Precise excision of bacteriophage Mu DNA.

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.

Cycle-regulated genes and cell cycle regulation.

The transcriptional profile of the entire Caulobacter crescentus genome over a synchronous cell cycle was recently described. The analysis reveals a stunning 553 cell-cycle-regulated genes or orfs, nearly 19% of the genome, including putative functions in virtually all biological activities. Over a quarter of these genes/orfs respond to the Caulobacter master regulator, CtrA, most of them apparently indirectly. The analysis confirms and extends earlier observations showing that many proteins involved in cell cycle functions are expressed at the cell age when they are needed. Conversely, the data suggest that proteins specifically expressed at a particular age may be involved in a process taking place then.

An algorithm for targeted convergence of Euler or Newton iterations.

The concept of multistationarity has become essential for understanding cell differentiation. For this reason theoretical biologists have more and more frequently to determine the steady values, often multiple, of systems of non-linear differential equations. It is well known that iteration processes of current use converge or not towards a fixed point depending on the absolute value of the slope of the iteration function in the vicinity of the considered fixed point. A number of methods have been developed to obtain or accelerate convergence. As biologists, we do not pretend to review these works. Rather, we propose here a simple algorithm which permits to converge at will towards a chosen type of steady state. Others and we have used this procedure extensively for years for the analysis of complex biological systems. A compact program (using Mathematica) is available.

The GemA protein of phage Mu and the GyrB gyrase subunit of Escherichia coli: the search for targets and interactions leading to the reversion of Mu-induced mutations.

The mutant bacteriophage Mugem2(Ts), known to synchronize the division of infected cells, to relax DNA supercoiling and, as prophage, to give rise to precisely excised revertants, has been thought to overexpress the gemA-mor operon, and genetic evidence suggests that the B subunit of DNA gyrase (GyrB) is the target of action of GemA. In two different double hybrid tests presented here, we find no evidence of GemA-GyrB protein-protein interaction. We do observe a GemA-GemA interaction, however, indicating that GemA can dimerize. In lacZ::Mu lysogens, overexpression of the gemA-mor operon from a plasmid, under control of the L-arabinose inducible p(araBAD) promoter, does not permit the recovery of Lac(+) revertants. These observations suggest that GyrB is not the direct target of GemA action and that the various phenotypes of Mugem2(Ts) are not caused by overexpression of the gemA-mor operon.

Selected amplification of the cell division genes ftsQ-ftsA-ftsZ in Escherichia coli.

Rapidly growing Escherichia coli is unable to divide in the presence of the antibiotic mecillinam, whose direct target is penicillin-binding protein 2 (PBP2), responsible for the elongation of the cylindrical portion of the cell wall. Division can be restored in the absence of PBP2 activity by increasing the concentration of the cell division proteins FtsQ, FtsA, and FtsZ. We tried to identify regulators of the ftsQ-ftsA-ftsZ operon among mecillinam-resistant mutants, which include strains overexpressing these genes. By insertional mutagenesis with mini-Tn10 elements, we selected for insertions that conferred mecillinam resistance. Among 15 such mutants, 7 suppressed the thermosensitivity of the ftsZ84(Ts) mutant, strongly suggesting that they had increased FtsZ activity. In all 7 cases, however, the mutants resulted from a duplication of the ftsQAZ region. These duplications seemed to result from multiple events, suggesting that no simple insertional inactivation can result in a mutant with sufficiently amplified ftsQAZ expression to confer mecillinam resistance. The structure of the duplications suggests a general method for constructing directed duplications of precise sequences.

RecA-independent pathways of lambdoid prophage induction in Escherichia coli.

Two Escherichia coli genes, expressed from multicopy plasmids, are shown to cause partial induction of prophage lambda in recA mutant lysogens. One is rcsA, which specifies a positive transcriptional regulator of the cps genes, which are involved in capsular polysaccharide synthesis. The other is dsrA, which specifies an 85-nucleotide RNA that relieves repression of the rcsA gene by histone-like protein H-NS. Genetic contexts known to increase Cps expression also cause RecA-independent lambda induction: the rcsC137 mutation, which causes constitutive Cps expression, and the lon and rcsA3 mutations, which stabilize RcsA. Lambdoid phages 21, phi80, and 434 are also induced by RcsA and DsrA overexpression in recA lysogens. Excess lambda cI repressor specifically blocks lambda induction, suggesting that induction involves repressor inactivation rather than repressor bypass. RcsA-mediated induction requires RcsB, the known effector of the cps operon, whereas DsrA-mediated induction is RcsB independent in stationary phase, pointing to the existence of yet another RecA-independent pathway of prophage induction.

