Map location of arginyl-tRNA synthetase mutations in Escherichia coli K-12

Summary Mutants of Escherichia coli K-12 previously isolated in the authors' laboratory have reduced arginyl-tRNA synthetase activity. The mutants fall into two classes. All mutants grow slowly on arginine-free medium. On arginine-supplemented medium some mutants grow at a normal rate (Class I) while others still grow slowly (Class II). Matings were performed to located a Class I and a Class II mutation on the E. coli chromosome map, and on the basis of our results we have assigned both to one locus, argS.     

Temperature-sensitive mutants in cysteinyl-tRNA ligase of E. coli K-12.

Summary Two mutants with a defective cysteinyl-tRNA synthetase have been found in a collection of spontaneous temperature sensitive mutants. The mutated gene, which is designated cysS, is closely contransduced with purE.     

Isolation of lysine codon suppressors inEscherichia coli

Starting with anEscherichia coli strain containingglyT56, a glycine transfer RNA suppressor of the arginine codons AGA and AGG, and atrpA mutant containing lysine at position 211 of the tryptophan synthetase alpha chain, we have isolated AAG-suppressors that fall into two classes. In class 1 are dominant suppressors that arose with the simultaneous loss ofglyT56 activity. They are approximately 50% cotransducible withargE, as isglyT, and appear to be derived fromglyT56. Class 2 suppressors, located betweenpurE andtrp on theE. coli map, are not near any glycine tRNA genes, and may represent novel missense suppressors.     

A Third Kasugamycin Resistance Locus, ksgC, Affecting Ribosomal Protein S2 in Escherichia coli K-12

A third kasugamycin-resistant mutant affecting ribosomal protein S2 has been isolated from Escherichia coli K-12. Mating and transduction revealed that this newly recognized kasugamycin resistance locus, designated as ksgC, is located at 0.1 to 0.2 min from purE.     

Isolation and genetic mapping of Escherichia coli aminopeptidase mutants

Summary Many mutant strains devoid of aminopeptidase activity have been isolated in Escherichia coli. All of them produce cross-reacting material when tested against specific antiaminopeptidase antibody. The map position of the locus specifying this enzyme has been determined by three conjugations and two P₁ mediated transduction experiments. By analogy with Salmonella typhimurium this locus has been called pepN (Miller, 1975). Mutations in pepN are jointly transduced with fabA and pyrD at high frequency. These data and conjugation results suggest a location between 20.5 and 22.5 minutes on E. coli genetic map.     

The use of suicide substrates to select mutants of Escherichia coli lacking enzymes of alcohol fermentation

Summary Mutants of Escherichia coli resistant to chloroethanol or to chloroacetaldehyde were selected. Such mutants were found to lack the fermentative coenzyme A (CoA) linked acetaldehyde dehydrogenase activity. Most also lacked the associated fermentative enzyme alcohol dehydrogenase. Both types of mutants, those lacking acetaldehyde dehydrogenase alone or lacking both enzymes, mapped close to the regulatory adhC gene at 27 min on the E. coli genetic map. The previously described acd mutants which lack acetaldehyde dehydrogenase and which map at 63 min were shown to be pleiotropic, affecting respiration and growth on a variety of substrates. It therefore seems likely that the structural genes for both the acetaldehyde and alcohol dehydrogenases lie in the adhCE operon. This interpretation was confirmed by the isolation of temperature sensitive chloracetaldehyde-resistant mutants, some of which produced thermolabile acetaldehyde dehydrogenase and alcohol dehydrogenase and were also found to map at the adh locus. Reversion analysis indicated that mutants lacking one or both enzymes carried single mutations. The gene order in the adh region was determined by three point crosses to be trp - zch:: Tn10 - adh - galU- bglY - tyrT - chlC.     

Regulatory gene mutations affecting arginine biosynthesis in Escherichia coli

Summary Mutants of Escherichia coli with altered regulation of arginine biosynthesis were isolated. The alterations in all of the mutants with increased levels of the biosynthetic enzymes were found to map in the argR locus. The mutants were grouped into three classes based on their effect on the regulatory behavior. Complementation studies with stable merodiploid strains demonstrated that the derepressed synthesis in the mutants was recessive to wild-type regulation.     

Syntheses and Plant Growth Retardant Activities of Hydrazones Containing a Terpenoid Moiety Derived from Girard’s Reagent T

The genetic background of the protein-leaky mutant of Escherichia coli (PE4LA) was examined. This mutant preferentially excretes periplasmic proteins and outer membrane components. The results obtained from various crosses indicated that the mutation responsible for protein leakage was located near the purE gene. The amount of protein excreted decreased to about half whenever PE4LA was converted from Pur^? to Pur⁺ either by transduction or spontaneous reversion. When purine or pyrimidine auxotrophs were obtained from purE⁺ revertant of PE4LA, only auxotrophs whose blockages were located at a step identical or close to that governed by purE gene excreted proteins to the same extent as PE4LA. Excess adenine in the culture medium repressed exprotein accumulation by PE4LA. These results suggest that purE is involved in some way with protein excretion, and perhaps the accumulating metabolic intermediate(s) causes protein excretion due to a defect in purE enzyme.     

