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. 2023 Dec;15(6):656-668.
doi: 10.1111/1758-2229.13203. Epub 2023 Oct 4.

Role of natural transformation in the evolution of small cryptic plasmids in Synechocystis sp. PCC 6803

Fabian Nies  1 , Tanita Wein  1 , Dustin M Hanke  1 , Benjamin L Springstein  1 , Jaime Alcorta  2 , Claudia Taubenheim  1 , Tal Dagan  1
Affiliations

Role of natural transformation in the evolution of small cryptic plasmids in Synechocystis sp. PCC 6803

Fabian Nies et al. Environ Microbiol Rep. 2023 Dec.

Abstract

Small cryptic plasmids have no clear effect on the host fitness and their functional repertoire remains obscure. The naturally competent cyanobacterium Synechocystis sp. PCC 6803 harbours several small cryptic plasmids; whether their evolution with this species is supported by horizontal transfer remains understudied. Here, we show that the small cryptic plasmid DNA is transferred in the population exclusively by natural transformation, where the transfer frequency of plasmid-encoded genes is similar to that of chromosome-encoded genes. Establishing a system to follow gene transfer, we compared the transfer frequency of genes encoded in cryptic plasmids pCA2.4 (2378 bp) and pCB2.4 (2345 bp) within and between populations of two Synechocystis sp. PCC 6803 labtypes (termed Kiel and Sevilla). Our results reveal that plasmid gene transfer frequency depends on the recipient labtype. Furthermore, gene transfer via whole plasmid uptake in the Sevilla labtype ranged among the lowest detected transfer rates in our experiments. Our study indicates that horizontal DNA transfer via natural transformation is frequent in the evolution of small cryptic plasmids that reside in naturally competent organisms. Furthermore, we suggest that the contribution of natural transformation to cryptic plasmid persistence in Synechocystis is limited.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Cryptic plasmid annotation and phylogeny of the pCA2.4 and pCC5.2 replication initiation proteins. (A) Annotations of open reading frames (ORFs) in pCA2.4, pCB2.4, and pCC5.2. The annotations were generated combining previous reports in the literature (Xu & McFadden, ; Yang & McFadden, 1993, 1994) and the reference genome (GCA_000340785.1). Plasmid encoded Reps are identified in at least one source and are highlighted in pink. (B) Constrained phylogeny of pCA2.4 Rep using representative homologues (see full list in Supplementary File S1). (C) Constrained phylogeny of pCC5.2 Rep using representative homologues (see full list in Supplementary File S1). The operational taxonomic units (OTUs) are coloured according to the host phylum (see legend on the left). Red branches in the tree correspond to branches having a low bootstrap support (<70%). Homologues encoded in small plasmids (<25 kbp) are marked by filled circles.
FIGURE 2
FIGURE 2
Comparison of transcription level among cryptic plasmid rep genes and three housekeeping genes. The underlying data corresponds to four transcriptomics studies (i.e., NCBI BioProjects) of Synechocystis cultured under various growth conditions in different laboratories (Bi et al., ; Cheng et al., ; García‐Cañas et al., ; Lau et al., ; see Table S1). Data points show transcript per million (TPM) per gene. Error bars show the interquartile range.
FIGURE 3
FIGURE 3
Gene transfer in Synechocystis cultures via natural transformation. (A) An illustration of our experimental setup for testing and quantifying plasmid‐encoded gene transfer in Synechocystis. The donor and recipient were co‐cultivated for 7 days. The donor was deficient in natural competence; thus, the transfer was unidirectional from donor to recipient. After 7 days the co‐culture was plated on non‐selective media for cell number determination. The marker gene transfer into the recipient was quantified by plating on double selective plates. (B) Transfer frequency of pCA or pCB encoded SmR marker, or a chromosomal encoded CmR marker from donor to the recipient WT* with or without DNase treatment (n = 3). Transfer frequency is calculated by the number of transformed recipient cells (double resistance to streptomycin/spectinomycin or chloramphenicol and kanamycin) divided by total cell number. Boxes indicate one standard error; whiskers indicate two standard errors. (C) An illustration of the DNA uptake machinery in Synechocystis. Type IV pilus (purple) and competence proteins (green) are essential for DNA transport across the outer and inner cell membranes. Knockout mutants of pilA1 (major pilus subunit), pilQ (outer membrane pore), comEA (DNA binding in periplasm), and comEC (inner membrane transport) as recipient cells are deficient in DNA uptake in marker gene transfer experiments.
FIGURE 4
FIGURE 4
Transfer frequency of pCA and pCB encoded antibiotic resistance genes from donor to recipient strains. Transfer frequency is shown according to recipient genotype (left: WT* or ∆recJ) or labtype (right: Kiel or Sevilla) and the donor plasmid (top: pCA; bottom: pCB). The Kiel WT* is the same recipient as in Figure 3B. Transfer frequency is calculated by the number of transformed recipient cells (double resistant to streptomycin/spectinomycin and kanamycin) divided by total cell number. The results of two independent experiments (solid lines and squares; dashed lines and triangles) are presented, with three or five technical replicates, respectively.
FIGURE 5
FIGURE 5
Transfer frequencies for plasmid uptake and horizontal transfer of plasmid and chromosome encoded traits during cocultivation of the same or different species. Transfer frequencies from this study and literature are ordered by the recipient from high to low on a logarithmic scale. Sensitivity to DNase is the most common control (conjugation or transduction are not sensitive to DNase added to the media). For transfer of chromosomal markers in several studies the DNase treatment was not successful or not applied. For the estimation of plasmid‐mediated transfer frequency, controls to exclude other routes of horizontal transfer are essential, which are not necessarily required for chromosome‐mediated transfer (previous studies on plasmids missing these controls were not included). Additional controls are functional knockouts of the natural competence machinery (NC‐KO), plasmid isolation from the recipient, digestion of restriction enzymes (RE), missing mob genes on the transferred plasmid, comparison of transformation characteristics with naked plasmid, experiments in dependency of the competence state, southern blot. Species‐specific properties in natural competence and differences in experimental settings (e.g., transformation protocol, incorporated DNA, cultivation in biofilms or suspension cultures) should be taken into account during the comparison. Studies in which transfer was not quantified as a ratio of living cells were not included in this summary (e.g., studies with transfer frequencies given as ratio of transformed cells to culture volume). We note that the combination of the generally lower transfer frequencies for both plasmid pCB and Sevilla labtype could lead to an underestimation of the general plasmid uptake ability of Synechocystis. References: 1. (Zhang et al., 2018), 2. (Graham & Istock, 1978), 3. (Wang et al., 2007), 4. (Stewart et al., 1983), 5. (Rochelle et al., 1988), 6. (Vakeria et al., 1985), 7. (Williams et al., 1996), 8. (Wang et al., 2002), 9. (Albritton et al., 1982), 10. (Paul et al., 1992).
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References

