The Stringent Response and Cell Cycle Arrest in Escherichia coli

Abstract

The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions.

Author Summary

Management of cell growth and division in response to environmental conditions is important for all cells. In bacteria, nutritional downturns are signaled by accumulation of the nucleotide ppGpp. Amino acid starvation causes a programmed change in transcription, known as the “stringent response”; ppGpp also causes an arrest of cell cycle in bacteria, whose mechanism has not been thoroughly investigated. Here, we show that E. coli cells, when the stringent response is in effect, complete chromosomal replication but do not initiate new rounds and arrest with an integer number of chromosomes. The number of chromosomes corresponds to the growth rate prior to arrest. In polyploid arrested cells, the chromosomal regions at which replication initiates are segregated, whereas the termini regions remain colocalized. The E. coli chromosome remains decondensed and unsegregated during arrest and rapidly resumes replication and segregation, concomitant with chromosome condensation, upon release. The protein SeqA, a DNA binding protein and negative regulator of replication, is necessary for enforcing this arrest.