Extracellular protease as a reversible adhesion regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2-3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h-1, the fraction of proteins in cells being almost the same (60-70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3-5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20C and pH 69 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20 degrees C and pH 6.9 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR

To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest.     

Nitrogenase activity and growth of Frankia in batch and continuous culture.

Frankia grown in batch culture was unable to maintain a high rate of nitrogenase activity and, once a peak level was reached, activity rapidly declined. Addition of 5 mM carbon source of cultures or transfer to fresh medium was followed by brief recovery of nitrogenase activity. The extent of recovery decreased as additions or transfers were made to progressively older cultures. Daily addition of fresh medium (dilution rate = 0.125 day-1) allowed Frankia to be maintained in continuous, derepressed culture with stable rates of growth and nitrogenase activity for more than 30 days. The proportion of active, mature vesicles also remained constant in continuous culture but decreased with time in batch culture.     

Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi

Indigenous actinomycetes isolated from rhizosphere soils were assessed for in vitro antagonism against Colletotrichum gloeosporioides and Sclerotium rolfsii. A potent antagonist against both plant pathogenic fungi, designated SRA14, was selected and identified as Streptomyces hygroscopicus. The strain SRA14 highly produced extracellular chitinase and β-1,3-glucanase during the exponential and late exponential phases, respectively. Culture filtrates collected from the exponential and stationary phases inhibited the growth of both the fungi tested, indicating that growth suppression was due to extracellular antifungal metabolites present in culture filtrates. The percentage of growth inhibition by the stationary culture filtrate was significantly higher than that of exponential culture filtrate. Morphological changes such as hyphal swelling and abnormal shapes were observed in fungi grown on potato dextrose agar that contained the culture filtrates. However, the antifungal activity of exponential culture filtrates against both the experimental fungi was significantly reduced after boiling or treatment with proteinase K. There was no significant decrease in the percentage of fungal growth inhibition by the stationary culture filtrate that was treated as above. These data indicated that the antifungal potential of the exponential culture filtrate was mainly due to the presence of extracellular chitinase enzyme, whereas the antifungal activity of the stationary culture filtrate involved the action of unknown thermostable antifungal compound(s).     

Purification, characterization and synergistic activity of β-1,3-glucanase and antibiotic extract from an antagonistic Bacillus subtilis NSRS 89-24 against rice blast and sheath blight

Antifungal compounds in the culture filtrate from Bacillus subtilis NSRS 89-24 that inhibited the growth of Pyricularia grisea and Rhizoctonia solani were mainly heat stable as the filter sterilized culture filtrate showed higher activity than an autoclaved one. The heat stable and labile components were due to an antibiotic and a β-1,3-glucanase, respectively. This β-1,3-glucanase was purified and characterized. Glucanase activity in the culture medium of B. subtilis NSRS 89-24 was inducible in the presence of 0.3% chitin, reaching a maximum on day 5. After purification, activity was associated with a protein of molecular mass of approximately 95.5 kDa by both gel filtration and native PAGE. Two major bands of M_r 64.6 and 32.4 kDa were revealed by SDS–PAGE. The enzyme had a K_m of 0.9 mg/ml, and V_max of 0.11 U, the optimal pH was 6.5–9.5 and was stable up to 50 °C. Both the pure enzyme and the antibiotic extract from the culture filtrate of the B. subtilis separately inhibited R. solani and P. grisea with MIC values of 12.5 and 6.25 mU/ml and 3.13 and 1.56 μg/ml, respectively. The glucanase enzyme in combination with the antibiotic showed a strong synergistic inhibitory effect on the hyphal growth of both fungi.     

