Genetic analysis of gene 1.2 of bacteriophage T7: isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants.

The product of gene 1.2 of bacteriophage T7 is not required for the growth of T7 in wild-type Escherichia coli since deletion mutants lacking the entire gene 1.2 grow normally (Studier et al., J. Mol. Biol. 135:917-937, 1979). By using a T7 strain lacking gene 1.2, we have isolated a mutant of E. coli that was unable to support the growth of both point and deletion mutants defective in gene 1.2. The mutation, optA1, was located at approximately 3.6 min on the E. coli linkage map in the interval between dapD and tonA; optA1 was 92% cotransducible with dapD. By using the optA1 mutant, we have isolated six gene 1.2 point mutants of T7, all of which mapped between positions 15 and 16 on the T7 genetic map. These mutations have also been characterized by DNA sequence analysis, E. coli optA1 cells infected with T7 gene 1.2 mutants were defective in T7 DNA replication; early RNA and protein synthesis proceeded normally. The defect in T7 DNA replication is manifested by a premature cessation of DNA synthesis and degradation of the newly synthesized DNA. The defect was not observed in E. coli opt+ cells infected with T7 gene 1.2 mutants or in E. coli optA1 cells infected with wild-type T7 phage.     

Cloning and expression in Escherichia coli of a phoA gene encoding a phosphate-irrepressible alkaline phosphatase of Zymomonas mobilis

The Zymomonas mobilis phoA gene, encoding a phosphate-irrepressible alkaline phosphatase (ZAPase), was cloned and its expression was studied in phoA mutants of Escherichia coli. The ZAPase was recovered in the soluble fraction of E. coli. The enzyme was synthesized constitutively and its synthesis not repressed by phosphate, unlike the phoA gene of E. coli. The phoA gene of Z. mobilis was mutagenized by Mini Mu PR13 and the mutated gene crossed into Z. mobilis in order to obtain phoA mutants by reverse genetics. Although Z. mobilis mutants with Mini Mu PR13 integrated in the chromosome were obtained, none had an allele replacement for none was defective in ZAPase.     

A Col E1 hybrid plasmid containing Escherichia coli genes complementing d-xylose negative mutants of Escherichia coli and Salmonella typhimurium

The Clarke and Carbon bank of Col El - Escherichia coli DNa hybrid plasmids was screened for complementation of d-xylose negative mutants of E. coli. Of several obtained, the smallest, pRM10, was chosen for detailed study. Its size was 16 kilobases (kb) and that of the insert was 9.7 kg. By transformation or F'-mediated conjugation this plasmid complemented mutants of E. coli defective in either D-xylose isomerase or D-xylulose kinase activity, or both. The activity of D-xylulose kinase in E. coli transformants which bear an intact chromosomal gene for this enzyme was greater than that for the host, due to a gene dosage effect. The plasmid also complemented D-xylose negative mutants of Salmonella typhimurium by F'-mediated conjugation between E. coli and S. typhimurium. Salmonella typhimurium mutants complemented were those for D-xylose isomerase and for D-xylulose kinase in addition to pleiotropic D-xylose mutants which were defective in a regulatory gene of the D-xylose operon. In addition, the plasmid complemented the glyS mutation in E. coli and S. typhimurium. The glyS mutant of E. coli was temperature sensitive, indicating that the plasmid carried the structural gene for glycine synthetase. The glyS mutation in E. coli maps at 79 min, as do the xyl genes. The behaviour of the plasmid is consistent with the existence of a d-xylose operon in E. coli. The data also suggest that the plasmid carries three of the genes of this operon, specifically those for D-xylose isomerase, D-xylulose kinase, and a regulatory gene.     

