Temperature-sensitive mutants of Escherichia coli K-12 with low activities of the L-alanine adding enzyme and the D-alanyl-D-alanine adding enzyme

A number of properties of temperature-sensitive mutants in murein synthesis are described. The mutants grow at 30 C but lyse at 42 C. One mutant possesses a temperature-sensitive d-alanyl-d-alanine adding enzyme, has an impaired rate of murein synthesis in vivo at both 30 and 42 C, and contains elevated levels of uridine diphosphate-N-acetyl-muramyl-tripeptide (UDP-MurNAc-l-Ala-d-Glu-m-diaminopimelic acid) at 42 C. The other mutant possesses an l-alanine adding enzyme with a very low in vitro activity at both 30 and 42 C. Its in vivo rate of murein synthesis is almost normal at 30 C but is much less at 42 C. When the murein precursors were isolated after incubation of the cells in the presence of (14)C-l-alanine, they contained only a fraction of the radioactivity that could be obtained from a wild-type strain. A genetic nomenclature for genes concerned with murein synthesis is proposed.     

Temperature-sensitive autolysis-defective mutants of Escherichia coli.

Two independently isolated temperature-sensitive autolysis-defective mutants of Escherichia coli LD5 (thi lysA dapD) were characterized. The mutants were isolated by screening the survivors of a three-step enrichment process involving sequential treatments with bactericidal concentrations of D-cycloserine, benzyl-penicillin, and D-cycloserine at 42 degrees C. Cultures of the mutants underwent autolysis during beta-lactam treatment, D-cycloserine treatment, or diaminopimelic acid deprivation at 30 degrees C. The same treatments at 42 degrees C inhibited growth but did not induce lysis of the mutants. The minimum inhibitory concentrations of selected beta-lactam antibiotics and D-cycloserine were identical for the parent and mutant strains at both 30 and 42 degrees C. Both mutants failed to form colonies at 42 degrees C, and both gave rise to spontaneous temperature-resistant revertants. The revertants exhibited the normal lytic response when treated with D-cycloserine and beta-lactams or when deprived of diaminopimelic acid at 42 degrees C. The basis for the autolysis-defective phenotype of these mutants could not be determined. However, a nonspecific in vitro assay for peptidoglycan hydrolase activity in cell-free extracts indicated that both mutants were deficient in a peptidoglycan hydrolase. Both mutations were localized to the 56- to 61-min region of the E. coli chromosome by F' complementation.     

Analysis of autolysins in temperature-sensitive morphological mutants of Bacillus subtilis.

The content and distribution of autolysin were measured in temperature-sensitive morphological mutants of Bacillus subtilis. Strains RUB1000 and RUB1012 grew as rods at 30 C. At 45 C the mutants contained disproportionately less teichoic acid than peptidoglycan and grew as irregular spheres. The amount of enzyme that could be extracted from rods was at least 31 times the amount extracted from spheres. The rate of autolysis of cell walls was 7- to 28-fold greater in rods than in spheres. The low activity found associated with the cell walls of spheres was not compensated for by larger amounts of autolytic activity in the cytoplasm. No activity was found in the growth medium at either temperature. The failure of the mutant cells to autolyze was due to low amidase activity and relatively resistant cell walls. Revertants of RUB1012 were isolated that had 13, 23, and 55% of the normal proportions of teichoic acid when grown at the nonpermissive temperature. Cell walls from the revertants were as sensitive to added amidase as the wild-type strain. None of the revertant strains regained the wild-type ability to produce more amidase at 45 C. However, the deficiency in autolysin observed with RUB1012 was partially restored in revertants containing higher proportions of teichoic acid.     

Isolation and characterization of revertants from four different classes of aryl hydrocarbon hydroxylase-deficient hepa-1 mutants.

Revertants were selected from aryl hydrocarbon hydroxylase (AHH)-deficient recessive mutants belonging to three complementation groups and from a dominant mutant of the Hepa-1 cell line. The recessive mutants had low spontaneous reversion frequencies (less than 4 X 10(-7] that were increased by mutagenesis. The majority of these revertants also had reacquired only partial AHH activity. Revertants of group A mutants were identical to the wild type with respect to both in vivo and in vitro enzyme stability and the Km for the substrate, benzo [alpha]pyrene, and therefore failed to provide evidence that gene A is the AHH structural gene. Group B and group C mutants are defective in the functioning of the Ah receptor required for AHH induction. Revertants of these groups were normal with respect to in vivo temperature sensitivity for AHH induction and for the 50% effective dose for the inducer, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and thus provided no evidence that the B and C genes code for components of the receptor. Two rare group C revertants possessed AHH activity in the absence of induction. The phenotype of one of these was shown to be recessive to the wild type. Spontaneous revertants of the dominant mutant occurred at a frequency 300-fold greater than those of the recessive mutants, and this frequency was not increased by mutagenesis. These revertants all displayed complete restoration of AHH activity to wild type levels. These observations and the results from cell hybridization studies suggest that the dominant revertants arose by a high frequency event leading to functional elimination of the dominant mutation.     

