Complex secondary structure in small Rep_3 plasmids of Acinetobacter spp.

Bacterial plasmids are important mobile genetic elements which often carry specific genes important for bacterial successful survival under various inhospitable environmental conditions. Most of the previous research has focused on large plasmids providing these beneficial traits to their host cells. In this study, small cryptic plasmid pALK1 (3 051 bp) was isolated from metallotolerant and alkalitollerant strain Acinetobacter sp. K1. The plasmid encodes Rep_3 family replication protein and MobM mobilization protein but none pALK1-like plasmids were detected in other Acinetobacter strains of environmental and animal origin. The secondary structure of the pALK1 plasmid is characterized by the complexity of multiple sets of direct and inverted repeats in its nucleotide sequence. Comparative genomics was used to hypothesize the biological functions of these repeats in Acinetobacter spp., whereas several similar plasmids with a related organization of direct repeats and palindromes are known in this genus.


Introduction
There are various natural extreme environmental conditions on the Earth.These inhospitable environments provide the habitats for many extremophilic organisms, such as bacteria which cannot survive under normal physiological conditions, as well as for extremotolerant microorganisms able to survive under both extreme and physiological conditions (Pikuta et al. 2007).These bacteria have developed various adaptation mechanisms during evolution enabling them to withstand harsh conditions.However, new types of unnatural environments also arise due to massive industrial activities often leading to the presence of heavy metals, dangerous organic or other toxic compounds in soil or water with unfavorable, negative effect to all living organisms.Specific adaptation to changing environmental conditions can be associated with the presence of extrachromosomal plasmid DNA.Plasmids frequently harbor genes encoding proteins involved in important metabolic processes, antibiotic resistance, virulence or tolerance to high concentrations of heavy metals or other toxic compounds (Elwell and Shipley 1980;Silver 1992;Russell 1996;Bennet 2008;El-Deeb and Altalhi 2009;Mustapha and Halimoon 2015).Plasmids, as mobile genetic elements, can be spread by horizontal gene transfer and thus they play an important role in the evolution and development of bacterial tolerance and adaptation to extreme environments.Horizontal gene transfer can be accomplished through conjugation, transformation or phage transduction (Thomas and Nielsen 2005).

