Potentiality of artificial sea water salts for the production of carrageenase by a marine Cytophaga sp

Production of an extracellular enzyme complex (carrageenase) was studied by examining cell-free fluids from cultures of a marine Cytophaga, 1k-C783, growing on different media. Among artificial sea water salts, only NaCl and MgCl2 were utilized by the organism to produce carrageenase. The minimal concentrations of suitable combinations of NaCl and MgCl2 were found to be 0.05 M NaCl plus 0.25 M MgCl2, and 0.15 M NaCl plus 0.15 M MgCl2. KCl and CaCl2 did not have any role in carrageenase production in ZoBell 2216 E broth medium. Carrageenase was synthesized continuously within the resting cells and was released from the cells as well as in the growing cells, when nutrient had been supplied.     

Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA.

The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA.     

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.     

Transformation and fusion of Streptococcus faecalis protoplasts.

Nonconjugative plasmids were transferred by protoplast fusion among Streptococcus faecalis strains and from Streptococcus sanguis to S. faecalis. S. faecalis protoplasts were also transformed with several different plasmids, including the Tn917 delivery vehicle pTV1. Transformation was reproducible, but low in frequency (10(-6) transformants per viable protoplast). A new shuttle vector (pAM610), able to replicate in Escherichia coli and S. faecalis, was constructed and transformed into S. faecalis protoplasts. pAM610 was mobilized by the conjugative plasmid pAM beta 1 in matings among S. faecalis strains and from S. sanguis to S. faecalis. Chimeric derivatives of pAM610 were also transformed into S. faecalis.     

Production of staphylococcal enterotoxin in mixed cultures

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Production of staphylococcal enterotoxin in mixed cultures.

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Transformation of Pseudomonas aeruginosa strain PAO with bacteriophage and plasmid DNA.

A procedure has been developed which allows transformation of P. aeruginosa strain PAO with plasmid and bacteriophage DNA at a frequency of 10(-6) per recipient cell. The method is similar in outline to that developed for Escherichia coli. It involves growing the recipient cells to 3-5 x 10(8) per ml in nutrient broth, washing the cells with 0.1 M MgCl2, resuspending in 0.175 M CaCl2 for 20 min, exposing to DNA for 1 h and then heat pulsing at 42 degrees C for 1 min. Some plasmid markers are expressed immediately, whereas others require time for phenotypic expression.     

应用矿物盐改良池塘水质的研究

为了研究矿物盐对淡水养殖池塘水质的改良效果及应用前景,在12口土质池塘中对淡水养殖斑点叉尾鲴(Ictalurus punctatus Rafinesque,1818)施用三组矿物盐:NaNO3、KCl+MgCl2、NaNO3+KCl+MgCl3的对照实验,对照组池塘不施用任何矿物盐类;在182d的养殖期间,对所有处理组和对照组池塘的17项水质和5项底质因子进行了定期监测.结果表明,各处理组均显著降低了池塘水中的蓝藻相对密度;KCl+MgCl2的施用可促进水中硝化作用的进行;NaNO3+KCl+MgCl2的施用,显著提高了养殖池塘底泥表层的氧化还原电位;NaNO3+KCl+MgCl2组的池塘鱼产量比对照组显著提高6.6%,而饲料系数显著降低6.2%;NaNO3与KCl+MgCl2的共同施用起到了协同改良池塘水质和底质的作用.因此,NaNO3+KCl+MgCl2的应用可为淡水池塘水质的改良开辟一个新的途径,具有一定的应用前景.     

Plasmid-associated bacteriocin production by a strain of Carnobacterium piscicola from meat

Carnobacterium piscicola LV17 isolated from vacuum-packed meat produces bacteriocin(s) that is active against closely related lactic acid bacteria, Enterococcus spp., and a strain of Listeria monocytogenes but not against gram-negative bacteria. The bacteriocin has a bactericidal mode of action, is heat resistant, and is stable over a wide range of pH but is inactivated by proteolytic enzymes. Sensitive and resistant cells were shown to adsorb the bacteriocin, but cell death depended on contact of the bacteriocin with the cell membrane. Bacteriocin production is detected early in the growth cycle of the organism in APT broth, but it is not produced in APT broth adjusted to pH 5.5. Bacteriocin production and resistance to the bacteriocin produced are associated with two plasmids of 40 and 49 megadaltons. The possibility that two bacteriocins are produced is indicated because the inhibitory substances of the mutant strains containing either the 40- or 49-megadalton plasmids have different antimicrobial spectra.     

