Pleiotropic Properties and Genetic Organization of the tolA, B Locus of Escherichia coli K-12

Colicin-tolerant mutants of Escherichia coli K-12, which map near gal at 17 min (tolA, B mutants), have been isolated and characterized. These mutants exhibited a very broad spectrum of phenotypic changes consistent with the interpretation that they are cell surface mutants. In addition to being colicintolerant and sensitive to deoxycholate and ethylenediaminetetraacetic acid, tolA, B mutants are sensitive to vancomycin, bacitracin, and dodecyl sulfate. The tolA, B mutants from most strains also formed mucoid colonies at 30 C on nutrient agar plates and had a greatly increased plating efficiency for lysisdefective S mutants of bacteriophage lambda. Complementation analysis showed that the four phenotypic groups of tol mutants that map near gal fall into three complementation groups: tolP, tolA, and tolB. Recombination analysis by three-factor crosses established the order of the three groups as tolP-tolA-tolB-gal. Because of the wide variety of phenotypic changes that accompanies mutation to colicin tolerance, revertants were isolated to test whether single or multiple mutations were involved. The reversion analysis, as well as other genetic criteria, confirmed that only single mutations were involved, suggesting that these pleiotropic changes are a consequence of a single change in the E. coli cell surface.     

The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo

Saccharomyces cerevisiae Dna2 protein is required for DNA replication and repair and is associated with multiple biochemical activities: DNA-dependent ATPase, DNA helicase, and DNA nuclease. To investigate which of these activities is important for the cellular functions of Dna2, we have identified separation of function mutations that selectively inactivate the helicase or nuclease. We describe the effect of six such mutations on ATPase, helicase, and nuclease after purification of the mutant proteins from yeast or baculovirus-infected insect cells. A mutation in the Walker A box in the C-terminal third of the protein affects helicase and ATPase but not nuclease; a mutation in the N-terminal domain (amino acid 504) affects ATPase, helicase, and nuclease. Two mutations in the N-terminal domain abolish nuclease but do not reduce helicase activity (amino acids 657 and 675) and identify the putative nuclease active site. Two mutations immediately adjacent to the proposed nuclease active site (amino acids 640 and 693) impair nuclease activity in the absence of ATP but completely abolish nuclease activity in the presence of ATP. These results suggest that, although the Dna2 helicase and nuclease activities can be independently affected by some mutations, the two activities appear to interact, and the nuclease activity is regulated in a complex manner by ATP. Physiological analysis shows that both ATPase and nuclease are important for the essential function of DNA2 in DNA replication and for its role in double-strand break repair. Four of the nuclease mutants are not only loss of function mutations but also exhibit a dominant negative phenotype.     

The isolation and genetic analysis of V79-derived etoposide sensitive Chinese hamster cell mutants: two new complementation groups of etoposide sensitive mutants

Using a replica plating microwell method, three Chinese hamster V79-derived cell lines, designated ETO1, ETO2 and ETO3, which exhibit hypersensitivity to the non-intercalating topoisomerase II inhibitor etoposide have been isolated. Mutant lines ETO2 and ETO3 are cross-sensitive to the topoisomerase II inhibitors adriamycin and streptonigrin; however, neither mutant is sensitive to the topoisomerase I inhibitor camptothecin, the bifunctional alkylating agent mitomycin C, nor hydrogen peroxide. In contrast, ETO1 is cross-sensitive to camptothecin but displays only slight sensitivity to adriamycin, streptonigrin and hydrogen peroxide, and is not sensitive to mitomycin C. It has been established through extensive cell fusion studies that all three mutants are genetically distinct, and that ETO2 and ETO3 genetically complement all other known etoposide-sensitive Chinese hamster cell mutants (i.e., irs1, XR-1, xrs1, V3, BLM2, ADR1, ADR3, ADR4 and ADR5) thus defining two new complementation groups of etoposide sensitive mutants. Interestingly, the hybrids created by the fusion irs2TOR (thioguanine and ouabain resistant)xETO1 and the reciprocal cross ETO1TORxirs2 both exhibited a response to camptothecin intermediate with respect to V79 and ETO1. It has been hypothesised that this partial complementation may be the result of intragenic complementation and that both ETO1 and irs2 result from mutations in the gene XRCC8. This study indicates that cellular responses to topoisomerase II inhibitors are complex and hypersensitivity may result from mutations in many different genes.     

Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system.

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.     

Mutations determining mitomycin resistance in Bacillus subtilis

Iyer, V. N. (Microbiology Research Institute, Canada Department of Agriculture, Ottawa, Canada). Mutations determining mitomycin resistance in Bacillus subtilis. J. Bacteriol. 92:1663-1669. 1966.-The pattern of development of genetic resistance in Bacillus subtilis to mitomycin C was studied, and spontaneous single and multistep mutants were obtained. The transmission and expression of these mutations in sensitive strains proved possible by means of genetic transformation. The mutations were genetically studied in relation to a chromosomal mutation, mac-1, which confers resistance to the macrolide antibiotic erythromycin and which has been previously localized in the early-replicating segment of the B. subtilis chromosome. The results indicate that all of three primary mutations studied in this manner, as well as a secondary and tertiary mutation derived from one of the primary mutations, are clustered in this early-replicating segment. It appears that the secondary and tertiary mutations enhance the resistance conferred by the primary mutation, apparently without themselves conferring any resistance.     

Fe(II)/Cu(I)-dependent P-type ATPase activity in the liver of Long-Evans cinnamon rats

This study examined Fe(II)-dependent ATPase activity in OTG (octylthioglucoside) -treated microsomes isolated from Wistar and LEC rats. The ATPase activity of the liver OTG-microsomes from Wistar rats increased sharply in the 5-150 microM range of Fe(II) with a K0.5 value of 23.9+/-3.6 microM, while the activity of LEC rat liver microsomes increased with increasing Fe(II) up to 500 microM with a K0.5 value of 64.4+/-8.1 microM. The K0.5 values for Fe(II)-dependent ATPase activity of spleen OTG-microsomes were nearly identical at 59.3 microM in the Wistar rat and 63.7 microM in the LEC rats with a similar level of activity at each Fe(II) concentration in both strains of animals. These results indicated that there are two types of Fe(II)-dependent ATPase with different Fe(II) sensitivity, a high sensitive (H) and a low sensitive (L) type, and that the H-type activity was specific to the liver. The H-type activity was, however, deficient in the liver of LEC rats that accumulate copper and iron in hepatocytes as a result of mutations in the Wilson's disease protein (WNDP). On the basis of these results, together with the similarity in optimal conditions required for full activity of the enzyme, we conclude that the Fe(II)-dependent ATPase (H-type) and WNDP may be identical.     

The endogenous nuclease sensitivity of repaired DNA in human fibroblasts

The limited DNA excision repair that occurs in the chromatin of UV-irradiated growth arrested cells isolated from a xeroderma pigmentosum (XP) complementation group C patient is clustered in localized regions. The repaired DNA was found to be more sensitive to nicking by endogenous nucleases than the bulk of the DNA. The extra-sensitivity does not change with increasing amounts of DNA damage or repair activity in the locally-repaired regions and is retained through a 24-h chase period. We suggest that these results are due to the occurrence of DNA repair limited to pre-existing, non-transient chromatin fractions that contain actively transcribed DNA. A similar extra-sensitivity of repaired DNA was not detected in cells of normal or XP complementation group A strains that exhibit either normal or limited repair located randomly throughout their genomes. The association between endogenous nuclease sensitivity and clustered repair probably defines a normal excision repair pathway that is specific for selected chromatin domains. The repair defect in XP-C strains may be one in pathways targeted for other endogenous nuclease-resistant domains.     

