Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature

Clostridium perfringens 11268 CDR (Rifr Tcs), the strain transformed in our experiments, was generated by curing a spontaneous, rifampicin-resistant mutant of C. perfringens 11268 (Rifr Tcr). High-temperature growth yielded tetracycline-sensitive, rifampicin-resistant cells which no longer contained pCW3, a 42.8-kilobase plasmid. The tetracycline-sensitive, rod-shaped cell was then converted to an L-phase variant by growth in the presence of penicillin G (10 micrograms/ml) and 0.4 M sucrose. After several passages, the antibiotic was removed from the medium, and cells continued to grow as L-phase variants. Another large plasmid, pJU124 (38.8 kilobases), which confers tetracycline resistance, was used for transformation. Transformation of L-phase variants of C. perfringens 11268 CDR (Rifr Tcs) was mediated by polyethylene glycol. Transformation frequency is a nonlinear function of DNA concentration. Restriction analysis showed that the plasmid isolated from the transformants was identical to that supplied. Stable L-phase variants do not revert to rod-shaped cells, but autoplasts can be both transformed and reverted.     

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.     

Superoxide dismutase activity during recovery of thermally stressed Staphylococcus aureus MF-31.

Superoxide dismutase (SOD) activity was determined during the growth cycle of unheated and heat-injured cells of Staphylococcus aureus MF-31. SOd activity levels dropped in unheated cells during the lag phase, increased during logarithmic phase, and became constant in the stationary phase. Cells which were sublethally heated (52 degrees c, 20 min) in 100 mM phosphate buffer and subsequently allowed to recover in tryptic soy broth demonstrated an 85% decrease in SOD activity upon inoculation into recovery medium. As the injured cells repaired the heat-induced lesions and entered logarithmic growth, SOD levels rapidly increased. Heat-injured cells allowed to recover in tryptic soy broth plus 10% NaCl showed similar decreases in SOD activity levels. However, no subsequent increase was observed when specific activity was calculated based on milligrams of protein.     

Catalase activity during the recovery of heat-stressed Staphylococcus aureus MF-31.

Sublethal heating of Staphylococcus aureus produced little loss of catalase activity, but incubation of the injured cells in tryptic soy broth, with or without 10% NaCl added, produced an 85% decrease in catalase activity. Cells recovered in tryptic soy broth demonstrated increases in catalase levels after approximately 5 h, whereas in tryptic soy broth with 10% NaCl the levels remained low for at least 12 h. Thus, the loss of catalase activity during the recovery period was greater than during the heat treatment.     

Catalase activity during the recovery of heat-stressed Staphylococcus aureus MF-31

Sublethal heating of Staphylococcus aureus produced little loss of catalase activity, but incubation of the injured cells in tryptic soy broth, with or without 10% NaCl added, produced an 85% decrease in catalase activity. Cells recovered in tryptic soy broth demonstrated increases in catalase levels after approximately 5 h, whereas in tryptic soy broth with 10% NaCl the levels remained low for at least 12 h. Thus, the loss of catalase activity during the recovery period was greater than during the heat treatment.     

Electron microscopic study of cell surface rings during cell division and morphogenesis of Arthrobacter crystallopoietes.

The whole cell ultrastructure during cell division and morphogenesis of Arthrobacter crystallopoietes was monitored using electron microscopic techniques. Glucose-grown spherical cells were inoculated into succinate-based medium. In this medium, the organism undergoes a morphogenetic cycle consisting of elongation of spheres to rods, exponential growth as rods, and fragmentation of rods to spherical cells. Raised bands or rings that encircled the cells were evident on the cell surface of both sphere- and rod-shaped cells. Many rod-shaped cells possessed two or more rings arranged adjacent to each other in a parallel orientation. At each cell division a new ring was formed on both siblings. However, as predicted by the proposed model of unidirectional cell growth and by maintaining a ring from the previous generation, unequal numbers of rings were observed on sibling cells. Only one ring was visible on most of the spherical inoculum cells, but in some cases a second ring perpendicular to the other ring was observed. Parallel rings were found on spherical cells resulting from fragmentation or reductive cell division of rods during the stationary growth phase. Thus, these spheres could be distinguished from inoculum spheres containing a single ring or perpendicular orientation of rings. The number of rings per cell and arrangement of rings on the cell surface of sibling cells after cell division, but before cell separation, are discussed with respect to cell age, cell division, and sphere-rod-sphere morphogenesis of A. crystallopoietes.     