Effect of growth conditions on the molecular weight of poly-3-hydroxybutyrate produced by Azotobacter chroococcum 7B

It has been shown that poly-3-hydroxybutyrate (PHB) of predetermined molecular weight can be obtained by varying the growth conditions of the producer strain, Azotobacter chroococcum 7B: pH, temperature, aeration, presence of sodium acetate as an additional carbon source, or growth on crude complex carbon sources (molasses, vinasse, or starch). High-molecular-weight polymer can be obtained at pH 7.0, optimal for the culture (1485 kDa), temperature 30-37 degrees C (1600-1450 kDa, respectively), and low aeration (2215 kDa). The following factors decrease PHB MW: pH deviation to the acidic (pH 6.0, 476 kDa) or alkaline (pH 8.0, 354 kDa) range or lower temperature (20 degrees C, 897 kDa). Introduction of additional carbon source (sodium acetate) at concentrations in the medium varying from 0 to 5 g/l provides an original method of production of PHB with predetermined MW in a wide range, from 270 to 1515 kDa, with high PHB content in the cell.     

Extracellular Protease as a Reversible Adhesion Regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2–3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h^–1, the fraction of proteins in cells being almost the same (60–70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3–5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.     

High-molecular-mass multicatalytic proteinase complexes produced by the nitrogen-fixing actinomycete Frankia strain BR.

A major-high-molecular mass proteinase and seven latent minor proteinases were found in cell extracts and in concentrates of culture medium from Frankia sp. strain BR after nondenaturing electrophoresis in mixed gelatin-polyacrylamide gels. All of these complexes showed multicatalytic properties. Their molecular masses and their sedimentation coefficients varied from 1,300 kDa (28S) to 270 kDa (12S). The electroeluted 1,300-kDa proteinase complex dissociated into 11 low-molecular-mass proteinases (40 to 19 kDa) after sodium dodecyl sulfate activation at 30 degrees C and electrophoresis under denaturing conditions. All of these electroeluted proteinases hydrolyzed N-carbobenzoxy-Pro-Ala-Gly-Pro-4-methoxy-beta- naphthylamide, D-Val-Leu-Arg-4-methoxy-beta-naphthylamide, and Boc-Val-Pro-Arg-4-methyl-7-coumarylamide, whereas Suc-Leu-Leu-Val-Tyr-4-methyl-7-coumarylamide was cleaved only by the six lower-molecular-mass proteinases (27.5 to 19 kDa). Examination by electron microscopy of uranyl acetate-stained, electroeluted 1,300- and 650-kDa intracellular and extracellular proteinase complexes showed ring-shaped and cylindrical particles (10 to 11 nm in diameter, 15 to 16 nm long) similar to those of eukaryotic prosomes and proteasomes. Polyclonal antibodies raised against rat skeletal muscle proteasomes cross-reacted with all of the high-molecular-mass proteinase complexes and, after denaturation of the electroeluted 1,300-kDa band, with polypeptides of 35 to 38, 65, and 90 kDa. Electrophoresis of the activated cell extracts under denaturing conditions revealed 11 to 17 gelatinases from 40 to 19 kDa, including the 11 proteinases of the 1,300-kDa proteinase complex. The inhibition pattern of these proteinases is complex. Thiol-reactive compounds and 1-10-phenanthroline strongly inhibited all of the proteinases, but inhibitors against serine-type proteinases were also effective for most of them.     

Growth, pigment production and protease activity of Monascus purpureus as affected by salt, sodium nitrite, polyphosphate and various sugars

The effect of different levels of salt, sodium nitrite, polyphosphate and various sugars on growth, pigment production, protease activity and culture pH caused by Monascus purpureus was studied in broth medium and ground meat. The addition of sodium chloride (> 50.0 g l(-1)) and polyphosphate (> 3.0g l(-1)) to broth medium decreased mycelial growth, pigment production and protease activity of M. purpureus, whereas low concentrations of sodium nitrite (< 0.2 g l(-1)) promoted mycelial growth and pigment production. When the basal medium and ground meat contained salt, 150.0 g l(-1), the mould growth was stopped. The medium with fructose as carbon source proved to be the most suitable for mycelium growth and pigment production, with maltose and glucose being the second most productive. When sucrose and lactose were used as carbon sources, mycelium growth and pigment production were inhibited but the protease activity increased significantly. The mould showed more tolerance to salt and polyphosphate in ground meat than in broth medium and used sucrose as a carbon source as well as glucose for growth and pigment production in the meat mixture.     