Repair-defective mutants of Alteromonas espejiana, the host for bacteriophage PM2

The in vivo repair processes of Alteromonas espejiana, the host for bacteriophage PM2, were characterized, and UV- and methyl methanesulfonate (MMS)-sensitive mutants were isolated. Wild-type A. espejiana cells were capable of photoreactivation, excision, recombination, and inducible repair. There was no detectable pyrimidine dimer-DNA N-glycosylase activity, and pyrimidine dimer removal appeared to occur by a pathway analogous to the Escherichia coli Uvr pathway. The UV- and MMS-sensitive mutants of A. espejiana included three groups, each containing at least one mutation involved with excision, recombination, or inducible repair. One group that was UV sensitive but not sensitive to MMS or X rays showed a decreased ability to excise pyrimidine dimers. Mutants in this group were also sensitive to psoralen plus near-UV light and were phenotypically analogous to the E. coli uvr mutants. A second group was UV and MMS sensitive but not sensitive to X rays and appeared to contain mutations in a gene(s) involved in recombination repair. These recombination-deficient mutants differed from the E. coli rec mutants, which are MMS and X-ray sensitive. The third group of A. espejiana mutants was sensitive to UV, MMS, and X rays. These mutants were recombination deficient, lacked inducible repair, and were phenotypically similar to E. coli recA mutants.     

Isolation of catalase-deficient Escherichia coli mutants and genetic mapping of katE, a locus that affects catalase activity

A number of catalase-deficient mutants of Escherichia coli which exhibit no assayable catalase activity were isolated. The only physiological difference between the catalase mutants and their parents was a 50- to 60-fold greater sensitivity to killing by hydrogen peroxide. For comparison, mutations in the xthA and recA genes of the same strains increased the sensitivity of the mutants to hydrogen peroxide by seven- and fivefold, respectively, showing that catalase was the primary defense against hydrogen peroxide. One class of mutants named katE was localized between pfkB and xthA at 37.8 min on the E. coli genome. A second class of catalase mutants was found which did not map in this region.     

Method for isolating mutants overproducing nicotinamide adenine dinucleotide and its precursors

A procedure has been developed for isolating mutants which are defective with respect to nicotinamide adenine dinucleotide (NAD) metabolism. It is based on the well known V-factor requirement of Haemophilus parainfluenzae. This procedure was used to isolate a series of mutants from Escherichia coli. The pyridine metabolism of wild-type and mutant E. coli cells falls in one of four distinct classes. Class A includes wild-type E. coli and represents strains that are normal with respect to pyridine metabolism. Class B mutants have altered internal pools of NAD. The intracellular NAD concentration of different class B mutants varies over a 10-fold range. Class C mutants excrete pyridine mononucleotides, and class D mutants excrete NAD. The production of pyridine nucleotides by class C and D mutants exceeds that of wild-type E. coli by a factor of at least ten. The mutant strains generally have normal generation times and achieve normal cell densities in minimal medium.     

Conditional lethal mutants of bacteriophage T4 unable to grow on a streptomycin resistant mutant of Escherichia coli.

Sixteen conditional lethal mutants of bacteriophage T4D have been isolated which grow on Escherichia coli CR63 (a su+ streptomycin-sensitive K12 strain) but are restricted by CR/s (a streptomycin-resistant derivative of CR63). These mutants have been given the prefix str. Four of these mutants are amber and 12 appear to be missense. Eleven of the 12 missense mutants appear to be "pseudo-amber" (i.e. they are restricted by a su- E. coli B strain but not by a su- K12 strain); the other missense mutant was not restricted by either B or K12. The str mutations mapped in 12 different genes. Most were clustered in a region of early genes (gene 56 to gene 47). Fifty-eight amber and 10 "pseudo-amber" mutants isolated previously for their inability to grow on E. coli B were tested for restriction by CR/s. All the amber mutants grew normally on CR/s, whereas all 10 "pseudo-amber" mutants were restricted by CR/s. This implies that the phenotype of the "pseudo-amber" mutants is the result of a ribosomal difference between the permissive host CR63 and the restrictive hosts B and CR/s. These str mutants should prove to be useful alternatives to amber mutants for genetic and biochemical studies of bacteriophage T4 and for studies of the E. coli ribosome. It should be possible ot isolate similar mutants in other bacteriophages provided that streptomycin resistant hosts are available.     

