Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk.

The consumption of amino acids and peptides was monitored during growth in milk of proteinase-positive (Prt+) and -negative (Prt-) strains of Lactococcus lactis. The Prt- strains showed monophasic exponential growth, while the Prt+ strains grew in two phases. The first growth phases of the Prt+ and Prt- strains were in same, and no hydrolysis of casein was observed. Also, the levels of consumption of amino acids and peptides in the Prt+ and Prt- strains were similar. At the end of this growth phase, not all free amino acids and peptides were used, indicating that the remaining free amino acids and peptides were unable to sustain growth. The consumption of free amino acids was very low (about 5 mg/liter), suggesting that these nitrogen sources play only a minor role in growth. Oligopeptide transport-deficient strains (Opp-) of L. lactis were unable to utilize oligopeptides and grew poorly in milk. However, a di- and tripeptide transport-deficient strain (DtpT-) grew exactly like the wild type (Opp+ Dtpt+) did. These observations indicate that oligopeptides represent the main nitrogen source for growth in milk during the first growth phase. In the second phase of growth of Prt+ strains, milk proteins are hydrolyzed to peptides by the proteinase. Several of the oligopeptides formed are taken up and hydrolyzed internally by peptidases to amino acids, several of which are subsequently released into the medium (see also E.R.S. Kunji, A. Hagting, C.J. De Vries, V. Juillard, A.J. Haandrikman, B. Poolman, and W.N. Konings, J. Biol. Chem. 270:1569-1574, 1995).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The penicillin G acylase production by B. megaterium is amino acid consumption dependent

Aiming at to enhance the production of penicillin G acylase (PGA) by Bacillus megaterium, we have performed flasks experiments using different medium composition. Using 51 g/L of casein hydrolyzed with Alcalase and 2.7 g/L of phenylacetic acid (PhAc), the following carbon substrates were tested, individually and combined: glucose, glycerol, and lactose (present in cheese whey). Glycerol and glucose showed to be effective nutrients for the microorganism growth but delayed the PGA production. Cheese whey always increased enzyme production and cell mass. However, lactose (present in cheese whey) was not a significant carbon source for B. megaterium. PhAc, amino acids, and small peptides present in the hydrolyzed casein were the actual carbon sources for enzyme production. Replacement of hydrolyzed casein by free amino acids, 10.0 g/L, led to a significant increase in enzyme production (app. 150%), with a preferential consumption of alanine, aspartic acid, glycine, serine, arginine, threonine, lysine, and glutamic acid. A decrease of the enzyme production was observed when 20.0 g/L of amino acids were used. Using the single omission technique, it was shown that none of the 18 tested amino acids was essential for enzyme production. The use of a medium containing eight of the preferentially consumed amino acids lead to similar enzyme production level obtained when using 18 amino acids. PhAc, up to 2.7 g/L, did not inhibit enzyme production, even if added at the beginning of the cultivation.     

不同磷源对金龟子绿僵菌CQMa102生物量及生成胞外磷酸酶的影响(英文)

金龟子绿僵菌Metarhizium anisopliae是一种广泛分布于世界各地土壤中的重要的昆虫病原真菌。已有研究表明,胞外磷酸酶在绿僵菌侵染并致死寄主过程中发挥了重要作用。利用摇瓶培养方法探究了无机磷(KH2PO4)、简单有机磷(植酸钠、磷酸苯二钠)和蛋白有机磷(酪蛋白)为单独磷源条件下,绿僵菌生物量、产胞外酸性磷酸酶以及酪氨酸蛋白磷酸酶、丝/苏氨酸蛋白磷酸酶活性的变化。实验结果显示:加入酪蛋白的培养基,最有利于绿僵菌生长、胞外蛋白的分泌和产酸性磷酸酶;其次为加入KH2PO4和磷酸苯二钠的培养基;加入植酸钠的培养基不利于绿僵菌的生长代谢。然而,加入磷酸苯二钠的培养基,最有利于酪氨酸蛋白磷酸酶、丝/苏氨酸蛋白磷酸酶的分离纯化。     

Effect of heavy water on the growth, glucose assimilation and stability of Escherichia coli to freezing-thawing]

Growth and resistance to freezing--thawing of Escherichia coli B-1640 were investigated during cultivation in synthetic media prepared with H2O and D2O. It is found that during cultivation in D2O the maximum specific growth rate decreases and the duration of the exponential growth phase increases. During the growth in D2O the glucose consumption rate drops in the exponential growth phase, the lactate content in the culture liquid is lower by two orders than that in H2O; the resistance to freezing--thawing is lower than that in H2O. After leaving the exponential phase the culture in D2O restores specific growth rate, glucose consumption rate and resistance to freezing--thawing up to the values obtained during the growth in H2O. The translation ability of ribosomes isolated from cells grown in D2O and H2O is the same. We conclude that the culture adapts to D2O during the exponential growth phase. It is suggested that during the adaptation the second carbon source is used which compensates the consequences of the disturbances of glucose metabolism and transport caused by deuteration of the cell content in the adaptation to D2O.     

