Plasmid Localization and Partition in Enterobacteriaceae.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Three-component ParABS partition systems ensure stable inheritance of many bacterial chromosomes and low-copy-number plasmids. ParA localizes to the nucleoid through its ATP-dependent nonspecific DNA-binding activity, whereas centromere-like parS-DNA and ParB form partition complexes that activate ParA-ATPase to drive the system dynamics. The essential parS sequence arrangements vary among ParABS systems, reflecting the architectural diversity of their partition complexes. Here, we focus on the pSM19035 plasmid partition system that uses a ParBpSM of the ribbon-helix-helix (RHH) family. We show that parSpSM with four or more contiguous ParBpSM-binding sequence repeats is required to assemble a stable ParApSM-ParBpSM complex and efficiently activate the ParApSM-ATPase, stimulating complex disassembly. Disruption of the contiguity of the parSpSM sequence array destabilizes the ParApSM-ParBpSM complex and prevents efficient ATPase activation. Our findings reveal the unique architecture of the pSM19035 partition complex and how it interacts with nucleoid-bound ParApSM-ATP. Editor's evaluation The work by Volante et al. studied a plasmid partition system, in which the authors discovered that four or more contiguous ParS sequence repeats are required to assemble a stable partitioning ParAB complex and activate the ParA ATPase. The work reveals a plasmid partitioning mechanism in which the mechanic property of DNA and its interaction with the partition complex may drive the directional movement of the plasmid. https://doi.org/10.7554/eLife.79480.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Faithful chromosome segregation is essential for the proliferation of bacterial cells, and low-copy-number plasmids also need a robust partition mechanism for their stable inheritance. However, prokaryotes do not possess the mitotic machinery of eukaryotes: instead, alternative active DNA partition systems have evolved, among which ParABS systems (also called class I partition systems) are the most widespread. Basic ParABS systems consist of three components, a partition ATPase (ParA), a ‘centromere’-binding protein (ParB), and a cis-acting centromere-like DNA site (parS). ATP-activated ParA dimers bind nonspecific DNA (nsDNA) and localize to the nucleoid in vivo (Ebersbach and Gerdes, 2001; Ebersbach and Gerdes, 2004; Pratto et al., 2008; Ringgaard et al., 2009). The parS sites, often composed of multiple tandem repeats of binding consensus sequences for ParBs, demark the DNA-cargos that are translocated and positioned into the two halves of the cell before cell division by recruiting ParB molecules to assemble partition complexes (PCs). ParB proteins fall into two structurally unrelated groups, dimeric helix-turn-helix (HTH), and dimeric ribbon-helix-helix (RHH) DNA-binding proteins. HTH-ParBs have been shown to bind not only site-specifically to their cognate parS sequences but also to spread many kilobase pairs into the DNA neighboring the parS sites (Murray et al., 2006; Graham et al., 2014; Soh et al., 2019; Rodionov et al., 1999; Lynch and Wang, 1995; Breier and Grossman, 2007; Jalal et al., 2020; Osorio-Valeriano et al., 2019; Sanchez et al., 2015). Therefore, they form large PCs containing many ParB molecules bound to condensed DNA around parS, and ParB spreading activity is essential for their partition function (Rodionov et al., 1999; Debaugny et al., 2018). In contrast, RHH-ParB does not spread beyond parS site judged by the absence of ParB-mediated silencing of parS-proximal gene expression (J. C. Alonso, unpublished observation), unlike HTH-ParBs (Lynch and Wang, 1995; Rodionov et al., 1999), but like HTH-ParBs, they interact with their cognate ParA proteins via their N-terminus (Radnedge et al., 1998; Figge et al., 2003; Barillà