Abbreviations
According to classical theoretical models, the maintenance of plasmids in bacterial cells is possible through bacterial conjugation.However, two types of situations may arise.First, plasmids with beneficial genes allow bacteria to survive under selection pressure (e.g., antibiotics, heavy metals etc.).Second, without selective pressure, plasmid maintenance increases the energy requirements in bacteria and decreases bacterial fitness (Baltrus 2013;Vogwill and MacLean 2014).These situations occur in the case of plasmids carrying genes beneficial or vital to the bacteria.Thus, the conjugation provides very important gene transfer in the pan-genome of the environment, such as spread of antibiotic resistance in the bacterial population.This fact is one of the reasons causing significant problems in the treatment of bacterial infections in clinical practice.On the other hand, the existence of the horizontal gene transfer leads to the spread of very interesting genes involved in the tolerance and/or detoxification of various environmental contaminants (Dong et al. 1998;Leungtongkam et al. 2018;Sultan et al. 2020).The study of the genetic organization of plasmids originating from extremotolerant bacteria is helpful for better understanding of adaptation mechanisms, and/ or their evolution, and for possible incorporation of bacteria into various types of biotechnologies and bioremediation processes.
Conjugative plasmids often consist of a primary "backbone" which includes rep genes for independent replication, cop genes for plasmid maintenance (stable copy number of plasmids in cell), genes for conjugation transfer mob and tra, par and mrs modules for stable vertical transmission and segregation fidelity and accessory elements with beneficial functions.Some plasmids have stb modules to kill plasmid-free segregants (Norman et al. 2009).On the other hand, many cryptic plasmids without any beneficial genes can be found in various bacterial strains and some of them do not contain all genes important for conjugation.This is the case of a small cryptic mobilizable plasmid pALK1 (3 051 bp) isolated from metallotolerant and alkalitolerant strain Acinetobacter sp.K1 originally found in industrial area near Ziar nad Hronom (Slovakia) contaminated with high concentrations of several heavy metals (Kopcakova et al. 2014).
Members of the genus Acinetobacter are strictly aerobic, Gram-negative, oxidase-negative, indole-negative and catalase-positive coccobacilli ubiquitous in various environments including soil and wastewaters, human and animal body, hospital environment, different types of foodstuffs and extreme environments (Ventosa et al. 1998;Doughari et al. 2011;Ghaima et al. 2018;Gonzalez-Martinez et al. 2018;Ekwanzala et al. 2020).Even though acinetobacters are an important component of various bacterial communities, their ecological roles in environment are still poorly understood (Veress et al. 2020;Zhao et al. 2023).Much attention is paid especially to clinically important species (e.g., A. baumannii) characterized by high antibiotic multiresistance and pathogenic potential (Vázquez-López et al. 2020).
Plasmids found in Acinetobacter spp.could play an important role in environmental adaptation and enhance the accumulation and horizontal transfer of various accessory genes (Maslova et al. 2022;Moran et al. 2022).Why bacteria maintain cryptic non-conjugative plasmids even though they do not contain any beneficial genes remains an unanswered question.According to Iranzo et al. (2016), they act as parasites because they use cellular energy for their synthesis.Despite the great interest in the study of large plasmids encoding antibiotic resistance genes, Lean and Yeo (2017) suggested in their review that small cryptic plasmids should not be forgotten in investigations.Studying of their secondary structure may provide interesting insights into their replication, mobilization or simply their existence.
The aim of this study was to clarify using comparative genomics the biological role of the multiple direct and inverted repeats found in the nucleotide sequence of the pALK1 cryptic plasmid isolated from the environmental Acinetobacter strain K1.

Origin of bacterial isolates and their identification
Bacterial isolates of the genus Acinetobacter were collected from various environmental and animal sources (Table 1).
Cultivation was carried out under aerobic conditions on Tryptic soy agar (TSA) (BD Difco, USA) or in liquid Luria-Bertani (LB) medium (BD Difco, USA) at 25 °C in the case of environmental isolates and at 37 °C in the case of animal isolates in all performed experiments.The identification of environmental isolates was performed in previous studies by Kopcakova et al. (2014) and Pristas et al. (2015).In the current study, all animal isolates were identified using Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS).Bacterial samples were prepared for analysis according to Ferreira et al. (2011) and were analyzed by the Microflex LT MALDI-TOF MS system with FlexControl v.3.0 (Bruker Daltonics GmbH, Germany).Protein profiles were analyzed using the Biotyper v.2.0 software against the reference library v.3.0.

