A comparison of DNA content between two substrains of Escherichia coli B/r.

The average DNA content per cell was measured in steady-state cultures of two substrains of $\text{E. coli}\text{B}\text{r}$ growing at various rates at 37°C. The DNA content of substrain $\text{B}\text{r}$ F was consistently lower than that of substrain $\text{B}\text{r}$ A. It is suggested that the differences in DNA contents are consequences of strain-specific differences in the relationship between chromosome replication and the division cycle of $\text{E. coli.}$     

Initiation of chromosome replication in Escherichia coli. I. Requirements for RNA and protein synthesis at different growth rates

The effects of inhibition of protein and RNA synthesis on initiation of chromosome replication in Escherichia coli$\text{B}\text{r}$ were determined by measuring rates of DNA synthesis during the division cycle before and after addition of chloramphenicol and rifampicin. The ability of cells to initiate a round of replication depended upon the pattern of chromosome replication during the division cycle. Initiation in the presence of chloramphenicol (200 μ/ml) and rifampicin (100 gmg/ml) was observed only in slowly growing cells which normally initiated a new round between the end of the previous round and the subsequent division (i.e. in the D period of the division cycle). The cells that initiated were in the D period at the time of addition of the drugs. Rapidly growing cells which normally initiated before the D period and slowly growing cells which normally initiated after the D period did not initiate in the presence of the drugs. The contrasting effects of the drugs in cells possessing different chromosome replication patterns, and the coupling between septum-crosswall formation (the D period) and initiation suggest that the timing of initiation of chromosome replication in E. coli is controlled by the cell envelope.     

Variation in periodic replication of the chromosome in Escherichia coli B/rTT.

A method using 5-bromouracil photolysis induction with 313 nm radiation was employed to estimate the variation in the period between successive rounds of DNA replication in rapidly growing cultures of Escherichia coli$\text{B}\text{rTT}$ The coefficient of variation of this period was 9.3%, which is significantly less than the corresponding value of about 20% reported for variation in the cell interdivision period. Thus chromosome replication is much more tightly controlled than is cell division. The reduced variability of the DNA replication cycle indicates that the period (D) between termination of a round of DNA replication and cell division and the following period ending in initiation of the next round of DNA replication (B) are riot independent of each other but tend to have compensatory variations. The results suggest that other events in the cell cycle are related more closely to DNA replication rather than to the much less regular event of cell division.     

The mechanism of integration of phage Mu in the chromosome of Escherichia coli.

Cultures of synchronized $\text{E. coli}$ were infected with phage Mu at various stages of the division cycle. Phage integration at a given locus on the chromosome was measured by the lost of the corresponding gene function. For several loci, maximal integration occured at gene locus during replication of that locus.     

Non-random segregation of DNA strands in Escherichia coli B-r

The segregation of DNA strands during growth of Escherichia coli$\text{B}\text{r}$ has been studied under conditions in which the chromosomal configuration and the ancestry of the cells during growth and division were known. Cells containing either one or two replicating chromosomes were pulse-labeled with [³H]thymidine, and the location of the radioactivity within chains of cells formed by growth in methylcellulose was determined by autoradiography. The locations of the radioactive cells within chains obtained after the second, third and fourth divisions were consistent with the co-segregation of only one of the replicating strands of each chromosome and a fixed region of the cell into daughter cells. The attachment of this strand to the region appeared to become permanent at the time the strand was used for the first time as a template. It is concluded that the segregation of DNA molecules into daughter cells is non-random in E. coli B/r.     

Evolution of the three overlapping gene systems in G4 and phi X174.

Bacteriophage G4 has the same $\text{A}\text{B}$ and $\text{D}\text{E}$ overlapping gene systems as φX174 and together with the A and $\text{C}\text{K}$ overlapping gene system (Shaw et al., 1978), 7 of the 11 G4 and φX174 genes are involved in overlaps. The nucleotide differences between G4 and φX174 in the overlapping portions of the A, C and D genes are 23%, 27% and 21%, respectively, compared with 32%, 36% and 34% in the non-overlapping portions of the same genes. The amino acid differences between the G4 and φX174 overlapping B, K and E proteins, are 44%, 39% and 44%, respectively, compared with 28%, 26% and 16% in the regions of genes A, A and C, and D which contain genes B, K and E. These results suggest that the nucleotide sequences of overlapping genes evolve at almost the same rate as in non-overlapping genes, and that this is made possible by a lower amino acid sequence stringency of one of the pairs of proteins. The overlapping $\text{D}\text{E}$ and A and $\text{C}\text{K}$ gene systems may have originated by taking advantage of a high incidence of T nucleotides in the second codon position to produce a hydrophobic protein and the $\text{A}\text{B}$ gene system may have evolved by read-through of the A gene into the B gene. From the nucleotide sequences, other overlapping genes appear to be possible in these bacteriophages.     