Diauxic growth of Penicillium camembertii on glucose and arginine

The growth of Penicillium camembertii during batch culture in a synthetic medium containing glucose and arginine was examined. The diauxic growth observed can be well characterized. Indeed, in a first phase, glucose and arginine were, respectively, assimilated as carbon and nitrogen sources, with an acidification of the medium (until 3.5), since arginine was taken up in exchange for protons. During this phase of growth, arginine, in addition to glucose, was also assimilated as an energy source, resulting in the release of the arginine carbon content as CO₂. Then, in a second phase, characterized by reduced growth rates after glucose depletion, arginine was assimilated as a carbon and nitrogen source, as well as an energy source, resulting in ammonium release which raised the pH (final pH 6.3), despite the amino acid/H⁺ exchange, since amino acids contain excess nitrogen in relation to their carbon content for fungi.     

Nutritional and physiological factors affecting germination of heterothallic Saccharomyces cerevisiae ascospores.

Saccharomyces cerevisiae strain AP-3 was examined with respect to those nutritional requirements and physiological conditions which influence its germination rate. It was found that glucose as a carbon source supported the most rapid rate of germination for this heterothallic strain. In contrast, strain AP-3 spore germination was supported the least by the carbon sources potassium acetate and lactose. Of the nitrogen sources tested in culture medium containing glucose, the complex nitrogen sources peptone and casein hydrolysate appeared to be capable of stimulating germination better than a control culture containing ammonium sulphate. None of the amino acids screened were found to stimulate strain AP-3 germination compared with ammonium sulphate. The optimal culture medium pH for ascospore germination was 4.5 although spore germination could still be initiated by glucose between pH 3.0 and pH 7.5. Germination initiation by glucose was observed over a temperature range from 25 degrees C to 50 degrees C, but the optimal temperature appeared to be 40 degrees C.     

Influence of the Crc global regulator on substrate uptake rates and the distribution of metabolic fluxes in Pseudomonas putida KT2440 growing in a complete medium

When the soil bacterium Pseudomonas putida grows in a complete medium, it prioritizes the assimilation of preferred carbon sources, optimizing its metabolism and growth. This regulatory process is orchestrated by the Crc and Hfq proteins. The present work examines the changes that occur in metabolic fluxes when the crc gene is inactivated and cells grow exponentially in LB complete medium. Analyses were performed at three different moments during exponential growth, examining the assimilation rates for the compounds present in LB, changes in the proteome, and the changes in metabolic fluxes predicted by the iJN1411 metabolic model for P. putida KT2440. During the early exponential phase, consumption rates for sugars, many organic acids and most amino acids were higher in a Crc-null strain than in the wild type, leading to an overflow of the metabolic pathways and the leakage of pyruvate and acetate. These accelerated consumption rates decreased during the mid-exponential phase, when cells mostly used sugars and alanine. At later times, pyruvate was recovered from the medium and utilized. The higher consumption rates of the Crc-null strain reduced the growth rate. The lack of the Crc/Hfq regulatory system thus led to unbalanced metabolism with poorly optimized metabolic fluxes.     

Effect of Na Concentration and Nutritional Factors on the Lag Phase and Exponential Growth Rates of the Marine Bacterium Deleya aesta and of Other Marine Species

Growth of the marine bacterium Deleya aesta in a succinate minimal medium showed increasingly long lag phases as Na was decreased below the optimum (200 to 500 mM). The minimum Na concentration permitting growth consistently was 15 mM. Supplementation of the medium with KHCO(3) (as a source of CO(2)) or yeast extract, especially in combination, reduced the lag phase, increased the rate of exponential growth, and allowed growth at 8 mM Na. KHCO(3) did not reduce the lag period but did increase the rate of exponential growth of Deleya venusta, Deleya pacifica, and Alteromonas haloplanktis 214. Yeast extract was active for all three. The effect of yeast extract on D. aesta could be reproduced by a mixture of amino acids approximating its amino acid composition. l-Alanine, l-aspartate, and l-methionine, in combination, were the most effective in reducing the lag phase, although not as effective as the complete mixture. Succinate, l-aspartate, and l-alanine were transported into the cells by largely independent pathways and oxidized at rates which were much lower at 10 than at 200 mM Na. l-Methionine was transported at a low rate in the absence of Na and at a higher rate at 10 mM but was not oxidized. Above 25 mM Na, the rate of transport of the carbon source was not the rate-limiting step for growth. It is concluded that a combination of transportable carbon sources reduced the lag period and increased the rate of exponential growth because they can be taken up independently and at low Na utilized simultaneously.     