The recA gene of Chlamydia trachomatis: Cloning, sequence, and characterization in Escherichia coli

Abstract The recA gene of Chlamydia trachomatis was isolated by complementation of an Escherichia coli recA mutant. The cloned gene restored resistance to methyl methanesulfonate in E. coli recA mutants. The DNA sequence of the chlamydial gene was determined and the deduced protein sequence compared with other RecA proteins. In E. coli recA deletion mutants, the cloned gene conferred moderate recombinational activity as assayed by Hfr matings. The chlamydial recA gene was efficient in repairing alkylated DNA but less so in repairing of UV damage when compared with the E. coli homologue. As detected by an SOS gene fusion, a small but measurable amount of LexA co-cleavage was indicated.     

Cloning of the Serratia marcescens recA gene and construction of a Serratia marcescens recA mutant

A recombinant plasmid, pSM2513, containing an 8.5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2.7 kb Bg/II-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.     

Chromosomal mutations that increase the production of a plasmid-encoded haemolysin in Escherichia coli

Transposon mutagenesis was used to isolate two Escherichia coli mutants which express very large amounts of haemolysin when carrying the multicopy plasmid pANN202-312. E. coli strain Hha-2 was isolated by Mud1 mutagenesis, and strain Hha-3 by Tn5 mutagenesis. The transposon insertion was chromosomal in both mutants and could be demonstrated to be unrelated to the haemolytic region of the plasmid. The substantial increase in both extracellular and intracellular haemolysin production was dependent upon plasmid copy number and was drastically reduced when either mutant carried the low-copy-number haemolytic plasmid pHly152. In both mutants, the marked increase in extracellular production was dependent upon the specific haemolysin transport genes, hlyB and hlyD. The lack of either gene function resulted in no external haemolysin production. SDS-PAGE analysis showed no change in the pattern of outer-membrane proteins of the mutants, although changes (differing between the two mutants) were seen in their periplasmic proteins. The mutations of both strains (termed hha-2 and hha-3) were mapped at minute 10.5 of the E. coli chromosome. No relation to any known gene affecting gene regulation in E. coli could be found.     

Control of methionine biosynthesis in Escherichia coli K12: a closer study with analogue-resistant mutants

Control of methionine biosynthesis in Escherichia coli K12 was reinvestigated by using methionine-analogue-resistant mutants. Norleucine (NL) and alpha-methylmethionine (MM) were found to inhibit methionine biosynthesis directly whereas ethionine (Et) competitively inhibited methionine utilization. Adenosylation of Et to generate S-adenosylethionine (AdoEt) by cell-free enzyme from E. coli K12 was demonstrated. Tolerance of increasing concentrations of NL by E. coli K12 mutants is expressed serially as phenotypes NLR, NLREtR, NLRMMR and finally NLREtRMMR. All spontaneous NLR mutants had a metK mutation, whereas NTG-induced mutants had mutations in both the metK and metJ genes. The kinetics of methionine adenosylation by the E. coli K12 cell-free enzyme were found to be similar to those reported for the yeast enzyme, showing the typical lag phase at low methionine concentration and disappearance of this phase when AdoMet was included in the incubation mixture. NL extended the lag phase, and lowered the rate of subsequent methionine adenosylation, but did not affect the shortening of the lag phase of adenosylation by AdoMet.     

Genetic and biochemical analyses of pantothenate biosynthesis in Escherichia coli and Salmonella typhimurium.

Pantothenate (pan) auxotrophs of Escherichia coli K-12 and Salmonella typhimurium LT2 were characterized by enzymatic and genetic analyses. The panB mutants of both organisms and the pan-6 ("panA") mutant of S. typhimurium are deficient in ketopantoate hydroxymethyltransferase, whereas the panC mutants lack pantothenate synthetase. panD mutants of E. coli K-12 were previously shown to be deficient in aspartate 1-decarboxylase. All mutants showed only a single enzyme defect. The finding that the pan-6 mutant was deficient in ketopantoate hydroxymethyltransferase indicates that the genetic lesion is a panB allele. The pan-6 mutant therefore is deficient in the utilization of alpha-ketoisovalerate rather than the synthesis of alpha-ketoisovalerate, as originally proposed. The order of the pan genes of E. coli K-12 was determined by phage P1-mediated three-factor crosses. The clockwise order was found to be aceF panB panD panC tonA on the genetic map of E. coli K-12. The three-factor crosses were greatly facilitated by use of a closely linked Tn10 transposon as the outside marker. We also found that supplementation of E. coli K-12 auxotrophs with a high concentration of pantothenate or beta-alanine increased the intracellular coenzyme A level two- to threefold above the normal level. Supplementation with pantoate or ketopantoate resulted in smaller increases.     

