Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.     

Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperatures

AIMS: To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. METHODS AND RESULTS: Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, -18 and -75 degrees C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain-heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. CONCLUSIONS: Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen.     

Mannheimia haemolytica leukotoxin-induced increase in leukotriene B4 production by bovine neutrophils is mediated by a sustained and excessive increase in intracellular calcium concentration

Isolated bovine neutrophils were used to study the relationship between the duration and magnitude of the Mannheimia haemolytica leukotoxin-induced increase in intracellular calcium concentration and leukotriene B4 synthesis. In contrast to recombinant human C5a, which caused a transient, small increase in intracellular calcium concentration and no effects on leukotriene B4 synthesis, exposure of neutrophils to leukotoxin resulted in a rapid, sustained, large increase in intracellular calcium concentration, followed by leukotriene B4 synthesis. This leukotoxin-induced response was similar to those produced by the calcium ionophore, A23187, and phorbol myristate acetate, which also caused significant leukotriene B4 production. Manipulation of the duration and magnitude of leukotoxin- and A23187-induced intracellular calcium concentration increase confirmed that a high and sustained intracellular calcium concentration was necessary to stimulate production of leukotriene B4, which is believed to play an important role in the pathogenesis of pulmonary M. haemolytica infection.     

Diversity of temperate bacteriophages induced in bovine and ovine Mannheimia haemolytica isolates and identification of a new P2-like phage

The diversity of temperate bacteriophages was examined in 32 Mannheimia haemolytica, six Mannheimia glucosida and four Pasteurella trehalosi isolates. Phage particles were induced and identified by electron microscopy in 24 (75%) M. haemolytica isolates, but in only one (17%) M. glucosida and one (25%) P. trehalosi isolate. The M. haemolytica phages were relatively diverse as seven Siphoviridae, 15 Myoviridae and two Podoviridae-like phages were identified; the Myoviridae-type phages also exhibited structural variation of their tails. The bacteriophages induced in M. glucosida and P. trehalosi were of the Myoviridae type. Restriction endonuclease (RE) analysis identified nine distinct RE types among the M. haemolytica bacteriophages, providing further evidence of their relative diversity. A limited number of phages caused plaques on indicator strains and the phages exhibited a narrow host range. A subgroup of 11 bovine serotype A1 and A6 isolates contained Myoviridae-type phages of the same RE type (type A), but these differed in their abilities to infect and form plaques on the same panel of indicator strains. A P2-like phage (phiPHL213.1), representative of the RE type A phages, was identified from the incomplete M. haemolytica genome sequence. The phiPHL213.1 genome contains previously unidentified genes and represents a new member of the P2 phage family.     

Kinetics of growth and leukotoxin production by <Emphasis Type="Italic">Mannheimia haemolytica</Emphasis> in continuous culture

The growth and product formation kinetics of the bovine pathogen Mannheimia (Pasteurella) haemolytica strain OVI-1 in continuous culture were investigated. The leukotoxin (LKT) concentration and yield on biomass could substantially be enhanced by supplementation of a carbon-limited medium with an amino acid mixture or a mixture of cysteine and glutamine. Acetic acid was a major product, increasing to 1.66 g l(-1) in carbon-limited chemostat culture at intermediate dilution rates and accounting for more than 80% of the glucose carbon, whereas in amino acid-limited cultures high acetic acid concentrations were produced at low dilution rates, suggesting a carbon-overflow metabolism. The maintenance coefficients of carbon-limited and carbon-sufficient cultures were 0.07 and 0.88 mmol glucose g(-1) h(-1), respectively. LKT production was partially growth-associated and the LKT concentration was maximised to 0.15 g l(-1) and acetic acid production minimised by using a carbon-limited medium and a low dilution rate.     

分批培养时pH和温度对重组大肠杆菌生产谷胱甘肽合成酶系的影响

研究了2．5L罐分批培养时pH和温度对重组大肠杆菌生产谷胱甘肽合成酶系的影响,确定了分批培养时生产谷胱甘肽合成酶系的最佳pH和最佳温度。研究结果表明：在发酵液的pH为7．2和温度为37℃时,谷胱甘肽合成酶系产量和细胞干重达到最大,分别为690．6U／L和3．77g／L。采用变温控制对菌体的生长和谷胱甘肽合成酶系的合成并没有明显的优点。     

