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 IICB(Glc) (PtsG). Here we demonstrate that inactivation of Mlc is achieved by sequestration of Mlc to membranes containing dephosphorylated Enzyme IICB(Glc). We show that Mlc binds specifically to membrane fractions which carry PtsG and that excess Mlc can inhibit Enzyme IICB(Glc) phosphorylation by the general PTS proteins and also Enzyme IICB(Glc)-mediated phosphorylation of alpha-methylglucoside. Binding of Mlc to Enzyme IICB(Glc) 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 IIB(Glc) 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.

A new essential gene of the 'minimal genome' affecting cell division.

The complete sequencing of bacterial genomes has offered new opportunities for the identification of essential genes involved in the control and progression of the cell cycle. For this purpose, we have disrupted ten E. coli genes belonging to the so-called 'minimal genome'. One of these genes, yihA, was necessary for normal cell division. The yihA gene possesses characteristic GTPase motifs and its homologues are present in eukaryotes, archaea and most prokaryotes. Depletion of YihA protein led to a severe reduction in growth rate and to extensive filamentation, with a block beyond the stage of nucleoid segregation. Filamentation was correlated with reduced FtsZ levels and could be specifically suppressed by overexpression of ftsQI, ftsA and ftsZ, and to some extent by ftsZ alone. We hypothesize that YihA, like the Era GTPase, may participate in a checkpoint mechanism that ensures a correct coordination of cell cycle events.

An Escherichia coli gene (yaeO) suppresses temperature-sensitive mutations in essential genes by modulating Rho-dependent transcription termination.

An extragenic multicopy suppressor of the cell division inhibition caused by a MalE-MinE fusion protein in Escherichia coli has been mapped and identified as yaeO, one of the two short open reading frames (ORFs) of an operon located at 4.6 min. Overexpressed yaeO also suppressed some temperature-sensitive mutations in division genes ftsA and ftsQ, in chaperone gene groEL and in co-chaperone gene grpE. Gene yaeO, whose expression is regulated by growth rate, codes for a 9 kDa acidic protein with no obvious resemblance to other proteins. Transcription termination protein Rho co-purified with a histidine-tagged derivative of YaeO protein on Ni2+-NTA agarose columns in a manner that suggested direct YaeO-Rho interaction. In vivo, yaeO expression reduced termination at rho-dependent bacteriophage terminator tL1 and at the terminator of autogenously regulated gene rho. The suppression of temperature-sensitive phenotypes was a consequence of anti-termination, as it could be mimicked by a Prho::Tn10 mutation that reduces the expression and activity of gene rho. Our data indicate that the suppression is not caused by overexpression of the mutated genes, but presumably by indirect stabilization of the mutated proteins.

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 multi-nucleated cells appears to favour division at non-polar PDS related to the most ancient replication/DNA segregation events.

MinCD-independent inhibition of cell division by a protein that fuses MalE to the topological specificity factor MinE.

We report that MinE, the topological specificity factor of cell division in Escherichia coli, inhibits septation when fused to the C terminus of the maltose-binding protein MalE. This contrasts with overexpression of MinE alone, which affects growth but has no effect on division. Inhibition by MalE-MinE was minCD independent and depended on MinE segments involved in dimerization and prevention of MinCD division inhibition. The SOS and the heat shock responses were not involved, suggesting that the inhibition comes from a direct interaction of MalE-MinE with the septation apparatus. MalE-MinE lethality was suppressed by overexpression of ftsZ, as well as by overexpression of ftsN, a suppressor of temperature-sensitive mutations in genes ftsQ, ftsA, and ftsI. We also report that high-level synthesis of MalE disturbs nucleoid partitioning.

Identification of a new inhibitor of essential division gene ftsZ as the kil gene of defective prophage Rac.

