The Streptomyces lividans 66 chromosome contains a 1 MB deletogenic region flanked by two amplifiable regions

Genetic instability in Streptomyces species often involves large deletions sometimes accompanied by DNA amplification. Two such systems in Streptomyces lividans 66 involve the production of mutants sensitive to chloramphenicol and the production of mutants resistant to the galactose analogue 2-deoxygalactose, respectively. Overlapping cosmids were isolated that span the ca. 1 Mb region between the two amplifiable regions. The structure of the region was confirmed by restriction mapping using the rarely cutting enzymes AseI, BfrI and DraI and pulsed-field gel electrophoresis. The region contains a non-clonable gap flanked by inverted repeats; the structure is consistent with the presence of a physical gap, i.e. a linear chromosome.     

Optimization of culture parameters for extracellular protease production from a newly isolated Pseudomonas sp. using response surface and artificial neural network models

Radial basis function (RBF) artificial neural network (ANN) and response surface methodology (RSM) were used to build a predictive model of the combined effects of independent variables (pH, temperature, inoculum volume) for extracellular protease production from a newly isolated Pseudomonas sp. The optimum operating conditions obtained from the quadratic form of the RSM and ANN models were pH 7.6, temperature 38 °C, and inoculum volume of 1.5 with 58.5 U/ml of predicted protease activity within 24 h of incubation. The normalized percentage mean squared error obtained from ANN and RSM models were 0.05 and 0.1%, respectively. The results demonstrated an higher prediction accuracy of ANN compared to RSM. This superiority of ANN over other multi factorial approaches could make this estimation technique a very helpful tool for fermentation monitoring and control.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Optimization of medium composition and culture conditions for agarase production by Agarivorans albus YKW-34

Effect of medium composition and culture conditions on agarase production by Agarivorans albus YKW-34 was investigated in shake flasks. The most suitable carbon source, nitrogen source, and culture temperature were agar, yeast extract, and 25 °C, respectively, for agarase production by one-factor-at-a-time design. The nutritional components of the medium and culture conditions were analyzed by Plackett–Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had significant effects on agarase production (p < 0.05). The optimum levels of these key variables were further determined using a central composite design. The highest agarase production was obtained in the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization strategy enhanced the agarase production from 0.23 U/ml to 0.87 U/ml. The economic medium composition and culture condition as well as the dominant occupation of agarase with high activity in culture fluid enlighten the potential application of A. albus YKW-34 for the production of agarase.     

Organisation and functions of the actV A region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor

Summary Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and-ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.     

Cloning of the glucose isomerase (D-xylose isomerase) and xylulose kinase genes of Streptomyces violaceoniger

Summary Xylose utilization mutants of Streptomyces violaceoniger were isolated lacking one or both of the enzymes, glucose isomerase (xylose isomerase) and xylulose kinase. Using pUT206 as a cloning vector, complementation of the glucose isomerase negative phenotype with fragments of the S. violaceoniger chromosome permitted isolation of two recombinant plasmids, designated pUT220 and pUT221, which contained 10.6 and 10.1 kb of chromosomal DNA, respectively. Both of these plasmids complemented all three different classes of xylose negative mutants and also provoked an increase of glucose isomerase and xylulose kinase activity in the mutant and wild-type strains. Plasmid pUT220 was chosen for detailed study by subcloning experiments. The putative glucose isomerase gene was localized to a 2.1 kb segment of the 10.6 kb chromosomal DNA fragment. The putative xylulose kinase gene resides nearby. Thus both genes seem to be clustered at a single chromosomal localization. This organization appears similar to that of the xylose utilization pathway in Escherichia coli, Salmonella typhimurium and Bacillus subtilis.     

Accurate intraocular pressure prediction from applanation response data using genetic algorithm and neural networks

The fact that Goldmann applanation tonometry does not accurately account for individual corneal elastic stiffness often leads to inaccuracy in the measurement of intraocular pressure (IOP). IOP should account not only for the effect of central corneal thickness (CCT) but should also account for other corneal biomechanical factors. A computational method for accurate and reliable determination of IOP is investigated with a modified applanation tonometer in this paper. The proposed method uses a combined genetic algorithm/neural network procedure to match the clinically measured applanation force-displacement history with that obtained from a nonlinear finite element simulation of applanation. An additional advantage of the proposed method is that it also provides the ability to determine CCT and material properties of the cornea from the same applanation response data. The performance of the proposed method has been demonstrated through a parametric study and via comparison with a well known clinical case. The proposed method is also shown to be computationally efficient, which is an important practical consideration for clinical application.     

