A rapid and efficient method of lysis of Listeria and other gram-positive bacteria using mutanolysin.

A rapid and simple procedure is described for cell lysis for preparation of nucleic acids and intact ribosomal RNA from Gram-positive bacteria. Commercial mutanolysin (purified from Streptomyces globisporus) was used for inducing lysis. Listeria, Lactobacillus and Lactococcus strains were very sensitive to mutanolysin when compared to lysozyme. Susceptibility to mutanolysin was improved by a preliminary treatment with acetone, and sodium dodecyl sulfate reduced the efficiency of lysis when used together with mutanolysin. The procedure was also effective for recovering plasmids from these bacteria.     

Use of enzyme preparation from Streptomyces recifensis subsp. lyticus for isolation of membrane substances from staphylococcal cells

A procedure for isolating staphylococcal membranes including preprocessing of the cells with 0.1 M solution of cysteine hydrochloride and subsequent differential centrifugation was developed. The procedure is based on enzymatic lysis with an enzyme preparation from Streptomyces recifensis subsp. lyticus 2435. The membrane preparations had oxidase and dehydrogenase activity and were characterized by a high specific activity of the membrane-bound ATPase. Determination of the cytochrome differential spectra revealed the presence of cytochromes a, b and o in the membrane preparations.     

Use of a lysoenzyme preparation from Streptomyces recifensis subsp. lyticus 2435 for isolating DNA from staphylococcal cells

It was shown that the preparation 2435 from Streptomyces recifensis subsp. lyticus, including a complex of bacteriolytic and concomitant enzymes provided lysis of thick staphylococcal suspensions within 15 to 20 minutes under optimal conditions after preliminary treatment of the cells with 0.1 M cystein-HCl. A procedure was developed for isolating DNA from the cells of staphylococci and other microorganisms based on enzymatic lysis. In terms of major physicochemical properties, the preparations of DNA were not inferior to the preparations of DNA isolated by the classical Marmur technique with Kirbi's deproteinization and had transforming activity. The developed procedure for isolation of DNA with using the lysoenzyme preparation widened the possibilities of investigating the genetics of staphylococci and other microorganisms.     

Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli

Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids.     

Genetic determinants of active antibiotic-producing soil streptomycetes.

Plasmids or covalently closed circular (CCC)-DNA molecules are abundant in the genus Streptomyces, and have been suggested to be involved in the genetic control of the production of many antibiotics in these organisms. In this study, 21 active antibiotic-producing Streptomyces isolates were screened for their plasmid content by an alkaline lysis method which revealed the presence of a small plasmid DNA in the positive control Streptomyces lividans ATCC 35287, containing pIJ702 plasmid (5.65 kb in size). However, no low molecular weight plasmids were observed in the tested antibiotic-producing Streptomyces strains suggesting that antibiotic production in these strains is likely chromosomally encoded DNA. Treatment of 2 Streptomyces strains with 10 mM ethidium bromide (EB) resulted in the failure to produce aerial mycelia and antibiotic activity.     

Detection and quantification of Streptomyces violaceolatus plasmid DNA in soil

Two methods for the direct extraction of DNA from soil were investigated using soil inoculated with Streptomyces violaceolatus ISP5438 harbouring the multicopy plasmid pIJ673. Detection limits for plasmid DNA were determined by Southern blot technique. An SDS/heat lysis method gave approximately two orders of magnitude less sensitivity than lysis and extraction by bead-beating soil inoculated with spores. The use of these two methods allowed differentiation between spore- and mycelial-borne DNA. This was due to the resistance of the spores to lysis when subjected to SDS/heat lysis.     

Purification and characterization of a Streptomyces albus endo-N-acetylmuramidase lytic for group A and other beta haemolytic streptococci.

The purification and characterization of the streptolytic exo-enzyme from the Maxted-McCarty strain of Streptomyces albus is described. This enzyme was shown to be an endo-N-acetylmuramidase with a molecular weight of 10 to 12,000 and optimal activity at pH 8 and 45 degrees C. The enzyme is lytic for streptococci of various groups, Micrococcus lysodeikticus, Staphylococcus aureus, as well as Escherichia coli. It closely resembles the F1 endo-N-acetylmuramidase described by Ghuysen et al. (1966) except for small differences in the products of lysis of streptococcal cell walls and the resistance of Escherichia coli to lysis by the F1 enzyme. Lysates of group A and A variant streptococcal cell walls prepared with purified Streptomyces albus muramidase contained serologically active M protein and C carbohydrate-peptidoglycan complexes. The chemical and immunological characteristics of these enzymmatic products of streptococcal cell walls are reported and their utility as immunologic reagents is described.     

