Malate Dehydrogenase Mutants in Escherichia coli K-12

Mutants devoid of malate dehydrogenase activity have been isolated in Escherichia coli K-12. They do not possess detectable malate dehydrogenase when grown aerobically or anaerobically on glucose as sole carbon source. All mutants revert spontaneously; a few partial revertants have been found with a malate dehydrogenase exhibiting altered electrophoretic mobility. Therefore, only one such enzyme appears to exist in the strains examined. No evidence could be obtained for the presence of a malate dehydrogenase not linked to nicotinamide adenine dinucleotide. Mutants deficient in both malate dehydrogenase and phosphoenol pyruvate carboxylase activities will grow anaerobically on minimal glucose plus succinate medium; also, malate dehydrogenase mutants do not require succinate for anaerobic growth on glucose. The anaerobic pathway oxaloacetate to succinate or succinate to aspartate appears to be accomplished by aspartase. Malate dehydrogenase is coded for by a locus somewhere relatively near the histidine operon, i.e., a different chromosomal location than that known for other citric acid cycle enzymes.     

Isolation of Temperature-Sensitive Membrane Mutants of Escherichia coli K-12

Mutants with impaired biosynthesis of unsaturated fatty acids or altered metabolism of the phospholipids were isolated at a rather high frequency from a set of temperature-sensitive lysis mutants. It is suggested that preselection for the lysis phenotype makes it possible to isolate several kinds of mutants affected in the integrity of the cytoplasmic membrane.     

Properties of Mitomycin C-sensitive Mutants of Escherichia coli K-12

Strains hypersensitive to mitomycin C (MC) were isolated from Escherichia coli K-12 after treatment with nitrosoguanidine. Of 43 MC-sensitive strains tested for their ultraviolet light (UV) sensitivity and for their ability to reactivate UV-inactivated λ phage, 38 were found to be insensitive to UV irradiation and to be able to reactivate UV-irradiated bacteriophage λ. Some properties of the MC-sensitive, uvr⁺ mutants were analyzed. Synthesis of deoxyribonucleic acid (DNA) in MC-sensitive, uvr⁺ mutants was inhibited at a lower concentration of MC than in the wild-type strain. Mutant cells, labeled with ³H-thymidine and then exposed to MC, released radioactivity as low molecular weight compounds. The amount of radioactivity released was the same as that from the wild-type strain. MC-sensitive, uvr⁺ mutants, as well as the corresponding wild-type strain, were equally susceptible to induction of prophage 80 by UV irradiation. However, MC induction of prophage was achieved in MC-sensitive, uvr⁺ mutants at a lower concentration of the antibiotic than in the wild-type strain. Genetic experiments indicated that a gene controlling MC sensitivity is located close to that determining lactose fermentation of E. coli. It is situated on episome F′13, and the wild type is dominant to the MC-sensitive allele.     

Isolation of Deoxyribonucleic Acid Methylase Mutants of Escherichia coli K-12

Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N(6)-methyladenine (N(6)-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict lambda. None of the mutations had the effect of being lethal.     

Escherichia coli K-12 Mutants Altered in the Transport of Branched-Chain Amino Acids

Two mutants of Escherichia coli K-12 are described which are resistant to the inhibition that valine exerts on the growth of E. coli. These mutants have lesions at two different loci on the chromosome. One of them, brnP, is linked to leu (87% cotransduction) and is located between leu and azi represented on the map at 1 min; the other, brnQ, is linked to phoA (96% cotransduction), probably between proC and phoA and represented at 10 min. These mutants are resistant to valine inhibition but are sensitive to dipeptides containing valine. Since it is known that dipeptides are taken up by E. coli through a transport system(s) different from those used by amino acids, this sensitivity to the peptides suggests an alteration in the active transport of valine. The mutants are resistant to valine only if leucine is present in the growth medium; the uptake of valine is less in both mutants than it is in wild-type E. coli, and it is reduced even further if leucine is present. Under these conditions the total uptake of valine is almost completely abolished in the brnQ mutant. The brnP mutant takes up about 60% as much valine as does the wild type, but no exogenous valine is incorporated into proteins. The apparent K(m) and V(max) of isoleucine, leucine, and valine for the transport system are reported; the brnP mutant, when compared to the wild type, has a sevenfold higher K(m) for isoleucine and a 17-fold lower K(m) for leucine; the V(max) for the three amino acids is reduced in the brnQ mutant, up to 20-fold for valine. The transport of arginine, aspartic acid, glycine, histidine, and threonine is not altered in the brnQ mutant under conditions in which that of the branched amino acids is. Evidence is reported that O-methyl-threonine enters E. coli through the transport system for branched amino acids, and that thiaisoleucine does not.     

