Screening of Actinomycetes from mangrove ecosystem for L-asparaginase activity and optimization by response surface methodology

Marine actinomycetes were isolated from sediment samples collected from Pitchavaram mangrove ecosystem situated along the southeast coast of India. Maximum actinomycete population was noted in rhizosphere region. About 38% of the isolates produced L-asparaginase. One potential strain KUA106 produced higher level of enzyme using tryptone glucose yeast extract medium. Based on the studied phenotypic characteristics, strain KUA106 was identified as Streptomyces parvulus KUA106. The optimization method that combines the Plackett-Burman design, a factorial design and the response surface method, which were used to optimize the medium for the production of L-asparaginase by Streptomycetes parvulus. Four medium factors were screened from eleven medium factors by Plackett-Burman design experiments and subsequent optimization process to find out the optimum values of the selected parameters using central composite design was performed. Asparagine, tryptone, d) extrose and NaCl components were found to be the best medium for the L-asparaginase production. The combined optimization method described here is the effective method for screening medium factors as well as determining their optimum level for the production of L-asparaginase by Streptomycetes parvulus KUAP106.     

Effect of Streptococcus parvulus and Peptostreptococcus magnus on cytotoxin levels of Clostridium difficile in anaerobic continuous flow culture

An anaerobic continuous flow (CF) culture method was used in order to study the effect of Peptostreptococcus magnus and Streptococcus parvulus, anaerobic gram-positive cocci which are members of intestinal bacterial flora, on growth and cytotoxin-activity of Clostridium difficile. The growth- and the cytotoxin activity-patterns of C. difficile in an established CF culture of P. magnus were similar to those of C. difficile alone. On the other hand, in the mixed culture system of C. difficile and S. parvulus, the cytotoxin levels were significantly lower as compared with C. difficile alone in spite of the fact that no differences existed between growth of C. difficile in mixed and single culture systems. The culture filtrate of P. magnus did not influence the growth and cytotoxin production of C. difficile, nor did that of S. parvulus have any effect on growth of C. difficile in static culture. The cytotoxin activity of C. difficile was, however, suppressed by the culture filtrate of S. parvulus. Furthermore, when P. magnus or S. parvulus was statically cultured in a medium containing cytotoxic culture filtrate of C. difficile, the toxin in the medium was not inactivated.     

High-frequency fusion of Streptomyces parvulus or Streptomyces antibioticus protoplasts induced by polyethylene glycol.

Conditions were established for the regeneration of protoplasts of Streptomyces parvulus and Streptomyces antibioticus to the mycelial form. Regeneration was accomplished with a hypertonic medium that contained sucrose, CaCl2, MgCl2, and low levels of phosphate. High-frequency fusion of protoplasts derived from auxotrophic strains of S. parvulus or S. antibioticus was induced by polyethylene glycol 4,000 (42%, wt/vol). The frequency of genetic transfer by the fusogenic procedure varied with the auxotrophic strains examined. Fusion with auxotrophic strains of S. parvulus resulted in the formation of true prototrophic recombinants. Similar studies with S. antibioticus revealed that both stable prototrophic recombinants and heterokaryons were formed.     

Metabolic regulation in Streptomyces parvulus during actinomycin D synthesis, studied with 13C- and 15N-labeled precursors by 13C and 15N nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry.

