Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

NAD⁺-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD⁺ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD⁺ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.     

Novel lignocellulolytic ability of Candida utilis during solid-substrate cultivation on apple pomace

Lignocellulolytic enzymes from conventional and non-conventional yeasts are not commonly studied, and they have never been described for Candida utilis species. After solid-substrate cultivation of C. utilis (CCT 3469) on apple pomace, degradation of cellulose, pectin and lignin fragments was observed. Production of the main lignocellulolytic enzymes by C. utilis was investigated and high activity for pectinase (239 U ml^–1) as well as a significant manganese-dependent peroxidase (19.1 U ml^–1) activity was found. Low cellulase (3.0 U ml^–1) and xylanase (1.2 U ml^–1) activities were also observed suggesting that C. utilis may have lignocellulose degradation ability.     

Antigenic relationships between petunia peroxidase a and specific peroxidase isoenzymes in other Solanaceae

Summary A highly specific rabbit antiserum raised against peroxidase (PRXa) from petunia (Petunia hybrida) was used to investigate the antigenic relatedness of peroxidases in the Solanaceae. After SDS-PAGE of crude leaf extracts from a large number of species of this family, immunoblotting revealed that cross-reacting protein bands were present in all species tested. In order to determine whether these protein bands represent peroxidases, the peroxidase isoenzymes in thorn apple (Datura stramonium L.), tobacco (Nicotiana tabacum L.), sweet pepper (Capsicum annuum L.), potato (Solanum tuberosum L.), and tomato (Lycopersicon esculentum Mill.) were further analyzed. Immunoblots obtained after native PAGE revealed that the antiserum only recognized fast-moving peroxidase isoenzymes that are localized in the apoplast. Despite their serological relatedness, these peroxidases differed with respect to heat stability and apparent molecular weight. Differences in avidity for the petunia PRXa antiserum were suggested by immunoprecipitation with antibodies bound to protein A-Sepharose. The antiserum did not react with peroxidases from horseradish (Armoracea rusticana Gaertn., Mey and Scherb), turnip (Brassica napus L.), African marigold (Tagetes cresta L.), maize (Zea mays L.), and oats (Avena sativa L.). Apparently, the Solanaceae contain orthologous genes encoding the fast-moving anionic peroxidases homologous to petunia PRXa.     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Molecular approaches of a mitochondrial mutation inCandida utilis

Analysis of the cytochrome spectra of a mitochondrial mutant ofCandida utilis showed complete absence of apocytochromeb; this suggests a certain degree of damage, probably a small deletion in themit genes of mitochondrial DNA. Oxygen uptake measurements with and without cyanide of the respiratory-competentCandida utilis parent strain and its derivative mitochondrial mutant P_1,2 indicated the absence of the cyanide-sensitive or normal respiratory chain and a lowered rate of cyanide-insensitive or alternate respiration. Mitochondrial profiles and distribution of parental and mutant cells account for an altered mitochondrial DNA which affects mitochondria in the latter cell shape and function. The mutant cells ofCandida utilis were considered asmit ^– mutants from the observations reported here.     

Serological Diversity of Nitrobacter spp. from Soil and Aquatic Habitats

Serotypic diversity among Nitrobacter spp. isolates is greater than previously reported. Typing with fluorescent antibodies prepared against 11 Nitrobacter spp. cultures isolated from soil and water placed the isolates into six serogroups. When these fluorescent antibodies were applied to a group of 16 additional isolates, 8 were identifiable by cross-reaction to 3 of the 11 fluorescent antibodies. Some nitrite-oxidizing enrichment cultures from different habitats contained cross-reacting strains of Nitrobacter spp.     

Immunofluorescence of sulphate-reducing bacteria

An indirect fluorescent antibody technique was used as a method of rapidly assessing and identifying sulphate-reducing bacteria. Five specific antisera and one polyvalent serum were raised and tested against 44 strains of the genera Desulfovibrio and Desulfotomaculum along with 4 control organisms. Immunofluorescence was found to be mainly strain specific with the sulphate-reducing bacteria although weak fluorescence was seen both within and between recognised groups. A polyvalent antiserum was successfully used to detect sulphate-reducing bacteria. No interference from 4 control organisms was found.     

