Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli

A study is described of the regulation of porphyrin synthesis in Escherichia coli using a heme-permeable, hemH deletion mutant, designated VS212. This strain utilizes only exogenous hemin that is supplied in the medium and accumulates porphyrins since the final step in the synthesis of heme is genetically blocked. It is possible, therefore, to monitor the rate of synthesis of heme by examining the accumulation of porphyrins. Using this system, we found that the rate of production of porphyrins depended on the availability of heme. The lower the concentration of hemin in the medium, the higher the level of porphyrins that accumulated. We next examined the mechanism responsible for the activation of porphyrin synthesis upon starvation for heme. The main activation occurred at the step that leads to the synthesis of 5-aminolevulinic acid (ALA). Starvation for heme induced the expression of a hemA-lacZ fusion gene, as previously reported, but an activation pathway that is independent of the hemA promoter was also identified. We found that starvation for heme caused the stringent response, and such starvation promoted the synthesis of porphyrins without having any effect on the expression of the hemA-lacZ fusion gene. We suggest a model for the regulation of porphyrin synthesis whereby the synthesis of porphyrins is coordinated with that of proteins. Received: 28 January 1997 / Accepted: 13 March 1997     

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.     

Escherichia coli mutants temperature-sensitive for DNA synthesis

Summary A series of mutants of E. coli temperature-sensitive for DNA synthesis has been studied. The temperature-sensitive DNA mutations map in seven distinct genetic loci most of which have not been previously reported. Mutations in dnaA and in dnaC affect the initiation of DNA replication; those at the remaining loci affect chain elongation. A temperature-sensitive Flac is shown to suppress a group A mutant with somewhat less efficiency than other F factors previously reported by others. The gene products rendered temperaturesensitive by the mutations have not been identified for any of the loci.     

Escherichia coli mutants with temperature-sensitive synthesis of DNA

Summary Seven mutants of E. coli with temperature-sensitive synthesis of DNA have been isolated. Synthesis of RNA, protein and DNA precursors does not appear to be directly affected. The mutants can be divided into at least two groups on the basis of their pattern of DNA synthesis, their ability to support phage growth at 41° and their genetic mapping.Mutants of the first group are heterogeneous in their pattern of DNA synthesis at 40°. Some mutants cease DNA synthesis abruptly upon transfer to 40° and any residual DNA synthesis is barely detectable. In others there is substantial residual synthesis at 40°. All these Group 1 mutants are alike, however, in that they support the growth of phage T4 but not Lambda at 41°. Two mutants with barely detectable residual DNA synthesis carry DNA mutations which have been mapped by P1 transduction and show about 72% linkage to the malB locus. It has not yet proved possible to map accurately the mutants showing substantial residual synthesis, and the possibility that these mutations are in a different gene(s) has not been excluded.A single mutant has been placed in a second group. Like some Group 1 mutants it synthesizes substantial amounts of DNA at 40° before synthesis stops. However, unlike them it supports the growth of T4 and Lambda at 41°. The DNA mutation maps near the leu locus. Certain properties of this mutant are consistent with the idea that initiation of DNA synthesis is temperature-sensitive in this strain.Adapted from a dissertation presented in partial fulfillment of the degree of Doctor of Philosophy. This investigation was supported in part by U.S. Public Health Services Grant 5-TO1-GM00829 from the National Institute of General Medical Sciences and in part by U.S.P.H.S. research grant GM12524.     

Hyper-recombination in dam mutants of Escherichia coli K-12

Summary F-prime heterogenotes of dam-3 bacteria segregate F-prime homogenotes at a frequency 30–200 times higher than the isogenic dam ⁺ strain. A hyperrecombination mutant which shows increased recombination between chromosomal duplications was characterized as a dam mutant. The dam-3 allele causes a reduction in linkage of proximal unselected markers in transconjugants and increases the recombination frequency between a pair of closely linked markers. It is concluded that dam mutations confer a hyperrecombination phenotype to the cell.     

Hyper-recombination in uvrD mutants of Escherichia coli K-12

Summary A mutant strain of E. coli which was isolated initially because of its strong hyper-recombination phenotype was shown to carry a lesion in uvrD. The presence of this mutation, designated uvrD210, increased the frequency of recombination between chromosomal duplications in F-prime repliconant cells and reduced linkage between closely linked markers in crosses with Hfr donors. A comparable hyper-rec phenotype was demonstrated in strains carrying other alleles of uvrD previously referred to as mutU4, uvr502 and recL152. The recombination activity of a uvrD210 strain was abolished by mutation of recA but the mutator activity associated with this allele proved to be independent of recA. It is suggested that uvrD mutations reduce the fidelity of DNA replication and that the accumulation of lesions in the newly synthesized strand provides additional sites for initiating recombination.     