Isolation and characterization of autolysis-defective mutants of Escherichia coli that are resistant to the lytic activity of seminalplasmin.

Two temperature-sensitive autolysis-defective mutants of Escherichia coli were isolated and shown to be resistant to lysis induced by seminalplasmin, an antimicrobial protein from bovine seminal plasma, as well as to lysis induced by ampicillin, D-cycloserine and nocardicin, at 37 or 42 degrees C but not at 30 degrees C. The mutants were, however, sensitive to inhibition of RNA synthesis by seminalplasmin even at the nonpermissive temperature. Temperature-resistant revertants of the mutants were sensitive to lysis induced by the various antibiotics at 37 or 42 degrees C. The mutations in both strains were mapped at 58 min on the E. coli linkage map. The lysis resistance of the mutants was phenotypically suppressed by the addition of NaCl. Partial suppression of the lysis-resistant phenotype was also observed in a relA genetic background.     

Characterization of revertants of stmF mutants of Dictyostelium discoideum: evidence that stmF is the structural gene of the cGMP-specific phosphodiesterase

stmF mutants of Dictyostelium discoideum produce long, banded aggregation streams on growth plates and exhibit altered cGMP metabolism. To learn more about the role of cGMP in chemotaxis and the nature of the defect in these mutants, 15 nonstreaming (Stm+) revertants of two stmF mutants were isolated and characterized. Fourteen of the revertants continued to show the elevated cAMP-induced cGMP response and very low cGMP-specific phosphodiesterase (cGPD) activity characteristic of their stmF parents. Parasexual genetic analysis revealed that many of these Stm+ revertants carried phenotypic suppressors unlinked to stmF. One Stm+ revertant, strain HC344, exhibited a low, prolonged cGMP response and relatively high cGPD activity throughout development. To determine whether the elevated cGPD activity in this revertant resulted from increased enzyme production or enhanced enzyme activity, cGPDs were partially purified from the wild-type strain, the stmF parent and revertant HC344, and properties of the enzymes were compared. cGPDs from the stmF mutant and the revertant showed similar differences from the wild-type enzyme in kinetic properties, thermal stability, and sensitivity to certain inhibitors. These results suggest that stmF is the structural gene of the cGPD. In addition, the unusual cGMP response in revertant HC344 appeared to be due to increased production of an altered cGPD.     

Water activity,temperature, and pH effects on growth of the biocontrol agent Pantoea agglomerans CPA-2

The growth response of the biocontrol agent Pantoea agglomerans to changes in water activity (a(w)), temperature, and pH was determined in vitro in nutrient yeast extract-sucrose medium. The minimum temperature at which P. agglomerans was able to grow was 267-272 kelvins (-6 to -1 degrees C), and growth of P. agglomerans did not change at varying pH levels (4.5-8.6). The minimum a(w) for growth was 0.96 in media modified with glycerol and 0.95 in media modified with NaCl or glucose. Solute used to reduce water activity had a great influence on bacterial growth, especially at unfavourable conditions (e.g., low pH or temperature). NaCl stimulated bacterial growth under optimum temperatures but inhibited it under unfavourable pH conditions (4.5 or 8.6). In contrast, the presence of glucose in the medium allowed P. agglomerans to grow over a broad range of temperature (3-42 degrees C) or pH (5-8.6) regimes. This study has defined the range of environmental conditions (a(w), pH, and temperature) over which the bacteria may be developed for biological control of postharvest diseases.     

