Introduction of air in the anaerobic culture of Streptococcus pneumoniae serotype 23F induces the release of capsular polysaccharide from bacterial surface into the cultivation medium

AIM: An approach to increase Streptococcus pneumoniae capsular polysaccharide (CPS) in the culture medium during fed-batch cultivation in bioreactor. METHODS AND RESULTS: Streptococcus pneumoniae serotype 23F was cultivated in a 5-l bioreactor with nitrogen-sparging and followed by addition of air in the stationary phase. The amount of CPS released in the supernatant progressively increased under air sparging. The profile of cellular viability and optical density was similar in both cultures. Immunoelectron microscopy showed that the amount of tightly cell-bound CPS was higher in bacteria cultivated under nitrogen than under air. CONCLUSIONS: The stress caused by the addition of air at the stationary phase promoted a large increase of free CPS into the medium, as a consequence of the morphologic change in the capsule. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of air in the stationary phase of the culture would greatly simplify the subsequent downstream process, allowing CPS purification from the supernatant. The direct consequence of this process improvement is the reduction of vaccine production costs.     

Effects of nutrients on exopolysaccharide production and surface properties of Aeromonas salmonicida.

Extracellular polysaccharide (EPS) and capsular polysaccharide (CPS) production by Aeromonas salmonicida A450 and the influence of the capsule on cell surface properties were studied. A. salmonicida did not produce CPS or EPS when glucose, phosphate, magnesium chloride, or trace mineral components were absent from the medium. The addition of yeast extract improved capsule production. Neither EPS nor CPS formation depended on the C/N ratio, although it appeared to be influenced by the level of carbon and nitrogen in the culture. Both EPS and CPS production started at the end of the logarithmic growth phase. The amounts of EPS and CPS produced were not influenced by temperature changes between 15 and 20 degrees C and was maximal from pH 7 to 7.5. Cell surface properties were strongly influenced by capsule production; high CPS production was associated with enhanced cell hydrophilicity and autoagglutination. The effect of CPS on cell surface properties was independent of the presence of the surface protein array (A-layer).     

Development and trifoliin A-binding ability of the capsule of Rhizobium trifolii.

The age-dependent lectin-binding ability of Rhizobium trifolii 0403 capsular polysaccharide (CPS) was examined by following the development of the capsule and its ability to interact with the white clover lectin trifoliin A. Bacteria grown on agar plates for 3, 5, 7, 14, and 21 days were examined by electron microscopy and immunofluorescence microscopy with antibodies prepared against either R. trifolii 0403 CPS or trifoliin A after pretreatment with the lectin. The capsule began to develop at one pole by day 3 and completely surrounded the cells in cultures incubated for 5 days or longer. The capsular polysaccharide on cells cultured for 3 and 5 days was completely reactive with trifoliin A, became noticeably less reactive by day 7, and was only reactive with the lectin at one pole of a few cells after that time. The quantity and location of lectin receptors on bacteria of different ages directly correlated with their attachment in short-term clover root hair-binding studies. Cells from 3- or 21-day-old cultures attached almost exclusively in a polar fashion, whereas cells grown for 5 days attached to root hairs randomly and in the highest numbers. CPS isolated from a 5-day-old culture had higher lectin-binding ability than CPS from 3- and 7-day-old cultures, whereas the CPS from a 14-day-old culture had the lowest. Chemical analyses of the isolated CPS showed changes in the levels of uronic acids (as glucuronic acid), pyruvate, and O-acetyl substitutions with culture age, but the neutral sugar composition remained relatively constant. These results provide evidence that the age-dependent distribution of lectin receptors dictates the level and orientation of attachments of R. trifolii 0403 to clover root hairs.     

Electron microscopic visualization of capsular material of Pasteurella multocida types A and D labeled with polycationic ferritin.

The presence of capsular material on cells of Pasteurella multocida types A and D was determined by transmission electron microscopy after polycationic ferritin labeling. The capsule of agar-grown isolates of P. multocida type A was thick (70 to 90 nm) and regular, whereas that of type D isolates was thinner (20 to 30 nm) and irregular. Such layers were seen on cells from 4- to 6-h broth cultures, but cells from older cultures (12 to 18 h) had very little cell-associated capsular material. Our data indicate that the capsular material of P. multocida types A and D is morphologically different and that capsule production in broth culture is maximal during early log phase.     