3-hydroxypyruvate substitutes for pyridoxine in serC mutants of Escherichia coli K-12.

Escherichia coli K-12 mutants with serC genotype required pyridoxine and serine for normal growth, as do E. coli B mutants of this type. Mutants of the K-12 strain, however, reverted easily to pyridoxine independence without regaining activity in the 3-phosphoserine oxoglutarate transaminase coded for by the serC gene. Both these revertants and the parental type synthesized pyridoxine in normal amounts when 3-hydroxypyruvate was used as a supplement, although neither of these mutants could use this compound to satisfy their serine requirement. Since serine alone was inadequate to provide the nutritional requirement of serC mutants, these mutants must have been unable to synthesize 3-hydroxypyruvate from serine. We suggest that 3-phosphoserine oxoglutarate transaminase in normal E. coli serves as a catalyst for transaminating small amounts of serine to 3-hydroxypyruvate, which is then used in pyridoxine biosynthesis. In serC mutants, this activity is blocked, and these mutants then show a double requirement for serine and pyridoxine.     

Inactivation of the ampD gene causes semiconstitutive overproduction of the inducible Citrobacter freundii beta-lactamase.

In Citrobacter freundii and Enterobacter cloacae, synthesis of AmpC beta-lactamase is inducible by the addition of beta-lactams to the growth medium. Spontaneous mutants that constitutively overproduce the enzyme occur at a high frequency. When the C. freundii ampC beta-lactamase gene is cloned into Escherichia coli together with the regulatory gene ampR, beta-lactamase expression from the clone is inducible. Spontaneous cefotaxime-resistant mutants were selected from an E. coli strain carrying the cloned C. freundii ampC and ampR genes on a plasmid. Virtually all isolates had chromosomal mutations leading to semiconstitutive overproduction of beta-lactamase. The mutation ampD2 in one such mutant was caused by an IS1 insertion into the hitherto unknown ampD gene, located between nadC and aroP at minute 2.4 on the E. coli chromosome. The wild-type ampD allele cloned on a plasmid could fully trans-complement beta-lactamase-overproducing mutants of both E. coli and C. freundii, restoring the wild-type phenotype of highly inducible enzyme synthesis. This indicates that these E. coli and C. freundii mutants have their lesions in ampD. We hypothesize that induction of beta-lactamase synthesis is caused by blocking of the AmpD function by the beta-lactam inducer and that this leads directly or indirectly to an AmpR-mediated stimulation of ampC expression.     

Functional Interchangeability of DNA Replication Genes in SALMONELLA TYPHIMURIUM and ESCHERICHIA COLI Demonstrated by a General Complementation Procedure

Twenty-four genes from Salmonella typhimurium that affect DNA replication were isolated from a lambda-Salmonella genomic library by lysogenic complementation of temperature-sensitive mutants of Salmonella or E. coli, using a new plaque complementation assay. The complementing lambda clones, which make red plaques in this assay, and noncomplementing mutant derivatives, which make uncolored plaques, were used to further characterize the temperature-sensitive Salmonella mutants and to establish the functional similarity of E. coli and Salmonella DNA replication genes. For 17 of 18 E. coli mutants representing distinct loci, a Salmonella gene that complemented the mutant was found. This result indicates that single Salmonella replication proteins are able to function in otherwise all E. coli replication complexes and suggests that the detailed properties of Salmonella and E. coli replication proteins are very similar. The other seven Salmonella genes that were cloned were unrelated functionally to any E. coli genes examined. --As an aid to the derivation of chromosomal mutations affecting some of the cloned genes, a general method was developed for placing a transposon in the Salmonella chromosome in a segment corresponding to cloned DNA. Chromosomal mutations were derived in Salmonella affecting a gene (dnaA) that was cloned by complementation of an E. coli mutant by using the transposon-encoded drug resistance as a selectable marker in local mutagenesis.     

