High concentration culture of Bacillus subtilis spo− mutant strain carrying a recombinant plasmid

A Bacillus subtilis spo⁻ mutant strain harboring a recombinant plasmid was cultured at a high cell density concentration by using a cross-flow filtration system. The cell concentration obtained was 300 as an optical density at 570 nm, which was six times as high as that of a fed-batch culture without using the cross-flow filtration system. However, the specific activity of plasmid-encoded β-galactosidase decreased by filtration. To improve the specific activity, the carbon source was stepwisely changed from glucose to starch. By using a fed-batch culture combined with cross-flow filtration and the stepped change of carbon source, the production of the plasmid-encoded enzyme was improved 3 times compared with that of the fed-batch culture.     

Functional Activity of Exoglycans from Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Resistant Mutant M-71 during the Formation of Legume–Rhizobia Symbiosis against a High-Nitrogen Background

The functional activity of the exoglycan complex (EGC) polysaccharides from Rhizobium leguminosarum bv. viciae 250a and its nitrogen-resistant mutant M-71, capable of inducing the formation of nitrogen-fixing nodules on pea roots against a high-nitrogen background (4.8 mM NO₃ ^–), was studied in vegetation tests. For this purpose, the bacterial inoculum washed free of its own exoglycans was supplemented with EGC of the same or another strain grown in the presence of 6 or 20 mM nitrate. The best symbiotic characteristics (nodule number and nitrogenase activity, mass of the roots and aerial parts of plants) were recorded when the inoculum cells and exoglycans were obtained from strain M-71 grown in the presence of 20 mM nitrate. When the plants were inoculated with the cells (grown at 6 mM nitrate) + EGC (obtained at 6 mM nitrate) of this strain, the nodulation characteristics and the effectiveness of symbiosis decreased 1.5- to 2-fold. Partial recovery of the symbiotic potential of strain M-71 was observed when EGC (obtained at 20 mM nitrate) was substituted for its exoglycans (obtained at 6 mM nitrate). In the presence of exoglycans of the parent strain 250a (obtained at 6 or 20 mM nitrate), the mutant formed a substantially lesser number of nodules with a very low nitrogen-fixing activity. In turn, the mutant exoglycans synthesized in medium with either high or low nitrate nitrogen concentration did not recover the fix⁺ phenotype of strain 250a, capable of forming symbiosis with pea plants only against a low-nitrogen background. In study of the relative content of high-molecular-weight exopolysaccharide components and low-molecular-weight glycans in the exoglycan complex, it was established that, in strain 250a (grown at 6 and 20 mM nitrate), as well as in its mutant M-71 (grown at 6 mM nitrate), exopolysaccharides prevailed, accounting for 72–75% of the sum of both types of glycopolymers, while low-molecular-weight glycans accounted for 25–28%. In contrast, in the EGC of strain M-71 obtained at 20 mM nitrate, which was the most active inducer of the formation of the symbiotrophic system by strain M-71 in the presence of a high mineral nitrogen concentration, low-molecular-weight glycans were the main component, accounting for 61% of total glycopolymers, while the polysaccharide content was 39%. Low-molecular-weight exoglycans are supposed to be involved in maintaining the physiological activity and the symbiotic status of rhizobia under unfavorable environmental conditions.     

Proline excretion in Escherichia coli: A comparison of an argD + strain and a proline-excreting argD − derivative

In an attempt to deduce the physiological basis of proline excretion in argD ^– strains of Escherichia coli K12, several properties of an argD ⁺ (nonexcreting) and an argD ^– (excreting) derivative were compared. No difference was found in the transport or in the utilization of either proline or its immediate precursor, ¹-pyrroline-5-carboxylate (PCA). Furthermore, no differences were found in the physical or kinetic properties of partially purified preparations of the enzyme mediating the final step in proline biosynthesis, PCA reductase. The specific activity of PCA reductase was, however, consistently higher in crude extracts prepared from the argD ^– mutant.This work was supported by grants from the National Institutes of Allergy and Infectious Diseases (Public Health Service No. AI-10862) and The University of Connecticut Research Foundation (to C. M. B.). J. J. R. was supported by an NDEA Predoctoral Fellowship.     

