Inactivation and proteolysis of heat-sensitive adenylate kinase of Escherichia coli CR341 T28

Adenylate kinase from Escherichia coli K12 (strains CR341 and CR341 T28, a temperature-sensitive mutant) was purified by a two-step chromatographic procedure. Denaturation by heat above 60 degrees C of pure or crude preparations of adenylate kinase from both strains of bacteria was shown to be "reversible" if the enzyme was converted to the random coiled state by guanidinium chloride after heat treatment. Like other small monomeric proteins, adenylate kinase refolded rapidly to the native active state by dilution of guanidinium chloride. Adenylate kinase from the mutant strain was irreversibly inactivated by exposure of crude extracts at 40 degrees C. This inactivation is due to proteolysis which follows thermal denaturation (or transconformation) of mutant adenylate kinase at 40 degrees C. ATP, P1, P5-di(adenosine 5')-pentaphosphate, and anti-adenylate kinase antibodies protected the thermosensitive adenylate kinase in crude extracts against denaturation and proteolysis at 40 degrees C.     

2-keto-3-deoxygluconate transport system in Erwinia chrysanthemi.

In Erwinia chrysanthemi, the gene kdgT encodes a transport system responsible for the uptake of ketodeoxyuronates. We studied the biochemical properties of this transport system. The bacteria could grow on 2,5-diketo-3-deoxygluconate but not on 2-keto-3-deoxygluconate. The 2-keto-3-deoxygluconate entry reaction displayed saturation kinetics, with an apparent Km of 0.52 mM (at 30 degrees C and pH 7). 5-Keto-4-deoxyuronate and 2,5-diketo-3-deoxygluconate appeared to be competitive inhibitors, with Kis of 0.11 and 0.06 mM, respectively. The 2-keto-3-deoxygluconate permease could mediate the uptake of glucuronate with a low affinity. kdgT was cloned on an R-prime plasmid formed by in vivo complementation of a kdgT mutation of Escherichia coli. After being subcloned, it was mutagenized with a mini-Mu-lac transposable element able to form fusions with the lacZ gene. We introduced a kdgT-lac fusion into the E. chrysanthemi chromosome by marker exchange recombination and studied its regulation. kdgT product synthesis was not induced by external 2-keto-3-deoxygluconate in the wild-type strain but was induced by galacturonate and polygalacturonate. Two types of regulatory mutants able to grow on 2-keto-3-deoxygluconate as the sole carbon source were studied. Mutants of one group had a mutation in the operator region of kdgT; mutants of the other group had a mutation in kdgR, a regulatory gene controlling kdgT expression.     

Secondary structure and thermostability of the phage P22 tailspike. XX. Analysis by Raman spectroscopy of the wild-type protein and a temperature-sensitive folding mutant

The thermostable tailspike endorhamnosidase of bacteriophage P22 has been investigated by laser Raman spectroscopy to determine the protein's secondary structure and the basis of its thermostability. The conformation of the native tailspike, determined by Raman amide I and amide III band analyses, is 52 to 61% beta-sheet, 24 to 27% alpha-helix, 15 to 21% beta-turn and 0 to 10% other structure types. The secondary structure of the wild-type tailspike, as monitored by the conformation-sensitive Raman amide bands, was stable to 80 degrees C, denatured reversibly between 80 and 90 degrees C, and irreversibly above 90 degrees C. The purified native form of a temperature-sensitive folding mutant (tsU38) contains secondary structures virtually identical to those in the wild-type in aqueous solution at physiological conditions (0.05 M-Na+ (pH 7.5], at both permissive (20 degrees C) and restrictive (40 degrees C) temperatures. This supports previous results showing that the mutational defect at 40 degrees C affects intermediates in the folding pathway rather than the native structure. At temperatures above 60 degrees C the wild-type and mutant forms were distinguishable: the reversible and irreversible denaturation thresholds were approximately 15 to 20 degrees C lower in the mutant than in the wild-type protein. The irreversible denaturation of the mutant tailspikes led to different aggregation/polymerization products from the wild-type, indicating that the mutation altered the unfolding pathway. In both cases only a small percentage of the native secondary structure was altered by irreversible thermal denaturation, indicating that the aggregated states retain considerable native structure.     

