A Marine Isolate of Bacillus pumilus Secretes a Pumilacidin Active against Staphylococcus aureus
<p>Antimicrobial plate assays. Fractions of <span class="html-italic">Bacillus pumilus</span> SF214 supernatant were tested against target bacteria. The fraction containing molecules smaller than 3 kDa was active against <span class="html-italic">S. aureus</span> and not against <span class="html-italic">L. monocytogenes</span>, while the fraction containing molecules larger than 10 kDa was active against <span class="html-italic">L. monocytogenes</span> and not against <span class="html-italic">S. aureus</span>.</p> "> Figure 2
<p>Production of the antimicrobials during growth. SF214 was grown in minimal (S7) medium at 25 °C and the growth curve reported (white symbols). Aliquots of the cell culture were collected at various times, size-fractionated, and tested against the target bacteria. Dashed lines indicate the antimicrobial activity (diameter in mm of inhibition halo) of the fraction larger than 10 kDa against <span class="html-italic">L. monocytogenes</span> (<b>A</b>) and of the fraction smaller than 3 kDa against <span class="html-italic">S. aureus</span> (<b>B</b>).</p> "> Figure 3
<p>NMR spectrum. <sup>1</sup>H NMR spectrum of the active fraction, recorded at 298 K, in DMSO, at 600 MHz.</p> "> Figure 4
<p>Positive ions MALDI-TOF mass spectrum of the active fraction.</p> "> Figure 5
<p>Positive ion MALDI-TOF MS/MS spectrum of the pumilacidin precursor ion at <span class="html-italic">m</span>/<span class="html-italic">z</span> 1058.7. b<sup>+</sup> and y<sup>+</sup> fragment ions are reported for pumilacidin isoforms in the insert.</p> "> Figure 6
<p>The <span class="html-italic">srfA-sfp</span> locus of surfactant-producing bacilli. Arrows of the same color indicate orthologous genes.</p> "> Figure 7
<p>The <span class="html-italic">srfA</span> operon of SF214 and its modular products. The prediction diagram was obtained by using the HMMER tool (<a href="https://www.ebi.ac.uk/Tools/hmmer/" target="_blank">https://www.ebi.ac.uk/Tools/hmmer/</a>) [<a href="#B23-marinedrugs-16-00180" class="html-bibr">23</a>]. The same colors indicate the same module.</p> "> Figure 8
<p>Phylogenetic trees. Diagrams were obtained on the basis of 16S rRNA (<b>A</b>) and gyrase B subunit (<b>B</b>) gene sequences (<a href="http://www.phylogeny.fr/phylogeny.cgi" target="_blank">http://www.phylogeny.fr/phylogeny.cgi</a>) [<a href="#B24-marinedrugs-16-00180" class="html-bibr">24</a>].</p> ">
Abstract
:1. Introduction
2. Results and Discussion
2.1. SF214 Secretes Two Antimicrobials Active Against Staphylococcus aureus or Listeria monocytogenes
2.2. Production of Both Antimicrobials Is Strictly Regulated
2.3. The Anti-Staphylococcus Molecule Was Stable to Heat, pH, and Treatments with Chemicals and Enzymes
2.4. Purification and Chemical Characterization of the Anti-Staphylococcus Molecule
2.5. The srfA-sfp Locus of SF214
3. Experimental Section
3.1. Bacterial Strains
3.2. Filter-Sterilized and Size-Fractionated Supernatants
3.3. Antimicrobial Plate Assay
