Genetic and molecular studies of the regulation of atypical citrate utilization and variable Vi antigen expression in enteric bacteria.

1. The atypical citrate-utilizing ability to two strains of E. coli has been shown to be plasmid-encoded. Strain V414 carries a 130 Mdal conjugative Cit+ plasmid that also specifies Tcr and Cmr. Strain V517 carries 9 different plasmid species but only the 36 Mdal species is correlated with Cit+ ability. These plasmids are different from previously reported Cit+ plasmids of E. coli and Salmonella, which express thermosensitive conjugal transfer systems. 2. A 9 kb Pstl fragment, carrying the Cit+ genes of pWR60, has been cloned into the pBR325 plasmid. 3. Metabolic studies indicate that intact citrate is not incorporated directly into whole cells. Rather, atypical citrate utilization by these E. coli strains appears to involve partial metabolism of citrate at the cell surface before or during uptake. 4. The expression of atypical Cit+ ability by the parental pWR60 plasmid or by the recombinant pWR61 plasmid appears reversible and may involve an expression switch mechanism (i.e., insertion sequence element). 5. Two widely separated genetic loci, viaA and viaB, are necessary for Vi antigen synthesis in Salmonella and Citrobacter. In some strains of C. freundii, Vi antigen expression is reversible, a phenomenon which can be visualized by a colonial morphology transition between Vi-expressing and -nonexpressing forms. 6. The C. freundii viaB locus appears to encode the Vi antigen as well as the genetic "switch" mechanism controlling reversible Vi antigen expression. The viaA locus, which is found in several different bacterial species, may encode some common property (e.g., cell surface structure or enzymatic activity) that is needed for Vi antigen expression. 7. S. typhi and E. coli K12 hybrid strains which carry the C. freundii viaB locus have been constructed. These hybrid strains express reversible Vi antigen expression, even in the absence of general recombination (i.e., functional recA gene product). 8. The C. freundii viaB locus was transposed via Mu-mediated events to an F'lac plasmid in the E. coli K12 hybrid strain WR2376. F' plasmids carrying the viaB locus should serve as a highly enriched source of viaB DNA for physical examination of the switch mechanism. 9. Genetic manipulations such as those described herein can be used to study virtually any plasmid, viral, or chromosomally-encoded property. The resultant better understanding of biochemical pathways and of genetic regulatory control systems, and the isolation of desired gene sequences should provide ample information and materials for improving chemical processes and constructing vaccines against various organisms.

Mannitol and fructose catabolic pathways of Pseudomonas aeruginosa carbohydrate-negative mutants and pleiotropic effects of certain enzyme deficiencies.

Mutant strains of Pseudomonas aeruginosa PAO were isolated on the basis of their inability to utilize mannitol as sole carbon source for growth. Four linkage groups (I through IV) among these mutant strains were resolved by two-factor crosses using the general transducing phage F116, and the strains appeared to contain point mutations as evidenced by ability to give rise to spontaneous revertants with wild phenotype on mannitol minimal agar. Group I strains were affected only in ability to grow on mannitol; all were deficient in inducible mannitol dehydrogenase activity, and all but one were deficient in inducible mannitol transport activity. Fructokinase was induced in group I strains and in wild-type bacteria during growth in the presence of mannitol but not fructose, indicating the presence of a pathway specific for endogenously generated fructose. Cells grown on fructose contained phosphoenolpyruvate:fructose-1-phosphotransferase activity, and mannitol-grown cells contained a lower level of this activity. Group II mutants were deficient in constitutive phosphoglucoisomerase, failed to grow on mannitol, grew very slowly on glycerol and fructose, but grew normally on glucose and gluconate. Group III strains were deficient in both nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase activities that reside in a single enzyme species. 6-Phosphogluconate appeared to be the inductive effector for this enzyme, which was not required for aerobic growth on glucose or gluconate. A single mannitol-negative mutant in group IV also failed to grow on glycerol and glucose, but no biochemical lesion was identified.

Regulation of alanine dehydrogenase in Bacillus (licheniformis).

Cell extracts of Bacillus licheniformis were found to contain nicotinamide adenine dinucleotide (NAD)-dependent l-alanine dehydrogenase (ADH) (l-alanine: NAD oxidoreductase, EC 1.4.1.1). High specific activities (3.5 to 6.0 IU/mg of protein) were found in extracts of cells throughout growth cycles only when l-alanine served as the primary source of carbon or carbon and nitrogen. Specific activities were minimal (0.02 to 0.04 IU/mg of protein) during growth on glucose, but increased at least sevenfold during the first 5 h of postlogarithmic-phase metabolism. Addition of 10 mM glucose to cultures during logarithmic-phase growth on l-alanine resulted in a rapid decrease in enzyme activity. Addition of 20 mM l-alanine to cells near the completion of log-phase growth on glucose resulted in a 20-fold increase in ADH specific activity during less than one cell generation. Extracts of postlogarithmic-phase cells cultured on glucose, malate, l-glutamate, or Casamino Acids contained intermediate levels of ADH activity. The enzyme was partially purified from crude extracts of B. licheniformis, and apparent kinetic constants were estimated. A role for ADH in the catabolism of l-alanine to pyruvate during vegetative growth on l-alanine and during sporulation of cells cultured on glucose is proposed on the basis of these experimental results.