Streptomyces spheroides mutant deprepressed for exoprotease biosynthesis

The effect of carbon, nitrogen and sulfur sources on the biosynthesis of exoproteases was studied with the parent Streptomyces spheroides strain 35 and its mutant M8-2. Addition of a carbon, nitrogen and sulfur source to the medium deficient in one of these elements did not repress the synthesis of exoproteases by the washed mycelium of the mutant as compared to the parent strain. Protein as a sole source of carbon, nitrogen and sulfur had no effect on the biosynthesis of exoproteases by the mutant. In contrast to the parent strain, the biosynthesis of exoproteases in the mutant was not controlled by metabolite repression.     

Mutant Strains of Rhodopseudomonas spheroides Lacking δ-Aminolevulinate Synthase: Growth, Heme, and Bacteriochlorophyll Synthesis

Two mutant strains of Rhodopseudomonas spheroides were described which lacked delta-aminolevulinate synthase activity. They required delta-aminolevulinate for growth; they did not respond to protoporphyrin or magnesium photoporphyrin, and only poorly to hemin. Synthesis of cytochromes and heme by mutant H-4 was dependent upon delta-aminolevulinate; this strain did not form bacteriochlorophyll either with or without delta-aminolevulinate and, consequently, grew only under aerobic conditions. Mutant H-5 formed bacteriochlorophyll in response to delta-aminolevulinate and grew both anaerobically in the light and aerobically in the dark; the amount of delta-aminolevulinate needed for optimal anaerobic growth was higher than that required aerobically. Synthesis of bacteriochlorophyll and heme by suspensions of mutant H-5 incubated anaerobically in the light was dependent upon delta-aminolevulinate; bacteriochlorophyll production was completely inhibited by high aeration and by puromycin. The mutants differed in their ability to take up radioactive delta-aminolevulinate from the external environment; mutant H-5 was less active than mutant H-4 or the wild type. It was suggested that R. spheroides made only one form of delta-aminolevulinate synthase, which provided delta-aminolevulinate for bacteriochlorophyll and heme synthesis.     

Bacteriochlorophyll Synthesis and the Ultrastructure of Wild Type and Mutant Strains of Rhodopseudomonas spheroides

The ultrastructure of sectioned cells of mutant and wild type Rhodopseudomonas spheroides has been examined by electron microscopy. The characteristic vesicles associated with the presence of bacteriochlorophyll were found in wild type cells grown with low aeration. These were also found in mutant TA-R which forms bacteriochlorophyll under high aeration. None of the mutants with blocks in bacteriochlorophyll synthesis contained intracytoplasmic membrane. These included mutant 8-17 which accumulates bacteriochlorophyllide but fails at the phytolation step. We conclude that the intact bacteriochlorophyll molecule, or some particular membrane protein associated with it, is needed for the development of the characteristic intracytoplasmic membrane system in R. spheroides.     

Isolation and Characterization of a Temperate Bacteriophage Specific for Rhodopseudomonas spheroides

The isolation of a temperature phage specific for the photosynthetic bacterium Rhodopseudomonas spheroides is reported. This phage, Rphi-1, establishes a state of lysogeny and can be induced from the prophage state by exposure to mitomycin C or UV irradiation. Mutants of Rphi-1 which grow on a standard laboratory strain (2.4.1) of Rhodopseudomonas spheroides were isolated. Although the original Rphi-1 isolated was chloroform sensitive, the mutant which plates on strain 2.4.1 is chloroform resistant. Rphi-1 does not grow on closely related bacteria, such as Rhodopseudomonas palustris or Rhodopseudomonas capsulata. Rphi-1 mutants forms plaques with the same efficiency whether the plates are incubated under aerobic conditions in the dark or under anaerobic conditions in the light (phototropic conditions).     