The roles of isomers of phytoene,phytofluene and ζ-carotene in carotenoid biosynthesis by a mutant strain of Scenedesmus obliquus

Considerable changes in pigment composition accur during a period of 10 h when dark-grown cultures of PG1, a -carotenic strain of Scenedesmus obliquus, are illuminated. These changes are consistent with a biosynthetic pathway in which 15-cis-phytoene is converted via 15-cis-phytofluene and 15-cis--carotene into all-trans-gz-carotene and trans-bicyclic carotenoids.The findings also support the view that the xanthophylls lutein and zeaxanthin are formed from the corresponding carotenes and are especially important in the development of a normal chloroplast structure.Non-Standard Abbreviations TLC thin-layer chromatography     

Generation of a stable non-reverting Leu− mutant of Kluyveromyces lactis by gene disruption

The LEU2 gene coding for -isopropylmalate dehydrogenase of the yeast Kluyveromyces lactis strain AWJ137 was disrupted. In the resulting Leu^– strain a 0.57 × 10³-base pairs PstI/BglII fragment of the LEU2 coding region was replaced by the TRP1 gene of Saccharomyces cerevisiae. The mutant strain was characterized by stability tests and a physical map of the disrupted region was established by restriction-enzyme analysis combined with hybridization experiments. The usefulness of the mutant strain as a recipient was shown by transformation experiments.     

α-Acetolactate overexpression from glucose in the diacetyl producer Lactococcus lactis ssp. lactis bv. diacetylactis B2103, a natural mutant lacking α-acetolactate decarboxylase

We investigated the effects of the oxygen supply rate on the activity of pyruvate metabolic pathways and their end products, the lactatedehydrogenase (LDH), pyruvateformiatelyase (PFL), pyruvatedehydrogenase (PDH) and acetolactatesynthase (ALS) pathways, in the Lactococcus lactis ssp. lactis bv. diacetylactis strain B2103/74. We found that this culture, apart from inactivated α-acetyldecarboxylase, also possesses a unique natural capacity to overexpress α-acetolactate (AL) up to 25–28 mM. Our search for similar properties among the diacetilicus bv. strains showed that this ability is quite rare. We identified a single additional strain, 7590 from the National Russian Collection of Industrial Microorganisms (NRCIM-7590), which displayed a similar capacity. However, unlike B2103/74, NRCIM-7590 has an active α-acetolactate decarboxylase and therefore can only produce acetoin. AL overexpression took place under conditions of intense aeration (K _L a ≥ 90–120 h^?1), and the composition of the medium played a decisive role in AL productivity. We found that AL overproduction is determined by a diversion of a portion of pyruvate flow from the LDH to the PDH and ALS pathways. We further found that all additional pyruvate, supplied from LDH, is utilized exclusively by the ALS pathway because of the restricted capacity of the PDH pathway. This shift in pyruvate metabolism in the B2103/74 strain, from LDH to PDH and ALS pathways, is associated with the initiation of an oxidation reaction that reduces oxygen to H₂O and sequesters NADH from the LDH pathway in the process. A specific manifestation of this reaction in B2103/74 and NRCIM-7590 cultures, which results in a profound shift of the pyruvate metabolism towards the production of α-acetolactate, is due to the function of a potent oxidative system that shifts 75–80% of NADH flow from LDH to the oxidative pathway, resulting in the regeneration of NAD⁺. The nature of this oxidative system is not known. Based on our studies, we propose that the structure of the newly discovered oxidative system is similar to a simple transmembrane electron transport chain.     