Nodulation of soybean byRhizobium japonicum mutants with altered capsule synthesis

Spontaneous mutants with altered capsule synthesis were isolated from a marked strain of the symbiont,Rhizobium japonicum. Differential centrifugation was used to enrich serially for mutants incapable of forming capsules. The desired mutants were detected by altered colony morphology and altered ability to bind host plant lectin. Three mutants failed to form detectable capsules at any growth phase when cultured in vitro or in association with the host (soybean,Glycine max (L.) Merr.) roots. These mutants were all capable of nodulating and attaching to soybean roots, indicating that the presence of a capsule physically surrounding the bacterium is not required for attachment or for infection and nodulation. Nodulation by several of the mutants was linearly proportional to the amount of acidic exopolysaccharide that they released into the culture medium during the exponential growth phase, indicating that such polysaccharide synthesis is important and perhaps required for nodulation. Two of the mutants appeared to synthesize normal lectin-binding capsules when cultured in association with host roots, but not when cultured in vitro. Nodulation by these mutants appeared to depend on how rapidly after inoculation they synthesized capsular polysaccharide.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - FITC fluorescein isothiocyanate Contribution No. 719 of the C.F. Kettering Research Laboratory     

Turnover of cell surface-bound capsular polysaccharide in Staphylococcus aureus

The capsular polysaccharide (CPS) of Staphylococcus aureus strain Smith was labelled by growth of bacteria in the presence of radioactive N-acetylglucosamine and was separated from labelled cell wall components by affinity chromatography on wheat germ agglutinin following dissolution of the cells by lysostaphin. The products were partially characterised chemically and immunochemically. Similar labelled components were found in the culture fluid during growth. In a pulse-chase experiment, cell-bound CPS was released continuously into the culture fluid at the same rate as cell wall turnover and there was no evidence of direct excretion of CPS.     

Capsular polysaccharide phase variation in Vibrio vulnificus

Commonly found in raw oysters, Vibrio vulnificus poses a serious health threat to immunocompromised individuals and those with serum iron overload, with a fatality rate of approximately 50%. An essential virulence factor is its capsular polysaccharide (CPS), which is responsible for a significant increase in virulence compared to nonencapsulated strains. However, this bacterium is known to vary the amount of CPS expressed on the cell surface, converting from an opaque (Op) colony phenotype to a translucent (Tr) colony phenotype. In this study, the consistency of CPS conversion was determined for four strains of V. vulnificus. Environmental conditions including variations in aeration, temperature, incubation time, oxidative stress, and media (heart infusion or modified maintenance medium agar) were investigated to determine their influence on CPS conversion. All conditions, with the exception of variations in media and oxidative stress, significantly affected the conversion of the population, with high ranges of CPS expression found even within cells from a single colony. The global quorum-sensing regulators RpoS and AI-2 were also examined. While RpoS was found to significantly mediate phenotypic conversion, quorum sensing was not. Finally, 12 strains that comprise the recently found clinical (C) and environmental (E) genotypes of V. vulnificus were examined to determine their rates of population conversion. C-genotype strains, which are most often associated with infection, had a significantly lower rate of population conversion from Op to Tr phenotypes than did E-genotype strains (ca. 38% versus ca. 14%, respectively). Biofilm capabilities of these strains, however, were not correlated with increased population conversion.     

INCORPORATION OF SULFATE INTO THE CAPSULAR POLYSACCHARIDE OF THE RED ALGA PORPHYRIDIUM

The accumulation of sulfate-³⁵S by Porphyridium aerugineum cells and subsequent appearance of solubilized capsular polysaccharide-³⁵S in the growth medium were examined The uptake of label by the cells was largely light dependent. Pulse-chase experiments using log phase cells revealed a rapid labeling of solubilized capsular polysaccharide, recovered from the medium as the cetylpyridinium chloride precipitate Polyacrylamide gel electrophoresis of the polysaccharide-³⁵S showed the sulfate to be firmly bound to an immobile fraction. Sephadex chromatography revealed the molecular weight of the polysaccharide to be in excess of 2 x 10⁵. Acid hydrolysis of the polysaccharide-³⁵S released sulfate-³⁵S ion as evidenced by radioautography of thin layer chromatographs Preliminary electron microscope evidence suggests that the synthesis, movement, and deposition of the capsular polysaccharide on the cell surface are Golgi complex-mediated processes     