Demonstration of a Ferric Vibrioferrin-Binding Protein in the Outer Membrane of Vibrio parahaemolyticus

Under iron-restricted conditions, Vibrio parahaemolyticus produces a siderophore, vibrioferrin, accompanying expression of two major outer membrane proteins of 78 and 83 kDa. Autoradiographic analysis of nondenaturing polyacrylamide gel electrophoregrams of outer membrane preparations previously incubated with [⁵⁵Fe]ferric vibrioferrin revealed a single radiolabeled band, in which the 78-kDa protein was detected predominantly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum against the purified 78-kDa protein partially inhibited Fe-VF binding to isolated OMPs. The 78-kDa protein was cleaved by the treatment of whole cells with proteinase K, indicating that a portion of this protein is exposed on the surface of the outer membrane. The treated cells lost most of their iron uptake activity mediated by vibrioferrin. These results suggest that the ferric vibrioferrin-binding protein of 78 kDa may function as the receptor for ferric vibrioferrin involved in the initial step of vibrioferrin-mediated iron uptake. Immunoblot analysis using the antiserum against the 78-kDa protein demonstrated that the molecular mass and antigenic properties of the protein were highly conserved among V. parahaemolyticus strains examined. The antiserum also recognized an iron-repressible outer membrane protein of 78 kDa from iron-restricted V. alginolyticus strains, some of which appeared to produce vibrioferrin.     

Comparison of various ways of extraction of nucleic acids and of preparation of yeast extract from saccharomyces cerevisiae and candida utilis

Six different variations of the extraction procedure applied to yeast cells of Saccharomyces cerevisiae and Candida utilis to optimize the production of yeast extract and isolation of nucleic acids were compared. The autolysis of C. utilis at 50 to 52°C without adding chemical agents was found to be the best for the production of yeast extract. The most suitable procedures used for the extraction of nucleic acids were those which were carried out from C. utilis at pH 7.5 (92°C) and the other with 0.4 M NH₄OH (40°C). Both these modifications yielded the highest amounts of polymer nucleic acids. Applying all procedures compared to S. cerevisiae an increased content of sterols (including Δ^5.7-sterols, predominantly ergosterol) was detected.     

Determination of live and dead Komagataeibacter xylinus cells and first attempt at precise control of inoculation in nanocellulose production

The timely enumeration of cells of nanocellulose-producing bacteria is challenging due to their unique growth properties. To better understand the metabolism of the bacteria and better control the concentration of living cells during cultivation, a prompt cell counting technology is crucial and urgently required. In this work, two fluorescent dyes, the asymmetrical anthocyanidin dye SYBR Green I (SG) and propidium iodide (PI), were first combined for Komagataeibacter xylinus species to determine live/dead bacterial cells quantitatively and promptly. The number of live and dead K. xylinus cells determined using an epifluorescence microscope corresponded well to the results obtained using a fluorescence microplate reader. The R² values were 0.9986 and 0.9920, respectively, and were similar to those obtained with the LIVE/DEAD^® BacLight^TM commercial kit. SG/PI double-staining showed proper efficiency in distinguishing live/dead cells for the K. xylinus strain (R² = 0.9898). The technology was applied to standardize four different K. xylinus strains, and the initial cell concentration of the strains was precisely controlled (no significant difference among the strains, P> 0.05). The cellulose yield per live cell was calculated, and significant differences (P < 0.05) were found among the four strains in the following order: DHU-ATCC-1> DHU-ZCY-1> DHU-ZGD-1> ATCC 23770. The study shows (i) the application of the SG/PI staining to standardizing inocula for bacterial cellulose production so that a more accurate comparison can be made between different strains, and (ii) the lower cost of using SG rather than the SYTO 9 of the commercially available LIVE/DEAD^® BacLight^TM kit.     

Analysis of the Antigenic Relationships Among Trichomonas,Histomonas, Dientamoeba and Entamoeba III. Immunoelectrophoresis Technics*

Antigens prepared from Trichomonas gallinae, Histomonas meleagridis, Dientamoeba fragilis, Entamoeba invodens and Entamoeba histolytica were separated by electrophoresis in agar gels and reacted with antisera prepared in rabbits against each of the 5 species. The most numerous and strongest precipitin lines were obtained from reactions between the homologous antigens and antisera. Direct and cross-absorption reaction methods were employed with each antiserum and the various antigens to ascertain quantitatively the immunologic relationships among the several organisms. Trichomonas shared many common antigens with Histomonas, fewer with Dientamoeba and none with either species of Entamoeba. Histomonas was more closely related antigenically to Dientamoeba than to Trichomonas. The histomonad had only a few weakly cross-reacting antigens in common with the 2 Entamoeba species. Dientamoeba shared the most common antigens with Histomonas, fewer with Trichomonas and the fewest with Entamoeba. Somewhat stronger cross-reactivity was obtained with anti-Dientamoeba serum and E. invadens than between this immune serum and E. histolytica. The 2 species of Entamoeba shared the largest number of common antigens with each other, and to a much lesser extent both species cross reacted with Dientamoeba. Anti-Entamoeba sera had only a few weak cross-reacting precipitins with Histomonas. No antigenic relationship was found between either species of Entamoeba and Trichomonas.     

