Eight structurally related halogenated aliphatic hydrocarbons mono-, di- and trichloroacetaldehyd... more Eight structurally related halogenated aliphatic hydrocarbons mono-, di- and trichloroacetaldehyde (the last in the anhydrous and hydrate form), moni-, di- and trichloroethanol and allyl chloride, were tested for their ability to induce gene mutations in prokaryotic and eukaryotic microorganisms. The genetic systems employed were the Salmonella reversion test with strain TA1535 and TA100, with and without metabolic activation, a forward and a back-mutation system in S. coelicolor and two forward mutation systems in A. nidulans. Each compound was tested with the spot and plate incorporation assay techniques. Mono-, di- and trichloroacetaldehyde were mutagenic in all the microorganisms employed; all the halogenated ethanols were positive in A. nidulans, while in S. typhimurium and in S. coelicolor the only active forms were respectively the mono- and dichloroderivatives. Allyl chloride was active in S. typhimurium and S. coelicolor and negative in A. nidulans. The technical approach a...
A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plas... more A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plasmid SCP1 susceptible to curing by UV irradiation. Lack of the SCP1 plasmid (in SCP1(-) strains) prevented the occurrence of the co-mutation process after MNNG treatment, although it left the susceptibility to the lethal effect of the mutagen virtually unaffected. SCP1(-) strains were, however, ultrasensitive to the lethal effect of UV. Curing a plc A(-) strain of its SCPI plasmid made it refractory to co-mutation by MNNG and sensitive to the lethal effect of UV; reinfected by the plasmid, the strain resumed both the co-mutation proficiency and the UV-resistance. The occurrence on the SCP1 plasmid of a gene comparable to the uvrE gene of E. coli (Nevers and Spatz 1975) was assumed.
The actinomycete Nonomuraea sp. ATCC 39727 produces the glycopeptide A40926, the precursor of dal... more The actinomycete Nonomuraea sp. ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by the dbv gene cluster which contains 37 protein coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926 biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and LC-MS analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation of dbv6 had no effect. In addition, over-expression of dbv3 led to higher levels of A40926 production. Transcriptional and quantitative RT-PCR analyses showed that Dbv4 is essential for the transcription of two operons, dbv14-dbv8 and dbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4, dbv29, dbv36, dbv37) and of six operons (dbv2-dbv1, dbv14-dbv8, dbv17-dbv15, dbv21-dbv20, dbv24-dbv28, dbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription of dbv4 and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 regulates directly biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions including the four cross-links, halogenation, glycosylation and acylation. This manuscript expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomycete Nonomuraea sp. ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of Gram positive bacterial skin infections. Therefore, the understanding of the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control other two similar glycopeptides (balhimycin and teicoplanin) have been elucidated and, beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.
We have further characterized the genomic region of Streptomyces coelicolor A3(2) that contains g... more We have further characterized the genomic region of Streptomyces coelicolor A3(2) that contains genes involved in the biosynthesis of histidine. A 2,357-base pair fragment contained in plasmid pSCH3328 that complemented hisD mutations has been sequenced. Computer analysis revealed an open reading frame that encodes a protein with significant homology to the Escherichia coli, Salmonella typhimurium and Mycobacterium smegmatis hisD product, Saccharomyces cerevisiae HIS4C, and Neurospora crassa his3 gene products. Two other contiguous open reading frames oriented divergently with respect to hisD did not show significant similarity with any of the his genes or to other sequences included in the gene bank. S1 nuclease mapping and primer extension experiments indicate that the transcription initiation site of the his-specific mRNA coincides with the GUG translation initiation codon of the hisD cistron.
The 'bald'(bld) mutants of filamentous bacterium Streptomyces coelicolor A3(2) are charac... more The 'bald'(bld) mutants of filamentous bacterium Streptomyces coelicolor A3(2) are characterized by a lack of aerial myceliumand spores. A 'bald' mutant was isolated exhibiting unusual features. It forms slightly sculptured colonies producing a red-orange mycelial pigment, large amounts of agarase and methylenomycin A; it is also highly resistant to U.V. killing. The bld mutation (bld F1) never reverted to bld+ phenotype and was localized very closed to met A.
