Biomedical subjects
Publications and source records attributed to R D'Ari.
Escherichia coli dam mutants, lacking the GATC DNA methylase, do not produce anucleate cells at high frequencies, suggesting that hemimethylation of the chromosome origin of replication, oriC, is not essential for correct chromosome partitioning.
From a gene bank of Klebsiella pneumoniae M5a1, a 1.7 kb gene fragment was isolated which was able to restore the Ntr+ phenotype and ammonium (methylammonium) transport, but not glutamate synthase in an Escherichia coli glt mutant (glutamate synthase deficiency). The fragment strongly hybridized with the gltF regulatory gene from E. coli. After subcloning the fragment into an overexpression vector, a protein with a molecular weight of 27,000 dalton was identified as the gene product. The results indicate that the fragment cloned contains the gltF gene from K.pneumoniae.
Cultures of the bacterium Escherichia coli were grown in the orbiting Biocosmos 2044 satellite in order to evaluate the effects of the space environment--weightlessness and heavy particle radiation--on growth parameters and energy metabolism, which have previously been reported to be affected, and on induction of the SOS response, which reflects DNA damage to the cell. We found no differences between the flight samples and control ground cultures in the growth yield per gram of carbon, in mean cell mass (from which we deduce that the growth rate was unaltered) or in the level of expression of the SOS response. These observations indicate that free-growing bacterial cells do not expend significant energy fighting gravity and that cosmic radiation within a space capsule does not produce significant levels of DNA damage.
The buoyant density of wild-type Escherichia coli cells has previously been reported not to vary with growth rate and cell size or age. In the present report we confirm these findings, using Percoll gradients, and analyze the recently described lov mutant, which was selected for its resistance to mecillinam and has been suggested to be affected in the coordination between mass growth and envelope synthesis. The average buoyant density of lov mutant cells was significantly lower than that of wild-type cells. Similarly, the buoyant density of wild-type cells decreased in the presence of mecillinam. The density of the lov mutant, like that of the wild type, was invariant over a 2.8-fold range in growth rate. In this range, however, the average cell volume was also constant. Analysis of buoyant density as a function of cell volume in individual cultures revealed that smaller (newborn) lov mutant cells had higher density than larger (old) cells; however, the density of the small cells never approached that of the wild-type cells, whose density was independent of cell size (age). A pattern similar to that of lov mutant cells was observed in cells carrying the mecillinam-resistant mutations pbpA(Ts) and rodA(Ts) and the division mutation ftsI(Ts) at nonpermissive temperatures as well as in wild-type cells treated with mecillinam, but not in mecillinam-resistant crp or cya mutants.
The chemical synthesis of 5-amino-1-(2'-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide, referred to as dZ, and of its 5'-triphosphate derivative (dZTP), from 2'-deoxyinosine is described. The polymerisation of dZTP using terminal deoxynucleotidyltransferase to give a homopolymer is also presented.
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The logic of Escherichia coli's responses to environmental changes gives hope that its cell cycle will be equally well designed. During growth in a constant environment, internal signals trigger cell-cycle events such as replication initiation and cell division. Internal signals must also provide the cell with information about its present state, enabling it to coordinate the synthesis of cytoplasm, DNA and cell wall and maintain proper cell shape and composition. How the cell regulates these aspects of its growth is a fascinating--and as yet unfinished--story.
While wild-type Escherichia coli K12 cannot grow with L-serine as carbon source, two types of mutants with altered methionine metabolism can. The first type, metJ mutants, in which the methionine biosynthetic enzymes are expressed constitutively, are able to grow with L-serine as carbon source. Furthermore, a plasmid carrying the metC gene confers ability to grow on L-serine. These observations suggest that in these mutants, L-serine deamination may be a result of a side-reaction of the metC gene product, cystathionine beta-lyase. The second type is exemplified by two newly isolated strains carrying mutations mapping between 89.6 and 90 min. These mutants use L-serine as carbon source, and also require methionine for growth with glucose at 37 degrees C and above. The phenotypes of the new mutants resemble those of both met and his constitutive mutants in some respects, but have been differentiated from both of them.
Synchronized cells of Escherichia coli were pulse-labeled with [3H]leucine and subjected to membrane fractionation to determine whether a fraction that is enriched for membrane-murein adhesion zones (fraction OML) was preferentially generated at specific times during the cell cycle, as previously suggested from studies of lkyD and cha mutants. Contrary to this prediction, the experiments showed that OML was formed continuously during the division cycle.