Relationship between oxygen and siderophore synthesis inPseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO1, growing in low-iron medium, produces two siderophores, pyochelin and pyoverdin, in massive bursts as the culture shifts from logarithmic phase to stationary phase. Two medium components, oxygen and iron, prolonged the logarithmic phase when they were added to the medium. Oxygen and iron appeared to be in demand during this period because, as heme synthesis increased in response to the low oxygen concentration in the medium, a situation resulting from the high density of bacteria present in the medium during late log phase, the iron content of the bacteria decreased. These phenomena resulted in the production of massive amounts of siderophores late in the log phase to supply iron for the increased heme synthesis.     

Inhibitory effect of capsular antigen of Vibrio vulnificus on bactericidal activity of human serum

Opaque (Op) and translucent (Tr) colonial variants were isolated from Vibrio vulnificus strains. Op-type variants were more resistant than the isogenic Tr-type variants, but the survival rate of the Op-type variants varied with the strains. Antisera were prepared by immunizing rabbit with whole cells of Op and Tr variants of some strains, in which the difference of the sensitivity between Op and Tr cells was remarkable. Then agglutination tests with their living and heat-killed cells were carried out. The results suggested the presence of capsular antigen in Op cells and its absence in Tr cells, with the exception of the existence of a slight amount of capsular material in Tr variants of strain L-180. The thin capsular layer of Tr cells of strain L-180 was also demonstrated electron microscopically, but the layer was thinner than that of the isogenic Op cells. Results of determination of sugar content in the extracted capsular fraction also showed that Op to Tr transformation was due to loss of capsular antigen of the cells. These results confirmed the morphological studies previously reported which suggested the prevention of host defense system by the capsular material of the vibrio.     

Capsular Polysaccharide Phase Variation in Vibrio vulnificus

Commonly found in raw oysters, Vibrio vulnificus poses a serious health threat to immunocompromised individuals and those with serum iron overload, with a fatality rate of approximately 50%. An essential virulence factor is its capsular polysaccharide (CPS), which is responsible for a significant increase in virulence compared to nonencapsulated strains. However, this bacterium is known to vary the amount of CPS expressed on the cell surface, converting from an opaque (Op) colony phenotype to a translucent (Tr) colony phenotype. In this study, the consistency of CPS conversion was determined for four strains of V. vulnificus. Environmental conditions including variations in aeration, temperature, incubation time, oxidative stress, and media (heart infusion or modified maintenance medium agar) were investigated to determine their influence on CPS conversion. All conditions, with the exception of variations in media and oxidative stress, significantly affected the conversion of the population, with high ranges of CPS expression found even within cells from a single colony. The global quorum-sensing regulators RpoS and AI-2 were also examined. While RpoS was found to significantly mediate phenotypic conversion, quorum sensing was not. Finally, 12 strains that comprise the recently found clinical (C) and environmental (E) genotypes of V. vulnificus were examined to determine their rates of population conversion. C-genotype strains, which are most often associated with infection, had a significantly lower rate of population conversion from Op to Tr phenotypes than did E-genotype strains (ca. 38% versus ca. 14%, respectively). Biofilm capabilities of these strains, however, were not correlated with increased population conversion.     

Osmotically-regulated trehalose accumulation and cyclic β-(1,2)-glucan excretion by Rhizobium leguminosarum biovar trifolii TA-1

Rhizobium leguminosarum biovar trifolii TA-1 produced high molecular weight extracellular (EPS) and capsular polysaccharides (CPS) as the main carbohydrate products in a medium (10 g of mannitol and 1 g of glutamic acid per liter) with low osmotic pressure of 0.20 MPa. By increasing the osmotic pressure of the medium with the addition of NaCl or other osmolytes up to 1.44 MPa, the synthesis of EPS and CPS was suppressed. Cyclic -(1,2)-glucans were excreted instead. Concentrations of over 1500 mg of glucans/l medium were produced by a biomass of 520 mg protein at 200 mM NaCl (1.20 MPa). Intracellular cyclic -(1,2)-glucan concentrations remained at 45 to 100 mg/g protein during the stationary phase, independent of the osmotic strength of the medium. Parallel to the increasing osmotic pressure of the medium, the disaccharide trehalose accumulated in the cells as osmo-protectant. Concentrations of up to 130 mg/g protein were reached. Strain TA-1 could tolerate 350 mM NaCl.Abbreviations CPS capsular polysaccharide - EPS extracellular polysaccharide - LM_r low molecular weight - HM_r high molecular weight     

Induction of siderophore activity in Anabaena spp. and its moderation of copper toxicity.