Analysis of the effect of ppGpp on the ftsQAZ operon in Escherichia coli.

Escherichia coli loses its rod shape by inactivation of PBP2 (penicillin-binding protein 2), target of the beta-lactam mecillinam. Under these conditions, cell division is blocked in rich medium. Division in the absence of PBP2 activity is restored (and resistance to mecillinam is conferred) when the three cell division proteins FtsQ, FtsA and FtsZ are overproduced, but not when only one or two of them are overproduced. Division in the absence of PBP2 activity is also restored by a doubling in the ppGpp pool, as in the argS201 mutant. However, the nucleotide ppGpp, a transcriptional regulator of many operons, does not govern any of the five promoters of the ftsQAZoperon, as shown by S1 mapping of ftsQAZ mRNA 5' ends in exponentially growing wild-type cells in the mecillinam-resistant argS201 mutant (intermediate ppGpp level) or during the stringent response elicited by isoleucine starvation (high ppGpp level). Furthermore, the concentration of FtsZ protein is not increased in exponentially growing mecillinam-resistant argS201 cells. These results show that the ftsQAZ operon is not the ppGpp target responsible for mecillinam resistance. We are currently trying to identify those targets that, at intermediate ppGpp levels, allow cells to divide as spheres in the absence of PBP2.

Lack of S-adenosylmethionine results in a cell division defect in Escherichia coli.

The enzyme S-adenosylmethionine (SAM) synthetase, the Escherichia coli metK gene product, produces SAM, the cell's major methyl donor. We show here that SAM synthetase activity is induced by leucine and repressed by Lrp, the leucine-responsive regulatory protein. When SAM synthetase activity falls below a certain critical threshold, the cells produce long filaments with regularly distributed nucleoids. Expression of a plasmid-carried metK gene prevents filamentation and restores normal growth to the metK mutant. This indicates that lack of SAM results in a division defect.

Underground metabolism.

All enzymes are able to use alternative substrates. When these are naturally occurring metabolites, an 'underground reaction' takes place. Examples are presented in which underground metabolism of this sort produces an observable phenotype. Although biological processes can be remarkably accurate, evolution has selected error rates far from perfect. It is suggested here that a certain level of metabolic inaccuracy, in addition to saving energy, may also confer an evolutionary advantage, for example by providing metabolic plasticity. Since underground reactions are unpredictable from DNA sequence data, caution is in order when interpreting correlations between genetic disorders and pathological syndromes.

Degradation of carboxy-terminal-tagged cytoplasmic proteins by the Escherichia coli protease HflB (FtsH).

Proteins with short nonpolar carboxyl termini are unstable in Escherichia coli. This proteolytic pathway is used to dispose of polypeptides synthesized from truncated mRNA molecules. Such proteins are tagged with an 11-amino-acid nonpolar destabilizing tail via a mechanism involving the 10Sa (SsrA) stable RNA and then degraded. We show here that the ATP-dependent zinc protease HflB (FtsH) is involved in the degradation of four unstable derivatives of the amino-terminal domain of the lambdacI repressor: three with nonpolar pentapeptide tails (cI104, cI105, cI108) and one with the SsrA tag (cI-SsrA). cI105 and cI-SsrA are also degraded by the ClpP-dependent proteases. Loss of ClpP can be compensated for by overproducing HflB. In an in vitro system, cI108 and cI-SsrA are degraded by HflB in an energy-dependent reaction, indicating that HflB itself recognizes the carboxyl terminus. These results establish a tail-specific pathway for removing abnormal cytoplasmic proteins via the HflB and Clp proteases.

Proteolysis and chaperones: the destruction/reconstruction dilemma.

Cytoplasmic proteases, although necessary for proper cell functioning, must be strictly regulated. In fact, they resemble chaperones, ancient protein folding devices. These molecules recognise exposed hydrophobic regions of unfolded or denatured proteins. For most substances it is not known how the cell chooses between the refolding and proteolytic pathways. In Escherichia coli, however, a carboxy-terminal proteolysis tag and binding site for the chaperone DnaK have recently been identified.

The Escherichia coli histone-like protein HU affects DNA initiation, chromosome partitioning via MukB, and cell division via MinCDE.

