Jean-Pierre Bouché

Signal transduction between a membrane‐bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli

The global regulator Mlc controls several genes implicated in sugar utilization systems, notably the phosphotransferase system (PTS) genes, ptsG, manXYZ and ptsHI, as well as the malT activator. No specific low molecular weight inducer has been identified that can inactivate Mlc, but its activity appeared to be modulated by transport of glucose via Enzyme IICBGlc (PtsG). Here we demonstrate that inactivation of Mlc is achieved by sequestration of Mlc to membranes containing dephosphorylated Enzyme IICBGlc. We show that Mlc binds specifically to membrane fractions which carry PtsG and that excess Mlc can inhibit Enzyme IICBGlc phosphorylation by the general PTS proteins and also Enzyme IICBGlc‐mediated phosphorylation of α‐methylglucoside. Binding of Mlc to Enzyme IICBGlc in vitro required the IIB domain and the IIC–B junction region. Moreover, we show that these same regions are sufficient for Mlc regulation in vivo, via cross‐dephosphorylation of IIBGlc during transport of other PTS sugars. The control of Mlc activity by sequestration to a transport protein represents a novel form of signal transduction in gene regulation.

Regulation of Escherichia coli cell division genes ftsA and ftsZ by the two-component system rcsC–rcsB

Genes rcsC and rcsB form a two‐component system in which rcsC encodes the sensor element and rcsB the regulator. In Escherichia coli, the system positively regulates the expression of the capsule operon, cps, and of the cell division gene ftsZ. We report the identification of the promoter and of the sequences required for rcsB‐dependent stimulation of ftsZ expression. The promoter, ftsA1p, located in the ftsQ coding sequence, co‐regulates ftsA and ftsZ. The sequences required for rcsB activity are immediately adjacent to this promoter.

Deletion analysis of gene minE which encodes the topological specificity factor of cell division in Escherichia coli

Division inhibition caused by the minCD gene products of Escherichia coli is suppressed specifically at mid‐cell by MinE protein expressed at physiological levels. Excess MinE allows division to take place also at the poles, leading to a minicell‐forming (Min−) phenotype. In order to investigate the basis of this topological specificity, we have analysed the ability of truncated derivatives of MinE to suppress either minCD‐dependent division inhibition in a chromosomal Δ(minB) background, or the division inhibition exerted by MinCD at the cell poles in a minB,+ strain. Our results indicate that these two effects are not mediated by identical interactions of MinE protein. In addition, gel filtration and the yeast two‐hybrid system indicated that MinE interacts with itself by means of its central segment. Taken together, our results favour a model in which wild‐type MinE dimer molecules direct the division inhibitor molecules to the cell poles, thus preventing polar divisions and allowing non‐polar sites to divide. This model explains how excess MinE, or an excess of certain MinE derivatives which prevent the accumulation of the division inhibitor at the poles, can confer a Min− phenotype in a minB+ strain.

Regulation of the expression of the cell-cycle gene ftsZ by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules

We show that the 53‐nucieotide RNA molecule encoded by gene dicF blocks cell division In Escherichia coli by inhibiting the translation of ftsZ mRNA. Such a role for dicF had been predicted on the basis of the complementarity of DicF RNA with the ribosome‐binding region of the ffsZ mRNA. An analysis of ftsZ expression at its chromosomal locus, and of an ftsZ–lacZ translational fusion controlled by promoters ftsZ1p and ftsZ2p only, indicates that ftsZ is not autoregulated. Partial inhibition of FtsZ synthesis leads to increased cell size. However, the number of FtsZ molecules per cell can be reduced threefold without affecting the division rate significantly. Our results suggest that septation is not triggered by a fixed number of newly synthesized FtsZ molecules per cell.

ColE1-type vectors with fully repressible replication

We have constructed two cloning vectors, pAM34 and pAM35, derived from pBR322, in which transcription of the replication primer RNA is under control of the lacZpo promoter/operator. These vectors contain a cloning cassette flanked by strong transcriptional terminators. They differ from each other by the presence (pAM34) or absence (pAM35) of gene lacIq. In the presence of repressor LacIq, replication is entirely dependent upon the addition of inducer. This feature allows the temporary maintenance of these plasmids, the construction of strains in which vector derivatives are stably integrated into the chromosome, and the recovery of nucleotide sequences adjacent to cloned fragments. Replication from the integrated plasmid can be adjusted to match the chromosome replication initiation rate required for cell growth in the absence of a functional origin, oriC.

Recovery of nanogram quantities of DNA from plasma and quantitative measurement using labeling by nick translation

A method which allows for the quantitative measurement of DNA in plasma is described. After treatment of plasma with phenol, DNA is precipitated by ethanol using gelatin as a coprecipitating agent. DNA is then measured by nick translation labeling. This assay takes a few hours. It is suitable for the measurement of DNA within a range of 0.02 to 20 ng in 10 microliters of plasma. For example, it is applied to the measurement of DNA in plasma from mice injected with bacterial lipopolysaccharide.

