Isolation and characterization of a slowly milk-coagulating variant of Lactobacillus helveticus deficient in purine biosynthesis

A slowly milk-coagulating variant (Fmc(-)) of Lactobacillus helveticus CRL 1062, designated S1, was isolated and characterized. Strain S1 possessed all the known essential components required to utilize casein as a nitrogen source, which include functional proteinase and peptidase activities as well as functional amino acid, di- and tripeptide, and oligopeptide transport systems. The amino acid requirements of strain S1 were similar to those of the parental strain. However, on a purine-free, chemically defined medium, the growth rate of the Fmc(-) strain was threefold lower than that of the wild-type strain. L. helveticus S1 was found to be defective in IMP dehydrogenase activity and therefore was deficient in the ability to synthesize XMP and GMP. This conclusion was further supported by the observation that the addition of guanine or xanthine to milk, a substrate poor in purine compounds, restored the Fmc(+) phenotype of L. helveticus S1.     

Resistance to Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. II. Permeability changes to amethopterin and other folates in the drug-resistant mutant.

the accumulation of amethopterin in a Pediococcus cerevisiae strain resistant to this analogue was about 30% of that in P. cerevisiae/PteGlu, the sensitive parent. The uptake in the resistant strain was strictly glucose dependent, whereas in the sensitive parent about 16% accumulation occurred in absence of glucose. The transport in both strains was inhibited by iodoacetate and KF. Amethopterin uptake exhibited saturation kinetics with an apparent Km of 5 muM in P. cerevisiae/AMr and 0.5 muM in P. cerevisiae/PteGlu. The apparent V was 0.2 nmol per min per mg cells (dry weight); the same for both strains. The optimum pH for the uptake of amethopterin by P. cerevisiae/AMr and P. cerevisiae/PteGlu was pH 6.0. Folate and methyltetrahydrofolate competitivity inhibited amethopterin uptake with apparent Ki values of 8 and 0.7 muM, respectively. The uptake of folate exhibited a slightly increased Km value as compared to that of the sensitive strain, whereas the uptake activity velocity was in the same range. Methyltetrahydrofolate accumulated up to about 60-fold higher intracellular concentration than that of the medium, which is a markedly lower accumulation from that in the sensitive strain. The uptake was glucose dependent and inhibited by iodoacetate and KF. The pH optimum for methyltetrahydrofolate uptake in the resistant strain was the same as that in the sensitive parent (pH 5.7--6). In contrast to the increase in the apparent Km value for amethopterin in the resistant strain, the affinity of the carrier for methyltetrahydrofolate was apparently unchanged, whereas the V value was about 16 times lower than that in the sensitive strain. The Ki for amethopterin when added to increasing concentrations of methyltetrahydrofolate was 5.2 muM, a value about the same as that of the Km.     

Studies on histoplasmosis I. Comparative virulence of variant and parent strain Histoplasma capsulatum in Hamsters

Summary 1. A variant strain ofHistoplasma capsulatum was isolated from a secondary colony in cultures of tissue from hamsters infected with the parent strain ofHistoplasma. The variant strain was found to differ from the parent strain in colonial morphology, stability of the yeast phase in subculture, and in growth rate.2. Results of virulence testing with these two strains showed the parent strain to be somewhat more virulent than the variant strain as measured by survival time and slope of the death rate curve over a 19 day period.3. Nevertheless, in view of the more rapid growth rate of the variant and its probable antigenic variation from the parent strain, the development of such a variant is viewed as an increased hazard to the host, particularly in a longer term infection.4. The repeated isolation of a variant strain ofHistoplasma suggests that strain variation may play a significant role in the outcome of naturally acquired infections.This work was initiated at the State University of New York, Downstate Medical Center, Brooklyn, N. Y., and supported in part by funds ((#12-1081) from the National Fund for Medical Education, grant # 12-1054.     