Analysis of Phaseolus–Rhizobium interactions in a subsistence farming system

Poor bean yields in the Cunha region of the Mata Atlântica ecosystem in the state of São Paulo, Brazil, are associated with low agronomic inputs, plant disease, and soil erosion. To identify sustainable farming practices that increase production and maximize biological N2 fixation (BNF), the effects of soil fertility and plant cultivar on seed yield and root nodule formation were measured under standard agronomic practices. Results from 16 sites showed that fertilizing with lime and molybdenum increased seed yields to 370% for the landrace Serro Azul. In addition to increased yields, plants grown with fertilizer had more nodules. Marked strains of Rhizobium tropici were tested under controlled environments. An indicator strain of Rhizobium containing the gusA marker gene was used. Our results verify that the indicator strain CM-255 GusA+Hup+ had a high capacity to associate with the five bean varieties tested. Fertilization with P, K, S + micronutrients and liming were essential for better nodulation by the indicator strain. Under low fertility conditions, the landrace variety Serro Azul was poorly nodulated, when associated with native strains or with the indicator strain. However, under better soil fertility conditions, nodulation of Serro Azul by the marked Rhizobium strain was increased. The commercial variety Carioca 80SH showed no increase in nodulation (nodule number).     

Evaluation of genetic diversity in a natural rosewood population (Dalbergia nigra Vell. Allemão ex Benth.) using RAPD markers

Dalbergia nigra (rosewood) is a long-lived leguminous species, which is endemic to the Brazilian Atlantic forest. Because of the high economic value of its wood, this species has been over-explored in recent years. Currently, rosewood is included in the IUCN Red List as vulnerable. We examined the genetic diversity of 87 specimens of D. nigra sampled from a continuous forest in the Veracel Reserve and Brazilwood Ecological Station, Porto Seguro, Bahia state, with random amplified polymorphic DNA markers. Grouping analyses were done using unweighted pair group method with arithmetic averages. Using the 16 most informative primers, 112 markers were obtained; 39% (44 bands) were polymorphic. A genetic similarity matrix was made based on the polymorphic bands. The dispersion graph and dendrogram analyses showed three distinct sub-populations. The degree of polymorphism was high, near that of other populations of similar species; however, it was considered low for the conservation of this species.     

A secondary effect of transformation in Rhizobium leguminosarum transgenic for Bacillus thuringiensis subspecies tenebrionisδ-endotoxin (cryIIIA) genes

 By introducing Bacillus thuringiensis subspecies tenebrionisδ-endotoxin genes (cryIIIA) into Rhizobium leguminosarum we have produced strains for the biological control of Sitona larvae. Comparisons between a transgenic and the parent strain show that transformation has induced changes not associated with the intended function of the transgene. Although growth rates in laboratory cultures are similar for both strains, the ability to compete for nodule occupancy is greater in the transgenic than in the non-transformed parent strain. This result demonstrates the importance of studying ecological and agronomic characters of transgenic micro-organisms that could have a bearing on the safety and success of their release into the environment, even if they are not thought to be connected with the transgenes introduced. Received: 20 April 1997 / Accepted: 2 June 1997     

Recombinant protein production in cultures of an Escherichia coli trp − strain

Fermentation conditions were developed in order to achieve simultaneously a high biomass concentration and high-level expression of a hybrid cI-human insulin B peptide gene. In our system, this hybrid gene is under control of the Escherichia coli trp promoter, in a trp ^– derivative strain of E. coli W3110. The dual role of tryptophan concentration on cellular growth and hybrid gene regulation was studied in 10-l batch fermentations. In the best batch conditions, a biomass concentration of 12 g dry weight/l can be obtained, and 0.53 g/l of cI-insulin B hybrid protein is produced. Tryptophan in the culture medium is consumed by the growing culture, until a level is reached that causes induction of the hybrid gene. Plasmid loss was detected, as only 62% of the cells retained the recombinant plasmid. In order to increase the hybrid protein production level, a fed-batch culture strategy was developed whereby the specific growth rate of the cells was restrained. Using the same amount of nutrients as in the batch fermentations, it was possible to increase the final biomass concentration to 20 g/l, plasmid-bearing cells in the population to 90% and recombinant hybrid protein to 1.21 g/l. Correspondence to: F. Bolivar     

Cloning of genes involved in the production of bacteriocin in Cicer–Rhizobium PR 2109a

The molecular basis of bacteriocin production by a Cicer–Rhizobium strain PR2109a was studied. The bacterial strain showed in vitro growth inhibition of non-bacteriocin producing strain of Cicer–Rhizobium PR2005b. Tn5 mutagenesis of the wild-type strain helped in the isolation of the bacteriocin-defective mutant JN365. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR1 and maintained in Escherchia coli background. Complementation analysis with the cosmid library resulted in the isolation of a cosmid clone which complemented the defective character in the mutant JN365. The size of the complementary DNA fragment was found to be 23 kb.     

Immobilization of β-glucosidase in calcium alginate gel using genipin as a new type of cross-linking reagent of natural origin

Summary -Glucosidase was immobilized in calcium alginate gel using genipin as the cross-linking reagent. The activity of this immobilized enzyme with genipin cross-linking was higher than that with other cross-linking reagents and no decrease in activity was observed even after using the gel 12 times.     

Some structural features of an insoluble α-D-glucan from a mutant strain of Leuconostoc mesenteroides NRRL B-1355

Leuconostoc mesenteroides strain NRRL B-1355 produces two soluble extracellular α-D-glucans from sucrose: alternan and dextran. An unusual mutant strain derived from NRRL B-1355 has recently been isolated which produces practically no soluble polysaccharide, but significant amounts of an insoluble D-glucan. Methylation analysis shows it contains linear (1→3) and (1→6) linkages as well as (1→2) and (1→3) branch linkages. The insoluble glucan was partially digestible by endodextranase, giving rise to a series of oligosaccharides, a high-molecular weight soluble fraction and an insoluble residue. Treatment of the soluble dextranase-limit fraction with an α(1→2) debranching enzyme led to further dextranase susceptibility. Methylation, FTIR and NMR analyses of the dextranase-treated fractions indicate a non-uniform structure with domains bearing similarities to L. mesenteroides strain NRRL B-1299 dextran and to insoluble streptococcal D-glucans. Received 05 November 1998/ Accepted in revised form 31 March 1999     

Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt −

The function of the eyespot in phototaxis of the flagellate green alga Chlamydomonas reinhardtii Dangeard was studied using quantitative reflection confocal laser scanning microscopy and photoelectric measurements. The reflective properties of the eyespot and the photoreceptor current of the C. reinhardtii eyespot mutant ey 627, mt ^– were compared with those of Chlamydomonas strains possessing a well-developed eyespot. Under growth conditions in which strongly disorganized eyespots were observed in the mutant by electron microscopy, there was a significant reduction in the reflection intensity of the eyespot and in the amplitude ratio (500440 nm) of photoreceptor currents induced by flashes of 500- and 440-nm light in non-oriented cells. Photoelectrical responses of pre-oriented cells revealed that the latter effect is caused by an altered directional sensitivity of the antenna complex, whereas the functional state of the photoreceptor pigment is not strongly affected in mutant cells. Both the reflection intensity and the amplitude ratio of photoreceptor currents increased to the level of reference strains under conditions supporting the development of a well-organized eyespot in the mutant. Furthermore, incubation of the mutant with high concentrations of all-trans-retinal (10 M), independent of whether carotenoid biosynthesis was inhibited or not, was found to increase the reflection intensity of the eyespot. An increase in the rate of photoorientation of the mutant occurred concomitant with the increase in the reflective properties of the mutant eyespot. These observations demonstrate the importance of an intact eyespot for interference reflection and absorption of phototactically active light, and thus for the directional sensitivity of the eyespot apparatus.Abbreviations HSM high-salt medium This study was supported by the Deutsche Forschungsgemeinschaft. O. A. Sineshchekov was supported by a Research Fellowship from the Alexander von Humboldt Foundation. The authors wish to thank U. Powalowski (Botanisches Institut, Universität zu Köln) for help with electron microscopy.