Biological activity ofTreponema hyodysenteriae hemolysin

The biological properties ofTreponema hyodysenteriae hemolysin (TH), which in some aspects resembles streptolysin S, were studied. Aside from different erythrocyte species, TH had no lytic activity on prokaryotic (protoplasts and spheroplasts) and eukaryotic (Chinese hamster ovary and spleen) cells. However, TH decreased the response of spleen cells to concanavalin A and lipopolysaccharide. In vivo, TH was found to induce fluid accumulation in ligated rat ileal loops as well as histological modifications similar to those observed in the swine dysentery. These results further differentiate TH from streptolysin S, and suggest that it is a new oxygen-resistant hemolysin produced by a Gram-negative bacterium.     

Streptolysin S: Improved purification and characterization

A simple and efficient procedure for the production and purification of streptolysin S has been developed. Maximal production of the hemolysin occurred when rapidly grown streptococci were harvested 1 h after reaching the stationary phase and incubated with the inducer oligoribonucleotide. Ammonium acetate at 0.1 m in 50 mm potassium phosphate buffer, pH 6.8, was found to effectively protect streptolysin S from thermal inactivation, and was used in the buffer throughout purification. The three-step purification procedure resulted in preparations with the highest specific activity (1.2 × 10⁷ HU per mg protein) ever reported, in recoveries ranging from 35 to 45%. The apparent molecular weights of streptolysin S and the carrier oligonucleotide were estimated as 15,000 and 7,100, respectively. The peptide nature of the active principle was confirmed by studies of the effects of various hydrolytic enzymes on streptolysin S; only pronase, chymotrypsin and subtilisin were found to inactivate the hemolysin. Amino acid analyses indicated that the active peptide consisted of 32 amino acid residues.     

Lethal Toxin of Bacillus cereus I. Relationships and Nature of Toxin, Hemolysin, and Phospholipase

Bacillus cereus phospholipase was characterized as a phospholipase C by the analysis of lecithin degradation products by thin-layer and paper chromatography. Methanol in the growth menstruum inhibited completely the synthesis of phospholipase C, whereas the synthesis of lethal toxin and hemolysin were only partially inhibited. Dialysis of preformed B. cereus products against ethyl alcohol and methanol did not inactivate hemolytic, phospholipase C, or lethal activity. The hemolytic and lethal activities of culture filtrates were completely abolished by trypsin, but phospholipase C activity was resistant to inactivation. Lethal and phospholipase C properties of culture filtrates were resistant to inactivation at 45 C, whereas the hemolytic activity was completely destroyed. Lethal, hemolytic, and phospholipase C activities appeared simultaneously in a complex growth menstruum, but the kinetics of synthesis were different in all cases. Resolution of B. cereus filtrates on columns of Sephadex showed that the phospholipase C, hemolysin, and lethal toxin are distinct proteins. Evidence is also presented which suggests a correlation between the synthesis of B. cereus toxin and the period of transition from vegetative growth to sporulation. The activity of each B. cereus product was cation-independent, as opposed to cation-dependency of the phospholipase C and lethal activities of Clostridium perfringens alpha-toxin. Immunological cross-reactivity between the B. cereus products and C. perfringens alpha-toxin was not apparent; indeed, they were shown to be antigenically distinct.     

猪源链球菌溶血性的检测

用平板法和上清法检测了19株猪源链球菌的溶血性,并通过加入还原剂或氧化剂分别作活化和抑制试验,对各菌株溶血素的性质做了初步鉴定,其中8株猪链球菌2型菌株在血平板上的溶血性较弱,仅为α溶血或弱β溶血,但在THB和5%血清THB中都可产生很强的溶血性,其溶血性可被还原剂活化,被氧化剂和胆固醇所抑制,卵磷脂对其活性则没有影响,属于巯基活化类（类SLO）溶血素,9株马链球菌兽疫亚种菌株在血平板上呈显的β溶血,在不含血清THB中不能产生溶血性,在含血清的THB中可产生较强的溶血性,其活性不被还原剂活化,不被氧化剂抑制,胆固醇能抑制大部分活性,卵磷脂则可全部抑制,属于类SLS溶血素,两株非典型兰氏C群链球菌菌株在血平板上呈显的β溶血,在THB中显示了非常强的溶血性,其活性不被还原剂活化,不被氧化剂抑制或卵磷脂抑制,但被胆固醇强烈抑制,属于非SLO,非SLS溶血素。     