Plasmid-associated bacteriocin production by a strain of Carnobacterium piscicola from meat.

Carnobacterium piscicola LV17 isolated from vacuum-packed meat produces bacteriocin(s) that is active against closely related lactic acid bacteria, Enterococcus spp., and a strain of Listeria monocytogenes but not against gram-negative bacteria. The bacteriocin has a bactericidal mode of action, is heat resistant, and is stable over a wide range of pH but is inactivated by proteolytic enzymes. Sensitive and resistant cells were shown to adsorb the bacteriocin, but cell death depended on contact of the bacteriocin with the cell membrane. Bacteriocin production is detected early in the growth cycle of the organism in APT broth, but it is not produced in APT broth adjusted to pH 5.5. Bacteriocin production and resistance to the bacteriocin produced are associated with two plasmids of 40 and 49 megadaltons. The possibility that two bacteriocins are produced is indicated because the inhibitory substances of the mutant strains containing either the 40- or 49-megadalton plasmids have different antimicrobial spectra.     

The influence of hydrostatic pressure and urethane on the thermal inactivation of bacteriophage

In Difco nutrient broth, containing 0.5 per cent NaCl, pH 6.6, Escherichia coli phages T1, T2, and T5 were inactivated at 66 degrees C., and T7 at 60 degrees C., at nearly the same rate. In each case the rate of destruction was not uniform but more or less decreased with time of heating. With T2 there was an initial increase in number of infective centers after heating for several minutes at 66 degrees C. Hydrostatic pressures up to 10,000 pounds per square inch retarded the thermal destruction of T1, T2, and T5, but accelerated that of T7, while small concentrations of urethane accelerated the rate of each. The rate of inactivation was increased by the addition of 0.005 M phosphate, and was decreased by 0.005 M MgCl(2) in all but T7, whose rate was unaffected by this amount of Mg. The influence of Ca was similar to that of Mg. The addition of 0.005 M MgCl(2) to the broth medium resulted in a first order rate of destruction of T5 at either normal or increased pressure, and with as well as without urethane. Analysis of data obtained under these conditions indicated that the thermal inactivation proceeds with a volume increase of activation of 113 cc. per mol, and with a heat and entropy of 170,000 calories and 425 E. U., respectively, in the rate-limiting reaction. In the presence of 0.1 M urethane the heat and volume change of activation are apparently slightly greater. The relation between concentration of urethane and the amount of acceleration in rate of destruction at normal pressure and 66 degrees C. indicated that the total rate involves at least two first order rate processes: the thermal inactivation itself and a urethane-catalyzed reaction, the latter involving the combination of an average of 2.3 molecules of urethane in the activated state of the bacteriophage molecule whose destruction results in loss of phage activity.     

Insertion of Tn916 into Bacillus pumilus plasmid pMGD302 and evidence for plasmid transfer by conjugation.

As part of an effort to develop systems for genetic analysis of strains of Bacillus pumilus which are being used as a microbial hay preservative, we introduced the conjugative Enterococcus faecalis transposon Tn916 into B. pumilus ATCC 1 and two naturally occurring hay isolates of B. pumilus. B. pumilus transconjugants resistant to tetracycline were detected at a frequency of approximately 6.5 x 10(-7) per recipient after filter mating with E. faecalis CG110. Southern hybridization confirmed the insertion of Tn916 into several different sites in the B. pumilus chromosome. Transfer of Tn916 also was observed between strains of B. pumilus in filter matings, and one donor strain transferred tetracycline resistance to recipients in broth matings at high frequency (up to 3.4 x 10(-5) per recipient). Transfer from this donor strain in broth matings was DNase-resistant and was not mediated by culture filtrates. Transconjugants from these broth matings contained derivatives of a cryptic plasmid (pMGD302, approx 60 kb) from the donor strain with Tn916 inserted at various sites. The plasmids containing Tn916 insertions transferred to a B. pumilus recipient strain at frequencies of approx 5 x 10(-6) per recipient. This evidence suggests that pMGD302 can transfer by a process resembling conjugation between strains of B. pumilus.     

