The survival and growth of Yersinia pestis cells within mouse peritoneal cavities and within mouse peritoneal macrophages maintained in vitro was examined. Two strains were used which differed only in that one (KIM) contained the 47-megadalton plasmid associated with virulence and the second (KIM1) lacked this plasmid. The KIM cells, but not the KIM1 cells, acquired some resistance to phagocytosis during growth at 37°C which was not evident when cells were grown at 26°C. Whether previously grown at 26 or 37°C, however, a substantial portion of the cells of either strain which were phagocytized were apparently killed after phagocytosis in vivo, although this was not observed in vitro. KIM cells which survived phagocytosis proliferated within macrophages in vivo, but no increase in viable cells was seen with the KIM1 cells. Growth of the KIM1 cells within macrophages in vitro required that a complex supportive medium be used in which the bacteria could have grown if extracellular. This was not the case for the KIM cells which proliferated within macrophages supported in medium not permissive to bacterial growth. After phagocytosis of cells of either strain by macrophages maintained in vitro, phagolysosome formation occurred normally, as shown by the acridine orange dye staining technique. KIM and KIM1 cells were equally sensitive to hydrogen peroxide and superoxide anion, although the sensitivity in each case varied with growth temperature. The oxidative burst, as determined by the luminol chemiluminescence assay, was low when compared with that seen after phagocytosis of Escherichia coli cells. Chemiluminescence after phagocytosis of yeast cells by macrophages which had engulfed KIM or KIMI was also low. We conclude that survival within macrophages is substantially independent of the 47-megadalton plasmid and may be a consequence, as least in part, of blockage of the oxidative burst or rapid removal of the oxidizing compounds formed. The 47-megadalton plasmid is apparently required for subsequent proliferation within the macrophage.

A 37 but not 26 degrees C virulent Yersinia pestis is known to require at least 2.5 mM Ca2+ for growth; this requirement is potentiated by Mg2+. After shift of log-phase cells (doubling time of 2 h) from 26 to 37 degrees C in Ca2+-deficient medium, shutoff of net ribonucleic acid synthesis preceded that of protein and cell mass. With 2.5 mM Mg2+, about two doublings in cell mass and number occurred before restriction with synthesis of sufficient deoxyribonucleic acid to account for initiation and termination of two postshift rounds of chromosome replication. Temperature shift with 20 mMMg2+ resulted in a single doubling of cell mass and number with one round of chromosome replication. Subsequent to shutoff of ribonucleic acid accumulation, ribonucleoside but not deoxyribonucleoside triphosphate pools became reduced to about 50% of normal values and the adenylate energy change fell from about 0.8, typical of growing cells, to about 0.6. Excretion of significant concentrations of adenine nucleotides under both permissive and restrictive conditions was observed. Only trace levels (less than 0.01 microM ol/g [dry weight]) of guaninosine 5'-diphosphate 3'-diphosphate accumulated under restrictive or permissive conditions; guanosine 5'-triphosphate 3'-diphosphate was not detected. Return of fully restricted cells from 37 to 26 degrees C with Ca2+ resulted in prompt growth, whereas addition of Ca2+ at 37 degrees C was ineffective. This finding indicates that the observed temperature-sensitive lesion in ribonucleic acid synthesis that results in restriction can be prevented but not reversed by cultivation with Ca2+.

Isocitrate lyase and malate synthase, the two unique enzymes of the glyoxylate cycle, were detected in crude extracts of Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica. Y. pestis, unlike Escherichia coli and the other yersiniae tested, yielded two forms of isocitrate lyase during growth on acetate. These forms differed in electrophoretic mobility and temperature optima. One form (A) was present during growth on acetate, but was absent during growth on alternate carbon sources such as glucose. The second form (B) was not constitutive, but was found during growth on acetate, glucose, xylose, or other complex carbon sources. Itaconate, a succinate analog which inhibited both forms of isocitrate lyase in crude extracts, did not affect the growth of Y. pestis under conditions where little isocitrate lyase activity was detected. This inhibitor, however, retarded the growth of Y. pestis under conditions where acetate was provided as the primary carbon and energy source as well as under all conditions in which either form of isocitrate lyase was evident. This suggests that the B form may play an important role in the growth of this bacterium under conditions where a requirement for the classical anaplerotic sequence involving this enzyme is not apparent.

The protein composition of the outer membrane of Yersinia pestis grown at 26 and at 370C was examined. The outer membrane was isolated by isopycnic sucrose density centrifugation, and its degree of purity was determined with known inner and outer membrane components. Using two-dimensional gel electrophoresis, we identified a large number of heat-modifiable proteins in the outer membrane of cells grown at either incubation temperature. One-dimensional

In Klebsiella aerogenes W70, there is an inducible pathway for the catabolism of ribitol consisting of at least two enzymes, ribitol dehydrogenase (RDH) and D-ribulokinase (DRK). These two enzymes are coordinately controlled and induced in response to D-ribulose, an intermediate of the pathway. Whereas wild-type K. aerogenes W70 are unable to utilize xylitol as a carbon and energy source, mutants constitutive for the ribitol pathway are able to utilize RDH to oxidize the unusual pentitol, xylitol, to D-xylulose. These mutants are able to grow on xylitol, presumably by utilization of the D-xylulose produced. Mutants constitutive for L-fucose isomerase can utilize the isomerase to convert Darabinose to D-ribulose. In the presence of i-ribulose, RDH and DRK are induced, and such mutants are thus able to phosphorylate the D-ribulose by using the DRK of the ribitol pathway. Derivatives of an L-fucose isomerase-constitutive mutant were plated on D-arabinose, ribitol, and xylitol to select and identify mutations in the ribitol pathway. Using the transducing phage PW52, we were able to demonstrate genetic linkage of the loci involved. Three-point crosses, using constitutive mutants as donors and RDH -, DRK-double mutants as recipients and selecting for DRK+ transductants on D-arabinose, resulted in DRK + RDH +-constitutive, DRK + RDH +-inducible, and DRK + RDH --inducible transductants but no detectable DRK + RDHconstitutive transductants, data consistent with the order rbtC-rbtD-rbtK, where rbtC is a control site and rbtD and rbtK correspond to the sites for the enzymes RDH and DRK, respectively. 162 on August 1, 2020 by guest