The role of protease in streptolysin S formation

Production of streptolysin S by streptococci was found to be inhibited by treatment with protease inhibitors, tosylphenylalanine chloromethyl ketone (TPCK), tosyllysine chloromethyl ketone (TLCK), or phenylmethylsulfonyl fluoride (PMSF), even in the presence of the inducer oligonucleotides. Other protease inhibitors, antipain, leupeptin, or pepstatin were found to have little or no effect. Trypsin reversed the effect of TPCK or TLCK. The reversal was dependent upon the amount of added trypsin and the incubation time at 37 degrees C, suggesting that a protease activity was involved in the hemolysin formation. The effect of trypsin was not observed if chloramphenicol was also added, suggesting that a precursor of streptolysin S was processed as it was synthesized and released into medium as the active hemolysin, by the concerted action of a protease and inducer oligonucleotides. Experiments with the subcellular fractions of streptococci indicated that the streptolysin precursor was localized in the insoluble fraction and the "processing" protease in the supernatant fraction.     

Differential inhibition of bacterial growth and hemolysin production by lincosamide antibiotics.

Lincomycin and clindamycin, at concentrations below those which partially inhibited bacterial growth, completely suppressed the production of streptolysin S. Chloramphenicol and erythromycin had no effect on hemolysin production.     

The differentiating agent, retinoic acid, causes an early inhibition of inositol lipid-specific phospholipase C activity in HL-60 cells

Retinoic acid, a derivative of vitamin A, is shown to inhibit the levels of inositol phosphates and diacylglycerol by 25-30% when added to intact HL-60 cells at concentrations which induce differentiation. The onset of inhibition occurs after 10 min and reaches a maximum at 45 min. To study the mechanism and the site of action of retinoic acid, the activity of the phosphatidylinositol bisphosphate-specific phospholipase C was studied in cells permeabilized with streptolysin O and in membrane preparations. Phospholipase C activity was stimulated either via the guanine nucleotide regulatory protein (G-protein) or directly by Ca2+. Retinoic acid treatment, in a time- and concentration-dependent manner, led to a decrease in phospholipase C activity when stimulated with either GTP gamma S or NaF, both of which activate the enzyme via the G-protein. By contrast, it had no effect on the enzyme activity when stimulated with Ca2+ alone. This indicates that retinoic acid interferes with the coupling of the G-protein and phospholipase C. A relationship between the inhibition of phospholipase C activity and the induction of differentiation by retinoic acid was investigated. Only a small inhibition of GTP gamma S-stimulated phospholipase C activity was observed when an analogue of retinoic acid, etretine or Ro10-1670, with low differentiating activity, was used. Moreover, no inhibition of the GTP gamma S-stimulated phospholipase C activity was observed in an HL-60 sub-line resistant to retinoic acid. These results suggest that phospholipase C inhibition is an important step in the induction of differentiation.     

Comparison of bacterial cardiotoxins: thermostable direct hemolysin from Vibrio parahaemolyticus, streptolysin O and hemolysin from Listeria monocytogenes.

Some properties of the bacterial cardiotoxins, thermostable direct hemolysin from Vibrio parahaemolyticus (vibriolysin), and streptolysin O and hemolysin from Listeria monocytogenes (listeriolysin), were compared. These toxins had rapid lethal effects on mice when injected intravenously. The electrocardiographic changes of rats after intravenous injections of these toxins were very similar, showing bradycardia and inhibition of atrio-ventricular conduction. These toxins also caused cessation of the spontaneous beating and degeneration of cultured foetal mouse heart cells. When equal hemolytic units of these three toxins were administered, vibriolysin had the most potent effects on mice and cultured mouse heart cells. Differences in the kinetics of the hemolysis by each toxin and in the effects of cholesterol of their hemolytic actions suggest that the mode of action of vibriolysin is different from those of streptolysin O and listeriolysin.     