Effects of the temperature range and the lack of beta-ketoacyl acyl-carrier protein synthase II on fatty acid synthesis in Escherichia coli K12 after shifts in temperature

Escherichia coli K12 cells grown at higher temperatures and then subjected to lower temperatures produce fatty acids with higher unsaturated/saturated ratios than cells completely adapted to the lower temperatures (Okuyama et al. (1982) J. Biol. Chem. 257, 4812-4817). This hyper-response was not an artefact of chloramphenicol treatment and was observed when the shift-down was more than 20 degrees C in the cells grown at either 40 degrees C or 35 degrees C. In contrast, cells grown at either 25 degrees C or 30 degrees C showed no appreciable hyper-response in terms of unsaturated/saturated ratio on temperature shifts to as low as 10 degrees C. By combining shift-down and shift-up experiments, we could show the presence of different types of temperature dependency in the fatty acid-synthesizing systems of cells grown at various temperatures. Contrary to wild-type cells which synthesized mainly cis-vaccenate on down-shift to 10 degrees C, a mutant strain lacking beta-ketoacyl acyl-carrier protein synthase II synthesized more palmitoleate (16:1) and less palmitate at 10 degrees C than at 40 degrees C. The average chain lengths of saturated and unsaturated fatty acids also changed, but differently, between the mutant and wild-type cells on shifts of temperature. Thus, the mutant strain has a temperature-dependent fatty acid-synthesizing system qualitatively different from that seen in a wild-type strain.     

Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction

By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change delta Happ on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 +/- 0.2 degrees C and a delta Happ of 86.8 +/- 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 degrees C and 96 +/- 2 kcal/mol. The heat capacity change on denaturation delta Cp was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of delta Happ and delta Sapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated delta Cp for the mutant proteins and the d(delta Gapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.     

Identification of tms-26 as an allele of the gcaD gene, which encodes N-acetylglucosamine 1-phosphate uridyltransferase in Bacillus subtilis.

The temperature-sensitive Bacillus subtilis tms-26 mutant strain was characterized biochemically and shown to be defective in N-acetylglucosamine 1-phosphate uridyltransferase activity. At the permissive temperature (34 degrees C), the mutant strain contained about 15% of the wild-type activity of this enzyme, whereas at the nonpermissive temperature (48 degrees C), the mutant enzyme was barely detectable. Furthermore, the N-acetylglucosamine 1-phosphate uridyltransferase activity of the tms-26 mutant strain was much more heat labile in vitro than that of the wild-type strain. The level of N-acetylglucosamine 1-phosphate, the substrate of the uridyltransferase activity, was elevated more than 40-fold in the mutant strain at the permissive temperature compared with the level in the wild-type strain. During a temperature shift, the level of UDP-N-acetylglucosamine, the product of the uridyltransferase activity, decreased much more in the mutant strain than in the wild-type strain. An Escherichia coli strain harboring the wild-type version of the tms-26 allele on a plasmid contained increased N-acetylglucosamine 1-phosphate uridyltransferase activity compared with that in the haploid strain. It is suggested that the gene for N-acetylglucosamine 1-phosphate uridyltransferase in B. subtilis be designated gcaD.     

Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphosphate synthase.

The screening of a collection of highly mutagenized strains of Escherichia coli for defects in isoprenoid synthesis led to the isolation of a mutant that had temperature-sensitive farnesyl diphosphate synthase. The defective gene, named ispA, was mapped at about min 10 on the E. coli chromosome, and the gene order was shown to be tsx-ispA-lon. The mutant ispA gene was transferred to the E. coli strain with a defined genetic background by P1 transduction for investigation of its function. The in vitro activity of farnesyl diphosphate synthase of the mutant was 21% of that of the wild-type strain at 30 degrees C and 5% of that at 40 degrees C. At 42 degrees C the ubiquinone level was lower (66% of normal) in the mutant than in the wild-type strain, whereas at 30 degrees C the level in the mutant was almost equal to that in the wild-type strain. The polyprenyl phosphate level was slightly higher in the mutant than in the wild-type strain at 30 degrees C and almost the same in both strains at 42 degrees C. The mutant had no obvious phenotype regarding its growth properties.     

Isolation and characterization of a Clo DF13::Tn901 plasmid mutant with thermosensitive control of DNA replication.

After nitrosoguanidine mutagenesis, a mutant Escherichia coli strain harboring the Clo DF13::Tn901 plasmid pJN03 was isolated that is thermosensitive (Ts) for growth at 43 degrees C. The mutation responsible for this thermosensitive phenotype resides on the pJN03 plasmid genome. Cells harboring the pJN03 cop-1(Ts) plasmid mutant showed a large increase in plasmid copy number at 43 degrees C accompanied by an increase in the synthesis of plasmid-specified gene products like cloacin DF13 and beta-lactamase. The pJN03 cop-1(Ts) mutant showed uncontrolled plasmid DNA replication at the nonpermissive temperature. Analysis of plasmid deletions showed that the mutation is located in the Clo DF13 map interval from 0 to 12% or 29 to 45%. This implies that native cloacin DF13 and the Clo DF13-specified polypeptides B, C, D, E, and G are not involved in the pleiotropic phenotype of the plasmid mutant pJN03 cop-1(Ts).     

