Zum Nachweis der Virulenz bei Agrobacterium tumefaciens

Zusammenfassung Der auf der bakteriellen Umwandlung von Lactose in 3-Ketolactose basierende Test von Bernaerts u. de Ley (1963 a) zum Nachweis von Agrobacterium tumefaciens ist nicht spezifisch und bedarf zusätzlicher Sicherungen. Die Korrelation zwischen Virulenz und Bildung von 3-Ketoglykosiden erscheint anfechtbar.

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Summary The test for Agrobacterium tumefaciens by means of a reaction based on the bacterial conversion of lactose to 3-ketolactose (Bernaerts and de ley 1963 a) is not specific, and therefore requires additional proof. The correlation between virulence and production of 3-ketoglykosides appears to be questionable.

     

Conversion of disaccharides to 3-ketodisaccharides by nongrowing and immobilized cells ofAgrobacterium tumefaciens

High-performance liquid chromatography was used to estimate 3-ketolactose and 3-ketosucrose in cultures of agrobacteria. The activities of enzymes that convert the disaccharide substrate were evaluated during batch cultivation ofAgrobacterium tumefaciens on sucrose, maltose, and lactose. The highest activity of glucoside 3-dehydrogenase and a slight activity of disaccharide-hydrolyzing enzymes were found in cells grown on lactose. Nongrowing cells converted lactose to 3-ketolactose faster than immobilized cells did. Immobilization of cells into polysaccharide gels did not stabilize the activity of glucoside 3-dehydrogenase. Glutaraldehyde cross-linking of the cell content led to an inactivation of the respiratory chain but Fe³⁺ could be used as an electron acceptor. Cells treated with glutaraldehyde converted lactose faster than nongrowing ones but the activity of glucoside 3-dehydrogenase was not stable.     

Utilization of oligosaccharides by three imperfect fungi

Summary The utilization of oligosaccharides (maltose, sucrose, lactose and raffinose) by three imperfect fungi viz.Alternaria tenuis Auct.,Colletotrichum capsici (Syd.)Butler &Bisby andColletotrichum gloeosporioides Penz. isolated from diseased leaves of tomato,Scindapsus pictus andPolyscias balfuriana respectively, was studied chromatographically. Except for lactose, all the sugars were utilizzed through a hydrolytic pathway and almost all proved to be a suitable source of carbon for the growth of fungi. Along with the utilization of sucrose, a simultaneous synthesis of an oligosaccharide was also observed in case of two species ofColletotrichum. C. gloeosporioides, however, also synthesized an oligosaccharide, maltotriose if it is grown in the medium containing maltose. During the utilization of raffinose, melibiose and fructose were detectted in the culture medium ofA. tenuis. Melibiose was further broken down into glucose and galactose by the two species ofColletotrichum.All the sugars were consumed from the media within 15 days except for lactose which persisted in the media even after 15 days.     

Influence of 2,4-d and lactose on pollen embryogenesis in anther culture of potato

Anthers of diploid genotypes of Solanum tuberosum capable of androgenesis were cultured on different media to examine the effect on induction of pollen embryogenesis of 2,4-d and lactose. Anthers cultured in callogenic medium with 2,4-d and sucrose produced pollen derived embryoids only exceptionally. When sucrose was replaced by lactose the frequency of embryogenesis was as high or higher than in embryogenic auxin-free medium. Substitution of lactose for sucrose in the embryogenic medium had no effect. Supplementing the embryogenic medium with 2,4-d strongly reduced the frequency of pollen embryoids in the presence of sucrose but not with lactose.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid     

Role of lactose on the production of d-arabitol by Kluyveromyces lactis grown on lactose

