Isolation and characterization of mutations in the structural gene for protease III (ptr).

Escherichia coli mutants defective in protease III were isolated by enzyme assays of heavily mutagenized colones. One mutant produced thermolabile enzyme, and it is presumed to have a mutation in the structural gene of protease III. Two other mutants mapping at the same site had less than 5% of the wild-type protease III level. The genetic locus of these mutations, designated ptr, was located at approximately 60 min on the E. coli linkage map based on its high frequency (70%) of contransduction by P1 with argA. Strains with less than 5% of the wild-type protease III activity grew normally and degraded nonsense fragments of beta-galactosidase at wild-type rates.     

Disruption of the structural gene for farnesyl diphosphate synthase in Escherichia coli

The chromosomal ispA gene encoding farnesyl diphosphate synthase of Escherichia coli was disrupted by inserting a neo gene cassette. The null ispA mutants were viable. The growth yield of the mutants was 70% to 80% of that of the wild-type strain under aerobic conditions, and was almost the same as the wild-type under anaerobic conditions. The levels of ubiquinone-8 and menaquinone-8 were both significantly lower (less than 13% and 18% of normal, respectively) in the mutants than in the wild-type. The undecaprenyl phosphate level in the mutants was modestly lower (40% to 70% of normal) than in the wild-type strain. Thus the synthesis of all-E-octaprenyl diphosphate, the precursor of ubiquinone-8 and menaquinone-8, was decreased more severely than that of Z,E-mixed undecaprenyl diphosphate, the precursor of undecaprenyl monophosphates, under the conditions where the synthesis of farnesyl diphosphate was decreased. The condensation of isopentenyl diphosphate with dimethylallyl diphosphate was detected in the cell-free extracts of the mutants, although it was 5% of that in the wild-type strain. A low level of farnesyl diphosphate seems to be synthesized in the mutants by other prenyltransferases such as octaprenyl diphosphate synthase or undecaprenyl diphosphate synthase.     

Properties of Haemophilus influenzae mutants that are slightly recombination deficient and carry a mutation in the rec-1 gene region.

The highly recombination-deficient rec-1 mutants of Haemophilus influenzae are, as far as tested, equivalent to recA mutants of Escherichia coli. By selection for mutations in the rec-1 gene of H. influenzae, mutants designated ird (intermediary recombination-deficient) mutants were isolated; these mutants were much less recombination deficient (degree of transformability, 0.2 to 30% of wild-type value) than previously isolated rec-1 mutants (degree of transformability, 0.0001% of wild-type value). The ird mutants were more sensitive to ultraviolet irradiation and mytomycin C treatment than the wild type, but less sensitive than rec-1 mutants. Spontaneous production of phage HP1c1 by lysogenic MC11 cells and prophage induction by mitomycin C or ultraviolet irradiation were the same as in the wild type. In the ird mutants endogenous deoxyribonucleic acid was degraded both spontaneously and after ultraviolet irradiation to the same extent as in the wild type. Examination of one of the ird mutants revealed that recombination could be enhanced by ultraviolet irradiation, possibly because of an increased synthesis of the rec-1 gene product induced by ultraviolet irradiation.     

Aspartase-hyperproducing mutants of Escherichia coli B

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutants in a Nonessential Gene of Bacteriophage T4 Which Are Defective in the Degradation of Escherichia coli Deoxyribonucleic Acid

Wild-type bacteriophage T4 was enriched for mutants which fail to degrade Escherichia coli deoxyribonucleic acid (DNA) by the following method. E. coli B was labeled in DNA at high specific activity with tritiated thymidine ((3)H-dT) and infected at low multiplicity with unmutagenized T4D. At 25 min after infection, the culture was lysed and stored. Wild-type T4 degrades the host DNA and incorporates the (3)H-dT into the DNA of progeny phage; mutants which fail to degrade the host DNA make unlabeled progeny phage. Wild-type progeny are eventually inactivated by tritium decay; mutants survive. Such mutants were found at a frequency of about 1% in the survivors. Eight mutants are in a single complementation group called denA located near gene 63. Four of these mutants which were examined in detail leave the bulk of the host DNA in large fragments. All eight mutants exhibit much less than normal T4 endonuclease II activity. The mutants produce somewhat fewer phage and less DNA than does wild-type T4.     

Aspartase-hyperproducing mutants of Escherichia coli B.

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenotypic expression of ammonia-excreting mutants of Anabaena 7120 under nitrogen limitation

Mutants of Anabaena 7120 defective in glutamine synthetase (GS) activity were isolated following transposon mutagenesis. Mutants M11, M55 and M73 showed about 60% less GS activity in N₂-grown aerobic cultures than the wild-type strain and were resistant to the glutamate analogue l-methionine-dl-sulphoximine (MSX). These mutants had the capacity to excrete N₂-fixed ammonia continuously into the culture medium and showed an enhanced level of aerobic nitrogenase activity. The intracellular ammonium pool generated in N₂-grown cells of mutants was found to be less than that of the wild-type strain. Similarly, ammonium uptake by these mutants was 50% less in mutants compared to the wild-type, suggesting a possible role of GS in controlling this function.     

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, less than 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to beta-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.     

Role for fadR in unsaturated fatty acid biosynthesis in Escherichia coli

Escherichia coli K-12 mutants constitutive for the synthesis of the enzymes of fatty acid degradation (fad) synthesize significantly less unsaturated fatty acid (UFA) than do wild-type (fadR+) strains. The constitutive fadR mutants synthesize less UFA than do fadR+) strains both in vivo and in vitro. The inability of fadR strains to synthesize UFAs at rates comparable to those of fadR+ strains is phenotypically asymptomatic unless the fadR strain also carries a lesion in fabA, the structural gene for beta-hydroxydecanoyl-thioester dehydrase. Unlike fadR+ fabA(Ts) mutants, fadR fabA(Ts) strains synthesize insufficient UFA to support their growth even at low temperatures and, therefore, must be supplemented with UFA at both low and high temperatures. The low levels of UFA in fadR strains are not due to the constitutive level of fatty acid-degrading enzymes in these strains. These results suggest that a functional fadR gene is required for the maximal expression of UFA biosynthesis in E. coli.     

Intracellular expression of Vitreoscilla hemoglobin modifies microaerobic Escherichia coli metabolism through elevated concentration and specific activity of cytochrome o

The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth.     

Subunit-specific phenotypes of Salmonella typhimurium HU mutants.

Salmonella hupA and hupB mutants were studied to determine the reasons for the high degree of conservation in HU structure in bacteria. We found one HU-1-specific effect; the F'128 plasmid was 25-fold less stable in hupB compared with hupA or wild-type cells. F' plasmids were 120-fold more unstable in hupA hupB double mutants compared with wild-type cells, and the double mutant also had a significant alteration in plasmid DNA structure. pBR322 DNA isolated from hupA hupB strains was deficient in supercoiling by 10 to 15% compared with wild-type cells, and the topoisomer distribution was significantly more heterogeneous than in wild-type or single-mutant strains. Other systems altered by HU inactivation included flagellar phase variation and phage Mu transposition. However, Mu transposition rates were only about fourfold lower in Salmonella HU double mutants. One reason that Salmonella HU double mutants may be less defective for Mu transposition than E. coli is the synthesis in double mutants of a new, small, basic heat-stable protein, which might partially compensate for the loss of HU. The results indicate that although either HU-1 or HU-2 subunit alone may accommodate the cellular need for general chromosomal organization, the selective pressure to conserve HU-1 and HU-2 structure during evolution could involve specialized roles of the individual subunits.     

Intracellular expression of Vitreoscilla hemoglobin modifies microaerobic Escherichia coli metabolism through elevated concentration and specific activity of cytochrome o

The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth. (c) 1996 John Wiley & Sons, Inc.     

16S rRNA mutations that confer tetracycline resistance in Helicobacter pylori decrease drug binding in Escherichia coli ribosomes

Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.     

Positive control of sulphate reduction in Escherichia coli. The nature of the pleiotropic cysteineless mutants of E. coli K 12

1. The function of the wild-type alleles of the pleiotropic mutants cysB and cysE of Escherichia coli was investigated. 2. The wild-type allele cysB(+) is dominant to the mutant allele cysB in stable and transient heterozygotes. 3. The wild-type allele cysE(+) is dominant to the mutant allele cysE, as predicted. 4. Sulphur-starved cultures of cysB or cysE strains contain less than 0.2nmole of free cysteine/mg. dry wt. 5. Complementation in vitro is not observed between extracts of cysB mutants and mutants lacking sulphite reductase only. 6. A scheme, involving positive control of the enzymes of sulphate activation and reduction, is suggested to account for the control of cysteine biosynthesis.     

Comparison of active mutants and wild-type aspartate transcarbamoylase of Escherichia coli

Two active mutants of aspartate transcarbamoylase from Escherichia coli have been purified from strains which produce large quantities of enzyme. Each enzyme was isolated from a different spontaneous revertant of a pyrimidine auxotrophic strain produced by mutagenesis with nitrogen mustard. Both enzymes exhibit allosteric properties with one having significantly less and the other more cooperativity than wild-type enzyme. Isolated catalytic subunits had different values of Km and Vmax. Studies on hybrids constructed from mutant catalytic and wild-type regulatory subunits (and vice versa) indicate that catalytic chains encoded by pyrB and not the regulatory chains encoded by pyrI were affected by the mutations. Differential scanning calorimetry experiments support these conclusions. Both mutant enzymes undergo ligand-promoted conformational changes analogous to those exhibited by wild-type enzyme: a 3% decrease in the sedimentation coefficient and a 5-fold increase in the reactivity of the sulfhydryl groups of the regulatory chains. Interactions between catalytic and regulatory chains in the mutants are weaker than those in the wild-type enzyme. The gross conformational changes of the mutants upon adding the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, in the presence of the substrate, carbamoylphosphate, and the activator, ATP, correlate with differences in cooperativity. The mutant with lower cooperativity is more readily converted from the low-affinity, compact, T-state to the high-affinity, swollen, R-state than is wild-type enzyme; this conversion for the more cooperative enzyme is energetically less favorable.     

DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling

Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon.     

Role of the Escherichia coli glnALG operon in regulation of ammonium transport.

Escherichia coli expresses a specific ammonium (methylammonium) transport system (Amt) when cultured with glutamate or glutamine as the nitrogen source. Over 95% of this Amt activity is repressed by growth of wild-type cells on media containing ammonia. The control of Amt expression was studied with strains containing specific mutations in the glnALG operon. GlnA- (glutamine synthetase deficient) mutants, which contain polar mutations on glnL and glnG genes and therefore have the Reg- phenotype (fail to turn on nitrogen-regulated operons such as histidase), expressed less than 10% of the Amt activity observed for the parental strain. Similarly, low levels of Amt were found in GlnG mutants having the GlnA+ Reg- phenotype. However, GlnA- RegC mutants (a phenotype constitutive for histidase) contained over 70% of the parental Amt activity. At steady-state levels, GlnA- RegC mutants accumulated chemically unaltered [14C]methylammonium against a 60- to 80-fold concentration gradient, whereas the labeled substrate was trapped within parental cells as gamma-glutamylmethylamide. GlnL Reg- mutants (normal glutamine synthetase regulation) had less than 4% of the Amt activity observed for the parental strain. However, the Amt activity of GlnL RegC mutants was slightly higher than that of the parental strain and was not repressed during growth of cells in media containing ammonia. These findings demonstrate that glutamine synthetase is not required for Amt in E. coli. The loss of Amt in certain GlnA- strains is due to polar effects on glnL and glnG genes, whose products are involved in expression of nitrogen-regulated genes, including that for Amt.     

Conformational stability and activity of ribonuclease T1 and mutants. Gln25----Lys, Glu58----Ala, and the double mutant

Ribonuclease T1 (RNase T1) and mutants Gln25----Lys, Glu58----Ala, and the double mutant were prepared from a chemically synthesized gene, cloned and expressed in Escherichia coli. The wild-type RNase T1 prepared from the cloned gene was identical in every functional and physical property examined to RNase T1 prepared from Aspergillus oryzae. Urea and thermal unfolding experiments show that Gln25----Lys is 0.9 kcal/mol more stable and Glu58----Ala is 0.8 kcal/mol less stable than wild-type RNase T1. In the double mutant, these contributions cancel and the stability does not differ significantly from that of wild-type RNase T1. For the double mutant, the dependence of delta G on urea concentration is significantly greater than for wild-type RNase T1 or the single mutants. This suggests that the double mutant unfolds more completely in urea than the other proteins. The activity of Gln25----Lys is identical with that of wild-type RNase T1. The activities of Glu58----Ala and the double mutant are 7% of wild-type when GpC hydrolysis is measured (due to a 35-fold decrease in kcat), and 37% of wild-type when RNA hydrolysis is measured. Thus, Glu58 is important, but not essential to the activity of RNase T1.     

A gene affecting accumulation of the RNA moiety of the processing enzyme RNase P.

The level of 10Sb (M1) RNA, the RNA of RNase P, is very low in growing cultures of rnpB mutants. Northern transfer experiments suggested that these strains accumulate no more than 10% of the wild-type level of 10Sb RNA. However, there is no indication that there is a limiting amount of RNase P activity in these mutants in vivo. A plasmid that directs the synthesis of 10Sb RNA does not complement the rnpB mutants, even though there is only a single gene for 10Sb RNA in the Escherichia coli genome. The 10Sb RNA synthesized from this plasmid is equivalent to wild-type 10Sb RNA since it can replace it in the reconstitution of RNase P. The 10Sb RNA, which is a rather stable molecule, is unstable in the presence of the rnpB mutation. This could explain why rnpB mutants do not accumulate 10Sb RNA. An F' plasmid that contains DNA from the rnpB region of the chromosome complements an rnpB mutant in vivo and in vitro, and it also contains the 10Sb RNA gene. A number of possible explanations for these phenomena are discussed.     

Complementation of Methylobacterium organophilum mutants affected in pyrroloquinoline quinone biosynthesis genes pqqE and pqqF by cloned Escherichia coli chromosomal DNA

The hybrid plasmid pBGT3, a derivative of pLA2917 containing a 7.8-kb fragment of Escherichia coli DNA, was found to complement pqqE and pqqF mutants of Methylobacterium organophilum, both impaired in PQQ biosynthesis. The cloned fragment of E. coli DNA did not hybridize with DNA fragments containing pqqE or pqqF previously cloned from M. organophilum. Yet, in M. organophilum mutants, expression of pqqE and pqqF genes from E. coli resulted in a PQQ production estimated at 9-16% of the production observed in M. organophilum wild-type. The growth rate in methanol medium of the complemented M. organophilum mutants was about 60% of that of the wild-type.