Bacterial longevity requires protein synthesis and a stringent response

Abstract

Gram-negative bacteria in infections, biofilms and industrial settings often stop growing due to nutrient depletion, immune responses or environmental stresses. Bacteria in this state tend to be tolerant to antibiotics and are often referred to as dormant. Rhodopseudomonas palustris, a phototrophic α-proteobacterium, can remain fully viable for more than four months when growth is arrested. Here, we show that protein synthesis, specific proteins involved in translation and a stringent response are required for this remarkable longevity. Because it can generate ATP from light during growth arrest, R. palustris is an extreme example of a bacterial species that will stay alive for long periods of time as a relatively homogeneous population of cells and it is thus an excellent model organism for studies of bacterial longevity. There is evidence that other Gram-negative species also continue to synthesize proteins during growth arrest and that a stringent response is required for their longevity as well. Our observations challenge the notion that growth-arrested cells are necessarily dormant and metabolically inactive, and suggest that such bacteria may have a level of metabolic activity that is higher than many would have assumed. Our results also expand our mechanistic understanding of a crucial but understudied phase of the bacterial life cycle.IMPORTANCEWe are surrounded by bacteria; but they do not completely dominate our planet despite the ability of many to grow extremely rapidly in the laboratory. This has been interpreted to mean that bacteria in nature are often in a dormant state. We investigated life in growth arrest of Rhodopseudomonas palustris, a proteobacterium that stays alive for months when it is not growing. We found that cells were metabolically active; they continued to synthesize proteins and mounted a stringent response, both of which were required for their longevity. Our results suggest that long-lived bacteria are not necessarily inactive but have an active metabolism that is well adjusted to life without growth.

Metabolism of non-growing bacteria

“…Yet, the majority of bacteria exist in a non-or slow-growing state, either because nutrients are lacking or other conditions are unfavorable (Bergkessel et al 2016;Gray et al 2019). However, even in the absence of biomass formation, non-growing bacteria synthetize macromolecules and they have a basal metabolism (Anderson and Domsch 2010;Yin et al 2019). For example, bacteria that account for the absolute minority of species in the biosphere have continued metabolic activity at zero growth (Hausmann et al 2019).…”

Diversity in Starvation Survival Strategies and Outcomes among Heterotrophic Proteobacteria

“…All of these studies were carried out using E. coli as a model organism. Studies of carbon starvation in other organisms have also been carried out; notably, Tn-Seq screens have identified genetic determinants of fitness during carbon starvation in Pseudomonas aeruginosa [Basta et al, 2017] and also in the photoheterotroph Rhodopseudomonas palustris [Pechter et al, 2017;Yin et al, 2019]. In both cases a minimal medium with a low starting concentration of a single carbon source was used (although R. palustris was also able to generate energy by photosynthesis), and most cells remained viable for an impressive length of time (30 days for P. aeruginosa and >120 days for R. palustris).…”

“…Adaptations occurring during the initial nutrient downshift have been the best-studied aspect of starvation survival. While the physiology of the long-term starvation survival state is different in many ways from the physiology of cells just entering stationary phase, the adaptations that occur during this transition appear to be important for survival later [Babin et al, 2016;Basta et al, 2017;Yin et al, 2019;Biselli et al, 2020], and several global regulators that have been extensively studied in E. coli are highly conserved across the Proteobacteria. These include the stress sigma factor referred to as RpoS or σ 38 (recently comprehensively reviewed by Gottesman [2019]) and the mediators of stringent response: the small alarmone (p)ppGpp and DksA [Hauryliuk et al, 2015].…”

“…Although new protein synthesis occurs at much slower average rates during starvation compared to growth [Bergkessel, 2020], it has been shown to be required for optimal survival [Yin et al, 2019]. Replacement of damaged proteins and responding to new environmental threats or opportunities are likely important functions of this low-level new protein synthesis.…”

“…For example, BONCAT (bio-orthogonal non-canonical amino acid tagging, a method to label and enrich for proteins that were newly synthesised during a defined period), applied during oxygen starvation in P. aeruginosa, identified translation elongation factor P (Efp) and a factor that enhances transcription of multiple housekeeping genes (SutA) [Babin et al, 2016]. The precise roles of the major regulators of the transition to stationary phase, RpoS and DksA/(p)pp-Gpp, during protracted starvation also remain to be determined [Bergkessel et al, 2016;Yin et al, 2019]. Under carbon starvation stress, bacterial regulatory strategies must either decrease activity to the greatest extent possible to best conserve limited resources, or maintain some ongoing metabolic activity to increase responsiveness to the environment [Bergkessel, 2020].…”