Temperature-sensitive Escherichia coli mutant with an altered initiation codon of the uncG gene for the H+-ATPase gamma subunit.

A mutant of Escherichia coli showing temperature-sensitive growth on succinate was isolated, and its mutation in the initiation codon (ATG to ATA) of the uncG gene (coding for the gamma subunit of H+-ATPase F0F1) was identified. This strain could grow on succinate as the sole carbon source at 25 and 30 degrees C, but not at 37 or 42 degrees C. When this strain was grown at 25 degrees C on succinate or glycerol, its membranes had about 15% of the ATPase activity of wild-type membranes, whereas when it was grown at 42 degrees C, its membranes had about 2% of the wild-type ATPase activity. Membranes of the mutant grown at 25 or 42 degrees C could bind F1 functionally, resulting in about 40% of the specific activity of wild-type membranes. The gamma subunit was identified in an EDTA extract of membranes of the mutant grown at 25 degrees C, but was barely detectable in the same amount of extract from the mutant grown at 42 degrees C. These results indicate that initiation of protein synthesis from the AUA codon is temperature sensitive and that the gamma subunit is essential for assembly of F1 in vivo as shown by in vitro reconstitution experiments (S. D. Dunn and M. Futai, J. Biol. Chem. 255:113-118, 1980).     

The C-terminal region of subunit 4 (subunit b) is essential for assembly of the F0 portion of yeast mitochondrial ATP synthase.

The role of the C-terminal part of yeast ATP synthase subunit 4 (subunit b) in the assembly of the whole enzyme was studied by using nonsense mutants generated by site-directed mutagenesis. The removal of at least the last 10 amino-acid residues promoted mutants which were unable to grow with glycerol or lactate as carbon source. These mutants were devoid of subunit 4 and of another F0 subunit, the mitochondrially encoded subunit 6. The removal of the last eight amino-acid residues promoted a temperature-sensitive mutant (PVY161). At 37 degrees C this strain showed the same phenotype as above. When grown at permissive temperature (30 degrees C) with lactate as carbon source, PVY161 and the wild-type strain both displayed the same generation time and growth yield. Furthermore, the two strains showed identical cellular respiration rates at 30 degrees C and 37 degrees C. However, in vitro the ATP hydrolysis of PVY161 mitochondria exhibited a low sensitivity to F0 inhibitors, while ATP synthesis displayed the same oligomycin sensitivity as wild-type mitochondria. It is concluded that, in this mutant, the assembly of the truncated subunit 4 in PVY161 ATP synthase is thermosensitive and that, once a functional F0 is formed, it is stable. On the other hand, the removal of the last eight amino-acid residues promoted in vitro a proton leak between the site of action of oligomycin and F1.     

Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2.

The temperature- and solvent-induced denaturation of both the SCP2 wild-type and the mutated protein c71s were studied by CD measurements at 222 nm. The temperature-induced transition curves were deconvoluted according to a two-state mechanism resulting in a transition temperature of 70.5 degrees C and 59.9 degrees C for the wild-type and the c71s, respectively, with corresponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stability parameters characterizing the guanidine hydrochloride denaturation curves were also calculated on the basis of a two-state transition. The transitions of the wild-type occurs at 0.82 M GdnHCl and that of the c71s mutant at 0.55 M GdnHCl. These differences in the half denaturation concentration of GdnHCl reflect already the significant stability differences between the two proteins. A quantitative measure are the Gibbs energies DeltaG(0)(D)(buffer) at 25 degrees C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for the mutant. We characterized also the alkyl chain binding properties of the two proteins by measuring the interaction parameters for the complex formation with 1-O-Decanyl-beta-D-glucoside using isothermal titration microcalorimetry. The dissociation constants, K(d), for wild-type SCP2 are 335 microM at 25 degrees C and 1.3 mM at 35 degrees C. The corresponding binding enthalpies, DeltaH(b), are -21. 5 kJ/mol at 25 degrees C and 72.2 kJ/mol at 35 degrees C. The parameters for the c71s mutant at 25 degrees C are K(d)=413 microM and DeltaH(b)=16.6 kJ/mol. These results suggest that both SCP2 wild-type and the c71s mutant bind the hydrophobic compound with moderate affinity.     