Exopolysaccharide production by Acremonium diospyri in continuous culture

Production of an exopolysaccharide (glucan) by Acremonium diospyri was not markedly affected by its specific growth rate, the culture pH or the stirrer speed under NH ₄ ⁺ -limiting chemostat conditions. The exopolysaccharide was also detected in the medium under conditions of NH ₄ ⁺ excess.P. Wood and R.J. Seviour are with the Biotechnology Research Centre, La Trobe University College of Northern Victoria, Bendigo, Victoria 3550, Australia     

Serotypes A1 and A2 of Mannheimia haemolytica are susceptible to genotypic, capsular and phenotypic variations in contrast to T3 and T4 serotypes of Bibersteinia (Pasteurella) trehalosi

Mannheimia haemolytica and Bibersteinia (Pasteurella) trehalosi are the most common bacterial isolates that cause pulmonary diseases in ruminants worldwide. The disease is determined by specific serotypes found in cattle and small ruminants. The molecular epidemiology of strains involved in disease is important in the control of outbreaks as well as in the preparation of vaccines. This study aimed to detect the instability and variations of bacterial strains that may affect the analysis of epidemic strains, or the stability of vaccinal strains. Eight strains of M. haemolytica belonging to serotypes A1 and A2 and three B. trehalosi strains of the T3 and T4 serotypes were used. Strains were subjected to pulsed field gel electrophoresis (PFGE) and capsular and phenotypic typing at each round of a total of 50 successive subcultures. Remarkable stability was found in all selected strains of B. trehalosi in contrast to M. haemoltyica, in which strains of both serotypes showed pattern variations produced by PFGE and capsular and phenotypic analysis. Objective criteria for M. haemolytica and B. trehalosi typing are consequently addressed.     

温度和营养盐限制对网状原角藻生长与产毒的影响

网状原角藻(Protoceratium reticulatum)是能够形成有毒赤潮的海洋甲藻之一,所产毒素为虾夷扇贝毒素(yessotoxins,YTXs),该藻在全球很多海域中普遍存在,其生长与产毒特征表现出较强的海域差异性。以分离自我国北黄海海域的网状原角藻为实验对象,研究了温度和营养盐(N、P)限制对该藻生长与产毒的影响。研究发现:温度和营养盐限制对藻细胞的生长和产毒均有影响,但营养盐限制影响更为显著。较低的温度更适宜P.reticulatum的生长,15℃时无营养盐限制的L1-Si培养基中藻细胞生长最好。营养盐限制尤其是P限制能够显著降低藻细胞的比生长速率和细胞密度(P0.01),缩短藻细胞指数生长期和稳定期的持续时间。所有温度下N、P限制均有利于藻细胞内毒素累积,15℃P限制培养基中单个藻细胞中YTX毒素最高,达到92.6 pg/细胞,分别是相同培养温度下N限制和L1-Si中藻细胞毒素含量的3.8倍和7.1倍。温度变化对N和P限制下藻细胞毒素含量影响不同:在5—15℃范围内,随温度升高,N限制培养基中藻细胞YTX含量增幅逐渐下降,而P限制条件下反之。在所有培养条件下,滤液中毒素含量在稳定期后开始增多,与L1-Si相比,N、P限制不利于毒素的释放。高温能促进L1-Si培养基和N限制培养基中毒素的释放,但对P限制影响不显著。     

The effects of nutrient,pH and herbicide levels on algal growth

Growth inhibition of algae increased as herbicide concentrations increased, particularly with prometryn and fluometuron. However, picloram had no effect on algal growth while dinoseb inhibited only Lyngbya. There were no differences in growth rate of algae treated with different levels of potassium or phosphorus. High levels of calcium or magnesium increased growth rate of the algae tested. High levels of nitrogen or pH increased growth rates except when combined with prometryn or fluometuron.     

On multiple-nutrient-limited growth of microorganisms,with special reference to dual limitation by carbon and nitrogen substrates

Simultaneous limitation of microbial growth by two or more nutrients is discussed for dual carbon/nitrogen-limited growth in continuous culture. The boundaries of the zone where double-limited growth occurs can be clearly defined from both cultivation data and cellular composition and they can be also predicted from growth yield data measured under single-substrate-limited conditions. It is demonstrated that for the two nutrients carbon and nitrogen the zone of double nutrient limitation is dependent on both the C:N ratio of the growth medium and the growth (dilution) rate. The concept on double-(carbon/nitrogen)-limited growth presented here can be extended to other binary and multiple combinations of nutrients.     