3.4. Stability of Antimicrobials at Different pH, Temperature, Chemical, and Enzyme Conditions
3.5. Lipopeptide Purification and Preliminary Analysis
3.6. Characterization of Lipopeptide
3.7. Bioinformatic Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Medium | Temperature (°C) | Supernatant Activity a > 10 kDa against L. monocytogenes | Supernatant Activity a < 3 kDa against S. aureus |
---|---|---|---|
LB (Luria-Bertani) | 25 | 7 | 5 |
BHI (Brain Heart Infusion) | 25 | 10 | 5 |
DS (Difco Sporulation) | 25 | 7 | 5 |
S7 | 25 | 10 | 10 |
S7 | 30 | 10 | 7 |
S7 | 37 | 5 | 5 |
pH | Supernatant Activity a < 3 kDa against S. aureus | |
---|---|---|
1 h | 5 h | |
2 | 7 | 7 |
4 | 10 | 10 |
7 | 10 | 10 |
10 | 10 | 10 |
13 | 10 | 10 |
Treatment | Supernatant Activity b < 3 kDa against S. aureus |
---|---|
None | 10 |
Trypsin a | 10 |
Proteinase K a | 10 |
DNase a | 10 |
Ribonuclease A a | 10 |
Acetone c | 10 |
Ethyl alcohol c | 10 |
Chloroform c | 10 |
Toluene c | 10 |
Incubation (15 min) at: | |
60 °C | 10 |
80 °C | 10 |
100 °C | 5 |
Lipopeptide | [M + Na]+ (m/z) | Isoforms |
---|---|---|
Pumilacidin | 1030.6 | C13 [Val7] |
1044.7 | C14 [Val7], C13 [Leu/Ile7] | |
1058.7 | C15 [Val7] (pumilacidin B) a, C14 [Leu/Ile7] | |
1072.7 | C16 [Val7] (pumilacidin F/G) a, C15 [Leu/Ile7] (pumilacidin A) a | |
1086.7 | C17 [Val7] (pumilacidin D) a, C16 [Leu/Ile7] (pumilacidin E) a | |
1100.7 | C18 [Val7], C17 [Leu/Ile7] (pumilacidin C) a |
Genes | Putative Encoded Protein | Protein Identity (%) a | ||
---|---|---|---|---|
B. pumilus SF214 vs. B. pumilus SAFR-032 | B. pumilus SF214 vs. B. safensis U17-1 | B. pumilus SAFR-032 vs. B. safensis U17-1 | ||
srfAA | surfactin synthase subunit 1 | 95 | 91 | 90 |
srfAB | surfactin synthase subunit 2 | 94 | 91 | 90 |
srfAC | surfactin synthase subunit 3 | 95 | 93 | 91 |
orfX | nonribosomal peptide synthetase | 94 | 91 | 89 |
orfY | nonribosomal peptide synthetase | 94 | 92 | 90 |
srfAD | surfactin synthase thioesterase subunit | 94 | 89 | 87 |
1 | hypothetical protein | - | 87 | - |
2 | hypothetical protein | - | 89 | - |
3 | hypothetical protein | - | 90 | - |
4 | hypothetical protein | - | 84 | - |
5 | hypothetical protein | - | 90 | - |
ycxC | transporter | 96 | 93 | 92 |
ycxD | transcriptional regulator | 95 | 93 | 93 |
sfp | 4′-phosphopantetheinyl transferase | 94 | 88 | 88 |
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Saggese, A.; Culurciello, R.; Casillo, A.; Corsaro, M.M.; Ricca, E.; Baccigalupi, L. A Marine Isolate of Bacillus pumilus Secretes a Pumilacidin Active against Staphylococcus aureus. Mar. Drugs 2018, 16, 180. https://doi.org/10.3390/md16060180
Saggese A, Culurciello R, Casillo A, Corsaro MM, Ricca E, Baccigalupi L. A Marine Isolate of Bacillus pumilus Secretes a Pumilacidin Active against Staphylococcus aureus. Marine Drugs. 2018; 16(6):180. https://doi.org/10.3390/md16060180
Chicago/Turabian StyleSaggese, Anella, Rosanna Culurciello, Angela Casillo, Maria Michela Corsaro, Ezio Ricca, and Loredana Baccigalupi. 2018. "A Marine Isolate of Bacillus pumilus Secretes a Pumilacidin Active against Staphylococcus aureus" Marine Drugs 16, no. 6: 180. https://doi.org/10.3390/md16060180