Recent studies have suggested that the onset of synthesis of actinomycin D in Streptomyces parvulus is due to a release from L-glutamate catabolic repression. In the present investigation we showed that S. parvulus has the capacity to maintain high levels of intracellular glutamate during the synthesis of actinomycin D. The results seem contradictory, since actinomycin D synthesis cannot start before a release from L-glutamate catabolic repression, but a relatively high intracellular pool of glutamate is needed for the synthesis of actinomycin D. Utilizing different labeled precursors, D-[U-13C]fructose and 13C- and 15N-labeled L-glutamate, and nuclear magnetic resonance techniques, we showed that carbon atoms of an intracellular glutamate pool of S. parvulus were not derived biosynthetically from the culture medium glutamate source but rather from D-fructose catabolism. A new intracellular pyrimidine derivative whose nitrogen and carbon skeletons were derived from exogenous L-glutamate was obtained as the main glutamate metabolite. Another new pyrimidine derivative that had a significantly reduced intracellular mobility and that was derived from D-fructose catabolism was identified in the cell extracts of S. parvulus during actinomycin D synthesis. These pyrimidine derivatives may serve as a nitrogen store for actinomycin D synthesis. In the present study, the N-trimethyl group of a choline derivative was observed by 13C nuclear magnetic resonance spectroscopy in growing S. parvulus cells. The choline group, as well as the N-methyl groups of sarcosine, N-methyl-valine, and the methyl groups of an actinomycin D chromophore, arose from D-fructose catabolism. The 13C enrichments found in the peptide moieties of actinomycin D were in accordance with a mechanism of actinomycin D synthesis from L-glutamate and D-fructose.     

13C nuclear magnetic resonance and gas chromatography-mass spectrometry studies of carbon metabolism in the actinomycin D producer Streptomyces parvulus by use of 13C-labeled precursors.

Fructose and glutamate metabolism was monitored in cell suspensions of streptomyces parvulus by 13C nuclear magnetic resonance. The experiments were performed for cells grown with various 13C sources in a growth medium containing D-[U-13C]fructose, L-[13C]glutamate, or L-[U-13C]aspartate and with nonlabeled precursors to compare intracellular pools in S. parvulus cells at different periods of the cell life cycle. The transport of fructose into the cells was biphasic in nature; during rapid transport, mannitol, fructose, and glucose 6-phosphate were accumulated intracellularly, whereas during the passive diffusion of fructose, the intracellular carbohydrate pool comprised mainly trehalose (1,1'-alpha-alpha-D-glucose). The regulation of fructokinase activity by the intracellular intermediates may play an important role in fructose catabolism in S. parvulus. Transaldolase activity in S. parvulus was determined from the 13C nuclear magnetic resonance labeling pattern of trehalose carbons obtained from cells grown in medium containing either L-[U-13C]aspartate or L-[U-13C]glutamate. Only carbons 4, 5, and 6 of the disaccharide were labeled. Isotopomer analysis of the trehalose carbons led us to conclude that the flux through the reverse glycolytic pathway, condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate, makes at best a minor contribution to the 13C-labeled glucose units observed in trehalose. The pentose pathway and transaldolase activity can explain the labeling pattern of 4,5,6-13C3 of trehalose. Moreover, the transfer of the 13C label of L-[U-13C]aspartate into the different isotopomers of trehalose C4, C5, and C6 by the transaldolase activity allowed us to calculate the relative fluxes from oxaloacetate via gluconeogenesis and through the tricarboxylic acid cycle. The ratio of the two fluxes is approximately 1. However, the main carbon source for trehalose synthesis in S. parvulus is fructose and not glutamate or aspartate. The 13C enrichment and isotopomer population, measured by nuclear magnetic resonance and gas chromatography-mass spectrometry, of the actinomycin D peptide ring enabled us to specify the origins of the five amino acids of actinomycin D. Threonine and proline exhibited isotopomer populations similar to that of the extracellular L-[13C]glutamate, indicating that protein catabolism is the origin of their 13C label, whereas the isotopomer populations of sarcosine and N-methylvaline were similar to those of the new intracellular pool of S. parvulus that originated from D-[U-13C]fructose during the production of actinomycin D.     

Protoplast fusion permits high-frequency transfer of a Streptomyces determinant which mediates actinomycin synthesis.

