Mutant of Escherichia coli K-12 Deficient for Detergent-Resistant Phospholipase A

A mutant deficient for detergent-resistant (DR) phospholipase A was isolated from Escherichia coli K-12. Because the enzyme is membrane-bound and the substrate is a lipid, a special procedure was developed for isolating mutants deficient for the enzyme from agar plates. A sodium dodecyl sulfate (SDS)-sensitive mutant was used as a parental strain for the isolation of DR phospholipase A-deficient mutant. Soft agar containing an unsaturated fatty acid auxotroph and SDS was poured over colonies of the parental strain. The cells were easily solubilized with SDS, and phospholipids were efficiently digested by DR phospholipase A from the colonies on an agar plate. Fatty acids released supported the growth of the indicator bacteria. After the cells of the parent were mutagenized with nitrosoguanidine, colonies which could not support the growth of an unsaturated fatty acid auxotroph in the presence of SDS were selected. Four mutants were isolated after in vitro scre[UNK]ning of DR phospholipase A activity of 30 halo-less clones. Since an extract of the parent strain mixed with that of a mutant strain was still active, it was concluded that the inability to hydrolyze phospholipids was not due to the accumulation of inhibitory substance; the activity of DR phospholipase A in the mutant was less than 1% of the parental activity. Physiological studies indicated that DR phospholipase A is not essential for the growth of E. coli.     

Threonine Locus of Escherichia coli K-12: Genetic Structure and Evidence for an Operon

Three genes, thrA, thrB, and thrC, were previously defined and localized in the threonine locus of Escherichia coli K-12. thrA, thrB, and thrC specify the enzymes aspartokinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase, respectively. A complementation analysis of the threonine cluster using derivatives of a lambda phage carrying the threonine genes (lambdadthr(c)) demonstrates that: (i) thrB and thrC each consist of a single cistron; and (ii) thrA is composed of two cistrons, thrA(1) and thrA(2), although it specifies a single polypeptide chain. thrA(1) and thrA(2) correspond to aspartokinase I and homoserine dehydrogenase I, respectively. Their relative order is established. The demonstration of polar effects of mutations (nonsense or induced by phage Mu) in thrA and thrB is taken as evidence for the existence of a thrA thrB thrC operon, transcribed in this order.     

Isolation and Characterization of a Phosphonomycin-Resistant Mutant of Escherichia coli K-12

A mutant was isolated from Escherichia coli K-12 which showed increased resistance towards phosphonomycin, a new bactericidal antibiotic recently isolated from strains of Streptomyces. Evidence is presented which suggests that this mutant is resistant to lysis by phosphonomycin because of a lower affinity of phosphoenolpyruvate: uridine diphospho-N-acetylglucosamine enolpyruvyl transferase for this antibiotic. This mutant was also found to be temperature-sensitive in growth. At 42 C mutant cells grew poorly, and the rate of incorporation of (3)H-diaminopimelic acid into trichloroacetic acid-insoluble material was also greatly reduced. Genetic studies indicate that the increased resistance toward phosphonomycin and temperature sensitivity in growth of this mutant are probably the consequences of a single mutation.     

NAD-dependent Recovery of Cell Division in UV-irradiated Escherichia coli B and Lon- Mutant of Escherichia coli K-12

We found that both benzyl isothiocyanate (ITC) and phenyl ITC inhibited respiration in the mitochondria in an electrophilic reaction-dependent manner. ITC-induced mitochondrial swelling and cytochrome c release were prevented by cyclosporin A, indicating that they are mediated through the ITC moiety-dependent reaction to critical thiol groups for the opening of membrane permeability transition-dependent pores.     

Mutant of Escherichia coli K-12 Defective in the Transport of Basic Amino Acids

Escherichia coli K-12 possesses two active transport systems for arginine, two for ornithine, and two for lysine. In each case there is a low- and a high-affinity transport system. They have been characterized kinetically and by response to competitive inhibition by arginine, lysine, ornithine and other structurally related amino acids. Competitors inhibit the high-affinity systems of the three amino acids, whereas the low-affinity systems are not inhibited. On the basis of kinetic evidence and competition studies, it is concluded that there is a common high-affinity transport system for arginine, ornithine, and lysine, and three low-affinity specific ones. Repression studies have shown that arginine and ornithine repress each other's specific transport systems in addition to the repression of their own specific systems, whereas lysine represses its own specific transport system. The common transport system was found to be repressible only by lysine. A mutant was studied in which the uptake of arginine, ornithine, and lysine is reduced. The mutation was found to affect both the common and the specific transport systems.     

