The initial adaptive responses to nutrient depletion in bacteria often occur at the level of gene expression. Hfq is an RNA-binding protein present in diverse bacterial lineages and contributes to many different aspects of RNA metabolism during gene expression. Using photoactivated localization microscopy (PALM) and single molecule tracking, we demonstrate that Hfq forms a distinct and reversible focus-like structure in Escherichia coli specifically experiencing long-term nitrogen (N) starvation. Using the ability of T7 phage to replicate in N-starved bacteria as a biological probe of E. coli cell function during N starvation, we demonstrate that Hfq foci have a role in the adaptive response of E. coli to long-term N starvation. We further show that Hfq foci formation does not depend on gene expression once N starvation has set in and occurs independently of the transcription factor N-regulatory protein C (NtrC), that activates the initial adaptive response to N starvation in E. coli. These results serve as a paradigm to demonstrate that bacterial adaptation to long-term nutrient starvation can be spatiotemporally coordinated and can occur independently of de novo gene expression during starvation.

Under conditions of nutrient adversity, bacteria adjust metabolism to minimize cellular energy usage. This is often achieved by controlling the synthesis and degradation of RNA. In Escherichia coli, RNase E is the central enzyme involved in RNA degradation and serves as a scaffold for the assembly of the multiprotein complex known as the RNA degradosome. The activity of RNase E against specific mRNAs can also be regulated by the action of small RNAs (sRNA). In this case, the ubiquitous bacterial chaperone Hfq bound to sRNAs can interact with the RNA degradosome for the sRNA guided degradation of target RNAs. The RNA degradosome and Hfq have never been visualized together in live bacteria. We now show that in long‐term nitrogen starved E. coli, both RNase E and Hfq co‐localize in a single, large focus. This subcellular assembly, which we refer to as the H‐body, forms by a liquid‐liquid phase separation type mechanism and includes components of the RNA degradosome, namely, the helicase RhlB and the exoribonuclease polynucleotide phosphorylase. The results support the existence of a hitherto unreported subcellular compartmentalization of a process(s) associated with RNA management in stressed bacteria.

Bacteria initially respond to nutrient starvation by eliciting large-scale transcriptional changes. The accompanying changes in gene expression and metabolism allow the bacterial cells to effectively adapt to the nutrient starved state. How the transcriptome subsequently changes as nutrient starvation ensues is not well understood. We used nitrogen (N) starvation as a model nutrient starvation condition to study the transcriptional changes in Escherichia coli experiencing long-term N starvation. The results reveal that the transcriptome of N starved E. coli undergoes changes that are required to maximise chances of viability and to effectively recover growth when N starvation conditions become alleviated. We further reveal that, over time, N starved E. coli cells rely on the degradation of allantoin for optimal growth recovery when N becomes replenished. This study provides insights into the temporally coordinated adaptive responses that occur in E. coli experiencing sustained N starvation. IMPORTANCE Bacteria in their natural environments seldom encounter conditions that support continuous growth. Hence, many bacteria spend the majority of their time in states of little or no growth due to starvation of essential nutrients. To cope with prolonged periods of nutrient starvation, bacteria have evolved several strategies, primarily manifesting themselves through changes in how the information in their genes is accessed. How these coping strategies change over time under nutrient starvation is not well understood and this knowledge is not only important to broaden our understanding of bacterial cell function, but also to potentially find ways to manage harmful bacteria. This study provides insights into how nitrogen starved Escherichia coli bacteria rely on different genes during long term nitrogen starvation.

26Bacteria initially respond to conditions that attenuate their growth by eliciting large-scale 27 transcriptional changes. The accompanying changes in gene expression and metabolism allow 28 the bacterial cells to effectively adapt to the growth attenuated state. How the transcriptome 29 subsequently changes as growth attenuation ensues is not well understood. We used nitrogen 30 (N) starvation as a model nutrient starvation condition to study the transcriptome of growth 31 attenuated Escherichia coli. The results reveal that the transcriptome of nitrogen starvation-32 induced growth attenuated E. coli remains dynamic and perturbations to it compromise the 33 viability of growth attenuated bacteria and their ability to effectively recover growth when N 34 starvation conditions become alleviated. We further reveal that, over time, N starvation-35 induced growth attenuated bacteria rely on the degradation of allantoin for optimal growth 36 recovery when N becomes replenished. This study provides insights into the temporally 37 coordinated adaptive responses that occur in E. coli experiencing sustained N starvation. 38 39 IMPORTANCE 40Bacteria in their natural environments seldom encounter conditions that support continuous 41 growth. Hence, many bacteria spend the majority of their time in states of little or no growth 42 due to starvation of essential nutrients. To cope with prolonged periods of nutrient starvation, 43 bacteria have evolved several strategies, primarily manifesting themselves through changes in 44 how the information in their genes is accessed. How these coping strategies change over time 45 under nutrient starvation is not well understood and this knowledge is not only important to 46 broaden our understanding of bacterial cell function, but also to potentially find ways to 47 manage harmful bacteria. This study provides insights into how nitrogen starved Escherichia 48 coli bacteria rely on different genes during long term nitrogen starvation. 49 50 3 KEYWORDS 51