A gene function carried by a plasmid, causing arrest of cell division in Escherichia coli, has been identified as the product of a short open reading frame of the prophage Rac, previously designated orfE, expressed only under conditions of prophage induction. Because Rac carries a killing function expressed under conditions of zygotic induction, an orfE-defective Rac+ strain was constructed. This strain had lost the killing function, indicating that orfE is kil. Division inhibition by kil was specifically relieved by overexpression of essential division gene ftsZ. The kil gene product acts independently of the min operon, and its effects are increased in conditions of high cyclic AMP (cAMP) receptor protein-cAMP complex levels in the cell. Furthermore, at high levels of expression, kil product distorts the rod shape of the cells. These features distinguish kil-encoded protein from the inhibitory product of gene dicB, which occupies a similar genetic location in Kim (Qin), another defective prophage of Escherichia coli.

RNase E processing of essential cell division genes mRNA in Escherichia coli.

The ratio of the FtsZ to FtsA proteins determines the correct initiation of cell division in Escherichia coli. The genes for these proteins are contiguous on the chromosome. Although both genes are transcribed from common promoters, the presence of ftsZ-specific promoters, along with differences in the efficiency of translation of their respective mRNAs, contribute to the increased relative expression of ftsZ. We report here that the polycistronic ftsA-ftsZ transcripts are cleaved by RNase E and that this cleavage affects the decay of ftsA and ftsZ mRNA. As a consequence of the cleavage, RNase E also contributes to the differential expression of the two genes.

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 delta(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.

Sigma S-dependent overexpression of ftsZ in an Escherichia coli K-12 rpoB mutant that is resistant to the division inhibitors DicB and DicF RNA.

The Escherichia coli genes dicF and dicB encode division inhibitors, which prevent the synthesis and activity, respectively, of the essential division protein FtsZ. A mutation at the C-terminal end of the RNA polymerase beta subunit renders cells resistant to both inhibitors. In the mutant strain the level of the ftsZ gene product is higher than in the wild type. Disruption of rpoS, which encodes the stationary phase sigma factor sigma S, lowers FtsZ protein levels in the mutant, and partially restores sensitivity to the inhibitors.

The min locus, which confers topological specificity to cell division, is not involved in its coupling with nucleoid separation.

In Escherichia coli, nucleoid separation and cell constriction remain tightly linked when division is retarded by altering the level of synthesis of the protein FtsZ. In this study, we have examined the role of the min locus, which is responsible for the inactivation of polar division sites, in the partition-septation coupling mechanism. We conclude that the coupling persists in a delta min strain and that its timing relative to replication remains dependent on the level of FtsZ synthesis. We suggest that the retarded nucleoid segregation observed in min mutants is the result of this coupling in cells with a perturbed pattern of nonpolar divisions.

Cell cycle control: prokaryotic solutions to eukaryotic problems?

Regulation of the eukaryotic cell cycle involves calcium- and lipid-stimulated kinases acting on cytoskeletal structures; there are two principal reasons for supposing that the regulation of the prokaryotic cell cycle may be fundamentally the same. First, evidence for their fundamental difference is still missing and, second, evidence for prokaryotic homologues of eukaryotic cell cycle proteins is accumulating. Such proteins include those involved in calcium regulation, such as calmodulin and calcium-dependent kinases, and those involved in lipid regulation, such as protein kinase C. Proteins identified as candidates for cytoskeletal elements now include MukB, a putative contractile protein responsible for chromosome segregation, and FtsZ, the key constituent of the "cytokinetic" ring. These similarities allow the application of powerful prokaryotic model systems to one of biology's most profound, complex and urgent problems: the nature of the regulation of the eukaryotic cell cycle.

Division inhibition gene dicF of Escherichia coli reveals a widespread group of prophage sequences in bacterial genomes.

