Jean Meury

Glycine betaine reverses the effects of osmotic stress on DNA replication and cellular division in Escherichia coli.

The accumulation of glycine betaine to a high internal concentration by Escherichia coli cells in high osmolarity medium restores, within 1 h, a subnormal growth rate. The experimental results support the view that cell adaptation to high osmolarity involves a decrease in the initiation frequency of DNA replication via a stringent response; in contrast, glycine betaine transport and accumulation could suppress the stringent response within 1–2 min and restore a higher initiation frequency. High osmolarity also triggers the cells to lengthen, perhaps via an inhibition of cellular division; glycine betaine also reverses this process. It is inferred that turgor could control DNA replication and cell division in two separate ways. Glycine betaine action is not mediated by K+ ions as the internal level of K+ ions is not modified significantly following glycine betaine accumulation.

Co‐ordination between membrane oriC sequestration factors and a chromosome partitioning protein, TolC (MukA)

oriC DNA in the hemimethylated (but not in the fully methylated) state reacts with an Escherichia coli K‐12 outer membrane preparation. This reaction is drastically reduced when the membrane preparation of a seqA null mutant is used. An in vitro reconstitution of the activity was undertaken by adding a partially purified SeqA protein to a seqA mutant membrane without success. A possible reason for this failure might be a profound modification of the outer membrane of the seqA mutant (as revealed by the fact that membrane from the mutant sediments more slowly than that from the wild type during ultracentrifugation). There is also a reduction in the content of OmpF protein. Moreover, one of the minor outer membrane proteins involved in partitioning of newly synthesized chromosomes, the TolC (MukA) protein, was also found to be downregulated in the seqA mutant. This is also true of the hobH mutant grown in a high‐osmolarity medium. Mutants of both seqA and hobH stop dividing after hyperosmotic shock, forming filaments (as observed in dam mutants).

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K+ up to 3.6 M. This creates a very large K+ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K+ but the residual K+ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K+ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a Km of between 1 to 2 mM. The driving force for K+ transport has been explored. Internal K+ concentration is not in equilibrium with ΔΨm suggesting that K+ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N′-dicyclohexylcarbodiimide inhibits by 100% K+ transport even though membrane potential ΔΨm is maintained under these conditions. By contrast, the necessity of a ΔΨm for K+ accumulation has not yet been clearly demonstrated. K+ transport in H. volcanii can be compared with K+ transport via the Trk system in Escherichia coli.

Importance of the replication origin sequestration in cell division of Escherichia coli

The DNA adenine methyltransferase of Escherichia coli methylates adenines at GATC sequences. The mutant deficient in this methylase has no apparent deficiency in the cell division process in spite of the absence of both synchrony in initiations of chromosomal DNA replication and sequestration of replication origin (oriC) at hemimethylated state. However, the dam mutant cannot resume cell division after hyperosmotic shock differing from the wild-type strain. This inhibition is not provoked by induction of the cell division inhibitor, SfiA protein. Although the FtsZ protein is present in the dam mutant in a reduced amount compared to wild-type, the quantitative difference of this protein is not the main reason of division arrest provoked by hyperosmotic shock. This observation supports the idea of oriC-membrane interaction playing a role both in chromosome partitioning and cell division as predicted by replicon theory.

Increased expression of a hemimethylated oriC binding protein, SeqA, in an aphA mutant

In Escherichia coli, the origin of DNA replication, oriC, becomes transiently hemimethylated at the GATC sequences immediately after initiation of replication and this hemimethylated state is prolonged because of its sequestration by a fraction of outer membrane. This sequestration is dependent on a hemimethylated oriC binding protein such as SeqA. We previously isolated a clone of phage lambda gt11 called hobH, producing a LacZ fusion protein which recognizes hemimethylated oriC DNA. Very recently, Thaller et al. (FEMS Microbiol. Lett. 146 (1997) 191-198) found that the same DNA segment encodes a non-specific acid phosphatase, and named the gene aphA. We show here that the interruption of the aphA reading frame by kanamycin resistance gene insertion, abolishes acid phosphatase (NAP) activity. Interestingly, in the membrane of the null mutant, the amount of SeqA protein is about six times higher than that in the parental strain, suggesting the existence of a regulatory mechanism between SeqA and NAP expression.