Temperature-Sensitive Cell Division Mutants of Escherichia coli with Thermolabile Penicillin-Binding Proteins

The thermostability of the penicillin-binding proteins (PBPs) of 31 temperature-sensitive cell division mutants of Escherichia coli has been examined. Two independent cell division mutants have been found that have highly thermolabile PBP3. Binding of [(14)C]benzylpenicillin to PBP3 (measured in envelopes prepared from cells grown at the permissive temperature) was about 30% of the normal level at 30 degrees C, and the ability to bind [(14)C]benzylpenicillin was rapidly lost on incubation at 42 degrees C. The other PBPs were normal in both mutants. At 30 degrees C both mutants were slightly longer than their parents and on shifting to 42 degrees C they ceased dividing, but cell mass and deoxyribonucleic acid synthesis continued and long filaments were formed. At 42 degrees C division slowly recommenced, but at 44 degrees C this did not occur. The inhibition of division at 42 degrees C was suppressed by 0.35 M sucrose, and in one of the mutants it was partially suppressed by 10 mM MgCl(2). PBP3 was not stabilized in vitro at 42 degrees C by these concentrations of sucrose or MgCl(2). Revertants that grew as normal rods at 42 degrees C regained both the normal level and the normal thermostability of PBP3. The results provide extremely strong evidence that the inactivation of PBP3 at 42 degrees C in the mutants is the cause of the inhibition of cell division at this temperature and identify PBP3 as an essential component of the process of cell division in E. coli. It is the inactivation of this protein by penicillins and cephalosporins that results in the inhibition of division characteristic of low concentrations of many of these antibiotics.     

Interaction of FtsA and PBP3 proteins in the Escherichia coli septum.

Mutations in the ftsA gene of Escherichia coli conferred a higher resistance to lysis induced by penicillin or by a combination of cefsulodin and furazlocillin. The ftsA2 allele codes for an FtsA protein which is inactive at 42 degrees C but is able to regain its activity once it is transferred back to 30 degrees C; ftsA2 filaments formed at 42 degrees C in the presence of penicillin divided once the penicillin was removed and the temperature was lowered to 30 degrees C. Potential septation sites in the filaments of wild-type cells treated in the same way remained inactive. The binding of a radioactively labeled derivative of ampicillin to penicillin-binding protein 3 (PBP3) was significantly decreased in strain D-3, containing the mutant allele ftsA3, when the binding assay was performed at the restrictive temperature. A molecular species able to cross-react with an anti-PBP3 serum was nevertheless found to be present in the envelope of D-3 cells. These observations suggested that the FtsA protein, a protein with a structural and regulatory role in septation, and PBP3, a protein enzymatically active in the synthesis of murein for septation, interact with each other.     

Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism.

DnaK is a major heat shock protein of Escherichia coli and has been previously reported to be essential for growth at high temperatures. We systematically investigated the role of DnaK in cellular metabolism at a wide range of growth temperatures by analyzing cellular defects caused by deletion of the dnaK gene (delta dnaK52). At intermediate temperatures (30 degrees C), introduction of the delta dnaK52 allele into wild-type cells caused severe defects in cell division, slow growth, and poor viability of the cells. delta dnaK52 mutants were genetically unstable at 30 degrees C and frequently acquired secondary mutations. At high (42 degrees C) and low (11 and 16 degrees C) temperatures the delta dnaK52 allele could only be introduced into the subpopulation of wild-type cells that had duplicated the dnaK region of their chromosome. delta dnaK52 mutants isolated at 30 degrees C were cold sensitive as well as temperature sensitive for growth. Cell division defects of delta dnaK52 mutants at 30 degrees C were largely suppressed by overproduction of the FtsZ protein, which is normally required for septation during cell division; however, slow growth and poor viability at 30 degrees C and cold sensitivity and temperature sensitivity of growth were not suppressed, indicating that delta dnaK52 mutants had additional defective cellular functions besides cell division.     

Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring

The FtsA protein is a member of the actin superfamily that localizes to the bacterial septal ring during cell division. Deletions of domain 1C or the S12 and S13 beta-strands in domain 2B of the Escherichia coli FtsA, previously postulated to be involved in dimerization, result in partially active proteins that do not allow the normal progression of septation. The truncated FtsA protein lacking domain 1C (FtsADelta1C) localizes in correctly placed division rings, together with FtsZ and ZipA, but does not interact with other FtsA molecules in the yeast two-hybrid assay, and fails to recruit FtsQ and FtsN into the division ring. The rings containing FtsADelta1C are therefore incomplete and do not support division. The production of high levels of FtsADelta1C causes filamentation, an effect that has been reported to result as well from the imbalance between FtsA+ and FtsZ+ molecules. These data indicate that the domain 1C of FtsA participates in the interaction of the protein with other FtsA molecules and with the other proteins that are incorporated at later stages of ring assembly, and is not involved in the interaction with FtsZ and the localization of FtsA to the septal ring. The deletion of the S12-S13 strands of domain 2B generates a protein (FtsADeltaS12-13) that retains the ability to interact with FtsA+. When the mutated protein is expressed at wild-type levels, it localizes into division rings and recruits FtsQ and FtsN, but it fails to sustain septation at normal levels resulting in filamentation. A fivefold overexpression of FtsADeltaS12-13 produces short cells that have normal division rings, but also cells with polar localization of the mutated protein, and cells with rings at abnormal positions that result in the production of a fraction (15%) of small nucleoid-free cells. The S12-S13 strands of domain 2B are not essential for septation, but affect the localization of the division ring.     

Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression.

Aminoacyl-tRNA synthetase mutants of Escherichia coli are resistant to amdinocillin (mecillinam), a beta-lactam antibiotic which specifically binds penicillin-binding protein 2 (PBP2) and prevents cell wall elongation with concomitant cell death. The leuS(Ts) strain, in which leucyl-tRNA synthetase is temperature sensitive, was resistant to amdinocillin at 37 degrees C because of an increased guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool resulting from partial induction of the stringent response, but it was sensitive to amdinocillin at 25 degrees C. We constructed a leuS(Ts) delta (rodA-pbpA)::Kmr strain, in which the PBP2 structural gene is deleted. This strain grew as spherical cells at 37 degrees C but was not viable at 25 degrees C. After a shift from 37 to 25 degrees C, the ppGpp pool decreased and cell division was inhibited; the cells slowly carried out a single division, increased considerably in volume, and gradually lost viability. The cell division inhibition was reversible when the ppGpp pool increased at high temperature, but reversion required de novo protein synthesis, possibly of septation proteins. The multicopy plasmid pZAQ, overproducing the septation proteins FtsZ, FtsA, and FtsQ, conferred amdinocillin resistance on a wild-type strain and suppressed the cell division inhibition in the leuS(Ts) delta (rodA-pbpA)::Kmr strain at 25 degrees C. The plasmid pAQ, in which the ftsZ gene is inactivated, did not confer amdinocillin resistance. These results lead us to hypothesize that the nucleotide ppGpp activates ftsZ expression and thus couples cell division to protein synthesis.     

Morphology of an Escherichia coli mutant with a temperature-dependent round cell shape.

