Carbohydrate Accumulation and Metabolism in Escherichia coli: Characteristics of the Reversions of ctr Mutations

The reversion behavior of pleiotropic carbohydrate mutants, previously designated as ctr, was studied. The mutants revert to complete restoration of the wild-type phenotype, as well as to a spectrum of partial wild-type phenotypes. Lac⁺ reversions were found in the lac region (11 min) and some Mal⁺ reversions occurred at malB (79 min), at a distance from the site of the ctr mutations (46 to 47 min). About one-third of Lac⁺ and Mal⁺ revertants were constitutive for uptake of their respective substrates, and one-third modified for inducibility. The remaining third were not distinguishable from wild type. Induction of a ctr mutation in a lac constitutive strain, either operator or repressor mutant, did not affect lactose metabolism. A polar-like ctr mutant, deficient in both enzyme I and heat-stable protein of the phosphoenolpyruvate-dependent phosphotransferase strain was also described. Partial revertants of ctr were still found to lack enzyme I.     

The Bifidobacterium longum NCIMB 702259T ctr Gene Codes for a Novel Cholate Transporter

Preexposure of Bifidobacterium longum NCIMB 702259^T to cholate caused increased resistance to cholate, chloramphenicol, and erythromycin. The B. longum ctr gene, encoding a cholate efflux transporter, was transformed into the efflux-negative mutant Escherichia coli KAM3, conferring resistance to bile salts and other antimicrobial compounds and causing the efflux of [¹⁴C]cholate.     

Apparent non-involvement of transfer RNA nucleotidyltransferase in the biosynthesis of Escherichia coli suppressor transfer RNAs

Transfer RNA nucleotidyltransferase has previously been shown to be required for the normal growth of Escherichia coli and for the biosynthesis of some bacteriophage T4 tRNAs. In order to obtain information about the involvement of this enzyme in E. coli tRNA biosynthesis we have measured the level of activity of suppressors 1 to 6 in strains carrying either a cca⁺ or cca allele. We found that cca strains, deficient in tRNA nucleotidyltransferase, contained the same amount of suppressor activities as the wild-type cca⁺ strains as determined by suppression of nonsense mutations in both E. coli alkaline phosphatase and in genes of bacteriophage T4. The results suggest that tRNA nucleotidyltransferase is not required for the biosynthesis of tRNAs specified by suppressors 1 to 6.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.     

Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1

Mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1 and PMS2 are associated with Lynch Syndrome (LS), a familial predisposition to early-onset cancer of the colon and other organs. Because not all LS families carry mutations in these four genes, the search for cancer-associated mutations was extended to genes encoding other members of the mismatch repairosome. This effort identified mutations in EXO1, which encodes the sole exonuclease implicated in MMR. One of these mutations, E109K, was reported to abrogate the catalytic activity of the enzyme, yet, in the crystal structure of the EXO1/DNA complex, this glutamate is far away from both DNA and the catalytic site of the enzyme. In an attempt to elucidate the reason underlying the putative loss of function of this variant, we expressed it in Escherichia coli, and tested its activity in a series of biochemical assays. We now report that, contrary to earlier reports, and unlike the catalytic site mutant D173A, the EXO1 E109K variant resembled the wild-type (wt) enzyme on all tested substrates. In the light of our findings, we attempt here to reinterpret the results of the phenotypic characterization of a knock-in mouse carrying the E109K mutation and cells derived from it.     

Novel cell line models to study mechanisms and overcoming strategies of proteasome inhibitor resistance in multiple myeloma

Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.     

Functional expression of cloned yeast DNA in Escherichia coli: specific complementation of argininosuccinate lyase (argH) mutations.

Segments of yeast (Saccharomyces cerevisiae) DNA cloned on various plasmid vectors in Escherichia coli can be functionally expressed to produce active enzymes. We have identified several ColE1-DNA(yeast) plasmids capable of complementing argH mutations, including deletions, in E. coli. Variants of the original transformants that grow faster on selective media and contain higher levels of the complementing enzyme activity (argininosuccinate lyase) can be readily isolated. The genetic alterations leading to increased expression of the yeast gene are associated with the cloned yeast DNA segment, rather than the host genome. The yeast DNA segment cloned in these plasmids also specifies a suppressor of the leuB6 mutation in E. coli. The argH and leuB6 complementing activities are expressed from discrete regions of the cloned yeast DNA segment, since the two genetic functions can be separated on individual recloned restriction fragments. The ease with which the bacterial cell can achieve functional high-level gene expression from cloned yeast DNA indicates that there are no significant barriers preventing expression of many yeast genes in E. coli.     

