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Genetic approach for the fast discovery of phenazine producing bacteria.

Schneemann I, Wiese J, Kunz AL, Imhoff JF - Mar Drugs (2011)

Bottom Line: The genetic potential for phenazine production was shown for four type strains belonging to the genera Streptomyces and Pseudomonas as well as for 13 environmental isolates from marine habitats.Phenazine production was demonstrated for the type strains known to produce endophenazines, 2-hydroxy-phenazine, phenazine-1-carboxylic acid, phenazine-1,6-dicarboxylic acid, and chlororaphin as well as for members of marine Actinobacteria.Interestingly, a number of unidentified phenazines possibly represent new phenazine structures.

View Article: PubMed Central - PubMed

Affiliation: Kieler Wirkstoff-Zentrum (KiWiZ) am Leibniz-Institut für Meereswissenschaften (IFM-GEOMAR), Am Kiel-Kanal 44, 24106 Kiel, Germany.

ABSTRACT
A fast and efficient approach was established to identify bacteria possessing the potential to biosynthesize phenazines, which are of special interest regarding their antimicrobial activities. Sequences of phzE genes, which are part of the phenazine biosynthetic pathway, were used to design one universal primer system and to analyze the ability of bacteria to produce phenazine. Diverse bacteria from different marine habitats and belonging to six major phylogenetic lines were investigated. Bacteria exhibiting phzE gene fragments affiliated to Firmicutes, Alpha- and Gammaproteobacteria, and Actinobacteria. Thus, these are the first primers for amplifying gene fragments from Firmicutes and Alphaproteobacteria. The genetic potential for phenazine production was shown for four type strains belonging to the genera Streptomyces and Pseudomonas as well as for 13 environmental isolates from marine habitats. For the first time, the genetic ability of phenazine biosynthesis was verified by analyzing the metabolite pattern of all PCR-positive strains via HPLC-UV/MS. Phenazine production was demonstrated for the type strains known to produce endophenazines, 2-hydroxy-phenazine, phenazine-1-carboxylic acid, phenazine-1,6-dicarboxylic acid, and chlororaphin as well as for members of marine Actinobacteria. Interestingly, a number of unidentified phenazines possibly represent new phenazine structures.

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Alignment of known phzE gene sequences. Marked blocks served as the basis for primer construction.
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f2-marinedrugs-09-00772: Alignment of known phzE gene sequences. Marked blocks served as the basis for primer construction.

Mentions: Former investigations were able to associate different steps of the phenazine biosynthetic pathway with the corresponding genes, e.g., phzC, phzD, phzE and phzF. The transformation from chorismate to 2-amino-2-deoxyisochorismic acid (ADIC) is necessary for the formation of the core structure of phenazines and is catalyzed by the enzyme PhzE. Thus, PhzE is a key enzyme in phenazine biosynthesis and the corresponding gene phzE is suitable for primer design. Sequences from the phenazine biosynthetic pathway for Alpha-, Beta- and Gammaproteobacteria, Actinomycetes and Firmicutes are available at the homepage of the National Centre for Biotechnology Information (NCBI) and known from literature [29,30]. To ensure the inclusion of only true phenazine sequences, oligonucleotide primers were constructed only from those genes known to be involved in the biosynthesis of corresponding chemical substances. Two conserved sites occurred within the alignment of phzE sequences (Figure 2), which had a distance to each other to produce fragments of an appropriate length. The degenerated primers phzEf (5′-GAA GGC GCC AAC TTC GTY ATC AA-3′) and phzEr (5′-GCC YTC GAT GAA GTA CTC GGT GTG-3′) were designed to amplify a highly conserved stretch of the phzE gene of approximately 450 bp. The comparison of the oligonucleotide sequences from designed phzEf and phzEr primers with known phenazine genes verified this stretch as highly specific for phzE genes. Because the basic phenazine gene cluster including the phzE gene is highly conserved and derivatization of the basic phenazine structure are made at a later stage in the biosynthesis, the constructed phzE primers are expected to detect genes of a large variety of different phenazine structures and are appropriate to search for unknown bacteria producing novel phenazines.