... production in pleiotropic bld mutants of Streptomyces coelicolor A3(2) ROSA PASSANTINO,ANNA-M... more ... production in pleiotropic bld mutants of Streptomyces coelicolor A3(2) ROSA PASSANTINO,ANNA-MARIA PUGLIA' and KEITH CHATER** ... However, a bald colony producing large amounts of blue pigment (presumed to be actinorhodin) was isolated. ...
ABSTRACT The process leading to gene recombination can be interrupted in the filamentous bacteria... more ABSTRACT The process leading to gene recombination can be interrupted in the filamentous bacteria Streptomyces coelicolor by growing mixed cultures on cellophane disks lying on complete medium. The mycelium is harvested, broken, diluted and the broken hyphae plated at different time intervals. By this means some markers can be excluded from heteroclones or from recombinant progeny in early samples. The recombinant pattern clearly changes with time, with an increase of markers contributed to the recombinant progeny. In crosses between male (NF) and female (UF) strains, the maleness is the first donor trait to appear in the cells of the recipient parent. The fertility factor does not produce a transfer origin on the donor chromosomes; the donor contribution may extend on either side or on both sides of the factor which appears to be compulsory for zygote formation. The longer the time of contact between parental cells, the longer the segment of the donor chromosome contributing to the recombinant progeny. When spores are formed they contain almost exclusively recombinant nuclei derived from segregation processes.
ABSTRACT Initial Fertility (IF) strains of Streptomyces coelicolor are able to convert recipient ... more ABSTRACT Initial Fertility (IF) strains of Streptomyces coelicolor are able to convert recipient strains (UF) to the IF condition by contact, without concomitant transfer of chromosomal markers. The conversion is prevented by the presence of acridine orange in the medium of the mixed culture. Acridine orange is also moderately effective in inducing the formation of UF variants from IF-treated strains. No effect of the drug is observed on UF variant formation from Normal Fertility (NF) strains nor on the behaviour of the fertility factor in NF × UF mixed cultures. The hypothesis is put forward that the fertility factor works as an episome in S. coelicolor, fixed to the chromosome in the NF strains, free in the IF strains and missing in the UF strains.
The molecular mechanisms regulating tryptophan biosynthesis in actinomycetes are poorly understoo... more The molecular mechanisms regulating tryptophan biosynthesis in actinomycetes are poorly understood; similarly, the possible roles of tryptophan in the differentiation program of microorganism life-cycle are still underexplored. To unveil the possible regulatory effect of this amino acid on gene expression, an integrated study based on quantitative teverse transcription-PCR (qRT-PCR) and proteomic approaches was performed on the actinomycete model Streptomyces coelicolor. Comparative analyses on the microorganism growth in a minimal medium with or without tryptophan supplementation showed that biosynthetic trp gene expression in S. coelicolor is not subjected to a negative regulation by the presence of the end product. Conversely, tryptophan specifically induces the transcription of trp genes present in the biosynthetic gene cluster of the calcium-dependent antibiotic (CDA), a lipopeptide containing D- and L-tryptophan residues. In addition, tryptophan stimulates the transcription of...
We report the genome sequence of Planobispora rosea ATCC 53733, a mycelium-forming soil-dweller b... more We report the genome sequence of Planobispora rosea ATCC 53733, a mycelium-forming soil-dweller belonging to one of the lesser studied genera of Actinobacteria and producing the thiopeptide GE2270. The P. rosea genome presents considerable convergence in gene organization and function with other members in the family Streptosporangiaceae, with a significant number (44%) of shared orthologs. Patterns of gene expression in P. rosea cultures during exponential and stationary phase have been analyzed using whole transcriptome shotgun sequencing and by proteome analysis. Among the differentially abundant proteins, those involved in protein metabolism are particularly represented, including the GE2270-insensitive EF-Tu. Two proteins from the pbt cluster, directing GE2270 biosynthesis, slightly increase their abundance values over time. While GE2270 production starts during the exponential phase, most pbt genes, as analyzed by qRT-PCR, are down-regulated. The exception is represented by pbtA, encoding the precursor peptide of the ribosomally synthesized GE2270, whose expression reached the highest level at the entry into stationary phase.