Cell division inhibition gene dicB is regulated by a locus similar to lambdoid bacteriophage immunity loci.

SummaryA mutation (dicA1) of a repressor gene located in the terminus region of the Escherichia coli chromosome has previously been shown to lead to temperature-dependent inhibition of division, and to be complemented by plasmids carrying either dicA or an adjacent gene dicC. In this study, operon fusions in the region coding for the division inhibition gene dicB have been used to show that temperature sensitivity does not result from high temperature inactivation of the dicA repressor. Sequence comparisons indicate that dicA and dicC are similar to genes c2 and cro respectively of bacteriophage P22, and carry similarly organized tandem operators, indicating a common evolutionary origin for dicAC and P22 immC. Nevertheless, the consensus half-operator sequence of dicAC, TGTTAGYYA, differs significantly from that of P22 immC (ATTTAAGAN). an analysis of the in vivo control of promoters dicAp, dicBp and dicCp placed upstream of malQ shows that the dicAC system is functionally similar to that of an immunity region, with the possible exception of an absence of pairwise cooperative binding. Our results also indicate that the dicA1 mutation causes a switch to permanent control by dicC at all temperatures.

Genetic inactivation of topoisomerase I suppresses a defect in initiation of chromosome replication in Escherichia coli

SummaryA strain of Escherichia coli K12 harboring simultaneously the temperature-sensitive dnaA46 mutation and a deletion of the trp-topA-cysB region plates with the same full efficiency at 30° C and 42° C. We have analyzed the possible involvement of the gene coding for topoisomerase I, topA, in this suppression phenomenon. The Ts phenotype was retrieved upon introduction of a plasmid-borne DNA fragment including an active topA gene into this strain, but not upon introduction of the same fragment harboring a topA::Tn1000 insertion. Replication seems to remain DnaA-dependent in the Δ(topA) strain, however, since we have been unable to introduce a dnaA::Tn10 allele. We propose either that the dnaA46 gene product is overproduced and compensates for its thermal inactivation, or that initiation at oriC demands less DnaA protein in the absence of topoisomerase I.

Involvement of FtsZ in coupling of nucleoid separation with septation

The cell‐cycle parameters of an Escherichia coli strain expressing essential division gene ftsZ at one‐fifth of its normal level, because of antisense regulation by DicF RNA, have been analysed. Inhibition of FtsZ expression affects neither the generation time nor the replication initiation mass, the C period, or the constriction period, but it does dramatically retard the initiation of constriction relative to replication termination. Separation of the nucleoids is equally postponed, indicating that division is not coupled to termination of replication, but to partitioning. The severe inhibition of nucleoid separation by DicF RNA, and its suppression by overproduction of FtsZ, suggest a role for FtsZ in the control of separation, and consequently in the coupling of separation and division. We suggest that the normal pattern of nucleoid separation previously found in cells deficient in ftsZ function was a consequence of the loss of a negative effect exerted by FtsZ on separation. In agreement with this view, we find that nucleoid separation is temporarily inhibited after arrest of FtsZ synthesis, but is later resumed as FtsZ is further diluted into the elongating filaments.

On the birth and fate of bacterial division sites

Thanks to genetics, to the study of protein–protein interactions and to direct viewing of subcellular structures by the use of immunofluorescence and green fluorescent protein (GFP) fusions, the organization of the constriction apparatus of walled bacteria is gradually coming to light. The tubulin‐like protein FtsZ assembles as a ring around the site of constriction and operates as an organizer and activator of septum‐shaping proteins. Much less is known about the factors specifying the location of FtsZ rings. Circumstantial evidence favours the presence at future ring positions of fixed elements, the potential division sites (PDS), before FtsZ assembles. FtsZ polymerization is initiated from a point on a PDS, the nucleation site, still to be identified, and proceeds bidirectionally around the cell. We hypothesize that new PDS are specified in a manner that depends on the functioning of an active chromosome partition apparatus. This view is supported by the fact that formation of mid‐cell PDS requires initiation of DNA replication, and by recent studies supporting the existence of a specialized partition apparatus in a variety of microorganisms. Although PDS may be specified directly by the partition apparatus, indirect localization linked to compartmentalized gene expression during chromosome segregation is also possible. Once created, PDS are used in a regulated manner, and several mechanisms normally operate to direct constriction to selected PDS at the correct time. One, dedicated to the permanent suppression of polar PDS, rests on the minicell suppression system and involves a protein that is able to discriminate between polar and non‐polar sites. Another is involved in asymmetric site selection at the early stages of sporulation in Bacillus subtilis. Finally, a mechanism observed only in certain multinucleated cells appears to favour division at non‐polar PDS related to the most ancient replication/DNA segregation events.