Synthesis of recA protein and induction of bacteriophage lambda in single-strand deoxyribonucleic acid-binding protein mutants of Escherichia coli.

We investigated the capacity of Escherichia coli mutants defective in the single-strand deoxyribonucleic acid (DNA)-binding protein to amplify the synthesis of the recA protein, induce prophage lambda, and degrade their DNA after treatment with ultraviolet radiation, mitomycin C, or bleomycin. The thermosensitive ssbA1 strain induced recA protein and lambda phage normally at 30 degrees C, but no induction was observed at 42 degrees C when ultraviolet radiation or mitomycin C was used. The lexC113 mutant did not amplify recA protein synthesis or induce phage lambda at either 30 or 42 degrees C with those agents. Bleomycin was able to elicit induction of recA and phage lambda in both mutants at any temperature. After induction with ultraviolet radiation at the elevated temperature, no DNA degradation was observed for 40 min, but at later times there was increased degradation in the lexC113 strain, compared with the wild type, and even greater degradation in the ssbA1 mutant. We discuss the role of single-strand DNA-binding protein in induction and the possibility that the lexC product may exert its influence on recA and lambda induction at the level of the single-strand DNA gap.     

Physiological and genetic regulation of the aldohexuronate transport system in Escherichia coli.

In Escherichia coli K-12, the specificity of the aldohexuronate transport system (THU) is restricted to glucuronate and galacturonate. There is a relatively high basal-level activity in uninduced wild-type or isomeraseless strains. Supplementary activity is obtained with the inducers mannonic amide (five-fold), galacturonate (fourfold), fructuronate (fivefold), and tagaturonate (sevenfold). Specific THU- mutants were selected as strains unable to grow on either aldohexuronate but able to grow on fructuronate or tagaturonate. The remaining transport activity in uninduced and induced THU- starins represents less than 20% of that found in the wild type. Conjugation and transduction experiments indicate that all of the THU- mutations are located in a unique locus, exuT, half-way between the tolC (59 min) and argG (61 min) markers. exuT is closely linked to the uxaC-uxaA operon (60 min) and to the regulatory gene exuR (60 min), which controls the above-mentioned operon and the uxaB operon (45 min). Growth on either aldohexuronate and transport activity are fully recovered when exuT mutants are allowed to revert to exuT+ on galacturonate or glucuronate. Reversion on glucuronate alone may lead to the mutational derepression of the 2-keto-3-deoxygluconate transport system, which is uninducible in the wild type, which also takes up glucuronate, and whose structural gene belongs to the kdg regulon. Such strains, which remain unable to grow on galacturonate, are exuT and kdgR (constitutive allele of the regulatory gene kdgR of the kdg regulon). THU activity is superrepressed in an exuR mutant in which the uxaC-uxaA operon and the uxaB operon are superrepressed; exuR+/exuR merodiploids are also superrepressed. In a thermosensitive exuR mutant in which the above-mentioned operons are constitutive at 42 degrees C, the THU activity is fully derepressed at this temperature. On the basis of these and other results, it is concluded that THU is coded for by the structural gene exuT, which is negatively controlled by the exuR gene product and which probably belongs to an operon distinct from the uxaA-uxaC operon.     

Conversion of wild-type p53 core domain into a conformation that mimics a hot-spot mutant

The wild-type p53 protein can be driven into a conformation corresponding to that adopted by structural mutant forms by heterodimerization with a mutant subunit. To seek partially folded states of the wild-type p53 core domain (p53C) we used high hydrostatic pressure (HP) and subzero temperatures. Aggregation of the protein was observed in parallel with its pressure denaturation at 25 and 37 degrees C. However, when HP experiments were performed at 4 degrees C, the extent of denaturation and aggregation was significantly less pronounced. On the other hand, subzero temperatures under pressure led to cold denaturation and yielded a non-aggregated, alternative conformation of p53C. Nuclear magnetic resonance (1H15N-NMR) data showed that the alternative p53C conformation resembled that of the hot-spot oncogenic mutant R248Q. This alternative state was as susceptible to denaturation and aggregation as the mutant R248Q when subjected to HP at 25 degrees C. Together these data demonstrate that wild-type p53C adopts an alternative conformation with a mutant-like stability, consistent with the dominant-negative effect caused by many mutants. This alternative conformation is likely related to inactive forms that appear in vivo, usually driven by interaction with mutant proteins. Therefore, it can be a valuable target in the search for ways to interfere with protein misfolding and hence to prevent tumor development.     