Prototrophic recombinants and heterocaryotic colonies developed at high frequency when protoplasts of nutritionally complementary actinomycin-producing and nonproducing strains of Streptomyces antibioticus were fused in the presence of polyethylene glycol and plated on minimal regeneration medium. Of the spores obtained from aerial hyphae of a single heterocaryotic colony, 99% carried the act+ character regardless of whether the nutritional markers of the spore were derived from the act+ or the act parent. Similarly, a high-frequency transfer (68% in S. antibioticus, 48% in Streptomyces parvulus) of act+ determinant(s) to act was achieved by protoplast fusion. Protoplasts of a doubly auxotrophic act strain of S. parvulus were efficiently transformed in the presence of polyethylene glycol with respect to the auxotrophic markers by DNA of an act+ auxotrophic strain with complementary nutritional requirements. The transformation frequency of the nutritional (chromosomal) markers was 17%. In contrast, the transformation frequency for actinomycin synthesis was less than 1%.     

Bacteria Responsible for the Retting of Brazilian Flax

Twenty-two species of bacteria were isolated from Linum usitatissimum stored for retting. Achromobacter parvulus, Clostridium beijerinckii, C. saprogenes, C. saccharoacetoperbutylicum, C. perenne, and Pseudomonas aeruginosa and its achromogenic variety are retting agents. The last species mentioned performs the retting in only 72 hr. This is the first time A. parvulus has been shown to be a retting agent.     

Action of polycarbon compounds on the oxidation of methanol and other Cl compounds by methylotrophic bacteria

Cells of the facultative methylotrophic bacteria Achromobacter parvulus 1T, Pseudomonas sp AM1, Ps. methylica 2K, Ps. methylica 20T, Ps. oleovorans 52Z, Ps. fluorescens 45K exhibit the ability to oxidize methanol only on the medium with methanol. At the growth of various methylotrophic bacteria on the media containing both methanol and succinate their ability to methanol oxidation is lost, and oxidation of formaldehyde and formate occur at a low rate. Glucose, citrate, acetate and glycerol produce no repressive effect on the ability of the cells to oxidize methanol and other C1-compounds.     

苏铁属花粉萌发及保存条件研究

以不同浓度梯度的蔗糖与硼酸组合在不同pH条件下用悬浮培养法测定德保苏铁、叉叶苏铁、元江苏铁和越南篦齿苏铁花粉的活力;将元江苏铁和越南篦齿苏铁花粉分别保存在不同低温、不同湿度的环境中,研究温度和湿度对保存花粉的影响。结果表明:(1)最适合苏铁属植物花粉萌发的培养液配方为蔗糖(1%～2.5%)+硼酸(100～500 mg/L),pH6.0～7.0;(2)在室温下,将苏铁花粉密封保存在有干燥剂的容器中,可存活30 d以上;(3)在0℃条件下,不加干燥剂,花粉可保存4个月以上;(4)用液氮保存后的越南篦齿苏铁花粉进行人工授粉,结实率高达90.3%,与用新鲜花粉人工授粉的结实率无明显差异;(5)将花粉含水率降低到15.5%～13.2%后,能在液氮中进行长期保存,表明花粉液氮保存可以作为苏铁花粉长期和超长期保存的方法。     

Purification and characterization of tryptophan dioxygenase from Streptomyces parvulus

Tryptophan dioxygenase, derived from Streptomyces parvulus, was purified to near homogeneity and shown to have a native Mr of 88,000. Kinetic parameters of the enzyme were determined and evidence suggesting that it is a hemoprotein was obtained. Tryptophan dioxygenase has a high specificity toward L-tryptophan with an apparent Km of 0.3 mM. L-3-Hydroxykynurenine was a competitive inhibitor with respect to L-tryptophan with a Ki of 0.16 mM. In vitro, the enzyme displayed little activity in the absence of a reducing agent; ascorbate, at 50 mM, was the preferred reductant providing almost a 50-fold increase in enzyme activity. The regulation of tryptophan dioxygenase synthesis and activity is described. The expression of the enzyme is correlated with the biosynthesis of actinomycin D in S. parvulus. These results support the hypothesis that tryptophan dioxygenase functions as the first enzyme in the sequence converting L-tryptophan to the chromophore of this antibiotic.     

Cloning, sequence analysis and expression of the F1F0-ATPase beta-subunit from wine lactic acid bacteria

The nucleotide sequences of the genes encoding the F1F0-ATPase beta-subunit from Oenococcus oeni, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Lactobacillus brevis and Lactobacillus hilgardii were determined. Their deduced amino acid sequences showed homology values of 79-98%. Data from the alignment and ATPase tree indicated that O. oeni and L. mesenteroides subsp. mesenteroides formed a group well-separated from P. damnosus and P. parvulus and from the group comprises L. brevis and L. hilgardii. The N-terminus of the F1F0-ATPase beta-subunit of O. oeni contains a stretch of additional 38 amino acid residues. The catalytic site of the ATPase beta-subunit of the investigated strains is characterized by the two conserved motifs GGAGVGKT and GERTRE. The amplified atpD coding sequences were inserted into the pCRT7/CT-TOPO vector using TA-cloning strategy and transformed in Escherichia coli. SDS-PAGE and Western blot analyses confirmed that O. oeni has an ATPase beta-subunit protein which is larger in size than the corresponding molecules from the investigated strains.     

Occurrence of the caryophyllid cestode Lytocestus parvulus Furtado, 1963 in Clarias batrachus (L.) in a tropical environment, Kedah, Malaysia

Clarias batrachus (L.) and C. macrocephalus Gunther were collected monthly between October 1982 and September 1984 from Pendang, Kedah, Malaysia. The caryophyllid cestode Lytocestus parvulus Furtado, 1963 was found in 37.7% of C. batrachus examined, with a mean intensity of 11.3. Infection of the cestode in C. macrocephalus was minimal, so detailed study was not attempted. In C. batrachus , no seasonal cycles of prevalence, mean intensity or maturation of the cestode were detected. The prevalence and mean intensity of L. parvulus decreased with increasing size of the fish. The first segment of the intestine was the preferred site where larger numbers of the cestodes and the greatest numbers of mature parasites were found. The number and maturation decreased towards the posterior end of the intestine.     

Respiratory resistance of methylotrophic bacteria to formate and cyanide

Whole cells and cell-free preparations of the methylotrophic bacteria, Pseudomonas sp. AM 1 and Achromobacter parvulus, can oxidize formate at tis concentration in the reaction medium up to 1 M. The respiration of whole cells is registered at a concentration of formate greater than 10(-2) M, while that of cell-free extracts at a formate concentration greater than 5 X 10(-5) M. This seems to be due to the presence of a permeability barrier in cells for formate. The oxidation of reduced TMPD and exogenous cytochrome c by the membrane preparations of the two bacteria is inhibited by formate and cyanide; Ki50% = 2.5 X 10(-2) and 10(-6) M, respectively. The oxidation of NADH by the membrane preparations of the bacteria is not inhibited by 1 M formate and 5 X 10(-4) M cyanide but is inhibited by formaldehyde with Ki50% = 3 X 10(-2) M. Formaldehyde has no effect on the oxidation of reduced TMPD and cytochrome c at concentrations greater than 2 X 10(-1) M. These data indicate that respiration of the studied methylotrophic bacteria in the presence of high formate concentrations should be attributed in the presence of a branched electron transport chain in them; one branch of the chain is resistant to formate and cyanide, but is sensitive to formaldehyde.     

Overseeding endophytic perennial ryegrass into stands of Kentucky bluegrass to manage bluegrass billbug (Coleoptera: Curculionidae)