Effect of Cultivation Conditions on the Composition of Extracellular Polysaccharide-Containing Substances in the Bacterium Azospirillum brasilense

Maintenance of pH 7.0 during the fermentation period favors accumulation of high molecular weight polysaccharide-containing components called lipopolysaccharide–protein and polysaccharide–lipid complexes in the capsules and culture medium. Increased pH of the culture medium to 8.0 reduced the period of exponential growth and the yield of polysaccharide-containing complexes as compared to optimal conditions. Maintenance of pH 5.5 suppressed the culture growth and polysaccharide production. The polysaccharide–lipid complexes obtained when pH was stabilized at the level of 7.0–8.0 had relatively low molecular weights and included only acidic polysaccharides. The use of potassium gluconate instead of sodium malate as a source of carbon in the culture medium changed the polysaccharide composition and increased the content of glucosamine, which increased the affinity of polysaccharides for wheat germ agglutinin. Prolongation of Azospirillum cultivation to five days introduced new glucose-containing polysaccharide components in the capsule.     

Release of proteinase from mycelium of Mucor hiemalis

When Mucor hiemalis NRRL 3103 was grown in soybean medium, only a small fraction of the proteinase produced by the organism appeared in the culture filtrate, whereas the bulk of the enzyme was bound to the mycelial surface. Optimal pH of the proteinase ranged from 3.0 to 3.5. Inclusion of sodium chloride or other ionizable salts in the growth medium, however, resulted in the liberation from the mycelium of the loosely bound enzyme as it was formed. Maximal release of proteinase was achieved at a sodium chloride concentration of 0.5 m. The loosely bound proteinase was eluted also from intact resting mycelium by ionizable salts but not by water or by nonionizable substances. The amount of enzyme eluted from the mycelium depended upon the concentration of sodium chloride up to 0.3 m. Since liberation took place rapidly even at 0 C, a loose ionic linkage must exist rather than a biochemical binding of the enzyme to the mycelium. The recovery of proteolytic activity from repeated salt extractions was greater than that originally detected in the intact mycelium, possibly owing to unmasking of more active enzymes or functional groups. Further proteinase activity was released when salt-extracted mycelium was ruptured. Part of the proteinase thus observed was firmly attached to the cell fraction, and part of it appeared in the supernatant fluid. These conditions implied the presence of intracellular or firmly attached proteinase which could be partially released.     

Extracellular fibrinogenolytic enzyme of Aspergillus fumigatus: substrate-dependent variations in the proteinase synthesis and characterization of the enzyme

Abstract To get a better understanding of the role of the previously reported fibringenolytic enzyme of Aspergillus fumigatus , we investigated the in vitro conditions of enzyme synthesis and attempted to characterize it. Modification of the nitrogen source did not influence the extracellular serine-proteinase profile, but resulted in important quantitative differences in the yields in batch cultures. The enzyme synthesis appeared to be an inducible phenomenon in A. fumigatus since it was initiated exclusively in the presence of proteins or protein hydrolysate. Free amino acids or inorganic nitrogen compounds could not promote significant enzyme production. Moreover, peptone at a concentration of 0.1% appeared to be the best inducer of enzyme synthesis. Conversely, modification of the carbon source did not affect fungal growth or enzyme synthesis. However, the production of chymotrypsin was highly sensitive to the carbohydrate level in the culture medium and, with peptone as nitrogen source, highest yields were obtained in the presence of 0.3 or 0.5% glucose. Culture filtrates of A. fumigatus CBS 113.26 grown with peptone or nitrate as nitrogen source were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the protein patterns suggested for the proteinase a molecular mass of 33 kDa which was confirmed by chromatographic purification of the enzyme through (Nα-CBZ)- d -phenylalanine agarose.     