Asparagine-Requiring Tumor Cell Lines and Their Non-Requiring Variants: Cytogenetics, Biochemistry and Population Dynamics

Asparagine-requiring Jensen and Walker rat tumor cells and their asparagine-independent variants have been analyzed. The following results were obtained: (1) Both cell lines have very low levels of asparagine synthetase, and non-requiring revertants isolated from these lines have elevated levels of the enzyme. (2) No differences in chromosome number were detected between the parent Jensen line and five Jensen non-requiring revertants isolated from it. (3) Both Jensen and Walker cells undergo asparagineless death when deprived of this amino acid, although the Jensen cells do so at a more rapid rate. (4) Jensen requiring lines are at a selective advantage when grown in competition with non-requiring variants in complete medium, and their growth rate is more rapid when grown separately. The selective coefficients for the variant with respect to the asparagine-requiring parent ASN(-) line were 0.94 for the competition experiments and 0.83 for growth rate estimates. (5) A somatic cell hybrid between Chinese hamster cells (which require asparagine at low densities, and posses measurable synthetase activity) and the Walker line was found to be asparagine-independent, and it possessed enzyme levels equivalent to the hamster parent. The results of these investigations suggest a parallel with microbial auxotrophic mutants and can be understood in terms of alterations within nuclear structural genes.     

Frequency of bacteriocin resistance development and associated fitness costs in Listeria monocytogenes

Bacteriocin-producing starter cultures have been suggested as natural food preservatives; however, development of resistance in the target organism is a major concern. We investigated the development of resistance in Listeria monocytogenes to the two major bacteriocins pediocin PA-1 and nisin A, with a focus on the variations between strains and the influence of environmental conditions. While considerable strain-specific variations in the frequency of resistance development and associated fitness costs were observed, the influence of environmental stress seemed to be bacteriocin specific. Pediocin resistance frequencies were determined for 20 strains and were in most cases ca. 10(-6). However, two strains with intermediate pediocin sensitivity had 100-fold-higher pediocin resistance frequencies. Nisin resistance frequencies (14 strains) were in the range of 10(-7) to 10(-2). Strains with intermediate nisin sensitivity were among those with the highest frequencies. Environmental stress in the form of low temperature (10 degrees C), reduced pH (5.5), or the presence of NaCl (6.5%) did not influence the frequency of pediocin resistance development; in contrast, the nisin resistance frequency was considerably reduced (<5 x 10(-8)). Pediocin resistance in all spontaneous mutants was very stable, but the stability of nisin resistance varied. Pediocin-resistant mutants had fitness costs in the form of reduction down to 44% of the maximum specific growth rate of the wild-type strain. Nisin-resistant mutants had fewer and less-pronounced growth rate reductions. The fitness costs were not increased upon applying environmental stress (5 degrees C, 6.5% NaCl, or pH 5.5), indicating that the bacteriocin-resistant mutants were not more stress sensitive than the wild-type strains. In a saveloy-type meat model at 5 degrees C, however, the growth differences seemed to be negligible. The applicational perspectives of the results are discussed.     

Defects of two temperature-sensitive lysyl-transfer ribonucleic acid synthetase mutants of Bacillus subtilis

Two temperature-sensitive mutants (lysS1 and lysS2) of the lysyl-transfer ribonucleic acid synthetase (l-lysine:tRNA ligase [adenosine 5'-monophosphate], EC 6.1.1.6) of Bacillus subtilis have been isolated. Although protein synthesis is inhibited in both mutants at the restrictive temperature (42 to 45 C), the mutants remain viable in a minimal medium. In comparison with the wild-type lysyl-tRNA synthetase, the l-lysine-dependent exchange of [(32)P]pyrophosphate with adenosine 5'-triphosphate (ATP) for both mutant enzymes is decreased. The lysS1 enzyme is completely defective in the ATP-dependent attachment of l-lysine to tRNA, whereas the lysS2 enzyme has 3- to 10-fold reduced levels of this activity. Temperature-resistant transformants have wild-type enzyme levels, whereas partial revertants to temperature resistance have varied levels of enzyme activity. The attachment and exchange activities of the lysS2 enzyme are more heat labile in vitro than the wild-type enzyme, as is the attachment activity of a partial revertant of the lysS1 mutant. The lysS1 and the lysS2 lysyl-tRNA synthetases have higher apparent K(m) values for lysine and ATP, in both the activation and the attachment reactions. The lysS2 enzyme has a V(max) for tRNA(lys) one-third that of the wild-type enzyme. Molecular weights of approximately 150,000 for the wild-type and lysS2 enzymes and approximately 76,000 for the lysS1 enzyme were estimated from sedimentation positions in sucrose density gradients assayed by the ATP-pyrophosphate exchange activity. We propose that the two mutations (lysS1 and lysS2) directly affect the sites for exchange activity, but indirectly alter attachment activity as a consequence of defective subunit association.     