Mutants of Escherichia coli capable of growing in the presence of 10 microgram of mecillinam per ml were selected after intensive mutagenesis. Of these mutants, 1.4% formed normal, rod-shaped cells at 30 degrees C but grew as spherical cells at 42 degrees C. The phenotype of one of these rod(Ts) mutants was 88% cotransducible with lip (14.3 min), and all lip+ rod(Ts) transductants of a lip recipient had the following characteristics: (i) growth was relatively sensitive to mecillinam at 30 degrees C but relatively resistant to mecillinam at 42 degrees C; (ii) penicillin-binding protein 2 was present in membranes of cells grown at 30 degrees C in reduced amounts and was undetectable in the membranes of cells grown at 42 degrees C. The mecillinam resistance, penicillin-binding protein 2 defect, and rod phenotypes all cotransduced with lip with high frequency. Thus the mutation [rodA(Ts)] is most likely in the gene for penicillin-binding protein 2 and causes the organism to grow as a sphere at 42 degrees C, although it grows with normal rodlike morphology at 30 degrees C. At 42 degrees C, cells of this strain were round with many wrinkles on their surfaces, as revealed by scanning electron microscopy. In these round cells, chromosomes were dispersed or distributed peripherally, in contrast to normal rod-shaped cells which had centrally located, more condensed chromosomes. The round cells divided asymmetrically on solid agar, and it seemed that the plane of each successive division was perpendicular to the preceding one. On temperature shift-down in liquid medium many cells with abnormal morphology appeared before normal rod-shaped cells developed. Few abnormal cells were seen when cells were placed on solid medium during temperature shift-down. These pleiotropic effects are presumably caused by one or more mutations in the rodA gene.     

Regulation of Cell Division in Escherichia coli: Characterization of Temperature-Sensitive Division Mutants

A temperature-sensitive division mutant of Escherichia coli was isolated by using differential filtration to select for filaments at 42 C and normal cells at 30 C. Cells shifted from 30 to 42 C stop dividing almost immediately, suggesting the temperature-sensitive element is required for cell division late in the cell cycle. Cells returned to 30 from 42 C divide abruptly, suggesting accumulation of division potential at 42 C. Inhibitors of protein, deoxyribonucleic acid, and ribonucleic acid synthesis do not block division during the recovery period at 30 C. Cycloserine does not stop cell division, vancomycin shows some effect on cell division, whereas penicillin completely stops cell division during this period. The addition of high concentrations of NaCl to filaments at 42 C results in a burst of cell division. The final cell number is equivalent to the control which is grown at 30 C if sufficient salt is added (11 g/liter, final concentration). After the original burst, cell division ceases at the nonpermissive temperature even at increased osmolality. Chloramphenicol, puromycin, vancomycin, and penicillin prevent division during the recovery in the presence of NaCl. Kinetic data indicate division potential decays to a reversible inactive intermediate which rapidly decays to an irreversible inactive form. Conversion of division potential to the inactive form is correlated with a 100- to 1,000-fold derepression of the synthesis of division potential. The mutation appears to involve a stage in cross-wall synthesis which is required during the terminal stages of division.     

Physiological Effects of Growth of an Escherichia coli Temperature-Sensitive dnaZ Mutant at Nonpermissive Temperatures

The physiological effects of incubation at nonpermissive temperatures of Escherichia coli mutants that carry a temperature-sensitive dnaZ allele [dnaZ(Ts)2016] were examined. The temperature at which the dnaZ(Ts) protein becomes inactivated in vivo was investigated by measurements of deoxyribonucleic acid (DNA) synthesis at temperatures intermediate between permissive and nonpermissive. DNA synthesis inhibition was reversible by reducing the temperature of cultures from 42 to 30 degrees C; DNA synthesis resumed immediately after temperature reduction and occurred even in the presence of chloramphenicol. Inasmuch as DNA synthesis could be resumed in the absence of protein synthesis, we concluded that the protein product of the dnaZ allele (Ts)2016 is renaturable. Cell division, also inhibited by 42 degrees C incubation, resumed after temperature reduction, but the length of time required for resumption depended on the duration of the period at 42 degrees C. Replicative synthesis of cellular DNA, examined in vitro in toluene-permeabilized cells, was temperature sensitive. Excision repair of ultraviolet light-induced DNA lesions was partially inhibited in dnaZ(Ts) cells at 42 degrees C. The dnaZ(+) product participated in the synthesis of both Okazaki piece (8-12S) and high-molecular-weight DNA. During incubation of dnaZ(Ts)(lambda) lysogens at 42 degrees C, prophage induction occurred, and progeny phage were produced during subsequent incubation at 30 degrees C. The temperature sensitivity of both DNA synthesis and cell division in the dnaZ(Ts)2016 mutant was suppressed by high concentrations of sucrose, lactose, or NaCl. Incubation at 42 degrees C was neither mutagenic nor antimutagenic for the dnaZ(Ts) mutant.     