Genetic approach for the fast discovery of phenazine producing bacteria.

Schneemann I, Wiese J, Kunz AL, Imhoff JF - Mar Drugs (2011)

Alignment of known phzE gene sequences. Marked blocks served as the basis for primer construction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3111181&req=5

f2-marinedrugs-09-00772: Alignment of known phzE gene sequences. Marked blocks served as the basis for primer construction.
Mentions: Former investigations were able to associate different steps of the phenazine biosynthetic pathway with the corresponding genes, e.g., phzC, phzD, phzE and phzF. The transformation from chorismate to 2-amino-2-deoxyisochorismic acid (ADIC) is necessary for the formation of the core structure of phenazines and is catalyzed by the enzyme PhzE. Thus, PhzE is a key enzyme in phenazine biosynthesis and the corresponding gene phzE is suitable for primer design. Sequences from the phenazine biosynthetic pathway for Alpha-, Beta- and Gammaproteobacteria, Actinomycetes and Firmicutes are available at the homepage of the National Centre for Biotechnology Information (NCBI) and known from literature [29,30]. To ensure the inclusion of only true phenazine sequences, oligonucleotide primers were constructed only from those genes known to be involved in the biosynthesis of corresponding chemical substances. Two conserved sites occurred within the alignment of phzE sequences (Figure 2), which had a distance to each other to produce fragments of an appropriate length. The degenerated primers phzEf (5′-GAA GGC GCC AAC TTC GTY ATC AA-3′) and phzEr (5′-GCC YTC GAT GAA GTA CTC GGT GTG-3′) were designed to amplify a highly conserved stretch of the phzE gene of approximately 450 bp. The comparison of the oligonucleotide sequences from designed phzEf and phzEr primers with known phenazine genes verified this stretch as highly specific for phzE genes. Because the basic phenazine gene cluster including the phzE gene is highly conserved and derivatization of the basic phenazine structure are made at a later stage in the biosynthesis, the constructed phzE primers are expected to detect genes of a large variety of different phenazine structures and are appropriate to search for unknown bacteria producing novel phenazines.

Bottom Line: The genetic potential for phenazine production was shown for four type strains belonging to the genera Streptomyces and Pseudomonas as well as for 13 environmental isolates from marine habitats.Phenazine production was demonstrated for the type strains known to produce endophenazines, 2-hydroxy-phenazine, phenazine-1-carboxylic acid, phenazine-1,6-dicarboxylic acid, and chlororaphin as well as for members of marine Actinobacteria.Interestingly, a number of unidentified phenazines possibly represent new phenazine structures.

View Article: PubMed Central - PubMed

Affiliation: Kieler Wirkstoff-Zentrum (KiWiZ) am Leibniz-Institut für Meereswissenschaften (IFM-GEOMAR), Am Kiel-Kanal 44, 24106 Kiel, Germany.

ABSTRACT
A fast and efficient approach was established to identify bacteria possessing the potential to biosynthesize phenazines, which are of special interest regarding their antimicrobial activities. Sequences of phzE genes, which are part of the phenazine biosynthetic pathway, were used to design one universal primer system and to analyze the ability of bacteria to produce phenazine. Diverse bacteria from different marine habitats and belonging to six major phylogenetic lines were investigated. Bacteria exhibiting phzE gene fragments affiliated to Firmicutes, Alpha- and Gammaproteobacteria, and Actinobacteria. Thus, these are the first primers for amplifying gene fragments from Firmicutes and Alphaproteobacteria. The genetic potential for phenazine production was shown for four type strains belonging to the genera Streptomyces and Pseudomonas as well as for 13 environmental isolates from marine habitats. For the first time, the genetic ability of phenazine biosynthesis was verified by analyzing the metabolite pattern of all PCR-positive strains via HPLC-UV/MS. Phenazine production was demonstrated for the type strains known to produce endophenazines, 2-hydroxy-phenazine, phenazine-1-carboxylic acid, phenazine-1,6-dicarboxylic acid, and chlororaphin as well as for members of marine Actinobacteria. Interestingly, a number of unidentified phenazines possibly represent new phenazine structures.

Show MeSH