Dinitrophenylation of rabbit skeletal sarcoplasmic reticulum ATPase protein

The ATPase (ATP phosphohydrolase (EC 3.6.1.3)) protein of rabbit skeletal sarcoplasmic reticulum rapidly incorporated three mol of 1-fluoro-2,4-dinitrobenzene per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-dependent ATPase activity was inhibited. The dinitrophenyl group was located mainly in the ATPase protein and a small amount of the label was found in the proteolipid component of the ATPase preparation as judged by dissociation experiments in sodium dodecyl sulfate. Cysteine and tyrosine residues were dinitrophenylated in the modified ATPase protein. Thiolysis of the dinitrophenylated ATPase protein with 2-mercaptoethanol under various conditions did not restore the Ca2+-dependent ATPase activity. Solubilization of the ATPase protein with sodium deoxycholate increased the reactivity of the reagent and the Ca2+-dependent ATPase activity was inhibited to a greater extent. Dinitrophenylation of the ATPase protein was Ca2+-dependent; in the presence of high Ca2+ the incorporation increased by 50% and a large decrease in the Ca2+-ATPase activity was noted. The half-maximal changes for the incorporation of the reagent and the inhibition of the Ca2+-ATPase activity occurred at 3--4 microgram Ca2+-concentration, consistent with the binding of Ca2+ to high affinity sites on the ATPase protein. These results indicate that the ATPase protein as a Ca2+-free and a Ca2+-bound conformation. The reagent, 1-fluoro-2,4-dinitrobenzene reacts differentially and thus characterizes these two conformations.     

Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca+ + Mg2+)-dependent adenosine triphosphatase.

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte membranes was solubilized with deoxycholate and purified by calmodulin affinity chromatography to yield a functional enzyme. The method gave an enzyme purified 207-fold as compared with that of the erythrocyte membranes. The molecular weight of the ATPase was in the range 135 000-150 000, as revealed by a single major band after electrophoresis on dodecyl sulphate/polyacrylamide gels. The isolated enzyme was highly sensitive to calmodulin, since the activity was increased about 9-fold. At 37 degrees C and in the presence of calmodulin the purified ATPase had a specific activity of 10.1 mumol/min per mg of protein. Triton X-100 or deoxycholate stimulated the calmodulin-deficient enzyme in a concentration-dependent fashion whereby the calmodulin-sensitivity was lost. The purification method is suitable for studying the lipid-sensitivity of the ATPase, since the lipids can easily be exchanged without a significant loss of activity. A purification procedure described by Niggli, Penniston & Carafoli [(1979) J. Biol. Chem. 254, 9955-9958] resulted in an enzyme that indeed was pure but was lacking a predominant feature, namely the modulation by calmodulin.     

Genetic complementation of propionyl-CoA carboxylase deficiency in cultured human fibroblasts.

Propionyl-CoA carboxylase (PCC) deficiency is an inherited metabolic disorder showing considerable variability of expression. We have investigated the possibility that there is a genetic basis for the clinical heterogeneity in this disorder by examining complementation in Sendai virus mediated heterokaryons of mutant fibroblast strains. Restoration of PCC activity was monitored in individual multinucleate cells in situ using a radioautographic procedure which detects the incorporation of 14C-propionate into trichloracetic acid precipitable material. Each mutant strain incorporated negligible amounts of radioactivity compared to control strains. Activity was not restored when different mutants were mixed without virus or when homokaryons were produced by self-fusion. Seven mutant strains were fused in all pairwise combinations and examined for increased 14C-propionate incorporation in heterokaryons. Two main complementation groups were revealed. One group was composed of three mutants. The other was a complex group composed of four mutants in which intragroup complementation was demonstrated. Two mutants showing excellent complementation by radioautography were examined for complementation by the direct assay of PCC ACTIVITY. The enzyme activity of virus-treated preparations with 23% multinucleate cells was 183 U (pmol/min/mg protein) compared to 16 U for the untreated mixture (normal range 450-850 u). We conclude that PCC deficiency resulted from mutations of heterogeneous origin, although the classification of mutants into complementation groups did not correlate with patterns of clinical heterogeneity.     

A yeast DNA repair gene partially complements defective excision repair in mammalian cells.

