Inhibition of an early event in the cell division cycle of Escherichia coli by FL1060, an amidinopenicillanic acid.

Analysis of exponential and synchronous cultures of Escherichia coli B/r after the addition of FL1060 indicates a block point for division by this agent some 15 to 20 min before the end of the preceding cell division cycle, a time corresponding to the beginning of the C period of the cell division cycle. Morphological examination of FL1060-treated synchronous cultures of E. coli /r was consistent with inhibition by FL1060 of a very early event in the cell division cycle. This event appears to be essential for normal cell surface elongation in a rod configuration. Temporary treatment of synchronous cultures of E. coli B/r with FL1060 resulted in division delay, the extent of which was a function of the duration of exposure to FL1060. However, even after relatively long times of FL1060 treatment the delayed divisions were still synchronous. Although FL1060 had no direct effect on deoxyribonucleic acid (DNA) synthesis, the synchronous delayed division occuring after temporary treatment with FL1060 were accompanied by a delay in the attainment of resistance of cell division to inhibitors of DNA, ribonucleic acid, and protein synthesis. These results suggest aht an FL1060-sensitive event initiates at the beginning of the C period of the cell division cycle of E. coli and is responsible for normal cell elongation. This cell elongation pathway procedes independently of DNA synthesis, but there is an interaction between this pathway and termination of a round of DNA replication in which a normal rod configuration is necessary to allow a signal for cell division to be generated upon completion of DNA replication.     

Electron Microscope Study of Septum Formation in Escherichia coli Strains B and B/r During Synchronous Growth

The formation of cell wall septa was monitored in Escherichia coli B and B/r during synchronous growth in glucose media at 37 C by means of electron microscopy. The visible events of septation comprised the following sequence, starting at about 30 min of incubation: (a) bleb formation of the outer membrane; (b) invagination of mucopeptide and cytoplasmic membrane (with associated mesosomes); the outer membrane is excluded from the septum; (c) formation of a cross-wall; (d) ingrowth of the outer membrane during cell separation. The septum is composed of a fold of cytoplasmic membrane plus mucopeptide, and the latter is a double structure, composed of two opposed lamellae separated by an electron-transparent gap. Experiments with chloramphenicol and nalidixic acid suggested that division could occur in the presence of these inhibitors once a round of deoxyribonucleic acid replication is completed. The initial stages of septation, as estimated by the potential of the cells to produce bulges in the presence of ampicillin, may involve the modification of mucopeptide by hydrolases at the end of the C period. Assembly of the septum may occur during the first half of the D period by means of precursors synthesized during the preceding C period.     

Effect of Nalidixic Acid and Hydroxyurea on Division Ability of Escherichia coli fil+ and lon− Strains

Short periods of incubation in medium containing nalidixic acid or hydroxyurea, followed by a return to normal growth conditions, induced filament formation in Escherichia coli B (fil(+)) and AB1899NM (lon(-)) but not in B/r (fil(-)) and AB1157 (lon(+)). These drugs reversibly stopped deoxyribonucleic acid (DNA) synthesis with little or no effect on ribonucleic acid (RNA) synthesis or mass increase. The initial imbalance caused by incubation in these drugs was the same for B and B/r as was macromolecular synthesis following a return to normal growth conditions. DNA degradation caused by nalidixic acid was measured and found to be the same for B and B/r. Hydroxyurea caused no DNA degradation in these two strains. Survival curves as determined under various conditions by colony formation suggested that the property of filament formation was responsible for the extrasensitivity of fil(+) and lon(-) strains to either nalidixic acid or hydroxyurea. E. coli B was more sensitive to either drug than was B/r or B(s-1). Pantoyl lactone or liquid holding treatment aided division and colony formation of nalidixic acid-treated B but had no effect on B/r. Likewise, the filament-former AB1899NM was more sensitive to nalidixic acid than was the non-filament-former AB1157. The sensitivity of B/r and B(s-1) to nalidixic acid was nearly the same except at longer times in nalidixic acid, when B(s-1) appeared more resistant. Even though nalidixic acid, hydroxyurea, and ultraviolet light may produce quite different molecular alterations in E. coli, they all cause a metabolic imbalance resulting in a lowered ratio of DNA to RNA and protein. We propose that it is this imbalance per se rather than any specific primary chemical or photochemical alterations which leads to filament formation by some genetically susceptible bacterial strains such as lon(-) and fil(+).     