Genetics and function of DNA ligase in Escherichia coli

The characterization of two classes of DNA ligase mutants in Escherichia coli is described. The first class consists of three mutations coding for a temperature-sensitive ligase and defines the structural gene for DNA ligase (lig). The second class of mutants (lop) overproduces an apparently wild-type enzyme; a genetic diploid analysis implies that these are promoter or operator mutations, lig and lop are cotransduced by phage P1 and map at 46 minutes on the E. coli map. Detailed studies of two lig mutants (lig4 and lig ts7) are reported, lig ts7 is a conditionally lethal mutation, proving the essential nature of the ligase gene product. Neither mutant has a major defect in recombination or ultraviolet-repair, but both show retarded sealing of 10 S pulse-labeled DNA (Okazaki fragments).     

Purification,kinetic studies,and homology model of Escherichia coli fructose-1,6-bisphosphatase

Previous kinetic characterization of Escherichia coli fructose 1,6-bisphosphatase (FBPase) was performed on enzyme with an estimated purity of only 50%. Contradictory kinetic properties of the partially purified E. coli FBPase have been reported in regard to AMP cooperativity and inactivation by fructose-2,6-bisphosphate. In this investigation, a new purification for E. coli FBPase has been devised yielding enzyme with purity levels as high as 98%. This highly purified E. coli FBPase was characterized and the data compared to that for the pig kidney enzyme. Also, a homology model was created based upon the known three-dimensional structure of the pig kidney enzyme. The k_cat of the E. coli FBPase was 14.6 s⁻¹ as compared to 21 s⁻¹ for the pig kidney enzyme, while the K_m of the E. coli enzyme was approximately 10-fold higher than that of the pig kidney enzyme. The concentration of Mg²⁺ required to bring E. coli FBPase to half maximal activity was estimated to be 0.62 mM Mg²⁺, which is twice that required for the pig kidney enzyme. Unlike the pig kidney enzyme, the Mg²⁺ activation of the E. coli FBPase is not cooperative. AMP inhibition of mammalian FBPases is cooperative with a Hill coefficient of 2; however, the E. coli FBPase displays no cooperativity. Although cooperativity is not observed, the E. coli and pig kidney enzymes show similar AMP affinity. The quaternary structure of the E. coli enzyme is tetrameric, although higher molecular mass aggregates were also observed. The homology model of the E. coli enzyme indicated slight variations in the ligand-binding pockets compared to the pig kidney enzyme. The homology model of the E. coli enzyme also identified significant changes in the interfaces between the subunits, indicating possible changes in the path of communication of the allosteric signal.     

Improvement of thermostable aldehyde dehydrogenase by directed evolution for application in Synthetic Cascade Biomanufacturing

The aldehyde dehydrogenase from Thermoplasma acidophilum, which was previously implemented as a key enzyme in a synthetic cell-free reaction cascade for the production of alcohols, was optimized by directed evolution. Improvements have been made to enhance reaction velocity and solubility. Using a random approach followed by site-directed and saturation mutagenesis, three beneficial amino acid mutations were found after screening of ca. 20,000 variants. Mutation Y399C enhanced the protein solubility after recombinant expression in Escherichia coli 6-fold. Two further mutations, F34M and S405N, enhanced enzyme activity with the cofactor NAD⁺ by a factor of eight. Impacts on enzyme stability and substrate specificity were negligible.     

Genetic characterization of early amber mutations in the Escherichia coli polA gene and purification of the amber peptides

The PolA1 mutation of Escherichia coli K12 and two further mutations. resA1 and resA2. characterized in E. coli B have been shown to produce enzymatically active nonsense (amber) peptides. These enzymes can be purified to virtual homogeneity by use of the λpolA transducing phage system. The peptides are immunologically related and react weakly hut specifically with antibody to whole DNA polymerase I. In their purified form the peptides are less heat-labile than the whole enzyme or the Klenow fragment produced by proteolysis. Physiological studies indicate that all three alleles are compatible with a number of different streptomycin resistance mutations (rpsL alleles) in a variety of genetic backgrounds. There is, however, clear evidence for slight amounts of “readthrough” of these mutations under these conditions. DNA sequence studies have indicated the exact nucleotides that have been mutated to produce the amber alleles. The resA1 and resA2 alleles appear to be independent isolates of the same mutation both resulting in CAG (Gln) → TAG (amber) at amino acid residue 298. The polA1 mutation results in TGC (Trp) → TAG (amber) at amino acid residue 342. The significance of these findings is discussed with reference to the structure of the whole enzyme as shown by the DNA sequence data of Joyce et al. (1982) and protein chemistry of Brown et al. (1982).     