Conditional lethal phosphatidylserine decarboxylase mutants of Escherichia coli

Summary The final step in the biosynthesis of phosphatidylethanolamine, the major membrane lipid of Escherichia coli, is catalyzed by the membrane-bound enzyme, phosphatidylserine decarboxylase. A variation of a procedure for localized mutagenesis (Hong and Ames, 1971) was employed to generate conditional lethal mutants in phosphatidylserine decarboxylase. In our modification, an episome carrying the psd gene closely linked to purA ⁺ was heavily mutagenized in vivo in a strain also lysogenic for phage P1 CMclr100. After induction of a phage lytic cycle, the purA ⁺ marker was transduced to a purA ^- recipient. A majority of the Pur⁺ transductants thus contained a psd gene originating from the heavily mutagenized episomal strain. Three mutants were isolated in which temperature-sensitive growth is caused by thermosensitive phosphatidylserine decarboxylase activity that is defective in vivo at the non-permissive temperature. All 3 mutations were mapped at the same location as psd1, being cotransduced with melA, purA, and ampA. The gene order in this region, as determined by a phage P1-mediated, three-factor cross is ampA-psd-purA. psd ⁺ is dominant to the psd mutant alleles.     

Ribosomal protein modification in Escherichia coli

Summary A mutant of Escherichia coli K12 has been isolated which shows an alteration in the ribosomal protein S18. Genetic analyses have revealed that the mutation causing this alteration maps at 99.3 min of the E. coli genetic map, between dnaC and deo. This indicated that the mutation has occurred in a gene different from the structural gene for this protein which has been located at 94 min. From the N-terminal amino acid sequence analysis it is concluded that the mutation has resulted in loss of the N-terminal acetyl group of this protein. The gene which is affected in this mutant is termed rimI that most likely specifies an enzyme acetylating the N-terminal alanine of protein S18. The mutation does not affect the acetylation of two other ribosomal proteins, S5 and L12, both of which are known to be acetylated in wild-type E. coli K12.     

Inducible sfi dependent division inhibition in Escherichia coli

Summary Brief exposure of an Escherichia coli tif lon strain to 40° results in subsequent prolonged inhibition of cell division (part of the SOS response), which is completely and specifically suppressed by sfiA and sfiB mutations. This sfi dependent division inhibition requires protein synthesis during the 40° incubation period, implying the existence of a tif-inducible protein which results in cell division arrest. sfi dependent division inhibition is also induced early during thymine starvation in tif ⁺ cells; at later times a sfi independent mechanism of division arrest is invoked as well.In lon mutants, known to lack a protease, the sfi dependent division inhibition is amplified, perhaps due to stabilization of the inducible protein involved in division arrest. In these strains the P1 lysogenization defect and the filamentation observed after a nutritional shift-up are sfi dependent, suggesting that P1 infection and nutritional shift-up may also induce the protein involved in division arrest. Bacteria are known to increase in size following a shift-up. Thus the latter observation suggests that the SOS response may be not only a last resort in time of distress but also a means permitting better adaptation of the cells to their environment.After five years of heroic struggle against cancer, Jacqueline George passed away 14 August 1979. Despite weakened health and debilitating therapy she continued to stimulate and participate in the work of the microbial genetics group which she had created     

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)     

Development of a downstream process for the isolation of Staphylococcus aureus arsenate reductase overproduced in Escherichia coli

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite). In order to study the structure of ArsC and to unravel biochemical and physical properties of this redox enzyme, wild type enzyme and a number of cysteine mutants were overproduced soluble in Escherichia coli. In this paper we describe a novel purification method to obtain high production levels of highly pure enzyme. A reversed-phase method was developed to separate and analyze the many different forms of ArsC. The oxidation state and the methionine oxidized forms were determined by mass spectroscopy.     

Control of ribosomal RNA synthesis in Escherichia coli

Summary The ribosomal RNA synthesis in a cell-free system containing the nucleoids and the cytoplasmic fraction prepared from Escherichia coli cells has been investigated. The addition of the 4S fraction from the cytoplasm to the isolated nucleoids induces RNA synthesis by a new chain initiation. In this system a preferential initiation of rRNA chains occurs. The experimental results suggest that the 4S fraction contains at least two activities, one for releasing RNA-polymerases from the nucleoids, and another for the frequent initiation of rRNA chains. No restriction of the rRNA synthesis has been observed in the nucleoids and the 4S fraction from the amino acidstarved rel ⁺ cells. The rRNA synthesized in the above system is detected at about 23S and 16S rRNA regions.     

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.     

Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis

Summary Uvm mutants of Escherichia coli K12 selected for defective UV reversion induction have previously been reported to differ considerably from the UV-reversion-less recA and lexA mutants with regard to survival or mutagenic response to UV, X-rays and alkylating agents. In the present study, the phenotypic characterization of uvm mutants was extended to investigate several cellular processes which also may be related to or involved in UV mutagenesis. Like recA and lexA mutations, the uvm mutations exhibit highly reduced Weigle reactivation and normal host cell reactivation of UV irradiated phage . But unlike recA and lexA, the uvm mutations do not impair genetic recombination, UV induction of prophage or R plasmid-mediated UV resistance and mutagenesis. These phenotypical characteristics and preliminary results of genetic mapping lend further support to the assumption that the uvm site may be a novel locus affecting, apart from the recA and lexA loci, the error-prone repair pathway in E. coli.     

Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis

Summary Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis.     

Regulatory mutants of dihydrofolate reductase in Escherichia coli K12

Summary Trimethoprim inhibits dihydrofolate reductase. Mutations conferring trimethoprim-resistance on E. coli K12 result in either an altered reductase with decreased affinity for the drug, or in 2–30 fold higher levels of the enzyme. Studies of the latter class of mutants indicate that dihydrofolate reductase is regulated by a diffusible molecule, and is probably under negative control. The regulatory mutants, some of which are temperature-sensitive, act cis.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.