et al., 2007). RHH-ParB proteins also control the expression of the proteins involved in the partition system and plasmid copy number control by binding parS sites, which overlaps promoters of their genes (de la Hoz et al., 2000). ParA–ParB interaction leads to activation of the ParA-ATPase, which is most efficient in the presence of nsDNA and parS DNA (Ah-Seng et al., 2009; Chu et al., 2019; Pratto et al., 2008; Taylor et al., 2021) and leads to dissociation of ParA from nsDNA. Interaction dynamics between the nucleoid-bound ParA and ParB in the PC prior to ATP hydrolysis and ParA dissociation determine the dynamics of the PC relative to the nucleoid. The common results of most systems in vivo appear to be the establishment of equidistant distribution of two or more PCs along the nucleoid(s) so that at cell division each daughter cell inherits at least one copy of the plasmid DNA (Sengupta et al., 2010; Ringgaard et al., 2009; Lioy et al., 2015; McLeod et al., 2017). With biochemical findings and observations from live-cell imaging approaches accumulating in the field, combined with experiments using reconstituted cell-free reaction systems, a diffusion-ratchet mechanism of the ParABS partition was proposed. Here, the driving force for the DNA-cargo motion is generated by a propagating nucleoid-bound ParA distribution gradient (Vecchiarelli et al., 2010; Hwang et al., 2013; Vecchiarelli et al., 2013; Vecchiarelli et al., 2014; Sugawara and Kaneko, 2011). Additional models related to the diffusion-ratchet mechanism have also been proposed based on high-resolution imaging observations (Lim et al., 2014; Le Gall et al., 2016). While findings supporting diffusion-ratchet-type models for ParABS systems accumulate, many molecular details required to put the model on quantitatively solid ground are lacking. Large number of HTH-ParB dimers load onto a PC by ParB ‘spreading’ to facilitate partitioning. Resulting high concentration of ParB at the PC assures near saturation ParB-binding to the local nucleoid-bound ParA molecules, leading to efficient sensing of the local ParA distribution gradient by the PC. Combined with relatively short lifetime of the ParA/ParB-mediated cargo-nucleoid bridges and sufficient ParA-ATP reloading rate to the nucleoid would maintain enough cargo-nucleoid bridges needed to significantly suppress thermal diffusion of the cargo without completely blocking the motion of the cargo. Proper balance among these parameters is needed for efficient plasmid partition by diffusion-ratchet mechanism. Insufficient free diffusion suppression results in random diffusion of the cargo, and oversuppression by too many or too stable bridges blocks the cargo motion altogether (Hu et al., 2015; Hu et al., 2017; Taylor et al., 2021). However, it is unclear how a system with RHH-ParBs, which cannot spread ParB beyond the parS sites and thus can load only limited number of ParB dimers to a PC, can fit into the diffusion-ratchet paradigm. Nevertheless, live-cell imaging studies of systems that use RHH-ParB proteins showed an oscillating dynamic ParA distribution pattern on the nucleoid and PC chasing the receding tail of the ParA distribution (Ringgaard et al., 2009; Lioy et al., 2015; McLeod et al., 2017) similar to observations with the F-plasmid partition system and other related systems involving HTH-ParB proteins (Hatano and Niki, 2010; Schofield et al., 2010) for which there is accumulating evidence supporting the diffusion-ratchet mechanism. Therefore, we suspect systems with RHH-ParBs also operate via a diffusion-ratchet-type mechanism. A better understanding of how the PCs are organized for this group and a quantitative understanding of the interaction dynamics of these PCs with nucleoid-bound ParA molecules are critical for advancing our mechanistic understanding of these systems. In this study, we focused on the pSM19035 