高温和干旱胁迫对西红柿幼苗生长、养分含量及元素利用效率的影响

全球气候变化将增加未来高温与干旱的发生频率和强度,然而高温与干旱的交互作用对农作物生长、养分含量及其利用效率的影响还不甚清楚。因此,研究高温与干旱交互作用对农作物生理生态的影响将为准确评价农作物对未来极端气候条件的响应提供科学依据。选取全球第四大经济作物——西红柿为研究对象,在人工智能气候箱中模拟高温和干旱环境。共设置两个水分处理(正常浇水;干旱)与两个温度处理(常温-26℃/19℃(白天/夜间);高温-42℃/35℃(白天/夜间)(7d))。主要测定指标包括生物量以及生物量分配、比叶面积、养分含量(全氮、全磷)、光合元素利用效率(光合氮素利用效率、光合磷素利用效率)。研究表明,高温、干旱单独作用以及交互作用均显著降低了根、茎、叶生物量以及总生物量,并且高温干旱交互作用使总生物量降低最多。在生物量分配方面,高温单独作用显著降低了根质量分数以及根冠比,而干旱单独作用增加了根质量分数、茎质量分数以及根冠比,但降低了叶质量分数。在养分含量方面,高温单独作用导致叶片全氮、全磷含量显著降低、茎全磷含量显著增加、根全磷含量显著降低。干旱单独作用导致叶片、茎全磷含量显著降低、根全氮含量显著升高。高温与干旱交互作用对生物量分配及养分含量的影响与干旱胁迫单独作用类似。在光合元素利用效率方面,高温、干旱单独作用均降低了幼苗光合氮素利用效率、光合磷素利用效率,并且高温加剧了干旱对光合磷素利用效率的影响。因此,在未来气候变化情况下,高温与干旱交互作用可能会对农作物产生更大威胁。     

Effect of ionophores and pH on growth of Streptococcus bovis in batch and continuous culture.

Batch cultures (pH 6.7) of Streptococcus bovis JB1 were severely inhibited by 1.25 and 5 microM lasalocid and monensin, respectively, even though large amounts of glucose remained in the medium. However, continuous cultures tolerated as much as 10 and 20 microM, respectively, and used virtually all of the glucose. Although continuous cultures grew with high concentrations of ionophore, the yield of bacterial protein decreased approximately 10-fold. When pH was decreased from 6.7 to 5.7, the potency of both ionophores increased, but lasalocid always caused a larger decrease in yield. The increased activity of lasalocid at pH 5.7 could largely be explained by an increased binding of the ionophore to the cell membrane. Because monensin did not show an increased binding at low pH, some other factor (e.g., ion turnover) must have been influencing its activity. There was a linear increase in lasalocid binding as the concentration increased, but monensin binding increased markedly at high concentrations. Based on the observations that (i) S. bovis cells bound significant amounts of ionophore (the ratio of ionophore to cell material was more important than the absolute concentration), (ii) batch cultures responded differently from continuous cultures, and (iii) pH can have a marked effect on ionophore activity, it appears that the term "minimum inhibitory concentration" may not provide an accurate assessment of microbial growth inhibition in vivo.     

Effect of different carbohydrates on growth, polysaccharidase and glycosidase production by Bacteroides ovatus, in batch and continuous culture

Bacteroides ovatus was grown in batch culture on 12 different carbon sources (five polysaccharides, seven monosaccharides and disaccharides). Specific growth rates were determined for each substrate together with polysaccharidase and glycosidase activities. Growth rates on polymerized carbohydrates were as fast or faster than on corresponding simple sugars, demonstrating that the rate of polysaccharide depolymerization was not a factor limiting growth. Bacteroides ovatus synthesized a large range of polymer-degrading enzymes. These polysaccharidases and glycosidases were generally repressed during growth on simple sugars, but arabinose was required for optimal production of α-arabinofuranosidase. Polysaccharidase and glycosidase activities were measured in continuous cultures grown with either xylan or guar gum under putative carbon limitation. With the exception of β-xylosidase, activities of the polymer-degrading enzymes were inversely related to growth rate. This correlated with polysaccharide utilization which was greatest at low dilution rates. These results show that Bact. ovatus is highly adapted for growth on polymerized carbohydrate in the human colon and confirm that the utilization of polysaccharides is partly regulated at the level of enzyme synthesis. and accepted 8 June 1989     

Effect of different carbohydrates on growth, polysaccharidase and glycosidase production by Bacteroides ovatus, in batch and continuous culture

