Aerobic growth on nitroglycerin as the sole carbon, nitrogen, and energy source by a mixed bacterial culture.

Nitroglycerin (glycerol trinitrate [GTN]), an explosive and vasodilatory compound, was metabolized by mixed microbial cultures from aeration tank sludge previously exposed to GTN. Aerobic enrichment cultures removed GTN rapidly in the absence of a supplemental carbon source. Complete denitration of GTN, provided as the sole C and N source, was observed in aerobic batch cultures and proceeded stepwise via the dinitrate and mononitrate isomers, with successive steps occurring at lower rates. The denitration of all glycerol nitrate esters was found to be concomitant, and 1, 2-glycerol dinitrate (1,2-GDN) and 2-glycerol mononitrate (2-GMN) were the primary GDN and GMN isomers observed. Denitration of GTN resulted in release of primarily nitrite-N, indicating a reductive denitration mechanism. Biomass growth at the expense of GTN was verified by optical density and plate count measurements. The kinetics of GTN biotransformation were 10-fold faster than reported for complete GTN denitration under anaerobic conditions. A maximum specific growth rate of 0.048 +/- 0.005 h-1 (mean +/- standard deviation) was estimated for the mixed culture at 25 degreesC. Evidence of GTN toxicity was observed at GTN concentrations above 0. 3 mM. To our knowledge, this is the first report of complete denitration of GTN used as a primary growth substrate by a bacterial culture under aerobic conditions.

Fusarium polycaprolactone depolymerase is cutinase.

Polycaprolactone (PCL), a synthetic polyester, is degraded by a variety of microorganisms, including some phytopathogens. Many phytopathogens secrete cutinase, a serine hydrolase that degrades cutin, the structural polymer of the plant cuticle. We compared wild-type strains and a cutinase-negative gene replacement mutant strain of Fusarium solani f. sp. pisi (D. J. Stahl and W. Schäfer, Plant Cell 4:621-629, 1992) and a wild-type strain of Fusarium moniliforme to show that Fusarium cutinase is a PCL depolymerase. The wild-type strains, but not the mutant strain, (i) degraded PCL and used it as a source of carbon and energy, (ii) showed induction of secreted PCL depolymerase and an esterase activity of cutinase when grown in the presence of cutin, and (iii) showed induction of PCL depolymerase and an esterase activity of cutinase when grown in the presence of a hydrolysate of PCL, which contains PCL oligomers that are structurally similar to the natural inducers of cutinase. These results together with other details of regulation and conditions for optimal enzyme activity indicate that the Fusarium PCL depolymerase, required for degradation and utilization of PCL, is cutinase.

Genetic locus, distant from ptsM, affecting enzyme IIA/IIB function in Escherichia coli K-12.

Most strains of Escherichia coli K-12 are unable to use the enzyme IIA/IIB (enzyme IIMan) complex of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in anaerobic growth and therefore cannot utilize glucosamine anaerobically. Introduction into these strains of a ptsG mutation, which eliminates activity of the enzyme IIIGlc/IIB' complex of the PTS, resulted in inability to grow anaerobically on glucose and mannose. Derivative strains able to grow anaerobically on glucosamine had mutations at a locus close to man, the gene coding for phosphomannose isomerase, and had higher enzyme IIA/IIB activities during anaerobic growth than did the parental strain. These results establish a locus affecting function of enzyme IIA/IIB that maps distant from ptsM, the probable structural gene for enzyme IIB.

New maltose Blu mutations in Escherichia coli K-12.

Mutations in the genes pgi, pfkA, and ptsG resulted in a maltose Blu phenotype in Escherichia coli K-12, bringing the number of known Blu alleles to six. The Blu phenotype, as visualized by staining with iodine vapor, is a convenient mutant isolation technique.

On the analysis of unrestricted mixed cultures in determining the fitness of microbial mutants.

Equations describing the growth of two microbial strains in unrestricted mixed culture are developed and their use described. With this treatment, mixed cultures maintained in growth by periodic dilution or by use of a turbidostat may be used to obtain a quantitative measure of the adaptive or maladaptive effects of specific mutant alleles.

Lack of glucose phosphotransferase function in phosphofructokinase mutants of Escherichia coli.

Phosphofructokinase (pfkA) mutants of Escherichia coli are impaired in growth on all carbon sources entering glycolysis at or above the level of fructose 6-phosphate (nonpermissive carbon sources), but growth is particularly slow on sugars, such as glucose, which are normally transported and phosphorylated by the phosphoenolpyruvate, (PEP)-dependent phosphotransferase system (PTS).

PfkB and pfkC loci of Escherichia coli.