Plasmid isolation, restriction analysis, recombination DNA techniques and PCR
The presence of plasmid DNA in Acinetobacter isolates was examined using a modified alkaline lysis method (Irawati et al. 2016) followed by electrophoresis in a 1% agarose gel.Isolated plasmid DNAs were digested with several restriction endonucleases (BamHI, EcoRI, EcoRV, HindIII, PaeI, PvuI, PstI, SacI) and obtained fragments were separated using electrophoresis in 1.5% agarose gel.Agarose gels were stained with ethidium-bromide (0.5 µg/mL) and DNA fragments were visualized under UV light using the Gel Logic 212 PRO Imaging System (Carestream, Health Inc., Rochester, NY, USA).
We selected the Acinetobacter sp.K1 isolate to evaluate the potential biological role of plasmid DNAs based on the presence of multiple plasmid DNAs found in its genome and its high tolerance to extreme conditions detected in our previous research (Siposova et al. 2017).To clone the pALK1 plasmid obtained from Acinetobacter sp.K1, the 2 800 bp EcoRI (Fermentas, LT) restriction fragment was cut out from the 1.5% agarose gel and purified using Wizard® SV Gel and PCR Clean-Up System (Promega, USA).Subsequently, five microliters of purified DNA were ligated into plasmid vector pUC118 EcoRI/BAP (Amp r ) (Takara, Japan) using T4 DNA ligase, 5 x concentrated T4 DNA Ligase Buffer and nuclease-free water according to manufacturer instructions (InsTAclone™ PCR Cloning Kit; Thermo-Scientific, USA).The reaction mixture was incubated for 1 h at 25 °C, then overnight at 4 °C and subsequently used to transform E. coli MC 1061 (Str r ) cells using the heat shock method of bacterial transformation (Chang et al. 2017).
Recombinant plasmids were isolated using GenEl-ute™ Plasmid Miniprep Kit (Sigma-Aldrich, USA) and sequenced using the Sanger dideoxy termination method by GATC Biotech (Konstanz, Germany).Subsequently, based on the sequences obtained, MC9outF/MC9outR primers were designed for PCR amplification of the remaining part of the pALK1 plasmid in all isolates listed in Table 1.
The PCR product obtained using total DNA of the K1 isolate as a template was purified, ligated into the plasmid vector pTZ57R/T (Amp r ) (Thermo-Scientific, USA), cloned and sequenced similarly as described above.After sequencing, the pALK1 sequence was completed and deposited under accession number ON109139 to the Genbank Brevundimonas spp.and Acinetobacter spp.(Pristas et al. 2015).
Seven animal isolates of Acinetobacter spp.were obtained from the cloaca and internal organs of exotic reptile and mammal species reared in large-scale farming outside the European Union, which died during the transport to Poland (see Table 1).
In our set of environmental samples, A. calcoaceticus was found to be prevalent species in environmental Acinetobacter communities and A. baumannii dominated among animal isolates.The K1 isolate from the brown mud was identified as Acinetobacter spp.and selected for further analysis (this study) based on the presence of complex plasmid population observed in our previous research.At least four plasmids in size from 3 kbp to more than 25 kbp were detected in this strain (Siposova et al. 2017).Since the K1 isolate showed a MALDI TOF MS score below the reliable species identification threshold (classified as Acinetobacter lwoffii with the score of 1.96), its 16 S rRNA gene sequence was analyzed.The 16 S rDNA sequence showed 99.8% similarity with the Acinetobacter lwoffii strain ZS207 (CP019143.2) (Kopcakova et al. 2014).However, complete genome comparisons using the Type (Strain) Genome Server (TYGS) (https://tygs.dsmz.de/;Meier-Kolthoff and Göker 2019) did not cluster the K1 isolate to Acinetobacter lwoffii species (Petrová et al. 2023), therefore we use Acinetobacter sp.K1 nomenclature for this strain in recent study.
Environmental or clinical acinetobacters often contain plasmid DNAs providing specific benefits depending on the environment in which they live, such as high tolerance to toxic heavy metals or antibiotic resistance.Plasmid DNA analysis indicated the presence of extrachromosomal DNA in five bacterial strains analyzed in this study.Multiple plasmids have already been detected in Acinetobacter baumannii, the most important clinical species of the genus Acinetobacter (Towner 2009).However, many plasmids have also been found in environmental acinetobacters, such as in the study of Midlin et al. (2016).They demonstrated four of the five permafrost strains (including Acinetobacter lwoffii) containing 8-12 plasmids with various lengths (4 135-287 630 bp), of which one or two plasmids carried different combinations of genes involved in the mechanisms of heavy-metal and arsenic resistance.Based on these findings, the authors hypothesized that not only mercury, but also the presence of other metals have played an important role in the evolution of the genus Acinetobacter (Midlin et al. 2016).
database (https://www.ncbi.nlm.nih.gov/genbank/).In addition, the pALK1 sequence was also confirmed by the whole genome sequence analysis of Acinetobacter sp.K1 and this sequence is available in the GenBank database under accession number NZ_JALGQY010000067 (Petrová et al. 2023).