Structure and function of the mitochondrial bc1 complex. Properties of the complex in temperature-sensitive cor1 mutants

The properties of the ubiquinol-cytochrome c reductase complex (bc1 complex) have been studied in respiratory defective mutants of Saccharomyces cerevisiae bearing lesions in the core 1 subunit. All the cor1 mutants examined have greatly reduced concentrations of mitochondrial cytochrome b and display succinate-cytochrome c reductase activities near the limits of detection. Two mutants (E576 and C7), however, had 5% of wild type activity when the cells were grown at 23 degrees C, but not at 37 degrees C. The temperature-sensitive phenotype was determined to result from substitution of either Arg or Glu for Gly68 of the core 1 subunit. The respiratory competent revertants E576/R8 and C7/R4 derived from E576 and C7 retain the temperature sensitivity of the original mutants. Both revertants are temperature sensitive in vivo, but only mitochondria isolated from E576/R8 are temperature sensitive in vitro. The bc1 complex of mitochondria isolated from this revertant displays a normal value of the ratio Kcat/Km for cytochrome c and four times higher than the wild type for duroquinol. The succinate-cytochrome c reductase activity of E576/R8 is almost completely abolished after incubation at 37 degrees C for 90 min. It is inferred that the quaternary structure of ubiquinol-cytochrome c reductase complex is more labile at the nonpermissive temperature in the mutant and undergoes an alteration such that cytochrome b is no longer able to receive electrons through either the "o" or the "i" site pathway. The temperature lability and kinetic properties of the mutant enzyme point to a requirement of the core 1 not only for assembly but also for the catalytic activity of the complex.     

Characterization of lactose-fermenting revertants from lactose-negative Streptococcus lactis C2 mutants

Partial lactose-fermenting revertants from lactose-negative (lac(-)) mutants of Streptococcus lactis C2 appeared on a lawn of lac(-) cells after 3 to 5 days of incubation at 25 C. The revertants grew slowly on lactose with a growth response similar to that for cryptic cells. In contrast to lac(+)S. lactis C2, the revertants were defective in the accumulation of [(14)C]thiomethyl-beta-d-galactoside, indicating that they were devoid of a transport system. Hydrolysis of o-nitrophenyl-beta-d-galactoside-6-phosphate by toluene-treated cells confirmed the presence of phospho-beta-d-galactosidase (P-beta-gal) in the revertant. However, this enzyme was induced only when the cells were grown in the presence of lactose; galactose was not an inducer. In lac(+)S. lactis C2, enzyme induction occurred in lactose- or galactose-grown cells. The revertants were defective in EII-lactose and FIII-lactose of the phosphoenolpyruvate-dependent phosphotransferase system. Galactokinase activity was detected in cell extracts of lac(+)S. lactis C2, but the activity was 9 to 13 times higher in extracts from the revertant and lac(-), respectively. This suggested that the lac(-) and the revertants use the Leloir pathway for galactose metabolism and that galactose-1-phosphate rather than galactose-6-phosphate was being formed. This may explain why lactose, but not galactose, induced P-beta-gal in the revertants. Because the revertant was unable to form galactose-6-phosphate, induction could not occur. This compound would be formed on hydrolysis of lactose phosphate. The data also indicate that galactose-6-phosphate may serve not only as an inducer of the lactose genes in S. lactis C2, but also as a repressor of the Leloir pathway for galactose metabolism.     

Osmotic Reversal of Temperature Sensitivity in Escherichia coli

Forty temperature-sensitive mutants, unable to grow on tryptone or nutrient agar at 42 C, were isolated from Escherichia coli K-12. When 0.5% NaCl was added to the medium, 32 grew at the nonpermissive temperature. Several were tested with different amounts of NaCl added to tryptone broth; all grew best when the osmolality of the medium was between 400 and 1,000 milliosmolal. One of the mutants was studied in more detail. Sucrose, inositol, KCl, and MgCl(2), as well as NaCl, permitted growth at 42 C. Glycerol, however, had no effect. When shifted from 30 to 42 C without osmotic protection, the mutant stopped growing but did not lyse, die, or leak significant amounts of intracellular material. In a similar shift experiment, a second mutant leaked all of its trichloroacetic acid-soluble pools into the medium. The majority of the mutants were hypersensitive to certain antibiotics, indicating possible cell envelope defects.     

