Phosphorylation of human CDC25B phosphatase by CDK1/cyclin A triggers its proteasome-dependent degradation.

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.

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.

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.

SummaryGenes 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 identi®cation 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.

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The replication cycle of Escherichia coli strains duplicating their chromosome from the same plasmid origin placed at various locations or of strains having undergone a major inversion event along the origin-to-terminus axis was studied by marker-frequency analysis. It was observed that replication forks are unidirectionally inhibited at two loci of the termination region: counterclockwise-moving forks are inhibited at terminator Ti (28.5 min), and forks moving in the opposite direction are inhibited at terminator T2 (33.5 min). By determining the strand preference of Okazaki fragments that are specific for markers from the Tl-T2 interval, it was shown that this interval is replicated in either direction, depending upon the strain analyzed. In addition, we also observed that forks moving in the "unnatural" direction along each onC-TI or-T2 arm are very slow, especially in the one-third portion of the chromosome around the terminators. We propose that this phenomenon is a consequence of nucleoid organization, which is proposed to be symmetrical on the two oriC-TI or -T2 arms and polarized with respect to the direction of replication. We also propose that Ti and T2 are the terminal limits of these two polarized half-nucleoid bodies.Replacement of the chromosomal origin of replication oriC by either a phage or a plasmid origin has allowed Kuempel et al. (1) and Louarn et al. (2) to demonstrate the invariance of the chromosomal region where opposite replication forks meet. When initiated at oriC, replication was found to terminate between rac (30 min) and manA (35.7 min) at position 31.2 ± 0.2 min. This position was defined statistically but not functionally (3, 4). Other experiments indicated that the terminus region does not prevent replication forks from moving beyond it after some delay (1,(5)(6)(7).Further analysis of the termination region is reported here. By determining the replication pattern of chromosomes that were placed under the control of an integrated plasmid or had undergone a major inversion event along the origin-toterminus axis, we have been able to show that replication forks are inhibited at two positions: the first, at 28.5 min, inhibits counterclockwise-moving forks; and the second, at about 33.5 min, inhibits clockwise-moving forks.

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.Three defective lambdoid prophages, Rac, QSRЈ, and Kim (Qin), have been identified in the genome of Escherichia coli K-12 (3, 21). Prophage Kim encodes two division inhibitors, DicB (4) and DicF (25), both expressed from a p L -like promoter under control of an immunity region related to that of phage P22 (1). DicF is a 53-nucleotide-long antisense RNA generated in part by RNase III processing in two regions with extensive secondary structures, R 1-2 and R 3-4 , located upstream and downstream respectively from the dicF gene (12).Southern blot hybridization revealed that a part of the Rac prophage hybridizes specifically to a probe containing dicF and the flanking R 1-2 and R 3-4 sequences (3). The sequence of that part of Rac has been established by Chu et al., and its resemblance to dicF has been confirmed (5). In another study, a fragment of E. coli B which hybridized to a R 1-2 -dicF-R 3-4 probe was cloned and sequenced. This fragment turned out to contain Rac DNA nearly identical to that of K-12 strains. Sequence analysis indicated that the fragment contains the 5Ј part of a gene related to sieB, followed by sequences similar to those of Kim R 1-2 , dicF, and R 3-4 , then by an open reading frame called orfE, and by the 5Ј region of another open reading frame, orfF (11) (Fig. 1).We noticed that the Rac fragment cloned in one orientation under control of P lac in a high-copy-number plasmid yielded a cellular filamentation phenotype under some conditions, suggesting the presence of a cell division inhibition gene. We report the identification and some properties of this gene. We also demonstrate that this gene expresses the killing function of the Rac prophage revealed by the study of Feinstein and Low (13). MATERIALS AND METHODSBacterial strains and media. The bacterial strains used in this study are described in Table 1. The construction of JS1478 is described in detail below. Strains were grown in Luria broth. Solid and soft agar contained 1.5% and 0.9% (wt/vol) agar, respectively. When appr...