Radiometric Detection of Some Food-Borne Bacteria

Studies on detection of bacteria by radiometric techniques have been concerned primarily with aerobic species in clinical specimens. The data presented here are related to detection of aerobic and anaerobic species that are of significance in foods, by measurement of (14)CO(2) evolved from the metabolism of (14)C-glucose. Salmonella typhimurium and Staphylococcus aureus were inoculated into tryptic soy broth containing 0.0139 muCi of (14)C glucose/ml of medium. Detection times ranged from 10 to 3 hr for inocula of 10(0) to 10(4) cells/ml of broth. Heat-shocked spores of Clostridium sporogenes or C. botulinum were incubated in tryptic soy broth supplemented with Thiotone and NaHCO(3). The medium was rendered anaerobic with N(2). Spores were detected when 0.0833 muCi of labeled glucose was available/ml of medium but not when 0.0139 muCi of glucose was present/ml. The spores required 3 to 4 hr longer for detection than did comparable numbers of aerobic vegetative cells. The results demonstrate the importance of availability of sufficient label in the media and the potential of the application of this technique for sterility testing of foods.     

Transformation of Haemophilus influenzae by plasmid RSF0885.

Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximum, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.     

Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia sp. strain DNT for enhancement of 2,4-dinitrotoluene degradation

The gene (vgb) encoding the hemoglobin (VHb) of Vitreoscilla sp. was cloned into a broad host range vector and stably transformed into Burkholderia (formerly Pseudomonas) sp. strain DNT, which is able to degrade and metabolize 2,4-dinitrotoluene (DNT). Vgb was stably maintained and expressed in functional form in this recombinant strain (YV1). When growth of YV1, in both tryptic soy broth and minimal salts broth containing DNT and yeast extract, was compared with that of the untransformed strain, YV1 grew significantly better on a cell mass basis (A(600)) and reached slightly higher maximum viable cell numbers. YV1 also had roughly twice the respiration as strain DNT on a cell mass basis, and in DNT-containing medium, YV1 degraded DNT faster than the untransformed strain. YV1 cells pregrown in medium containing DNT plus succinate showed the fastest degradation: 100% of the initial 200 ppm DNT was removed from the medium within 3 days.     

GROWTH RESPONSE OF SALMONELLA SPP. TO CYCLOHEXIMIDE AMENDMENT IN MEDIA

The present study was designed to evaluate cycloheximide as a potential media amendment to prevent fungal overgrowth on selective media for salmonellae enumeration. The objectives were to determine the effect of cycloheximide on Salmonella spp growth rates and to determine the effect of cycloheximide addition on Salmonella enumeration in selective media. The bacteria tested included two strains of Salmonella typhimurium (NO/NA and LT2) and one strain of Salmonella arizonae. All strains were grown in tryptic soy broth containing cycloheximide to determine the effect of cycloheximide on bacterial specific growth rates. The growth rate of all strains grown in tryptic soy broth were not significantly influenced by addition of cycloheximide at concentrations up to 1,000 mg/L. Growth rates of S. typhimurium NO/NA in minimal media were significantly decreased by addition of cycloheximide aerobically (300 mg/L) and anaerobically (600 mg/L). However, S. typhimurium NO/NA populations on brilliant green agar, MacConkey agar, and from selenite cysteine broth and tetrathionate broth were not affected by cycloheximide additions at concentrations up to 1,000 mg/L. Cycloheximide has potential as a fungistat additive for salmonellae selective media.     

Adenosine triphosphate pool levels and endogenous metabolism in Arthrobacter crystallopoietes during growth and starvation

The adenosine triphosphate (ATP) content of Arthrobactery crystallopoietes was measured during growth, starvation and recovery from starvation. During exponential growth of the cells as spheres in a glucose salts medium, the level of ATP per cell remained constant at 8.0×10^-10 g/cell. Morphogenesis to rodshaped cells and an increased growth rate following addition of casein hydrolysate was accompanied by an almost two-fold increase in the ATP level. As division of the rod-shaped cells proceeded, the level of ATP declined. After growing as rods for 12–14 h the cells underwent fragmentation to spheres during which time the ATP level again increased to the original value of 8.0×10^-10 g/cell. As the spherical cells resumed growth on the residual glucose, their ATP content declined for a short period and then remained relatively constant. During starvation of sphere or rod-shaped cells for one week, the ATP level declined by approximately 70% during the first 40–50 h and then remained constant. The endogenous metabolism rate of spherical cells declined during the first 10–20 h of starvation and then remained constant at approximately 0.02% of the cell carbon being utilized per h. Addition of glucose to spherical cells which had been starved for one week increased both the ATP content per cell and their rate of endogenous metabolism. The ATP content fluctuated and then remained at a level higher than maintained during starvation while endogenous metabolism quickly declined.Non-Standard Abbreviations ATP adenosine triphosphate - GS glucose mineral salts - HC casein hydrolysate - PVP polyvinylpyrrolidone - DMSO dimethylsulfoxide - MOPS morpholinopropane sulfonic acid - EDTA ethylene diaminetetraacetic acid     