Multiple myeloma-associated hDIS3 mutations cause perturbations in cellular RNA metabolism and suggest hDIS3 PIN domain as a potential drug target

hDIS3 is a mainly nuclear, catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) active domains. Mutations in hDIS3 have been found in ∼10% of patients with multiple myeloma (MM). Here, we show that these mutations interfere with hDIS3 exonucleolytic activity. Yeast harboring corresponding mutations in DIS3 show growth inhibition and changes in nuclear RNA metabolism typical for exosome dysfunction. Construction of a conditional DIS3 knockout in the chicken DT40 cell line revealed that DIS3 is essential for cell survival, indicating that its function cannot be replaced by other exosome-associated nucleases: hDIS3L and hRRP6. Moreover, HEK293-derived cells, in which depletion of endogenous wild-type hDIS3 was complemented with exogenously expressed MM hDIS3 mutants, proliferate at a slower rate and exhibit aberrant RNA metabolism. Importantly, MM mutations are synthetically lethal with the hDIS3 PIN domain catalytic mutation both in yeast and human cells. Since mutations in PIN domain alone have little effect on cell physiology, our results predict the hDIS3 PIN domain as a potential drug target for MM patients with hDIS3 mutations. It is an interesting example of intramolecular synthetic lethality with putative therapeutic potential in humans.     

Efficient production of recombinant T7 endonuclease I using silkworm-baculovirus expression vector system

Recently, T7 Endonuclease I (T7E1) cleavage assay has been widely employed as an efficient approach for detecting mutations from CRISPR/Cas9 targeted samples. This enzyme is sufficient to detect single- and multiple-base mismatches from various heteroduplex DNA samples. However, T7E1 is quite expensive for researchers to use it only for screening mutations, especially in the condition of a large number of test samples. Regarding the production of this enzyme, to data, only the E. coli system has been reported and the highly overexpressed T7E1 seems toxic to the E. coli host cells. Thus, in this study, we tested whether the silkworm-baculovirus expression vector system (BEVS) is suitable to produce recombinant T7 Endonuclease I (rT7E1). The rT7E1 with N- or C-tags in cultured silkworm cells and silkworm pupae were successfully expressed. Our results demonstrated that the rT7E1-Ntag was highly expressed in silkworm pupae and we obtained rT7E1 proteins in high purity. Moreover, rT7E1 from silkworm-BEVS sufficiently recognized and cleaved the mismatches of designed and CRISPR/Cas9-mediated DNA substrates, which was equivalent to the commercial rT7E1 of the E. coli system. Taken together, our study would greatly support the genome-editing research by providing a cost-effective and active rT7E1 enzyme.     

Evolved Cobalamin-Independent Methionine Synthase (MetE) Improves the Acetate and Thermal Tolerance of Escherichia coli

Acetate-mediated growth inhibition of Escherichia coli has been found to be a consequence of the accumulation of homocysteine, the substrate of the cobalamin-independent methionine synthase (MetE) that catalyzes the final step of methionine biosynthesis. To improve the acetate resistance of E. coli, we randomly mutated the MetE enzyme and isolated a mutant enzyme, designated MetE-214 (V39A, R46C, T106I, and K713E), that conferred accelerated growth in the E. coli K-12 WE strain in the presence of acetate. Additionally, replacement of cysteine 645, which is a unique site of oxidation in the MetE protein, with alanine improved acetate tolerance, and introduction of the C645A mutation into the MetE-214 mutant enzyme resulted in the highest growth rate in acetate-treated E. coli cells among three mutant MetE proteins. E. coli WE strains harboring acetate-tolerant MetE mutants were less inhibited by homocysteine in l-isoleucine-enriched medium. Furthermore, the acetate-tolerant MetE mutants stimulated the growth of the host strain at elevated temperatures (44 and 45°C). Unexpectedly, the mutant MetE enzymes displayed a reduced melting temperature (T_m) but an enhanced in vivo stability. Thus, we demonstrate improved E. coli growth in the presence of acetate or at elevated temperatures solely due to mutations in the MetE enzyme. Furthermore, when an E. coli WE strain carrying the MetE mutant was combined with a previously found MetA (homoserine o-succinyltransferase) mutant enzyme, the MetA/MetE strain was found to grow at 45°C, a nonpermissive growth temperature for E. coli in defined medium, with a similar growth rate as if it were supplemented by l-methionine.     