There are remarkably few reports on d-arabitol production from lactose. Previous studies in our laboratory have shown that the osmophilic yeast Kluyveromyces lactis NBRC 1903 convert lactose to extracellular d-arabitol. The present study was undertaken to determine the participation of osmotic stress caused by lactose on d-arabitol production by K. lactis NBRC 1903 and to provide the information on the kinetics of d-arabitol production from lactose by K. lactis NBRC 1903. It was confirmed that d-arabitol production was triggered when an initial lactose concentration was above 278 mmol L⁻¹. d-Arabitol yield increased with an increase in initial lactose concentration. The highest d-arabitol concentration of 79.5 mmol L⁻¹ was achieved in the cultivation of K. lactis NBRC 1903 in a medium containing 555 mmol L⁻¹ lactose and 40 g L⁻¹ yeast extract. Lactose was found to play two important roles in d-arabitol production by K. lactis NBRC 1903 grown on lactose. First, lactose was assimilated as the substrate both for cell growth and d-arabitol production. Second, a high lactose concentration induced cellular response to high osmotic stress and up-regulated the flow from d-glucose-6-phosphate to d-arabitol. The arrest of cell growth triggered d-arabitol production.     

Sorbitol production from lactose by engineered Lactobacillus casei deficient in sorbitol transport system and mannitol-1-phosphate dehydrogenase

Sorbitol is a sugar alcohol largely used in the food industry as a low-calorie sweetener. We have previously described a sorbitol-producing Lactobacillus casei (strain BL232) in which the gutF gene, encoding a sorbitol-6-phosphate dehydrogenase, was expressed from the lactose operon. Here, a complete deletion of the ldh1 gene, encoding the main l-lactate dehydrogenase, was performed in strain BL232. In a resting cell system with glucose, the new strain, named BL251, accumulated sorbitol in the medium that was rapidly metabolized after glucose exhaustion. Reutilization of produced sorbitol was prevented by deleting the gutB gene of the phosphoenolpyruvate: sorbitol phosphotransferase system (PTS^Gut) in BL251. These results showed that the PTS^Gut did not mediate sorbitol excretion from the cells, but it was responsible for uptake and reutilization of the synthesized sorbitol. A further improvement in sorbitol production was achieved by inactivation of the mtlD gene, encoding a mannitol-1-phosphate dehydrogenase. The new strain BL300 (lac::gutF Δldh1 ΔgutB mtlD) showed an increase in sorbitol production whereas no mannitol synthesis was detected, avoiding thus a polyol mixture. This strain was able to convert lactose, the main sugar from milk, into sorbitol, either using a resting cell system or in growing cells under pH control. A conversion rate of 9.4% of lactose into sorbitol was obtained using an optimized fed-batch system and whey permeate, a waste product of the dairy industry, as substrate.     

Variation of antimetabolite sensitivity with different carbon sources inBacillus subtilis

The susceptibility ofBacillus subtilis to amino acid analogues was found to be markedly influenced by the carbon source used in the test media. Thialysine inhibited the bacterium with a greater number of carbon sources than the other two analogues tested. 5-Hydroxylysine was inhibitory with glycerol, lactose,D-xylose,L-arabinose and soluble starch while ethionine showed toxicity with lactose,D-xylose andL-arabinose. None of these analogues were toxic at the levels tested whenD-galactose was used as carbon source. The bacterium was not susceptible to thialysine with glycerol, to 5-hydroxylysine withL-arabinose and to ethionine with lactose.     

Utilization of oligosaccharides by three pathogenic isolates of colletotrichum gloeosporioides

Summary Utilization of six oligosaccharides (viz., sucrose, maltose, cellobiose, lactose, raffinose and melezitose) by the isolates ofColletotrichum gloeosporioides Penz. causing leaf-spot diseases ofCarissa carandas L.,Eucalyptus robusta Sm. andBougainvillaea glabra Choizy has been studied. Daily chromatographic analysis of the culture medium revealed that the utilization of sucrose, maltose, cellobiose and raffinose was through a hydrolytic pathway. In case of lactose and melezitose no hydrolytic product could be detected. During the utilization of sucrose two oligosaccharides (Rf 0.27 and 0.2) made their appearance. TheBougainvillaea-isolate synthesized two oligosaccharides (Rf 0.18 and 0.1) in a maltose medium although no glucose could be traced, while in case of the other two isolates, in addition to these synthetic oligosaccharides, glucose also appeared. When theCarissa-isolate ofColletotrichum gloeosporioides was grown on a cellobiose medium, in addition to glucose, two synthetic oligosaccharides (Rf 0.15 and 0.11) were formed. Other two isolates showed the formation of one oligosaccharide (Rf 0.15) and no glucose. The dry weight of mycelium recorded an increase on oligosaccharides with slow rate of consumption, while on oligosaccharides with rapid rate of utilization it tended to become constant or showed a decline towards the end of the incubation.     