Pullulanase synthesis in Klebsiella (Aerobacter) aerogenes strains growing in continuous culture

1. Pullulanase synthesis was studied in 16 classified (N.C.I.B.) strains and in an industrial strain (R) of Klebsiella aerogenes grown in chemostats containing maltose as inducer and sole carbon source. 2. Maximum synthesis was associated with carbon-limited growth at a low dilution rate (about 0.2h(-1)). The enzyme remained firmly cell-bound and seemed to be located on the cell surface. 3. Three strains had high activity (R, N.C.I.B. 5938, 8017), twelve were intermediate, and two (N.C.I.B. 8153, 9146) had negligible activity but were inducible with pullulan. 4. Pullulan similarly induced low, but adequate, activity in the other strains in conditions (nutrient limitation other than carbon-limitation) in which pullulanase was otherwise very seriously repressed. Nevertheless, in carbon limitation pullulan induced no more enzyme than did maltose, maltotriose or oligosaccharide mixtures, and ;hyperactivity' never developed on protracted culture. 5. Cyclic AMP relieved the transient repression produced by adding glucose to maltose-limited cultures and a further change to glucose-limited conditions led to constitutive pullulanase synthesis. 6. Amylomaltase and alpha-glucosidase activities were also examined but in less detail. 7. The presence of pullulanase in maltose-limited growth is discussed, but no clear function can be assigned to it at present. The molar growth yields for all the strains were very similar, and no correlation was found between the overgrowth of one strain by another and pullulanase activity. Further, any function as a general branching enzyme in polysaccharide synthesis seems unlikely.     

Temperature-sensitive beta-lactam-tolerant mutants of Escherichia coli

Seven temperature-sensitive penicillin-tolerant mutants of Escherichia coli strain LD5 (thi lysA dapD) were isolated and characterized. Treatment with beta-lactams caused lysis of the mutants at 30 degrees C. Although growth of the mutants at 42 degrees C was inhibited by beta-lactams, no lysis occurred. The mutants were also slightly tolerant to D-cycloserine at 42 degrees C but lysed readily when deprived of diaminopimelate or when treated with moenomycin. The minimum inhibitory concentrations of various antibiotics were the same for the mutants and their parent. The mutations conferring penicillin tolerance were phenotypically suppressed in the presence of a variety of compounds which may act as chaotropic or antichaotropic agents. No defects in penicillin-binding proteins and peptidoglycan hydrolases were detected. Temperature-resistant revertants of the mutants were no longer tolerant to penicillin-induced autolysis at 42 degrees C. The mutations in five isolates were localized to the 56 to 61 min region of the E. coli linkage map and to the 44 to 51 min region in the case of two other isolates.     

Temperature-Sensitive Mutants of Vesicular Stomatitis Virus: In Vitro Studies of Virion-Associated Polymerase

The temperature dependence of the virion-associated polymerase activity of six temperature-sensitive (ts) mutants of vesicular stomatitis virus (tsW10, 11, 14, 16B, 28, and 29) has been examined in vitro and compared to the heat-resistant parent (HR). The polymerase of five of the mutants (tsW10, 11, 14, 16B, and 28) appears to be significantly more ts than that of HR. Because certain pairs of these five mutants can complement each other's in vitro polymerase activity, it appears that in vitro some components involved in the polymerase of one virion can be utilized by another virion. Examination of 19 revertants of tsW11 and tsW16B which had regained their ability to replicate at 38 C showed that their in vitro polymerase activity had also become less ts. Furthermore, it was found that the pairs of mutants which showed in vitro complementation of polymerase activity at 38 C were those which had shown complementation in yielding infectious progeny in mixedly infected cells. These two observations suggest that the ts behavior of the in vitro polymerase activity of the five mutants is related to their inability to replicate at the nonpermissive temperature.     

Characterization of Mutants of Escherichia coli Temperature-Sensitive for Ribonucleic Acid Regulation: an Unusual Phenotype Associated with a Phenylalanyl Transfer Ribonucleic Acid Synthetase Mutant

A mutant strain AA-522, temperature-sensitive for protein synthesis, was isolated from a stringent strain (CP-78) of Escherichia coli K-12. The mutant strain has a relaxed phenotype at the nonpermissive growth temperature. Protein synthesis stops completely at 42 C, whereas the rate of ribonucleic acid (RNA) synthesis is maintained at 20% of the 30 C rate. Sucrose-gradient centrifugation analysis of RNA-containing particles formed at 42 C indicated the presence of “relaxed particles.” These particles possess 16S and 23S RNA and are precursors to normal 50S and 30S ribosomal subunits. A search for the temperature-sensitive protein responsible for the halt in protein synthesis implicated phenylalanyl transfer RNA (tRNA) synthetase. Essentially no enzyme activity is detected in vitro at 30 or 40 C. Analysis of phenylalanyl tRNA synthetase activity in revertants of strain AA-522 indicated the presence of intragenic suppressor mutations. Revertants of strain AA-522 analyzed for the relaxed response at 42 C were all stringent; strain AA-522 was stringent at 30 C. These data indicate that a single mutation in phenylalanyl tRNA synthetase is responsible for both a block in protein synthesis and the relaxed phenotype at 42 C.     

