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Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques.

Petříčková K, Chroňáková A, Zelenka T, Chrudimský T, Pospíšil S, Petříček M, Krištůfek V - Front Microbiol (2015)

Bottom Line: Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains.Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms.Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, Czech Academy of Sciences, v. v. i. Prague, Czech Republic.

ABSTRACT
A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

No MeSH data available.


Related in: MedlinePlus

Cladogram of hemA was constructed as specified in the Section “Materials and Methods,” posterior probability values are given when higher than 0.90. All branches encompassing genes encoding putative cyclizing type of ALAS (cALAS) are color-shaded, types of secondary metabolites based on characterized producers are indicated in each branch. Reference sequences – genes of primary metabolism ALASs (“blue”) and of secondary metabolite-connected cALASs (“purple” and “green”), are shown in bold. Orange branch covers strains carrying cALAS-encoding hemA genes without a close phylogenetic relationship to any characterized producer strains. Original organisms are abbreviated as follows: S. – Streptomyces, K. – Kitasatospora, A. – Amycolatopsis, N. – Nocardia, Sac. – Saccharothrix, P. – Pseudomonas, Ch. – Chromobacterium, R. – Rhodobacter, Asp. – Aspergillus, Schiz. – Schizosaccharomyces, H. – Homo, M. – Mus. Organization of the C5N biosynthetic gene triad, encoding amide synthase (AMS), cALAS, and aminolevulinate ligase (ALL), conserved within the two major producer groups, designated as “green” and “purple,” is illustrated.
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Figure 2: Cladogram of hemA was constructed as specified in the Section “Materials and Methods,” posterior probability values are given when higher than 0.90. All branches encompassing genes encoding putative cyclizing type of ALAS (cALAS) are color-shaded, types of secondary metabolites based on characterized producers are indicated in each branch. Reference sequences – genes of primary metabolism ALASs (“blue”) and of secondary metabolite-connected cALASs (“purple” and “green”), are shown in bold. Orange branch covers strains carrying cALAS-encoding hemA genes without a close phylogenetic relationship to any characterized producer strains. Original organisms are abbreviated as follows: S. – Streptomyces, K. – Kitasatospora, A. – Amycolatopsis, N. – Nocardia, Sac. – Saccharothrix, P. – Pseudomonas, Ch. – Chromobacterium, R. – Rhodobacter, Asp. – Aspergillus, Schiz. – Schizosaccharomyces, H. – Homo, M. – Mus. Organization of the C5N biosynthetic gene triad, encoding amide synthase (AMS), cALAS, and aminolevulinate ligase (ALL), conserved within the two major producer groups, designated as “green” and “purple,” is illustrated.

Mentions: Sequences of all hemA gene homologs, identified by genome scanning of actinomycete genomic data using BLASTP search with AsuD2 cALAS as a query and listed in Supplementary Table S3, were added to the alignment and a cladogram constructed to illustrate phylogenetic relationships among the genes. Referential genes of the characterized producers (type producer strains) and a few representatives of primary metabolism hemA genes were also included. As expected, the cALAS-encoding representatives were clearly separated from “classical” ALAS. As C5N-carrying compounds display remarkable structural variability and diversity of relevant genetic information, we expected hemA genes evolution to follow that of entire biosynthetic gene clusters. Indeed, the genes show apparent clustering based on the type of C5N-containing compound produced (Figure 2). Two major producer groups were identified: The first, designated as “a purple group,” encompasses producers of simple linear polyketides and reductiomycin, which are characterized by head-to-tail organization of the C5N-encoding gene triplet ams-hemA-all and by direct attachment of the C5N unit to a polyketide chain. The second, “a green group,” represents producers of complex compounds such as sugar-containing polyketides or macrolides. In this case, the hemA gene is located upstream of the ams-all pair in the divergent orientation (Figures 2 and 4). Only two putative hemA loci of Saccharothrix espanaensis show different arrangement of the C5N-encoding genes (discussed later, Figure 6B). Contrary to original expectations, substantial fraction of identified genes (15%) clustered clearly with typical primary metabolism hemA representatives and their products did not show cALAS sequence characteristics.


Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques.

Petříčková K, Chroňáková A, Zelenka T, Chrudimský T, Pospíšil S, Petříček M, Krištůfek V - Front Microbiol (2015)

Cladogram of hemA was constructed as specified in the Section “Materials and Methods,” posterior probability values are given when higher than 0.90. All branches encompassing genes encoding putative cyclizing type of ALAS (cALAS) are color-shaded, types of secondary metabolites based on characterized producers are indicated in each branch. Reference sequences – genes of primary metabolism ALASs (“blue”) and of secondary metabolite-connected cALASs (“purple” and “green”), are shown in bold. Orange branch covers strains carrying cALAS-encoding hemA genes without a close phylogenetic relationship to any characterized producer strains. Original organisms are abbreviated as follows: S. – Streptomyces, K. – Kitasatospora, A. – Amycolatopsis, N. – Nocardia, Sac. – Saccharothrix, P. – Pseudomonas, Ch. – Chromobacterium, R. – Rhodobacter, Asp. – Aspergillus, Schiz. – Schizosaccharomyces, H. – Homo, M. – Mus. Organization of the C5N biosynthetic gene triad, encoding amide synthase (AMS), cALAS, and aminolevulinate ligase (ALL), conserved within the two major producer groups, designated as “green” and “purple,” is illustrated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525017&req=5

Figure 2: Cladogram of hemA was constructed as specified in the Section “Materials and Methods,” posterior probability values are given when higher than 0.90. All branches encompassing genes encoding putative cyclizing type of ALAS (cALAS) are color-shaded, types of secondary metabolites based on characterized producers are indicated in each branch. Reference sequences – genes of primary metabolism ALASs (“blue”) and of secondary metabolite-connected cALASs (“purple” and “green”), are shown in bold. Orange branch covers strains carrying cALAS-encoding hemA genes without a close phylogenetic relationship to any characterized producer strains. Original organisms are abbreviated as follows: S. – Streptomyces, K. – Kitasatospora, A. – Amycolatopsis, N. – Nocardia, Sac. – Saccharothrix, P. – Pseudomonas, Ch. – Chromobacterium, R. – Rhodobacter, Asp. – Aspergillus, Schiz. – Schizosaccharomyces, H. – Homo, M. – Mus. Organization of the C5N biosynthetic gene triad, encoding amide synthase (AMS), cALAS, and aminolevulinate ligase (ALL), conserved within the two major producer groups, designated as “green” and “purple,” is illustrated.
Mentions: Sequences of all hemA gene homologs, identified by genome scanning of actinomycete genomic data using BLASTP search with AsuD2 cALAS as a query and listed in Supplementary Table S3, were added to the alignment and a cladogram constructed to illustrate phylogenetic relationships among the genes. Referential genes of the characterized producers (type producer strains) and a few representatives of primary metabolism hemA genes were also included. As expected, the cALAS-encoding representatives were clearly separated from “classical” ALAS. As C5N-carrying compounds display remarkable structural variability and diversity of relevant genetic information, we expected hemA genes evolution to follow that of entire biosynthetic gene clusters. Indeed, the genes show apparent clustering based on the type of C5N-containing compound produced (Figure 2). Two major producer groups were identified: The first, designated as “a purple group,” encompasses producers of simple linear polyketides and reductiomycin, which are characterized by head-to-tail organization of the C5N-encoding gene triplet ams-hemA-all and by direct attachment of the C5N unit to a polyketide chain. The second, “a green group,” represents producers of complex compounds such as sugar-containing polyketides or macrolides. In this case, the hemA gene is located upstream of the ams-all pair in the divergent orientation (Figures 2 and 4). Only two putative hemA loci of Saccharothrix espanaensis show different arrangement of the C5N-encoding genes (discussed later, Figure 6B). Contrary to original expectations, substantial fraction of identified genes (15%) clustered clearly with typical primary metabolism hemA representatives and their products did not show cALAS sequence characteristics.

Bottom Line: Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains.Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms.Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, Czech Academy of Sciences, v. v. i. Prague, Czech Republic.

ABSTRACT
A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

No MeSH data available.


Related in: MedlinePlus