Mutants of Escherichia coli K-12 with an Altered Glutamyl-Transfer Ribonucleic Acid Synthetase

Three streptomycin-suppressible lethal mutants of Escherichia coli K-12 have been shown to possess structurally altered glutamyl-transfer ribonucleic acid (tRNA) synthetases. Each mutant synthetase displays a K(m) value for glutamate which is 10-fold higher than the parental value, and the mutations reside in two widely separate loci on the genetic map. Mixing of the mutant extracts in pairs gave no indication of in vitro complementation. All three enzymes charge the minor tRNA(glu) fraction identically, but one (EM 120) charges the major fraction at a twofold lower rate than do the other two (EM 102 and EM 111). Possible explanations for the existence of the two synthetase loci are presented.     

Genetic Studies on Ribose 5-Phosphate Isomerase Mutants of Escherichia coli K-12

A gene for the constitutive ribosephosphate isomerase (rpiA) is highly cotransducible with serA at 56.2 min on the genetic linkage map of Escherichia coli K-12. Suppression of ribosephosphate isomerase A-negative mutants can occur by a regulator gene mutation permitting constitutive synthesis of the normally inducible ribosephosphate isomerase B.     

Mutants of Escherichia coli K-12 which synthesize the pyruvate dehydrogenase complex constitutively

Regulatory mutants of E. coli which synthesize the pyruvate dehydrogenase complex constitutively can be selected in strains lacking phosphoenol pyruvate synthase by taking advantage of the regulatory properties of the glyoxylate cycle operon. Constitutivity can lead to production of still more pyruvate dehydrogenase complex than has been found before as “fully-induced” synthesis; in one constitutive mutant about 5% of the total soluble protein is enzyme complex. The altered regulatory element in all nine mutants is closely linked to the structural genes for the pyruvate dehydrogenase complex. There does not appear to be a common regulatory element involved in the control of glyoxylate cycle enzymes and pyruvate dehydrogenase synthesis. Both systems share only the same effector, pyruvate, which represses the synthesis of glyoxylate cycle enzymes and induces that of the pyruvate dehydrogenase complex.     

Excision Repair Properties of Isogenic rec− Mutants of Escherichia coli K-12

We have examined the excision repair properties of isogenic rec⁻ and uvr⁻ strains of Escherichia coli K-12. A recB⁻recC⁻ strain excises dimers at a rate nearly that of the rec⁺ parent, reaching the same extent of excision after a 1-hr postirradiation incubation. recA⁻ and recA⁻recB⁻ strains excise 75 to 80% of the dimers excised by their rec⁺ parent, whereas a uvrB⁻ strain excises no dimers during a 1-hr incubation. The doses of ultraviolet light (254 nm) required to reduce survival to 37% of the original population are 8 ergs/mm² for recA or recA recB mutants, 5 ergs/mm² for the uvrB⁻ strain, 30 ergs/mm² for the recB recC mutant, and 230 ergs/mm² for the wild-type parent. From these data one cannot account for the ultraviolet light sensitivity of rec⁻ strains on the basis of their excision repair properties. We conclude that rec gene products play no significant role in the early steps of excision repair. The assay we have used for excision of thymine dimers is a modification of the Carrier-Setlow technique, and is described in detail in the Appendix to this paper. To show the properties and validity of this method, results of experiments with thymine dimers formed in vitro and in vivo in E. coli K-12 are presented. These results show our method to be reproducible and sensitive to 0.005% of the total radioactive thymine present in thymine-containing dimers.     