Growth of Anabaena sp. strain 7120 (in the absence of chelators or added iron) was inhibited by the addition of 2.1 to 6.5 microM copper and was abolished by copper concentration of 10 microM or higher. When the copper was chelated to schizokinen (the siderophore produced by this organism in response to iron starvation), the toxic effects were eliminated. Analysis of culture filtrates showed that the cupric schizokinen remains in the medium, thereby lowering the amount of copper taken up by the cells. Although this organism actively transports ferric schizokinen, it apparently does not recognize the cupric complex. Thus, Anabaena sp. is protected from copper toxicity under conditions in which siderophore is being produced. For cells grown in low iron, the accumulation of extracellular schizokinen was observed to parallel cell growth and continue well into stationary phase. The actual iron status of the organism was monitored by using iron uptake velocity as an assay. Cultures grown on 0.1 microM added iron were found to be severely iron limited upon reaching stationary phase, thus explaining the continued production of schizokinen. These data show that the siderophore system in Anabaena spp. has developed primarily as a response to iron starvation and that additional functions such as alleviation of copper toxicity or allelopathic inhibition of other algal species are merely secondary benefits.     

Induction of siderophore activity in Anabaena spp. and its moderation of copper toxicity

Growth of Anabaena sp. strain 7120 (in the absence of chelators or added iron) was inhibited by the addition of 2.1 to 6.5 microM copper and was abolished by copper concentration of 10 microM or higher. When the copper was chelated to schizokinen (the siderophore produced by this organism in response to iron starvation), the toxic effects were eliminated. Analysis of culture filtrates showed that the cupric schizokinen remains in the medium, thereby lowering the amount of copper taken up by the cells. Although this organism actively transports ferric schizokinen, it apparently does not recognize the cupric complex. Thus, Anabaena sp. is protected from copper toxicity under conditions in which siderophore is being produced. For cells grown in low iron, the accumulation of extracellular schizokinen was observed to parallel cell growth and continue well into stationary phase. The actual iron status of the organism was monitored by using iron uptake velocity as an assay. Cultures grown on 0.1 microM added iron were found to be severely iron limited upon reaching stationary phase, thus explaining the continued production of schizokinen. These data show that the siderophore system in Anabaena spp. has developed primarily as a response to iron starvation and that additional functions such as alleviation of copper toxicity or allelopathic inhibition of other algal species are merely secondary benefits.     

How does iron deficiency disrupt the electron flow in photosystem I of lettuce leaves?

The changes observed photosystem I activity of lettuce plants exposed to iron deficiency were investigated. Photooxidation/reduction kinetics of P700 monitored as ΔA₈₂₀ in the presence and absence of electron transport inhibitors and acceptors demonstrated that deprivation in iron decreased the population of active photo-oxidizable P700. In the complete absence of iron, the addition of plant inhibitors (DCMU and MV) could not recover the full PSI activity owing to the abolition of a part of P700 centers. In leaves with total iron deprivation (0 μM Fe), only 15% of photo-oxidizable P700 remained. In addition, iron deficiency appeared to affect the pool size of NADP⁺ as shown by the decline in the magnitude of the first phase of the photooxidation kinetics of P700 by FR-light. Concomitantly, chlorophyll content gradually declined with the iron concentration added to culture medium. In addition, pronounced changes were found in chlorophyll fluorescence spectra. Also, the global fluorescence intensity was affected. The above changes led to an increased rate of cyclic electron transport around PSI mainly supported by stromal reductants.     

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.     