The RAD10 gene of Saccharomyces cerevisiae is required for nucleotide excision repair of DNA. Expression of RAD10 mRNA and Rad10 protein was demonstrated in Chinese hamster ovary (CHO) cells containing amplified copies of the gene, and RAD10 mRNA was also detected in stable transfectants without gene amplification. Following transfection with the RAD10 gene, three independently isolated excision repair-defective CHO cell lines from the same genetic complementation group (complementation group 2) showed partial complementation of sensitivity to killing by UV radiation and to the DNA cross-linking agent mitomycin C. These results were not observed when RAD10 was introduced into excision repair-defective CHO cell lines from other genetic complementation groups, nor when the yeast RAD3 gene was expressed in cells from genetic complementation group 2. Enhanced UV resistance in cells carrying the RAD10 gene was accompanied by partial reactivation of the plasmid-borne chloramphenicol acetyltransferase (cat) gene following its inactivation by UV radiation. The phenotype of CHO cells from genetic complementation group 2 is also specifically complemented by the human ERCC1 gene, and the ERCC1 and RAD10 genes have similar amino acid sequences. The present experiments therefore indicate that the structural homology between the yeast Rad10 and human Ercc1 polypeptides is reflected at a functional level, and suggest that nucleotide excision repair proteins are conserved in eukaryotes.     

Similarity in properties and mapping of three Rec mutants of Haemophilus influenzae.

Three Rec- mutants of Haemophilus influenzae have been studied with respect to their transformability, ultraviolet and mitomycin C sensitivities, spontaneous and ultraviolet-induced deoxyribonucleic acid breakdown, inducibility of lysogens, and the linkage of the three mutations to a streptomycin resistance marker. The data indicate that the three mutations cause the same phenotypic changes, and that they are all on the same gene. Transformability of the mutants is different when two different media are used for competence development, although transformability with the two competence methods is not different in a Rec- strain that is mutant at another gene.     

Ultraviolet light-induced mutation in UV-resistant, thermosensitive derivatives of lexA-strains of Escherichia coli K-12

It was shown previously that a major class of UV-resistant derivatives of lexA- strains of E. coli K-12 is defective in cell division at 42.5 degrees. The thermosensitive mutations, judging by genetic mapping and complementation tests, are believed to be intragenic suppressor mutations that lower the activity of the diffusible product that results in the LexA- phenotype (Mount et al., 1973). Several thermosensitive derivatives have been characterized in regard to their susceptibility to mutation induction by UV at the permissive growth temperature (30 degrees). Although the strains tested are approximately as resistant to UV as lexA+ strains, they showed a level of mutation induction that was considerably lower. By means of genetic complementation tests it was demonstrated that the low levels of UV mutagenesis in lexA- strains and their thermosensitive derivatives result from the synthesis of a diffusible product. One possible interpretation of these results is that a diffusible product in lexA- strains prevents the induction of error-prone repair. Altering the activity of this product by tsl mutations can lead to increased, but not normal, levels of error-prone repair.     

Isolation and characterization of mitomycin-C-sensitive mouse lymphoma cell mutants

26 mutants with increased sensitivity to the lethal effects of mitomycin C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Most of them were about 5-10 times more sensitive in terms of D37 values to MMC than were parental cells. 5 of the MMC-sensitive mutants isolated from independently mutagenized cell populations were further analyzed. They were highly sensitive to the killing by decarbamoyl (DC) MMC, a monofunctional derivative of MMC, but were not sensitive to ultraviolet radiation, X-rays, 4-nitroquinoline-1-oxide or methyl methanesulfonate. These 5 mutants were classified into at least 2 genetic complementation groups. The implication of these mutations in cross-link and mono-adduct repair of DNA damage induced by MMC and DCMMC is discussed.     

Cross talk between the nuclease and helicase activities of Dna2: role of an essential iron-sulfur cluster domain

Dna2 nuclease/helicase is a multitasking protein involved in DNA replication and recombinational repair, and it is important for preservation of genomic stability. Yeast Dna2 protein contains a conserved putative Fe-S (iron-sulfur) cluster signature motif spanning the nuclease active site. We show that this motif is indeed an Fe-S cluster domain. Mutation of cysteines involved in metal coordination greatly reduces not just the nuclease activity but also the ATPase activity of Dna2, suggesting that the nuclease and helicase activities are coupled. The affinity for DNA is not significantly reduced, but binding mode in the C to A mutants is altered. Remarkably, a point mutation (P504S), proximal to the Fe-S cluster domain, which renders cells temperature sensitive, closely mimics the global defects of the Fe-S cluster mutation itself. This points to an important role of this conserved proline residue in stabilizing the Fe-S cluster. The C to A mutants are deficient in DNA replication and repair in vivo, and, strikingly, the degree to which they are defective correlates directly with degree of loss of enzymatic activity. Taken together with previous results showing that mutations in the ATP domain affect nuclease function, our results provide a new mechanistic paradigm for coupling between nuclease and helicase modules fused in the same polypeptide.     

Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal beta-subunits.

To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.     

All Three Subunits of RecBCD Enzyme Are Essential for DNA Repair and Low-Temperature Growth in the Antarctic Pseudomonas syringae Lz4W

Background

The recD mutants of the Antarctic Pseudomonas syringae Lz4W are sensitive to DNA-damaging agents and fail to grow at 4°C. Generally, RecD associates with two other proteins (RecB and RecC) to produce RecBCD enzyme, which is involved in homologous recombination and DNA repair in many bacteria, including Escherichia coli. However, RecD is not essential for DNA repair, nor does its deletion cause any growth defects in E. coli. Hence, the assessment of the P. syringae RecBCD pathway was imperative.

Methodology/Principal Findings

Mutational analysis and genetic complementation studies were used to establish that the individual null-mutations of all three genes, recC, recB, and recD, or the deletion of whole recCBD operon of P. syringae, lead to growth inhibition at low temperature, and sensitivity to UV and mitomycin C. Viability of the mutant cells dropped drastically at 4°C, and the mutants accumulated linear chromosomal DNA and shorter DNA fragments in higher amounts compared to 22°C. Additional genetic data using the mutant RecBCD enzymes that were inactivated either in the ATPase active site of RecB (RecB^K29Q) or RecD (RecD^K229Q), or in the nuclease center of RecB (RecB^D1118A and RecB^Δnuc) suggested that, while the nuclease activity of RecB is not so critical in vivo, the ATP-dependent functions of both RecB and RecD are essential. Surprisingly, E. coli recBCD or recBC alone on plasmid could complement the defects of the ΔrecCBD strain of P. syringae.

Conclusions/Significance

All three subunits of the RecBCD^Ps enzyme are essential for DNA repair and growth of P. syringae at low temperatures (4°C). The RecD requirement is only a function of the RecBCD complex in the bacterium. The RecBCD pathway protects the Antarctic bacterium from cold-induced DNA damages, and is critically dependent on the helicase activities of both RecB and RecD subunits, but not on the nuclease of RecBCD^Ps enzyme.     

Expression of Serratia marcescens extracellular proteins requires recA.

A previously described regulatory mutation which abolishes expression of the extracellular nuclease of Serratia marcescens is shown to be a mutation of the Serratia recA gene. The defect in nuclease expression could be restored by introducing a plasmid carrying the recA gene of Escherichia coli. The DNA sequence of the Serratia gene is very similar to that of the E. coli gene. The putative LexA-binding site of the Serratia recA gene is almost identical to that of E. coli, along with the promoter. A similar LexA-binding site can also be found upstream of the nuclease gene. As expected from this finding, we show that nuclease expression can be induced by SOS-inducing agents such as mitomycin C. Although inducible in S. marcescens, the nuclease was expressed only at the uninduced levels in E. coli and could not be induced by mitomycin C. The extracellular chitinase and lipase were similarly affected by the mutations altering nuclease expression and were also induced by mitomycin C.     

Differential effects of lipid depletion on membrane sodium-plus-potassium ion-dependent adenosine triphosphatase and potassium ion-dependent phosphatase.

The phospholipid-dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) and associated K-+-dependent phosphatase activity (EC 3.6.1.7) have been compared. Unlike the (Na-++K-+)-dependent ATPase activities, the K-+-dependent phosphatase activities of a number of different preparations were not closely correlated with their total phospholipid contents. After partial lipid depletion with a single extraction in Lubrol W the residual ATPase and phosphatase activities were correlated, but their magnitudes were quite different: on average only about 5% of the former remained compared with 50% of the latter. A similar differential effect on these activities was found after extraction with deoxycholate. In contrast with the ATPase, consistent restoration of the phosphatase activity of Lubrol-extracted enzymes by added exogenous phospholipids was not observed. We conclude that, although the K-+-dependent phosphatase may be lipid-dependent, the lipid requirement must be different from that of the complete ATPase system, and this difference should help investigations of their relationship.