Osmotic Properties of Spheroplasts from Saccharomyces cerevisiae Grown at Different Temperatures

Spheroplasts were prepared from cells of Saccharomyces cerevisiae NCYC 366, grown at 30 or 15 C, by incubating cells with snail-gut juice after pretreatment with 2-mercaptoethanol. Walls of cells grown batchwise or in continuous culture at 15 C were more resistant to digestion with snail juice than walls on cells grown under the same conditions as 30 C. Spheroplasts lysed when suspended in hypotonic solutions of mannitol. The resistance of spheroplasts to osmotic lysis tended to increase when the test temperature was lowered below 30 C. The increased resistance was greater with spheroplasts from cells grown at 15 C. Cations, especially Ca²⁺, protected spheroplasts against osmotic lysis. In general, the protective effects, measured at 30 C, were smaller with spheroplasts from cells grown at 15 C compared with 30 C. Citrate and ethylenediaminetetraacetate (EDTA) decreased the resistance of spheroplasts to osmotic lysis. On the whole, the decrease was greater with spheroplasts from cells grown at 30 C rather than 15 C. In the presence of EDTA, spheroplasts from cells grown at 30 C were less resistant to osmotic lysis at 5 C than at 30 C; when spheroplasts from cells grown at 15 C were similarly examined, they were more resistant to lysis at 5 C than at 30 C. Spheroplast membranes from cells grown at 15 C had slightly but significantly greater contents of Mg²⁺, Ca²⁺, K⁺, and Na⁺ compared with spheroplast membranes from cells grown at 15 C. Mg²⁺ and Ca²⁺ were more easily extracted with EDTA from membranes of 30 C-grown cells than from 15 C-grown cells.     

Phorbol myristate acetate inhibits phosphoinositol lipid-specific phospholipase C activity via protein kinase C activation in conditions inducing differentiation in HL-60 cells.

We have studied, in streptolysin O-permeabilized HL-60 cells and in HL-60 membrane preparations, the effects of phorbol 12-myristate 13-acetate (PMA) on polyphosphoinositide-specific phospholipase C (PLC) activity and on terminal differentiation towards macrophagic-like cells. We showed that terminal differentiation was induced when differentiating concentrations of the drug were present for only 1-2 h in the culture medium. Conditions inducing differentiation also inhibited PLC activity for a long lasting period (at least 5 h). When terminal differentiation affected only part of the cell population, inhibition of phospholipase C activity was found to be less marked and reversible over the period studied. Moreover in experiments done in an HL-60 clone resistant to PMA, no inhibition of PLC activity was provoked by this tumour promotor. In order to study the involvement of protein kinase C in this process, we measured modifications of PLC activity by PMA in the presence of two different protein kinase C inhibitors, staurosporine and H-7. They both prevented the inhibition of PLC activity by PMA indicating that this inhibition is likely to be related to the effect of PMA on protein kinase C activity. This was also confirmed by the fact that active protein kinase C, by itself, was able to decrease PLC activity when added to membrane preparations or to streptolysin O-permeabilized control HL-60 cells. These results indicate that PMA acts in inhibiting phospholipase C activity through its effect on protein kinase C activation and/or on protein kinase C translocation to the plasma membrane and that terminal differentiation, might be related to changes in both protein kinase C and PLC activities.     

Insertional inactivation of streptolysin S expression in Streptococcus pyogenes

The inactivation of a genetic determinant critical for streptolysin S production was accomplished by transfer and insertion of the transposon Tn916 into the DNA of a group A streptococcal strain. The group D strain CG110 was able to efficiently transfer Tn916 into the group A strain CS91 when donor and recipient cells were concentrated and incubated together on membrane filters. Among tetracycline-resistant transconjugants, nonhemolytic mutants that no longer produced streptolysin S and retained the capacity to produce streptolysin O were discovered. Hemolytic revertants from these mutants regained tetracycline sensitivity; other revertants still retained a tetracycline resistance phenotype. Hybridization studies employing Tn916 DNA located Tn916 sequences in EcoRI and HindIII fragments of DNA from mutants devoid of streptolysin S; one carried a single copy of Tn916, and the other two carried multiple copies of the transposon.     