Characterization of bacterial isolates
Multiple bacterial isolates were identified in the brown mud created by the sintering method of aluminum production near Ziar nad Hronom (Slovakia) in the previous research (Kopcakova et al. 2014).The brown mud was characterized by extreme pH 11.6 and high heavy metal concentrations at the time of sampling (Schwarz and Lalik 2012).A combination of diverse extreme conditions led to the considerably low bacteria counts (3 500 cfu/g) using a non-selective cultivation medium.The bacterial community of the brown mud include various species belonging to the genera such as Acinetobacter, Bacillus, Kocuria, Isoptericola, Arthrobacter, or Streptomyces (Kopcakova et al. 2014;Pristas et al. 2015).
Four 15-nucleotide long direct repeats designated DR1 are located 68 nucleotides upstream of the replication protein Rep_3 in the pALK1 plasmid.This structure can be recognized by a replication protein as the Rep_3 family includes the E. coli protein RepA which binds to repetitive DNA sequences flanking the gene encoding this protein.Huang et al. (2014) described oriV region of the A. baumannii plasmid pABTJ2 which consists of four time repeating imperfectly conserved direct repeats of 22 bp called iterons (Chattoraj 2000).The part of the sequence that encodes the Rep_3 protein is localized immediately downstream of oriV.This composition is typical of theta-type replication, but a rolling-circle replication mechanism is more characteristic of small multicopy plasmids (Espinosa et al. 2000;Krüger et al. 2004;Khan 2005).Iterons (3-6 repetitions) in Acinetobacter strains with Rep_3 superfamily replicons are 19-22 bp long and average distance between iterons and rep gene is 10-200 bp.Repetitions are organized in a row without any gaps (Lean and Yeo 2017).No such DNA organization was observed in the set of plasmids analyzed in this study.Repetitions in pALK1-like plasmids are shorter and separated by 6-18 nucleotides.
Similar organization of DR1 to that of pALK1 can be observed in pALK1-like and unnamed-like plasmids.The only difference is that the unnamed-like plasmids possess one repetition of the four repeats localized in pALK1-like plasmids.Distances between repetitions and between repetitions and rep gene are highly conserved.RepM plasmids are characterized by different structure of DR1, while repetitions are organized in a row (Fig. 3a).
The putative role of these repeats in replication initiation could be confirmed by the low GC content in the DR1-rich region of the pALK1 plasmid.Plasmid ori regions are generally characterized by a low GC content because of stronger chemical bond between these bases.Cleavage of the AT base pair bond requires less energy and the ori region often consists mainly of them.The AT-rich region in the pALK1 nucleotide sequence was found around 2 716 bp and the second lowest GC content was observed around 590 bp, the region downstream of the DR1 and upstream of the nucleotide sequence of replication protein.
Searching for small Rep_3/RepM plasmids that do not encode mobM showed that only RepM plasmid sequences

Sequence analysis of the pALK1 plasmid
We obtained the complete sequence of a small plasmid isolated from the strain Acinetobacter sp.K1 using recombinant DNA techniques.The pALK1 plasmid is 3 051 bp long and shows significant similarity to five small plasmid sequences available in the GenBank database (Online resource 1: Figure S1).
The sequence analysis shows that the pALK1 plasmid sequence contains two ORFs encoding proteins involved in plasmid replication and mobilization.The ORF1 is 1 134 bp long (377 amino acids) and encodes the mobilization protein Pre/MobM (plasmid recombination enzyme, pfam01076).The second ORF2 is 939 bp long (312 amino acids) and encodes the replication protein belonging to the Rep_3 protein family (pfam01051).The pALK1 plasmid sequence lacks tra genes involved in bacterial conjugation, therefore, it is probably transferred to other bacteria through the conjugative systems of larger conjugative plasmids.Thus, pALK1 is a mobilizable plasmid whose transfer depends on the genetic equipment of another mobile genetic element.This observation is consistent with the findings of Fondi et al. (2010), who demonstrated that most of the analyzed plasmids lack the genes necessary for their transmission (tra genes) or mobilization (mob genes).Therefore, the authors hypothesized that horizontal gene transfer was realized probably by natural transformation or transduction with the participation of bacteriophages.The size of the pALK1 plasmid is consistent with the trend that mobilizable plasmids are smaller than conjugative ones (Smillie et al. 2010).Genes encoding mobilization and replication proteins show high similarities with the genes of Acinetobacter spp.and other genera such as Psychrobacter.All plasmids of similar size to pALK1 (Online resource 1: Fig. S1) contain Rep_3 or RepM and MobM proteins and show high similarity to pALK1 proteins (Tables 2 and 3).
No other putative protein-coding genes were found in the nucleotide sequence of the pALK1 plasmid, indicating that the pALK1 plasmid is cryptic and provides no selection benefits to the host cell.
Blastn analysis revealed the presence of a highly conserved DNA sequence (240-327 nr) in the pALK1 plasmid, which is present in multiple plasmids of Acinetobacter, Psychrobacter and Alcaligenes species (data not shown).Its sequence is characterized by complex secondary structures (Fig. 1).

