Quorum-sensing acts at initiation of chromosomal replication in Escherichia coli - PubMed (original) (raw)

Quorum-sensing acts at initiation of chromosomal replication in Escherichia coli

H L Withers et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Chromosomal replication in Escherichia coli was studied by flow cytometry and was found to be inhibited by an extracellular factor present in conditioned media collected during late exponential and early stationary phase, i.e., via a quorum-sensing mechanism. Our results suggest that the inhibitory activity of the extracellular factor is exerted during initiation of DNA replication rather than during elongation. Furthermore, we present evidence that this interaction may occur directly at each of the replication forks. Unlike other quorum-sensing systems described so far for Gram-negative bacteria, this inhibitory activity does not require transcription or translation to be effective. Implications of quorum-sensing regulation of DNA replication are discussed.

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Figures

Figure 1

Figure 1

MG1655_dnaC2_ was synchronized for replication by incubation at nonpermissive temperature. At 0 min the culture was split into two and either no addition (solid line) was made or 1 ml of cCM per 10 ml of culture (dashed line) was added before the cultures were shifted to the permissive temperature. Samples were taken for flow cytometry at the times indicated.

Figure 2

Figure 2

MG1655 was grown at 37°C to an OD600 of 0.1. The culture was split into two and either no addition (A) was made or 1 ml of cCM per 10 ml of culture (B) was added. Samples were taken for flow cytometry at the times indicated.

Figure 3

Figure 3

MG1655 was grown in M9 medium supplemented with glucose and Casamino acids at 37°C to the indicated OD (↓), whereupon the culture was split into two. Either 1 ml of sterile distilled water (○) or 1 ml of cCM (▵) per 10 ml of culture was added. Samples were taken at regular intervals throughout the experiment, and the OD was measured at 600 nm.

Figure 4

Figure 4

MG1655_dnaC2_ (A) and MG1655_dnaC2tus_ (B) were synchronized for replication by incubation at nonpermissive temperature. Both cultures were handled as follows. At 0 min the cultures were split into equal portions and either no addition was made or cCM was added to a final concentration of 2×. All cultures then were shifted to the permissive temperature for 10 min before being returned to the nonpermissive temperature for the duration of the experiment. Peaks 1, 2, and 3 referred to in the text are indicated on graphs where appropriate. Samples were taken for flow cytometry at the times indicated.

Figure 5

Figure 5

MG1655_dnaC2_ was synchronized by incubation at the nonpermissive temperature. At 0 min, the culture was divided into two equal parts. Either no addition (A) or cCM was added to a final concentration of 2× after the cultures were shifted to the permissive temperature (30°C) for 10 min and returned to the nonpermissive temperature (38°C) for a further 5-min incubation (B). Samples were taken for flow cytometry at the times indicated.

Figure 6

Figure 6

Analysis of transcriptional and translational requirements for inhibition of DNA replication by an extracellular factor. MG1655_dnaC2_ was synchronized at 38°C for 120 min. (A) After synchronization the culture was subdivided, and rifampin and cephalexin were added to both. At the same time cCM was added to one aliquot (Lower) to a final concentration of 2×; the other formed the control (Upper). In both cases, aliquots were incubated at 38°C for 0 or 15 min before being shifted to 30°C and incubated for a further 120 min before sampling for flow cytometry. (B) Same as for A except that chloramphenicol was added instead of rifampin.

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