Usefulness of strb1 and 16S rDNA-targeted PCR for detection of Streptomyces spp. in environmental samples

In this study, we revealed rapid detection of streptomycin-producing Streptomyces spp. by extraction of total soil DNA from 14 soil samples using a modified lysis method followed by PCR amplification ofa genus-specific sequence in the Streptomyces' 16S rDNA gene. DNA band of the expected size (438 bp) was seen with all the samples. Additionally, specific amplification of the streptomycin-coding gene (strb1) directly from soil revealed the presence of a single DNA band of 940 bp. These results indicate that PCR-amplification of Streptomyces specific genes could be used for direct detection of streptomycin-producing Streptomyces species from soil.     

Extracellular sugar phosphates are assimilated by Streptomyces in a PhoP-dependent manner

Filamentous microorganisms of the bacterial genus Streptomyces have a complex life cycle that includes physiological and morphological differentiations. It is now fairly well accepted that lysis of Streptomyces vegetative mycelium induced by programmed cell death (PCD) provides the required nutritive sources for the bacterium to erect spore-forming aerial hyphae. However, little is known regarding cellular compounds released during PCD and the contribution of these molecules to the feeding of surviving cells in order to allow them to reach the late stages of the developmental program. In this work we assessed the effect of extracellular sugar phosphates (that are likely to be released in the environment upon cell lysis) on the differentiation processes. We demonstrated that the supply of phosphorylated sugars, under inorganic phosphate limitation, delays the occurrence of the second round of PCD, blocks streptomycetes life cycle at the vegetative state and inhibits antibiotic production. The mechanism by which sugar phosphates affect development was shown to involve genes of the Pho regulon that are under the positive control of the two component system PhoR/PhoP. Indeed, the inactivation of the response regulator phoP of Streptomyces lividans prevented the 'sugar phosphate effect' whereas the S. lividans ppk (polyphosphate kinase) deletion mutant, known to overexpress the Pho regulon, presented an enhanced response to phosphorylated sugars.     

Induction of programmed lysis in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> culture by the inhibitors of eukaryotic type serine/threonine protein kinases

Programmed death (PD) of the mycelium of Streptomyces lividans, namely, its delayed lysis in response to treatment with indolylmaleimide derivatives, which inhibit actinobacterial serine/threonine protein kinases (STPK), is described. Delayed lysis of mycelial cell was accompanied by DNA damage similar to PD in differentiating S. lividans mycelium. Two-dimensional electrophoresis and mass spectrometry were used to identify proteins up-regulated by a PD-inducing STPK inhibitor. Most of these proteins are known to be implicated in responses to various stress stimuli. Thus, our model of delayed cell lysis of actinobacteria upon STPK inhibition may serve for unveiling the molecular mechanisms of bacterial PD and for antimicrobial drug design.     

Depolarization of the membrane potential by beta-lactams as a signal to induce autolysis.

The effect of beta-lactam antibiotics that are known to inhibit cell wall biosynthesis and induce cell wall autolysis on the electrophysiological state of the plasma membrane in Streptomyces griseus was studied. Addition of various beta-lactam antibiotics induced a dose- and growth-stage-dependent depolarization of the membrane potential of Streptomyces griseus. The hydrolyzed biologically inactive derivative penicilloic acid had no depolarizing effect on the membrane potential. The ionophore gramicidin D, while depolarizing the membrane potential, also induced a dose-dependent increase in cell wall lysis. These observations suggest that alteration of the transmembrane potential could be an important signal in triggering cell wall autolysis of S. griseus.     

The properties of cell wall hydrolysates of Streptococcus group A]

Products obtained from lysis in the cell wall of group A streptococcus have been studied in different growth phases: at the end of the exponential phase and in the stationary one. Endo-beta-N-acetylmuramidase extracted from the culture liquid of Streptomyces levoris 96 has been used for lysis of streptococcus. It is stated that streptococcus cell walls isolated at different growth stages differ in the protein and polysaccharide content. High content of protein in the cell wall of a young culture makes lower the initial rate of the walls' hydrolysis by endo-beta-N-acetylmuramidase. However, with the enzyme penetration into peptidoglycan the rate of hydrolysis of cell walls gets higher and after four-hour incubation the lysis degree of walls of the 16- and 8-hour cultures reaches the equal value (63%). Studies in the protein composition of lysates of the streptococcus cell walls have shown that they contain at least 12 proteins most of which are acid and neutral ones.     

