Novel Selectable Marker Sesquiterpenoid Antibiotic Pentalenolactone

Abstract

Antibiotic resistance has been and remains a major problem in our society. The main solution to this problem is to search and study the mechanisms of antibiotic action. Many groups of secondary metabolites, including antimicrobial ones, are produced by the Actinomycetota phylum. The actinobacterial strains isolated from habitats that have not been well studied are of great interest. Due to high resource competition, antibiotics are now considered a 'trump card in the game of life' due to their presence in natural substrates with limited nutrients. Potentially, strains isolated from such habitats can be producers of novel or poorly studied antibiotics. In the current research, we identified the strain Streptomyces sp. AP22 from the soils of the Akhshatyrsky Gorge, which is capable of producing pentalenolactone. This study describes the phenotypic and morphological characteristics of Streptomyces sp. AP22 and its biological activity. Pentalenolactone is a known inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important enzyme involved in glycolysis. We identified a previously unknown mutation in the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase that confers resistance to this antibiotic compound. This antibiotic is not used in clinical practice, so its application as a selectable marker will not lead to the creation of pathogens resistant to clinically relevant antibiotics. In this case, the selectable marker is based on a genetic construct containing the glyceraldehyde-3-phosphate dehydrogenase gene with a resistance mutation. The use of this selectable marker can be applied to various genetic and molecular techniques, such as cloning and transformation. This can help to facilitate genetic and molecular biology studies of strains resistant to standard selectable markers such as kanamycin or ampicillin.

Keywords: antibiotics; pentalenolactone; selectable marker.

Figure A1

UV spectrum of the peak after HPLC purification of the most active LPS fraction (10% acetonitrile).

Figure A2

Mass spectrum (positive) of the compound with retention time 5.63 min.

Figure A3

Mass spectrum (negative) of the compound with retention time 5.63 min.

Figure A4

Fragmentation of [M + H]⁺ ion with m/z 277.106 (pentalenolactone).

Figure A5

Fragmentation of [M − H]⁻ ion with m/z 275.092 (pentalenolactone).

Figure A6

Mass spectrum (positive) of the compound with retention time 5.72 min.

Figure A7

Mass spectrum (negative) of the compound with retention time 5.72 min.

Figure A8

Fragmentation of [M + H]⁺ ion with m/z 279.122 (pentalenolactone F).

Figure A9

Fragmentation of [M − H]⁻ ion with m/z 277.107 (pentalenolactone F).

Figure A10

The gapA gene contains a replacement fragment, which resulted in a chromatogram from a Sanger capillary sequencer for a mutant strain.

Figure 1

In vitro test for the antagonistic activity of the HPLC fraction on E. coli strain JW5503 ΔtolC. Erythromycin (Ery, 5 ug) and levofloxacin (Lev, 25 ng) were used as positive controls.

Figure 2

Phylogenetic tree based on six concatenated housekeeping gene sequences (16S rRNA, atpD, gyrB, recA, rpoB, trpB, Streptomyces sp. AP22, and related type strains) performed using neighbor-joining maximum-likelihood tree-making algorithms after CLUSTAL W alignment [11] by using MEGA software version XI [12].

Figure 3

Scanning electron micrograph of the strain Streptomyces sp. AP22, showing the spore surface after incubation on ISP 3 medium at 28 °C for 14 days. Microphotographs show that most hyphae have a spiral shape. Some hyphae have a smooth surface, but the predominant majority of the aerial mycelium becomes warty and rough, due to the fact that it is covered with special surface proteins where spore-bearing hyphae are formed. Various magnification options are shown in the figure: (A) ×4500 (B) ×6500, and (C) ×12,000.

Figure 4

Chromatogram of the most active LPS fraction (10% acetonitrile). The active peak is marked by a red circle.

Figure 5

Proposed structures of the isolated compounds.

Figure 6

Genetic organization of pentalenolactone synthesis cluster in producer Streptomyces sp. AP22 (top) and Streptomyces sp. NBC_01231 (bottom). Homologous genes filled with the same colors, GenBank accession numbers are listed in Data Availability Statement.

Figure 7

Alignment of wild-type and resistant-to-pentalenolactone mutated GapA protein structures. The location of the replacement is indicated by the red circle.

Figure 8

Alignment of 6 different protein sequences of bacterial glyceraldehyde-3-phosphate dehydrogenase. Red color indicates positively charged amino acid residues, purple—negatively charged, green—polar, orange—glycines, blue—hydrophobic, and cyan—aromatic. In mutated E. coli GapA protein, threonine at alignment position 178 (in E. coli protein sequence the position is 175) is replaced by serine, and this mutation makes bacteria resistant to pentalenolactone. What is more, the mutation cannot be observed in pentalenolactone producer species (such as S. griseoviridis and S. gvermitilis). The multiple sequence alignment was performed using the CLUSTALW program, and the results were visualized and edited using the Jalview program.

Figure 9

Verification of selective marker. On all plates, E. coli DH5α cells were plated after transformation of pCDF_PLR or pRFPCER, and as a control, non-transformed cells were used. (A) Plate with 1000 μg/mL of pentalenolactone, (B) plate with 100 μg/mL of ampicillin, (C) plate without antibiotics.