A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plas... more A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plasmid SCP1 susceptible to curing by UV irradiation. Lack of the SCP1 plasmid (in SCP1(-) strains) prevented the occurrence of the co-mutation process after MNNG treatment, although it left the susceptibility to the lethal effect of the mutagen virtually unaffected. SCP1(-) strains were, however, ultrasensitive to the lethal effect of UV. Curing a plc A(-) strain of its SCPI plasmid made it refractory to co-mutation by MNNG and sensitive to the lethal effect of UV; reinfected by the plasmid, the strain resumed both the co-mutation proficiency and the UV-resistance. The occurrence on the SCP1 plasmid of a gene comparable to the uvrE gene of E. coli (Nevers and Spatz 1975) was assumed.
Actinomycetes, filamentous Gram-positive bacteria, are usually exploited as bio-farms naturally p... more Actinomycetes, filamentous Gram-positive bacteria, are usually exploited as bio-farms naturally producing a wide range of small biologically active metabolites, such as antibiotics, extensively used in medicine, food-industry, chemistry and bio-remediation strategies. The development of high throughput technologies, like proteomics, allows functional genomic studies aimed at shedding light on molecular mechanisms controlling the production of useful compounds and macromolecules. Differential proteomic analyses, performed by using Two Dimensional PolyAcrylamide Gel Electrophoresis (2D-PAGE) coupled to mass spectrometry (MS) procedures, revealed novel links between balhimycin production (a vancomycin-like antibiotic) and metabolic pathway regulation in Amycolatopsis balhimycina DSM5908. In particular, our investigation, performed by combining data from differential proteomic analyses carried-out using wild-type (Wt) and non-producing strains incubated in different growth conditions, s...
Eight structurally related halogenated aliphatic hydrocarbons mono-, di- and trichloroacetaldehyd... more Eight structurally related halogenated aliphatic hydrocarbons mono-, di- and trichloroacetaldehyde (the last in the anhydrous and hydrate form), moni-, di- and trichloroethanol and allyl chloride, were tested for their ability to induce gene mutations in prokaryotic and eukaryotic microorganisms. The genetic systems employed were the Salmonella reversion test with strain TA1535 and TA100, with and without metabolic activation, a forward and a back-mutation system in S. coelicolor and two forward mutation systems in A. nidulans. Each compound was tested with the spot and plate incorporation assay techniques. Mono-, di- and trichloroacetaldehyde were mutagenic in all the microorganisms employed; all the halogenated ethanols were positive in A. nidulans, while in S. typhimurium and in S. coelicolor the only active forms were respectively the mono- and dichloroderivatives. Allyl chloride was active in S. typhimurium and S. coelicolor and negative in A. nidulans. The technical approach a...
A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plas... more A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plasmid SCP1 susceptible to curing by UV irradiation. Lack of the SCP1 plasmid (in SCP1(-) strains) prevented the occurrence of the co-mutation process after MNNG treatment, although it left the susceptibility to the lethal effect of the mutagen virtually unaffected. SCP1(-) strains were, however, ultrasensitive to the lethal effect of UV. Curing a plc A(-) strain of its SCPI plasmid made it refractory to co-mutation by MNNG and sensitive to the lethal effect of UV; reinfected by the plasmid, the strain resumed both the co-mutation proficiency and the UV-resistance. The occurrence on the SCP1 plasmid of a gene comparable to the uvrE gene of E. coli (Nevers and Spatz 1975) was assumed.
