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A Jaffé

Publications and source records attributed to A Jaffé.

At least 37 records · Page 2Linked to original sources
PubMed1992

Penicillin binding protein 2 is dispensable in Escherichia coli when ppGpp synthesis is induced.

Mecillinam, a beta-lactam antibiotic which specifically inactivates penicillin binding protein 2 (PBP2) in Escherichia coli, prevents lateral cell wall elongation, inducing spherical morphology and cell death. Two mecillinam resistant mutants, lov-1 and lovB, both able to dispense entirely with PBP2, are shown here to be affected in the aminoacyl-tRNA synthetase genes argS and alaS, respectively. Although the argS and alaS mutants grow slowly, we show that there is no correlation between mecillinam resistance and either growth rate or translation speed. A role of the ribosomes in mecillinam sensitivity, suggested by our earlier report that the lov-1 mutation is suppressed by certain rpsL(StrR) alleles affecting ribosomal protein S12, is supported by the present observation that a pseudo-streptomycin dependent mutant is mecillinam resistant in the presence of streptomycin. The argS and alaS mutants have high pools of the nucleotide ppGpp (effector of the stringent response) and the mecillinam resistance of both mutations is suppressed by a relA mutation, inactivating the ribosome-associated ppGpp synthetase and preventing ppGpp synthesis in response to aminoacyl-tRNA starvation. Furthermore, a ptacrelA' multicopy plasmid makes a wild type strain mecillinam resistant. The effect of ppGpp is probably mediated by RNA polymerase, since sublethal doses of the polymerase inhibitor rifampicin suppress mecillinam resistance in argS, alaS and ptacrelA'-bearing strains. We conclude that ppGpp regulates the transcription of a gene whose product is involved in mecillinam sensitivity, possibly as part of a chain of interacting elements which coordinate ribosomal activity with that of the PBPs.

Alanine-tRNA Ligase↗
PubMed1991

The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli.

An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated. The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome. The mukB gene codes for a large protein (approximately 180 kd). A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene. Computer analysis revealed that the predicted MukB protein has distinct domains: an amino-terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha-helical coiled-coil domains and one globular domain, and a carboxyl-terminal globular domain which is rich in Cys, Arg and Lys. A 180 kd protein detected in wild-type cell extracts by electrophoresis is absent in mukB null mutants. Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning. At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells. We conclude that the MukB protein is required for chromosome partitioning in E. coli.

Amino Acid Sequence↗
PubMed1990

Rule governing the division pattern in Escherichia coli minB and wild-type filaments.

Escherichia coli minB mutants form anucleate minicells and multinucleate filaments. We show here that the overwhelming majority of nucleate cells contain 2n (n = 0, 1, 2, ...) nucleoids, as determined by 4',6-diamidino-2-phenylindole staining, and 2n (n = 1, 2, 3, ...) copies of the replication origin, as determined by flow cytometry. This shows that division sites are not chosen randomly among the available sites in minB filaments. Similarly, wild-type cells contain 2n nucleoids, both during cell division inhibition and when furazlocillin-induced filaments are allowed to divide. We conclude that the min+ function is only to prevent septation only at polar sites; the placement of internal cell division sites must obey strict rules, which are the same in minB and wild-type cells.

Cell Division↗
PubMed1989

The Escherichia coli lov gene product connects peptidoglycan synthesis, ribosomes and growth rate.

Mecillinam, a beta-lactam antibiotic which binds specifically to penicillin-binding protein 2 (PBP2), blocks lateral cell-wall elongation, induces spherical morphology and ultimately kills bacteria. We describe here a new mecillinam-resistant mutant of Escherichia coli, the lov mutant. It possesses active PBP2, as evidenced by its rod shape in the absence of mecillinam (but not in its presence), its ability to filament when septation is inhibited, and its penicillin-binding ability. The lov mutant grows slowly but seems to regulate its macromolecular parameters properly: cell volume, RNA content (ribosome concentration), and DNA content are appropriate for the growth rate, and the growth yield is identical to that of wild type. The lov mutation is located at 41 min on the E.coli genetic map and is recessive. Certain rpsL (StrR) mutations suppress the lov mutant's mecillinam resistance. The allele specificity of the suppression suggests that the lov gene product may interact directly with the ribosomes. The lov gene product thus seems to define a link between PBP2 (the mecillinam target) and the ribosomes; we propose that this link is involved in transmitting information on the growth rate (ribosome concentration) to the peptidoglycan synthesizing apparatus.

