Regulation of spore-formation in Streptomyces galbus mutants

Some asporogenous strains of Streptomyces galbus recover the capacity to form aerial mycelium and spores when they are grown together with the parent sporulating strain under the conditions of superficial cultivation. The sporulating strain of Str. griseus that produces the A factor, a bioregulator, does not restore sporogenesis in asporogenous variants of Str. galbus, in contrast to Str. griseus. Therefore, Str. galbus must liberate a specific compound which controls spore formation in this species.     

Isolation and characterization of melanin-producing (mel) mutants of Vibrio cholerae

Vibrio cholerae strain Htx-3, a hypertoxinogenic mutant of V. cholerae 569B Inaba, produces a dark brown pigment under certain growth conditions, whereas strain 569B does not. We investigated the biochemical basis for this pigment production and the possible relationships between pigmentation and other phenotypic properties in V. cholerae. After mutagenesis of V. cholerae 569B with N-methyl-N'-nitro-N-nitrosoguanidine, 28 independently derived pigment-forming (mel) mutants were isolated and characterized. The mel mutants frequently differed from wild type in toxinogenicity or motility and occasionally differed in other phenotypic traits. Individual mel mutants differed from wild type both in the amount of toxin produced and in the growth conditions optimal for toxin production. It has not yet been established whether multiple phenotypic changes in individual mel mutants represent pleiotropic effects of single mutations or induction of multiple mutations by N-methyl-N'-nitro-N-nitrosoguanidine or both. Production of pigment by mel mutants occurred at temperatures from 22 to 40 degrees C, was inhibited by anaerobiosis, and was stimulated by supplementation of growth media with the amino acid precursors of melanin (l-phenylalanine, l-tyrosine, or l-tyrosine plus l-cysteine). The pigment possessed several other properties reported for microbial melanins. We conclude that a biochemical pathway for melanin production is present in V. cholerae, that this pathway cannot be fully expressed in wild-type strain 569B, and that mutations in the gene(s) which we have designated mel can permit hyperproduction of melanin under appropriate conditions.     

Phenotypic heterogeneity of the progeny of Streptomyces griseus conidia

In order to understand the complex ontogenetical processes, the development of Streptomyces (S.) griseus was applied as a model. The developmental cycle of S. griseus starts and ends as a conidium. In between, coenocytic mycelium develops which, if studied by cytomorphological or biochemical methods, exhibits conspicious heterogeneity. The hyphae develop into young, transient and old vegetative hyphae and different stages of reproductive forms. In developmentally blocked mutants these sequences of events appear mixed in all possible associations. It seems as if the program of development could be divided into several subprograms. The quantitative evaluation of the results show that the individual morphological markers exhibit certain independence from each other realized with a given probability. The conidia of S. griseus are also heterogeneous concerning all morphological and physiological traits examined so far (shape, size, light refraction, staining and shape of nucleoids with Feulgen, methyl green--pyronine, intensity and form of polysaccharide distribution, heat resistance, etc.). Kinetics of the survival curves of two S. griseus strains--a well-sporulating and its developmentally blocked mutant /24/--are different from each other, one has many more heat resistant conidia than the other but the kinetics of the survival curves of the two S. griseus strains indicate that spore populations of both react differently to heat treatment and heat resistance can be modeled by assuming the presence of two independent subpopulations of spores with different heat sensitivity. The emergence of two distinct subpopulations with (possibly) the same genetic make-up is designated: phenotypic segregation. Heat resistance is first of all species specific (genetically determined) but the epigenetic segregation seems to be characteristic of the developmental process. This process can in certain mutants be affected by environmental conditions and more importantly by the so-called autoregulators (A-factor and factor C). Factor C and A-factor are needed to normal development, if their quantity or the time of addition to the culture was not optimal, the quantity of spores decreased.     