Occurrence of direct and inverted repeats in the pALK1 sequence
A detailed analysis of the pALK1 nucleotide sequence revealed the set of multiple direct (DR1 -DR5) and inverted 1 3

Table 2
Structural comparisons of DR1-DR5 within the Rep_3/RepM-MobM group of small Acinetobacter plasmids Rep_3/RepM-MobM plasmids of Acinetobacter spp.The second interesting direct repeat DR4 is composed of 21 nucleotides with one part of this repetition located in a highly conserved sequence (240-327 nr) widely presented in Acinetobacter species (Fig. 1).Two hundred and seventynine Acinetobacter strains contain this 21 nr sequence at least once in the plasmid or in nine cases on chromosomal DNA.Two to seven repetitions are found in 37 Acinetobacter species.These plasmids encode a replication protein of the Rep_3 protein family similar to the pALK1 plasmid or the RepM protein (Online resource 1: Table S3).Some of them also encode an uncharacterized protein rep_pAB02_ ORF2.We hypothesize that this sequence might be helpful in natural transformation in Acinetobacter spp.It is known are deposited in the database at the time of research.The secondary structure of DR1 but not the sequence of repeats is conserved in Rep_3/RepM-(non-MobM) plasmids and they contain other repeats in the same position.MobM-(non-Rep_3/RepM) plasmids completely lack the DR1-5 structure (Online resource 1: Table S1).

Plasmid
Direct repeats DR2, DR2-like and DR3 occur in all analyzed plasmids of the Rep_3/RepM-MobM group (Table 2.), while their organization within the plasmid DNA is similar to pALK1 (Fig. 3b, c).DR2-like structure was found just in two plasmids of Rep_3/RepM-(non MobM) group; DR3 is present in all plasmids of this group, with one exception (Online resource 1: Table S1).The pALK1 sequence also contains 14 nr palindrome IR1 likely forming a stem loop structure to which the Mob protein can bind.This palindrome is also found in plasmid pAL_065 − 11.Plasmids unnamed8, p6_010030, unnamed2, unnamed2, pXBB1-1 and p6_060092 contain different inverted repeat at the same position.Almost all these plasmids have this palindrome at the same distance (77 bp) upstream of the mob gene.This distance is shorter by one nucleotide only in plasmid pXBB1-1.In addition, pE47_008, unnamed8, p6_010030, unnamed2, unnamed2 and p6_060092 contain inverted repeats IR4 and IR6 (Table 3).

The occurrence of pALK1-like plasmids in Acinetobacter spp. from various environments
The presence of pALK1 related plasmids in Acinetobacter strains from industrial areas in Ziar nad Hronom, Sered and animal isolates was examined using PCR reactions with primers MC9outF and MC9outR, specifically constructed for the amplification of this part of the pALK1 plasmid.Sequences similar to the pALK1 plasmid were not detected in other isolates of Acinetobacter spp.found in the brown sludge dump or in a similar environment of a landfill waste mud from the nickel production.Similarly, no pALK1-like sequences were detected in animal isolates.This finding is quite surprising because we found several plasmids from different environments with similar size and secondary structure in the NCBI database.

Conclusions
This study was focused on the analysis of the secondary structure of a small cryptic plasmid pALK1 isolated from the bacterium living in the extreme environment and comparative analyses with similar small plasmids from bacteria belonging to the genus Acinetobacter.Based on our results, examined plasmids were divided into three main groups as Rep_3/RepM-MobM plasmids, Rep_3/RepM-(non MobM) plasmids and MobM-(non-Rep_3/RepM) plasmids.Each group has a specific arrangement of direct and inverted repetitions.Repetition DR1 is probably associated with the Rep_3/RepM protein and may function as iteron sequence.DR4 present in multiple plasmids of Acinetobacter species that some Gram-negative bacteria such as Haemophilus influenzae or Neisseria gonorrhoeae are highly selective for DNA acquired by natural transformation processes.Mell and Redfield (2014) identified a short 9 nr DNA segment AAGTGCGGT in H. influenzae named as uptake signal sequence (USS) and a 10 nr DNA segment GCCGTCTGAA in N. gonorrhoeae named as DNA uptake sequence (DUS) by sequencing of preferentially absorbed sequences.These sequences are widely represented in their genomes and are recognized by naturally competent bacteria of the same bacterial species through bacterial surface receptor proteins.However, in the case of N. gonorrhoeae, DNA uptake may even occur when the DUS sequence is not present in donor cells.For example, this was confirmed by the presence of a human gene fragment in the genome of N. gonorrhoeae (Seitz and Blokesch 2012;Anderson and Seifert 2011).Acinetobacter spp.are well studied as natural competent Gram-negative bacteria that are widespread in natural soil and aquatic environments, industrial areas contaminated with toxic substances or in human hosts.
The organization of DR4 and the Rep_3 protein was analyzed in plasmids listed in Online resource 1: Table S3 and in the Rep_3/RepM-MobM group of plasmids (Table 2).The results of this analysis show that the organization of the DR4 repetition and the replication protein changes with plasmid length.Plasmid p6_010030 (CP029394.1)has an almost identical organization and the same arrangement of repeats to pALK1.The plasmids analyzed could be classified into three groups according to distances between DR4 and Rep_3/RepM and between DR4 repeats.First group includes plasmids with the length of 2 000-5 000 bp.In this case, two repetitions are located upstream of the Rep_3 or RepM protein, while distances between them are similar among all plasmids examined.Figure 3d demonstrates small differences compared to the pALK1 plasmid.Although, unnamed-like plasmids encode the Rep_3 protein, they retain different direct repeats upstream of its gene at the similar position to DR1.Larger plasmids (5 000-15 000 bp) encoding the Rep_3 protein contain two repetitions with conserved distances between them.An exception is the plasmid p7_010062 containing four repetitions in front of the Rep_3.RepM plasmids contain only one DR4 sequence which is characteristic of all larger plasmids (15 000-74 000 bp) encoding several RepM proteins (Online resource1: Fig. S2 and Fig. S3).
1 3 has probably another important biological function, which is currently unknown.The MobM protein is probably linked to IR1 palindrome in studied plasmid, because distances between them is conserved in all analyzed plasmids in the Rep_3/RepM-MobM group.On the other hand, further analyses will be necessary to completely understand the biological functions of highly conserved repeating structures found in Acinetobacter plasmids and for better understanding of the existence of small cryptic plasmids in bacteria.
acid sequences; b the sequence is not repeating, DR -direct repeat, + the presence of DR 1 3

3 Fig. 2
Fig. 1 The secondary structure of part (259-327 nr) of the conserved pALK1 sequence (240-327 nr) including the IR6 palindrome and two of the four DR3 repetitions localized 305 bp upstream of the rep gene

Table 1
Origin of isolates used in this study Sampling source