Characteristics of Methylobacillus flagellatum KT mutants, deficient for phosphoglucoisomerase, an enzyme of the ribulose monophosphate cycle of obligate methylotrophic bacteria

The activities of the key enzymes of ribulose monophosphate cycle for formaldehyde oxidation and assimilation were tested in crude extracts from temperature sensitive mutants of obligatemethylotroph M. flagellatum KT. Two mutants deficient in phosphoglucoisomerase activity were identified during this screening. Phosphoglucoisomerase of T525 pgi-1 mutant was active both at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. Complete inactivation of the enzyme at 42 degrees C occurred in 2 h in vitro, while in vivo incubation at nonpermissive temperature for more than 10 h was required for the enzyme inactivation. Phosphoglucoisomerase activity of T566 pgi-2 was 5-fold lower as compared with the one from the parent strain incubated at 30 degrees C. The enzyme was inactivated in 2 min. in crude extract at nonpermissive temperature.     

Structural inhibition and reactivation of Escherichia coli septation by elements of the SOS and TER pathways.

The inhibition of cell division caused by induction of the SOS pathway in Escherichia coli structurally blocks septation, as deduced from two sets of results. Potential septation sites active at the time of SOS induction became inactivated, while those initiated during the following doubling time were active. Penicillin resistance increased in wild-type UV light-irradiated cells, a behavior similar to that observed in mutants in which structural blocks were introduced by inactivation of FtsA. Potential septation sites that have been structurally blocked by either the SOS division inhibitor, furazlocillin inhibition of PBP3, or inactivation of a TER pathway component, FtsA3, could be reactivated one doubling time after removal of the inhibitory agent in the presence of an active lon gene product. Reactivation of potential septation sites blocked by the presence of an inactivated FtsA3 was significantly lower when the lon protease was not active, suggesting that Lon plays a role in the removal of inactivated TER pathway products from the blocked potential septation sites.     

Process of Infection with Bacteriophage φX174: XXIV. New Type of Temperature-sensitive Mutant

A group of temperature-sensitive mutants of phiX174 has been isolated which can go through a single, normal one-step growth cycle at 40 C but fail to form plaques at this temperature. Such mutants fail to initiate a second cycle at 40 C; however they can gain the capacity to infect at 40 C, upon incubation for 10 min in broth at 30 C. In regaining the ability to infect, the phage appear to undergo a temperature-dependent conformational alteration. The inverse process, a reversible loss of ability to infect at 40 C, is observed when such phage produced at 30 C are incubated for 2 hr at 40 C. The defect in initiation of a second cycle of infection appears to be in the injection of viral deoxyribonucleic acid. A two-step complementation test has been used to identify the cistron coding for the affected function. Such mutants are also unusually sensitive to an irreversible thermal inactivation when incubated at 40 C.     

Escherichia coli dnaK null mutants are inviable at high temperature.

DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on rapid reversible and non-reversible inactivation of fructose-1,6-bisphosphatase and malate dehydrogenase in wild-type and glycolytic block mutants of Saccharomyces cerevisiae

