Genetic Approach for the Fast Discovery of Phenazine Producing Bacteria
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design of Oligonucleotides to Search for phzE Phenazine Gene Fragments
2.2. Screening for phzE Gene Fragments with the Constructed Primers
2.3. Detection of Phenazines in the phzE Positive Strains
3. Experimental Section
3.1. Bacterial Strains and Their Phylogenetic Affiliation
3.2. Design of Oligonucleotides for Molecular Detection of phzE Phenazine Gene Fragments
3.3. Amplification and Identification of the Phenazine Gene Fragments
3.4. Cultivation of phzE Strains
3.4.1. Cultivation of phzE Positive Strain
3.4.2. Cultivation of phzE Negative Strains
3.5. Culture Extracts of phzE Positive and Negative Strains
3.6. Chemical Analysis of phzE Positive and Negative Strains
3.7. Nucleotide Sequence Accession Numbers
4. Conclusions
Acknowledgments
- Samples Availability: Available from the authors.
References
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Phylogenetic group | Number of strains | ||
---|---|---|---|
Analyzed | PCR amplification phzE gene positive | Producing phenazines in culture | |
Actinobacteriaa | 76 | 11 | 11 |
Bacteroidetes | 2 | 0 | 0 |
Firmicutes | 28 | 1 | 0 |
Alphaproteobacteria | 21 | 2 | 0 |
Betaproteobacteria | 2 | 0 | 0 |
Gammaproteobacteriab | 39 | 3 | 3 |
In total | 168 | 17 | 14 |
Strain no. | Next relative type strain and acc.-no.a | Phylumb | Sequence lengthc | Related phenazine gene, acc.-no.d, similarity and producer |
---|---|---|---|---|
Strains used as DSM 1042T | Positive control Streptomyces cinnamonensis DSM 1042T; DQ462657 | A | 127 | phzE; CAL34110; 100%; S. cinnamonenis |
DSM 6698T | Pseudomonas chlororaphis subsp. aureofaciens DSM 6698T; AY509898 | GP | 139 | phzE: ADP21173; 100% P. chlororaphis phzF: ADP21174; 100% P. chlororaphis |
DSM 19603T | Pseudomonas chlororaphis subsp. aurantiaca DSM 19603T; DQ682655 | GP | 137 | phzE: ADP21173; 98% P. chlororaphis phzF: ADP21174 49% P. chlororaphis |
DSM 50083T | Pseudomonas chlororaphis subsp. chlororaphis DSM 50083T; Z76673 | GP | 125 | phzE; AAF17499; 92% P. chlororaphis phzF: AAF17500; 99% P. chlororaphis |
Environmental | Isolates | |||
AB108 | Pseudovibrio ascidiaceicola F423T; AB175663 | AP | 144 | phzE; CAL34110; 95%; S. cinnamonenis |
HB117 | Streptomyces fulvorobeus LMG 19901T; AJ781331 | A | 141 | phzE; AAF17499; 73%; P. chlororaphis |
HB122 | Streptomyces luridiscabiei S63T AF361784 | A | 141 | phzE; AAF17499; 74%; P. chlororaphis |
HB202 | Streptomyces mediolani LMG 20093T; AJ781354 | A | 91 | phzE; NP_252903; 84%; P. aeruginosa |
HB253 | Micromonospora matsumotoense IMSNU 22003T; AF152109 | A | 144 | phzB; AAF17496; 67%; P. chlororaphis |
HB254 | Micromonospora matsumotoense IMSNU 22003T; AF152109 | A | 140 | phzE; AAF17499; 73%; P. chlororaphis |
HB291 | Streptomyces fulvorobeus LMG 19901T; AJ781331 | A | 140 | phzE; AAF17499; 73%; P. chlororaphis |
LB066 | Kiloniella laminariae LD81T; AM749667 | AL | 132 | phzE; CAL34110; 91%; S. cinnamonenis |
LB114 | Streptomyces flavogriseus DSM 40323T; AJ494864 | A | 141 | phzE; AAF17499; 79%; P. chlororaphis |
LB129 | Streptomyces fimicarius ISP 5322T; AY999784 | A | 145 | phzB; AAF17496; 75%; P. chlororaphis |
LB150 | Streptomyces luridiscabiei S63T; AF361784 | A | 132 | phzB; AAF17496; 74%; P. chlororaphis |
LB151 | Streptomyces griseus ATCC51928T; AF112160 | A | 133 | phzE; AAF17499; 65%; P. chlororaphis |
Strain no. | Next relative type strain | [M+] | UV absorption maxima (nm)a | Dereplication of phenazines |
---|---|---|---|---|
Strains used as DSM 1042T | positive control Streptomyces cinnamonensis DSM 1042T | 206 | 327, 249, 212 | no hit in database |
224 | 371, 249, 215 | phenazine-1-carboxylic acid [41] | ||
268 | 375, 256, 223 | phenazine-1,6-dicarboxylic acid [15] | ||
292 | 371, 254, 214 | endophenazine A [15] c | ||
306 | 387, 269, 211 | no hit in database | ||
308 | 372, 249, 212 | endophenazine C [15] | ||
322 | 375, 256, 223 | endophenazine B [15]c | ||
336 | 372, 249, 212 | no hit in database | ||
DSM 6698T | Pseudomonas chlororaphis subsp. aureofaciens DSM 6698T | 196 | 368, 257, 219 | 2-hydroxy-phenazine [42] |
