The physiology of growth arrest: uniting molecular and environmental microbiology

Abstract

Most bacteria spend the majority of their time in prolonged states of very low metabolic activity and little or no growth, in which electron donors, electron acceptors and/or nutrients are limited, but cells are poised to undergo rapid division cycles when resources become available. These non-growing states are far less studied than other growth states, which leaves many questions regarding basic bacterial physiology unanswered. In this Review, we discuss findings from a small but diverse set of systems that have been used to investigate how growth-arrested bacteria adjust metabolism, regulate transcription and translation, and maintain their chromosomes. We highlight major questions that remain to be addressed, and suggest that progress in answering them will be aided by recent methodological advances and by dialectic between environmental and molecular microbiology perspectives.

Figure 1 |. Metabolic rewiring during growth arrest.

Different organisms use distinct metabolic strategies to meet cellular requirements under growth-limiting conditions. Both Mycobacterium spp. and Pseudomonas aeruginosa must adjust their strategies for maintaining their membrane electrochemical gradient under oxygen-limited conditions. Mycobacterium spp. continue to use the electron transport chain, using either low levels of oxygen or fumarate as the terminal electron acceptor, and also contribute to the membrane electrochemical gradient by secreting succinate, which is generated through reversal of the tricarboxylic acid (TCA) cycle in Mycobacterium tuberculosis and by the glyoxylate shunt in Mycobacterium smegmatis. In P. aeruginosa, fluxes through the TCA cycle and electron transport chain drop close to zero under anoxic conditions, and substrate-level phosphorylation generates ATP to run the ATP synthase in reverse, thus pumping protons outward across the membrane. Under carbon-limiting conditions, fatty acid β-oxidation generates acetyl-CoA, which can be fed into biosynthetic pathways through the glyoxylate shunt; this generates the TCA cycle intermediates that are most useful as precursors without overproducing other intermediates. P_i, inorganic phosphate; Q, quinone.

Figure 2 |. Transcription and translation during different growth phases.

a | During exponential phase, rRNA genes are among the most highly transcribed in the cell, as ribosome biogenesis is a top biosynthetic priority. In addition, genes with promoters that have strong consensus sequences for RpoD binding are highly expressed and efficiently co-transcriptionally translated, aided by the transcription elongation factor NusG, which helps physically associate a ribosome with the RNA polymerase. The stationary phase sigma factor RpoS is synthesized to some extent but fails to compete with RpoD for RNA polymerase; consequently, stress-responsive genes with promoters that do not match the RpoD consensus sequence are not efficiently expressed. b | In the transition to stationary phase, limitation for amino acids activates RelA, which senses uncharged tRNAs and synthesizes the alarmone guanosine pentaphosphate ((p)ppGpp). (p)ppGpp, in conjunction with DksA, represses the transcription of rRNA by destabilizing the rRNA open promoter complex. The decrease in abundance of the nucleoid-associated protein Fis and the increase in abundance of leucine-responsive regulatory protein (Lrp) also contribute to rRNA repression. Hibernation promoting factor (HPF) and ribosome modulation factor (Rmf) are upregulated and lead to the dimerization of ribosomes to 100S complexes that are inactive for translation. RNA polymerase complexes with RpoD are selectively sequestered through several mechanisms, including binding to a small RNA (6S RNA), and levels of RpoS are also increased, which leads to increased transcription of stress-responsive genes in the RpoS regulon. RpoS can also drive transcription of housekeeping genes that have RpoD-consensus promoters, but does so at much lower levels than transcription of these genes that is mediated by RpoD. c | During growth arrest, overall gene expression activity is much lower than in exponential phase or the transition to stationary phase. Although much remains to be elucidated about how these very low levels of activity are regulated, the observations that several global regulators change in abundance suggest some possible mechanisms. Regulators that are important during the transition to stationary phase, such as DksA, (p)ppGpp, RpoS and HPF, seem to be downregulated during growth arrest. Also, the complement of nucleoid-associated proteins (NAPs) changes substantially, which probably affects the expression of rRNA and other genes, although details remain to be explored. In Pseudomonas aeruginosa, some factors that are thought to contribute to transcription and translation elongation (GreA, S10 and elongation factor P (Efp)) were upregulated, possibly suggesting that they could help buffer against pausing and arrest in severely substrate-limited conditions. Although some ribosomes are catabolized, with the dual benefit of decreasing the number of ribosomes that are competing for amino acid substrates and liberating nutrients to be used for energy and maintenance, the newly identified transcriptional regulator SutA, which is upregulated during growth arrest in P aeruginosa, enhances rRNA and ribosomal protein gene expression, which suggests that some repair and replacement of ribosomes may also be important.

Figure 3 |. Overview of cellular morphology with emphasis on nucleoid.

Cells undergo gross morphological changes in transitions between different growth states. a | In exponential phase, the chromosome has a high degree of negative supercoiling, owing to large amounts of active transcription. RNA polymerase is mostly bound to DNA and is gathered in large clusters of highly transcriptionally active genes, which tend to migrate to the periphery of the nucleoid region. Ribosomes are observed in the central portion of the cell, sharing space with the nucleoid. New rounds of replication, initiated by DnaA, begin even before previous rounds have completed. Cytokinesis is inhibited by interaction of an abundant metabolic enzyme (for example, OpgH in Escherichia coli) with FtsZ, thus maintaining a larger cell size. b | In the transition to stationary phase, decreases in total transcription, and rRNA transcription in particular, lead to less supercoiling of the chromosome but a more condensed overall morphology, with fewer RNA polymerases and ribosomes associated with the nucleoid region in the centre of the cell. New rounds of replication are inhibited by the decreased abundance and activity of DnaA, but cell division to segregate chromosomes that have already been replicated can still take place (facilitated, in part, by a decrease in OpgH, which releases FtsZ), leading to progeny with a small cell size. The abundance of the nucleoid-associated DNA-binding protein from starved cells (Dps) begins to increase, owing to transcriptional upregulation by the stationary phase sigma factor RpoS. c | During growth arrest, an extremely high abundance of Dps leads to a highly condensed, crystalline appearance of the nucleoid region. Reductive divisions in stationary phase, combined with catabolism of ribosomes and membranes, leads to much smaller cell sizes. Transcription and translation still occur, despite reduced ribosome abundance, but at greatly decreased rates.