 hours of culture at 25°C and 250 rpm rotational shaking. Similarly, optical density of B.subtilis NRS-762 also precipitously declined from 6.4 at 33 hours of culture to 1.8 at 51 hours post-inoculation at 37°C in TSB. This is in stark contrast to aerobic growth of Escherichia coli DH5α (ATCC 53868) in LB Lennox medium at 37°C and 230 rpm rotational shaking, where optical density remained stable during stationary phase. More importantly, observations of B.subtilis NRS-762 culture after autoclave decontamination revealed a lack of cellular debris; thereby, indicating massive cell lysis resulting in population collapse. Although B. subtilis is known to enter into various cellular differentiation programmes upon nutrient starvation, complete absence of cell debris that usually settle at the bottom of the shake flask after autoclave decontamination pointed to cannibalism or prophage induced cell lysis as key reasons underlying observed drastic decline in optical density of the culture. However, prophage induced cell lysis may be discounted as this would have led to rapid collapse of the entire cell population shortly after entry into stationary phase except during growth of B. subtilis NRS-762 at 37°C where a temperature sensitive sensor might have activated prophage entry into the lytic programme. Hence, cannibalism, where a subpopulation of B.subtilis NRS-762 cells secrete cell lysis factors which other B.subtilis NRS-762 cells are not resistant to, likely resulted in massive cell lysis that released cellular contents that served as nutrients for the surviving population. Collectively, B.subtilis NRS-762 is not suitable as model organism for microbial survivability studies given its tendency to undergo differentiation into the cannibalism programme, which in killing a significant fraction of cells upon nutrient starvation, would confound experiments aimed at understanding the survivability of the bacterium under a variety of environmental conditions.