Performance of bacterial strains used for distillery waste treatment on different substrates

Six bacterial strains were isolated and acclimatized on distillery waste. The performance of these bacterial strains in respect to growth, reduction in chemical oxygen demand (COD) values, carbon dioxide production and volatile acid production were studied on five different substrates. Glucose and xylose exhibited growth patterns similar to that on spentwash. Glucose, xylose, casein hydrolysate and amino acids led to very good reduction in COD values compared with glycerol. Rate of substrate consumption was maximum in the case of glucose followed by amino acids, casein hydrolysate, xylose and glycerol. Production of volatile acids and carbon dioxide from glucose amounted to ≈ 50% of the theoretical yield based on glycolysis and the tricarboxylic acid cycle. Production of carbon dioxide followed the usual microbiological growth pattern while volatile acids did not show any such pattern. Carbon dioxide and volatile acids appear to be the major degradation products in distillery waste treatment by these bacteria.     

Analysis of N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1 by a proteomic approach

Pirellula sp. strain 1 is a marine bacterium that can grow with the chitin monomer N-acetylglucosamine as sole source of carbon and nitrogen under aerobic conditions, and that is a member of the bacterial phylum Planctomycetes. As a basis for the proteomic studies we quantified growth of strain 1 with N-acetylglucosamine and glucose, revealing doubling times of 14 and 10 h, respectively. Studies with dense cell suspensions indicated that the capacity to degrade N-acetylglucosamine and glucose may not be tightly regulated. Proteins from soluble extracts prepared from exponential cultures grown either with N-acetylglucosamine or glucose were separated by two-dimensional gel electrophoresis and visualized by fluorescence staining (Sypro Ruby). Analysis of the protein patterns revealed the presence of several protein spots only detectable in soluble extracts of N-acetylglucosamine grown cells. Determination of amino acid sequences and peptide mass fingerprints from tryptic fragments of the most abundant one of these spots allowed the identification of the coding gene on the genomic sequence of Pirellula sp. strain 1. This gene showed similarities to a dehydrogenase from Bacillus subtilis, and is closely located to a gene similar to glucosamine-6-phosphate isomerase from B. subtilis. Genes of two other proteins expressed during growth on N-acetylglucosamine as well as on glucose were also identified and found to be similar to a glyceraldehyde-3-phosphate-dehydrogenase and a NADH-dehydrogenase, respectively. Thus the coding genes of three proteins expressed during growth of Pirellula sp. strain 1 on carbohydrates were identified and related by sequence similarity to carbohydrate metabolism.     

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.     

A Proposed Role for the Cuticular Fatty Amides of Liposcelis bostrychophila (Psocoptera: Liposcelidae) in Preventing Adhesion of Entomopathogenic Fungi with Dry-conidia

Maximum challenge exposure of Liposcelis bostrychophila to Beauveria bassiana, Paecilomyces fumosoroseus, Aspergillus parasiticus or Metarhizium anisopliae resulted in no more than 16% mortality. We investigated several of L. bostrychophila's cuticular lipids for possible contributions to its tolerance for entomopathogenic fungi. Saturated C14 and C16 fatty acids did not reduce the germination rates of B. bassiana or M. anisopliae conidia. Saturated C6 to C12 fatty acids that have not been identified in L. bostrychophila cuticular extracts significantly reduced germination, but the reduction was mitigated by the presence of stearamide. Cis-6-hexadecenal did not affect germination rates. Mycelial growth of either fungal species did not occur in the presence of caprylic acid, was reduced by the presence of lauric acid, and was not significantly affected by palmitic acid. Liposcelis bostrychophila is the only insect for which fatty acid amides have been identified as cuticular components. Stearamide, its major fatty amide, did not reduce germination of B. bassiana or M. anisopliae conidia or growth of their mycelia. Adhesion of conidia to stearamide preparations did not differ significantly from adhesion to the cuticle of L. bostrychophila. Pretreatment of a beetle known to be fungus-susceptible, larval Oryzaephilus surinamensis, with stearamide significantly decreased adhesion of B. bassiana or M. anisopliae conidia to their cuticles. This evidence indicates that cuticular fatty amides may contribute to L. bostrychophila's tolerance for entomopathogenic fungi by decreasing hydrophobicity and static charge, thereby reducing conidial adhesion.     