D609, an inhibitor of phosphatidylcholine-specific phospholipase C, inhibits group IV cytosolic phospholipase A2

As an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 has been widely used to explain the role of PC-PLC in various signal transduction pathways. This study shows that D609 inhibits group IV cytosolic phospholipase A2 (cPLA2), but neither secretory PLA2 nor a Ca2+ -dependent PLA2. Dixon plot analysis shows a mixed pattern of noncompetitive and uncompetitive inhibition with Ki = 86.25 microM for the cPLA2 purified from bovine spleen. D609 also time- and dose-dependently reduces the release of arachidonic acid from a Ca2+- ionophore A23187-stimulated MDCK cells. In the AA release experiment, IC50 of D609 was approximately 375 microM, suggesting that this reagent may not enter the cells easily. The present study indicates that the inhibitory effects of D609 on various cellular responses may be partially attributable to the inhibition of cPLA2.     

Purification of oligonucleotides with streptolysin S inducer activity and de Novo synthesis of the hemolysin

The oligoribonucleotide fraction containing the Streptolysin S inducer activity from the RNase digest of yeast RNA (active fraction; A. W. Bernheimer and M. J. Rodbart, 1948, J. Exp. Med., 80, 149–168) was purified by the oligo(dC)-cellulose affinity chromatography based on its high guanine content. A 20-fold purification of the inducer activity over that of AF the active fraction, and approximately 2000-fold over that of yeast RNA has been obtained. The purification oligonucleotide was found to contain several molecular species with 7–10 nucleotide residues, all apparently with inducer activity. Streptolysin S induced with this oligonucleotide preparation and gel filtered has a specific activity comparable to the highest value reported previously. Incorporation of amino acids into streptolysin S was observed upon induction with the purified oligonucleotide and paralleled the increase in the hemolysin activity. This and experiments with chloramphenicol indicated that streptolysin was synthesized de novo on stimulation with the oligonucleotide inducer. The pattern of amino acid incorporation was in good agreement with the amino acid composition of purified streptolysin reported earlier. No incorporation of glucose or mannose was observed.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Retinyl phosphate mannose synthesis in rat liver membranes. Phospholipase sensitivity and phospholipid requirement.

A remarkable and immediate decrease in GDP-mannose:retinyl phosphate mannosyltransferase activity was found on pre-incubation of rat liver postnuclear membranes with phospholipase A2 or phospholipase C. Under the same conditions of pre-incubation (1 min at 37 degrees C) trypsin did not affect the enzyme activity, whereas pre-incubation for 30 min with trypsin and Pronase abolished enzyme activity. The lipid extract of untreated rat liver membranes partially restored enzyme activity after phospholipase treatment. Sphingomyelin was as active as the endogenous lipids. Other phospholipids were less active in the following order: phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol = phosphatidylserine. Dolichyl phosphate mannose synthesis was inhibited less (33%) by phospholipase C than was Ret-P-Man synthesis (98.5%) under identical conditions of incubation, which included 0.025% Triton. However, retinyl phosphate mannose synthesis by purified endoplasmic reticulum was found to be resistant to phospholipase C. Mixing experiments failed to demonstrate an inhibitory effect of the phospholipase-treated postnuclear membrane fraction on the synthetic activity of the endoplasmic reticulum, thus excluding the release of an inhibitory factor from the postnuclear membranes.     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.     

The transport activity of the Na+-Ca2+ exchanger NCX1 expressed in HEK 293 cells is sensitive to covalent modification of intracellular cysteine residues by sulfhydryl reagents