Unique Properties of Four Lactobacilli in Amino Acid Production and Symbiotic Mixed Culture for Lactic Acid Biosynthesis

With four Lactobacilli--L. delbrueckii subsp. lactis (ATCC 12315), L. casei (NRRL-B1445), L. delbrueckii (NRRL-B445), and L. heveticus (NRRL-B1937)--the characteristics of cell growth and production of lactate and amino acids were investigated. Especially, the time-course variation in concentration of amino acids (classified into alanine, serine, aspartate, glutamate, aromatic amino acid, and histidine families) was estimated in detail, and the results were systematically compared. It was elucidated that L. delbrueckii (NRRL-B445) and L. helveticus (NRRL-B1937) had quite different characteristics in growth, lactic acid synthesis, and amino acid production. L. helveticus (NRRL-B1937) was superior in the production of amino acids as well as in cell growth, but showed very poor ability in lactic acid production. However, L. delbrueckii (NRRL-B445) showed higher yield of lactic acid despite repressed cell growth, but suffered from severe amino acid deficiency in culture. By modulating the initial concentration of each strain in the mixed culture containing both L. delbrueckii (NRRL-B445) and L. helveticus (NRRL-B1937), the lactic acid production (i.e., the amount of lactic acid produced and lactic acid yield to glucose consumed) was significantly improved, presumably via symbiotic interaction between the two strains.     

Characterization and infectivity of a spontaneous variant isolated fromFrankia sp. WEY 0131391

Summary A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the originalFrankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.     

Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.     

Effect of modified glucose uptake using genetic engineering techniques on high-level recombinant protein production in escherichia coli dense cultures

Reduction of acetate excretion using a modified cellular glucose uptake rate was examined. An Escherichia coli strain bearing a mutationin ptsG, a gene encoding enzyme II in glucose phosphotransferase system (PTS), was constructed and characterized. The growth rate of the mutant strain was slower than its parent in glucose defined medium, butwas not affected in complex medium. Experimental results using this mutant strain showed a significant improvement in culture performance in simple batch cultivations due to reduced acetate excretion through the modified glucose uptake. Both biomass and recombinant protein productivity were increased by more than 50% with the ptsG mutant when compared to the parent strain. Recombinant protein productivity by the newly constructed strain at a level of more than 1.6 g/L was attained consistently in a simple batch bioreactor. (c) 1994 John Wiley & Sons, Inc.     

携带新城疫病毒融合蛋白基因的重组减毒单核细胞增多性李斯特菌构建与鉴定

以鸡新城疫病毒F基因(NDV-F)为模式外源基因,通过基因切割-重叠延伸PCR法(SOE-PCR)将其插入到单核细胞增多性李斯特菌(Listeria monocytogenes)毒力基因hly的启动子和信号肽序列下游,并将该融合片段克隆入穿梭质粒pKSV7,随后将重组质粒电转李斯特菌进行同源重组。NDV-F基因的PCR扩增表明该重组菌构建成功,RT-PCR结果表明F基因在重组菌中得到了转录。比较了重组菌和野生型菌株的溶血性、黏附和侵袭力、对小鼠和鸡胚的毒力和生长特性以及重组菌的体内外稳定性,结果表明:hly基因中F片段的整合消除了单核细胞增多性李斯特菌溶血素基因的表达,其培养上清液没有溶血性,而野生型菌株的溶血价达24;细胞试验表明重组菌对细胞的黏附力和相对侵袭力均有不同程度的降低,而相对侵袭力与野生型菌株具有显著性差异(P<0.05);重组菌对小鼠及鸡胚的毒力(LD50)与野生型相比分别下降3.7和6.5个对数数量级;重组菌在BHI肉汤和小鼠体内连续5次后,仍然可以扩增出目的基因NDV-F,初步表明该重组菌较为稳定。     

一多重耐药单增李斯特菌株的生长特性及毒力分析

为了解单增李斯特菌株耐药后可能发生的生物学变化,以哈市生肉中分离到的1株对17种抗生素耐受的单增李斯特菌株L.M.B8为研究对象,对其生长及毒力特性进行研究。结果显示,L.M.B8的生长及毒力特性均与标准菌株有明显差异。在NaC l浓度为0.5%~5%、pH值为4.0~10.0及温度为20~45℃范围内,L.M.B8的生长速度均明显高于标准菌株。L.M.B8对高浓度盐的敏感性高于标准菌株,且对温度的适应能力强于标准菌株。从生长曲线看,L.M.B8的对数生长期与稳定期均较标准菌株提前2~3 h,且其稳定期较标准菌株明显缩短。L.M.B8小鼠腹腔注射半数致死量（LD50）较标准菌株明显降低。该研究为进一步探讨单增李斯特菌的耐药性与其他生物学特性的相关性奠定基础。     