    1. Albritton, W.L. , Setlow, J.K. & Slaney, L. (1982) Transfer of Haemophilus influenzae chromosomal genes by cell‐to‐cell contact. Journal of Bacteriology, 152, 1066–1070. - PMC - PubMed
    1. Andrews, S. , Krueger, F. , Segonds‐Pichon, A. , Biggins, L. , Krueger, C. & Wingett, S. (2010) FastQC: a quality control tool for high throughput sequence data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
    1. Armshaw, P. , Carey, D. , Sheahan, C. & Pembroke, J.T. (2015) Utilising the native plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC6803 as a cloning site for enhanced product production. Biotechnology for Biofuels, 8, 201. - PMC - PubMed
    1. Barry, K.E. , Wailan, A.M. , Sheppard, A.E. , Crook, D. , Vegesana, K. , Stoesser, N. et al. (2019) Don't overlook the little guy: an evaluation of the frequency of small plasmids co‐conjugating with larger carbapenemase gene containing plasmids. Plasmid, 103, 1–8. - PubMed
    1. Berla, B.M. & Pakrasi, H.B. (2012) Upregulation of plasmid genes during stationary phase in Synechocystis sp. strain PCC 6803, a cyanobacterium. Applied and Environmental Microbiology, 78, 5448–5451. - PMC - PubMed
-