Effects of temperature on the transport of nucleosides in guinea pig erythrocytes

The initial rate of [14C]uridine transport by guinea pig erythrocytes was investigated at different temperatures. At 37, 22, and 10 degrees C the concentration dependence of uridine zero-trans influx and equilibrium exchange influx was resolved into two components; (a) a saturable component which followed simple Michaelis-Menten kinetics and which was inhibited by nitrobenzylthioinosine, and (b) a linear component of low magnitude and insensitive to nitrobenzylthioinosine inhibition. The maximum velocity, Vmax, of zero-trans uridine influx for the saturable transport system was 70-fold higher at 37 than 10 degrees C (1.24, 0.20, and 0.018 mmol/L of cells per hour at 37, 22, and 10 degrees C, respectively). Similarly, the apparent affinity, Km, for zero-trans influx decreased as the temperature was lowered (0.27, 0.066, and 0.038 mM at 37, 22, and 10 degrees C, respectively). In contrast, uridine equilibrium exchange influx was less temperature dependent (Vmax, 2.80, 0.89, and 0.14 mmol/L of cells per hour; apparent Km 0.61, 0.36, and 0.24 mM at 37, 22, and 10 degrees C, respectively). These results demonstrate that the mobility of the empty carrier is impaired to a greater extent than the mobility of the loaded carrier temperature decreased. However, the kinetic constants for zero-trans uridine influx and efflux at 37 degrees C were similar, indicating that the nucleoside transporter exhibited directional symmetry at 37 degrees C. Arrhenius plots of the maximum velocity for equilibrium exchange and zero-trans uridine influx were discontinuous above 25 degrees C, but between 20 and 5 degrees C the plots were linear (Ea = 22 and 30 kcal/mol for equilibrium exchange and zero-trans influx, respectively.     

Phosphatidylinositol is an essential phospholipid of mycobacteria

Phosphatidylinositol (PI) and metabolically derived products such as the phosphatidylinositol mannosides and linear and mature branched lipomannan and lipoarabinomannan are prominent phospholipids/lipoglycans of Mycobacterium sp. believed to play important roles in the structure and physiology of the bacterium as well as during host infection. To determine if PI is an essential phospholipid of mycobacteria, we identified the pgsA gene of Mycobacterium tuberculosis encoding the phosphatidylinositol synthase enzyme and constructed a pgsA conditional mutant of Mycobacterium smegmatis. The ability of this mutant to synthesize phosphatidylinositol synthase and subsequently PI was dependent on the presence of a functional copy of the pgsA gene carried on a thermosensitive plasmid. The mutant grew like the control strain under permissive conditions (30 degrees C), but ceased growing when placed at 42 degrees C, a temperature at which the rescue plasmid is lost. Loss of cell viability at 42 degrees C was observed when PI and phosphatidylinositol dimannoside contents dropped to approximately 30 and 50% of the wild-type levels, respectively. This work provides the first evidence of the essentiality of PI to the survival of mycobacteria. PI synthase is thus an essential enzyme of Mycobacterium that shows promise as a drug target for anti-tuberculosis therapy.     

Characterization of a cycloheximide-resistant Tetrahymena thermophila mutant which also displays altered growth properties.

A cycloheximide-resistant strain of Tetrahymena thermophila, expressing a mutant chx-B gene (Ares and Bruns, Genetics 90:463-474, 1978), displayed very different temperature-dependent growth characteristics than either wild-type cells or another cycloheximide-resistant strain expressing a different mutant gene. Whereas wild-type cells showed an immediate decline in ribosome translocation rates when shifted from 30 to 38 or 40 degrees C, this mutant strain (X-8) showed no such decline. These results directly correlated with the growth rate differences we found for these cells at these temperatures. By genetic analysis, we showed that the phenotype of cycloheximide resistance cosegregated with the ability to grow rapidly at 40 degrees C. Analyses, both direct and indirect, suggested that a number of functional and structural characteristics of the ribosomes from strain X-8 cells are most likely conformationally different from those of wild-type ribosomes.     

DSC studies of the conformational stability of barstar wild-type.

The temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of delta HvH/delta Hcal near 1 that denaturation follows a two-state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol-1) exceeds that of the C82A mutant (276 kJ mol-1) by approximately 10%. The heat capacity changes show a similar difference, delta Cp being 5.3 +/- 1 kJ mol-1 K-1 for the wild-type and 3.6 +/- 1 kJ mol-1 K-1 for the C82A mutant. The extrapolated stability parameters at 25 degrees C are delta G0 = 23.5 +/- 2 kJ mol-1 for barstar wt and delta G0 = 25.5 +/- 2 kJ mol-1 for the C82A mutant.