Role of poly-β-hydroxybutyric acid in cyst formation byAzotobacter

Several parameters associated with the growth ofAzotobacter vinelandii in liquid culture were examined in order to investigate the relationship between the accumulation and degradation of poly-β-hydroxybutyric acid (PHB), the development of viscous capsular components, and cyst formation. The amount of intracellular PHB, which increased markedly during the log phase of growth, reached a maximum during the early stationary phase and subsequently declined. During polymer degradation there was a concurrent increase in the extent of encystment in the cultures supplemented with CaCO₃. An increase was noted in the viscosity of culture supernatants during polymer degradation when CaCO₃ was deleted from the medium and the culture pH was controlled by the periodic addition of 0.1m KOH. The extent of encystment and the amount of PHB accumulated were directly proportional to the substrate concentration. The PHB was selectively labeled by the addition of sodium acetate-2-¹⁴C to late log-phase cells. During polymer utilization in either encysting or nonencysting cultures 20% of the label was evolved as CO₂. In the nonencysting cultures, 45% of the radioactivity was distributed between residual PHB and other cellular components, and 35% was in the supernatant polysaccharide-like material. Intact cysts retained 80% of the label. Experiments with ruptured cysts indicated that about 35% of the radioactivity was present in the intine material.     

大规模茶叶细胞悬浮培养茶氨酸合成工艺优化研究

以婺源绿茶为材料进行茶叶愈伤组织悬浮培养,采用正交实验设计进行了大规模茶叶细胞悬浮培养合成茶氨酸工艺条件优化研究。结果显示,NH4+/NO-30.0/60.0mmol/L、K+100.0mmol/L、Mg++3.0mmol/L、H2PO-43.0mmol/L、蔗糖30.0g/L、水解酪蛋白2.0g/L条件下,茶叶细胞生长量(速率)和茶氨酸积累量均达到最高值;提高培养基中蔗糖和水解酪蛋白浓度可延长细胞对数生长期和稳定生长期,从而有利于茶氨酸积累;H2PO-4浓度主要影响细胞生长速率和茶氨酸积累速率的同步性,低H2PO4-浓度环境中茶氨酸积累速率峰值滞后于细胞增长速率峰值,高H2PO4-浓度环境中早于细胞生长速率峰值出现时间;K+和Mg++对细胞生长的影响不明显,但影响茶氨酸合成酶活性,维持适量的K+和Mg++有利于茶氨酸积累。先加入一定量盐酸乙胺再每天进行少量补充,茶氨酸合成量比一次性加入的效果要好。从生产效率考虑,培养周期以19～22d为宜。     

Expression of insulin-like growth factor binding protein-5 by ovine granulosa cells is regulated by cell density and programmed cell death in vitro

In vivo, in the sheep ovary, the expression of insulin-like growth factor binding protein (IGFBP)-2 and particularly IGFBP-5 has been shown to increase dramatically in apoptotic granulosa cells from atretic follicles. The aim of this work was to study the relationship between apoptosis induced by serum starvation in vitro and expression of IGFBP-2 and -5 by ovine granulosa cells. For this purpose, granulosa cells from follicles 1–3 mm in diameter were cultured in the presence of serum for 2 days, then cultured in the presence or absence of serum for 24, 48, or 72 hr. At the end of the culture, cells were counted, cell viability was assessed by studying DNA fragmentation, and IGFBPs expression was studied by quantitative autoradiography, Western-ligand blotting, immunoblotting, and quantitative in situ hybridization. In vitro, IGFBP-2 and particularly IGFBP-5 were the main IGFBPs secreted by ovine granulosa cells. Serum starvation provoked (i) apoptosis of granulosa cells within 48 hr, (ii) a marked decrease in cell density, and (iii) a marked increase in the amount of IGFBP-5 associated with cell membranes and with the walls of culture wells, but no change in culture medium. The increase in the amount of cell- and wall-associated IGFBP-5 after serum starvation was essentially due to the consecutive decrease in cell density rather than to an increase in cell apoptosis. Indeed, irrespective of the presence or absence of serum, the amount of IGFBP-5 associated to cell membranes was inversely correlated to cell density. In contrast, the amount of IGFBP-5 present in culture medium was positively correlated to cell density. Furthermore, expression of IGFBP-5 mRNA was shown to increase with both cell density and cell death. Indeed, the expression of IGFBP-5 mRNA dramatically increased with cell density, irrespective of the presence or absence of serum, but at a similar cell density, expression was higher in serum-free than in serum conditions. Overall, these results indicate that, in vitro, the localization of IGFBP-5 on ovine granulosa cell membranes and in culture medium, respectively, was mainly dependent on cell density, whereas expression of IGFBP-5 mRNA was related to both cell density and cell death. These data suggest that IGFBP-5 is involved in both growth arrest and apoptosis of granulosa cells in the sheep. J. Cell. Physiol. 177:13–25, 1998. © 1998 Wiley-Liss, Inc.