Cloning of xylanase gene of Streptomyces flavogriseus in Escherichia coli and bacteriophage lambda-induced lysis for the release of cloned enzyme.

The xylanase gene of Streptomyces flavogriseus was cloned in pUC8 plasmid and expressed in Escherichia coli lysogenic for lambda cI857. lambda-Induced lysis of E. coli at 42 degrees C allowed efficient release of cloned enzyme activity in extracellular environment. The xylanase gene was located in the 0.8-kb HindIII fragment and coded for 18,000 Mr xylanase.     

Mutant strains ofStreptomyces cinnamonensis protoplasts

Mycelium of Streptomyces cinnamonensis mutant strains cultivated in a synthetic medium with glycine produced protoplasts after lysis of cell walls with lysozyme. The protoplast yield was up to 95%. The protoplasts could revert and mycelial forms were thus regenerated. In a sucrose-containing medium the protoplasts stored at 4 degrees C were stable for 2 d.     

Properties of a bacteriocin-like substance produced byStreptomyces virginiae

Streptomyces virginiae produces both spontaneously and after irradiation a substance of polypeptide nature which kills streptomycetes without lysis. In its increased synthesis after U.V.-irradiation, its narrow spectrum of action restricted to the streptomycetes, and its specific adsorption on living sensitive cells, this inhibitor resembles the bacteriocins.     

Method for isolating cyanobacteria-lysing streptomycetes from soil

A new technique is described for the isolation and enumeration of cyanobacteria-lysing Streptomyces spp from soil or water. Two cyanobacteria, Anabaena cylindrica (ACTT 27899) and Tolypothrix tenuis (ATCC 27914) were used as the test organisms. Ten-day-old cyanobacterial cultures were vacuum-filtered to form a lawn on a 7 cm Whatman No. 1 filter paper which was then supported on Allen's agar. When the lawn was inoculated with dilutions of a heavy clay prairie soil and incubated at 27 PT 1dEC under constant illumination, white or grey colonies of streptomycetes grew. These colonies were surrounded by zones where a yellowing and lysis of the algal cells occurred. Streptomyces achromogenes was identified as a major lytic species within a population of 5 times 10₃ plaque-producing streptomycetes/g (dry weight) soil.     

Transformation of Arthrobacter and studies on the transcription of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli.

We report the development of a plasmid-mediated transformation system for Arthrobacter sp. NRRLB3381, using the Streptomyces cloning vector pIJ702. Our procedure gives a transformation frequency of 10(3)/micrograms of plasmid DNA. In addition we have explored the expression of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli, and shown that the ermA promoter is recognized in S. lividans not E. coli. The relationship between Arthrobacter, Streptomyces and E. coli promoters is discussed.     

Isolation of high quality RNA from Streptomyces

Several molecular techniques require high quality RNA, completely free of DNA. Standard methods to isolate total RNA from Streptomyces spp. are based on the application of a 'Modified Kirby Mix' [Practical Streptomyces Genetics. The John Innes Foundation, Norwich, pp. 613]. Here we present an alternative procedure using Triton X-100 and EDTA for the isolation of total RNA from Streptomyces.     

High-throughput screening for Streptomyces antibiotic biosynthesis activators

A genomic cosmid library of Streptomyces clavuligerus was constructed and transferred efficiently by conjugation to Streptomyces lividans, and 12 distinct groups of overlapping cosmid clones that activated the silent actinorhodin biosynthesis gene cluster were identified. This generally applicable high-throughput screening procedure greatly facilitates the identification of antibiotic biosynthesis activators.     

Chitinase production by Streptomyces viridificans: its potential in fungal cell wall lysis

R. GUPTA, R.K. SAXENA, P. CHATURVEDI AND J.S. VIRDI. 1995. Streptomyces viridificans was found to be a good chitinase producer among nine species of Streptomyces screened. Minimum levels of constitutive enzyme were observed with both simple and complex carbon substrate. Arabinose doubled the enzyme production amongst the various pentoses and hexoses used with chitin. However, with glucose end-product inhibition and catabolite repression were observed. The enzyme tolerated a wide range of temperature (30–55°C) and pH (3–7˙5). Among various divalent cations Mn²⁺ and Hg²⁺ completely inhibited the purified enzyme while β-mercaptoethanol stimulated its activity. Crude and purified enzyme had potential for cell wall lysis of many fungal pathogens tested.