partition system of Streptococcus pyogenes. This plasmid harbors a ParABS system composed of ParApSM (also called Delta), an RHH ParBpSM (also called Omega), and six parSpSM sites, each comprising 7–10 consecutive non-palindromic 7-bp-long sequence repeats (5′-WATCACW-3′, symbolized by →) that overlap the promoter regions of copS, δ (coding ParApSM) and ω (coding ParBpSM) genes. Each ParBpSM dimer binds one copy of the 7 bp parSpSM consensus sequence. However, the affinity for a single repeat is low, while two dimers bind with high affinity to two direct (→→) or inverted (→←) repeats forming dimers of dimers (de la Hoz et al., 2004; Weihofen et al., 2006; Welfle et al., 2005). Within the structures of these ParBpSM-parSpSM complexes, DNA does not show significant curvature, and although the protein dimers bound to each DNA sequence repeat slightly deviates from dyad symmetry due to the bound DNA sequence asymmetry, full-size parSpSM-ParBpSM complexes could be modeled as nearly straight DNA wrapped by left-handed spiral arrangement of ParBpSM dimers (Weihofen et al., 2006). Atomic force microscopy images of the complex involving seven parSpSM consensus sequence repeats supported its straight arrangement and the lack of spreading (Pratto et al., 2009). ParApSM, unlike most other ParAs, forms dimers in solution in the absence of ATP (Pratto et al., 2008). Like other ParAs, it also undergoes a conformational transition upon binding ATP that increases its affinity for nsDNA (Soberón et al., 2011; Pratto et al., 2008). In the ATP-bound form, ParApSM has been shown to bind nsDNA forming limited size patches containing several ParApSM dimers at random location, instead of individual dimers independently distributed on the nsDNA (Pratto et al., 2009). In the presence of parSpSM DNA and ParBpSM, several parSpSM-ParBpSM mini-filaments and ParApSM-nsDNA patches appeared to bind together to form large protein-DNA complexes bridging multiple DNA molecules (Pratto et al., 2008; Pratto et al., 2009; Soberón et al., 2011; Lioy et al., 2015). Interactions among these components fueled by ATP hydrolysis are thought to drive dynamic oscillations of the nucleoid-bound ParApSM in vivo, which resembles those observed for the TP228 ParABS system (Lioy et al., 2015; McLeod et al., 2017). Despite accumulating information summarized above, how the observed inter-molecular interactions coordinate the in vivo system dynamics resulting in robust plasmid partitioning remains a mystery. To approach this puzzle, here we studied functional requirements of the parSpSM sequence-structure necessary for ParBpSM-mediated activation of the ParApSM ATPase. We found a minimum of four contiguous repeats of the parSpSM heptad consensus sequence without a gap is necessary for full activity. Kinetics of the nsDNA-bound parSpSM-ParBpSM-ParApSM complex formation and disassembly indicated the presence of a complex multistep process involved in ATPase activation. Results ParApSM ATPase is synergistically activated by nsDNA, ParBpSM, and parSpSM-DNA To define the requirements for stimulation of the ParApSM ATPase activity by ParBpSM, the steady-state ParApSM ATP turnover rate was measured with varying concentrations of ParBpSM in the presence or absence of different duplex DNA cofactors. The turnover rate of ParApSM ATPase alone is very low at 37°C (0.9 ± 0.1 ATP/ParA-dimer/h, N = 3; Figure 1A; no DNA). In the presence of a saturating concentration of double-stranded DNA (40 μg/ml pBR322 plasmid DNA plus 23–38 μg/ml double-stranded oligonucleotide with or without parSpSM sequence), to which ATP-ParApSM dimers can bind to support ATPase activation by ParBpSM (see below), no significant rate change was observed (1.0 ± 0.1 h–1, N = 46, Figure 1A, pool of all measurements at [ParBpSM] = 0). Next, effects of the parSpSM-DNA in addition to 40 μg/ml nsDNA and ParBpSM were examined. ParApSM