Mutant of Rhodopseudomonas spheroides Unable to Grow Aerobically

We isolated a mutant of Rhodopseudomonas spheroides that grows normally under photosynthetic conditions but is unable to grow exponentially under aerobic conditions. Photosynthetically grown cultures of the mutant increase in mass and cell number for about 10 hr after transfer to aerobic conditions. During this time, no heme pigments are synthesized and the Q(O(2)) declines. In the mutant, synthesis of heme pigments is obligatorily coupled to synthesis of bacteriochlorophyll.     

Control of porphyrin biosynthesis in Rhodopseudomonas spheroides and Propionibacterium shermanii. A direct 13C nuclear-magnetic-resonance spectroscopy study.

The facultative anaerobes Rhodopseudomonas spheroides and Propionibacterium shermanii were grown under anaerobic and aerobic conditions. The effect of light was studied with the photosynthetic R. spheroides, and the adaptation of both species to dark anaerobic life was monitored by direct observation of 5-amino[5-13C]laevulinic acid metabolism by using 13C nuclear-magnetic-resonance spectroscopy.     

Comparative study of proteolytic enzyme preparations of the actinomycete Streptomyces spheroides and its mutant

Preparations of proteolytic enzymes with high fibrinolytic and thrombolytic activities were isolated from the cultural broth filtrates of Streptomyces spheroides and its mutant by precipitation with ammonium sulfate, dialysis and freeze-drying. The fibrinolytic and thrombolytic activities of the enzymes produced by the mutant are 5.6 and 6 times higher, respectively, than those of the parent strain. Moreover, the enzymes of the mutant have an elevated stability in the alkaline pH region, the optimum of their activities is also shifted to the alkaline pH region, they are more resistant to the action of blood plasma inhibitors and more sensitive to the action of high temperatures. As was shown using isoelectric focusing in a sucrose density gradient, the preparations of both the parent and mutant strains contain several proteolytic enzymes (or their groups) with acid, neutral and alkaline isoelectric points.     

Bacteriophages of Rhodopseudomonas spheroides: Isolation and Characterization of a Rhodopseudomonas spheroides Bacteriophage

A DNA-containing bacteriophage, designated RS1, infecting Rhodopseudomonas spheroides 2.4.1, has been isolated from sewage. The buoyant density of RS1 in CsCl equilibrium centrifugation is 1.50 g/cm(3), and the buoyant density of RS1 DNA is 1.706. The phage possesses a polyhedral head, approximately 65 nm in diameter, and a tail 60 nm long. When grown on aerobic cells, RS1 has a latent period of 120 min and an average burst size of 20. When grown on anaerobic cells, RS1 has a latent period of 150 min, and a burst size similar to that observed during aerobic infection. The adsorption rate constant of RS1 to aerobic cells is 1.2 x 10(-9) ml/min, and 0.58 x 10(-9) ml/min to anaerobic cells. Adsorption of RS1 to R. spheroides requires the presence of divalent cations.     

Natural variability of Streptomyces spheroides--a producer of extracellular proteolytic enzymes possessing fibrinolytic action

Streptomyces spheroides M 8-2 was obtained using UV irradiation of spores taken from the parent culture. It shows a high activity of exocellular proteases which are capable of fibrin hydrolysis and degradation of blood clots. On certain media, a population of S. spheroides M8-2 was shown to yield three variants differing in their morphologo-biochemical properties. During submerged cultivation, these variants produce different quantities of exocellular proteases (3 to 19 units per 1 mg of biomass) with the fibrinolytic activity. Variants I and III are most active; their proteolytic activity is 10-14 units per 1 mg of biomass, on the average, and can reach 18-19 units per 1 mg of biomass. Variants with a low activity (variant II) are accumulated when the actinomycets is kept as spores on a solid medium with corn extract and on a solid medium with fibrin. The high proteolytic activity of the strain can be preserved by selection on a diagnostic medium with fibrin taking account of the diameter of hydrolytic zones around the colonies and selecting solely the colonies of variants I and III.     

Control of δ-aminolaevulate synthetase activity in Rhodopseudomonas spheroides

1. delta-Aminolaevulate synthetase from Rhodopseudomonas spheroides grown semi-anaerobically undergoes a spontaneous activation during the first hour after the disruption of cells when homogenates are stored at 4 degrees . 2. After cultures of R. spheroides growing semi-anaerobically are oxygenated no activation of delta-aminolaevulate synthetase occurs in cell extracts. Cessation of activation in extracts is almost complete 10min. after oxygenation of cells has begun. 3. A heat-stable fraction of low molecular weight from semi-anaerobic cells reactivates delta-aminolaevulate synthetase in extracts of oxygenated cells and appears to contain a compound responsible for the spontaneous activation. 4. A heat-stable fraction of low molecular weight from oxygenated cells inhibits the spontaneous activation in extracts of semi-anaerobic cells. 5. The effect of oxygen on the rate of bacteriochlorophyll synthesis in R. spheroides may be mediated through alterations in the concentrations of a low-molecular-weight activator and inhibitor of delta-aminolaevulate synthetase.     

Deficiencies of Chromatophore Proteins in Some Mutants of Rhodopseudomonas spheroides with Altered Carotenoids

Chromatophore proteins of a wild type and three mutant strains of Rhodopseudomonas spheroides were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mutants consisted of a green and a blue-green one, whose phenotypes were essentially the same as those of known mutants, and a brown one, which may be a double mutant and represents a new phenotype. Wild-type chromatophores contained at least six major and seven minor protein bands, with molecular weights ranging from 10,000 to 65,000. The green mutant contained the same protein bands in the same relative quantities. The brown mutant had one protein completely missing and no other alterations. The blue-green mutant was deficient in a different protein, and had reduced quantities of all proteins with molecular weights less than 25,000. Chromatophores were separated into a fraction containing the reaction centers and a fraction containing the light-harvesting bacteriochlorophyll by treatment with sodium dodecyl sulfate. Eight of the proteins were found only in the reaction center fraction, one was only in the light-harvesting fraction, and the remainder were present in both fractions. The protein missing from the brown mutant was found to be a component of the reaction center fraction, whereas the proteins which were missing from the blue-green mutant were all components of the light-harvesting fraction. Some implications for the structure and biogenesis of chromatophores are discussed.     

Isolation and properties of carboxypeptidase from Streptomyces spheroides, strain 35

Extracellular carboxypeptidase was isolated from culture filtrates of Str. spheroides strain 35, using affinity chromatography on bacitracin-silochrome, bacitracin-Sepharose and CABS-Sepharose. The electrophoretically homogenous enzyme was obtained with a 44% yield and 4160-fold purification. The enzyme-molecular weight is 33,000 Da; pI is 4.7. The amino acid composition of carboxypeptidase is as follows: Asp43, Thr30, Ser35, Glu33, Pro30, Gly47-50, Ala38, 1/2 Cys5-6, Val16, Met2, Ile11, Leu15, Tyr8, Phe10, Lys10, His6, Arg9. The enzyme shows an activity optimum at pH 7.5 is stable at pH 6-8, is completely inhibited with EDTA and can be reactivated by Ca2+. The carboxypeptidase from Str. spheroides strain 35 has a dual substrate specificity, i. e., it splits N-substituted di-, three- and tetrapeptides having both neutral and basic amino acids at the C-ends similar to mammalian carboxypeptidases A and B. The enzyme belongs to the family of metallocarboxypeptidases; its properties are very similar to those of carboxypeptidase S from Str. griseus K-1 and of carboxypeptidase T from Thermoactinomyces sp.     

