DNA modifications impact natural transformation of Acinetobacter baumannii

Abstract

Acinetobacter baumannii is a dangerous nosocomial pathogen, especially due to its ability to rapidly acquire new genetic traits, including antibiotic resistance genes (ARG). In A. baumannii, natural competence for transformation, one of the primary modes of horizontal gene transfer (HGT), is thought to contribute to ARG acquisition and has therefore been intensively studied. However, knowledge regarding the potential role of epigenetic DNA modification(s) on this process remains lacking. Here, we demonstrate that the methylome pattern of diverse A. baumannii strains differs substantially and that these epigenetic marks influence the fate of transforming DNA. Specifically, we describe a methylome-dependent phenomenon that impacts intra- and inter-species DNA exchange by the competent A. baumannii strain A118. We go on to identify and characterize an A118-specific restriction-modification (RM) system that impairs transformation when the incoming DNA lacks a specific methylation signature. Collectively, our work contributes towards a more holistic understanding of HGT in this organism and may also aid future endeavors towards tackling the spread of novel ARGs. In particular, our results suggest that DNA exchanges between bacteria that share similar epigenomes are favored and could therefore guide future research into identifying the reservoir(s) of dangerous genetic traits for this multi-drug resistant pathogen.

Graphical Abstract

Figure 1.

Origin of the transforming DNA impacts natural transformability. (A) Transformability of all transformable strains tested (indicated on Y-axis) using gDNA from different A. baumannii strains as transforming material (indicated on X-axis). The heat map indicates the transformation frequencies according to the legend. (B) Transformation frequency of strain A118 after transformation with genomic DNA (gDNA) originating from different A. baumannii strains. Details of boxed area in panel A. (C) Transformability of strains using plasmid pABA derived from the different A. baumannii strains as transforming material. Heatmap as in panel A. (D) Transformation frequency of strain A118 when transformed with plasmid DNA (pABA) originating from different A. baumannii strains. Details of boxed area in panel C. The mean values (± standard deviation, SD) of three independent biological replicates are shown for panels B and D with each individual experiment being represented as a dot. <d.l., below detection limit. #, below detection limit in at least one experiments. Statistics were performed on log-transformed data, using two-way analysis of variance (ANOVA) with Dunnett's (A, C) or Tukey's (B, D) correction for multiple comparisons. Only statistically significant differences are shown. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 2.

SMRT sequencing reveals DNA modifications in diverse A. baumannii strains. (A) The heatmap shows the abundance of the 15 DNA modification motifs, which were detected on one or several of the six different A. baumannii strains. The color indicates the proportion of modified recognition sites, as shown by the color key (a detailed list is provided as Table S6). unk, unknown type of modification. (B, C) Spatial distribution of the DNA modification m6A:RGATCY:3 (B) and unk:TGGCCA:4 (C) on both strands of the A118 chromosome. Modified recognition sites are shown in color (red and blue for + and – strand, respectively), while unmodified sites are shown in black.

Figure 3.

Strain A118 carries unique RM system. (A) Comparison of A118 genomic region carrying the gene encoding the predicted methylase H0N27_10820 with the same region of other A. baumannii strains. Predicted annotations for gene products are represented with different colors, as shown in the legend. Genes that encode for defense systems according to predictions by PADLOC or DefenseFinder are defined underneath the gene number(s). (B) Zoom of the A118-specific genomic region, carrying the newly identified RM system genes H0N27_10820-30 (H0N27_10820 (MT) in dark red, H0N27_10825 (TR) in red and H0N27_10830 (RE) in light red).

Figure 4.

The RMC is responsible for source DNA-dependent transformability of A118. Transformability of strain A118 (WT) and A118ΔH0N27_10830 (ΔRE) using (A) gDNA of different A. baumannii strains, (B) PCR product amplifying Δhcp::aprR region or (C) plasmid pABA originating from different strains as transforming material. (D, E) Transformation of strain A118 with plasmid pABA originating from various strains grown with 2% L-arabinose: (D) A118 (WT), ΔRMC (A118ΔH0N27_10820-30), ΔRMC-Tn (ΔRMC carrying transposon control), or ΔRMC-TnMT (ΔRMC complemented with inducible H0N27_10820 (MT) on transposon); (E) A118, ATCC17978, ATCC17978 carrying empty transposon (Tn) or transposon with inducible copy of H0N27_10820 (TnMT) or H0N27_12600 (TnMT2). (F) The RMC cluster leads to m6A modified motif RGATCY. Heatmap showing the abundance of different DNA modification motifs in the RMC-related mutants of strain A118 (for the proportion of modified recognition sites see also Table S8). Details as described for Figure 2. The average values (± SD) of three independent biological replicates are shown (A–E). <d.l., below detection limit. #, below detection limit in at least one replicate. Statistical analyses were performed on log-transformed data using a two-way ANOVA with Šidák's correction for multiple comparisons (A, C), an unpaired t test (B), or a one-way ANOVA with Dunnett's correction for multiple comparisons (D, E). *P < 0.05, ***P < 0.001, ****P < 0.0001; n.s., not significant.

Figure 5.

TR deficiency can overcome DNA restriction. (A) Relative expression of the MT and the RE genes in the TR-deficient mutant strain (ΔTR) compared to the parental WT strain A118. (B) Heatmap showing the abundance of different DNA modifications in WT and ΔTR (see also Table S10). Details as described for Figure 2. (C) Transformability of strain A118 and its RMC-related derivatives using self and non-self plasmid DNA (isolated from A118 or ATCC17978) as transforming material. Transformation frequencies of RMC-related derivatives are compared to the WT within each set that tested the same plasmid source. Only statistically significant differences are shown. (D) Relative expression of the MT, RE, TR, and pilA genes in strain A118 after 2 h and 6 h of growth. (E) Transformation frequency of strain A118 with plasmid pABA originating from strains A118, E. coli TOP10 and E. coli INV110. Dam methylase-dependent in vitro methylation was performed where indicated (+methyl). The mean values (±SD) were calculated from three independent biological replicates (A, C–E). <d.l., below detection limit. The data was log-transformed and statistical differences were calculated using a two-way (A, C–D) or one-way (E) ANOVA corrected for multiple comparisons with Šidák's method. ****P < 0.0001; n.s., not significant.