Optimization of extraction process of Glycyrrhiza glabra polysaccharides by response surface methodology

Experimental design was used to investigate the effect of three parameters (extraction time, extraction number and ratio of water to raw material) on polysaccharides yields. The ranges of the factors investigated were 3.5–4.5 h for extraction time (X₁), 4–6 for extraction number (X₂), and 25–35 for ratio of water to raw material (X₃). The statistical analysis of the experiment indicated that extraction time and ratio of water to raw material had significant effect on Glycyrrhiza glabra polysaccharides yields. The central composite design showed that polynomial regression models were in good agreement with the experimental results with the coefficients of determination of 0.924 for Glycyrrhiza glabra polysaccharides yield. The optimal condition for Glycyrrhiza glabra polysaccharides yield within the experimental range of the variables studied was at 4.3 h, 6, and 35. At this condition, the predicted yield of polysaccharides extracted was 3.6%.     

Five transfer RNA genes lacking CCA termini are clustered in the chromosome of Streptomyces rimosus

Summary The nucleotide sequence of a 1105 by Streptomyces rimosus DNA fragment containing five transfer RNA genes was determined. Two tRNA^Gln (CUG) genes, differing by 1 by in the aminoacyl stem, and three identical tRNA^Glu (CUC) genes were identified. The five tRNA genes, arranged in the order: Gln1-Glul-Glu2-Gln2-Glu3, were separated by short, nonhomologous intergenic regions. Surprisingly, none of these tRNA genes encoded the CCA 3 terminus of mature tRNAs. All five encoded tRNAs for the translation of GC rich codons, which are preferentially used in Streptomyces genes (CAG and GAG, respectively). We recently reported nucleotide sequences of two initiator tRNA genes from S. rimosus, which also do not encode the CCA end of mature tRNAs. It is therefore very likely that S. rimosus represents an example of those eubacteria in which the majority of tRNA genes do not encode the 3 terminal CCA end of mature tRNAs. Evolutionary implications of this finding remain to be elucidated.     

Effect of biodelignification of rice straw on humification and humus quality by Phanerochaete chrysosporium and Streptomyces badius

The effect of biodelignification of rice straw by two different ligninolytic organisms, Phanerochaete chrysosporium (white-rot fungus) and Streptomyces badius (actinomycetes), on humus quality was investigated during a 56-day incubation at 30 °C. Lignin degradation, the release of humic extract (HE), humic acid (HA) and fulvic acid (FA), E4/E6 ratio of HA, and humification index (HI, HA/FA) were measured during the incubation. Lignin was degraded by both organisms, but to different extents. Lignin was degraded to 41% and 31% by P. chrysosporium and S. badius, respectively. HE released by P. chrysosporium and S. badius were, respectively, 2.10 and 2.13 times larger than that in the control at the maximum values. A significant correlation between lignin degradation and humus-related parameters involving HA fraction showed that both organisms are converting lignin to humic substances.     

Optimization of ultrasonic-assisted extraction process of Poria cocos polysaccharides by response surface methodology

Polysaccharides production from Poria cocos was carried out using aqueous NaOH with the assistance of ultrasonic. Experimental design was used to investigate the effect of three parameters (extraction time, extraction concentration of NaOH, and ratio of aqueous NaOH to raw material) on polysaccharides yields. The ranges of the factors investigated were 1–3 min for extraction time (X₁), 0.5–1.0 mol/L for extraction concentration of NaOH (X₂), and 30–50 for ratio of aqueous NaOH to raw material (X₃). The statistical analysis of the experiment indicated that extraction concentration of NaOH had significant effect on P. cocos polysaccharides yields. The central composite design showed that polynomial regression models were in good agreement with the experimental results with the coefficients of determination of 0.9935 for P. cocos polysaccharides yield. The optimal condition for P. cocos polysaccharides yield within the experimental range of the variables studied was at 2.44 min, 0.789 mol/L, and 53.0. At this condition, the predicted yield of polysaccharides extracted was 82.3%.     

Improvement of trypanocidal metabolites production by Aspergillus fumigatus using neural networks

An optimization procedure using artificial neural networks was developed to determine the optimal combination of parameters, such as medium culture, initial pH, temperature and time of fermentation for maximal trypanocidal metabolites production by Aspergillus fumigatus. A data set of 81 experiments was carried out and an artificial neural network was trained to identify the optimal conditions for this process. Good correlation was obtained between the experimental and predicted values of lysis of the trypomastigote forms of Trypanosoma cruzi (r2 = 0.9990). The simulations of fermentation performance were undertaken on combinations of input variables and the highest level of activity against T. cruzi was obtained from the chloroform extract of the modified Jackson medium culture, initial pH of 6.0, incubated at 40 degrees C for 144 h. It displayed lysis of 95% of the trypomastigote forms of T. cruzi and the red blood cells remained normal.     