16s and 23s components in the ribosomal ribonucleic acid of Rhodopseudomonas spheroides

1. Ribosomal RNA was extracted from lysates of Rhodopseudomonas spheroides without prior isolation of ribosomes. 2. The composition of this RNA was investigated by using gradient centrifugation, showing that the proportion present as 23s component depended on the method of extraction. 3. The highest proportion of 23s component was found when cells were disrupted by ultrasonic treatment in the presence of ribonuclease inhibitors. 4. The results indicated that a ribonuclease is active in the cell lysate; this could account for the previous report (Lessie, 1965) that ribosomes of Rhodopseudomonas spheroides do not contain a 23s component.     

Subtilisin-like proteinase SSPB from Streptomyces spheroides, strain 35

A serine proteinase possessing a fibrinolytic activity was isolated from a culture filtrate of Streptomyces spheroides, strain 35. A consecutive use of affinity chromatography on bacillichin-silochrome and bacitracin-sepharose and ion-exchange chromatography on anionie PAP and cationic KMT resulted in a homogeneous proteinase with 1060-fold purification and 19% yield. The enzyme has a molecular weight of 28000; its amino acid composition is Asp31, Ser28, Thr29, Glu9, Pro14, Gly35, Ala42, Val26, Ile14, Leu13, Met2, Tyr9, Phe4, Trp3, His6, Lys4, Arg10. The enzyme has a pI at pH greater than 10 and the activity optimum against Z-L-Ala-L-Ala-L-Leu-pNA at pH 10-11. The enzyme is stable within the pH range of 4-11 and in 6 M guanidinium chloride pH 8.0 in the presence of Ca2+. The enzyme is inhibited by diisopropylfluorophosphate and benzylsulfofluoride, specific inhibitors of serine proteinases as well as by potato proteinase inhibitor. The serine proteinase SSPB isolated from Str. spheroides, strain 35 can be related to subtilisin-like serine proteinase, especially to those of SGPD and SGPE of Str. griseus.     

A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg(2+) and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg(2+) caused the refolding of the initial products of unfolding and in the presence of Ni(2+) the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na(+)/Mg(2+) ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein-protein interactions in the structure of the R. spheroides ribosome.     

A 4-vinylprotochlorophyllide complex accumulated by "phofil" mutant of Rhodopseudomonas spheroides. An authentic intermediate in the development of the photosynthetic apparatus.

A photosynthetically competent mutant strain of Rhodopseudomonas spheroides was isolated. In addition to bacteriochlorophyll, this organism produced particle-bound precursor 4-vinylprotochlorophyllide. The spectral characteristics of the pigment complexes(es) accumulated in the culture medium were very variable. The spectral form occurring within the bacteria was characterized from fluorescence data. Its particle weight, 130 000, was determined by Sephadex G200 filtration. The main components of the complex were protein, lipid and pigment (6.8:61, w/w). As indicated by qualitative analysis, the lipid components were characteristic constituents of the photosynthetic membrane. Kinetics of pigments synthesis showed that the total pigment synthesis was not affected by the mutation; bacteriochlorophyll content was always lower in the mutant than in the parent strain. The repigmentation process was followed by fluorescence emission. The results indicated that the mutation affected membrane component synthesis required for the bacteriochlorophyll(ide) incorporation. The pigment complex was concluded to be an authentic intermediate in photosynthetic apparatus morphogenesis. The reasons for its excretion are discussed.     

Electron microscopy of chromatophores of Rhodopseudomonas spheroides

Gibson, K. D. (St. Mary's Hospital Medical School, London, England). Electron microscopy of Rhodopseudomonas spheroides. J. Bacteriol. 90:1059-1072. 1965.-Fixed and stained chromatophores and whole cells of anaerobically grown Rhodopseudomonas spheroides were examined in thin sections in the electron microscope. Both purified chromatophores and intracellular membrane-bound vesicles had exactly the same appearance, namely that of spheres or ellipsoids with a thin electron-dense shell surrounding an electron-lucent interior. The distribution of diameters in the two types of structure was also found to be the same, and was compatible with a normal distribution, with a mean of 570 A and a standard deviation 40 A. Negatively stained chromatophores appeared like discs or collapsed spheres. The presence of invaginations of the cytoplasmic membrane in this species was confirmed, and a new structure resembling a twin chromatophore was observed. The bearing of these results on theories of the origin of chromatophores is discussed, and it is concluded that they offer some support for each one of the three main theories about the origin of particulate organelles.     