Acyltransferases↗
PubMed1989

Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells.

To study the chromosomal partitioning mechanism in cell division, we have isolated a novel type of Escherichia coli mutants which formed anucleate cells, by using newly developed techniques. One of them, named mukA1, is not lethal and produces normal-sized anucleate cells at a frequency of 0.5 to 3% of total cells in exponentially growing populations but does not produce filamentous cells. Results suggest that the mutant is defective in the chromosome positioning at regular intracellular positions and fails frequently to partition the replicated daughter chromosomes into both daughter cells, resulting in production of one anucleate daughter cell and one with two chromosomes. The mukA1 mutation causes pleiotropic effects: slow growth, hypersensitivity to sodium dodecyl sulfate, and tolerance to colicin E1 protein, in addition to anucleate cell formation. Cloning of the mukA gene indicates that the mukA1 mutation is recessive and that the mukA gene is identical to the tolC gene coding for an outer membrane protein.

Bacterial Outer Membrane Proteins↗
PubMed1989

Penicillin-binding protein 2 is essential in wild-type Escherichia coli but not in lov or cya mutants.

Penicillin-binding protein 2 (PBP2), target of the beta-lactam mecillinam, is required for rod morphology and cell wall elongation in Escherichia coli. A new temperature-sensitive PBP2 allele and an in vitro-constructed insertion deletion allele were shown to be lethal in wild-type strains, establishing that the activity of this protein is essential. Mutations in the lov or cya genes, conferring mecillinam resistance, compensated for the deleterious effect of the absence of PBP2. The resulting double mutants grew as spheres. In a cya mutant lacking PBP2, the restoration of a Cya+ phenotype by addition of cyclic AMP caused lethality and a block in cell division. These results show that in wild-type cells, PBP2 is essential for growth and division.

Acyltransferases↗
PubMed1989

Multiple defects in Escherichia coli mutants lacking HU protein.

The HU protein isolated from Escherichia coli, composed of two partially homologous subunits, alpha and beta, shares some of the properties of eucaryotic histones and is a major constituent of the bacterial nucleoid. We report here the construction of double mutants totally lacking both subunits of HU protein. These mutants exhibited poor growth and a perturbation of cell division, resulting in the formation of anucleate cells. In the absence of HU, phage Mu was unable to grow, to lysogenize, or to carry out transposition.

Bacterial Proteins↗
PubMed1988

Cyclic AMP and cell division in Escherichia coli.

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.

Amdinocillin↗
PubMed1988

Minicell-forming mutants of Escherichia coli: production of minicells and anucleate rods.

The Escherichia coli minB mutant originally isolated is known to septate at cell poles to form spherical anucleate minicells. Three new minicell-producing mutants were isolated during a screening by autoradiography for chromosome partition mutants giving rise spontaneously to normal-sized anucleate cells. These min mutants were affected close to or in the minB locus. Autoradiography analysis as well as fluorescent staining of DNA showed that in addition to minicells, these strains and the original minB mutant also spontaneously produced anucleate rods of normal size and had an abnormal DNA distribution in filaments. These aberrations were not associated with spontaneous induction of the SOS response. Inhibition of DNA synthesis in these mutants gave rise to anucleate cells whose size was longer than unit cell length, suggesting that the min defect allows septation to take place at normally forbidden sites not only at cell poles but also far from poles. Abnormal DNA distribution and production of anucleate rods suggest that the Min product(s) could be involved in DNA distribution.

Autoradiography↗
PubMed1986

SOS-independent coupling between DNA replication and cell division in Escherichia coli.

Inhibition of DNA synthesis in Escherichia coli mutants in which the SOS-dependent division inhibitors SfiA and SfiC were unable to operate led to a partial arrest of cell division. This SOS-independent mechanism coupling DNA replication and cell division was characterized with respect to residual division, particle number, and DNA content. Whether DNA replication was blocked in the initiation or the elongation step, numerous normal-sized anucleate cells were produced (not minicells or filaments). Their production was used to evaluate the efficiency of this coupling mechanism, which seems to involve the cell division protein FtsZ (SulB), also known to be the target of the division inhibitors SfiA and SfiC. In the absence of DNA synthesis, the efficiency of coupling was modulated by the cyclic-AMP-cyclic-AMP receptor protein complex, which was required for anucleate cell production.

Adenylyl Cyclases↗
PubMed1986

F plasmid ccd mechanism in Escherichia coli.

