Abstract“Non-growing” is a dominant life form of microorganisms in nature, where available nutrients and resources are limited. In laboratory culture systems, Escherichia coli can survive for years under starvation, denoted as long-term stationary phase, where a small fraction of cells manages to survive by recycling resources released from nonviable cells. Although the physiology by which viable cells in long-term stationary phase adapt to prolonged starvation is of great interest, their genome-wide response has not been fully understood. In this study, we analyzed transcriptional profiles of cells exposed to the supernatant of 30-day long-term stationary phase culture and found that their transcriptome profiles displayed several similar responses to those of cells in the 16-h short-term stationary phase. Nevertheless, our results revealed that cells in long-term stationary phase supernatant exhibit higher expressions of stress-response genes such as phage shock proteins (psp), and lower expressions of growth-related genes such as ribosomal proteins than those in the short-term stationary phase. We confirmed that the mutant lacking the psp operon showed lower survival and growth rate in the long-term stationary phase culture. This study identified transcriptional responses for stress-resistant physiology in the long-term stationary phase environment.

18 Background 19 "Non-growing" is a dominant life form of microorganisms in nature, where available 20 nutrients and resources are extremely limited. However, the knowledge of the manner in 21 which microorganisms resist nutrient deficiency is still rudimentary compared to those of 22 the growing cells. In laboratory culture, Escherichia coli can survive for several years 23 under starvation, denoted as long-term stationary phase (LSP), where a small fraction of 24 the cells survive by recycling resources released from the starved nonviable cells and 25 constitute a model system for understanding survival mechanisms under long-term 26 starvation. Although the physiology by which viable cells in LSP adapt to long-term 27 starvation is of great interest, their genome-wide response has not yet been fully 28 understood. 29 30 Results 31To understand the physiological state of viable cells in the LSP environment, we analyzed 32 the transcriptional profiles of cells exposed to the supernatant of LSP culture. We found 33 that high expression of transporter genes and low expression of biosynthesis genes are 34 the primary responses of the cells in the LSP supernatant compared to growing cells, 35 which display similar responses to cells entering the stationary phase from the exponential 36 growth phase. We also revealed some specific transcriptional responses in the LSP 37 supernatant, such as higher expression of stress-response genes and lower expression of 38 translation-related genes, compared to other non-growing conditions. This suggests that 39 cells in LSP are highly efficient in terms of cellular survival and maintenance functions 40 under starvation conditions. We also found population-density-dependent gene 41 expression profiles in LSP, which are also informative to understand the survival 42 mechanism of bacterial population. 43 44 Conclusion 45 Our current comprehensive analysis of the transcriptome of E. coli cells provides an 46 overview of the genome-wide response to the long-term starvation environment. We 47 51 52 Keywords 53 Long-term stationary phase, Gene ontology, Transcriptome, Non-growing state, 54 Escherichia coli 55 56 Background 57 The physiology of growing microorganisms has been quantitatively characterized based 58 on stable and reproducible culture systems since the 1940s [1]. The quantitative 59 approaches have steadily revealed fundamental principles in exponentially growing 60 microorganisms, such as the growth rate's strong dependence on available nutrients in the 61 environment or the cellular macromolecular composition (e.g., ribosomes, DNA, and 62 RNA) [2-4]. The expression profiles of genes responsible for these interrelations have 63 also been characterized [5-7], enabling us to understand the physiology of growing cells 64 from systems-level mechanisms. However, most prokaryotes in nature live in nutrient-65 poor environments, such as deep biospheres, where they exhibit slow-or non-growing 66 states [8]. The physiology of such microbial cells has been appreciated as a general ...