Membrane permeable N-ethylmaleimide (NEM) and (2-aminoethyl)methanethiosulfonatehydrobromide (MTSEA) inhibited the rat brain Na(+)-Ca(2+) exchanger RBE-2 (NCX1.5) expressed in HEK 293 cells in a dose dependent manner. 50% inhibition was obtained at 1 mm MTSEA and 1.65 mm NEM. External application of membrane impermeable [2-(trimethylammonium) ethyl]methanethiosulfonatebromide (MTSET) and sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES) did not inhibit the transport activity in whole cells. Following reconstitution, however, of RBE-2 transfected cell proteins into proteoliposomes, external application of MTSET and MTSES led to a decrease in transport activity to 42.7 (S.D. = 9.1) and 51% (S.D. = 10.14), respectively. Similar results were obtained also when the rat heart isoform RHE-1 (NCX1.1) or the rat brain isoform RBE-1 (NCX1.4) was expressed. NEM and MTSEA inhibited Na(+) gradient-dependent Ca(2+) uptake also in HEK 293 cells expressing RBE-2/C14A/C20S/ C122S/C780S (numbering corresponds to RBE-2), a mutant in which all putative extracellular cysteines were exchanged. To study the accessibility of different cysteines to covalent modification, surface biotinylation of cells expressing the wild type exchanger and its mutants was carried out using 3-(N-maleimidylpropionyl)biocytin. Surface biotinylation revealed immunoreactive protein derived from the wild type Na(+)-Ca(2+) exchanger only if the transfected cells were exposed to the reducing agent Tris(2-carboxyethyl)phosphine. No reduction was needed when the single cysteine mutants of RBE-2, C14A, C20S, and C780S, were expressed. Treatment of the cells expressing these mutants with MTSET before biotinylation, led to a decrease in the amount of exchanger protein that was revealed. No immunoreactive protein was detected when the quadruple mutant RBE-2, C14A/C20S/C122S/C780S, was biotinylated, suggesting that no additional cysteines are accessible directly from the extracellular face of the membrane. Permeabilizing the cells expressing RBE-2/C14A/C20S/ C122S/C780S with streptolysin O resulted in biotinylation of the exchanger protein. Its amount decreased if exposure to NEM preceded streptolysin O treatment. Our results suggest that Na(+)-Ca(2+) exchange activity is inhibited by covalent modification with sulfhydryl reagents of cysteine residues that are accessible from the cytoplasmic face of the membrane.     

Purification and characterization of phospholipase C of Salmonella gallinarum

Phospholipase C was isolated from an outbreak strain of Salmonella gallinarum with ciprofloxacin extraction, dialysis, gel filtration, ion exchange chromatography and chromatofocussing. Purified phospholipase C (mol wt. 65 KDa; isoelectric point, pI 3.5) was resistant to pasteurization, stomach enzyme (pepsin), bacterial protease and lipase but lost its activity on trypsin and chymotrypsin treatment. It was sensitive to pH > or = 8.0. It was haemolytic, embryotoxic, enterohaemorrhagic, lethal to birds, cytotoxic to Vero and MDBK cells, dermonecrotoxic in rabbit and antigenically active protein. Antisera raised against purified phospholipase C neutralized its all biological activities and agglutinated the producer Salmonella strains. Serologically it was proved similar to phospholipase C of Klebsiella pneumoniae and S. weltevreden. Fluorescent antibody technique (FAT) was standardized to detect phospholipase producer strains.     

Catabolic activity of two phosphoric diester hydrolases in wheat leaves inoculated with brown rust,Puccinia recondita

Changes in total phospholipids and activities of two phosphoric diester hydrolases,i.e. phospholipase C and phospholipase D have been studied in inoculated wheat leaves of 25-day old plants, interacting differentially with three races of brown rust,Puccinia recondita, during early stages of pathogenesis. With progressive rust iafection the lipids and phospholipids were found to decrease in all the three interactions except at later stages (48 h and 72 h) in race 104-interaction. However, total phospholipids at various stages were found to be higher in resistant interaction (race 63) as compared to the other two interactions. An initial increase in activities of phospholipase C and D in all the three interactions was followed by a decline at the intermediate stages which continued in case of phospholipase C activity while a reverse trend was observed in phospholipase D activity towards later stages of infection. Further the enzymatic activities at various stages were found to be higher in the susceptible and intermediate interactions (with races 77 and 104 respectively) as compared to the resistant interaction.     

Active and inactive forms of hemolysin (HlyA) from Escherichia coli

The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W. & Hedgpeth, J. (1982) J. Bacteriol. 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle. However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase. External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication. The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished. On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA. Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes. Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein. An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product. This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase.