Diversity and bioactive potential of Actinomycetia from the rhizosphere soil of Juniperus excelsa

Phylogenetic characterisation of the actinomycete strains

In our previous study, 372 actinomycete-like strains were isolated from the rhizosphere soil of J. excelsa. Most of the isolated strains exhibited the typical characteristics of actinomycetes, including slow growth, substrate and aerial mycelium formation, sporulation and pigment production. The taxonomic identification of these strains was evaluated on the basis of their 16S rRNA gene sequences (Table S1). The isolated actinomycete strains were distributed among seven families (Kribbellaceae, Micrococcaceae, Micromonosporaceae, Nocardiaceae, Promicromonosporaceae, Pseudonocardiaceae and Streptomycetaceae) and eleven genera (Actinoplanes, Actinorectispora, Amycolatopsis, Kribbella, Micrococcus, Micromonospora, Nocardia, Promicromonospora, Rhodococcus, Saccharopolyspora and Streptomyces). However, the majority of the isolates (350 or 94.08% of all the isolated strains) belonged to the genus Streptomyces.

The evolutionary relationships among the isolated actinomycete strains are demonstrated in the phylogenetic tree of the 16S rRNA gene presented in Fig. 1. All of the isolated strains were grouped within the respective genus and formed close clades with the 16S rRNA gene of representatives of the respective genera (Figs. S1S6). Based on the evolutionary relationships of the 16S rRNA genes, the isolated strains of the genus Streptomyces were conditionally combined into seven groups. These groups formed clades consisting of both isolates and typical members of the genus Streptomyces and were named after them. Of the seven groups, the S. kanamyceticus group was the largest and included 103 isolated strains. There were also some strains among the isolates that were not combined into the formed groups. The non-streptomyce group included the group of isolates identified as non-streptomycetes and their closest neighbors, as shown in detail in Fig. S6.

Fig. 1figure 1

Scheme of phylogenetic relationships of the actinomycete strains isolated from the rhizosphere soil of J. excelsa based on the 16S rRNA sequences. A phylogenetic tree was constructed by means of MEGA 11 using the neighbour-joining method with 1000 bootstrap replicates. The colours show the groups of isolates that formed common clades with typical members of the genus Streptomyces and were named after them. The 16S rRNA sequence of the E. coli strain U5/41 (NR_024570.1) was used as an outgroup

Analysis of the antimicrobial activity of the actinomycete strains

All of the isolated actinomycete strains were tested with regard to their ability to produce antimicrobial metabolites against Gram-positive bacteria, Gram-negative bacteria and yeast using the spot inoculation technique. Among the 372 isolated actinomycete strains, 159 strains (42.74%) exhibited antimicrobial activity against at least one of the tested microbial strains. Most of the strains inhibited the growth of Gram-positive bacteria, including B. subtilis ATCC 31,324 (132 strains, 35.48%) and S. aureus ATCC 25,923 (73 strains, 19.62%). However, significantly fewer strains inhibited the growth of Gram-negative bacteria. Here, 19 strains (5.11%) produced antimicrobial compounds against E. coli ATCC 25,922, whereas 14 strains (3.76%) and 18 strains (4.84%) were active against K. pneumoniae ATCC 13,883 and P. vulgaris ATCC 29,905, respectively, and only seven strains (1.88%) exhibited antagonistic activity against P. aeruginosa ATCC 9027. In addition, 36 strains (9.67%) exhibited antimicrobial activity against C. albicans ATCC 885–653.

Aside from ascertaining the ability of the isolated strains to inhibit the growth of a particular test culture, we also evaluated the level of antibiotic activity of these strains, which was calculated as the AAI. For this purpose, we grouped the different AAI into three categories: < 3 (low), 3–6 (medium) and > 6 (high). Most of the studied strains (from 1.08% of K. pneumoniae antagonists to 23.65% of B. subtilis antagonists) had a low AAI, whereas significantly fewer strains had a medium AAI (0.54–10.5%). The exception was the K. pneumoniae antagonists, among which 1.08% had an AAI < 3 while twice as many strains had a medium AAI. Only 2.71% of the strains had an AAI greater than 6, with no AAI greater than 6 being found for the E. coli, P. aeruginosa or C. albicans antagonists (Fig. 2).

Fig. 2figure 2

Screening the antimicrobial activity of the actinomycete strains isolated from the rhizosphere soil of Juniperus excelsa. AAI Antimicrobial Activity Index, < 3 - grey, 3-6 light grey, > 6 - dark grey; Bs B. subtilis ATCC 31324, Sa S. aureus ATCC 25923; Ec E. coli ATCC 25922; Pa P. aeruginosa ATCC 9027, Kp K. pneumoniae ATCC 1388, Pv P. vulgaris ATCC 29905, Ca, C. albicans ATCC 29905

About half of the strains of the genus Streptomyces exhibited antimicrobial activity. Among the strains from other, less numerous genera, only a few showed antimicrobial activity. In particular, two representatives of the genus Amycolatopsis (strains Je 1–447 and Je 1–666) and the Actinorectispora sp. strain Je 1–571 suppressed the growth of the Gram-positive bacteria B. subtilis and S. aureus. Representatives of other genera of the isolated actinomycetes showed no inhibitory activity against the utilised test cultures.

Dereplication of the secondary metabolite profile of the Streptomyces sp. strain Je 1–651

After analysing the established collection of actinomycete strains, we identified several strains with a broad spectrum of antimicrobial activity, which captured both antibacterial and antifungal activities. In particular, the Streptomyces sp. Je 1–651 strain exhibited strong inhibitory activity (the AAI of this strain was at a medium level (3–6) or above) against all of the utilised microbial test cultures, except for P. aeruginosa. To better understand the nature of the compounds that may be responsible for the observed activity, we analysed the secondary metabolites produced by this strain. To accomplish this, we performed a dereplication analysis of the secondary metabolites of this strain within the DNP database (CRC Press). The ability of the strain Je 1–651 to produce secondary metabolites was studied by growing it in DNPM and SG liquid media. A total of 18 major secondary metabolite peaks were detected in the cultural liquid and biomass extracts of the Streptomyces sp. Je 1–651 cultivated in both media (Figs. 3, S7 and Table S2). Using the DNP database, seven of these peaks were identified as spiramycins. These peaks were present in all of the studied chromatograms, although their number and magnitude varied. In the crude biomass extract of the Je 1–651 strain, which was grown in SG medium, three large peaks were identified in addition to the spiramycins (Fig. 3b). These peaks were annotated as stambomycin A/B (retention time (tR) of 9.08; m/z 1376.9378 [M + H]+), stambomycin C/D (tR of 8.7; m/z 1362.9238 [M + H]+), stambomycin E (tR of 8.39; m/z 1348.9108 [M + H]+) and stambomycin F (tR of 9.66; m/z 1390.9558 [M + H]+) (Figs. 3b and S7).

Fig. 3figure 3

LC–MS chromatogram of the ethyl acetate a and aceton:methanol b extracts of the Streptomyces sp. strain Je 1–651 after cultivation in SG (above) and DNPM (bottom) media. The medium peaks and identified antibiotic spiramycins and stambomycins are shown in grey. Red stars indicate compounds not identified in the DNP database

In the crude extracts of the Je 1–651 strain grown on DNPM medium, seven peaks were identified that did not yield positive matches in the DNP database and so could not be annotated based on the available mass spectrometry data. The absence of matches in the database may indicate the novelty of the associated compounds. The characteristics of the identified peaks, which likely form unknown compounds, are shown in Table S2.

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