The actinomycete Nonomuraea sp. ATCC 39727 produces the glycopeptide A40926, the precursor of dal... more The actinomycete Nonomuraea sp. ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by the dbv gene cluster which contains 37 protein coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926 biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and LC-MS analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation of dbv6 had no effect. In addition, over-expression of dbv3 led to higher levels of A40926 production. Transcriptional and quantitative RT-PCR analyses showed that Dbv4 is essential for the transcription of two operons, dbv14-dbv8 and dbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4, dbv29, dbv36, dbv37) and of six operons (dbv2-dbv1, dbv14-dbv8, dbv17-dbv15, dbv21-dbv20, dbv24-dbv28, dbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription of dbv4 and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 regulates directly biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions including the four cross-links, halogenation, glycosylation and acylation. This manuscript expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomycete Nonomuraea sp. ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of Gram positive bacterial skin infections. Therefore, the understanding of the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control other two similar glycopeptides (balhimycin and teicoplanin) have been elucidated and, beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.
We have further characterized the genomic region of Streptomyces coelicolor A3(2) that contains g... more We have further characterized the genomic region of Streptomyces coelicolor A3(2) that contains genes involved in the biosynthesis of histidine. A 2,357-base pair fragment contained in plasmid pSCH3328 that complemented hisD mutations has been sequenced. Computer analysis revealed an open reading frame that encodes a protein with significant homology to the Escherichia coli, Salmonella typhimurium and Mycobacterium smegmatis hisD product, Saccharomyces cerevisiae HIS4C, and Neurospora crassa his3 gene products. Two other contiguous open reading frames oriented divergently with respect to hisD did not show significant similarity with any of the his genes or to other sequences included in the gene bank. S1 nuclease mapping and primer extension experiments indicate that the transcription initiation site of the his-specific mRNA coincides with the GUG translation initiation codon of the hisD cistron.
The 'bald'(bld) mutants of filamentous bacterium Streptomyces coelicolor A3(2) are charac... more The 'bald'(bld) mutants of filamentous bacterium Streptomyces coelicolor A3(2) are characterized by a lack of aerial myceliumand spores. A 'bald' mutant was isolated exhibiting unusual features. It forms slightly sculptured colonies producing a red-orange mycelial pigment, large amounts of agarase and methylenomycin A; it is also highly resistant to U.V. killing. The bld mutation (bld F1) never reverted to bld+ phenotype and was localized very closed to met A.
... production in pleiotropic bld mutants of Streptomyces coelicolor A3(2) ROSA PASSANTINO,ANNA-M... more ... production in pleiotropic bld mutants of Streptomyces coelicolor A3(2) ROSA PASSANTINO,ANNA-MARIA PUGLIA' and KEITH CHATER** ... However, a bald colony producing large amounts of blue pigment (presumed to be actinorhodin) was isolated. ...
ABSTRACT The process leading to gene recombination can be interrupted in the filamentous bacteria... more ABSTRACT The process leading to gene recombination can be interrupted in the filamentous bacteria Streptomyces coelicolor by growing mixed cultures on cellophane disks lying on complete medium. The mycelium is harvested, broken, diluted and the broken hyphae plated at different time intervals. By this means some markers can be excluded from heteroclones or from recombinant progeny in early samples. The recombinant pattern clearly changes with time, with an increase of markers contributed to the recombinant progeny. In crosses between male (NF) and female (UF) strains, the maleness is the first donor trait to appear in the cells of the recipient parent. The fertility factor does not produce a transfer origin on the donor chromosomes; the donor contribution may extend on either side or on both sides of the factor which appears to be compulsory for zygote formation. The longer the time of contact between parental cells, the longer the segment of the donor chromosome contributing to the recombinant progeny. When spores are formed they contain almost exclusively recombinant nuclei derived from segregation processes.
ABSTRACT Initial Fertility (IF) strains of Streptomyces coelicolor are able to convert recipient ... more ABSTRACT Initial Fertility (IF) strains of Streptomyces coelicolor are able to convert recipient strains (UF) to the IF condition by contact, without concomitant transfer of chromosomal markers. The conversion is prevented by the presence of acridine orange in the medium of the mixed culture. Acridine orange is also moderately effective in inducing the formation of UF variants from IF-treated strains. No effect of the drug is observed on UF variant formation from Normal Fertility (NF) strains nor on the behaviour of the fertility factor in NF × UF mixed cultures. The hypothesis is put forward that the fertility factor works as an episome in S. coelicolor, fixed to the chromosome in the NF strains, free in the IF strains and missing in the UF strains.