Lipopolysaccharides in four strains of the unicellular cyanobacterium Synechocystis

The results of the cross reactions of the 27 strains of Azospirillum spp. with 4 fluorescent antibodies (FA) show a neat differentiation between the two species. A. lipoferum represents a more homogenous group in respect to FA reactions and highly fluorescent preparations were obtained with strains from a large scope origin against Sp59 FA, the type strain. In contrast A. brasilense contains at least three sub groups in respect to FA reactions. The first includes all denitrifing strains (nir⁺) which react with FA from Sp7 the type strain. None of the nir^- strains reacted strongly with Sp7 FA. One part of the A. brasilense nir^- group which includes the strains isolated from well sterilized rice and wheat roots (Sp 107, 107 st, 106 and 109 st) reacts with FA of their reference strain Sp107 but not with that of Sp28 FA. The strains isolated from unsterilized roots and soils reacted with SP28 FA and not with that of Sp107 FA. In addition there were 3 strains (Sp A4, 34 and 67) which reacted with neither of the FAs.Abbreviations Fa fluorescent antibody - FITC fluorescein isothiocyanate - Rh ITC gelatin-rhodamine isothiocyanate - nir⁺ nitrite reductase positive - nir^- nitrite reductase negative     

Molecular cloning and characterization of the alcohol dehydrogenase ADH1 gene of Candida utilis ATCC 9950

The alcohol dehydrogenase gene (ADH1) of Candida utilis ATCC9950 was cloned and expressed in recombinant Escherichia coli. C. utilis ADH1 was obtained by PCR amplification of C. utilis genomic DNA using two degenerate primers. Amino acid sequence analysis of C. utilis ADH1 indicated that it contained a zinc-binding consensus region and a NAD(P)⁺-binding site, and lacked a mitochondrial targeting peptide. It has a 98 and 73% identity with ADH1s of C. albicans and Saccharomyces cerevisiae, respectively. Amino acid sequence analysis and enzyme characterization with various aliphatic and branched alcohols suggested that C. utilis ADH1 might be a primary alcohol dehydrogenase existing in the cytoplasm and requiring zinc ion and NAD(P)⁺ for reaction.     

川西米亚罗亚高山暗针叶林岷江冷杉和糙皮桦空间格局及其关联性分析

基于2019年9月完成的川西米亚罗亚高山暗针叶林9.6 hm~2动态样地的调查数据,分析了主要优势种岷江冷杉(Abies fargesii var.faxoniana)与糙皮桦(Betula utilis)的径级结构,并运用O-ring统计中的成对相关函数g(r)和g₁₂(r),研究了两个优势种的空间格局及其种内、种间的关联性。结果表明：岷江冷杉与糙皮桦径级分布连续,均呈倒“J”型。岷江冷杉与糙皮桦的不同径级个体主要呈现由聚集分布向随机分布发展的趋势。岷江冷杉的小树对幼树、中树对幼树、中树对小树以及大树对中树的种内相关性在所有空间尺度上均为显著正相关(P<0.05),而相同径级糙皮桦的种内相关性则表现为由显著正相关(P<0.05)逐渐向无显著相关(P>0.05)变化。随着空间尺度的增大,岷江冷杉的大树对幼树、大树和小树由无显著相关(P>0.05)逐渐向显著正相关(P<0.05)变化,糙皮桦的大树对幼树由显著负相关(P<0.05)逐渐向无显著相关(P>0.05)变化。岷江冷杉大树对糙皮桦所有径级个体的种间相关性主要呈现由为显...     

A Comparative Study of 14 Strains of Naegleria australiensis Demonstrates the Existence of a Highly Virulent Subspecies: N. australiensis italica n. spp.1

Fourteen strains of Naegleria australiensis, including the type strain, were compared for virulence for mice, maximum growth temperature, lectin agglutination, isoenzyme pattern, and total protein banding pattern. Their relation to other species of Naegleria also was compared by immunoelectrophoretic analysis. Strains with high virulence, comparable to that of N. fowleri, were found to be different in concanavalin A agglutination as well as with regard to zymograms and total protein patterns. Although serologically different from N. fowleri and reacting with N. australiensis antiserum in the fluorescent antibody test, these high-virulence strains differed in number of immunoelectrophoretic precipitin bands. Because of these results, the high-virulence strains are considered to be a subspecies of N. australiensis. The low-virulence strains showed minor differences from the type strain. Thus, N. australiensis does not appear to be as homogenous a species as N. fowleri. Pathogenic N. australiensis also seems to be more widespread than previously thought.     