Mutants of Bacillus species isolated on the basis of protonophore resistance are deficient in fatty acid desaturase activity.

The fatty acid desaturase activity in cell extracts of Bacillus subtilis was characterized and found to be O2 dependent, NADH dependent, and cyanide sensitive. In cell fractionation studies, only 10% of the desaturase activity was recovered in the membrane fraction; the addition of cytosolic factors, which by themselves were devoid of activity, restored membrane activity to the level found in the unfractionated cell extracts. NADH was preferred over NADPH as an electron donor, and palmitoyl-coenzyme A was used preferentially over stearoyl-coenzyme A as the straight-chain fatty acid substrate. An increase in desaturase activity was observed when either the growth or the assay temperature was lowered from 37 to 20 degrees C, although the assay temperature appeared to be the more important parameter. Three protonophore-resistant mutants of B. subtilis and a comparable mutant of Bacillus megaterium had been found to possess reduced levels of unsaturated fatty acids in their membrane phospholipids; their protonophore resistance was abolished when grown in the presence of an unsaturated fatty acid supplement. All of these strains were found to be either significantly deficient in or totally lacking desaturase activity in comparison with their wild-type parent strains. Full, protonophore-sensitive revertants of the mutants had levels of desaturase activity comparable to those of the wild-type. Temperature-sensitive revertants of two of the mutants, which grew at 32 degrees C but not at 26 degrees C in the presence of protonophore, exhibited desaturase activity comparable to that of the wild-type at 26 degrees C but lacked activity at 32 degrees C. These results indicate that the biochemical basis for protonophore resistance in these Bacillus mutants is a fatty acid desaturase deficiency.     

Characterization of revertants of stmF mutants of Dictyostelium discoideum: Evidence that stmF is the structural gene of the cgmp-specific phosphodiesterase

stmF mutants of Dictyostelium discoideum produce long, banded aggregation streams on growth plates and exhibit altered cGMP metabolism. To learn more about the role of cGMP in chemotaxis and the nature of the defect in these mutants, 15 nonstreaming (Stm⁺) revertants of two stmF mutants were isolated and characterized. Fourteen of the revertants continued to show the elevated cAMP-induced cGMP response and very low cGMP-specific phosphodiesterase (cGPD) activity characteristic of their stmF parents. Parasexual genetic analysis revealed that many of these Stm⁺ revertants carried phenotypic suppressors unlinked to stmF. One Stm⁺ revertant, strain HC344, exhibited a low, prolonged cGMP response and relatively high cGPD activity throughout development. To determine whether the elevated cGPD activity in this revertant resulted from increased enzyme production or enhanced enzyme activity, cGPDs were partially purified from the wild-type strain, the stmF parent and revertant HC344, and properties of the enzymes were compared. cGPDs from the stmF mutant and the revertant showed similar differences from the wild-type enzyme in kinetic properties, thermal stability, and sensitivity to certain inhibitors. These results suggest that stmF is the structural gene of the cGPD. In addition, the unusual cGMP response in revertant HC344 appeared to be due to increased production of an altered cGPD.     

Penicillin-resistant temperature-sensitive mutants of Escherichia coli which synthesize hypo- or hyper-cross-linked peptidoglycan

A group of Escherichia coli mutants which are ampicillin resistant at 32 C and which either are unable to grow or lyse at 42 C has been selected. These mutants have been classified by a number of characteristics: total peptidoglycan synthesis measured by [(14)C]diaminopimelic acid incorporation, extent of cross-linking of the peptidoglycan which is synthesized, growth characteristics at the two temperatures, and morphology. Two especially interesting groups of mutants have been described. In one of these, a hypo-cross-linked peptidoglycan was synthesized at the nonpermissive temperature. Most of these organisms lysed at 42 C. In another group, the peptidoglycan synthesized at 42 C was hyper-cross-linked. Many of these organisms were spherical. Studies of revertants indicated that ampicillin resistance, temperature sensitivity, cross-linking, growth characteristics, and morphological changes may be related to a single mutational event in both of these groups.