A genetic assay for transversion mutations in bacteriophage T4

Summary Seventy-two mutants deficient in formate-nitrate reductase activity were selected in Escherichia coli strain PK 27, by two different procedures. Forty-five strains were selected on the basis of chlorate resistance and 27 strains were selected by their inability to reduce nitrate with formate as an electron donor. Genetic analysis of these strains showed that the two techniques yield distinctly different distributions of mutants among the various controlling genetic loci. Chlorate resistance appears to select for severe alterations in the nitrate reductase system; 98% of these mutants fell into the pleiotropic chl A, B, D and E classes and are deficient in all the activities of the formate-hydrogenlyase pathway as well as formate-nitrate reductase pathway. In contrast, 48% of the mutants selected for their inability to reduce nitrate with formate as the electron donor were of the chl C class and two new classes were identified among mutants selected by this procedure. Chl F mutants are linked to tryptophan and the chl C locus. Chl G mutants map at zero minutes on the E. coli genetic map.     

Genetic analysis of lon mutants of strain K-12 of Escherichia coli

Summary Following UV irradiation of AB1157 31 mucoid ultraviolet light UV sensitive mutants were isolated. These were all induced to form filaments by UV irradiation, i.e. they had all the phenotypic properties of Lon mutants. These lon mutants fell into two phenotypic classes based on their sensitivity to UV. The gene determining UV sensitivity and mucoidy in all mutants of both Class A and Class B was cotransducible with proC. Intra-class crosses by Pl transduction yielded no UV resistant recombinants. Inter-class crosses yielded UV resistant nonmucoid recombinants, the frequency depending on the direction of the cross. The data imply two adjacent blocks in the lon region of E. coli and the order of markers in this region is probably proC tsx lon Class A lon purE Class B.This work was carried out under Public Health Service Grant CA 05687-08 from the National Cancer Institute.Recipient of a Public Health Service Career Development Award.     

Supersensitivity of Escherichia coli Cells to Several β-Lactam Antibiotics Caused by Rod− Morphology Mutations at the mrd Gene Cluster

Two kinds of spherical mutants, mrdA and mrdB mutants, have been isolated from Escherichia coli strain K12. The mrdA mutants have thermosensitive penicillin-binding protein 2, while the mrdB mutants have normal penicillin-binding proteins. Both kinds of mutants form spherical cells at 42°C and are resistant to the amidinopenicillin, mecillinam, at the same temperature. The two mutations have been mapped very close to lip at 14.2 min (revised chromosome linkage map, 1980) on the E. coli chromosome. Both mutations cause supersensitivities of cell growth to various β-lactam antibiotics, such as ampicillin, cephalexin, cefoxitin and nocardicin A at 42°C.     

Host interference with expression of the lambda N gene product

We have searched among E. coli M72 (D, bio11cI857H1) temperature resistant survivors and have found two bacterial mutants, gro100 and gro101 which block λiλ and λi⁴³⁴ phage development but allow growth of their N-independent derivatives λiλ nin and λi⁴³⁴nin. It is not known yet whether these two mutants interfere with the production of the N gene product or with its function. At least part of the gro genotype maps at 12′ of the E. coli genetic map and is co-transductible by Pl with the lac locus.     

Genetic characterization of the Escherichia coli cyclopropane fatty acid (cfa) locus and neighboring loci

Summary The phenotypically silent cyclopropane fatty acid synthesis (cfa) gene of Escherichia coli K-12 has been located on the genetic linkage map. This was accomplished by integrating (via homologous recombination) the selectable marker of a recombinant plasmid into the host chromosome near the cfa locus. This integration allowed the subsequent isolation of a cfa-linked transposon Tn10 insertion. Genetic mapping of the Tn10 insertion, using conventional techniques, placed the cfa locus at min 36.5 on the linkage map in the vicinity of several other non-selectable markers. We ordered cfa and these other loci by three-factor transductional analyses. Selection for excision of the Tn10 element resulted in several types of mutants which harbor mutations of cfa and of neighboring genes, presumably as a consequence of Tn10-catalyzed chromosomal rearrangements.     

Mechanism of conjugation

Summary A new conditional thermosensitive Hfr mutant of Escherichia coli K-12 was isolated. The ts mutation is cotransducible with purE and tsx loci on the E. coli chromosome. Upon temperature shift to 42° C the DNA synthesis and transfer of chromosome is stopped immediately and RNA, protein synthesis in about ten minutes.     