ATP hydrolysis was stimulated up to ~20-fold (kcat = 20.5 ± 2.9 h–1, N = 7) in the presence of ParBpSM and oligonucleotide duplex DNA containing one of the native arrangements containing seven parSpSM heptad-sequence-repeats (7R-parSpSM, →→←→→←←) (Figure 1A). The stimulation approached saturation around 2 μM ParBpSM at varying ParApSM concentrations (Figure 1—figure supplement 1A). In contrast, when the parSpSM DNA fragment was replaced with one having a scrambled sequence, ParBpSM stimulated ParApSM ATPase activity only to 2.4 ± 1.1 h–1 even in the presence of 8 μM ParBpSM that should have allowed non-parSpSM DNA binding (N = 3*) (Figure 1A, scram). Similarly, a low level of ParApSM ATPase stimulation was observed with ParBpSM1-27 peptide, which lacked the DNA-binding and dimerization domains (Figure 1—figure supplement 1B). These results demonstrated that full ParApSM-ATPase stimulation by ParBpSM requires specific parSpSM interactions. Even the low parSpSM-independent ATPase stimulation was not detected in the absence of DNA (Figure 1A, no DNA). Similarly, the low-level ATPase stimulation by non-parSpSM-binding ParBpSM1-27 peptide was not detected without DNA (Figure 1—figure supplement 1B). We conclude ParApSM-nsDNA binding is required for the ATPase activation by ParBpSM, as reported for ParAF ATPase activation by ParBF (Taylor et al., 2021). Consistently, in the absence of nsDNA, excess ParBpSM relative to the concentration of 7R-parSpSM in the reaction inhibited ATPase stimulation, presumably competing with ParApSM for parSpSM DNA binding (Figure 1—figure supplement 1C). Figure 1 with 3 supplements see all Download asset Open asset ParApSM ATPase activation by ParBpSM, parSpSM, and nsDNA. (A) Efficient activation of ParApSM ATPase by ParBpSM exhibits critical dependency on parSpSM heptad-sequence-repeat number. The ATPase reactions contained ParApSM (2 μM), pBR322 DNA (60 μM in bp, unless noted otherwise), ParBpSM (at the concentration indicated), and parSpSM duplex substrates (4.4 μM of the 7 bp consensus sequence repeats) or equal amount of a scrambled sequence duplex (scram). (B) Comparison of parSpSM containing four contiguous repeats with different heptad orientation arrangements. (C) parSpSM containing different heptad arrangements of three contiguous repeats and two contiguous triple heptad repeats with a gap fails to fully activate the ParApSM ATPase. (D) 7R-parSpSM concentration dependence of ParApSM ATPase activity. Reaction mixtures contained ParApSM (2 μM), ParBpSM (2 μM), pBR322 DNA (60 μM in bp), and increasing concentration of 7R-parSpSM duplex (duplex fragment concentration shown, the ratio of parSpSM heptad repeat sequence to ParBpSM dimers are indicated on top). (E) The numbers and arrangement of the heptad repeats of the parSpSM fragments used in this study (also see Figure 1—figure supplement 2). Data points represent means and standard errors of mean (SEM) of N repeated experiments (N* represents repeats for majority of data points, see Figure 1—source data 1 for details). Curves were fitted after subtraction of the background in the absence of ParApSM to an equation v-v0 = (vmax[B]n)/(KAn + [B]n). The maximum turnover rates (vmax) cited in the text represent mean ± 95% confidence intervals (symmetrized to the larger estimated errors from the mean for simplicity). Figure 1—source data 1 https://cdn.elifesciences.org/articles/79480/elife-79480-fig1-data1-v3.xlsx Download elife-79480-fig1-data1-v3.xlsx Formation of short clusters of ParApSM molecules bound to nsDNA might be taken as a hint for cytoskeletal filament treadmilling models for this class of partition systems, rather than diffusion-ratchet model. However, the low maximum ATP turnover rate (~0.3 /min) observed would be too slow for a treadmilling filament model. Combined with low abundance of ParApSM molecules inside a cell insufficient to form axial