Cloning and expression of the SalI restriction-modification genes of Streptomyces albus G

Summary The Streptomyces albus G genes (salR and salM) for the class II restriction enzyme SalI (SalGI) and its cognate modification enzyme were cloned in Streptomyces lividans 66. Selection was initially for the salR gene. From a library of S. albus G DNA in the high copy number plasmid pIJ486 several clones of S. lividans were obtained that were resistant to phage C31 unmodified at the many SalI sites in its DNA, but were sensitive to modified phages last propagated on a restriction-deficient, modification-proficient mutant of S. albus G. SalI activity was detected in cell-free extracts of the clones, though only at levels comparable with that in S. albus G. Five different recombinant plasmids were isolated, with inserts of 5.6, 5.7, 8.9, 10 and 18.9 kb that contained a common region of 4.5 kb. These plasmids could not be digested by SalI, although the vector has four recognition sites for this enzyme, indicating that the salM gene was also cloned and expressed. Subcloning experiments in S. lividans indicated the approximate location of salR and salM, and in Escherichia coli led to detectable expression of salM but not of salR. A variety of previously isolated S. albus G mutants affected in aspects of SalI-specific restriction and modification were complemented by the cloned DNA; they included a mutant temperature-sensitive for growth apparently because of a mutation in salM. Southern blotting showed that DNA homologous to the cloned sal genes was present in Xanthomonas and Rhodococcus strains, but not detectably in Herpetosiphon strains, all of which produce SalI isoschizomers.     

A mutation of Streptomyces lividans which prevents intraplasmid recombination has no effect on chromosomal recombination

Summary A mutation (rec-46) of Streptomyces lividans, previously shown to prevent (or greatly diminish) homologous and illegitimate intraplasmid recombination, was shown to have no effect on generalised chromosomal recombination occurring in matings or in protoplast fusions, nor to affect homologous recombination between a recombinant plasmid and the host chromosome. By comparison with Escherichia coli mutants defective in various aspects of recombination, the rec-46 mutation is similar to those in recF, recJ, recO and topA.     

SSRs transferability and genetic diversity of Tunisian Festuca arundinacea and Lolium perenne

SSR primers specific to Lolium perenne generated a total of 96 alleles and 124 genotypes within Festuca arundinacea and Lolium perenne accessions. Their highly transferability (100 %) across genera was evidenced. Six alleles specific to loci H01F02, H02C11 and K01A03 and only 5/96 common alleles between both species (60, 140, 144, 190 and 192) expressed the differentiation between species. Besides, based on the Wrights fixation indices, the genetic variation within each species was attributable to differences within populations with a significant deficiency of heterozygous. The unweighted pair group method with arithmetic averaging dendrogram based on the Nei’s distances and the principal coordinate analysis based on Jaccard coefficient similarity distinguished each genus independently of the geographical origin. However, typically continuous genetic diversity and a low level of gene flow (N_m: 0.29–2.47) expressed the relatively closely relationships of both genera and suggest a possible hybridization in nature.     

A genetic map of Xenopus tropicalis

We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132 cM in length, and 4 smaller linkage groups between 7 and 40 cM. The total effective size of the map is 1658 cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75 cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000 bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.     

Induction of anterior neural fates in the ascidian Ciona intestinalis

The sensory vesicle of ascidians is thought to be homologous to the vertebrate forebrain and midbrain (Development 125 (1998) 1113). Here we report the isolation of two sensory vesicle markers in the ascidian Ciona intestinalis, which are homologs of vertebrate otx and gsx homeobox genes. By using these markers to analyze the induction of anterior neural tissue in Ciona, we find that the restriction of anterior neural fate to the progeny of the anterior animal blastomeres is due to a combination of two factors. The vegetal blastomeres show a differential inducing activity along the anterior-posterior axis, while the competence to respond to this inducing signal is markedly higher in the anterior animal blastomeres than in the posterior animal blastomeres. This differential competence to respond is also observed in response to bFGF, a candidate neural inducer in ascidians (J. Physiol. 511.2 (1998) 347) and can be detected by the gastrula stage. Our results, however, indicate that bFGF can only induce a subset of the responses of the endogenous inducer, suggesting that additional signals in the embryo are necessary to induce a fully patterned nervous system.