Construction of an Escherichia coli K-12 Mutant for Homoethanologenic Fermentation of Glucose or Xylose without Foreign Genes

Conversion of lignocellulosic feedstocks to ethanol requires microorganisms that effectively ferment both hexose and pentose sugars. Towards this goal, recombinant organisms have been developed in which heterologous genes were added to platform organisms such as Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli. Using a novel approach that relies only on native enzymes, we have developed a homoethanologenic alternative, Escherichia coli strain SE2378. This mutant ferments glucose or xylose to ethanol with a yield of 82% under anaerobic conditions. An essential mutation in this mutant was mapped within the pdh operon (pdhR aceEF lpd), which encodes components of the pyruvate dehydrogenase complex. Anaerobic ethanol production by this mutant is apparently the result of a novel pathway that combines the activities of pyruvate dehydrogenase (typically active during aerobic, oxidative metabolism) with the fermentative alcohol dehydrogenase.     

Isolation and Characterization of an Escherichia coli K-12 Mutant with a Temperature-Sensitive RecA− Phenotype

A mutation that causes a temperature-sensitive RecA(-) phenotype was identified in a derivative of a PolA(-) strain that failed to grow at high temperature. The mutant allele (recA200) was shown to be linked to cysC, conferred a sharply temperature-sensitive, ultraviolet-sensitive Rec(-) phenotype in the range 35 to 42 C, and in crosses failed to show complementation at 42 C with Hfr's that transferred recA(-). Double mutants that carried both recA200 and polA were examined for ability to grow and synthesize DNA at restrictive temperatures.     

Isolation of an Escherichia coli K-12 dnaE mutation as a mutator.

Using a papillation method, a large number of Escherichia coli K-12 mutator mutations have been isolated. Only one of these (out of 1,250) mutator mutations has proved to be conditionally lethal at high temperatures. In vivo complementation tests indicated that this mutation, dnaE9, lies in dnaE, the structural gene for DNA polymerase III. The dnaE9 polymerase was not thermolabile in vitro; however, it showed a slow decline in specific activity in vivo at the nonpermissive temperature. Cultures of this mutant exhibited a comparably slow shutoff of DNA synthesis on shift to a nonpermissive temperature. dnaE9 showed temperature-sensitive mutator activity, which is not dependent on recA.     

Isolation and Characterization of a Regulatory Mutant of an Aminoacyl-Transfer Ribonucleic Acid Synthetase in Escherichia coli K-12

From Escherichia coli strain K28, which is temperature sensitive for growth because of a mutation in its seryl-transfer ribonucleic acid (tRNA) synthetase gene (serS), temperature-resistant mutants were selected which were found to have a fivefold higher level of seryl-tRNA synthetase than the parent strain. The "high-level" character was found to be genetically stable and is due to a mutation in a locus denoted serO. This locus was found to be very closely linked to serS on the genetic map, and the relative gene order was concluded to be serS-serO-serC. In a serO(-) strain, the normal dependence of seryl-tRNA synthetase (SerRS) activity on changes of exogenous serine concentration was not observed. In a stable heterozygous merodiploid, the serO(-) mutation is still expressed, i.e., it is cis dominant. These results strongly suggest that serO is an operator site involved in the control of the serS gene.     