protein filaments, the treadmilling model is highly unlikely to fit this system. Efficient ParApSM-ATPase stimulation requires ParBpSM bound to parSpSM-DNA with at least four contiguous heptad-sequence-repeats We asked whether entire 7R-parSpSM is required for the full stimulation of ParApSM ATPase by ParBpSM. A series of deletions of parSpSM heptad-sequence-repeats were made and their ATPase stimulation activities were tested (Figure 1E). Efficient ATPase stimulation was observed when 6R (→←→→←←), 5R (→→→←←), or 4R (→→←←) was added along ParBpSM (Figure 1A). No significant difference was observed among 5R, 6R, or 7R; both half-saturation concentrations and the apparent kcat were comparable (Figure 1A). ParBpSM-4R-parSpSM also induced similar rates of ParApSM ATP hydrolysis, but a significantly higher concentration was required for full stimulation (Figure 1A). Different heptad orientation arrangements of 4R-parSpSM (→→←→ and →→→→) stimulated ParApSM ATPase to a similar extent (Figure 1B). In contrast, 3R- (→→←), 2R- (→←), and 1R- (→) parSpSM were poor cofactors for ParBpSM-dependent ATPase stimulation (apparent kcat = 3.2–3.6 ± 1.3–2.1 h–1, N = 4–7*, Figure 1A), not significantly different from the scrambled sequence DNA. 3R-parSpSM fragments with different repeat arrangements behaved similarly to each other (Figure 1C). The ATPase stimulation by parSpSM DNA with 1–3 parSpSM repeats also appeared to saturate at around 2 μM ParBpSM, indicating that the affinity of the nsDNA-bound ParApSM to ParBpSM in the presence of truncated parSpSM was not limiting above ~2 μM ParBpSM. Previously it has been shown that ParBpSM bound 3R, 4R DNA fragments of different sequence orientation combinations, or a 10R DNA fragment with roughly similar affinity that was >50-fold higher than nsDNA or 1R DNA (de la Hoz et al., 2004). We confirmed that ParBpSM binding was similarly strong for 4R and 3R duplex DNA (KD ~ 17 nM) and weak for nsDNA (KD ~ 1 μM, Figure 1—figure supplement 3). Therefore, the affinity of ParBpSM for the 3R-parSpSM DNA is not limiting the ParApSM ATPase stimulation. In the above experiment, the length of parSpSM DNA fragments decreased as the number of parSpSM repeats decreased (Figure 1E, Figure 1—figure supplement 2). We tested longer 3R-parSpSM DNA fragments containing an additional non-parSpSM heptamer sequence (non-consensus, nc) (3R-1nc) and confirmed that parSpSM-DNA fragment size was not a significant factor for the ATPase stimulation efficiency (Figure 1C). These results suggested that ParApSM ATP turnover is fine-tuned by the structural arrangement of the ParBpSM and parSpSM within the PC. ParBpSM assembles as a left-handed spiral to wrap parSpSM DNA without significantly distorting the DNA backbone geometry (Weihofen et al., 2006), implying that after approximately four repeats, ParBpSM dimers would make a full turn around parSpSM, positioning themselves on the same face of the nucleoprotein filament. Thus, the position of the fourth repeats relative to that of the first repeat might be functionally important. To test this possibility, we examined the ParApSM ATPase stimulation efficiency of parSpSM DNA fragments containing two copies of 3R sequences separated by 7 bp or 14 bp of non-consensus sequence (3R-1nc-3R and 3R-2nc-3R) (Figure 1E). The 3R-1nc-3R-parSpSM fragments showed only slightly higher stimulation (kcat = 8.3 ± 3.5 h–1, N = 8*) compared to the single 3R-parSpSM fragment, and the 3R-2nc-3R was functionally indistinguishable from the single 3R-parSpSM fragment (Figure 1C). We conclude that disrupted 6R-parSpSM fragments are unable to recover the full stimulation of ParApSM ATPase activity. These findings indicate that the ParBpSM-ParApSM interactions differ when ParBpSM dimers are bound to ≥4 contiguous heptad repeats compared to ParBpSM dimers unbound to the repeats or bound to fewer or a disrupted array of heptad