Utilization of lactose in non-respiring cells of the yeast Debaryomyces polymorphus

The facultative anaerobic yeast Debaryomyces polymorphus ferments glucose and galactose but does not utilize the disaccharide lactose under anaerobic conditions. The activity of the intracellulary located -galactosidase was not affected by anaerobiosis. Hence, the transport of lactose appears to be limiting for lactose utilization. The uptake of lactose (and of its metabolizable analogue, 4-nitrophenol--d-galactoptranoside) was mediated by an inducible transport system and it was strictly dependent on metabolic energy. Anaerobic conditions inhibited the transport of lactose completely as did the uncoupler carbonylcyanide-m-chlorophenyl-hydrazone, the electron transport chain inhibitors rotenone, antimycin A, potassium cyanide and the ATPase inhibitor diethylstilbestrol under aerobic conditions. Transport inhibited by antimycin A was resumed by adding ascorbate+tetramethyl-p-phenylenediamine. Glucose was taken up by a constitutive transport system, even in anaerobic cells it was still about five times faster than the uptake of lactose in respiring cells. Thus, monosaccharides can energize their uptake by glycolysis and represent, in contrast to lactose, fermentable, substrates in D. polymorphus.Abbreviations 4NPßgal 4-nitrophenol--d-galactopyranoside - TMPD tetramethyl-p-phenylenediamine Dedicated to Professor Augustin, Betz at the occassion of his 65th birthday.     

An Evaluation of the Nile Blue Test for Differentiating Rhizobia from Agrobacteria

The ability of agrobacteria to reduce Nile Blue more strongly than do rhizobia is the basis of a test for separating these two groups (Hamdi 1969). In a modified test using only 35 parts 10° of Nile Blue in the medium, 89 of 90 rhizobia ( Rhizobium japonicum, R. leguminosarum, R. lupini, R. phaseoli, R. trifolii , cowpea, groundnut and Lotus rhizobia) failed to reduce the dye whereas all 24 strains of agrobacteria ( Agrobacterium radiobacter var. radiobacter, A. r. var. tumefaciens and A. r. var. rhizogenes ) reduced it to the colourless state. Only one Rhizobium strain formed 3-ketolactose from lactose, but 13 agrobacteria produced it. Rhizobium meliloti strains (12) gave variable reactions in both tests. The Nile Blue Test detected rapidly, but not slowly growing, strains of agrobacteria present as contaminants of rhizobia cultures even when their initial numbers were small.     

Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems

Protein production under the control of lac operon regulatory elements using autoinduction is based on diauxic growth of Escherichia coli on lactose after consumption of more preferred carbon substrates. A novel simple and cost-effective defined autoinduction medium using a mixture of glucose, glycerol, and lactose as carbon substrate and NH₄⁺ as sole nitrogen source without any supplementation of amino acids and vitamins was developed for T7-based E. coli expression systems. This medium was successfully employed in 96-well microtiter plates, test tubes, shake flasks, and 15-L bioreactor cultivations for production of different types of proteins achieving an average yield of 500 mg L⁻¹ product. Cell-specific protein concentrations and solubility were similar as during conventional isopropyl β-d-1-thiogalactopyranoside induction using Luria-Bertani broth. However, the final yield of target proteins was about four times higher, as a higher final biomass was achieved using this novel defined autoinduction broth.     

Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

Effect of glucose on sporulation and extracellular amylase production byClostridium perfringens type A in a defined medium

The effect of glucose and other sugars on sporulation and extracellular amylase production byClostridium perfringens NCTC 8679 type A in a defined medium was studied. Cells grown in the presence of glucose and mannose yielded the highest levels of amylase activity, while disaccharides such as lactose, maltose, and sucrose resulted in moderate amylase production. Little amylase activity was detected in the medium in the presence of ribose or galactose. The concentration of each sugar resulting in highest amylase production was between 6 and 10mm except for fructose (25mm). Levels of heat-resistant spores decreased as sugar concentrations increased. The addition of even small amounts of glucose to the medium before exponential growth suppressed sporulation but maximized amylase activity. The addition of glucose after the initiation of sporulation did not inhibit spore formation. However, its addition to 3-h amylase-producing cells did inhibit subsequent sporulation but promoted the continued excretion of amylase. The different response to glucose between sporulating cells and amylase-producing cells suggests that the mechanisms of catabolite repression of extracellular amylase production and sporulation are distinct in this strain ofC. perfringens.     

Evidence for two β-galactosidase activities inStreptomyces violaceus

The kinetics of β-galactosidase synthesis induced by galactose and lactose inStreptomyces violaceus, as well as the pattern ofo-nitrophenyl-β-d-galactoside-positive bands observed after electrophoresis of both crude extracts, showed the presence of different β-galactosidase activities in the two cellular extracts. It is postulated that the lactase activity induced by lactose is the physiological enzyme responsible for lactose utilization. The possible function of the galactose-induced activity is also discussed.     

Influence of reaction conditions on the selectivity of the synthesis of lactulose with microbial β-galactosidases

Commercial β-galactosidase preparations from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae were evaluated as catalysts for the synthesis of lactulose. Among them, the enzyme from A. oryzae was selected for further studies. The effect of reaction conditions was then studied on product composition during the kinetically controlled synthesis of lactulose by transgalactosylation with A. oryzae β-galactosidase. Product composition was not affected by pH, temperature, total initial concentration of sugar (lactose plus fructose) and enzyme to substrate ratio within the ranges studied. However, lactose to fructose ratio strongly influenced product composition being then possible to control the lactulose to galacto-oligosaccharide ratio within ample margins. Maximum lactulose yield (0.282 g of lactulose per g initial lactose) was obtained using 1/8 lactose to fructose molar ratio, 50% (w/w) total initial sugars, 40 °C, pH 4.5 and enzyme to initial lactose ratio equivalent to 200 IU/g.     

Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula

We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°_20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K _m=0.75 mm and molecular activity (k _cat)= 63.1 s^–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K _m=0.04 mm k _cat= 55.8 s^–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl₂) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl₂). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose. Correspondence to: T. Nakayama     

Proton-linked sugar transport systems in bacteria

The cell membranes of various bacteria contain proton-linked transport systems ford-xylose,l-arabinose,d-galactose,d-glucose,l-rhamnose,l-fucose, lactose, and melibiose. The melibiose transporter ofE. coli is linked to both Na⁺ and H⁺ translocation. The substrate and inhibitor specificities of the monosaccharide transporters are described. By locating, cloning, and sequencing the genes encoding the sugar/H⁺ transporters inE. coli, the primary sequences of the transport proteins have been deduced. Those for xylose/H⁺, arabinose/H⁺, and galactose/H⁺ transport are homologous to each other. Furthermore, they are just as similar to the primary sequences of the following: glucose transport proteins found in a Cyanobacterium, yeast, alga, rat, mouse, and man; proteins for transport of galactose, lactose, or maltose in species of yeast; and to a developmentally regulated protein of Leishmania for which a function is not yet established. Some of these proteins catalyze facilitated diffusion of the sugar without cation transport. From the alignments of the homologous amino acid sequences, predictions of common structural features can be made: there are likely to be twelve membrane-spanning -helices, possibly in two groups of six, there is a central hydrophilic region, probably comprised largely of -helix; the highly conserved amino acid residues (40–50 out of 472–522 total) form discrete patterns or motifs throughout the proteins that are presumably critical for substrate recognition and the molecular mechanism of transport. Some of these features are found also in other transport proteins for citrate, tetracycline, lactose, or melibiose, the primary sequences of which are not similar to each other or to the homologous series of transporters. The glucose/Na⁺ transporter of rabbit and man is different in primary sequence to all the other sugar transporters characterized, but it is homologous to the proline/Na⁺ transporter ofE. coli, and there is evidence for its structural similarity to glucose/H⁺ transporters in Plants.In vivo andin vitro mutagenesis of the lactose/H⁺ and melibiose/Na⁺ (H⁺) transporters ofE. coli has identified individual amino acid residues alterations of which affect sugar and/or cation recognition and parameters of transport. Most of the bacterial transport proteins have been identified and the lactose/H⁺ transporter has been purified. The directions of future investigations are discussed.     