D-Amino acid dehydrogenase of Escherichia coli K12: positive selection of mutants defective in enzyme activity and localization of the structural gene

Summary A method for the positive selection of dadA mutants defective in Dolor-amino acid dehydrogenase has been devised. It consists in isolating mutants resistant to -chroro-Dolor-alanine and screening for mutant colony color on a special agar medium. All 70 Escherichia coli K12 dadA mutants isolated either by this method or by other selection procedures map at a locus which is near to hemA and closely linked with dadR. Since some of the dadA mutants are thermosensitive in Dolor-methionine utilization in vivo and have thermolabile Dolor-amino acid dehydrogenase in vitro, it is proposed that the dadA gene codes for the enzyme structure. The broad substrate specificity, apparent membrane localization, inducibility by alanine, and repressibility by glucose strongly suggest that the Dolor-amino acid dehydrogenase coded by the dadA gene is a species variant of the enzyme described under the same name in Salmonella typhimurium. It may be identical or homologous with the enzymes described under the names alaninase, Dolor-alanine oxidase or Dolor-alanine dehydrogenase in E. coli K12 or B.     

Ribosomal protein modification in Escherichia coli

Summary Among mutants of E. coli selected for temperaturesensitive growth, four were found to possess alterations in ribosomal proteins L7/L12. Of these, three apparently lack protein L7, the acetylated form of protein L12. Genetic analyses have revealed that the mutation responsible for this alteration maps at a locus around 34 min of the current E. coli genetic map, which is clearly different from the location for the structural gene for protein L7/L12 which is situated at 89 min. Hence, the gene affected in these mutants was termed rimL. Tryptic and thermolysin fingerprints of the protein L12 purified from the rimL mutants showed a profile indistinguishable from that of wild-type protein. It was found that the acetylase activity specific for protein L12 was negligible, when assayed in vitro, in the high-speed supernatant prepared from mutant cells. These results indicated that the three mutants contain mutations in the gene rimL that codes for an acetylating enzyme specific for ribosomal protein L12.Previous paper in this series is Isono and Isono (1980)     

Molecular cloning of genes involved in purine biosynthetic and salvage pathways of Salmonella typhimurium

Summary Genes have been cloned from Salmonella typhimurium which when present on the multicopy plasmid pBR322 in the E. coli strain NT31 confer a Gua⁺ phenotype on this strain. NT31 is a purE gpt double mutant and it was expected that a Gua⁺ phenotype could be conferred on it by the cloning of either gpt or purE. It was, however found that in addition to these two loci the molecular cloning of another gene, which has been identified as hpt, in pBR322 confers a Gua⁺ phenotype on NT31. This result is explained by the overproduction of the hpt gene product, hypoxanthine phosphoribosyl transferase, which compensates for the lack of the gpt product guanine-xanthine phosphoribosyl transferase. Restriction analysis of the three loci, gpt, hpt and purE is also presented.Abbreviations Kb kilobase pairs - Tc tetracyline - m.o.i multiplicity of infection - 8AG 8-azaguanine     

The absence of a surface protease, OmpT, determines the intercellular spreading ability of Shigella: the relationship between the ompT and kcpA loci

A large plasmid-encoded protein, VirG, on the bacterial surface is essential for the spreading of Shigella by eliciting polar deposition of filamentous actin In the cytoplasm of epithelial cells. VirG expression from the large plasmid is diminished greatly when it is introduced into Escherichia coli K-12 from Shigella. In an attempt to identify factors affecting VirG expression, we found that the absence of the ompT gene, encoding outer membrane protease OmpT, restored full production of VirG protein to E. coli K-12. Conversely, upon introduction of the ompT gene of E. coii K-12 into Shigella, spreading ability was completely abolished, probably because of the proteolytic degradation of VirG protein by OmpT. Analysis of the DNA sequence of the ompT region indicated that the absence of the ompT gene occurred in Shigella and enteroinvasive E. coli strains, and that the absent DNA segment corresponded to a remnant lambdoid phage structure found in E. coli K-12, which encompasses a 21 kb DNA segment spanning from argU through to the ompT genes. Since ompT is located near purE in E. coli K-12 and a virulence locus for provoking keratocon-junctivitis in the eyes of guinea-pigs, named kcpA is located near purE in S. fiexnerl, and the two loci are involved in VirG expression, the KcpA～ mutants of S. flexneri 2a constructed were examined for correlation between acquisition of ompT and VirG degradation. Our data suggest that the previous recognition of a kcpA locus in S. flexneri is the result of transfer of the ompr gene from E. coli K-12, giving rise to a KcpA phenotype. These results indicate that the lack of OmpT protease confers upon Shigella the ability to spread into adjacent epithelial cells.     

Genetic analysis of essential genes in the ftsE region of the Escherichia coli genetic map and identification of a new cell division gene, ftsS

Summary Several conditional lethal mutants of Escherichia coli have been analysed genetically using generalized transduction and lambda transducing vectors. Three temperature-sensitive ftsE mutants were found as was a cold-sensitive ftsE mutant. A new gene was foud which mapped close to ftsE, namely ftsS. Both cell division genes map close to the gene which controls the heat-shock regulon (htpR).