Mechanism of action of nalidixic acid on Escherichia coli. 3. Conditions required for lethality

Deitz, William H. (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), Thomas M. Cook, and William A. Goss. Mechanism of action of nalidixic acid on Escherichia coli. III. Conditions required for lethality. J. Bacteriol. 91:768-773. 1966.-Nalidixic acid selectively inhibited deoxyribonucleic acid (DNA) synthesis in cultures of Escherichia coli 15TAU. Protein and ribonucleic acid synthesis were shown to be a prerequisite for the bactericidal action of the drug. This action can be prevented by means of inhibitors at bacteriostatic concentrations. Both chloramphenicol, which inhibits protein synthesis, and dinitrophenol, which uncouples oxidative phosphorylation, effectively prevented the bactericidal action of nalidixic acid on E. coli. The lethal action of nalidixic acid also was controlled by transfer of treated cells to drug-free medium. DNA synthesis resumed immediately upon removal of the drug and was halted immediately by retreatment. These studies indicate that nalidixic acid acts directly on the replication of DNA rather than on the "initiator" of DNA synthesis. The entry of nalidixic acid into cells of E. coli was not dependent upon protein synthesis. Even in the presence of an inhibiting concentration of chloramphenicol, nalidixic acid prevented DNA synthesis by E. coli 15TAU.     

Bactericidal action of nalidixic acid on Bacillus subtilis

Cook, Thomas M. (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), Karen G. Brown, James V. Boyle, and William A. Goss. Bactericidal action of nalidixic acid on Bacillus subtilis. J. Bacteriol. 92:1510-1514. 1966.-Nalidixic acid at moderate concentrations exerts a bactericidal action upon the gram-positive bacterium Bacillus subtilis. The synthesis of deoxyribonucleic acid (DNA) in B. subtilis is selectively inhibited by nalidixic acid at concentrations approximating the minimal growth inhibitory concentration. Higher concentrations (25 mug/ml) result in a 30 to 35% degradation of DNA. After extended exposure to nalidixic acid, protein synthesis is also depressed. Cells of B. subtilis treated with nalidixic acid exhibit characteristic morphological abnormalities including cell elongation and development of gram-negative areas. From the results presented, it can be concluded that the mode of action of nalidixic acid upon susceptible bacteria is similar for both gram-positive and gram-negative species.     

Properties of a thermosensitive asporogenous filamentous mutant of Bacillus megaterium

Mutant TH14 of Bacillus megaterium ATCC 19213 is thermosensitive and defective in cell-division septation and spore formation at the restrictive temperature (39 C). As a consequence, the mutant forms multinucleate aseptate filaments and is asporogenic. The mutation does not result in any qualitative compositional changes in extractable membrane proteins. At the restrictive temperature, the mutant membrane has a reduced content of a small molecular weight protein(s). A membrane protein(s) with a molecular weight of nearly 80,000 appears to be partially derepressed in the mutant grown at the restrictive temperature. In addition, numerous unidentified spherical inclusions of fairly uniform size (diameter approximately 100 nm) are present in the cytoplasm at the restrictive temperature. They are especially concentrated at only one pole of each filament. Filamentous growth of the mutant is less sensitive to penicillin than growth in the rod form. Growth in either form is equally sensitive to d-cycloserine at the concentrations used for selection of the mutant. Temperature shift-up experiments suggest that one to two rounds of deoxyribonucleic acid (DNA) replication occur before the phenotypic expression of the mutation occurs. The septations after these replication events can be either two-division septations or a single-division septation plus a subsequent sporulation septation. This conclusion, coupled with previously reported work, supports the hypothesis that the early stages of sporulation represent a modified cell division.     