The effects of traT insertion mutations on detergent sensitivity and serum resistance of Escherichia coli and Salmonella typhimurium

The SS-A mutation of Salmonella typhimurium, which probably causes the production of a mutated TraT-like protein, sensitizes the bacteria to hydrophobic antibiotics. A similar phenotype is caused by insertion mutations in the cloned traT gene of R6-5. While the SS-A mutants are resistant to detergents and have unaltered serum resistance, the insertion mutations sensitize both S. typhimurium and Escherichia coli to detergents and abolish the increase in serum resistance caused by the wild-type traT gene product in E. coli.     

Glutamic acid residues as metal ligands in the active site of Escherichia coli alkaline phosphatase

Four independent mutations were introduced to the Escherichia coli alkaline phosphatase active site, and the resulting enzymes characterized to study the effects of Glu as a metal ligand. The mutations D51E and D153E were created to study the effects of lengthening the carboxyl group by one methylene unit at the metal interaction site. The D51E enzyme had drastically reduced activity and lost one zinc per active site, demonstrating importance of the position of Asp⁵¹. The D153E enzyme had an increased k_cat in the presence of high concentrations of Mg²⁺, along with a decreased Mg²⁺ affinity as compared to the wild-type enzyme. The H331E and H412E enzymes were created to probe the requirement for a nitrogen-containing metal ligand at the Zn₁ site. The H331E enzyme had greatly decreased activity, and lost one zinc per active site. In the absence of high concentrations of Zn²⁺, dephosphorylation occurs at an extremely reduced rate for the H412E enzyme, and like the H331E enzyme, metal affinity is reduced. Except at the 153 position, Glu is not an acceptable metal chelating amino acid at these positions in the E. coli alkaline phosphatase active site.     

Role of accessory DNA polymerases in DNA replication in Escherichia coli: analysis of the dnaX36 mutator mutant

The dnaX36(TS) mutant of Escherichia coli confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (−1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various E. coli DNA polymerases. The dnaX gene encodes the τ subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The dnaX36 defect resides in the C-terminal domain V of τ, essential for interaction of τ with the α (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered α-τ interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of dnaX36 with mutM, mutY, and recA defects, support our assumption that dnaX36-mediated mutations originate as errors of replication rather than DNA damage-related events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (dinB) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of τ subunit, in securing a high fidelity of replication.     

The molecular basis of aminoacylase 1 deficiency

Aminoacylase 1 is a zinc-binding enzyme which hydrolyzes N-acetyl amino acids into the free amino acid and acetic acid. Deficiency of aminoacylase 1 due to mutations in the aminoacylase 1 (ACY1) gene follows an autosomal-recessive trait of inheritance and is characterized by accumulation of N-acetyl amino acids in the urine. In affected individuals neurological findings such as febrile seizures, delay of psychomotor development and moderate mental retardation have been reported. Except for one missense mutation which has been studied in Escherichia coli, mutations underlying aminoacylase 1 deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase of the aminoacylase 1 activity of homogenized cells. Most mutations resulted in a nearly complete loss of enzyme function. Notably, the two newly discovered mutations p.Arg378Trp, p.Arg378Gln and the mutation p.Arg393His yielded considerable residual activity of the enzyme, which is tentatively explained by their intramolecular localization and molecular characteristics. In contrast to aminoacylase 1 variants which showed no detectable aminoacylase 1 activity, aminoacylase 1 proteins with the mutations p.Arg378Trp, p.Arg378Gln and p.Arg393His were also detected in Western blot analysis. Investigations of the molecular bases of additional cases of aminoacylase 1 deficiency contribute to a better understanding of this inborn error of metabolism whose clinical significance and long-term consequences remain to be elucidated.     

The biosynthesis of proline in Escherichia coli phosphate-dependent glutamate γ-semialdehyde dehydrogenase (NADP), the second enzyme in the pathway

Extracts of Escherichia coli contain an enzyme which catalyzes the oxidation of glutamic γ-semialdehyde. This reaction is dependent upon the presence of inorganic phosphate, and utilizes NADP⁺ as a cofactor. The enzyme is subject to neither end-product inhibition nor repression by proline, and strains which lack the second enzyme in the pathway, lack this reaction.     

Evolution of a regulated operon in the laboratory

The evolution of new metabolic functions is being studied in the laboratory using the EBG system of E. coli as a model system. It is demonstrated that the evolution of lactose utilization by lacZ deletion strains requires a series of structural and regulatory gene mutations. Two structural gene mutations act to increase the activity of ebg enzyme toward lactose, and to permit ebg enzyme to convert lactose into allolactose, an inducer of the lac operon. A regulatory mutation increases the sensitivity of the ebg repressor to lactose, and permits sufficient ebg enzyme activity for growth. The resulting fully evolved ebg operon regulates its own expression, and also regulates the synthesis of the lactose permease.