Effect of the interrupted adaptation to D-methionine on the efficiency of Rhizobium meliloti strains

Summary R. meliloti strains 107-1, 111 and 152 were adapted to D-methionine in three ways: a) consecutive transfer in the presence of increasing amounts of D-methionine, b) alternate transfer between D- and L-methionine-containing media followed by final cultivation in the presence of each isomer, c) alternate transfer between D-methionine and medium 79 followed by cultivation in medium 79 or in D-methionine-medium. At the end of the experiment efficiency of the strains was ascertained by a plant test.Strain 111 lost efficiency when it was adapted consecutively to 0.125% D-methionine or alternated between D-methionine and either L-methionine or medium 79-Strain 107-1 sucessively adapted to D-methionine lost efficiency within 16 weeks. On adaptation to D-methionine alternated with L-methionine, efficiency was retained in L-methionine medium and lost in D-methionine medium. On alternate adaptation between D-methionine and medium 79, strain 107-1 lost efficiency in the D-methionine-medium but not in medium 79. Efficiency of strain 152 was lost by adaptation to 0.125% D-methionine, but it was maintained on the alternate adaptation between D-methionine and L-methionine or medium 79.     

Klebsiella planticola sp. nov.: A new species of enterobacteriaceae found primarily in nonclinical environments

The nameKlebsiella planticola sp. nov. is proposed for a group of organisms isolated primarily from botanical and daquatic environments. Both numerical and molecular taxonomy techniques show that the species belongs within the genusKlebsiella and that it is distinct from other described species of the genus. The new species has 3 biogroups.K. planticolla is phenotypically identical toK. pneumoniae in classical biochemical tests, but it is distinguishable fromK. pneumoniae based on its ability to grow at 10°C and its inability to produce gas from lactose at 44.5°C. Additional reactions that can be used in combination with the temperature criteria includel-sorbose fermentation and ability to utilize hydroxy-l-proline as a sole carbon source. Strain V-236 (ATCC 33531; CDC 4245-72) is the type strain of the new species.     

Lactulose production by β-galactosidase in permeabilized cells of<Emphasis Type="Italic"> Kluyveromyces lactis</Emphasis>

Lactulose production from lactose and fructose was investigated with several commercial -galactosidases. The enzyme from Kluyveromyces lactis exhibited the highest lactulose productivity among the -galactosidases tested. The reaction conditions for lactulose production were optimized using cells that had been permeabilized by treatment with 50% (v/v) ethanol: cell concentration, 10.4 g l^–1; concentration of substrates, 40% (w/v) lactose and 20% (w/v) fructose; temperature, 60^°C; pH 7.0. Under these conditions, the permeabilized cells produced approximately 20 g l^–1 lactulose in 3 h with a lactulose productivity of 6.8 g l^–1 h^–1. These results represent 1.3- and 2.1-fold increases in lactulose concentration and productivity compared with untreated washed cells. This is the first reported trial of enzymatic synthesis of lactulose using permeabilized yeast cells.