Escherichia coli hupA hupB double mutants, lacking both subunits (HU1 and HU2) of the histone-like protein HU, accumulate secondary mutations. In some genetic backgrounds, these include mutations in the minCDE operon, inactivating this system of septation control and resulting in the formation of minicells. In the course of the characterization of hupA hupB mutants, we observed that the simultaneous absence of the HU2 subunit and the MukB protein, implicated in chromosome partitioning, is lethal for the bacteria; the integrity of either HU or MukB thus seems to be essential for bacterial growth. The HU protein has been shown to be involved in DNA replication in vitro; we show here that its inactivation in the hupA hupB double mutant disturbs the synchrony of replication initiation in vivo, as evaluated by flow cytometry. Our results suggest that global nucleoid structure, determined in part by the histone-like protein HU, plays a role in DNA replication initiation, in proper chromosome partitioning directed by the MukFEB proteins, and in correct septum placement directed by the MinCDE proteins.

The Escherichia coli cell cycle, cell division and ppGpp: regulation and mechanisms.

The literature demonstrating tight regulation of the Escherichia coli cell cycle is reviewed. Recent evidence is presented indicating that the normal rod cell shape can be abandoned, allowing growth as a coccus, either by increasing the amount of the division proteins FtsZ, FtsA and FtsQ, or by increasing the pool of the nucleotide ppGpp. It is argued that ppGpp may be a cell cycle signal in E. coli.

The HflB protease of Escherichia coli degrades its inhibitor lambda cIII.

The cIII protein of bacteriophage lambda is known to protect two regulatory proteins from degradation by the essential Escherichia coli protease HflB (also known as FtsH), viz., the lambda cII protein and the host heat shock sigma factor sigma32. lambda cIII, itself an unstable protein, is partially stabilized when the HflB concentration is decreased, and its half-life is decreased when HflB is overproduced, strongly suggesting that it is degraded by HflB in vivo. The in vivo degradation of lambda cIII (unlike that of sigma32) does not require the molecular chaperone DnaK. Furthermore, the half-life of lambda cIII is not affected by depletion of the endogenous ATP pool, suggesting that lambda cIII degradation is ATP independent (unlike that of lambda cII and sigma32). The lambda cIII protein, which is predicted to contain a 22-amino-acid amphipathic helix, is associated with the membrane, and nonlethal overproduction of lambda cIII makes cells hypersensitive to the detergent sodium dodecyl sulfate. This could reflect a direct lambda cIII-membrane interaction or an indirect association via the membrane-bound HflB protein, which is known to be involved in the assembly of certain periplasmic and outer membrane proteins.

Mecillinam resistance in Escherichia coli is conferred by loss of a second activity of the AroK protein.

Mecillinam, a beta-lactam antibiotic specific to penicillin-binding protein 2 (PBP 2) in Escherichia coli, blocks cell wall elongation and, indirectly, cell division, but its lethality can be overcome by increased levels of ppGpp, the nucleotide effector of the stringent response. We have subjected an E. coli K-12 strain to random insertional mutagenesis with a mini-Tn10 element. One insertion, which was found to confer resistance to mecillinam in relA+ and relA strains, was mapped at 75.5 min on the E. coli map and was located between the promoters and the coding sequence of the aroK gene, which codes for shikimate kinase 1, one of two E. coli shikimate kinases, both of which are involved in aromatic amino acid biosynthesis. The mecillinam resistance conferred by the insertion was abolished in a delta relA delta spoT strain completely lacking ppGpp, and it thus depends on the presence of ppGpp. Furthermore, the insertion increased the ppGpp pool approximately twofold in a relA+ strain. However, this increase was not observed in relA strains, although the insertion still conferred mecillinam resistance in these backgrounds, showing that mecillinam resistance is not due to an increased ppGpp pool. The resistance was also abolished in an ftsZ84(Ts) strain under semipermissive conditions, and the aroK::mini-Tn10 allele partially suppressed ftsZ84(Ts); however, it did not increase the concentration of the FtsZ cell division protein. The insertion greatly decreased or abolished the shikimate kinase activity of AroK in vivo and in vitro. The two shikimate kinases of E. coli are not equivalent; the loss of AroK confers mecillinam resistance, whereas the loss of Arol, does not. Furthermore, the ability of the aroK mutation to confer mecillinam resistance is shown to be independent of polar effects on operon expression and of effects on the availability of aromatic amino acids or shikimic acid. Instead, we conclude that the AroK protein has a second activity, possibly related to cell division regulation, which confers mecillinam sensitivity. We were able to separate the AroK activities mutationally with an aroK mutant allele lacking shikimate kinase activity but still able to confer mecillinam sensitivity.