The genomes of various eubacteria were analyzed by Southern blot hybridization to detect sequences related to the segment of the defective lambdoid prophage Kim which encodes DicF RNA, an antisense inhibitor of cell division gene ftsZ in Escherichia coli K-12. Among the homologous sequences found, one fragment from E. coli B, similar to a piece of Rac prophage, and two fragments from Shigella flexneri were cloned and sequenced. dicF-like elements similar to transcriptional terminators were found in each sequence, but unlike dicF these had no effect on division in E. coli K-12. Like dicF, these sequences are flanked by secondary structures which form potential sites for RNase III recognition. Coding sequences located upstream from the dicF-like feature in E. coli B are related to gene sieB of bacteriophage lambda, while sequences downstream of the S. flexneri elements are similar to the immunity region of satellite bacteriophage P4. Under hybridization conditions in which only strong sequence homologies were detected in E. coli B and S. flexneri, the genomes of a large variety of microorganisms, including some gram-positive bacteria, hybridized to the dicF probe. Our results suggest that dicF and its flanking regions are markers of a widespread family of prophage-like elements of different origins.

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.

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-nucleotide 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 ftsZ 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.

New minC mutations suggest different interactions of the same region of division inhibitor MinC with proteins specific for minD and dicB coinhibition pathways.

Proper positioning of division sites in Escherichia coli requires balanced expression of minC, minD, and minE gene products. Previous genetic analysis has shown that either MinD or an apparently unrelated protein, DicB, cooperates with MinC to inhibit division. We have isolated and sequenced minC mutations that suppress division inhibition caused by overproduction of either DicB or MinD proteins. Most missense mutations were located in the amino acid 160 to 200 region of MinC (231 amino acids). Some mutations exhibited preferential resistance to one or the other coinhibitor, suggesting that two distinct proteins, possibly MinD and DicB themselves, interact in slightly different manners with the same region of MinC to promote division inhibition.

[Comparative efficacy of sustained release verapamil and captopril in mild to moderate arterial hypertension by ambulatory measurement and occasional measurement]. / Efficacité comparée du vérapamil à libération prolongée et du captopril dans l'hypertension artérielle légére à modérée par la mesure ambulatoire et mesure "occasionnelle".

The evaluation of mild to moderate hypertension must be carried out under the conditions in which treatments are usually prescribed, i.e., in general practice. After specific training of the physicians in the methods used, we evaluated the efficacy and safety of a new formulation of verapamil by comparing it with a reference drug: captopril. The main assessment criterion was the restoration of normal blood pressure in mildly to moderately hypertensive patients (blood pressure in excess of 160/95 mmHg). Blood pressure was evaluated by two methods: a mercury column sphygmomanometer, after the patient had rested in a half-sitting position for 10 minutes, and the ambulatory measurement of blood pressure (AMBP) using the SpaceLabs system. The results of this study involving 40 patients followed up for 3 months by 8 GPs in collaboration with our blood pressure unit were as follows: on verapamil, 47% of patients recovered normal values after 30 days of treatment and 71% after 60 days (with no change in dosage). On captopril, the normalization rates were 22 and 27% respectively. The highly significant reduction of blood pressure found by the "occasional" measurement for both treatments (p less than 0.001) was only faintly reflected by AMBP. Verapamil induced a reduction of nighttime blood pressure with no significant impact on heart rate. The clinical, paraclinical and electrocardiographic safety of both treatments was good.

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.

Minicell-forming mutants of Escherichia coli: suppression of both DicB- and MinD-dependent division inhibition by inactivation of the minC gene product.

We have determined the nucleotide sequence of the minB operon of 10 min mutants of Escherichia coli, characterized by impaired inhibition of polar divisions. These mutants were either sensitive or resistant to the division inhibitor DicB. All the mutations were found to lie in minC or minD, confirming the requirement of both gene products in the process of inhibition of polar sites. Mutations conferring resistance to inhibitor DicB were found exclusively in minC. In agreement with de Boer et al. (P. A. J. de Boer, R. E. Crossley, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 87:1129-1133, 1990), these results provide evidence that, in addition to promoting division inhibition with MinD, protein MinC acts in concert with DicB to inhibit division by a second, MinD-independent process.