The ccd mechanism specified by the ccdA and ccdB genes of the mini-F plasmid determines fate of plasmid-free segregants in Escherichia coli (Jaffé et al., J. Bacteriol. 163:841-849, 1985). The killing function in plasmid-free segregants by the ccd mechanism did not affect cell growth of coexisting cells in the same culture. Elongated cells and anucleate cells caused by the ccd mechanism were clearly detected by flow cytometry in cultures of bacterial strains harboring Ccd+ Sop- mini-F plasmids defective in partitioning. This indicates that the defect in correct partitioning of plasmid DNA molecules into daughter cells also induces the ccd mechanism to operate.

Bacterial Proteins↗
PubMed1986

DNA replication termination in Escherichia coli parB (a dnaG allele), parA, and gyrB mutants affected in DNA distribution.

We investigated the Escherichia coli mutants carrying the parB, parA, and gyrB mutations, all of which display faulty chromosome partitioning at the nonpermissive temperature, to see whether their phenotype reflected a defect in the termination of DNA replication. In the parB strain DNA synthesis slowed down at 42 degrees C and the SOS response was induced, whereas in the parA strain DNA synthesis continued normally for 120 min and there was no SOS induction. To see whether replication forks accumulated in the vicinity of terC at the nonpermissive temperature, the mutants were incubated for 60 min at 42 degrees C and then returned to low temperature and pulse-labeled with [3H]thymidine. In all cases the restriction pattern of the labeled DNA was incompatible with that of the terC region, suggesting that replication termination was normal. In the parA mutant no DNA sequences were preferentially labeled, whereas in the parB and gyrB strains there was specific labeling of sequences whose restriction pattern resembled that of oriC. In the case of parB this was confirmed by DNA-DNA hybridization with appropriate probes. This test further revealed that the parB mutant over initiates at oriC after the return to the permissive temperature. Like dna(Ts) strains, the parB mutant formed filaments at 42 degrees C in the absence of SOS-associated division inhibition, accompanied by the appearance of anucleate cells of nearly normal size (28% of the population after 3 h), as revealed by autoradiography. The DNA in the filaments was either centrally located or distributed throughout. The parB mutation lies at 67 min, and the ParB- phenotype is corrected by a cloned dnaG gene or by a plasmid primase, strongly suggesting that parB is an allele of dnaG, the structural gene of the E. coli primase. It is thus likely that the parB mutant possesses an altered primase which does not affect replication termination but causes a partial defect in replication initiation and elongation and in chromosome distribution.

Alleles↗
PubMed1985

Growth of the Escherichia coli cell envelope.

The growth pattern of the Escherichia coli envelope was studied by immunoelectron microscopy, using the outer membrane protein LamB specifically labelled by a double antibody gold particle technique. An operon fusion placing the lamB gene under lac promoter control permitted rapid turn-off of LamB synthesis. In the generation following turn-off no lamB-free regions appeared, strongly suggesting that bulk outer membrane material is not inserted in restricted growth zones.

Bacterial Outer Membrane Proteins↗
PubMed1985

Effects of the ccd function of the F plasmid on bacterial growth.

The ccd segment of the mini F plasmid containing the ccdA and ccdB genes controls the coordination between plasmid proliferation and cell physiology and fate. When the DNA replication of a thermosensitive-replication plasmid carrying the ccd segment of mini F is blocked, plasmid DNA molecules are progressively diluted through cell division until the copy number reaches 1 per cell. From this time on, there is little increase in the number of viable cells, although cells continue to divide, resulting in a mixed population of viable cells (mostly plasmid containing), nonviable but residually dividing cells, and nonviable nondividing cells. Results are presented suggesting that plasmid-containing cells are viable and continue to divide, whereas plasmid-free segregants are nonviable and form filaments after a few residual divisions, with DNA synthesis reduced or arrested in the filaments. Although the ccd functions are known to induce the SOS response when plasmid replication is blocked, the production of nonviable plasmid-free segregants is independent of the SOS cell division inhibition mechanism determined by the sfiA and sfiC genes.

Cell Division↗
PubMed1983

Selection and characterization of beta-lactam-resistant Escherichia coli K-12 mutants.

beta-Lactam-resistant mutants of Escherichia coli K-12 were selected by using 12 different beta-lactam derivatives. The mutants fell into three categories showing (i) altered permeation through reduction or loss of outer membrane porin proteins (including ompF, ompR, and envZ alleles); (ii) increase in the rate of synthesis of chromosomally mediated beta-lactamase; or (iii) defective synthesis or action of cyclic adenosine 3',5'-phosphate (cya and crp alleles).

Anti-Bacterial Agents↗