Evaluation of enrichment broths for their ability to recover heat-injured Listeria monocytogenes

J.R. PATEL AND L.R. BEUCHAT. 1995. Listeria selective enrichment broth (LEB), University of Vermont (UVM) broth, modified UVM (MUVM) broth and Fraser broth (FB) were compared for their ability to recover cells of L. monocytogenes from heated tryptose phosphate broth. Three strains of L. monocytogenes were heated at 54C for 30 min, inoculated into enrichment broths supplemented with 400 µg catalase ml⁻¹, and incubated for 8 h at 30°C. After incubation for 4 h, the total viable cell populations either decreased or did not change, whereas the number of healthy (non-injured) cells of all strains increased significantly in all broths except FB inoculated with the LCDC strain. With an increase in incubation time to 8 h, the number of healthy cells of all strains increased in all broths. At 8 h, the difference between populations of total (injured plus healthy cells) and healthy cells detected in LEB inoculated with two strains was not significant. Overall, recovery of heat-treated cells was significantly higher in LEB, followed by MUVM broth, UVM broth and FB. The addition of catalase to enrichment broths significantly enhanced recovery of heat-injured cells. A slight reduction of catalase activity of heated cells of all test strains in all enrichment broths except FB was observed by extending the incubation period from 4 to 8 h. A test strain that produces relatively higher catalase activity compared to the other strains exhibited the greatest resistance to exogenous hydrogen peroxide. Enumeration of viable L. monocytogenes cells in heated foods should be done using LEB supplemented with 400 µg catalase ml⁻¹ to maximize the recovery of injured cells.     

Studies on interaction of 5 S RNA with ribosomal proteins

Proteins of the large ribosomal subunit of rat liver (TP 60) were immobilized by diffusion transfer onto nitrocellulose after two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Incubation of the TP 60 blots with 32P-labeled 5 S RNA under defined ionic conditions (300 mM KCl, 20 mM MgCl2) resulted in specific binding to a limited set of ribosomal proteins consisting of proteins L3, L4, L6, L13/15 and--to a lesser extent--L7 and L19. Under identical conditions, blots with proteins of the small ribosomal subunit (TP 40) did not bind 5 S RNA.     

Isolation and characterization of two alkaline ribonucleases from calf serum.

Treatment of calf serum at 60 degrees C and pH 3.5 followed by chromatography on carboxymethyl (CM) cellulose resulted in the separation of two major peaks of alkaline RNAse activity. One was eluted from CM-cellulose at 0.075 M KCl with an overall purification of 5400-fold and the other was eluted at 0.25 M KCl with a 6700-fold purification. The RNAse eluted from CM-cellulose at 0.075 M KCl was almost completely inhibited by anti-RNAse A serum and by the endogenous RNAse inhibitor and a 33% inhibition was observed in the presence of 5 mM MgCl2. This enzyme seems to be similar or identical to RNAse A. The other RNAse, eluted from CM-cellulose at 0.25 M KCl was not inhibited by anti-RNAse A or 5 mM MgCl2 and was much less sensitive to the endogenous inhibitor. Both enzymes degraded RNA endonucleolytically and the nucleoside monophosphates obtained after partial hydrolysis of RNA by the two serum RNAases were primarily 2'- or 3' -CMP and 2'- or 3' -UMP. Poly(A), native DNA and denatured DNA were degraded slowly or not at all. The RNAase A-like enzyme degraded poly(C) at a significantly faster rate, and poly(U) at a slower rate, than RNA. However, the other serum RNAase was more active with poly(U) than with RNA and almost inactive with poly(C) as the substrate.     