Genetic and enzymatic characterization of a conditional lethal mutant of Escherichia coli K12 with a temperature-sensitive DNA ligase

$\text{TAU}$ts7 an Escherichia coli 15 strain with a thermolabile DNA ligase, has previously been shown to be a temperature-sensitive conditional lethal mutant that is sensitive to methyl methane sulfonate and to ultraviolet irradiation; it also accumulates 10 S DNA fragments to an abnormal extent. When the ligase mutation is transferred to a wild-type E. coli K12 strain, the strain becomes temperature sensitive for growth and displays the same characteristics as $\text{TAU}$ts7. These findings show that a functional DNA ligase is essential for normal DNA replication and repair in E. coli.     

Selection and timing of replication of plasmids R1drd-19 and F'lac in Escherichia coli.

The selection and timing of plasmid replication was studied in exponentially growing cultures of Escherichia coli K-12 carrying the plasmid R1drd-19 and E. coli strains B/r A and B/r F carrying the plasmid F′lac. In all cases plasmid replication was studied by analysis of covalently closed circular (CCC) DNA. The turnover time of replicating plasmid DNA into CCC-DNA was found to be less than 4 min. Density shift experiments (from ¹⁵NH₄⁺, D₂O to ¹⁴NH₄⁺, H₂O) showed that plasmids R1drd-19 and F′lac are selected randomly for replication. This means that one of the plasmid copies in a cell is selected and replicated. There is no further plasmid replication in the cell until all plasmid copies, including the newly formed ones, have the same probability of being selected for replication. The early kinetics of the appearance of light plasmid DNA after the density shift showed that the time interval between successive replications of plasmids R1drd-19 and F′lac is $\text{τ}\text{n}$, where τ is the generation time and n is the average number of plasmid replications per cell and cell cycle. In a second type of experiment, exponentially growing cells were separated into a series of size classes by low-speed centrifugation in sucrose step gradients. Replication of plasmids R1drd-19 and F′lac was equally frequent in all size classes. This result is in accordance with the results of the density shift experiment. It can therefore be concluded that replication of plasmids R1drd-19 and F′lac is evenly spread over the whole cell cycle, which means that one plasmid replication occurs every time the cell volume has increased by one initiation mass.     

Dynamic aspects of membrane-bound DNA in Bacillus subtilis during the course of synchronous growth

Changes in the marker frequencies of membrane-bound DNA (M-DNA) were studied with special respect to the division cycle of $\text{B}\text{.}\text{subtilis}$ W23 cells. M-DNA was obtained by a sucrose density gradient centrifugation after a mild shearing of Brij-58 lysate from synchronized cells. It was found that the markers located in the replication point appeared in the M-DNA fraction successively in the order of the map position during the synchronous growth. The result suggests that the replication of DNA proceeds in the membrane-bound state during the whole course of the division cycle.     

Interaction between the oxidative phosphorylation genes of Escherichia coli k12 and the nitrogen fixation gene cluster of Klebsiella pneumoniae.

Hybrids were constructed between $\text{E. coli}$ K12 $\text{unc}$^? mutants uncoupled in oxidative phosphorylation, and thus defective in ATP biosynthesis, and an F′ plasmid carrying nitrogen fixation genes from $\text{Klebsiella pneumoniae}$. Examination of these hybrids showed that expression of $\text{nif}$⁺_Kp genes in $\text{E. coli}$ K12 does not require coupling of oxidative phosphorylation but needs the contribution of an anaerobic electron transport system involving fumarate reduction. The $\text{nif}$_Kp cluster of genes does not contain functions able to complement a defective Mg²⁺-ATPase aggregate but does contain a function(s) which appears to interact with the $\text{unc}$B^? mutant over the formation of a redox system.     

The origin of the carbon chain in the thiazole moiety of thiamine in Escherichia coli: incorporation of deuterated 1-deoxy-D-threo-2-pentulose

Non growing washed cells of Escherichia coli, derepressed for the biosynthesis of thiamine, have been incubated in the presence of glucose and either 1-deoxy-$\text{D}$-threo-2-pentulose $\text{1}$ or 1-déoxy-$\text{D}$-erythro-2-pentulose $\text{2}$ trideuterated on the methyl group. The incorporation of deuterium into the thiazole moiety of thiamine was measured by mass spectrometry. The label of the threo-compound was found in more than 40% of the thiazole biosynthesized in its presence; the label of the erythro-compound in less than 5%. Hence it is likely that the carbon chain of 1-deoxy-$\text{D}$-threo-2-pentulose is the precursor of the five carbons chain of the thiazole moiety of the thiamine molecule in E. coli.     

Genetic regulation of a constitutive operon in E. coli B-r

Complementation analysis, using a regulatory mutant in the constitutive d-ribose operon of $\text{E. coli}\text{B}\text{r}$, have shown that the genetic regulation of constitutive operon may follow a truly positive control mechanism whereby the expression of the operon requires an active constitutive initiation protein to allow the synthesis of the structural genes products.     

Cycle-specific replication of chromosomal and F-plasmid origins

There have been various proposals for the pattern of F-plasmid replication during the division cycle. Here we show that the recent studies of Gordon et al. (Cell 90, 1113–1121, 1997) on the duplication and segregation of green fluorescent protein (GFP) labeled replication origins of the Escherichia coli chromosome and the F plasmid during the division cycle support the proposal that the F plasmid replicates with a cell-cycle-specific (artiocyclic) pattern.     