Experimental conditions have been elaborated to test for reversibility of the malate dehydrogenase inactivation (E.C.1.1.1.37) after addition of glucose to derepressed yeast cells. Malate dehydrogenase inactivation was shown to be irreversible at all stages of inactivation. In contrast fructose-1,6-bisphosphatase inactivation (E.C.3.1.11) remained reversible for at least 30 min after addition of glucose. Rapid reversible inactivation of fructose-1,6-bisphosphatase and irreversible inactivation of malate dehydrogenase were additionally investigated in glycolytic block mutants. Normal inactivation kinetics were observed in mutants without catalytic activity of phosphoglucose isomerase (E.C.5.3.1.9), phosphofructokinase (E.C.2.7.1.11), triosephosphate isomerase (E.C.5.3.1.1) and phosphoglycerate kinase (E.C.2.7.2.3). Hence, neither type of inactivation depended on the accumulation of any glucose metabolite beyond glucose-6-phosphate. Under anaerobic conditions irreversible inactivation was completely abolished in glycolytic block mutants. In contrast rapid reversible inactivation was independent of energy provided by respiration or fermentation. Reversibility of fructose-1,6-bisphosphatase inactivation was tested under conditions which prevented irreversible malate dehydrogenase inactivation. In these experiments, fructose-1,6-bisphosphatase inactivation remained reversible for at least 120 min, whereas reversibility was normally restricted to about 30 min. This indicated a common mechanism between the irreversible part of fructose-1,6-bisphosphatase inactivation and irreversible malate dehydrogenase inactivation.     

An essential enzyme for phospholipid synthesis associates with the Bacillus subtilis divisome

The mechanism by which the membrane synthetic machinery might be co‐organized with the cell‐division architecture during the bacterial cell cycle remains to be investigated. We characterized a key enzyme of phospholipid and fatty acid synthesis in Bacillus subtilis, the acyl–acyl carrier protein phosphate acyltransferase (PlsX), and identified it as a component of the cell‐division machinery. Comprehensive interaction analysis revealed that PlsX interacts with FtsA, the FtsZ‐anchoring protein. PlsX mainly localized at the potential division site independent of FtsA and FtsZ and then colocalized with FtsA. By multidirectional approaches, we revealed that the Z‐ring stabilizes the association of PlsX at the septum and pole. The localization of PlsX is also affected by the progression of DNA replication. PlsX is needed for cell division and its inactivation leads to aberrant Z‐ring formation. We propose that PlsX localization is prior to Z‐ring formation in the hierarchy of septum formation events and that PlsX is important for co‐ordinating membrane synthesis with cell division in order to properly complete septum formation.     

Correlation between the structure and biochemical activities of FtsA, an essential cell division protein of the actin family.

Cell division protein FtsA, predicted to belong to the actin family, is present in different cell compartments depending on its phosphorylation state. The FtsA fraction isolated from the cytoplasm is phosphorylated and capable of binding ATP, while the membrane-bound form is unphosphorylated and does not bind ATP. A variant of the protein FtsA102, in which the nucleotide binding site was destroyed by mutagenesis of a highly conserved residue predicted to be needed for the binding, does not bind ATP. Another variant, FtsA104, cannot be phosphorylated because the predicted phosphorylatable residue has been replaced by a non-phosphorylatable one. This protein although unable to bind ATP in vitro, is able to rescue the reversible ftsA2, the irreversible ftsA3 and, almost with the same efficiency, the ftsA16 amber alleles. Consequently, phosphorylation and ATP binding may not be essential for the function of FtsA. Alternatively they may have a regulatory role on the action of FtsA in the septator.     

Cell division in Bacillus subtilis: FtsZ and FtsA association is Z-ring independent, and FtsA is required for efficient midcell Z-Ring assembly

The earliest stage in cell division in bacteria is the assembly of a Z ring at the division site at midcell. Other division proteins are also recruited to this site to orchestrate the septation process. FtsA is a cytosolic division protein that interacts directly with FtsZ. Its function remains unknown. It is generally believed that FtsA localization to the division site occurs immediately after Z-ring formation or concomitantly with it and that FtsA is responsible for recruiting the later-assembling membrane-bound division proteins to the division site. Here, we report the development of an in vivo chemical cross-linking assay to examine the association between FtsZ and FtsA in Bacillus subtilis cells. We subsequently use this assay in a synchronous cell cycle to show that these two proteins can interact prior to Z-ring formation. We further show that in a B. subtilis strain containing an ftsA deletion, FtsZ localized at regular intervals along the filament but the majority of Z rings were abnormal. FtsA in this organism is therefore critical for the efficient formation of functional Z rings. This is the first report of abnormal Z-ring formation resulting from the loss of a single septation protein. These results suggest that in this organism, and perhaps others, FtsA ensures recruitment of the membrane-bound division proteins by ensuring correct formation of the Z ring.     

