Investigation of N. gerenzanensis sporulation

Usually, the aerial hypha's differentiated spores are considered the ideal receipt cells for the conjugation transfer of filamentous microorganisms. However, mycelium was selected as the recipient cells in almost all published studies involving the genetic manipulation of N. gerenzanensis (Lo Grasso et al. 2015; Marcone et al. 2014; Stinchi et al. 2003). These studies appear to suggest that it is hard for this rare actinomycete to differentiate spores. To check the sporulation of N. gerenzanensis, we investigated the growth of N. gerenzanensis on nine agar media commonly used for Streptomyces. As shown in Additional file 1: Figure S2, the spores were not observed on any experimental agar medium. As a result, mycelium as recipient cell should be the unique option for the conjugation transfer of N. gerenzanensis. Moreover, it is interesting that N. gerenzanensis presents apparent morphology and color variations in the different media (Additional file 1: Figure S2), which reveals that N. gerenzanensis has plenty of secondary metabolites.

Sensitivity of N. gerenzanensis to antibiotics

In microbial pathogens, plasmid-mediated conjugation transfer is one of the primary causes of bacterial resistance to antibiotics (de Nies et al. 2022; Jamieson-Lane and Blasius 2022; Nohejl et al. 2022). In general, antibiotics targeting bacterial walls or membranes are more likely to cause drug resistance, which may be related to their ability to change the permeability of the cell barrier. Therefore, we infer that antibiotics such as glycopeptides or beta-lactams may affect conjugation transfer. Cephalosporins, ampicillin, vancomycin, and teicoplanin were chosen to assess their inhibition of N. gerenzanensis. As shown in Table 1 and Additional file 1: Figure S3, ampicillin and cephalosporin did not inhibit the growth of N. gerenzanensis in the concentration range of 1 μg mL−1 to 80 μg mL−1. In contrast, vancomycin and teicoplanin showed certain inhibitory effects. In particular, teicoplanin could inhibit the growth of N. gerenzanensis when its concentration increased to 2.5 μg mL−1. Moreover, the sensitivity of E. coli ET12567 to teicoplanin was assessed, and the result showed that the antibiotic had no inhibition on E. coli (Additional file 1: Figure S4).

Table 1 Sensitivity of N. gerenzanensis to antibioticsOptimization of the classic conjugation transfer protocol

The growth circumstances play an important role in the conjugation transfer between heterogeneous microorganisms. Here, four commonly used solid media MS, V0.1, TSBY, and R3 were tested to determine the optimal regenerative medium for conjugation. At the same time, 20 mM and 30 mM magnesium chloride were added to four kinds of media to evaluate their impact, respectively. The results shown in Fig. 1A demonstrated that the conjugation frequency reached the highest values of 1.67 × 10–4 (20 mM MgCl2) and 3.25 × 10–4 (30 mM MgCl2) on the V0.1 medium. In contrast, the conjugation frequency of the TSBY medium was less than 1.55 × 10–5 (30 mM MgCl2). No exconjugant colonies emerged on the R3 medium. Consequently, the V0.1 medium was determined to be the optimum conjugation medium.

Fig. 1

Screening and optimization of factors affecting conjugation frequency. A, effect of agar medium on conjugation frequency; B, effect of magnesium ion on conjugation frequency; C, effect of liquid medium on conjugation frequency; D, effect of cell growth state on conjugation frequency. Each point represents the mean (n = 3) ± standard deviation

The addition of magnesium has been proven to improve the conjugation transfer of actinomycetes effectively. Here, the optimal concentration of magnesium ions in the V0.1 medium was investigated, while the MS medium was used as a control. As shown in Fig. 1B and Additional file 1: Figure S5, the V0.1 medium containing 30 mM MgCl2 displayed the highest conjugation frequency of 2.64 × 10–4. In addition, the conjugation frequency decreased with the increase in concentration when the concentration of magnesium ions exceeded 30 mM. Thus, the V0.1 medium supplemented with 30 mM of MgCl2 was the optimal medium for the intergeneric conjugation of N. gerenzanensis.

Furthermore, we assessed the effect of mycelium state on the intergeneric conjugation of N. gerenzanensis in the different liquid media, such as R3, YEME, TSB, and VSP. The results indicated that the conjugation frequency of TSB medium (5%) was the highest, which was 7.35 × 10–2, while that of other media was less than 2 × 10–2 (Fig. 1C). Additionally, the culture time test showed that the optimal culture time was 24 h (Fig. 1D). In order to explore the reasons for the above results, we examined the growth and micromorphology of N. gerenzanensis. As shown in Additional file 1: Figure S6A and Fig. 1C, although the biomass in the VSP medium was significantly higher than that in other media, its conjugation frequency was the lowest. The results of micro- morphological observation suggested that the morphology of mycelium clusters in VSP, R3, and YEME medium was not conducive to conjugation, which means that a relatively small quantity of receipt cells is provided. On the contrary, the hyphae in the TSB medium were rod-shaped rather than clustered (Additional file 1: Figure S6B).

Finally, the antibiotic coverage time and donor-to-recipient ratio were optimized. As shown in Fig. 2A, almost no exconjugants were obtained when the coverage time was 15 h, and the conjugation frequency increased with the extension of the coverage time. The appropriate coverage time was about 27 h. The optimization result of the donor-to-recipient ratio showed that the highest conjugation frequency of 3.9 × 10–1 was obtained at the ratio of 10:1 (Fig. 2B).

Fig. 2

Effect of antibiotic coverage time and ratio of donor to recipient on intergeneric conjugation. A, antibiotic coverage time; B, ratio of donor to recipient. Each point represents the mean (n = 3) ± standard deviation

Effect of the autoinducer GBL on the growth and conjugation of N. gerenzanensis

The QS mechanism exists extensively in microorganisms and is closely related to the horizontal transfer of genetic elements through conjugation transfer (Banderas et al. 2020; Breuer et al. 2018; Lin et al. 2021). In the process of QS, autoinducers are key signal molecules, such as GBL biosynthesized by streptomyces (Barreales et al. 2020; Liu et al. 2021; Xu and Yang 2019). Here, the autoinducer GBL was used to assess its effect on the growth and conjugation of N. gerenzanensis. As shown in Fig. 3A, the addition of GBL with different concentrations in the growth medium had little on the growth of N. gerenzanensis. The autoinducer GBL promoted cell growth when the concentration was less than 80 µM, but when the concentration increased to 100 µM, the cell growth was inhibited. In addition, the effect of GBL on conjugation was evaluated. The results in Fig. 3B suggested that GBL had an evident effect on the conjugation frequency. The maximum conjugation frequency increased to 0.6 when the GBL concentration was 60 µM.

Fig. 3

Effect of GBL on strain growth and intergeneric conjugation. A, growth curve of N. gerenzanensis in 5% TSB medium with different concentrations of GBL; B, effect of GBL on conjugation frequency. Each point represents the mean (n = 3) ± standard deviation

Effect of antibiotics on the conjugation of N. gerenzanensis

As is well known, one of the main reasons for the emergence and spread of bacterial antibiotic resistance is the conjugational transfer of drug-resistant plasmids among closely related prokaryotic microorganisms. Some studies have confirmed that polypeptides can promote the plasmid transfer between drug-resistant bacteria (Vickerman and Mansfield 2019; Xiao et al. 2022). β-lactam and GPAs can target to destroy the wall of Gram-positive bacteria and further inhibit cell growth. Here, it is hypothesized that destroying the bacterial cell wall may promote the conjugation between bacteria. In this study, vancomycin, teicoplanin, cephalosporin, and ampicillin were selected to evaluate their effects on the growth of N. gerenzanensis. Vancomycin and teicoplanin could significantly inhibit the growth of N. gerenzanensis at concentrations of 20 µg mL−1 and 2.5 µg mL−1, respectively. However, cephalosporin and ampicillin could not inhibit the growth of N. gerenzanensis even if the concentration was more than 80 µg mL−1 (Additional file 1: Figure S3). Therefore, the GPA teicoplanin was used to evaluate its effect on the conjugation between N. gerenzanensis and E. coli. As shown in Fig. 4, teicoplanin could significantly increase the conjugation frequency at a concentration of 0.5 µg mL−1. However, no conjugator was found once the concentration reached 5 µg mL−1.

Fig. 4

Effect of GPA teicoplanin on intergeneric conjugation. A, effect of teicoplanin concentration on conjugation frequency; B, effect of teicoplanin treatment time on conjugation frequency. Each point represents the mean (n = 3) ± standard deviation

Furthermore, the treatment time of teicoplanin was investigated. The results demonstrated that the best processing time was 5 min. In order to reveal the mechanism of low concentration teicoplanin promoting conjugation transfer, the mycelia treated with 0.5 µg mL−1 teicoplanin for the different time were observed by SEM. The results demonstrated that the wrinkles and depressions on the surface of mycelium increased with the extension of treatment time, indicating that the cell wall of mycelium changed obviously (Fig. 5B). When the concentration of teicoplanin in the control sample was 80 µg mL−1, the cellular debris of mycelium caused by a high concentration of teicoplanin could be observed obviously. Accordingly, no conjugator was obtained from the control sample (Fig. 5A).

Fig. 5

Conjugator regeneration and morphological analysis of N. gerenzanensis recipient cells treated with teicoplanin at different time. A, Conjugator regeneration plates at different time; B, SEM analysis of mycelium. The control sample was treated with 80 μg mL−1 of teicoplanin for 30 min; the other samples were treated with 0.5 μg mL−1 of teicoplanin for different time

Confirmation of exconjugants.

The exconjugant was confirmed by antibiotic resistance, fluorescence observation, and amplification of the egfp gene. A large number of exconjugants showed resistance to the antibiotic apramycin (Additional file 1: Figure S7A), which proved that the genetic transformation was highly efficient. Subsequently, the mycelia of exconjugants cultivated in a liquid medium were collected and then observed by fluorescence microscope. As shown in Additional file 1: Figure S7B, apparent green fluorescence was observed from the mycelium. In addition, the egfp gene was amplified from exconjugants by PCR. Gel electrophoresis analysis showed that compared with the wild-type strain, all the selected exconjugants obtained the 720 bp PCR products of egfp gene (Additional file 1: Figure S7C).