Suitability of some enrichment broths and diluents for enumerating cold- and heat-stressed Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells were injured by chilling and heating, and their recovery was tested in glucose-salt-Teepol broth (GSTB), tryptic soy broth containing 7% NaCl (TSBS), Horie - arabinose - ethyl violet broth (HAEB), and water blue - alizarin yellow broth (WBAY). Exponential phase cells were more sensitive to cold shock than were stationary phase cells. Exposure of chill-injured V. parahaemolyticus to GSTB and TSBS resulted in 70 to 80% death; about 70% lethality was noted for heat-injured cells inoculated into TSBS. Neither HAEB nor WBAY enrichment media were lethal to stressed cells, although rates of growth were retarded. The 3% NaCl in 0.1 M potassium phosphate (pH 7.0) diluent proved to be most suitable for protecting against inactivation of cold- and heat-injured cells.     

High concentration of sodium chloride could induce the viable and culturable states of Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis

In the present study, Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis were transferred into Luria–Bertani medium without NaCl (LBWS) and adjusted to various pHs (4, 5, 6 and 7) with lactic acid containing 0·75, 5, 10 and 30% NaCl, and stored at 25°C until the bacterial populations reached below detectable levels on tryptic soy agar (TSA). Although E. coli O157:H7 and S. Enteritidis did not grow on TSA when incubated in LBWS with 30% NaCl for 35 and 7 days, more than 60 and 70% of the bacterial cells were shown to be viable via fluorescent staining with SYTO9 and propidium iodide (PI), respectively, suggesting that a number of cells could be induced into the viable but nonculturable (VBNC) state. These bacteria that were induced into a VBNC state were transferred to a newly prepared tryptic soy broth (TSB) and then incubated at 37°C for several days. After more than 7 days, E. coli O157:H7 and S. Enteritidis regained their culturability. We, therefore, suggest that E. coli O157:H7 and S. Enteritidis entered the VBNC state under the adverse condition of higher salt concentrations and were revived when these conditions were reversed.     

Transformation by the oncogene v-fms: The effects of castanospermine on transformation-related parameters

The effects of castanospermine on various parameters associated with transformation were examined in cells expressing the viral oncogene v-fms. Fischer rat embryo (FRE) cells transformed by the oncogene v-fms and grown in the presence of castanospermine reverted to a more normal cell morphology and accumulated fms protein within the endoplasmic reticulum. Treated cells attained contact inhibition of cell growth at a much lower cell density compared to the untreated controls. No effect of castanospermine on cell growth was observed for FRE cells transformed by a different oncogene v-fgr. Castanospermine-treated SM-FRE (v-fms transformed) cells reexpressed extracellular matrix fibronectin and exhibited an extensive actin-containing cytoskeleton similar to that of normal nontransformed FRE cells. Castanospermine treatment of SM-FRE cells resulted in a sixfold decrease in [3H]deoxyglucose uptake compared to that of the nonreverted SM-FRE cells. Again, no effect was observed in FRE cells transformed by the oncogene v-fgr (GR-FRE). These results further characterize the reversion caused by castanospermine and indicate that cell surface expression coordinately controls anchorage independent growth, cell morphology, contact inhibition of growth, and hexose uptake.     

Demonstration of a Bactericidal Substance Against β-Hemolytic Streptococci in Supernatant Fluids of Staphylococcal Cultures

Staphylococcal skin isolates belonging to phage type 71 were found to produce a bactericidal substance against some streptococci, pneumococci, and corynebacteria. Fifteen strains of group A streptococci belonging to 13 different M types, group C streptococci, and group D streptococci were uniformly inhibited on solid media and in broth by membrane-filtered supernatant fluids of the staphylococcal broth cultures. Inhibition of group G streptococci and other staphyloccoci was variable, and no inhibition of group B streptococci or of a variety of gram-negative rods was demonstrable. A quantitative variation observed to exist among susceptible organisms was a function of the inoculum size of the inhibited strains. The bactericidal substance could be detected best from 24 to 48 hr after inoculation of the staphylococci in tryptic soy broth or in a dialysate of tryptic soy broth. Little or no bactericidal activity was noted when the organisms were grown in several other liquid media. The bactericidal substance was nondialyzable and could be precipitated with ammonium sulfate. It was heat-stable and its activity was not altered within a pH range of 4.0 to 8.5. Pronase and three times crystallized trypsin totally abolished its activity. The concentrated ammonium sulfate precipitate could be fractionated on a Sephadex G-100 column into several peaks, with the bactericidal activity localized to a single peak.     

Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723

Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by Sphingobium chlorophenolicum, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and S. chlorophenolicum cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by S. chlorophenolicum. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.     

Genome Shuffling Improves Degradation of the Anthropogenic Pesticide Pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723

Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by Sphingobium chlorophenolicum, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and S. chlorophenolicum cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by S. chlorophenolicum. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.     

Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine

The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.