The ftsZ gene of Escherichia coli, which lies in a cluster of cell division genes at 2 min on the genetic map, codes for a protein which is thought to play a key role in triggering cell division. Using an ftsZ::lacZ operon fusion, we have studied the transcription of the ftsZ gene under conditions in which cell division was either inhibited or synchronized in the bacterial population. In ftsZ, ftsA, ftsQ, and ftsI (or pbpB) mutants, there was no change in the differential rate of expression of the ftsZ gene in nonpermissive conditions, when cell division was completely blocked. Although the FtsZ protein is thought to be limiting for cell division, in synchronized cultures the ftsZ gene was expressed not only at the moment of septation initiation but throughout the cell cycle. Its expression, however, was not exponential but linear, with a rapid doubling in rate at a specific cell age; this age, about 20 min after division in a 60-min cycle, was different from the age at which the ftsZ::lacZ operon was duplicated. However, it was close to the age at which replication initiated and at which the rate of phospholipid synthesis doubled. During the transient division inhibition after a nutritional shift-up, ftsZ transcription again became linear, with two doublings in rate at intervals equal to the mass doubling time in the rich medium; it adopted the exponential rate typical of rich medium about 60 min after the shift-up, just before the bacterial population resumed cell division. The doubling in the rate of ftsZ transcription once per cycle in synchronized cultures and once per mass doubling time during the transition period after a nutritional shift-up reflects a new cell cycle event.
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.
We have isolated and characterized a highly pleiotropic Escherichia coli mutant affected in the activity of a number of enzymes involved in different metabolic pathways, all of which are regulated by leucine. Selected for its ability to grow with L-serine as sole carbon source, the rbl-1::Tn10 mutant had high levels of L-serine deaminase activity (due to increased transcription of the structural gene) and of another amino acid-degrading enzyme, L-threonine dehydrogenase, and decreased transcription of the operons serA and ilvIH, coding for biosynthetic enzymes. The rbl mutation suppressed the slow growth of a metK mutant, deficient in S-adenosylmethionine synthetase. Furthermore, metK mutants spontaneously accumulated faster-growing rbl-like derivatives, and a commonly used metK strain, RG62, carries such a mutation. The rbl gene is located near 20 min on the E. coli genetic map. All phenotypes of the rbl mutant could be observed in rbl+ strains cultivated in the presence of L-leucine, and exogenous L-leucine had little further effect on the rbl strains. We propose that the rbl gene product is the regulator of a global response to leucine.
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.
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.
In growing cultures of Escherichia coli, simultaneous inhibition of penicillin-binding proteins 1a and 1b (PBPs 1) by a beta-lactam efficiently induces cell lysis. However, the lytic behavior of cultures initiating growth in the presence of beta-lactams specifically inhibiting PBPs 1 suggested that the triggering of cell lysis was a cell division-related event, at least in the first cell cycle after the resumption of growth (F. Garcia del Portillo, A. G. Pisabarro, E. J. de la Rosa, and M. A. de Pedro, J. Bacteriol. 169:2410-2416, 1987). To investigate whether this apparent correlation would hold true in actively growing cells, we studied the lytic behavior of cultures of E. coli aligned for cell division which were challenged with beta-lactams at different times after alignment. Cell division was aligned either by nutritional shift up or by chromosome replication alignment. Specific inhibition of PBPs 1 with the beta-lactam cefsulodin resulted in a delayed onset of lysis which was coincident in time with the resumption of cell division. The apparent correlation between the initiation of lysis and cell division was abolished when cefsulodin was used in combination with the PBP 2-specific inhibitor mecillinam, leading to the onset of lysis at a constant time after the addition of the beta-lactams. The results presented clearly argue in favor of the hypothesis that the triggering of cell lysis after inhibition of PBPs 1 is a cell division-correlated event dependent on the activity of PBP 2.
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.
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.
In synchronized culture of Escherichia coli, the specific arrest of phospholipid synthesis (brought about by glycerol starvation in an appropriate mutant) did not affect the rate of ongoing DNA synthesis but prevented the initiation of new rounds. The initiation block did not depend on cell age at the time of glycerol removal, which could be before, during, or after the doubling in the rate of phospholipid synthesis (DROPS) and as little as 10 min before the expected initiation. We conclude that the initiation of DNA replication is not triggered by the preceding DROPS but requires active phospholipid synthesis. Conversely, when DNA replication initiation was specifically blocked in a synchronized culture of a dnaC(Ts) mutant, two additional DROPS were observed, after which phospholipid synthesis continued at a constant rate for at least 60 min. Similarly, when DNA elongation was blocked by thymine starvation of a synchronized culture, one additional DROPS was observed, followed by linear phospholipid accumulation. Control experiments showed that specific inhibition of cell division by ampicillin, heat shock, or induction of the SOS response did not affect phospholipid synthesis, suggesting that the arrest of DROPS observed was due to the DNA replication block. The data are compatible with models in which the DROPS is triggered by an event associated with replication termination or chromosome segregation.