Genetic Fine Structure of the Leucine Operon of Escherichia coli K-12

The order of mutational sites in 10 independently isolated leucine auxotrophys of Escherichia coli K-12 was determined by three-point reciprocal transductions. The sites of mutation mapped in linear sequence in a cluster; all leucine auxotrophic mutations were cotransducible with mutations in the arabinose operon. The mutations were assigned to four complementation groups by abortive transduction tests, designated D, C, B, and A, reading in a clockwise direction from the arabinose operon. Enzyme analyses showed that strains with a mutation in gene A lacked alpha-isopropylmalate synthetase activity (EC 4.1.3), and those with a mutation in gene B lacked beta-isopropylmalate dehydrogenase activity (EC 1.1.1). It is concluded that the gross structure of the leucine operon in E. coli is closely similar to, if not identical with, the gross structure of the leucine operon in Salmonella typhimurium.     

Structure and function of an OmpC deletion mutant porin from Escherichia coli K-12.

Escherichia coli K-12 strain RAM122 contains a mutation in the ompC gene that results in an eight amino acid deletion, delta 103-110, in the porin protein. Since this strain is capable of growing on maltodextrins in the absence of a functional lamB gene, the mutant protein is thought to have a larger channel size. The stability and structure/function properties of the mutant OmpC porin were investigated and compared to wild-type porin. Isolated unheated RAM122 porin was characterized as a trimer on sodium dodecyl sulfate-polyacrylamide gels. The RAM122 trimer was less stable to temperature when compared to the wild-type porin. In addition, the overall enthalpy for thermal denaturation was lower for the mutant than the wild-type porin as determined by using differential scanning microcalorimetry. Both the proteins' secondary structures, monitored by circular dichroism, were high in beta-sheet content, but the spectra were slightly different in their crossover points as well as their minima. When the proteins were reconstituted and channel activity was assayed by using a liposome swelling technique, the size-exclusion limit of the mutant porin was twice that of the wild-type porin. Conductance measurements across bilayer lipid membranes showed that the mutant porin was voltage gated at much lower membrane potentials, 50 and 75 mV, than the wild-type sample. The closing events of the mutant porin were predominantly of monomer size. The channels detected by using the mutant protein were larger in size than those measured for the wild-type porin monomer. These data suggest that the OmpC mutant porin has a channel size capable of allowing maltodextrins to enter and that this channel is highly voltage regulated.     

Outer Penetration Barrier of Escherichia coli K-12: Kinetics of the Uptake of Gentian Violet by Wild Type and Envelope Mutants

Wild-type strains of Escherichia coli K-12 adsorb gentian violet to the cell surface, but the dye is not transported into the cytoplasm. However, when some mutants that have an altered outer membrane are exposed to gentian violet, the dye is also found in the ribosomal fraction. The transport into the cytoplasm is inhibited at 0 C and requires that the concentration of gentian violet exceeds a threshold value. The initial rate of uptake as well as the amount of gentian violet found in the cytoplasm increases with the concentration of the dye in the medium. The rate of transport of the dye into the cytoplasm is much lower for stationary mutant cells than for exponentially growing cells. The rate of uptake into the cytoplasm increases with increasing deficiency of carbohydrate in the lipopolysaccharide (carbohydrate content lpsB > lpsA > galU). However, other components are also responsible for the barrier since an envA mutant which is not altered in the lipopolysaccharide carbohydrates show an extremely rapid uptake of the dye. The rate of uptake for the envA mutant was the highest found and the same as that of spheroplasts. Growth in the presence of agents affecting the murein sacculus, e.g., lysozyme and sublethal concentrations of penicillin, increased the rate of uptake of gentian violet. Brief treatments with tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid drastically impaired the barrier function. Inhibition of protein synthesis by chloramphenicol also opened the barrier to gentian violet. In conclusion, the outer part of the bacterial envelope is a penetration barrier for gentian violet and probably also for other substances. The lipopolysaccharide, the murein and also other components are important for the function of this barrier. Resistance to gentian violet was found to be inversely correlated to the rate of penetration of the dye into the cytoplasm.     

Characterization and Genetic Analysis of a Mutant of Escherichia coli K-12 with Rounded Morphology

A morphological mutant of Escherichia coli K-12 that grows as round cells at 30, 37, or 42 C in a variety of complex and synthetic media has been isolated and characterized. The gene concerned, designated rodA, has been shown to be on the chromosome between the purE and pyrC loci and to be located at about minute 15. The rodA gene has been found to be co-transducible with the lip gene at a frequency of 95%. The rodA mutant showed an increased resistance to ultraviolet irradiation and a changed sensitivity to drugs. The resistance to ultraviolet irradiation and mitomycin C appears to be co-transducible with the rodA gene.     