The SIGNAL experiment in BIORACK: Escherichia coli in microgravity.

Microgravity affects certain physical properties of fluids, such as convection movement and surface tension. As a consequence, cells and living organisms may exhibit different behaviour in space, which may result from differences in the immediate environment of the cell or changes in the structure of the membrane in microgravity. Two experiments to examine the effects of microgravity on cell microenvironment and signal transduction through membranes were performed using a well-characterized system with different strains of the non-pathogenic Gram-negative bacterium Escherichia coli. Our results indicate that (i) microgravity appears to reduce the lag period of a non-motile culture of E. coli, and (ii) the ompC gene, regulated by the two-component system EnvZ-OmpR, is induced as well or better in microgravity than in ground controls.

Regulation of the heat-shock response depends on divalent metal ions in an hflB mutant of Escherichia coli.

HflB, also called FtsH, is an essential Escherichia coli protein involved in the proteolysis of the heat-shock regulator sigma 32 and of the phage regulator lambda cll. The hflB1(Ts) allele (formerly called ftsH1) conferring temperature-sensitive growth at 42 degrees C is suppressed by loss of the ferric-uptake repressor Fur and by anaerobic growth. We show here that suppression requires TonB-dependent Fe(III) transport in the hflB1(Ts) fur mutant during aerobic growth at 42 degrees C and Feo-dependent Fe(II) transport during anaerobic growth at 42 degrees C. Temperature-resistant growth of hflB1(Ts) strains is also observed at 42 degrees C in the presence of a high concentration of Fe(II), Ni(II), Mn(II) or Co(II) salts, but not in the presence of Zn(II), Cd(II), Cu(II), Mg(II), Ca(II) or Cr(III) salts. However, neither Ni(II) nor a fur mutation permits growth in the complete absence of HflB. The heat-shock response, evaluated by an htpG::lacZ fusion, is overinduced in hflB1(Ts) strains at 42 degrees C because of stabilization of sigma 32. Growth in the presence of Ni(II) or in the absence of the Fur repressor abolishes this overinduction in the hflB1(Ts) strain, and, in the hflB1(Ts) fur mutant, sigma 32 is no longer stabilized at 42 degrees C. These results reinforce the recent observation that HflB is a metalloprotease active against sigma 32 in vitro and suggest that it can associate functionally in vivo with Fe(II), Ni(II), Mn(II) and Co(II) ions.

Reversal of Mu gem2ts-induced mutations.

Mutations induced by the integration of a Mu gem2ts mutant prophage can revert at frequencies around 1 x 10(-6), more than 10(4)-fold higher than that obtained with Mu wild-type. Several aspects characterize Mu gem2ts precise excision: (i) the phage transposase is not involved; (ii) the RecA protein is not necessary; and (iii) revertants remain lysogenic with the prophage inserted elsewhere in the host genome. In addition, prophage re-integration seems to be non-randomly distributed, whereas Mu insertion into the host genome is a transposition event without any sequence specificity. In this paper, we describe that the site of re-integration somehow depends on the original site of insertion. Two alternative models are proposed to explain the strong correlation between donor and receptor sites.

Overview of controls in the Escherichia coli cell cycle.

The harmonious growth and cell-to-cell uniformity of steady-state bacterial populations indicate the existence of a well-regulated cell cycle, responding to a set of internal signals. In Escherichia coli, the key events of this cycle are the initiation of DNA replication, nucleoid segregation and the initiation of cell division. The replication initiator is the DnaA protein. In nucleoid segregation, the MukB protein, required for proper partitioning, may be a member of the myosin-kinesin superfamily of mechanoenzymes. In cell division, the FtsZ protein has a tubulin motif, is a GTPase and polymerizes in a ring around midcell during septation; the FtsA protein has an actin-like structure. The nature of the internal signals triggering these events is not known but candidates include cell mass, the superhelical density of the chromosome and the concentration of two regulatory nucleotides, cyclic AMP and ppGpp. The involvement of cytoskeletal-like proteins in key cycle events encourages the notion of a fundamental biological unity in cell cycle regulation in all organisms.