Ecology, community, and AIDS prevention

The A-factor receptor protein (ArpA) plays a key role in the regulation of secondary metabolism and cellular differentiation in Streptomyces griseus. ArpA binds the target DNA site forming a 22 bp palindrome in the absence of A-factor, and exogenous addition of A-factor to the ArpA-DNA complex immediately releases ArpA from the DNA. An amino acid (aa) replacement at Val-41 to Ala in an alpha-helix-turn-alpha-helix (HTH) motif at the N-terminal portion of ArpA abolished DNA-binding activity but not A-factor-binding activity, suggesting the involvement of this HTH in DNA-binding. On the other hand, an aa replacement at Trp-119 to Ala generated a mutant ArpA that was unable to bind A-factor, thus resulting in an A-factor-insensitive mutant that bound normally to its target DNA in both the presence and absence of A-factor. These data suggest that ArpA consisting of two functional domains, one for HTH-type DNA-binding at the N-terminal portion and one for A-factor-binding at the C-terminal portion, is a member of the LacI family. Consistent with this, two ArpA homologues, CprA and CprB, from Streptomyces coelicolor A3(2), each of which contains a very similar aa sequence of the HTH to that of ArpA, also recognized and bound the same DNA target. However, neither CprA nor CprB recognized A-factor, probably due to much less similarity in the C-terminal domains.     

Skeletal muscle triacylglycerol in the rat: methods for sampling and measurement, and studies of biological variability

dTDP-L-dihydrostreptose: streptidine-6-phosphate dihydrostreptosyltransferase, an enzyme involved in the biosynthesis of streptomycin, has been purified from Streptomyces griseus to near homogeneity by a six-step procedure involving chromatography on streptidine-6-phosphate-Sepharose. By gel filtration the apparent Mr of the enzyme was found to be about 63 000. The subunit Mr found on sodium dodecylsulfate gels is about 35 000. The transferase is dependent on Mn2+ or Mg2+ ions. Co2+ is as effective as Mg2+. From the substrates tested only streptidine 6-phosphate was an acceptor for dihydrostreptose in the synthesis of O-alpha-L-dihydrostreptose(1 leads to 4)-streptidine 6-phosphate. No activity was found with streptidine, 2-deoxystreptamine and 4-deoxy-streptamine. The activity of the transferase in the course of fermentation runs parallel to the activity of dTDP-dihydrostreptose synthase and reaches a maximum after around 50 h of fermentation, just before appearance of streptomycin in the medium.     

Ultrastructural changes in the liver in experimental diffuse peritonitis

Experiments were performed to detect "alkali" and "acid" carboxypeptidases in 39 enzymic preparations from fungi, yeast, actinomyces, bacteria and algae. Distribution of both types of carboxypeptidases is different: they are absent in the six of the studied sources, there are no "acid" carboxypeptidases in 11 sources and no "alkali" in nine ones. The largest amount of carboxypeptidases is in the objects from fungi and actinomycetes, the least, in those from bacteria and algae. There is no correlation between synthesis of these enzymes by one microorganism. Thus, fungi produce mainly "acid" enzymes, and actinomycetes only "alkali" ones. Asp. oryzae and Asp. flavus are powerful producers of the former, Streptomyces griseus of the latter. Specific activities 15-20 times as high as all the already studied ones are obtained for the preparations isolated from Str. griseus (protezym, proteinase-1, proteinase-3, crystal line complex of proteases). Carboxypeptidase of Str. griseus is relatively stable in comparison with "acid" ones in purification and concentration.     

Does afferent loop syndrome exist?

A comparison was made of the pigment-forming capacity and the enzymatic activity, growth rate, virulence and sensitivity to antibiotics and egg lysozyme in 397 pigment, 203 pigment-free strains of staphylococci and 24 pigment-free Staph aureus mutants. Pigment formation did not always correlate with the general biological activity of the cultures, the rate of their growth and the sensitivity to antibiotics and lysozyme. Pigment-free staphylococcus mutants retained the principal properties of the initial strains (including the virulence and sensitivity to antibiotics and egg lysozyme) more frequently. It is supposed that the marker determining the pigment formation in staphylococci was not connected with the markers determining the most significant pathogenicity properties and also antibiotic resistance.     

