Factors affecting protoplast formation and regeneration by four species of Streptomyces

Four species of Streptomyces, Streptomyces canescens, S. limosus, S. griseus and S. griseolus , were used to study the effects of glycerine and gelatin on the formation and regeneration of protoplasts. For each species efficient protoplast formation with high protoplast concentrations and low levels of non-protoplast units was obtained with mycelia grown in medium without glycerine. The low regeneration frequencies of protoplasts of S. canescens and S. limosus on R2 medium were increased substantially by the addition of 1% gelatin. The use of single colonies, rather than spores, to establish mycelial cultures was found routinely to produce good protoplast preparations.     

Bacterial protoplast fusion: recombination in fused protoplasts of Streptomyces coelicolor

Summary Numerous recombinants arose when protoplasts of S. coelicolor were treated with polyethylene glycol and regenerated on non-selective solid medium. In six-factor crosses, recombination frequencies of more than 10% (up to 17%) were routinely observed. This recombination did not require either of the known sex factors, SCP1 and SCP2.The proportion of multiple crossover classes was much higher than amongst recombinants produced by conjugation between mycelia. Analysis of the spatial distribution of crossovers in double and quadruple crossover recombinants showed only a slight tendency for crossovers to occur closer together than randomly on the complete linkage group. This suggests that genomes brought together by protoplast fusion are complete, or nearly so (in conjugation, in contrast, one genome is represented by a comparatively short fragment). Individual colonies arising from fused protoplasts did not contain different parental genomes without recombinants, but recombinants often occurred without parentals. Several recombinant genotypes often occurred in the same colony, showing a segregation of some, only, of the parental alleles. Complementary genotypes, parental or recombinant, did not occur in the same colony. It is postulated that complete genomes of fused protoplasts usually become fragmented and that crossing-over, often repeated, occurs between the fragments, to generate haploid recombinants.Analysis of fusions between protoplasts of four different genotypes indicated that the average number of protoplasts fusing together was low, but nevertheless appreciable numbers of fusions involved three or four genomes. Crossing-over between them produced recombinants inheriting markers from three or four parents.The generation of nearly random populations of recombinants between two or more parent strains by protoplast fusion under the conditions described appears to have simple applications in industrial and academic strain construction.     

Factors affecting protoplast electrofusion efficiency

The electrofusion efficiency of protoplasts isolated from a carrot (Daucus carota) suspension culture was increased by treatment with 0.1 mg/ml lysolecithin, 2.5% dimethylsulfoxide (DMSO), or 0.5 mM Ca²⁺. The lysolecithin and DMSO treatments substantially increased protoplast lysis, whereas calcium treatment did not. The enzymes used for protoplast isolation were also found to have a dramatic effect on the efficiency of fusion. A mixture of Cellulysin and Driselase led to a two-fold enhancement of fusion as compared with Driselase alone. The stimulation by Cellulysin appears to be due to enzymatic modification of the cell surface. However, comparison of the time course for wall digestion with the development of susceptibility to electrofusion suggests that the effect of Cellulysin is not simply due to removal of the cell wall. Brief treatment of the cells with pronase or proteinase K also doubled the efficiency of fusion. Taken together, these results indicate that electrofusion efficiency can be enhanced by the method used for protoplast isolation; they also suggest that modification of membrane/cell-surface proteins during protoplast isolation may be particularly important in determining electrofusion efficiencies.Abbreviations a.c. alternating current - d.c. direct current - DMSO dimethylsulfoxide - NAA naphthaleneacetic acid - PEG polyethylene glycol     

Glutamate synthesis in Streptomyces coelicolor.

Both glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) are involved in glutamate synthesis in Streptomyces coelicolor. The highest levels of GDH were seen in extracts of cells grown with high levels of ammonium as the nitrogen source. GOGAT activity was reduced two- to threefold in extracts of cells grown with good sources of glutamate. S. coelicolor mutants deficient in GOGAT (Glt-) required glutamate for growth with L-alanine, asparagine, arginine, or histidine as the nitrogen source but grew like wild-type cells when ammonium, glutamine, or aspartate was the nitrogen source. The glt mutations were tightly linked to hisA1. Mutants deficient in both GOGAT and GDH (Gdh-) required glutamate for growth in all media. The gdh-5 mutation was mapped to the left region of the S. coelicolor chromosomal map, between proA1 and uraA1.     

The phosphotransferase system of Streptomyces coelicolor.

We have investigated the crr gene of Streptomyces coelicolor that encodes a homologue of enzyme IIAGlucose of Escherichia coli, which, as a component of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays a key role in carbon regulation by triggering glucose transport, carbon catabolite repression, and inducer exclusion. As in E. coli, the crr gene of S. coelicolor is genetically associated with the ptsI gene that encodes the general phosphotransferase enzyme I. The gene product IIACrr was overproduced, purified, and polyclonal antibodies were obtained. Western blot analysis revealed that IIACrr is expressed in vivo. The functionality of IIACrr was demonstrated by phosphoenolpyruvate-dependent phosphorylation via enzyme I and the histidine-containing phosphoryl carrier protein HPr. Phosphorylation was abolished when His72, which corresponds to the catalytic histidine of E. coli IIAGlucose, was mutated. The capacity of IIACrr to operate in sugar transport was shown by complementation of the E. coli glucose-PTS. The striking functional resemblance between IIACrr and IIAGlucose was further demonstrated by its ability to confer inducer exclusion of maltose to E. coli. A specific interaction of IIACrr with the maltose permease subunit MalK from Salmonella typhimurium was uncovered by surface plasmon resonance. These data suggest that this IIAGlucose-like protein may be involved in carbon metabolism in S. coelicolor.     

Peptidases affecting recombinant protein production by Streptomyces lividans

The influence of peptidases on human interleukin-3 (rhIL-3) production by a recombinant Streptomyces lividans strain was investigated. The bacterium produced several general peptidases and tripeptidyl peptidases compromising the authenticity of rhIL-3. The level of peptidases depended on growth morphology. Growing S. lividans as compact pellets successfully reduced peptidase activity. Maximum general peptidase activity in pellet culture was delayed after maximum rhIL-3 concentration was achieved. The activity of the tripeptidyl peptidase was product (rhIL-3) associated.     

Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization

The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s?1 at 30 °C. Since adenine is deaminated ～103 slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism.     

Genetic analysis of intraspecies recombinant formation by protoplast fusion with three species of Streptomyces

Abstract Protoplast fusion was shown to produce high frequencies of recombinant progeny in intraspecies crosses with auxotrophic mutants of Streptomyces canescens, Streptomyces griseus and Streptomyces limosus . The fused protoplasts were regenerated on non-selective media and the progeny spores subsequently analysed on selective media to allow detection of all possible genotypes. Prototrophic recombinants arose with frequencies of between 1% and 8%. All 4 possible genotypes were recovered in a series of 2-factor crosses and 6 of the 8 possible genotypes were detected in a 3-factor cross. In spite of attempts to equalise the ratios of parental protoplasts in the fusion mixture, there were noticeable deviations from unity in the ratios of parental genotypes in the progeny.