Antigens of Bacillus cereus: A Comparison of a Parent Strain, an Asporogenic Variant and Cell Fractions

S ummary . The antigenic structure of a stable asporogenic variant of the M8 strain of Bacillus cereus has been compared with that of the parent strain. Ultrasonic extracts of cells of both parent strain and variant harvested at different ages have been analysed by immunoelectrophoresis against antisera prepared by injecting such extracts into rabbits.
Disintegrates of cells of the asporogenic variant were antigenically identical with disintegrates of vegetative cells of the parent strain. Disintegrates of cells in later stages of sporulation and of mature spores of the parent strain contained thermostable antigens which were never detected in the variant. Antigens of isolated cell walls, protoplasts and flagella were also studied.
Examination of esterase and catalase content of the two strains showed that although the variant had the same enzymes as the young vegetative cells of the parent strain it never developed the thermostable catalase found in disintegrated spores. Protein components of the two strains at different stages of growth and of the isolated cell fractions were studied by electrophoresis in polyacrylamide gels.     

保加利亚乳杆菌H+-ATPase缺陷型菌株的筛选

【目的】从传统乳制品中筛选具有新霉素抗性的H+-ATPase缺陷的德氏乳杆菌保加利亚亚种自发突变株,为最终开发弱后酸化的酸奶发酵剂奠定基础。【方法】利用API 50 CH细菌鉴定系统和16s rRNA基因序列分析对菌株进行鉴定。新霉素作为筛选压力,筛选具有新霉素抗性自发突变菌株,比较亲本和突变菌株的H+-ATPase活力及其代谢情况。【结果】从内蒙古地区的传统发酵酸奶中分离鉴定出一株德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii subsp. bulgaricus）,并命名为KLDS 1.9201。以此为出发菌株,筛选出两株H+-ATPase缺陷的自发突变株,分别命名为KLDS 1.9201-1、KLDS 1.9201-4,它们的H+-ATPase活力分别比亲本KLDS 1.9201降低了46%和60%。在MRS培养基中生长24 h后,KLDS 1.9201、KLDS 1.9201-1和KLDS 1.9201-4对初始葡萄糖的代谢率分别为65%、41%和31%,终产物中乳酸的浓度分别为26g/L、18g/L和15g/L,突变菌株的生物量均低于亲本。【结论】H+-ATPase活力降低的德氏乳杆菌保加利亚亚种的自发突变株具有较低的生长速率和弱产酸能力,它们可被用于制作弱后酸化的酸奶发酵剂。     

Surface of lactic acid bacteria: relationships between chemical composition and physicochemical properties

The surface chemical composition and physicochemical properties (hydrophobicity and zeta potential) of two lactic acid bacteria, Lactococcus lactis subsp. lactis bv. diacetilactis and Lactobacillus helveticus, have been investigated using cells harvested in exponential or stationary growth phase. The surface composition determined by X-ray photoelectron spectroscopy (XPS) was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbonlike compounds. The concentration of the last was always below 15% (wt/wt), which is related to the hydrophilic character revealed by water contact angles of less than 30 degrees. The surfaces of L. lactis cells had a polysaccharide concentration about twice that of proteins. The S-layer of L. helveticus was either interrupted or crossed by polysaccharide-rich compounds; the concentration of the latter was higher in the stationary growth phase than in the exponential growth phase. Further progress was made in the interpretation of XPS data in terms of chemical functions by showing that the oxygen component at 531.2 eV contains a contribution of phosphate in addition to the main contribution of the peptide link. The isoelectric points were around 2 and 3, and the electrophoretic mobilities above pH 5 (ionic strength, 1 mM) were about -3.0 x 10(-8) and -0.6 x 10(-8) m(2) s(-1) V(-1) for L. lactis and L. helveticus, respectively. The electrokinetic properties of the latter reveal the influence of carboxyl groups, while the difference between the two strains is related to a difference between N/P surface concentration ratios, reflecting the relative exposure of proteins and phosphate groups at the surface.     