金龟子绿僵菌中性海藻糖酶基因的克隆及其表达特性分析

根据中性海藻糖酶NTL基因的同源序列设计引物,PCR扩增出杀蝗专一菌株———金龟子绿僵菌CQMa102NTL基因片段,利用5′_RACE和3′_RACE扩增出NTLcDNA的5′和3′端序列,经拼接得到CQMa102NTL基因cDNA全长。根据其全长cDNA序列,设计引物PCR扩增出CQMa102NTL的完整基因。为了解该基因的上游调控信息,采用PanhandlePolymeraseChainReactionAmplification方法扩增其上游序列。序列分析表明,CQMa102NTL全长DNA3484bp,cDNA全长2385bp,编码737个氨基酸的蛋白,推测蛋白分子量为83.1kD;含有3个内含子,包含一个依赖于cAMP的磷酸化作用位点(RRGS)和一个钙附着位点(DTDGNMQITIED);上游序列含有一个压力反应元件(CCCCT);与金龟子绿僵菌广谱性菌株ME1NTL的核苷酸序列和氨基酸序列分别具有93%和99%同源性,由此确定该序列为金龟子绿僵菌中性海藻糖酶基因序列。Southern杂交表明,NTL基因在CQMa102基因组中为单拷贝。Northern杂交表明,NTL基因转录出约2.5kb的mRNA单带,在液体培养条件下,对数生长前期表达水平最高,对数生长后期降到最低,进入稳定生长期后表达水平又有所提高。金龟子绿僵菌CQMa102中性海藻糖酶基因DNA全长和cDNA全长登录GenBank,登录号分别为:AY557613,AY557612。     

Isolation, Characterization, and Transcriptional Analysis of the Chitinase chi2 Gene (DQ011663) from the Biocontrol Fungus Metarhizium anisopliae var. anisopliae

Metarhizium anisopliae infects arthropods via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the host penetration step and include chitinases. The characterization of the chi2 chitinase gene from M. anisopliae var. anisopliae is reported. The chi2 gene is interrupted by two short introns and is 1,542-bp long, coding a predicted protein of 419 amino acids with a stretch of 19 amino acid residues displaying characteristics of signal peptide. The predicted chitinase molecular mass is 44 kDa with a mature protein of 42 kDa and a theoretical pI of 4.8. The comparison of the CHI2 predicted protein to fungal orthologues revealed similarity to the glycohydrolase family 18 and a phylogenetic analysis was conducted. The chi2 gene is up-regulated by chitin as a carbon source and in conditions of fungus autolysis, and is down-regulated by glucose. This regulation is consistent with the presence of putative CreA/Crel/Crr1 carbon catabolic repressor binding domains on the regulatory sequence.     

Production of L-serine by the methanol utilizing bacterium,Pseudomonas 3ab

Summary A bacterium capable of growth on methanol and some organic acids as sole source of carbon and energy has been isolated and designated Pseudomonas 3ab. This facultative methylotrophic organism apparently utilizes the serine pathway of formaldehyde fixation.When methanol was used as the sole carbon source for growth, L-serine production by Pseudomonas 3ab occurred upon the addition of glycine and methanol at the end of the exponential growth phase. The maximum yield of L-serine (4.7 g/l) was obtained when 20 g/l glycine and 8 g/l methanol were added and the pH of the culture medium was changed to 8.5.Although Pseudomonas 3ab is unable to grow on L-serine or glycine, it is very active in decomposing these amino acids. The degradation of L-serine and glycine has been shown to be pH-dependent with a minimum at pH 8.5–9.0.     

Control of dimorphism in a biochemical variant of Candida albicans

The cellular morphology of a biochemical variant of Candida albicans could be controlled by the ratio of carbon dioxide to oxygen in the culture system or by individual amino acids. Predominantly pseudohyphal morphology was observed (i) at a CO(2) to O(2) ratio of 2:1 and (ii) without the addition of carbon dioxide, when either glycine, d- or l-ornithine, l-serine, l-methionine, l-phenylalanine, or l-tyrosine was the sole nitrogen source in the culture medium. When ammonium chloride, ammonium sulfate, l-glutamic acid, l-glutamine, or l-proline was the nitrogen source, yeastlike growth was observed in the presence or absence of CO(2). More adenosylmethionine was present in pseudohyphal than in yeastlike cells, and pseudohyphal cell wall preparations contained less methionine than cell walls from the yeastlike form. These results suggest a correlation between sulfur amino acid metabolism and dimorphism.