Mutants lacking Escherichia coli phosphofructokinase (pfkA, 78 min) are suppressed by the unlinked pfkB1 mutation, which restores some enzyme activity (Morrissey and Fraenkel, 1972). We here describe a secondary mutation at pfkB, "PFKB-," which abolishes the suppression as well as the low residual activity of unsuppressed pfkA mutants. pfkB is at about 33 min. with the gene order groD-pps-pheS-pfkB. A positive selection was found that yielded both the pfkB-mutations and a new similar mutation, pfkC-. pfkC is an early marker in Hfr HL16(ca. 50 to 55 min). Some pfkC-, but no pfkB-, mutations were amber. A temperature-sensitive pfkB- was also obtained. Strains carrying pfkB- or pfkC-, but wild type at pfkA, were not markedly affected in growth on sugars. A new search for suppressors such as pfkB1 gave five independent candidates, all of which suppressed both pfkA1 and pfkA2 and occurred in the pfkB region; none occurred at pfkC. Neither the pfkB nor the pfkC loci have assigned functions. It is likely that they are somehow involved in expression of phosphofructokinase activity 2 (Fraenkel, Kotlarz, and Buc, 1973).

PfkA locus of Escherichia coli.

pfkA was know, on the basis of three mutants, as the likely locus of phosphofructokinase in Escherichia coli, and the unlinked pfkB1 mutation suppressed these mutations by restoring some enzyme activity (Morrissey and Fraenkel, 1972). We now report a new search for the complete inactivation of pfkA (e.g., by deletion or amber mutation), done to assess whether the pfkB1 suppression is by an independent enzyme, phosphofructokinase activity 2 (Fraenkel, Kotlarz, and Buc, 1973). Ten new phosphofructokinase mutants all were at pfkA, rather than at pfkB or pfkC. One of them (pfkA9) gave temperature-sensitive reverants with heat-labile enzyme. Another (pfkA11) proved genetically to be a nonsense mutation, but showed no restored activity when suppressed by supF. However, even unsuppressed it was found to contain an enzyme related to phosphofructokinase activity 1 kinetically (more allosteric), physically (almot identical subunit), and antigenically. All the pfkA mutants apparently contained cross-reacting material to activity 1. All (including pfkA11) were suppressed by the pfkB1 mutation. Several results support the idea that pfkA is the structural gene for the main phosphofructokinase of E. coli (activity 1), but that there is some restriction to its complete inactivation.

New phosphoglucose isomerase mutants of Escherichia coli.

The mutants used to show that phosphoglucose isomerase, and glucose itself, are not essential components of Escherichia coli had not been characterized genetically, other than by mapping. We now describe two new pgi mutants, one amber and the other a Mu-phage insertion, presumably both complete inactivation mutations. The new mutations do not give a phenotype markedly different from those described earlier. However, they might be preferred for certain physiological studies, and we have prepared for this a new double mutant, strain DF214, with a Mu insertion in pgi and a deletion in zwf (flucose 6-phosphate dehydrogenase).

Phenotypic suppression of phosphofructokinase mutations in Escherichia coli by constitutive expression of the glyoxylate shunt.

Fructose-6-phosphate kinase (pfkA) mutants have impaired growth on carbon sources which enter glycolysis at or above the level of fructose-6-phosphate, but the degree of impairment depends on the carbon source (e.g., growth on glucose is very much slower than growth on glucose-6-phosphate). The present report contains considerable data on this complicated growth phenotype and derives mainly from the finding of a class of partial revertants which grow as fast on glucose as on glucose-6-phosphate; the reversion mutation is shown to be constitutivity of the glyoxylate shunt (iclR(c)). iclR(c) does not increase the fructose-6-phosphate kinase level in the mutants, and the exact mechanism of the partial phenotypic suppression is not understood. However, iclR(c) was already known to suppress some mutations which affected phosphoenolpyruvate levels, and H. L. Kornberg and J. Smith have suggested (1970) that the growth phenotype of pfkA mutants might be related to pathways of phosphoenolpyruvate formation. Surprisingly, the hexose-monophosphate shunt is not necessary for the suppression, which therefore must act to restore metabolism via the residual phosphofructokinase activity present in all pfkA mutants. A mutant totally lacking phosphofructokinase activity was not suppressed.

Selection of ribosomal mutants by antibiotic suppression in yeast.

Wild-type Saccharomyces cerevisiae is highly resistant to streptomycin. A histidine auxotroph was found which could grow without histidine in the presence of high concentrations of streptomycin. Selection for derivatives of this strain which could be suppressed by much lower concentrations of streptomycin yielded streptomycin-sensitive mutants which are cold-sensitive and have altered ribosomal profiles.