Studies were conducted to determine the utility of overseeding endophyte enhanced varieties of perennial ryegrass, Lolium perenne L., into existing stands of Kentucky bluegrass, Poa pratensis L., to manage bluegrass billbug, Sphenophorus parvulus Gyllenhal, larval populations and their damage. Two commercial varieties of endophyte enhanced perennial ryegrass (Repell II and Triple Play) were overseeded at two different rates in the field. S. parvulus larval populations, their damage, and turfgrass composition were monitored over the course of 3 yr. Significantly fewer larvae were found in plots overseeded with Repell II, whereas Triple Play had no significant effect on larval population density. Endophyte infection was confirmed in approximately 58% of all Repell II shoots, whereas only approximately 31% of Triple Play shoots were infected. Regression analysis, using a segmented model, demonstrated that larval populations declined as the proportion of endophyte-infected perennial ryegrass increased. However, no additional reductions in larval population density were achieved in stands containing >40% endophyte-infected perennial ryegrass. Visual damage varied with cultivar, seeding rate, and year, but generally decreased as the proportion of endophyte-infected perennial ryegrass increased. Although seeding rate had an initial effect on turfgrass composition, these differences were not detected after the first year of the study. Results indicate overseeding existing stands of Kentucky bluegrass with endophytic varieties of perennial ryegrass can reduce S. parvulus larval populations and their damage. However, plant genetic characteristics (i.e., different cultivars) may be overriding determinants of many aspects of plant performance including establishment, tolerance to insect feeding, and expression of endophyte-mediated resistance.     

Bacterial formate dehydrogenase. Substrate specificity and kinetic mechanism of S-formyl glutathione oxidation

The substrate specificity of NAD-dependent formate dehydrogenase from the methylotrophic bacterium Achromobacter parvulus T1 was studied. The kinetic mechanism of S-formyl glutathione oxidation was determined. The initial velocity studies and inhibition analysis were carried out. It was shown that the kinetic mechanism for the enzyme with S-formyl glutathione as a substrate is similar to that with formate and is rapid-equilibrium random. Using independent methods, it was found that formate dehydrogenase forms a binary complex with S-formyl glutathione (Kd = 2.5 mM).     

Overwintering,soil distribution and phenology of Childers canegrub,Antitrogus parvulus (Coleoptera: Scarabaeidae) in Queensland sugarcane

In this study, the question of whether Childers canegrub, Antitrogus parvulus (Britton) overwinters in the subsoil was addressed. Irrigated fields of sugarcane were sampled during a 2-year period near Bundaberg in southern Queensland. Antitrogus parvulus overwintered as second and third instars at each of three sites. During autumn and winter third instars of different allochronic (separated in age by 12 months) populations occurred together and could not be readily separated. Field-collected third instars were reared on ryegrass and separated into two age groups based on the date of pupation. Third instars in the first year of their life cycle (young third instars) remained at shallow depth (100-200 mm) and did not overwinter in the subsoil as once thought. Minimum temperatures during winter were 13-16 degrees C and did not prevent young third instars from feeding and gaining weight. Third instars in their second and final year moved downwards from late summer and pupated in the subsoil at 293-425 mm in spring. General phenology was as previously reported with first instar larvae occurring from January until April, second instars from January until November and third instar larvae throughout the year. Prepupae and pupae were found between October and December and adults occurred in soil during November and January. Batches of eggs occurred at a mean depth of 350 mm. First and second instars occurred predominantly at relatively shallow (100-200 mm) depths in the soil profile. All stages tended to be most common under rows of sugarcane rather than in the interrow.     

Heterologous production of polyketides by modular type I polyketide synthases in Escherichia coli

Heterologous production of polyketide compounds, an important class of natural products with complex chemical structures, was first demonstrated with Streptomyces parvulus in 1984. Although Streptomyces strains are good first options for heterologous polyketide biosynthesis, their slow growth kinetics prompt other hosts to also be considered. Escherichia coli provides key elements of an ideal host in terms of the growth rate, culture conditions, and available recombinant DNA tools. Here we review the current status and potential for metabolic engineering of polyketides in E. coli.     

Heterologous expression of a position 2-substituted (1-->3)-beta-D-glucan in Lactococcus lactis

Exopolysaccharides play an important role in the rheology and texture of fermented foods, and among these beta-glucans have immunomodulating properties. We show that the overproduction of the Pediococcus parvulus GTF glycosyltransferase in an uncapsulated Lactococcus lactis strain results in synthesis and secretion (300 mg liter(-1)) of a position 2-substituted (1-->3)-beta-D-glucan that has potential use as a food additive.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil.

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.