Protoplast fusion between a petite strain ofCandida utilis andSaccharomyces cerevisiae respiratory-competent cells

Prototrophic RD mutant cells ofCandida utilis NRRL-Y-1084 and auxotrophic mutant respiratory-competent cells ofSaccharomyces cerevisiae 4003-5Ba his ₄ leu ₂ can ^S meth ₂ trp ₅ ade ₁ ura ₃ gal were turned into protoplasts to be further fused with the aid of polyethylene glycol (PEG) and Ca²⁺ ions. Minimal medium containing glycerol as the carbon source was employed for fusion product selection. The respiratory-competent fusion products, mainly oval cells, resembledCandida utilis and had the fermentative abilities of this strain (dextrose, sucrose, raffinose). Five fusion products were analyzed as to their ability to metabolize dextrose, xylose, cellobiose, trehalose, glycerol, succinic acid, citric acid, salycin, and maltose. Fusion products partially restored the respiratory-competentCandida utilis capacity to grow by use of these carbon compounds, and none of theSaccharomyces cerevisiae fermenting abilities were found. Our results would suggest either a partial recombination between parental mitochondria or some occurring phenomenon affecting the cell, membrane function after somatic fusion without concomitant nuclear fusion.     

Nucleic acid reduction from yeast activation of intracellular rnase

The study of a heat-shock process for RNA reduction was carried out for different yeast strains. Different results were obtained from each of them. Candida utilis NRRL Y-660 shows its best performance after a 8-s. heat-shock in the presence of 3% NaCl. For commercial baker's yeast Saccharomyces cerevisiae and Kluyveromyces fragilis L-1930, similar results were obtained with only 1% of NaCl. The latter needed longer heat-shock periods. e.g. 15s. to give such an RNA reduction. Biomass recovery ranged from 60 to 75%, being higher for C. utilis and K. fragilis while excessive losses were observed in S. cerevisiae cells. No significant protein deterioration was obtained in the best performance samples. The aminoacid profile appears to be improved in comparison to the starting material in these strains after RNA reduction.     

Ethanol Production from Xylose by a Recombinant Candida utilis Strain Expressing Protein-Engineered Xylose Reductase and Xylitol Dehydrogenase

The industrial yeast Candida utilis can grow on media containing xylose as sole carbon source, but cannot ferment it to ethanol. The deficiency might be due to the low activity of NADPH-preferring xylose reductase (XR) and NAD⁺-dependent xylitol dehydogenase (XDH), which convert xylose to xylulose, because C. utilis can ferment xylulose. We introduced multiple site-directed mutations in the coenzyme binding sites of XR and XDH derived from the xylose-fermenting yeast Candida shehatae to alter their coenzyme specificities. Several combinations of recombinant and native XRs and XDHs were tested. Highest productivity was observed in a strain expressing CsheXR K275R/N277D (NADH-preferring) and native CsheXDH (NAD⁺-dependent), which produced 17.4 g/L of ethanol from 50 g/L of xylose in 20 h. Analysis of the genes responsible for ethanol production from the xylose capacity of C. utilis indicated that the introduction of CsheXDH was essential, while overexpression of CsheXR K275R/N277D improved efficiency of ethanol production.     

Production of single cell protein using waste capsicum powder produced during capsanthin extraction

Aims: To produce single cell protein (SCP) by using waste capsicum powder produced during capsanthin extraction as a substrate. Methods and results: The extraction [CPM (capsicum powder medium)] from waste capsicum powder was used as culture medium to cultivate four yeast strains. The main composition of CPM was determined. The average concentration of total sugar, total nitrogen and phosphorous of CPM were 16·3, 3·7 g l^?1 and 785·4 mg l^?1, respectively. Four yeast strains were cultured in two CPMs, and the cell mass, protein content of cells and specific growth rate of cells were determined. Addition of corn steep liquor significantly increased the cell mass production. Presence of capsaicin in CPM did not show inhibition of cell growth of yeast tested. Conclusions: CPM contained sufficient nutrients and could be used as a good medium to produce SCP. Candida utilis 1769 was chosen as the biomass producer because of its highest SCP formation (6·8 g l^?1) and higher specific growth rate (0·12 h^?1). The amino acid composition of its protein was well balanced. Significance and Impact of the Study: Utilization of waste capsicum powder can reduce environmental pollution and increase protein supply for animal feed.     

Serological studies with Agrobacterium radiobacter,A. tumefaciens,and Rhizobium strains

Summary Antisera were prepared against cell material from 3 strains of A. radiobacter and 6 of A. tumefaciens. Agar diffusion and immune absorption techniques revealed 3 antigens common to each strain of these organisms. However, 5 different lipopolysaccharide antigens occurred in the 9 test strains. There was no obvious species differences in the distribution of these antigens. Mannose, and possibly glucuronic acid were immunologically active in one of the lipopolysaccharides.The agrobacterial antisera were further crosstested with antigenic material for 34 strains of Rhizobium. Fast-growing rhizobia showed extensive cross-reaction, but only one of 7 R. lupini strains tested reacted with any antiserum.