FtsA is localized to the septum in an FtsZ-dependent manner.

The localization of the cell division protein FtsA in E. coli was examined. FtsA was found to localize to the septum in a ring pattern as previously shown for FtsZ. The localization of FtsA was completely dependent on the localization of FtsZ. Under a variety of conditions that prevented formation of the Z ring, FtsA was unable to localize. In mutants where FtsZ forms structures in addition to Z rings, the pattern of FtsA duplicated these structures. These results suggest that the Z ring recruits FtsA to the septum.     

Influence of polymolecular events on inactivation behavior of xylose isomerase from Thermotoga neapolitana 5068

The inactivation behavior of the xylose isomerase from Thermotoga neapolitana (TN5068 XI) was examined for both the soluble and immobilized enzyme. Polymolecular events were involved in the deactivation of the soluble enzyme. Inactivation was biphasic at 95 degrees C, pH 7.0 and 7.9, the second phase was concentration-dependent. The enzyme was most stable at low enzyme concentrations, however, the second phase of inactivation was 3- to 30-fold slower than the initial phase. Both phases of inactivation were more rapid at pH 7.9, relative to 7.0. Differential scanning calorimetry of the TN5068 XI revealed two distinct thermal transitions at 99 degrees and 109 degrees C. The relative magnitude of the second transition was dramatically reduced at pH 7.9 relative to pH 7.0. Approximately 24% and 11% activity were recoverable after the first transition at pH 7.0 and 7.9, respectively. When the TN5068 XI was immobilized by covalent attachment to glass beads, inactivation was monophasic with a rate corresponding to the initial phase of inactivation for the soluble enzyme. The immobilized enzyme inactivation rate corresponded closely to the rate of ammonia release, presumably from deamidation of labile asparagine and/or glutamine residues. A second, slower inactivation phase suggests the presence of an unfolding intermediate, which was not observed for the immobilized enzyme. The concentration dependence of the second phase of inactivation suggests that polymolecular events were involved. Formation of a reversible polymolecular aggregate capable of protecting the soluble enzyme from irreversible deactivation appears to be responsible for the second phase of inactivation seen for the soluble enzyme. Whether this characteristic is common to other hyperthermophilic enzymes remains to be seen.     

Dimerization or oligomerization of the actin-like FtsA protein enhances the integrity of the cytokinetic Z ring

In bacteria, the actin-like FtsA protein interacts with the tubulin-like FtsZ protein, helping to assemble the cytokinetic Z ring, anchor it to the cytoplasmic membrane and recruit other essential divisome proteins. FtsA also interacts with itself, but it is not clear whether this self-interaction is required for its full functionality. Here we describe new dominant negative missense mutations in Escherichia coli ftsA that specifically inhibit FtsA homodimerization and simultaneously cause disruption of Z rings. The negative effects of one mutation, M71A, were suppressed by altering levels of certain division proteins or by additional mutations in ftsA that promote increased integrity of the Z ring. Remarkably, when FtsA, FtsA-M71A, and other mutants of FtsA that compromise self-interaction were connected in a tandem repeat, they were at least partially functional and suppressed defects of an ftsZ84(ts) mutation. This gain of function by FtsA tandems further suggested that FtsA monomers cause deleterious interactions with FtsZ and that increased dimerization or oligomerization of FtsA enhances its ability to promote Z-ring integrity. Therefore, we propose that FtsZ assembly is regulated by the extent of FtsA oligomerization.