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.     

Inducible DNA polymerase I synthesis in a UV hyper-resistant mutant of Escherichia coli

A mutant of Escherichia coli which is more resistant to shortwave UV light than its wild-type parent strain and which can synthesise DNA polymerase I constitutively has been further analysed. It carries two mutational alleles which are located about 1.5 min apart and cotransducible by P1 with the argH locus. The two mutational alleles have been segregated and their analysis shows that one of them is responsible for UV hyper-resistance whereas the other mutation confers UV sensitivity. Recombinant plasmids carrying various sections of the polA regulatory region, linked to a galK gene, were introduced into the mutant strains. Analysis of galactokinase shows that the enzyme activity in the UV hyper-resistant mutant is increased. The results suggest that the synthesis of DNA polymerase I in E. coli is inducible.     

The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene

A DNA clone complementing the rne-3071 mutation has been expressed and localized in the physical map of Escherichia coli. The DNA fragment from this clone was localized to the region of the E. coli chromosome where the rne-3071 mutation has been mapped. The position of this DNA fragment in the E. coli chromosome, the size of the product directed by this DNA fragment (110,000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne-3071 and the ams mutations cause similar patterns of RNA synthesis, show that the rne gene--a gene specifying the processing endonuclease RNase E--and the ams gene--a gene that affects mRNA stability--are identical.     

Genetic mapping of ada and adc mutations affecting the adaptive response of Escherichia coli to alkylating agents.

The adaptive response to alkylating agents is an inducible repair system which protects Escherichia coli against the mutagenicity and toxicity of these agents. Four mutations, ada-3, ada-5, ada-6, and adc-1, which confer differing phenotypes as regards this response, were shown to be cotransducible with gyrA, and were located at 47 min on the E. coli genetic map. A mutation already shown on the map at 47 min as tag (B. J. Bachmann and K. B. Low, Microbiol. Rev. 44:1--56, 1980; Karran et al., J. Mol. Biol. 140:101--127, 1980) is now known to be an ada mutation (G. Evensen and E. Seeberg, personal communication).     

Method for localization of cloned DNA fragments on the Escherichia coli chromosome.

In exponentially growing cultures of Escherichia coli strains carrying the dnaC28 mutation, DNA replication can be synchronized by temperature changes (R. L. Rodriguez, M. S. Dalbey, and C. I. Davern, J. Mol. Biol. 74:599-604, 1973). We used this synchronization procedure and DNA-DNA hybridization to develop a technique for the localization of cloned chromosomal fragments on the genetic map. Because of the bidirectional nature of replication in E. coli, our method gave two possible positions (one on each replication arm). However because of the precision obtained for each position (+/- 1 map unit), the final mapping with various genetic techniques was greatly facilitated. Using this technique and a simple chromosomal mobilization test, we located at 93.2 +/- 1 min a cloned DNA fragment carrying an extragenic suppressor of dnaA46, a thermosensitive mutation in the dnaA initiation gene. Further analysis showed that the groES (mopA) and groEL (mopB) genes, both located at 94.2 min on the standard map, were indeed carried by the cloned suppressor fragment.     

Mutants in three genes affecting transport of magnesium in Escherichia coli: genetics and physiology.

Mutants in three genes affecting two Mg2+ transport systems are described. System I, for which Co2+, Mn2+, and Mg2+ are substrates, is inactive in corA mutants corB mutants express system I after growth on high (10 mM) Mg2+ but not low (0.1 mM) Mg2+. Both corA and corB mutants are resistant to Co2+ or Mn2+. corA mutants are sensitive to CA2+. Transport system II is specific for Mg2+ and is repressed by growth on 10 mM Mg2+. mgt mutations inactivate system II. Growth on mgt mutants in normal except on very low (1 muM) concentrations of Mg2+, corA mgt strains exhibit no high-affinity, energy-dependent transport of Mg2+ and require 10 mM Mg2+ for optimal growth. The three genes are not linked. The corA locus is contransducible with ilv at 75 min, corB is cotransducible with pyrB at 85 min, and mgt is cotransducible with malB and mel at 81 min on the genetic map.     