Macromolecular composition of bacteria

Equations are presented that describe the macromolecular composition in exponential bacterial cultures as functions of five parameters: doubling time of the culture (τ), protein per origin of replication (P₀), chromosome replication time (C-period), peptide chain elongation rate (c_p), and the time between termination of replication and cell division (D-period). Implicit in the value for some of these parameters is a specific macromolecular control system: the control of the growth rate (τ), the timing of initiation of rounds of chromosome replication (P₀), and the regulation of cell division (D). The utility of these relations is illustrated by using updated measurements of the macromolecular composition of E. coli B/r to calculate values for the fundamental parameters and to predict the composition of a mutant which has a defect in the control of DNA replication. Furthermore, the meaning of several often-cited physiological parameters (RNA/protein, RNA/cell and RNA/genome) is examined. The relations presented here show that these parameters and their variation with growth rate are not directly relevant to arguments about control of ribosome synthesis or culture growth.     

Chloramphenicol damages bacterial DNA.

L(+)-$\text{threo}$-chloramphenicol induces reversion of His^?$\text{Salmonella typhimurium}$ strains TA100 and TA1535 in the conventional Ames' assay without microsomal activation. Any mutagenicity of D(?)-$\text{threo}$-chloramphenicol was masked by toxicity. Similarly, a sensitive fluctuation test showed mutagenesis with L(+)-$\text{threo}$-chloramphenicol at concentrations of 0.5 μM and above but the D(?) isomer proved to be toxic even at these low levels. The L(+) isomer caused single strand breaks in the DNA of $\text{Escherichia coli}\text{B}\text{r}$ and $\text{Salmonella typhimurium}$ strains TA1535, TA100 and TA1976. The D(?) isomer caused breaks in $\text{Escherichia coli}\text{B}\text{r}$ and $\text{Salmonella typhimurium}$ TA1976 although it was less effective and it did not produce DNA breaks in TA1535 or TA100.     

Association of R6K plasmid replicative intermediates with the folded chromosome of Escherichiacoli

When $\text{Escherichia}\text{coli}$ strain CSH50(R6K) is lysed so as to preserve the folded chromosome structure approximately 9 of the 11 R6K molecules maintained per chromosomal equivalent cosediment with the host nucleoid on a neutral sucrose gradient; the remaining 2 plasmids sediment at their normal rate. When cells are briefly labeled with [³H]thymidine, the majority of plasmid replicative intermediates and nascent mature plasmids are found in the plasmid subpopulation that cosediments with host folded chromosomes. This finding suggests that plasmid replication occurs in a restricted cellular locus, perhaps even while in association with its host's folded chromosome.     

Synthesis of diphtheria toxin in E. coli cell-free lysate

An $\text{E. coli}$ cell-free lysate was used to translate $\text{C. diphtheriae}$ RNA from nontoxinogenic C₇(?), C₇ infected with β tox⁺ corynebacteriophage, and $\text{C. diphtheriae}$ strain PW8. De novo synthesis of toxin was detected by immune precipitation with antitoxin, ADP-ribosylation of mammalian elongation factor 2 and rabbit skin test. The results indicated that toxin is produced in the $\text{E. coli}$ protein synthesizing system primed with RNA from cells infected with tox⁺ bacteriophage and is absent in systems primed with RNA from C₇(?) cells.     

Estimation of the D period from residual division after exposure of exponential phase bacteria to chloramphenicol

Summary Values of the D period, between termination of chromosome replication and cell division, were determined from measurements of residual cell division after exposure of exponential phase cultures of Escherichia coli B/r and K12 and of Salmonella typhimurium to chloramphenicol. The results obtained by this method were compared with earlier results for E. coli B/r obtained from measurements of DNA content per cell and were found to be almost identical. For each, values of the D period were independent of growth rate, and the average value of D=26.1±1.2 min obtained by residual division is in good agreement with the value of 25 min obtained earlier. These results indicate that the method of residual division provides a good measure of the duration of the D period. Values of D were also independent of growth rate for each of the other strains.This work supported by the U.S. Atomic Energy Commission.     

Adenylate cyclase and cyclic AMP through the cell cycle of Tetrahymena.

Adenylate cyclase activity and 3′, 5′ cyclic adenosinemonophosphate (cAMP) have been followed through the heat-synchronized cell cycle of Tetrahymena pyriformis. While the specific activity of adenylate cyclase remained essentially constant throughout the cycle, cAMP oscillated (between 10 and 50 pmoles/mg protein) through two cycles. Minima were observed at each division ($\text{D}\text{S}$ border) and maxima at each $\text{S}\text{G}\text{2}$ border. Each heat shock caused slight temporary reduction in cyclase activity. Further observations suggest to us that adenylate cyclase shows conformational changes in response to temperature-induced alterations and to changes in lipid composition of membranes.