Ozone-induced damage of Escherichia coli K-12

Escherichia coli K-12 transformed with pACYC184 plasmid DNA was exposed to ozone (O₃) in aqueous solution. The damage to the membrane, protein, plasmid DNA, and cell survival were investigated. Cell viability was unaffected by short-term O₃ exposure (1–5 min) but membrane permeability was compromised as indicated by protein and nucleic acid leakage and lipid oxidation. The intracellular components, protein and DNA, remained intact. With longer durations of O₃ exposure (up to 30 min) cell viability decreased with a more significant increase in lipid oxidation and protein and nucleic acid leakage. The proteins leaking out were further oxidized by O₃. The total intracellular proteins run on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and plasmid DNA run on agarose gel, showed progressive degradation corresponding to the decrease in cell viability. The data indicate that membrane components are the primary targets of O₃ damage with subsequent reactions involving the intracellular components, protein and DNA. Received: 18 Apirl 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Flavodoxin mutants of Escherichia coli K-12

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.     

Potassium-dependant mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 that grow more slowly in media containing low concentrations of K have been isolated. All independent mutants of this type which have been studied carry a mutation in a small region of the bacterial chromosome between the supE and gal loci. The growth rate of the mutants is the same as that of the parental strains in medium containing more than 1 mm K, but is only 50% that of the parent when the K concentration is reduced to 0.1 mm. The mutants do not appear to have a primary alteration in K transport, and are therefore referred to as K-dependent. The abbreviation kdp is proposed for this class of mutant.     

L-Serine-sensitive mutants of Escherichia coli K-12

While attempting to isolate d-serine-sensitive mutants of Escherichia coli K-12, we found a class of mutants sensitive to low concentrations of l-serine (10 to 25 mug/ml).     

Novel UGA-suppressors in Escherichia coli K-12

UGA-specific nonsense suppressors from Escherichia coli K-12 were isolated and characterized. One of them (Su+UGA-11) was identified as a mutant of the prfB gene for the peptide releasing factor RF2. It appears that in this strain, while peptide release at sites of UGA mutations is retarded, the UGA stop codon is read through even in the absence of a tRNA suppressor, exhibiting a novel type of passive nonsense suppression. Three suppressors (Su+UGA-12, -16 and -34) were capable of restoring the streptomycin sensitive phenotype in resistant bacteria (strAr). Because of their drug-related phenotype, these are possibly mutations in the components of the ribosomal machinery, particularly those concerned with peptide release at UGA nonsense codons. A tRNA suppressor was also obtained which was derived from the tRNA(Trp) gene. In this strain, a long region between rrnC (84.5 min) and rrnB (89.5 min) was duplicated and one of the duplicated genes of tRNA(Trp) was mutated to the suppressor. The mechanism of UGA-suppression is discussed in terms of translation termination at the nonsense codon in both active and passive fashions.     

Escherichia coli K-12 Mutants Resistant to Nalidixic Acid: Genetic Mapping and Dominance Studies

Escherichia coli K-12 strains tested so far (approximately 20) can be separated into three groups on the basis of their abilities to form colonies on nutrient agar supplemented with nalidixic acid (NAL): (i) Nal(s) or wild type (no growth at 1 to 2 mug/ml); (ii) NalA(r) (growth at 40 mug/ml or higher); and (iii) NalB(r) (growth at 4 mug/ml, but no growth at 10 mug/ml). The NalA(r) group has a spectrum of sensitivity ranging from 60 to over 100 mug/ml. All Hfr strains of the NalA(r) and NalB(r) groups transfer NAL resistance to recipient cells at genetic loci which are at 42.5 +/- 0.5 and 51 +/- 1 min, respectively, on the Taylor-Trotter map. Some members of the NalA(r) group also have the genetic locus for NalB(r). The nalA(s) allele is completely dominant to nalA(r) in a partial diploid configuration. In haploids, nalA(r)-nalB(r) is phenotypically NalA(r); nalA(r)-nalB(s) is NalA(r); and nalA(s)-nalB(r) is NalB(r). The map location of nalA and the easy differentiation between NalA(r) and NalA(s) allow this marker to be used as a counterselector in bacterial conjugation experiments.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol     

Binding of Escherichia coli K-12 to cellulose

Abstract A mutant strain of Pseudomonas aeruginosa , PAC35, was shown to lack homoserine dehydrogenase activity. In minimal salt medium, with growth-limiting concentrations of homoserine, strain PAC35 excreted lysine into the medium and this did not occur when exogenous homoserine, or threonine, was in excess of requirements. The hom gene mapped at about 42 min on the PAO chromosome.