Polymer production by Klebsiella pneumoniae 4-hydroxyphenylacetic acid hydroxylase genes cloned in Escherichia coli

The expression of Klebsiella pneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin.     

Physicochemical parameters of the surface of bacteria and the penicillin-binding capacity in relation to the sensitivity to penicillins and streptomycin

Development of resistance to penicillins and streptomycin in bacteria results in an increase in the electrokinetic potential changes in the hydratation rate and isoelectric levels of pH. Changes in the physico-chemical properties of the surface of penicillin-resistant Escherichia depends mainly on the accompanying dissociation. The ability of Escherichia to bind labeled benzylpenicillin is shown. Binding of 35S-benzylpenicillin by Escherichia in S-form correlates with sensitivity of the culture to the antibiotic. The penicillin-resistant mutants in R-form bound the antibiotic to a greater extent than sensitive R-cultures. The role of penicillinase in binding 35S-benzylpenicillin by Escherichia was not shown experimentally. Only increased synthesis of the enzyme by the penicillin-resistant mutants was observed. The ability of Escherichia to bind 35S-benzylpenicillin does not depend on the physico-chemical properties of the cell surface and dissociation.     

Symbiotic effectiveness of antibiotic-resistant mutants of fast- and slow-growing strains of Rhizobium nodulating Lotus species

Mutants resistant ot 16 individual antibiotics were isolated from two fast-growing and two slow-growing strains of Lotus rhizobia and their symbiotic effectiveness on Lotus pedunculatus evaluated. Resistance to streptomycin, spectinomycin, chloramphenicol, and tetracycline (inhibitors of protein synthesis) was associated with little or no loss of effectiveness with all four strains but resistance to nalidixic acid and rifampicin (inhibitors of nucleic acid synthesis), and to D-cycloserine, novobiocin, and penicillin (inhibitors of cell wall-cell membrane synthesis) was associated with significant loss of effectiveness in 20-100% of the mutants. Resistance to viomycin, neomycin, kanamycin, and vibramycin was associated with loss of effectiveness with mutants of the two fast-growing strains but not with mutants of the two slow-growing strains. When tested on four alternate host legumes individual mutants of a slow-growing strain showed significantly different levels of effectiveness. The results suggest that both the inherent characteristics of the bacterium and of the host plant will influence the symbiotic effectiveness of antibiotic-resistant mutants of Rhizobium.     

A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.     

c-type cytochromes and manganese oxidation in Pseudomonas putida MnB1

Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides. We used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and we characterized some of these mutants. The mutants were divided into three groups: mutants defective in the biogenesis of c-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan. The mutants in the first two groups were cytochrome c oxidase negative and did not contain c-type cytochromes. Mn(II) oxidation capability could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation.     

Isolation and biological characterization of Legionella pneumophila mutants resistant to aminoglycoside antibotics and their protective action]

Preliminary estimation of virulence in some antibiotic resistant mutants of Legionella pneumophila, Philadelphia 1 in various models of infection revealed its decrease in the mutants resistant to azlocillin, cefotaxime, fluoroquinolone LIB-80, neamine and streptomycin. Detailed investigation of the neamine resistant mutants showed that in relation to streptomycin susceptibility such mutants could be divided into 3 classes: susceptible to streptomycin, resistant to high concentrations of streptomycin and resistant to moderate concentrations of streptomycin. Part of mutants Nea(r)Strr and Nea(r)Strr500 and all mutants Nea(r)Strr100 proved to be less virulent with respect to guinea pigs and chick embryos. The study of the spectinomycin resistant mutants of Legionella spp. did not reveal any changes in the virulence which made it possible to suggest that the influence of the mutations in the ribosomal protein genes determining resistance to streptomycin and neamine on virulence of L. pneumophila was based on the interdependence of the mutant effect on the suppression and the influence on the virulence detected by us in S. flexneri, Y. pseudotuberculosis, L. monocytogenes and F. tularensis. The Legionella mutants Nea(r)Strr100 were characterized by significant protective activity and protected immunized guinea pigs when tested in a model of their aerogenic infection.     