Genetic analysis of the biosynthesis of the pyrrole and carbamoyl moieties of coumermycin A1 and novobiocin

The aminocoumarin antibiotic coumermycin A(1) contains a central and two terminal pyrrole moieties. The coumermycin gene cluster in Streptomyces rishiriensis contains three genes (couN3, couN4 and couN5) that show sequence similarity to genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens and of undecylprodiginine in S. coelicolor. The gene couN3, which codes for a putative L-prolyl-S-PCP dehydrogenase, and the gene couN4, which encodes a putative L-prolyl-AMP ligase, were disrupted using in-frame deletion and insertional inactivation, respectively. HPLC analysis of culture extracts showed that formation of the two terminal pyrrole moieties was abolished in the couN3 (-) und couN4 (-) mutants. The mutants accumulated coumermycin D, which contains only the central pyrrole moiety. This result not only confirmed the involvement of couN3 and couN4 in the biosynthesis of the terminal pyrrole-2-carboxylic acid moieties of coumermycin A(1), but also indicated, for the first time, that the central 3-methylpyrrole-2,4-dicarboxylic acid unit of the coumermycins is formed by a biosynthetic pathway that differs from that used to assemble the terminal pyrrole moieties. novN, a putative carbamoyl transferase gene from the gene cluster for novobiocin biosynthesis in S. spheroides was expressed in the couN3 (-) mutant. This led to the formation of bis-carbamoylated coumermycin D, a novel compound of the coumermycin series.     

Temperature dependence of the kinetics of dark reduction of bacteriochlorophyll P870, oxidized by impulse laser and continuous light in photosynthetic reaction center preparations from Rhodosuedomonas spheroides strain 1760-1]

A comparative study was carried out of temperature dependence of kinetics of dark reduction of bacteriochlorophyll P870 oxidized both by pulsed laser and continuous actinic light in preparations of photosynthetic reaction centres-bacteriochlorophyll-protein complexes from Rhodopseudomonas spheroides, strain 1760-1. Half-time of the recombination of primary products--P870+ and reduced primary electron acceptor A1, which decreases with temperature lowering from 180-240 ms at 120K, is determined. Values of the rate constant of electron transfer from A1 to secondary acceptors at 29,K (2.7.10-1s-1) and the activation energy of this reaction in the range of 298-255K which is 11.8 kcal/mole are calculated.     

On the nature of the activating enzyme of the inactive form of delta-aminolevulinate synthetase in Rhodopseudomonas spheroides.

The activating enzyme of the inactive form of Fraction I of delta-aminolevulinate (ALA) synthetase [EC 2.3.1.37] in Rhodopseudomonas (R.) spheroides was purified about 1,000-fold from an extract of R. spheroides cells grown anaerobically in the light. The purification of the activating enzyme was achieved by fractionating the 100,000 X g supernatant fraction of the crude extract with ammonium sulfate and acetone, followed by Sephadex G-200 chromatography, pyridoxamine phosphate-Sepharose 4B chromatography, and preparative gel electrophoresis. The final preparation of the activating enzyme still contained a minor contaminant (less than 20%) as judged by disc gel electrophoresis. The activating enzyme exhibited cystathionase [EC 4.4.1.1] activity throughout the purification. These two enzyme activities were not separated at all during any step of the purification. An apparently homogeneous preparation of cystathionase [EC 4.4.1.8] purified from rat liver also exhibited activating activity in the presence of L-cystine. It was concluded that the activating enzyme is a cystathionase.