Plasmid Replication and the Induced Synthesis of Colicins E1 and E2 in Escherichia coli

Induction of colicins E1 and E2 in Escherichia coli occurs when plasmid synthesis has been inhibited either by nalidixic acid or by lack of deoxyribonucleic acid polymerase I. Moreover, colicin E1 and E2 synthesis induced by mitomycin C and exposure to chloramphenicol is not associated with a large increase in circular plasmid deoxyribonucleic acid. The mean plasmid content of cells in populations having a low spontaneous frequency of colicin-producing cells because of growth at low temperature or because of the presence of recA(-) or crp(-) alleles, is not significantly different to that in wild-type cells grown at 37 C.     

Effects of some platinum IV complexes on cell division of Escherichia coli.

A comparison was made of the effects of cis-tetrachlorodiaminoplatinum (IV) (cis-TCDPt), rans-TCDPt), and hexachloroplatinum (HCP) on growth and cell division of Escherichia coli strains D21 and D22. At or below 40 microgram/mL, cis-TCDPt inhibited cell division but not growth, DNA, or protein synthesis, although areas of increased electron density could be demonstrated in treated cells. In contrast, 40 microgram/mL of trans-TCDPt or HCP inhibited growth. Trans-TCDPt-treated cells developed condensed nucleoids; HCP-treated cells showed no obvious cytological changes to correlate with growth inhibition. Combination of cis-TCDPt with nalidixic acid, both at one-half the lowest filament-forming concentrations, resulted in formation of filaments, suggesting an additive effect. Combination of cis-TCDPt followed by ampicillin on E. coli B/r resulted in single bulges near the center of the filaments. Cis-TCDPt could therefore inhibit an initial step in the septation sequence, possibly at the level of the regulation of the hydrolytic enzymes. Whether cis-TCDPt exerts its effect by interreaction with DNA or with a membrane target is still uncertain.     

Membrane proteins correlated with expression of the polysialic acid capsule in Escherichia coli K1.

Growth of Escherichia coli K1 strains at 15 degrees C results in a defect in the synthesis or assembly of the K1 polysialic acid capsule. Synthesis is reactivated in cells grown at 15 degrees C after upshift to 37 degrees C, and activation requires protein synthesis (Whitfield et al., J. Bacteriol. 159:321-328, 1984). Using this temperature-induced defect, we determined the molecular weights and locations of membrane proteins correlated with the expression of K1 (polysialosyl) capsular antigen. Pulse-labeling experiments demonstrated the presence of 11 proteins whose synthesis was correlated with capsule appearance at the cell surface. Using the differential solubility of inner and outer membranes in the detergent Sarkosyl, we localized five of the proteins in the outer membrane and four in the inner membrane. The subcellular location of two of the proteins was not determined. Five proteins appeared in the membrane simultaneously with the initial expression of the K1 capsule at the cell surface. One of these proteins, a 40,000-dalton protein localized in the outer membrane, was identified as porin protein K, which previously has been shown to be present in the outer membrane of encapsulated E. coli. The possible role of these proteins in the synthesis of the polysialosyl capsule is discussed.     

Production of giant cells of Escherichia coli.

Giant cells, with volumes up to 500-fold those of normal cells, have been produced by both genetic and pharmacological means in Escherichia coli K-12. In the genetic approach, an envB or mon mutation (conferring rounded or irregular morphology) was combined with a lon mutation (block of septation after irradiation). UV irradiation and subsequent incubation for 2 to 5 h in a rich medium supplemented with 1% sodium chloride led t; production of polymorphic giant cells. In the pharmacological approach, incubation of several different strains of E. coli K-12 with the drug 6-amidinopenicillanic acid (FL1060) in the same rich medium gave rise to a homogeneous population of smoothly rounded giant cells.     

Mechanism of action of nalidixic acid on Escherichia coli. Vi. Cell-free studies

The effects of nalidixic acid in vitro on deoxyribonucleic acid (DNA)- polymerase (deoxyribonucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7), deoxyribonucleotide kinases (ATP: deoxymono- and diphosphate phosphotransferases), and deoxyribosyl transferase (nucleoside: purine deoxyribosyltransferase, EC 2.4.2.6) were examined employing partially purified and crude extracts of Escherichia coli ATCC 11229 and E. coli 15TAU. Nalidixic acid had no inhibitory effect on the DNA-polymerase of the wild-type strain E. coli ATCC 11229 at concentrations of 1.4 x 10(-3) to 2.8 x 10(-3)m. No inhibition of deoxyribonucleotide kinase activity was observed at concentrations of nalidixic acid ranging from 2 x 10(-3) to 8.6 x 10(-3)m. Nalidixic acid (0.43 x 10(-4) to 0.43 x 10(-3)m) had no inhibitory effect on the deoxyribosyl transferase activity of crude extracts obtained from E. coli ATCC 11229 or E. coli 15TAU. Analytical CsCl density gradient centrifugation demonstrated that the DNA obtained after treatment of E. coli 15TAU with nalidixic acid was not cross-linked. These results suggest that the prevention of DNA synthesis in vivo by nalidixic acid is not attributable to inhibition of DNA polymerase, deoxyribonucleotide kinase, deoxyribosyl transferase, or to cross-linking of the DNA of treated cells.     

Biosynthesis of the outer membrane receptor for vitamin B12, E colicins, and bacteriophage BF23 by Escherichia coli: kinetics of phenotypic expression after the introduction of bfe+ and bfe alleles.

The bfe locus codes for the cell surface receptor for vitamin B12, the E colicins, and bacteriophage BF23 in the Escherichia coli outer membrane. When the bfe+ allele, which is closely linked to the argH locus, was introduced into an argH bfe recipient by conjugation, arg+ recombinant cells rapidly and simultaneously acquired sensitivity to colicin E3 and phage BF23. In the reciprocal experiment introducing bfe into an argH bfe+ recipient, it was found that colicin E3-resistant, arg+ cells began to appear shortly after the arg+ recombinant population began to divide. This was far earlier than would have been predicted on the basis of 220 receptors per haploid cell. Moreover, there was a lag between the appearance of colicin resistance and the appearance of resistance to killing by phage BF23, and hence a period of time during which some arg+ recombinant cells were sensitive to the phage but resistant to the colicin. Colicin E3 added to cells during this period of time protected against phage killing, indicating that the colicin-resistant cells still had receptors capable of binding colicin on their surface. The modification of the phenotypic expression of colicin and phage resistance by inhibitors of deoxyribonucleic acid, ribonucleic acid, and protein synthesis was also investigated. The results obtained indicate that the receptor protein coded for by the bfe locus can exist on the cell surface in several different functional states.     

Gyrase inhibitors and thymine starvation disrupt the normal pattern of plasmid RK2 localization in Escherichia coli

Multicopy plasmids in Escherichia coli are not randomly distributed throughout the cell but exist as defined clusters that are localized at the mid-cell, or at the 1/4 and 3/4 cell length positions. To explore the factors that contribute to plasmid clustering and localization, E. coli cells carrying a plasmid RK2 derivative that can be tagged with a green fluorescent protein-LacI fusion protein were subjected to various conditions that interfere with plasmid superhelicity and/or DNA replication. The various treatments included thymine starvation and the addition of the gyrase inhibitors nalidixic acid and novobiocin. In each case, localization of plasmid clusters at the preferred positions was disrupted but the plasmids remained in clusters, suggesting that normal plasmid superhelicity and DNA synthesis in elongating cells are not required for the clustering of individual plasmid molecules. It was also observed that the inhibition of DNA replication by these treatments produced filaments in which the plasmid clusters were confined to one or two nucleoid bodies, which were located near the midline of the filament and were not evenly spaced throughout the filament, as is found in cells treated with cephalexin. Finally, the enhanced yellow fluorescent protein-RarA fusion protein was used to localize the replication complex in individual E. coli cells. Novobiocin and nalidixic acid treatment both resulted in rapid loss of RarA foci. Under these conditions the RK2 plasmid clusters were not disassembled, suggesting that a completely intact replication complex is not required for plasmid clustering.     