Bacteroides ovatus was grown in batch culture on 12 different carbon sources (five polysaccharides, seven monosaccharides and disaccharides). Specific growth rates were determined for each substrate together with polysaccharidase and glycosidase activities. Growth rates on polymerized carbohydrates were as fast or faster than on corresponding simple sugars, demonstrating that the rate of polysaccharide depolymerization was not a factor limiting growth. Bacteroides ovatus synthesized a large range of polymer-degrading enzymes. These polysaccharidases and glycosidases were generally repressed during growth on simple sugars, but arabinose was required for optimal production of alpha-arabinofuranosidase. Polysaccharidase and glycosidase activities were measured in continuous cultures grown with either xylan or guar gum under putative carbon limitation. With the exception of beta-xylosidase, activities of the polymer-degrading enzymes were inversely related to growth rate. This correlated with polysaccharide utilization which was greatest at low dilution rates. These results show that Bact. ovatus is highly adapted for growth on polymerized carbohydrate in the human colon and confirm that the utilization of polysaccharides is partly regulated at the level of enzyme synthesis.     

Effects of dissolved CO2 levels on the growth of Mannheimia succiniciproducens and succinic acid production

A capnophilic rumen bacterium Mannheimia succiniciproducens produces succinic acid as a major fermentation end product under CO(2)-rich anaerobic condition. Since succinic acid is produced by carboxylation of C3 compounds during the fermentation, intracellular CO(2) availability is important for efficient succinic acid formation. Here, we investigated the metabolic responses of M. succiniciproducens to the different dissolved CO(2) concentrations (0-260 mM). Cell growth was severely suppressed when the dissolved CO(2) concentration was below 8.74 mM. On the other hand, cell growth and succinic acid production increased proportionally as the dissolved CO(2) concentration increased from 8.74 to 141 mM. The yields of biomass and succinic acid on glucose obtained at the dissolved CO(2) concentration of 141 mM were 1.49 and 1.52 times higher, respectively, than those obtained at the dissolved CO(2) concentration of 8.74 mM. It was also found that the additional CO(2) source provided in the form of NaHCO(3), MgCO(3), or CaCO(3) had positive effects on cell growth and succinic acid production. However, growth inhibition was observed when excessive bicarbonate salts were added. By the comparison of the activities of key enzymes, it was found that PEP carboxylation by PEP carboxykinase (PckA) is the most important for succinic acid production as well as the growth of M. succiniciproducens by providing additional ATP.     

Efficient one-step production of astaxanthin by the microalga Haematococcus pluvialis in continuous culture

The performance of Haematococcus pluvialis in continuous photoautotrophic culture has been analyzed, especially from the viewpoint of astaxanthin production. To this end, chemostat cultures of Haematococcus pluvialis were carried out at constant light irradiance, 1,220 microE/m2.s, and dilution rate, 0.9/d, but varying the nitrate concentration in the feed medium reaching the reactor, from 1.7 to 20.7 mM. Both growth and biomass composition were affected by the nitrate supply. With saturating nitrate, the biomass productivity was high, 1.2 g/L.d, but astaxanthin accumulation did not take place, the C/N ratio of the biomass being 5.7. Under moderate nitrate limitation, biomass productivity was decreased, as also did biomass concentration at steady state, whereas accumulation of astaxanthin developed and the C/N ratio of the biomass increased markedly. Astaxanthin accumulation took place in cells growing and dividing actively, and its extent was enhanced in response to the limitation in nitrate availability, with a recorded maximum for astaxanthin cellular level of 0.8% of dry biomass and of 5.6 mg/L.d for astaxanthin productivity. The viability of a significant continued generation of astaxanthin-rich H. pluvialis cells becomes thus demonstrated, as also does the continuous culture option as an alternative to current procedures for the production of astaxanthin using this microalga. The intensive variable controlling the behavior of the system has been identified as the specific nitrate input, and a mathematical model developed that links growth rate with both irradiance and specific nitrate input. Moreover, a second model for astaxanthin accumulation, also as a function of irradiance and specific nitrate input, was derived. The latter model takes into account that accumulation of astaxanthin is only partially linked to growth, being besides inhibited by excess nitrate. Simulations performed fit experimental data and emphasize the contention that astaxanthin can be efficiently produced under continuous mode by adjustment of the specific nitrate input, predicting even higher values for astaxanthin productivity. The developed models represent a powerful tool for management of such an astaxanthin-generating continuous process, and could allow the development of improved systems for the production of astaxanthin-rich Haematococcus cells.     

Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara