Complete sequence of classic F-type plasmid pRK100 shows unique conservation over time and geographic location.

Plasmids exhibit great diversity of gene content and host ranges and are famous for quick adaptation to the genetic background of the bacterial host cell. In addition to observing ever evolving plasmids, some plasmids have conserved backbones: a stable core composition and arrangement of genes in addition to variable regions. There are a few reports of extremely conserved plasmids. Here we report the complete sequence of pRK100 plasmid - a large, well-characterized conjugative F-like plasmid found in an Escherichia coli strain isolated from a urinary tract infection patient in 1990. The sequence shows that the 142 kb-long pRK100 plasmid is nearly identical to plasmids circulating in distant geographical locations and found in different host E. coli strains between 2007 and 2017. We also performed additional functional characterization of pRK100. Our results showed that pRK100 does not have a strong pathogenicity phenotype in porcine primary bladder epithelial cell culture. Moreover, the conjugation of pRK100 seems to strongly depend on recipient characteristics. These observations and identification of the pRK100 plasmid in different strain genotypes leave the extreme sequence conservation and broad distribution of this plasmid unexplained.

Introduction

Antibiotic resistance of bacterial pathogens has become an urgent threat in current medicine. Genes encoding such antibiotic resistance are often carried by extrachromosomal elements - plasmids. Plasmids and plasmid transmission were recognized as the most important phenomena involved in transfer of resistance genes among bacterial strains (Aslam et al., 2018; Johnson and Nolan, 2009; Smillie et al., 2010). Apart from carrying antibiotic resistance genes plasmids can also encode a remarkably diverse array of phenotypic traits of medical, agricultural, environmental, and commercial importance, e.g. virulence-associated genes (Kado, 1998; Thomas and Nielsen, 2005).

One type of plasmids that tend to carry a broad repertoire of the virulence genes are the colicin V (ColV) plasmids that encode the production of the toxin colicin V (Johnson, 2021; Johnson et al., 2006; Waters and Crosa, 1991). Virulence determinants of ColV plasmids include siderophores (aerobactin, salmochelin), iron metabolism traits (ABC transporters like Sit and Eit, a hemoglobin protease), hemolysins (Hly), increased serum survival (Iss, Bor) (Di Lorenzo and Stork, 2014; Nolan et al., 2003; Nowrouzian et al., 2001a, 2001b; Searle et al., 2015). Consistently, prior studies showed that ColV plasmids are important for bacterial strains carrying these plasmids for colonization of the host intestine and for increased virulence in diverse infection models, such as urinary tract infection, neonatal rat sepsis, chicken embryos (Aguero et al., 1989; Lemaître et al., 2013; Nolan et al., 2003; Skyberg et al., 2006).

Despite the discovery of conjugative plasmids back in the mid twentieth century, many aspects of plasmid biology remain unclear. For example, it was long thought that presence of large conjugative plasmids is a burden to the bacterial host due to the necessity of maintaining the large, non-essential, DNA molecule and spending energy on expression of the conjugation-associated transfer genes (San Millan and MacLean, 2017). Therefore, the systems that ensure stable plasmid maintenance through addiction modules, partitioning and conjugation regulation have been investigated in great details (Ebersbach and Gerdes, 2005; Frost and Koraimann, 2010; Kroll et al., 2010; Sengupta and Austin, 2011; Sysoeva et al., 2020). But recently more studies show that plasmid-carrying cells (1) often have higher fitness and (2) can undergo rapid genetic changes in the plasmid and in the host chromosome(s) to minimize the cost of carrying the plasmid. This way novel next generation sequencing (NGS) methods now allow determination of complete plasmid sequences and measurement of host chromosome and plasmid co-evolution in almost real time. Rapid co-evolution of plasmids and their hosts was observed already within 100–200 generations and concerns both plasmid and chromosomal genes (Hughes et al., 2012; Hülter et al., 2017; Stalder et al., 2017). On other hand, there are known examples when a plasmid co-exists with its host for extended times without undergoing significant changes. Moreover, with current ability to establish complete sequences of plasmids reports appear on highly conserved plasmid sequences and backbone structures (Harmer and Hall, 2020; Schmitz-Esser et al., 2015; Weisberg et al., 2020).

The plasmid pRK100 was identified in 1990 in a clinical strain of Escherichia coli KS533 isolated from urine of a patient with urinary tract infection (UTI). Initial phenotypic and sequencing characterization of this plasmid found the presence of transfer genes responsible for conjugation of this plasmid, aerobactin iron uptake system, ColV colicin toxin and resistances to ampicillin and tetracycline (Žgur-Bertok et al., 1990). Later, the resistance genes were found to be part of the Tn5431 transposon, which arose by transposition of Tn3 carrying ampicillin resistance gene bla into Tn1721 with its tetracycline resistance gene tetA (Žgur-Bertok et al., 1996; Žgur-Bertok et al., 1994). Further research revealed that pRK100 plasmid also encodes the ColIa toxin, two replicons, and can integrate into the chromosome. Following studies measured the size of pRK100 at ~145 kB, constructed the restriction map by hybridization experiments, and established nucleotide sequences of several regions of interest (Ambrožič et al., 1998). pRK100 plasmid contains two functional replication regions that were identified by constructing and testing several minireplicon plasmids - an F plasmid related RepFIB and a R1 plasmid related RepFIIA replication region (Starčič Erjavec et al., 2003; Starčič Erjavec and Žgur-Bertok, 2006). PCR and hybridization experiments further demonstrated that pRK100 harbors multiple IS2 and IS3 insertion sequences (Starčič Erjavec et al., 2003). Further it was discovered that the pRK100 tra region is the most similar to the tra region of the prototypic F plasmid (Starčič Erjavec et al., 2002). Functional studies identified cyclic AMP (cAMP), H-NS and Lrp as regulators of the transfer genes expression (Starčič Erjavec et al., 2003; Starčič et al., 2003).

The evolutionary mechanisms underlying sequence conservation versus rapid evolution in different plasmids are unclear. Hence more functional studies are needed to understand plasmid-gene functions and behavior in different hosts in conjunction with the NGS methods to connect the phenotypes with genotypes. The aim of this work was to establish the complete nucleotide sequence of pRK100 plasmid and to perform its further phenotypic characterization to expand our knowledge on this ColV plasmid.

Material and methods

Bacterial culture conditions

All strains were grown in LB (liquid medium or solid plates). When needed the growth medium was supplemented with antibiotics (ampicillin (Amp) 100 μg/ml; tetracycline (Tc) 10 μg/ml; streptomycin (Sm) 150 μg/ml). When grown in liquid medium, the culture was aerated (180 rpm). Bacterial strains used in the study are listed in Table 1.

Table 1

Bacterial strains used in this study.

Bacterial strain	Phylogenetic group	Characteristics	Reference or source
KS533	B2	Natural Escherichia coli strain, isolated from urine of a patient with a urinary tract infection (UTI), original strain harboring pRK100	Žgur-Bertok et al., 1990
KS533-p	B2	KS533 strain without the plasmid pRK100 (KS533 was treated with SDS in order to lose the plasmid)	Ambrožič et al., 1998
CL225	A	E. coli HB101 strain with pRK100	Ambrožič et al., 1998
J96	B2	Natural E. coli strain, isolated from urine of a UTI patient, prototype of an uropathogenic E. coli	E. Moreno
MG1655	A	Model non-pathogenic laboratory K12 E. coli strain	C. Beloin
BJ69	B2	Commensal E. coli strain isolated from feces of a healthy person	Starčič Erjavec et al., 2010
BJ69 + p	B2	BJ69 strain with pRK100	This study
R33	B2	Clinical UTI E. coli isolate	M. Kuznetsova
R43	B1	Clinical UTI E. coli isolate	M. Kuznetsova
R53	C	Clinical UTI E. coli isolate	M. Kuznetsova
R55	B2	Clinical UTI E. coli isolate	M. Kuznetsova

DNA isolation and sequencing

Total genomic DNA from CL225 (HB101 strain carrying pRK100) was purified from 8 ml of dense LB-grown culture via MagAttract HMW DNA Kit (Qiagen, Germantown, MD). The obtained DNA was used by the Sequencing and Genomic Technologies Core Facility of the Duke University Center for Genomic and Computational Biology for library preparation and sequencing.

For short-read Illumina NextGen sequencing, 500 ng of genomic DNA was sheared to produce ~500 bp DNA fragments. DNA-Seq libraries were prepared using the Kapa BioSystem HyperPrep Library Kit (Cape Town, South Africa), amplified by PCR and purified using AMPure beads (Beckman Coulter, Indianapolis, IN). The library was sequenced on Illumina NextSeq 500 Mid-Output flow cell (Illumina, San Diego, CA, USA) configured for 150 bp reads.

For long-read PacBio sequencing, 500 ng total DNA was sheared to the average 10 kbp size and used for creating the multiplexed microbial SMRTbell Libraries for the PacBio Sequel system (Pacific Biosciences, Menlo Park, CA). The library was subjected to size selection using the BluePipin automated electrophoresis system with the lower cutoff of 4 kbp (SageScience, Beverly, MA) and subjected to sequencing on Sequel instrument. When two alternative assemblies for pRK100 revealed several single nucleotide mismatches, the consensus sequence was established via classic Sanger sequencing (Genewiz, NJ) using the specific primers (Table S1).

Assembly and annotation of the complete pRK100 sequence

Illumina and PacBio reads were used in hybrid data de novo assembly using default settings of the Unicycler program (Wick et al., 2017) at the HPC cluster of the Alabama Supercomputer. This assembly resulted in three contigs one of which represented the circularized sequence of pRK100 plasmid with 142,357 bp. The chromosome sequence was assembled as 2 contigs – 4,497,988 bp and 303 bp. The coverage of the pRK100 was at 1.73 copies per chromosomal contigs. The annotation of the resulting pRK100 sequence was done using automated NCBI Prokaryotic Genome Annotation Pipeline (PGAP) upon deposition of the assembled sequence onto GenBank database (Haft et al., 2018; Li et al., 2021; Tatusova et al., 2016).

Determination of the phylogenetic groups and plasmid similarity

The determination of the KS533 phylogenetic groups was performed according Clermont scheme (Clermont et al., 2013). The determination of the phylogenetic groups for other strains was done by the in silico Clermont typing using http://clermontyping.iame-research.center/ with default settings (Beghain et al., 2018; Clermont et al., 2019) and chromosomal assemblies of the relevant strains available online (2009_36 strain GCA_013780485.1, F2_14D strain GCA_013780445.1, 11.3_R3 strain GCA_002001945.1).

Comparison of plasmids was done by online BLASTn tool (Johnson et al., 2008), MAUVE program (Darling et al., 2004), and average nucleotide identity (ANI) calculator (Goris et al., 2007).

Solid medium conjugation assay

The strain CL225 (HB101) was used as the donor strain for the conjugative transfer of the pRK100 plasmid to recipient BJ69 strain and to MG1655 strain on an LB plate. The conjugative transfer was performed as described earlier (Starčič Erjavec et al., 2015), with the exception of using minimal A medium plate supplemented with Amp and Tc as the selection plate.

Quantitative conjugation assays

Single colonies of donor (CL225) and recipient (MG1655, R33, R43, R53, R55) strains were inoculated in 5 ml of liquid LB with the appropriate antibiotics and incubated at 37 °C and 160–180 rpm overnight. Overnight cultures were diluted in a 1:100 ratio in 5 ml of liquid LB without antibiotics and incubated at 37 °C and 160–180 rpm for 2 h. 1 ml of recipient strain culture was centrifuged at 5000 rpm for 10 min, the supernatant was discarded and the harvested cells were resuspended in 400 μl of the donor strain culture. The whole volume was spread over an LB plate and incubated at 37 °C for 24 h. Subsequently, conjugation mixture was collected from the LB plate by resuspension in 1 ml of 1 × dPBS. Tenfold serial dilutions were prepared with 1 × dPBS and plated on selective plates for CFU counts of the transconjugants (minimal medium with glucose and tetracycline), the recipient strain (MacConkey medium) and donor strain (LB medium with streptomycin). All plates were incubated at 37 °C overnight and CFUs of the transconjugants, the recipient and the donor strain were assessed. Selectivity of plates was tested by streaking the donor and the recipient strains separately on selective plates and incubating overnight at 37 °C. The conjugation frequency was calculated as CFU of transconjugants over the CFU of recipient cells. Experiments were done in biological triplicates with consecutive calculation of arithmetic means and standard deviations of the conjugation frequencies.

Urothelium cell viability assay

For the assessment of the possible change in pathogenicity of the E. coli strain BJ69 due to the presence of the pRK100 plasmid an in vitro biomimetic pathogenicity model based on the porcine bladder urothelium primary cell culture was used. Firstly, the NPU cells were cultured on multi-well microtiter plastic plates using initially medium for proliferation and then medium for the differentiation of the cells in later stages of growth. Cell culture model was prepared for the inoculation when the NPU cells finally formed highly differentiated multi-layer urothelium. This in vitro model of urothelium was inoculated with bacterial cultures (with multiplicity of infection 10:1) and incubated for 3 h. Subsequently, the plate was washed, treated with a commercially available TrypLE™ Select Enzyme and individual urothelial cells were stained using trypan blue dye. Suspension of contrasted NPU cells was transferred to hemocytometer and the NPU cells were counted manually using inverted-light microscope. Viability of the urothelial cells was the main measure of the pathogenicity of E. coli strains in this model (Predojević et al., 2018).

Statistical analysis

For statistical analysis, either viability values obtained in urothelium cell viability assays or conjugational frequencies of strains, Analysis ToolPak in Excel was used. Single factor or one-way ANOVA were used to test the null hypothesis that the data means obtained in the repeated assays with different strains were all equal. When the null hypothesis was rejected by the ANOVA analysis, the F-test (one-tail) was performed in order to test the null hypothesis that the variances of two data sets were equal. Finally, depending on the F-test results, either the t-Test (two-tail) assuming equal or unequal variances was performed to analyze the null hypothesis that the means of two data sets were equal.

Results and discussion

Overview of the complete pRK100 sequence

Using a hybrid sequencing and assembly approach, the complete sequence of the pRK100 plasmid was established. An annotated circular plasmid map of pRK100 is presented below (Fig. 1). The map shows the arrangement of key genes similar to the F plasmid with the transfer region spanning about 35 kb. Sequencing showed 1.73× coverage with the Illumina reads that is reflective of the low copy number of pRK100 predicted before.

Fig. 1.

Map of conjugative plasmid pRK100. Selected annotated open reading frames encoding functional elements are shown. The map is prepared using SnapGene software (from Insightful Science), which is freely available at https://www.snapgene.com/snapgene-viewer/download. The bla and tet genes are part of the Tn1207 (previously Tn5431) element spanning from 47,393 to 63,475 bp.

The complete sequence (Fig. 1) identified the positions of previously established partial sequences with the exception of the IS3 element (IS3 GenBank AY230885, Starčič Erjavec et al., 2003). This element was previously detected via PCR from pRK100 plasmid isolated from E. coli HB101 strain (Starčič et al., 2003). At this stage it is unclear whether this element was excised from the plasmid naturally or it was amplified in the prior PCR assay from contaminating chromosomal sequences, as the host strain HB101’s chromosome appears to carry at least three IS3 copies. Moran and Hall (2018) reported that in the pCERC4 plasmid (a related sublineage plasmid) the IS3 is inserted in the traS gene and due to the polar effects of this IS3 insertion the plasmid is not able to conjugate. Since pRK100 has already been reported to be capable of conjugative transfer (Žgur-Bertok et al., 1990), it is likely that no IS sequences were present disrupting the tra region of pRK100.

pRK100 plasmid is uniquely conserved and geographically widespread

BLAST search revealed that the complete pRK100 plasmid has four highly homologous plasmids in the GenBank: namely pCERC5, pRHBSTW-00004_2, p2009_36_F, and pF2_14D_F (Moran and Hall, 2018; Reid et al., 2019)(Table 2). In particular, pCERC5 plasmid carries only 16 single nucleotide polymorphisms and 2 gaps, being identical otherwise within the entire 142.4 kb sequences. pCERC5 is reported to have a slightly lower copy number ~ 1.3 in comparison with the estimated from sequence coverage 1.73 in this work. Strikingly, the homologous plasmids were found in strains isolated in Australia (2007–2010) and United Kingdom (2017) while pRK100 was isolated in Slovenia (1990). This shows a broad geographic distribution across continents with extreme sequence conservation for over two decades.

Table 2

List of plasmids that show the closest homology to pRKlOO plasmid.

Plasmid name	Size, bp	GenBank accession number	ANI[a], %	Query coverage, %	Percent identity	Differences with pRKlOO	Isolation source	Isolation location	Isolation year	Host Phylogroup	Co-habitant plasmids	Reference or source
pRKlOO	142,357	CP060383	na[b]	na	na	na	Urine from patient with UTI	Slovenia	1990	B2	none	Žgur-Bertok et al., 1990
pCERC5	142,359	KU664810.1	99.99	100	99.99% (46 mismatches)	No large rearrangements/insertions/deletions	Feces of a healthy human	Australia	2010	B2	none	Moran and Hall, 2018
pRHBSTW-00004 2	143,134	CP056915.1	99.99	100	99.99% (49 mismatches)	ISl-like element IS1A family transposase in between traG and traS ORFs	Wastewater influent	United Kingdom	2017	B2	none	Shaw L.P., unpublished,
p2009_36_F	143,671	MK461929.1	99.99	99	99.99% (48 mismatches)	IS3 transposase is inserted in a colicin gene breaking it into two ORFs	Human catheter stream urine from person with UTI	Australia	2009	B2	IncHI2	Reid et al., 2019
pF2_14D_F	139,372	MK46192.1	99.95	97	99.90%; (45 mismatches)	Insertion sequences in trbB ORF (ISEc23), modified traG; Tnl721- derivative transposon	Fecal swab of a healthy piglet	Australia	2007	B2	IncHI2	Reid et al., 2019

ANI - average nucleotide identity.

na - not applicable.

As the detailed comparisons of the pCERC5, p2009_36_F, and pF2_14D_F among themselves and with other closely related plasmids were recently published (Moran and Hall, 2018; Reid et al., 2019) we only briefly summarize the differences of the five most similar plasmids in the Table 2.

pCERC5, p2009_36_F, and pF2_14D_F were found in hosts with very close genotypes and thus Reid and co-workers hypothesized that the plasmid is unchanged because the host strains were closely related (Reid et al., 2019). pRK100 was found in B2 KS533 strain but then was propagated in HB101, which is a K12 clade strain.

Interestingly, p2009_36_F, and pF2_14D_F plasmids were found in strains with a larger, over 200 kb, IncHI2 plasmids, while the pCERC5 and pRK100 were the only plasmids in their respective carrier cells. The identified IncHI2 plasmids carry multiple mobile genetic elements (insertion elements and transposons) that could have potentially contributed to the observed changes in pRK100 homologues, but detailed studies will need to be conducted to test this hypothesis. In addition to this speculation, one can note (Table 2) that there are several changes in the transfer region of the pRK100 homologues that includes splitting of traG ORF, modifying traG ORF, inserting transposase or inverted repeats (between traG and traS; trbA and trbN). Additionally, two observed changes concern colicins’-proximal or interrupting locations. It is unclear at this point if these are just coincidences.

Phenotypic characterization of pRK100

pRK100 conjugation efficiency strongly depends on recipient cell identity

As recently it became evident that different E. coli strains can differ in their recipient efficiency of a plasmid via conjugal transfer (Sysoeva et al., 2020; Kuznetsova et al., 2021), conjugation experiments with pRK100 into five different E. coli strains, four clinical uropathogenic E. coli strains and one laboratory E. coli strain (MG1655), were performed.

Conjugation assays revealed differences in conjugation frequency in mating assays with different recipient strains (Fig. 2, Table S2). As seen from Fig. 2 we were able to successfully perform conjugal transfer of plasmid pRK100 into three different recipients (R43, R53 and MG1655), albeit with very different conjugation frequencies. The arithmetic means and standard deviations of the conjugation frequencies obtained from three independent experiments were as follows: 6.09E-04 ± 3.76E-04 for the mating pair CL225 × MG1655; 6.34E-02 ± 7.74E-02 for the mating pair CL225 × R43 and 7.99E-06 ± 9.07E-06 for the mating pair CL225 × R53. Into the two other strains, R33 and R55 plasmid pRK100 could not be transferred.

Fig. 2.

Recipient background strongly affects conjugative transfer of pRK100 plasmid. Four recent clinical isolates of uropathogenic E. coli and the laboratory MG1655 E. coli strain were used as recipients of pRK100 conjugation from CL225 strain. The average conjugation frequency, expressed as number of transconjugants (colony forming units/ml, CFU/ml) per number of recipients (CFU/ml), and respective standard deviation are calculated from three independent mating assays. In strains R33 and R53 the conjugation frequency was below the detection limit of the assay. Statistical analysis showed that the observed pairwise differences in conjugation frequencies were statistically significant (MG1655:R43 p = 0.0161; MG1655:R53 p = 0.004 and R43:R53 p = 0.0008). Presence of possibly excluding incF plasmid in recipient background is labeled based on PCR amplification data (Kuznetsova et al., 2021).

The mechanism behind the observed differences in conjugation frequencies could be related to presence of prophages that could be triggered by the conjugation-induced SOS response, synthesis of bacteriocins, differences in biofilm formation abilities, differences in restriction-modification systems, surface exclusion due to recipient’s possession of another conjugative plasmid or CRISPR-Cas system (Samson et al., 2015; Stalder and Top, 2016; Thomas and Nielsen, 2005). As the employed R strains were previously (Kuznetsova et al., 2021) analyzed for the presence of prophages, production of bacteriocins and presence of RepFIA sequences in the genome, it could be stated that both recipients into which pRK100 did not conjugate, harbored RepFIA sequence as revealed by PCR and hence probably the presence of another conjugative plasmid was either excluding or incompatible with the pRK100 conjugation. In addition, the phylogentic background of a strain could influence the ability to receive a plasmid. Recent study of transmission of plasmids conferring multi-drug resistance (MDR) suggested that acquisition and maintenance of MDR plasmids might be related to appearance of adaptive mutations in intergenic regions and selection on genes involved in anaerobic metabolism (Dunn et al., 2019). But further studies are needed to identify the underlying mechanisms.

pRK100 plasmid does not increase uropathogenicity of E. coli strains

As the pRK100 plasmid has several features that could be connected with host strain’s virulence, the KS533 strain with the plasmid and the KS533 strain that was cured of the plasmid (KS533-p) were tested for pathogenicity on an in vitro biomimetic pathogenicity model based on the porcine bladder urothelium primary cell culture (Predojević et al., 2018). In addition, a natural E. coli strain, isolated from feces of a healthy person (BJ69) and the BJ69 harboring pRK100 (BJ69 + p) were tested. For controls, we used the laboratory non-pathogenic E. coli strain MG1655 and highly uropathogenic strain J96 showing low and high cytotoxic activity, respectively (Blum et al., 1995).

As shown in Fig. 3 strains possessing the pRK100 plasmid (KS533 and BJ69 + p) caused a small, non-significant, increase in the viability of the urothelial cells of the in vitro model compared to the strains without the plasmid (KS533-p, BJ69). The pathogenicity level of all tested natural strains (KS533, KS533-p, BJ69, BJ69 + p) was moderate (Fig. 3) when compared to the two control MG1655 and J96 strains. This observation is in contrast with previously mentioned studies of other ColV plasmids that do increase virulence of carrying bacterial strain in diverse infection models including urinary tract colonization (Aguero et al., 1989; Lemaître et al., 2013; Nolan et al., 2003; Skyberg et al., 2006).

Fig. 3.

pRK100 plasmid does not increase virulence of E. coli bacteria towards urothelial cells. Viability of the porcine urothelial (NPU) cells was assessed after infecting them with various E. coli strains: non-pathogenic laboratory strain MG1655 (green); human uropathogenic strain J96 (red); human commensal strain BJ69 and urinary tract infection isolate KS533 with (solid fill) and without (slanted) pRK100 plasmid (blue). Unlike difference between non-pathogenic MG1655 and uropathogenic J96 strains, the differences between BJ69 with and without pRK100 as well as KS533 and KS533 without pRK100 were not statistically significant.

As presence of pRK100 plasmid in two different E. coli backgrounds does not increase their cytotoxic effect on the epithelial cells, it is possible that other properties of this plasmid are important for bacterial cells driving plasmid’s maintenance and selection in populations. The strain possessing pRK100 plasmid does not grow appreciably slower or faster (not shown) and, therefore, other properties should be at play. If one were to speculate perhaps presence of pRK100 plasmid helps with changed growth or metabolic capacity in urine, bacterial adhesion, or it has increased epithelial cell invasion, as it was shown for a well characterized uropathogenic strain of E. coli UTI89 carrying F-type plasmid pUTI89 (Cusumano et al., 2010).

Contemporary issues in plasmid classification, taxonomy, and naming

Recent efforts were made to review, to organize and to propose unifying nomenclature of the conjugative transfer or plasmid genes (Fernandez-Lopez et al., 2017; Orlek et al., 2017; Thomas et al., 2017). Several types of classification of plasmids are currently in use or proposed: (1) based on replication mechanisms and incompatibility groups (Carattoli et al., 2005); (2) Mob families for conjugative plasmids using conservation of key transfer protein – relaxase (Francia et al., 2004; Orlek et al., 2017); (3) partitioning mechanism (Bousquet et al., 2015), (4) based on ANI via a taxonomic classifier of plasmids tool (COPLA) for plasmid taxonomic units (PTUs) (Redondo-Salvo et al., 2021). Unfortunately, these classifications cannot capture all essential plasmid features, for example, Mob-based classification does not relate to nonconjugative plasmids; replicon typing gets complicated by abundant multireplicon plasmids, such as pRK100.

With current improved NGS and assembly methods, like the hybrid assembly used here, we are able to recover complete plasmid sequences (Douarre et al., 2020; Galata et al., 2018). It is highly likely that we will observe fast-changing, unique plasmids as well as those that are more well-conserved, similar to the pRK100 plasmid described here. Therefore, as a community, we need to discuss how to better classify plasmids based on their conserved backbone homology and how to treat highly similar homologues in terms of naming, database deposition, and literature discussions. While clearly a generalized classification akin 16S rRNA gene analysis is not possible for plasmids, some classification resembling viral taxonomy might benefit the field. In this example, sequences of two identical plasmids (barring the few substitutions and gaps) were established (CP060383 in current study and KU664810 from Moran and Hall, 2018). It seems prudent to keep one name and database entry to allow for easier literature searches or to have specific notations included into the database entry indicating that the same or nearly the same plasmid was identified independently in another project.

Conclusions

Complete sequence of the classic F-type plasmid pRK100 was established using hybrid sequencing approach and de novo assembly. Bioinformatic analysis showed that pRK100 sequence is extremely conserved and this plasmid was detected in different sources, locations, and times spanning two decades. Our targeted phenotypic characterization of pRK100 plasmid did not provide an explanation of why this plasmid has the extreme sequence conservation in different genotypic E. coli backgrounds. Therefore, as of now it appears that the well characterized antibiotic resistances, conjugative transfer system, encoded colicins and several toxin/antitoxin pairs, hemolytic factors, and enhanced iron scavenging systems provide sufficient benefit to carrying pRK100 plasmid and pressure to keep unchanged sequence throughout this large, 142 kb plasmid. Further studies are needed to trace epidemiology of this plasmid and understand why under some conditions this plasmid remains intact while in other plasmids quickly evolve.