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.     

sn-Glycerol-3-phosphate auxotrophy of plsB strains of Escherichia coli: evidence that a second mutation, plsX, is required.

sn-Glycerol-3-phosphate auxotrophs defective in phospholipid synthesis contain a Km-defective sn-glycerol-3-phosphate acyltransferase. Detailed genetic analysis revealed that two mutations were required for the auxotrophic phenotype. One mutation, in the previously described plsB locus (sn-glycerol-3-phosphate acyltransferase structural gene), mapped near min 92 on the Escherichia coli linkage map. Isolation of Tn10 insertions cotransducible with the auxotrophy in phage P1 crosses revealed that a second mutation was required with plsB26 to confer the sn-glycerol-3-phosphate auxotrophic phenotype. This second locus, plsX, mapped between pyrC and purB near min 24 on the E. coli linkage map. Tn10 insertions near plsX allowed detailed mapping of the genetic loci in this region. A clockwise gene order putA pyrC flbA flaL flaT plsX fabD ptsG thiK purB was inferred from results of two- and three-factor crosses. Strains harboring the four possible configurations of the mutant and wild-type plsB and plsX loci were constructed. Isogenic plsB+ plsX+, plsB+ plsX50, and plsB26 plsX+ strains grew equally well on glucose minimal medium without sn-glycerol-3-phosphate. In addition, plsX or plsX+ had no apparent effect on sn-glycerol-3-phosphate acyltransferase activity measured in membrane preparations. The molecular basis for the plsX requirement for conferral of sn-glycerol-3-phosphate auxotrophy in these strains remains to be established.     

Cloning the gene for ribonuclease E, an RNA processing enzyme

A transducing bacteriophage lambda Ch25rne+, which codes for ribonuclease E of E. coli, has been isolated. To achieve this a random library of Escherichia coli HindIII fragments was cloned in the lambda Charon 25 vector (prepared in F.R. Blattner's laboratory), and lambda Ch25rne+ was selected by its ability upon lysogenization to enable a temperature-sensitive (ts) rne-3071 mutant to grow and to exhibit normal RNA processing at the nonpermissive temperature of 45 degrees C. The level of RNase E was doubled in an rne+ strain lysogenized with lambda Ch25rne+. lambda Ch25rne+ directs the synthesis of a polypeptide of 71 000 m.wt., which is the size of RNase E. Restriction analysis and electron micrography of heteroduplexes suggested that the size of the host DNA insert is about 1.9 kb.     

Salmonella typhimurium mutants with reduced levels of transfer ribonucleic acid-inhibitable endodeoxyribonucleolytic activity.

Two methods of mutagenesis, chemical alkylation and insertion of the tetracycline resistance transposon Tn10, were used to generate mutants of Salmonella typhimurium which had reduced levels of endodeoxyribonucleolytic activity. The chemically induced mutations defined a locus, endA, which was cotransduced with serA at a low frequency and with metK at a high frequency. Three-factor crosses revealed that metK was between serA and endA. The major endodeoxyribonucleolytic activity in crude extracts of s. typhimurium was similar to the activity of Escherichia coli endonuclease I. A Tn10 insertion mutation of endA resulted in the most severe loss of endodeoxyribonucleolytic activity among the endA alleles studied. Two of the chemically induced mutations resulted in activities which were more thermolabile than the wild-type activity.     

Genetic mapping of a mutation that causes ribonucleases III deficiency in Escherichia coli.

the mutation that causes ribonuclease III (RNase III) deficiency in strain AB301-105 of Kindler et al. (1973) has been mapped by use of F' merodiploids, Hfr matings, and P1 transduction. This mutation, rnc-105, lies close to nadB, near 49 min on the genetic map of Escherichia coli. The rnc-105 mutation has been transferred from its original genetic background by transduction and conjugation, and these new strains have the same defects in ribonucleic acid processing reported previously for AB301-105. Strains that carry rnc-105 grow more slowly than parental rnc+ strains, but the difference in growth rate seems to depend on the genetic background of each strain. Bacteriophage T7 grows about equally well in RNase III+ and III- female strains of E. coli, even though the specific cuts that RNase III makes in T7 ribonucleic acid are not made in the RNase III- strains. A low-phosphate defined medium in which most E. coli strains seem to grow well was developed. This medium is equally useful for labeling ribonucleic acids with 32PO4 and as a selective medium for genetic manipulations. It was used to determine the growth requirements of strain AB301-105, which are biotin and succinate in addition to the methionine and histidine requirements of the parental strain. The biotin mutation lies near the position expected from known mutations of E. coli, but the succinate mutation apparently does not. The possibility that the succinate requirement could be due to the RNase III deficiency is discussed. A uraP mutation was isolated for use in transferring rnc-105 between strains by conjugation. It lies near 47 min, somewhat removed from the commonly accepted position for uraP.     

Crystal structure of RuvC resolvase in complex with Holliday junction substrate

The key intermediate in genetic recombination is the Holliday junction (HJ), a four-way DNA structure. At the end of recombination, HJs are cleaved by specific nucleases called resolvases. In Gram-negative bacteria, this cleavage is performed by RuvC, a dimeric endonuclease that belongs to the retroviral integrase superfamily. Here, we report the first crystal structure of RuvC in complex with a synthetic HJ solved at 3.75 Å resolution. The junction in the complex is in an unfolded 2-fold symmetrical conformation, in which the four arms point toward the vertices of a tetrahedron. The two scissile phosphates are located one nucleotide from the strand exchange point, and RuvC approaches them from the minor groove side. The key protein–DNA contacts observed in the structure were verified using a thiol-based site-specific cross-linking approach. Compared with known complex structures of the phage resolvases endonuclease I and endonuclease VII, the RuvC structure exhibits striking differences in the mode of substrate binding and location of the cleavage site.     