Strain improvement and metabolic flux analysis in the wild-type and a mutant Lactobacillus lactis strain for L(+)-lactic acid production

The effects of initial glucose concentration and calcium lactate concentration on the lactic acid production by the parent strain, Lactobacillus lactis BME5-18, were studied. The results of the experiments indicated that glucose and lactate repressed the cell growth and the lactic acid production by Lactobacillus lactis BME5-18. A L(+)-lactic acid overproducing strain, Lactobacillus lactis BME5-18M, was screened by mutagenizing the parent strain with ultraviolet (UV) light irradiation and selecting the high glucose and lactate calcium concentration repression resistant mutant. Starting with a concentration of 100g L(-1) glucose, the mutant produced 98.6 g L(-1) lactic acid after 60 h in flasks, 73.9% higher than that of the parent strain. The L(+)-lactic acid purity was 98.1% by weight based on the amount of total lactic acid. The culture of the parent strain could not be analyzed well by conventional metabolic flux analysis techniques, since some pyruvate were accumulated intracellularly. Therefore, a revised flux analysis method was proposed by introducing intracellular pyruvate pool. Further studies demonstrate that there is a high level of NADH oxidase activity (12.11 mmol mg(-1) min(-1)) in the parent strain. The molecular mechanisms of the strain improvement were proposed, i.e., the high level of NADH oxidase activity was eliminated and the uptake rate of glucose was increased from 82.1 C-mmol (g DW h)(-1) to 98.9 C-mmol (g DW h)(-1) by mutagenizing the parent strain with UV, and therefore the mutant strain converts mostly pyruvate to lactic acid with a higher productivity (1.76 g L(-1) h(-1)) than the parent strain (0.95 g L(-1) h(-1)).     

Ultrastructure and enzyme activities of a virulent and an avirulent variant of Bacteroides gingivalis W50

The ultrastructure and enzyme activity of an avirulent, weakly-pigmenting, colonial variant (W50/BE1) was compared with that of the highly-virulent parent strain, Bacteroides gingivalis W50, in an attempt to identify significant virulence factors. Electron microscopy of thin sections of the organisms showed strain W50 to possess a 3-4-fold thicker layer of material external to the outer membrane. No significant differences between the strains were found with respect to collagen- or hyaluronic acid-breakdown activities at assay pH 7.5. However, cultures of strain W50 had over 3-fold more trypsin-like activity (P less than 0.01) than the avirulent variant. These results, when taken with other data, suggest that a thick external layer on the cell surface together with high trypsin-like activity might be important virulence factors of B. gingivalis.     

Glycollate inhibition of growth of Pseudomonas aeruginosa on lactate medium

Glycollate inhibited growth of Pseudomonas aeruginosa in media containing either pyruvate or lactate as carbon sources. Glycollamide, but not glyoxylate, showed similar effects. Spontaneous mutants (L/G strains) were isolated that were able to grow on lactate medium in the presence of glycollate: their growth in pyruvate medium was still inhibited by glycollate. Synthesis of membrane-bound NAD+-independent D(-)- and L(+)-lactate dehydrogenase (iLDHs) was inducible by D- or L-lactate in the parent strain but was constitutive in the L/G strains. Glycollate inhibited induction of the synthesis of iLDHs in the parent strain growing in succinate medium but had no effect under the same conditions on strain L/G1. Glycollate was a competitive inhibitor of L(+)-iLDH (Ki = 11 mM). No differences were found in the kinetic properties of L(+)-iLDH in cell-free extracts from strain L/G1 and the parent organism. Glycollate appears to inhibit growth on lactate medium predominantly through prevention of lactate induction of iLDH synthesis.     