224 | 371, 249, 215 | phenazine-1-carboxylic acid [41] | ||
DSM 19603 T | Pseudomonas chlororaphis subsp. aurantiaca DSM 19603T | 196 | 368, 257, 219 | 2-hydroxy-phenazine [42]c |
224 | 371, 249, 215 | phenazine-1-carboxylic acid [41]c | ||
DSM 50083 T | Pseudomonas chlororaphis subsp. chlororaphis DSM 50083T | 223 | 370, 248, 213 | chlororaphin [43] |
Environmental | Isolates | |||
HB117 | Streptomyces fulvorobeus LMG 19901T | 494 | 370(br), 274, 224 | Senacarcin A |
512 | 370(br), 275, 230 | saphenyl ester D [29] | ||
HB122 | Streptomyces luridiscabiei S63T | 492 | 376, 275, 235sh | saphenyl ester D [29] |
496 | 438sh, 383(br), 276, 227 | no hit in database | ||
498 | 419sh, 393-325, 289, 253sh, 220 | no hit in database | ||
508 | 376, 275, 235sh | no hit in database | ||
510 | 430(br), 325, 224 | derivative of aestivophoenin C [44] | ||
512 | 432(br), 327, 226 | aestivophoenin C [44] | ||
HB202 | Streptomyces mediolani LMG 20093T | 396 | 368, 364sh, 351sh, 252, 218 | streptophenazines E [39]c |
410 | 371, 364sh, 354sh, 252, 213 | streptophenazines C [39] | ||
410 | 368, 364sh, 351sh. 252, 218 | streptophenazines D [39]c | ||
424 | 367, 363sh, 350sh, 252, 215 | streptophenazines A [39] | ||
424 | 368, 364sh, 351sh, 252, 218 | streptophenazines B [39]c | ||
438 | 368, 364sh, 353sh, 252, 215 | streptophenazines F [39]c | ||
438 | 368, 363sh, 351sh, 252, 214 | streptophenazines G [39] | ||
440 | 368, 363sh, 352sh, 252, 215 | streptophenazines H [39] | ||
HB253 | Micromonospora matsumotoense IMSNU 22003T | 260 | 458, 302sh, 261, 232 | no hit in database |
465 | 362sh, 345, 299, 221 | no hit in database | ||
566 | 362sh, 345, 299, 221 | no hit in database | ||
HB254 | Micromonospora matsumotoense IMSNU 22003T | 451 | 361, 343, 352, 301, 223 | no hit in database |
HB291 | Streptomyces fulvorobeus LMG 19901T | 492 | 376, 275, 235sh | saphenyl ester D [29] |
496 | 438sh, 383(br), 276, 227 | no hit in database | ||
498 | 419sh, 393-325, 289, 253sh, 220 | no hit in database | ||
508 | 376, 275, 235sh | no hit in database | ||
510 | 430(br), 325, 224 | derivative of aestivophoenin C [44] | ||
512 | 432(br), 327, 226 | aestivophoenin C [44] | ||
LB114 | Streptomyces flavogriseus DSM 40323T | n.d.b | 370, 270, 244 | no hit in database |
n.d.b | 419, 367, 305, 228 | no hit in database | ||
LB129 | Streptomyces fimicarius ISP 5322T | 296 | 366, 249, 214 | phencomycin methyl ester [9] |
238 | 366, 249, 214 | 1-carboxymethyl phenazine | ||
LB150 | Streptomyces luridiscabiei S63T | 510 | sh401, 378, 274, 227 | no hit in database |
LB151 | Streptomyces griseus ATCC 51928T | 510 | sh401, 378, 274, 227 | no hit in database |
Primer | Sequence | Function | Ref. |
---|---|---|---|
27f | 5′-GAGTTTGATCCTGGCTCAG-3′ | PCR of the 16S rRNA gene | [54] |
1492r | 5′-GGTTACCTTGTTACGACTT-3′ | PCR of the 16S rRNA gene | [54] |
534r | 5′-ATTACCGCGGCTGCTGG-3′ | Sequencing of the 16S rRNA gene | [55] |
342f | 5′-TACGGGAGGCAGCAG-3′ | sequencing of the 16S rRNA gene | [55] |
790f | 5′-GATACCCTGGTAGTCC-3′ | sequencing of the 16S rRNA gene | [50] |
phzEf | 5′-GAAGGCGCCAACTTCGTYATCAA-3′ | PCR and sequencing of phzE gene | this study |
phzEr | 5′-GCCYTCGATGAAGTACTCGGTGTG-3′ | PCR and sequencing of phzE gene | this study |
Ps_up1 | 5′-ATCTTCACCCCGGTCAACG-3′ | PCR and sequencing of phzF gene | [21] |
Ps_low1 | 5′-CCRTAGGCCGGTGAGAAC-3′ | PCR and sequencing of phzF gene | [21] |
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Schneemann, I.; Wiese, J.; Kunz, A.L.; Imhoff, J.F. Genetic Approach for the Fast Discovery of Phenazine Producing Bacteria. Mar. Drugs 2011, 9, 772-789. https://doi.org/10.3390/md9050772
Schneemann I, Wiese J, Kunz AL, Imhoff JF. Genetic Approach for the Fast Discovery of Phenazine Producing Bacteria. Marine Drugs. 2011; 9(5):772-789. https://doi.org/10.3390/md9050772
Chicago/Turabian StyleSchneemann, Imke, Jutta Wiese, Anna Lena Kunz, and Johannes F. Imhoff. 2011. "Genetic Approach for the Fast Discovery of Phenazine Producing Bacteria" Marine Drugs 9, no. 5: 772-789. https://doi.org/10.3390/md9050772