Inhibition of Escherichia coli Division by Protein X

We propose that protein X provides the connection between damage to Escherichia coli DNA and inhibition of septation and cell division. This connection is needed to guarantee that each new bacterium receives a complete DNA copy. We present several new experiments here which demonstrate that the degree to which septation is inhibited following damage to DNA is correlated with the amount of protein X that is produced. Rifampin selectively blocks protein X production. This drug was shown to allow cells whose DNA had been damaged by nalidixic acid to resume septation. Several mutants formed septa-less filaments and also produced protein X at 42 degrees C; rifampin both inhibited their production of protein X and permitted them to form septa and divide. Essentially complementary results were obtained with a dnaA mutant which at 42 degrees C stopped making DNA, did not produce protein X, and continued to divide; added bleomycin degraded DNA, induced protein X, and inhibited septation. These results, as well as previous observations, are all consistent with the proposal that protein X is produced as a consequence of DNA damage and is an inhibitor of septation. We suggest that septation could require binding of a single-stranded region of DNA to a septum site in the membrane. Protein X could block this binding by combining with the DNA. This control could provide an emergency mechanism in addition to the usually proposed coordination in which completion of DNA synthesis creates a positive effector for a terminal step of septation. Or it could be the sole coordinating mechanism, even under unperturbed growth conditions.     

Generation of buds, swellings, and branches instead of filaments after blocking the cell cycle of Rhizobium meliloti.

Inhibition of cell division in rod-shaped bacteria such as Escherichia coli and Bacillus subtilis results in elongation into long filaments many times the length of dividing cells. As a first step in characterizing the Rhizobium meliloti cell division machinery, we tested whether R. meliloti cells could also form long filaments after cell division was blocked. Unexpectedly, DNA-damaging agents, such as mitomycin C and nalidixic acid, caused only limited elongation. Instead, mitomycin C in particular induced a significant proportion of the cells to branch at the poles. Moreover, methods used to inhibit septation, such as FtsZ overproduction and cephalexin treatment, induced growing cells to swell, bud, or branch while increasing in mass, whereas filamentation was not observed. Overproduction of E. coli FtsZ in R. meliloti resulted in the same branched morphology, as did overproduction of R. meliloti FtsZ in Agrobacterium tumefaciens. These results suggest that in these normally rod-shaped species and perhaps others, branching and swelling are default pathways for increasing mass when cell division is blocked.     

The effect of nalidixic acid on the expression of some genes in Escherichia coli K-12.

The synthesis of maltodextrin phosphorylase and the phage λ receptor of Escherichia coli K-12 is substantially inhibited by the presence of 50 μg nalidixic acid/ml in the culture medium. β-galactosidase synthesis is inhibited to a lesser extent and no inhibition of L-tryptophanase synthesis is observed. The inhibition of enzyme synthesis is apparently not due to the effect of nalidixic acid on deoxyribonucleic acid synthesis.     

Studies on the inhibition of protein synthesis by selenodiglutathione.

Amino acid incorporation in a cell-free system derived from rat liver has previously been found to be inhibited by GSSeSG (selenodiglutathione). In the present experiments the effect of GSSeSG on protein synthesis in 3T3-f cells, on growth and protein synthesis in Escherichia coli, and on amino acid incorporation in a cell-free system derived from E. coli, was studied. GSSeSG inhibits the incorporation of [3H]leucine into protein by 3T3-f cells. This inhibition cannot be reversed by removing GSSeSG and is correlated with the uptake of GSSeSG. Sodium selenite (Na2SeO3) and oxidized glutathione had no inhibitory effect in this system. [3H]Uridine or [3H]thymidine incorporation into RNA or DNA was not inhibited, indicating that the primary action of GSSeSG was on protein synthesis. GSSeSG did not influence the growth of E. coli in a synthetic medium, although enhanced amino acid incorporation was observed. In the cell-free system derived from E. coli, amino acid incorporation was not changed by GSSeSG, indicating that elongation factor G, in contrast to elongation factor 2 of mammalian cell systems, is not blocked by GSSeSG.     

Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA

Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.