Cell wall composition of Streptomyces roseoflavus var. roseofungini and its Nocardia-like variant

The composition of cell walls was comparatively studied in Streptomyces roseoflavus var. roseofungini 1128 and in its variant 1-68. In the logarithmic phase of growth, the content of teichoic acid in the cell wall of the parent culture was four times as high as in the cell wall of the variant. The cell walls of the parent culture contained 5 to 7 times more O-lysyl residues not only due to a higher content of teichoic acid in the walls but also owing to a lower content of lysyl groups in the teichoic acid of the variant. An additional polysaccharide comprising galactose and glucosamine was found in the cell wall of the variant but not in the parent strain. The peptidoglycan of the both cultures had a structure typical of Streptomyces spp.; its content in the cell walls of the two cultures was identical (ca. 50% of the dry cell wall biomass weight). The results are discussed in connection with the peculiarities of the variant hyphal septation.     

Metabolic engineering of Lactobacillus helveticus CNRZ32 for production of pure L-(+)-lactic acid

Expression of D-(-)-lactate dehydrogenase (D-LDH) and L-(+)-LDH genes (ldhD and ldhL, respectively) and production of D-(-)- and L-(+)-lactic acid were studied in Lactobacillus helveticus CNRZ32. In order to develop a host for production of pure L-(+)-isomer of lactic acid, two ldhD-negative L. helveticus CNRZ32 strains were constructed using gene replacement. One of the strains was constructed by deleting the promoter region of the ldhD gene, and the other was constructed by replacing the structural gene of ldhD with an additional copy of the structural gene (ldhL) of L-LDH of the same species. The resulting strains were designated GRL86 and GRL89, respectively. In strain GRL89, the second copy of the ldhL structural gene was expressed under the ldhD promoter. The two D-LDH-negative strains produced only L-(+)-lactic acid in an amount equal to the total lactate produced by the wild type. The maximum L-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain. Furthermore, process variables for L-(+)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology. The temperature and pH optima were 41 degrees C and pH 5.9. At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86.     

高产耐热脂肪酶嗜热脂肪地芽孢杆菌的选育

采用氮离子注入技术对耐热脂肪酶产生菌嗜热脂肪地芽孢杆菌（Geobacillus stearothermophilus）L4进行诱变,筛选获得酶活力有较大提高且传代稳定的正突变菌株L4-3;再对L4-3进行紫外线诱变,得到脂肪酶活力提高的正突变菌株L4-3-2,其脂肪酶活力达25．71U／mL,较原始菌株M提高511．9％。高产突变株L4-3-2所产脂肪酶的最适作用温度为50℃,70℃保温60min的剩余酶活为82％,最适作用pH为7．0～8．0,为一种耐热碱性脂肪酶。     

Markers Differentiating Type 3 Parainfluenza Virus Grown at 25 and 37 C

Serial passage of the 64-2389 strain of type 3 parainfluenza virus in cercopithecus monkey kidney tissue cultures at low temperatures resulted in the selection of a variant which had a higher efficiency of plaque formation at 25 C than the parent line grown at 37 C. The cold variant, unlike the parent strain, plaqued readily at 25 C, and at 37 C it produced significantly larger plaques. Virus titers of the cold variant in hamster lungs were significantly lower and this was probably caused by the stimulation of interferon by the cold variant during the early phase of the infection. The cold variant, like the virus grown at 37 C, also induced the synthesis of interferon late in the infection. Hamsters responded to the intranasal inoculation of each virus line by the development of hemagglutinating-inhibiting antibodies in the sera.     

Evaluation of Culture Media for the Isolation of Salmonellae from Feces

Serial passage of the 64-2389 strain of type 3 parainfluenza virus in cercopithecus monkey kidney tissue cultures at low temperatures resulted in the selection of a variant which had a higher efficiency of plaque formation at 25 C than the parent line grown at 37 C. The cold variant, unlike the parent strain, plaqued readily at 25 C, and at 37 C it produced significantly larger plaques. Virus titers of the cold variant in hamster lungs were significantly lower and this was probably caused by the stimulation of interferon by the cold variant during the early phase of the infection. The cold variant, like the virus grown at 37 C, also induced the synthesis of interferon late in the infection. Hamsters responded to the intranasal inoculation of each virus line by the development of hemagglutinating-inhibiting antibodies in the sera.