Purification and characterization of an extracellular exochitinase,beta-N-acetylhexosaminidase,from the fungal mycoparasite Stachybotrys elegans

The mycoparasite Stachybotrys elegans produces two exo- and one endo-acting chitinases when grown on chitin. We purified to homogeneity one of the exo-acting chitinases, beta-N-acetylhexosaminidase and partially characterized its physical and biochemical properties. The native enzyme has a molecular mass of 120 kDa when determined by gel filtration and 68 kDa by sodium dodecyl sulfate - polyacrylamide gel electrophoresis indicating that the native protein probably occurs as a dimer in solution. The purified beta-N-acetylhexosaminidase is most active at pH 5.0 and 40 degrees C and hydrolyzes the p-nitrophenyl-N-acetyl-beta-D-glucosaminide with apparent Km of 84.6 microM. Polyclonal antibodies raised against the 68-kDa beta-N-acetylhexosaminidase (NAG-68) indicated that the antibody is highly specific and recognizes the protein in crude filtrate preparation. This suggests that the protein is a not a proteolytic product of another protein. Western blot analysis showed that the activity of NAG-68 was induced when S. elegans was grown on purified cell wall fragments of its host, Rhizoctonia solani, as well as during antagonistic interaction of the mycoparasite and host when both were grown on synthetic medium with or without supplemental carbon source.     

Regulation of proteinase synthesis in Lactococcus lactis

The synthesis of extracellular serine proteinase of Lactococcus lactis was studied during the growth in a batch and a continuous culture on chemically defined media. In a batch culture the proteinase synthesis started during the exponential phase of growth and the highest proteinase concentrations were found at the end of the exponential and beginning of the stationary phase of growth. During the growth in a lactose-limited chemostat with amino acids as the sole source of nitrogen, the specific rate of proteinase synthesis was maximal at a μof 0.23 h^?1. At higher growth rates the proteinase productin declined. The proteinase synthesis was dependent on the amino acid sources in the medium. In batch cultures of L. lactis grown on a chemically defined medium with amino acids, the proteinase production was increased four-fold compared to media containing casein or a tryptic digest of casein as the sole source of nitrogen. The inhibition of the rate of proteinase synthesis by casein and peptides was also observed during the growth in a chemostat. The addition of the dipeptide leucylproline (final concentration of 100 μM) to a lactose-limited continuous culture during the steady state (D = 0.23 h^?1) resulted in a transient inhibition of the rate of proteinase synthesis. This suggested that exogenously supplied peptides control the regulation of proteinase synthesis of L. lactis.     

Phosphate and carbon source regulation of alkaline phosphatase and phospholipase in Vibrio vulnificus

In this study, the effects of phosphate concentration and carbon source on the patterns of alkaline phosphatase (APase) and phospholipase (PLase) expression in Vibrio vulnificus ATCC 29307 were assessed under various conditions. The activities of these enzymes were repressed by excess phosphate (4 mM) in the culture medium, but this repression was reversed upon the onset of phosphate starvation in low phosphate defined medium (LPDM) containing 0.2 mM of phosphate at approximately the end of the exponential growth phase. The expressions of the two enzymes were also influenced by different carbon sources, including glucose, fructose, maltose, glycerol, and sodium acetate at different levels. The APase activity was derepressed most profoundly in LPDM containing fructose as a sole carbon source. However, the repression/derepression of the enzyme by phosphate was not observed in media containing glycerol or sodium acetate. In LPDM-glycerol or sodium acetate, the growth rate was quite low. The highest levels of PLase activity were detected in LPDMsodium acetate, followed by LPDM-fructose. PLase was not fully repressed by high phosphate concentrations when sodium acetate was utilized as the sole carbon source. These results showed that multiple regulatory systems, including the phosphate regulon, may perform a function in the expression of both or either APase and PLC, in the broader context of the survival of V. vulnificus.     

