A new cell division operon in Escherichia coli

Summary At 76 min on theE. coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division. A combination ofin-vivo andin-vitro genetic analysis of cell division mutants suggests that the cell division genefts E is the second gene in a 3 gene operon. A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division genefts X. Another cell division gene,fts S, is very close to, but distinct from, the 3 genes of the operon. Thefts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport. Both thefts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast. This common domain is indicative of a nucleotide-binding site.     

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (M_i) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only (M_i) but also D varied with growth rate at generation times () between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in , by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.Abbreviations OD₄₅₀ and OD₆₃₀ Optical density at a given wavelength in nm Dedicated to Dr. John Ingraham to honor him for his many contributions to Science     

The DD-carboxypeptidase activity encoded by pbp4B is not essential for the cell growth of Escherichia coli

The gene (pbp4B) encoding a putative DD-carboxypeptidase has been deleted in Escherichia coli and it is shown to be not essential for cell division. Disruption of the gene in a genetic background where all putative activities of DD-carboxypeptidases and/or DD-endopeptidases had been eliminated indicates that these activities are not required for cell growth in enterobacteria. The penicillin-binding capacity and a low DD-carboxypeptidase activity of PBP4B are demonstrated. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Localised mutagenesis of the fts YEX operon conditionally lethal missense substitutions in the FtsE cell division protein of Escherichia coli are similar to those found in the cystic fibrosis transmembrane conductance regulator protein (CFTR) of human patients

Summary After localised mutagenesis of the 76 min region of the Escherichia coli chromosome, we isolated a number of conditionally lethal mutants. Some of these mutants had a filamentation temperature sensitive (fts) phenotype and were assigned to the cell division genes ftsE of ftsX whereas others were defective in the heat shock regulator gene rpoH. Both missense and amber mutant alleles of these genes were produced. The missense mutant ftsE alleles were cloned and sequenced to determine whether or not the respective mutations mapped to the region of the gene encoding the putative nucleotide binding site. Surprisingly, most of these mutant FtsE proteins had missense substitutions in a different domain of the protein. This region of the FtsE protein is highly conserved in a large family of proteins involved in diverse transport processes in all living cells, from bacteria to man. One of the proteins in this large family of homologues is the human cystic fibrosis transmembrane conductance regulator (CFTR), and the FtsE substitutions were found to be in very closely linked, or identical, amino acid residues to those which are frequently altered in the CFTR of human patients. These results confirm the structural importance of this highly conserved region of FtsE and CFTR and add weight to the current structural model for the human protein.     

On the precision and accuracy achieved by Escherichia coli cells at fission about their middle

Length and width of each of the prospective siblings of constricted Escherichia coli cells from different strains and culture conditions were measured from electron micrographs. The data were statistically analyzed to investigate how equally the length and volume of one cell was divided into two. The analysis showed that, for all cultures, bipartition is unbiased or very nearly so, i.e. sibling cells were on the average equally long and large. The precision of bipartition attained by the cells was usually high; it was related to the average cell shape (length/width): slender E. coli cells divided into two less precisely than squat cells. Absolute size, growth rate and strain specificity affected the precision of bipartition only indirectly, i.e. in as much as they influenced cell shape.     

The Escherichia coli heat shock regulatory gene is immediately downstream of a cell division operon: the fam mutation is allelic with rpoH

Summary Several mutations which affect critical cell functions in Escherichia coli map at 76 min on the chromosome. The genes which map in this region are the cell division genes ftsY, E, X and S, the heat shock regulatory gene rpoH/htpR/hin, the lipoprotein biogenesis gene fam and another essential gene dnaM. We determined the relative positions of most of these genes and show that the rpoH gene lies immediately downstream of the last gene (ftsX) of a cell division operon and is transcribed in the same direction. We also show that the fam-715 mutation is allelic with rpoH and so the conditional lipoprotein deficiency of the fam mutation must be due to the pleiotropic nature of the heat shock response.     

Filamentous growth of Escherichia coli K12 elicited by dimeric,mixed-valence complexes of ruthenium

Dimeric, mixed-valence [(Ru(II), Ru(III)] compounds of ruthenium caused filament formation in growing cultures of Escherichia coli K12. Three compounds with the general formula Ru₂(NH₃)₆X₅ · H₂O (where X is a halide) were tested; in order of decreasing effectiveness (and with the concentration giving maximum effect), these were the bromo (10^-5M), chloro (10^-4 to 10^-5M), and iodo (10^-3 to 10^-4M) analogues. Filamentation elicited by the bromo and chloro compounds was spontaneously reversible after 3–4 h, and tentatively attributed to oxidation of the active mixed-valence form to inactive Ru(III) complexes. Several compounds known to accelerate division of filaments formed under other conditions were ineffective in reversing the filamentation, but the presence of 0,43 M-dimethylsulphoxide totally inhibited filamentation caused by the bromo or chloro compounds and by cis-Pt(NH₃)₂Cl₂ (cisplatin), an established filamenting and antitumour agent. The ruthenium complexes bound to mammalian DNA, but were without effect on the UV spectrum or cellular content of DNA in E. coli, despite showing marked mutagenic activity in reverse mutation tests with Salmonella typhimurium. Cells remained sensitive to inhibition of division by the ruthenium complexes until immediately prior to the division event. Possibilities for the (probably complex) mode of action and the potential of related compounds for therapeutic use are discussed.Non-standard abbreviation DMSO dimethylsulphoxide     

Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis

Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.     

Relationship between size of parent at cell division and relative size of its progeny in Escherichia coli

This article examines the empirical basis for the assumption of independence between the relative size (length or surface area) of a newborn cell w and the absolute size of its mother at cell division. Random samples from two strains of Escherichia coli B/r cells in steady-state exponential growth, covering a range of doubling times, were fixed in osmium tetroxide and prepared for electron microscopy by agar filtration. Length and diameter of over 3000 constricted cells were measured from the electron micrographs and cell surface area computed by assuming an idealized geometry of right circular cylinders with hemispherical polar caps. In general, these strains were found to divide into two daughter cells with a precision that is independent of the size of the mother. In addition, both a normal and a symmetrical beta-distribution were shown to fit the observed size distributions of w rather well; theoretical grounds for preferring the latter are discussed.     

Synergistic action of rapid chilling and nisin on the inactivation of Escherichia coli

The effect of rapid and slow chilling on survival and nisin sensitivity was investigated in Escherichia coli. Membrane permeabilization induced by cold shock was assessed by uptake of the fluorescent dye 1-N-phenylnapthylamine. Slow chilling (2°C min⁻¹) did not induce transient susceptibility to nisin. Combining rapid chilling (2,000°C min⁻¹) and nisin causes a dose-dependent reduction in the population of cells in both exponential and stationary growth phases. A reduction of 6 log of exponentially growing cells was achieved with rapid chilling in the presence of 100 IU ml⁻¹ nisin. Cells were more sensitive if nisin was present during stress. Nevertheless, addition of nisin to cell suspension after the rapid chilling produced up to 5 log of cell inactivation for exponentially growing cells and 1 log for stationary growing cells. This suggests that the rapid chilling strongly damaged the cell membrane by disrupting the outer membrane barrier, allowing the sensitization of E. coli to nisin post-rapid chilling. Measurements of membrane permeabilization showed a good correlation between the membrane alteration and nisin sensitivity. Application involving the simultaneous treatment with nisin and rapid cold shock could thus be of value in controlling Gram negatives, enhancing microbiological safety and stability.     

Effects of temperature on the size and shape of Escherichia coli cells

Two substrains of Escherichia coli B/r were grown to steady-state in batch cultures at temperatures between 22 and 42° C in different growth media. The size and shape of the cells were measured from light and electron micrographs and with the Coulter channelizer. The results indicate that cells are shorter and somewhat thicker at the lower temperatures, especially in rich growth media; cell volume is then slightly smaller. A lower temperature was further found to increase the relative duration of the constriction period. The shapes of the cell size distributions are indistinguishable, indicating that the pattern of growth of the cells is the same at all temperatures. The adaptation of the cells to a temperature shift lasted several generations, indicating that the morphological effects of temperature are mediated by the cell's physiology.     

Two streptokinase genes are expressed with different solubility in Escherichia coli W3110

The streptokinase (SK) gene from S. equisimilis H46A (ATCC 12449) was cloned in E. coli W3110 under the control of the tryptophan promoter. The recombinant SK, which represented 15% of total cell protein content, was found in the soluble fraction of disrupted cells. The solubility of this SK notably differed from that of the product of the SK gene from S. equisimilis (ATCC 9542) which had been cloned in E. coli W3110 by using similar expression vector and cell growth conditions, and occurred in the form of inclusion bodies.     

Pole cap formation in Escherichia coli following induction of the maltose-binding protein

After induction with maltose, 30–40% of the total protein in the osmotic shock fluid consist of maltose-binding protein while the induction ratio (maltose versus glycerol grown cells) for the amount of binding protein synthesized as well as for maltose transport is in the order of 10. Induction of maltose transport does not occur during all times of the cell cycle, but only shortly before cell division. Electronmicroscopic analysis of cells grown logarithmically on glycerol or maltose revealed in the latter the formation of large pole caps. These pole caps arise from an enlargement of the periplasmic space. Small cells contain one pole cap, large cells contain two. Pulse label studies with strain BUG-6, a mutant that is temperature sensitive for cell division reveal the following: Growth at the non-permissive temperature prevents maltose-binding protein synthesis and formation of new transport capacity.After shifting to the permissive temperature the cells regain both functions. Simultaneously, the newly formed cells exhibit pole caps.We conclude that the induction of maltose-binding protein is responsible for the formation of pole caps. In addition, beside the presence of inducer, cell cycle events occuring during division are necessary for the synthesis of maltose-binding protein.Non Standard Abbreviations GLPT periplasmic protein, related to transport of glycerolphosphate in Escherichia coli (Silhavy et al., 1976b)     

Lipopolysaccharide of Escherichia coli, polyamines, and acetic acid stimulate cell proliferation in intestinal epithelial cells

Summary Our aim was to examine whether lipopolysaccharide of Escherichia coli, polyamines of dietetic and/or bacterial origin, and products of the bacterial metabolism influence cell proliferation in epithelial cells from the colon and small intestine. Lipopolysaccharide of Escherichia coli 0111:B4 was incubated with cultures from human colonic mucosa. The mitoses were arrested with Vincristine and the total number of metaphases per crypt was counted. In addition, lipopolysaccharide was incubated with a human colonic epithelial cell line from adenocarcinoma (LS-123 cells) and with a nontransformed small intestinal cell line from germ-free rats (IEC-6 cells) for 24 h. In the last 4 h, the cells were labeled with tritiated thymidine. The cells were incubated with putrescine, cadaverine, and spermidine at 10⁻¹¹–10⁻³ M and with acetic acid (10⁻⁵–10⁻¹ M), acetaldehyde (10⁻¹⁰–10⁻⁴ M) and ammonium chloride (1–20 mM). Lipopolysaccharide of Escherichia coli increased the number of arrested metaphases in human colonic crypts and DNA synthesis in L-123 and IEC-6 cells (P<0.001). All polyamines increased DNA synthesis in the colonic and small intestinal cell lines, the effects being more marked for putrescine (P<0.001). The higher concentrations of acetic acid increased DNA synthesis in both epithelial cell lines (P<0.001). Acetaldehyde slightly decreased DNA synthesis in LS-123 cells at cytotoxic concentrations. Ammonium chloride did not significantly affect DNA synthesis. The final concentration of nonionized ammonia was less than 3%. It is concluded that lipopolysaccharides of Escherichia coli and intraluminal factors derived from microorganisms increase cell proliferation in human colonic crypts and intestinal epithelial cell lines.     

Glycine betaine reverses the effects of osmotic stress on DNA replication and cellular division in Escherichia coli

The accumulation of glycine betaine to a high internal concentration by Escherichia coli cells in high osmolarity medium restores, within 1 h, a subnormal growth rate. The experimental results support the view that cell adaptation to high osmolarity involves a decrease in the initiation frequency of DNA replication via a stringent response; in contrast, glycine betaine transport and accumulation could suppress the stringent response within 1–2 min and restore a higher initiation frequency. High osmolarity also triggers the cells to lengthen, perhaps via an inhibition of cellular division; glycine betaine also reverses this process. It is inferred that turgor could control DNA replication and cell division in two separate ways. Glycine betaine action is not mediated by K⁺ ions as the internal level of K⁺ ions is not modified significantly following glycine betaine accumulation.     

Using a kinetic model that considers cell segregation to optimize hEGF expression in fed-batch cultures of recombinant Escherichia coli

Growth inhibition of recombinant Escherichia coli during the expression of human epidermal growth factor was observed. The recombinant cells could be segregated into three populations based on their cell division and plasmid maintenance abilities: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable-but-non-culturable (VBNC) cells. Fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and foreign protein production. The results showed that a low concentration of inducer caused weak induction, whereas high levels cause strong induction, resulting in cells segregating into VBNC bacteria and producing a low foreign protein yield. A kinetic model for cell segregation was proposed and its predictions correlated well with experimental data for cell growth and protein expression. The optimal induction strategy could then be predicted by the model, and this prediction was then verified by experimentally deriving the conditions necessary for maximum expression of recombinant protein.     

Elongation and cell division in Bdellovibrio bacteriovorus

Elongation and division of Bdellovibrio bacteriovorus were studied in axenic synchronous cultures. The cells elongate unidirectionally from one end attaining a length of several unit cells, and then divide into the corresponding number of cells. The length of the filament and, consequently, the progeny number, vary within the range of two to several dozen cells, according to the conditions used. A protein and a low molecular weight component are required for normal division.     

Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli

2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli. The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR). The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations. Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.     

Resistance to trifluoroperazine, a calmodulin inhibitor, maps to the fabD locus in Escherichia coli

A mutant, tfpA1, resistant to the calmodulin inhibitor trifluoroperazine (TFP) at 30°C, was isolated in Escherichia coli. The mutant showed a reduced growth rate at 30°C and was temperature sensitive (ts) at 42°C for growth, forming short filaments. The mutation was mapped to the 24 min region of the chromosome and the gene was cloned by complementation of the is defect. Subsequent subcloning, complementation analysis, marker rescue mapping and sequencing, identified tfpA as fabD, encoding the 35 kDa, malonyl-coenzyme A transacylase (MCT) enzyme, required for the initial step in the elongation cycle for fatty acid biosynthesis. Resistance to TFP may result from altered permeability of the cell envelope, although the mutant remained sensitive to other calmodulin inhibitors and to other antibacterial agents. Alternatively, resistance may be more indirect, resulting from alterations in intracellular Ca⁺⁺ levels which affect the activity of the TFP target in some way.     

Expression, purification, and characterization of Leishmania donovani trypanothione reductase in Escherichia coli

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are K(m), 50 microM; k(cat), 18,181 min(-1); and k(cat)/K(m), 6.06x10(6) M(-1) s(-1). The yield of recombinant LdTR was approximately 16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.