Salmonella typhimurium mutants lacking protease II.

Mutants of Salmonella typhimurium lacking protease II, an endoprotease with trypsin-like specificity, have been isolated. These mutants can be identified by using the chromogenic substrate N-methyl-N-p-toluenesulfonyl-L-lysine beta-naphthyl ester to screen colonies growing on agar for the presence of the enzyme. All of the mutations isolated map at locus tlp (typsin-like protease) which is cotransducible (approximately 1%) using phage P1 with tre (trehalose utilization) at approximately 58 min on the Salmonella map. Double mutants lacking both protease I and protease II have been constructed. These strains grew normally. They were able to degrade abnormal proteins and to carry out protein turnover during carbon starvation at the same rate as the wild type.     

Properties of Escherichia coli mutants altered in calcium/proton antiport activity.

Mutants sensitive to growth inhibition by CaCl2 were found to have alterations in calcium uptake in everted membrane vesicles. These mutations map at different loci on the Escherichia coli chromosomes. A mutation at the calA locus results in vesicles which have two- to threefold higher levels of uptake activity than vesicles from wild-type cells. The calA mutation is phenotypically expressed as increased sensitivity to CaCl2 in a strain also harboring a mutation in the corA locus, which is involved in Mg2+ transport. The calA locus maps very close to purA and cycA at about min 97. The calB mutation results both in sensitivity to CaCl2 at pH 5.6 and in vesicles with diminished calcium transport capability. The CalB phenotype is also expressed only in a corA genetic background; the calB locus appears to map very near, yet separately from, the calA locus. When the cor+ allele is present, calA and calB mutations still result in a defect in calcium transport in vesicles. In addition, both calC and calD mutations result in vesicles with impaired calcium transport activity. calC is cotransducible with kdp and nagA, whereas calD is cotransducible with proC.     

Genetic mapping of the Salmonella typhimurium pepB locus.

Transposon technology has been used to map the pepB locus of Salmonella typhimurium. This locus is cotransducible by phage P22 with glyA and strB at min 56 on the Salmonella genetic map. The gene order is strB pepB glyA.     

Identification of a deoxyribonuclease implicated in genetic transformation of Diplococcus pneumoniae.

A mutation of Diplococcus pneumoniae, end-1, reduces the major deoxyribonuclease activity of the cell, an endonuclease, to 10% of its normal value without impairing transformation. Further mutations, called noz, abolish the residual endonuclease activity and block transformation. The residual endonuclease is similar to the wild-type enzyme in size, charge, divalent cation dependence, inhibition by ribonucleic acid, and formation of oligonucleotide products. However, the mutant endonuclease is more temperature sensitive, which suggests that the end-1 mutation occurred in a structural gene for the enzyme. Genetic analysis showed that the noz mutations occur at the same genetic locus. A number of new end mutants were analyzed. Those that retained more than 1.4% of the normal endonuclease activity were essentially normal in transformation; those with less than 1% were defective. The transformation-defective end mutants appear to be blocked in the entry of deoxyribonucleic acid (DNA) since they carry out the prior step of binding DNA to the outside of the cell. The major endonuclease of the cell may act as a DNA translocase by attacking and degrading one strand of DNA, thereby facilitating entry of the complementary strand into the cell.     

TolF locus in Escherichia coli: chromosomal location and relationship to loci cmlB and tolD.

The tentative map position on the Escherichia coli chromosome of the tolF locus, determining tolerance to colicins A, E2, E3, K, and L, has been confirmed by three-point transduction. It lies between the aroA and pyrD loci at about 21 min on the linkage map of Bachmann et al. (1976). The cmlB locus, determining increased resistance to the antibiotics chloramphenicol and tetracycline, also lies in this region (Reeve, 1966). Phenotypic and genetic comparison of isogenic strains that carry a mutation in either the tolF or cmlB locus makes it likely that these loci are closely related or identical. The tolD locus determining tolerance to colicins E2 and E3 as well as increased resistance to antibiotics has been reported to be located close to the aroA locus as a result of conjugation experiments (Eriksson-Grennberg et al. 1965). However, tolD did not cotransduce with any of several loci in this region, indicating that the mutation is not located within the region of the genetic map corresponding to approximately 19 to 22.5 min.