Enhancing the antimicrobial effects of bovine lactoferrin against Escherichia coli O157:H7 by cation chelation, NaCl and temperature

AIM: To evaluate the effect of NaCl, growth medium and temperature on the antimicrobial activity of bovine lactoferrin (LF) against Escherichia coli O157:H7 in the presence of different chelating agents. METHODS AND RESULTS: LF (32 mg ml(-1)) was tested against E. coli O157:H7 strain 3081 in Luria broth (LB) and All Purpose Tween (APT) broth with metal ion chelators sodium bicarbonate (SB), sodium lactate (SL), sodium hexametaphosphate (SHMP), ethylene diamine tetraacetic acid (EDTA) or quercetin at 0.5 and 2.5% NaCl at 10 and 37 degrees C. LF and the chelators were tested against four other E. coli O157:H7 strains in LB at 2.5% NaCl and 10 degrees C. LF alone was bacteriostatic against strains 3081 and LCDC 7283 but other strains grew. Antimicrobial effectiveness of LF was reduced in APT broth but enhanced by SB at 2.5% NaCl and 10 degrees C where 4.0 log(10) CFU ml(-1) inoculated cells were killed. EDTA enhanced antimicrobial action of the LF-SB combination. SL alone was effective against E. coli O157:H7 but a reduction in its activity at 2.5% NaCl and 10 degrees C was reversed by LF. The combinations LF-SHMP and LF-quercetin were more effective at 37 degrees C and NaCl effects varied. CONCLUSIONS: LF plus SB or SL were bactericidal toward the same 3/5 E. coli O157:H7 strains and inhibited growth of the others at 2.5% NaCl and 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of LF with either SL or SB shows potential for reducing viability of E. coli O157:H7 in food systems containing NaCl at reduced, but growth permissive temperature.     

Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.     

Injury and recovery of Bacillus megaterium from mild chlorhexidine treatment

Treatment of Bacillus megaterium cell suspensions with 12 /μmol/1 chlorhexidine diacetate for 5 min led to an approximate 50%, reduction in viability when plated onto tryptone soya agar (TSA). Fifty percent of the surviving fraction were unable to form colonies on TSA containing 5.5% w/v KCI. Such loss of KCl tolerance is indicative of membrane damage, and was recovered within 30 min of incubation in tryptone soya broth (TSB). Multiplication of the damaged organisms did not recommence in this medium until after 60 min. Inclusion of inhibitors of respiration, and of protein, RNA and DNA synthesis in the TSB recovery medium did not significantly affect either the rate or extent of the recovery of KCl tolerance by damaged organisms.     

Deoxyribonucleic Acid Breaks in Heated Salmonella typhimurium LT-2 After Exposure to Nutritionally Complex Media

Minimal medium recovery of heat-treated Salmonella typhimurium LT-2 has been expressed as the reduced viability on trypticase soy agar supplemented with 0.5% yeast extract (TSY) relative to a glucose-salts (M-9) agar. Incubation of S. typhimurium LT-2 in water at 50 C for 15 min did not change the sedimentation patterns of deoxyribonucleic acid (DNA) in alkaline sucrose gradients. The same pattern was obtained if the heated cells were further incubated for 15 min at 37 C in M-9 broth. However, a marked increase in DNA single-strand breakage accompanied by a loss of viability was observed after a similar incubation of heated bacteria in TSY broth. If heated bacteria were incubated in M-9 broth before TSY broth, there was a decrease in the single-strand breakage occurring in the TSY broth. This decrease is believed to be the result of repair of heat-induced damage. We conclude that minimal medium recovery after heat treatment is due to DNA damage caused by sequential exposure to heat and TSY medium, such damage not occurring after sequential exposure to heat and M-9 medium.     

The opposite effect of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase.

The effects of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase were studied with the proteinase-solubilized enzymes. Cytochrome b5 reduction by NADH:cytochrome b5 reductase was strongly inhibited by CaCl2 or MgCl2. When 1.2 microM-cytochrome b5 was used, the concentrations of CaCl2 and MgCl2 required for 50% inhibition (I50) were 8 and 18 mM respectively. The inhibition was competitive with respect to cytochrome b5. The extent of inhibition by CaCl2 or MgCl2 was much higher than that by KCl or other alkali halides. In contrast, cytochrome b5 reduction by NADPH:cytochrome c reductase was extremely activated by CaCl2 or MgCl2. In the presence of 5 mM-CaCl2, the activity was 24-fold higher than control when 4.4 microM-cytochrome b5 was used. The magnitude of activation by CaCl2 was 2-3-fold higher than that by MgCl2. The activation by these salts was much higher than that by KCl, indicating that bivalent cations play an important role in this activation. The mechanisms of inhibition and activation by bivalent cations of cytochrome b5 reduction by these two microsomal reductases are discussed.