Type 1 fimbriation and fimE mutants of Escherichia coli K-12.

We reexamined the influence of fimE, also referred to as hyp, on type 1 fimbriation in Escherichia coli K-12. We found that one strain used previously and extensively in the analysis of type 1 fimbriation, strain CSH50, is in fact a fimE mutant; the fimE gene of CSH50 contains a copy of the insertion sequence IS1. Using a recently described allelic exchange procedure, we transferred the fimE::IS1 allele from CSH50 to our present wild-type strain, MG1655. Characterization of this IS1-containing strain (AAEC137), together with another fimE mutant of MG1655 (AAEC143), led to two conclusions about the role of fimE. First, the formation of phase variant colony types, reported widely in strains of E. coli, depends on mutation of fimE, at least in K-12 strain MG1655. Here we showed that this phenomenon reflects the ability of fimE to stimulate the rapid inversion of the fim invertible element from on to off when the bacteria are grown on agar. Second, our analysis of fimE mutants, which is limited to chromosomal constructs, provided no evidence that they are hyperfimbriate. We believe that these results, which are at odds with a previous study using fim-containing multicopy plasmids, reflect differences in gene copy number.     

Some Effects of Nalidixic Acid on Conjugation in Escherichia coli K-12

The role of deoxyribonucleic acid (DNA) synthesis in the Escherichia coli conjugation system has been studied using nalidixic acid (NAL) to specifically inhibit DNA synthesis in matings between reciprocal combinations of male (Hfr) and female (F⁻) mutants resistant and sensitive to NAL; the physiological action of NAL on the strains utilized was also studied. Matings between combinations of mutants resistant (Nal^r) and sensitive (Nal^s) to NAL allow various parental functions to be established: pair formation studies show that the female cells are responsible for the slight decrease in pair formation when NAL is present in Hfr(Nal^s) × F⁻ (Nal^s) matings. Preformed mating pairs are stable in the presence of NAL. In matings between Hfr(Nal^s) and F⁻(Nal^r), the transfer gradient constant increases linearly with low NAL concentration (0.1 to 0.6 μg of NAL per ml). Higher concentrations of NAL (5 μg/ml) act on Nal^s males to rapidly stop chromosome transfer; under these conditions, however, DNA degradation is unmeasurable as determined from single-strand nicking in the male cells. This result is consistent with a model for chromosome transfer which requires DNA synthesis in the male cell. Inhibition of DNA synthesis (by 85%) in the female has no effect on conjugal chromosome transfer. High concentrations of NAL (>20 μg/ml) produce slight inhibition in chromosome transfer for the Hfr(Nal^r) × F⁻(Nal^r) mating tested; this effect is probably caused by action of NAL on the male. The inhibition of chromosomal transfer by NAL appears to be irreversible in the normal sense. A pulse of NAL, applied during transfer, immediately stops the transfer which is in progress. On removal of the NAL block, the temporal appearance of recombinants is consistent with the idea that a new round of transfer has commenced from the sex factor location on the male chromosome.     

Outer-membrane PhoE protein of Escherichia coli K-12 as an exposure vector: possibilities and limitations.

The phosphate-limitation-inducible outer-membrane protein (PhoE) of Escherichia coli K-12 can be used in an expression system as a carrier for foreign antigenic determinants, facilitating their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature can be made in different cell-surface-exposed regions of PhoE protein, without interfering with the assembly process into the outer membrane. Multiple insertions of an antigenic determinant can be made in the second and eighth exposed regions, resulting in a total insert length of up to 30 and 50 amino acid (aa) residues. Insertions can be made in two exposed regions, simultaneously. However, some limitations were encountered, e.g., insertion of eight or more hydrophobic aa residues affected both the translocation process across the inner membrane and the assembly process into the outer membrane. Also, the insertion of sequences containing many charged residues resulted in accumulation of precursor protein in the cytoplasm.