Response of nitrogenase to altered carbon supply in a Frankia-Alnus incana symbiosis

To study the effect of altered carbon supply on nitrogenase (EC 1.7.99.2), plants of Alnus incana (L.) Moench in symbiosis with the local source of Frankia were exposed to darkness for 2 days, and then returned to normal light/dark conditions. During the dark period nitrogenase activity in vivo (intact plants) and in vitro ( Frankia cells supplied with ATP and reductant), measured as acetylene reduction activity, was almost completely lost. Western blots for both the Fe-protein (dinitrogenase reductase) and the MoFe-protein (dinitrogenase) showed that, in particular, the amount of MoFe-protein was strongly reduced during darkness. Protein stained sodium dodecyl sulphate-polyacrylamide gels of Frankia protein showed that the nitrogenase proteins were the only abundant proteins that clearly decreased during darkness. During recovery, studied for 4 days, nitrogenase activity in vivo recovered to the level before dark treatment but was still only half of control activity, Nitrogenase activity in vitro and the amount of MoFe-protein, both expressed per Frankia protein, recovered and reached similar values in previously dark treated plants and in control plants. The rate of recovery was similar to the increase in activity of control plants, suggesting growth of Frankia in addition to synthesis of nitrogenase proteins during the recovery after carbon starvation.     

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790.

A substantial portion of the second peptidoglycan hydrolase (muramidase-2) activity of Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) is present in the supernatant culture medium. In contrast, nearly all muramidase-1 activity is associated with cells in the latent, proteinase-activatable form. Muramidase-2 activity is produced and secreted throughout growth, with maximal levels attained at or near the end of exponential growth in a rich organic medium. Muramidase-2 activity in the culture medium remained high even during overnight incubations in the absence of proteinase inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant culture medium concentrated by 60% saturated ammonium sulfate precipitation showed the presence of several Coomassie blue-staining bands. One intensely staining protein band, at about 71 kDa, selectively adsorbed to the insoluble peptidoglycan fraction of cell walls of E. hirae, retained muramidase-2 activity, and reacted in Western immunoblots with monoclonal antibodies to muramidase-2. The mobility of extracellular muramidase-2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from that of muramidase-2 extracted with 6 M guanidine hydrochloride from intact bacteria. Muramidase-2 appears to have only a limited number of binding sites on the peptidoglycan of E. hirae cell walls but binds with high affinity. Although high levels of muramidase-2 activity were present in supernatants of stationary-phase cultures, the bacteria were resistant to autolysis. Thus it appears that the peptidoglycan in walls of intact cells of E. hirae is somehow protected from the hydrolytic action of extracellular muramidase-2.     

Effect of growth conditions on the molecular weight of poly-3-hydroxybutyrate produced by <Emphasis Type="Italic">Azotobacter chroococcum</Emphasis> 7B

It has been shown that poly-3-hydroxybutyrate (PHB) of predetermined molecular weight can be obtained by varying the growth conditions of the producer strain, Azotobacter chroococcum 7B: pH, temperature, aeration, presence of sodium acetate as an additional carbon source, or growth on crude complex carbon sources (molasses, vinasse, or starch). High-molecular-weight polymer can be obtained at pH 7.0, optimal for the culture (1485 kDa), temperature 30–37°C (1600–1450 kDa, respectively), and low aeration (2215 kDa). The following factors decrease PHB MW: pH deviation to the acidic (pH 6.0, 476 kDa) or alkaline (pH 8.0, 354 kDa) range or lower temperature (20°C, 897 kDa). Introduction of additional carbon source (sodium acetate) at concentrations in the medium varying from 0 to 5 g/l provides an original method of production of PHB with predetermined MW in a wide range, from 270 to 1515 kDa, with high PHB content in the cell.