Identification of stsC, the gene encoding the L-glutamine:scyllo-inosose aminotransferase from streptomycin-producing Streptomycetes

Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins - together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster - show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the L-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.     

Pool of free amino acids in mycelium and culture fluid of Streptomyces galbus (F) subsp. achromogenes 695, a strain producing actinomycin complex and its variants

Free amino acids in the mycelium and culture fluid of Streptomyces galbus (F) subsp. achromogenes 695 and its active and inactive variants were comparatively studied. It was found that the amino acid pool in the mycelium of the highly productive variant was 14 per cent higher than that of the initial strain and 40 per cent higher than that of the inactive variant. Even so, the highest amounts of the synthesized protein in the three strains were about the same. The free amino acid composition of the mycelium of the active antibiotic-producing strains and the inactive variant was shown to be the same and included all the investigated 16 amino acids. Glutamic acid was the main amino acid. The contents of alanine, serine and valine were comparatively high. The contents of methionine, histidine and phenylalanine were the lowest. It was shown that the quantities of the amino acids or their precursors participating in the construction of the antibiotic molecule were to a higher extent determined by the strain development and growth rate than by the actinomycin biosynthesis. The qualitative and quantitative composition of the amino acid pool in the culture fluid of the active strains was inferior to that in the inactive variant.     

Acriflavine-resistant mutant of Streptococcus cremoris

Selection for resistance to acriflavine in Streptococcus cremoris resulted in cross-resistance to the drugs neomycin, streptomycin, ethidium bromide, mitomycin C, and proflavine. Furthermore, the mutants showed resistance to lytic bacteriophages to which the parental strain was sensitive, and, unlike the parent, the mutants grew well at higher temperatures (40 degrees C). Revertants selected independently either for temperature sensitivity or for acriflavine sensitivity lost resistance to all the drugs and dyes but retained the bacteriophage resistance phenotype. The acriflavine-resistant mutation resulted in an increase in resistance by the bacterial cells to sodium dodecyl sulfate, a potent solvent of lipopolysaccharide and lipoprotein. It is suggested that the acriflavine resistance mutation determines the synthesis of a membrane substance resistant to higher temperatures.     

Increased doxorubicin levels in hepatic tumors with reduced systemic drug exposure achieved with complete hepatic venous isolation and extracorporeal chemofiltration

FIZ15 phage of Pseudomonas aeruginosa causes lysogenic conversion in PAO1 strain. Lysogen shows increased adhesion to human buccal epithelial cells, increased resistance to 75 percent human serum bactericidal effect, and streptomycin resistant. These phenotypes apparently are due to a phage-induced superficial change on its own bacterial receptor, which probably is the O-antigen. In order to begin FIZ15 characterization, nitrous acid-induced clear-plaque mutants were obtained. They belonged to three complementation groups and mapping by two factor crosses revealed that they were closely linked. In a search for phage mutants that do not cause lysogenic conversion, two streptomycin-sensitive mutants were obtained by ethyl methane sulphonate mutagenesis of PAO1 lysogenic for FIZ15 (PIZ15 strain). One mutant (con1) showed and adhesion value similar to that of PAO1 and the other (con2) had an adhesion twofold and 1.3 times greater than PAO1 and PIZ15, respectively. con1 did not show increased serum resistance, whereas con2 was as resistant as PIZ15. Phages were isolated from the streptomycin sensitive mutants and used to relisogenize PAO1 to obtain the con1d and con2d lysogens. Adhesion and serum sensitivity of con1d was identical to that of PAO1 but con2d behaves like PIZ15. FIZ15 phage was unable to adsorb to PIZ15, con2 and con2d. On the other hand, FIZ15 phage adsorbs well to con1 and con1d but not to PIZ15. These results suggest that con1 mutation lies on the phage chromosome and con2 on the bacterial one. Finally, adhesion of all lysogens and PAO1 was stimulated 2-3 times by KCl and this effect was suppressed by and oxidative phosphorylation uncoupler.     