Isolation and characterization of autolysis-defective mutants of Escherichia coli that are resistant to the lytic activity of seminalplasmin.

Two temperature-sensitive autolysis-defective mutants of Escherichia coli were isolated and shown to be resistant to lysis induced by seminalplasmin, an antimicrobial protein from bovine seminal plasma, as well as to lysis induced by ampicillin, D-cycloserine and nocardicin, at 37 or 42 degrees C but not at 30 degrees C. The mutants were, however, sensitive to inhibition of RNA synthesis by seminalplasmin even at the nonpermissive temperature. Temperature-resistant revertants of the mutants were sensitive to lysis induced by the various antibiotics at 37 or 42 degrees C. The mutations in both strains were mapped at 58 min on the E. coli linkage map. The lysis resistance of the mutants was phenotypically suppressed by the addition of NaCl. Partial suppression of the lysis-resistant phenotype was also observed in a relA genetic background.     

Modification of the radiosensitivity of E. coli cells by factors affecting the yield of photoreactivated damage

A study was made of the influence of irradiation conditions on the yield of the photoreactivable damages in radiosensitive mutants of E. coli cells (E. coli WP2). Pyrimidine dimers were shown to occur in exrA- and recA- mutants irradiated under anoxic conditions, the survival of these mutants being modified depending on cell genotype. The processes of direct excitation of the molecules were involved in the formation of the damages observed. It can be assumed that the lesser oxygen effect observed in exrA- and partially in recA- mutants of E. coli WP2 cells is associated with a contribution of the photoreactivable damages to a lethal effect of ionizing radiation.     

Effect of the molecular weight of DNA on the effectiveness of plasmid transformation of E. coli K12

The level of plasmid transformation and transfection by the high molecular mass DNA was studied for Escherichia coli mutants having increased efficiency of plasmid transformation by low molecular mass DNA. Decreased level of plasmid transformation and transfection registered in some mutants as compared to the one in wild type strain suggests the specificity of Escherichia coli cells penetration for DNA of different molecular mass.     

Mutants in a Nonessential Gene of Bacteriophage T4 Which Are Defective in the Degradation of Escherichia coli Deoxyribonucleic Acid

Wild-type bacteriophage T4 was enriched for mutants which fail to degrade Escherichia coli deoxyribonucleic acid (DNA) by the following method. E. coli B was labeled in DNA at high specific activity with tritiated thymidine ((3)H-dT) and infected at low multiplicity with unmutagenized T4D. At 25 min after infection, the culture was lysed and stored. Wild-type T4 degrades the host DNA and incorporates the (3)H-dT into the DNA of progeny phage; mutants which fail to degrade the host DNA make unlabeled progeny phage. Wild-type progeny are eventually inactivated by tritium decay; mutants survive. Such mutants were found at a frequency of about 1% in the survivors. Eight mutants are in a single complementation group called denA located near gene 63. Four of these mutants which were examined in detail leave the bulk of the host DNA in large fragments. All eight mutants exhibit much less than normal T4 endonuclease II activity. The mutants produce somewhat fewer phage and less DNA than does wild-type T4.     

Isolation of Escherichia coli mutants with an adenosine triphosphatase insensitive to aurovertin.

Energy-transducing adenosine triphosphatase (ATPase) from Escherichia coli is inhibited by aurovertin. Aurovertin-resistant mutants were generated by nitrosoguanidine mutagenesis of E. coli AN180, whose growth on a nonfermentable carbon source was blocked by aurovertin. The ATPase activity of cell extracts from 15 different mutants (designated MA1, MA2, MA3, etc.) was found to be at least 20 times less sensitive to aurovertin than that from the parent strain. The aurovertin-resistant mutants did not show cross-resistance towards a number of ATPase inhibitors including azide, dicyclohexylcarbodiimide, quercetin, 7-chloro-4-nitrobenzofurazan, and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. Aurovertin inhibited the energization brought about by addition of ATP to E. coli AN180 membrane vesicles; it was without effect on MA1 and MA2 membrane vesicles energized by ATP. The mutation in MA1, like other mutations of the ATPase complex, maps in the unc region of the bacterial chromosome.