The molecular mechanisms regulating tryptophan biosynthesis in actinomycetes are poorly understoo... more The molecular mechanisms regulating tryptophan biosynthesis in actinomycetes are poorly understood; similarly, the possible roles of tryptophan in the differentiation program of microorganism life-cycle are still underexplored. To unveil the possible regulatory effect of this amino acid on gene expression, an integrated study based on quantitative teverse transcription-PCR (qRT-PCR) and proteomic approaches was performed on the actinomycete model Streptomyces coelicolor. Comparative analyses on the microorganism growth in a minimal medium with or without tryptophan supplementation showed that biosynthetic trp gene expression in S. coelicolor is not subjected to a negative regulation by the presence of the end product. Conversely, tryptophan specifically induces the transcription of trp genes present in the biosynthetic gene cluster of the calcium-dependent antibiotic (CDA), a lipopeptide containing D- and L-tryptophan residues. In addition, tryptophan stimulates the transcription of...
We report the genome sequence of Planobispora rosea ATCC 53733, a mycelium-forming soil-dweller b... more We report the genome sequence of Planobispora rosea ATCC 53733, a mycelium-forming soil-dweller belonging to one of the lesser studied genera of Actinobacteria and producing the thiopeptide GE2270. The P. rosea genome presents considerable convergence in gene organization and function with other members in the family Streptosporangiaceae, with a significant number (44%) of shared orthologs. Patterns of gene expression in P. rosea cultures during exponential and stationary phase have been analyzed using whole transcriptome shotgun sequencing and by proteome analysis. Among the differentially abundant proteins, those involved in protein metabolism are particularly represented, including the GE2270-insensitive EF-Tu. Two proteins from the pbt cluster, directing GE2270 biosynthesis, slightly increase their abundance values over time. While GE2270 production starts during the exponential phase, most pbt genes, as analyzed by qRT-PCR, are down-regulated. The exception is represented by pbtA, encoding the precursor peptide of the ribosomally synthesized GE2270, whose expression reached the highest level at the entry into stationary phase.
A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plas... more A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plasmid SCP1 susceptible to curing by UV irradiation. Lack of the SCP1 plasmid (in SCP1(-) strains) prevented the occurrence of the co-mutation process after MNNG treatment, although it left the susceptibility to the lethal effect of the mutagen virtually unaffected. SCP1(-) strains were, however, ultrasensitive to the lethal effect of UV. Curing a plc A(-) strain of its SCPI plasmid made it refractory to co-mutation by MNNG and sensitive to the lethal effect of UV; reinfected by the plasmid, the strain resumed both the co-mutation proficiency and the UV-resistance. The occurrence on the SCP1 plasmid of a gene comparable to the uvrE gene of E. coli (Nevers and Spatz 1975) was assumed.
Actinomycetes, filamentous Gram-positive bacteria, are usually exploited as bio-farms naturally p... more Actinomycetes, filamentous Gram-positive bacteria, are usually exploited as bio-farms naturally producing a wide range of small biologically active metabolites, such as antibiotics, extensively used in medicine, food-industry, chemistry and bio-remediation strategies. The development of high throughput technologies, like proteomics, allows functional genomic studies aimed at shedding light on molecular mechanisms controlling the production of useful compounds and macromolecules. Differential proteomic analyses, performed by using Two Dimensional PolyAcrylamide Gel Electrophoresis (2D-PAGE) coupled to mass spectrometry (MS) procedures, revealed novel links between balhimycin production (a vancomycin-like antibiotic) and metabolic pathway regulation in Amycolatopsis balhimycina DSM5908. In particular, our investigation, performed by combining data from differential proteomic analyses carried-out using wild-type (Wt) and non-producing strains incubated in different growth conditions, s...
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