The spermidine transport system is regulated by ligand inactivation, endocytosis, and by the Npr1p Ser/Thr protein kinase in Saccharomyces cerevisiae

We have characterized the regulation of spermidine transport in yeast and identified some of the genes involved in its control. Disruption of the SPE2 gene encoding S-adenosylmethionine decarboxylase, which catalyzes an essential step in polyamine biosynthesis, upregulated the initial velocity of spermidine uptake in wild-type cells as well as in the polyamine transport-deficient pcp1 mutants. Exogenous spermidine rapidly inactivated spermidine transport with a half-life of approximately 10-15 min via a process that did not require de novo protein synthesis but was accelerated by cycloheximide addition. Conversely, reactivation of spermidine influx upon polyamine deprivation required active protein synthesis. The stability of polyamine carrier activity was increased 2-fold in polyamine-depleted spe2 deletion mutants, indicating that endogenous polyamines also contribute to the down-regulation of spermidine transport. Ligand-mediated repression of spermidine transport was delayed in end3 and end4 mutants that are deficient in the initial steps of the endocytic pathway, and spermidine uptake activity was increased 4- to 5-fold in end3 mutants relative to parental cells, although the stability of the transport system was similar in both strains. Disruption of the NPR1 gene, which encodes a putative Ser/Thr protein kinase essential for the reactivation of several nitrogen permeases, resulted in a 3-fold decrease in spermidine transport in NH4(+)-rich media but did not prevent its down-regulation by spermidine. The defect in spermidine transport was more pronounced in NH4(+)- than proline-grown npr1 cells, suggesting that NPR1 protects against nitrogen catabolite repression of polyamine uptake activity. These results suggest that (a) the polyamine carrier is an unstable protein subject to down-regulation by spermidine via a process involving ligand inactivation followed by endocytosis and that (b) NPR1 expression fully prevents nitrogen catabolite repression of polyamine transport, unlike the role predicted for that gene by the inactivation/reactivation model proposed for other nitrogen permeases.     

Cellular retinaldehyde-binding protein ligand interactions. Gln-210 and Lys-221 are in the retinoid binding pocket

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known. We have characterized the interactions of retinoids with native bovine CRALBP, human recombinant CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or identify a Schiff base in the dark, under a variety of reducing, denaturing, and pH conditions were unsuccessful, suggesting the lack of covalent interactions between CRALBP and retinoid. Buried and solvent-exposed lysine residues were identified in bovine CRALBP by reductive methylation of the holoprotein followed by denaturation and reaction with [3H]acetic anhydride. Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteolysis and purification of modified peptides. Human rCRALBP mutants K152A, K221A, and K294A were prepared to investigate possible retinoid interactions with buried or partially buried lysines. Two other rCRALBP mutants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The structures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by UV-visible and fluorescence spectroscopy. All of the mutants bound 11-cis-retinal essentially like the wild type protein, indicating that the proteins were not grossly misfolded. Three of the mutants bound 9-cis-retinal like the wild type protein; however, Q210R and K221A bound less than stoichiometric amounts of the 9-cis-isomer and exhibited lower affinity for this retinoid relative to wild type rCRALBP. Residues Gln-210 and Lys-221 are located within a region of CRALBP exhibiting sequence homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein. The data implicate Gln-210 and Lys-221 as components of the CRALBP retinoid binding cavity and are discussed in the context of ligand interactions in structurally or functionally related proteins with known crystallographic structures.