repeats. Substoichiometric concentration of parSpSM relative to ParBpSM is sufficient to fully activate ParApSM ATPase We originally assumed that for efficient ParApSM ATPase activation by the ParBpSM-parSpSM complex all ParBpSM dimers need to be bound to parSpSM and accordingly maintained stoichiometrically excess parSpSM-sequence concentration relative to ParBpSM-dimers in the reaction. To test this assumption, we next changed the concentration of the 7R-parSpSM DNA fragment while keeping the concentrations of ParApSM and ParBpSM both at 2 μM. Contrary to our expectation, significantly lower heptad consensus sequence concentrations of the 7R-parSpSM DNA fragment compared to ParBpSM dimers (B2) were sufficient for ATPase activation (Figure 1D). If the original assumption was correct, 2 μM ParBpSM should have required 1 μM consensus sequence repeats (143 nM 7R-parSpSM) for full activation. According to the results of Figure 1A, at high 7R-parSpSM concentration, half-activation by ParBpSM required ~650 nM ParBpSM, which would have required ~325 nM parSpSM consensus sequence if full binding was needed. Observed half-saturation parSpSM repeat concentration was ~130 nM (~19 nM 7R-parSpSM) or less. Thus, assuming most of the ParBpSM molecules in our preparations are active, it appears that at any given time, less than half of the ParBpSM dimers need to be in complex with 7R-parSpSM to exert full ATPase activation. Stimulation of ParApSM release from nsDNA-carpet by ParBpSM requires parSpSM DNA with four or more heptad repeats Next, we examined how the dissociation of ParApSM-ATP dimers from nsDNA is influenced by ParBpSM in the presence of parSpSM DNA. For these experiments, we used an nsDNA-carpeted two-inlet flow cell observed under TIRF microscopy as described in and (Vecchiarelli et al., 2013; Figure We used a protein and a to to facilitate of protein the Stimulation of ATPase activity by ParBpSM and was comparable to ParApSM (Figure supplement we tested the of individual proteins at 1 the The affinity of to nsDNA was in the absence of ParApSM on the a of ± after of flow (Figure supplement the solution was to a without most ParBpSM with that can be fitted to a function = ± = ± N = 3; all reported in this study represent apparent rate (Figure supplement with ATP and a saturation on the nsDNA-carpet of (N = whereas in the absence of less than of ParApSM bound to the nsDNA-carpet (Figure supplement top). Next, 1 μM with ATP was onto the nsDNA-carpet of saturation at which the flow was to containing ATP without A of than within and the with of (N = Figure Figure 2 with 3 supplements see all Download asset Open asset Kinetics of ParApSM disassembly from the (A) of two-inlet flow cell used for the and dissociation of proteins on and each containing double-stranded DNA fragments or alone as were at different flow rates or from two on the into a flow the of the flow of the flow the observations are of the the the flow rates of the two protein binding to the nsDNA-carpet and dissociation the can be (B) ParBpSM in the presence of parSpSM with at least four contiguous ParBpSM-binding sequence repeats the dissociation from the with 1 ATP was into the nsDNA-carpeted flow cell at while the solution containing the components was at the on the nsDNA-carpet of the saturation = ParApSM the flow rates were to the with solution bp fragment nM in bp), or with ParBpSM without or with different parSpSM fragment μM parSpSM heptad repeat The the to that at = Each represents the mean with to the standard errors of mean (SEM) of N repeated Figure data 1 Download The addition of nsDNA nM scrambled bp duplex in the slightly the of majority of = ± N = in by competing with of to the nsDNA-carpet (Figure 1 μM ParBpSM or was added to the without nsDNA, of from nsDNA-carpet ± (N = Figure dissociation from the nsDNA-carpet was when solution contained nM 7R-parSpSM DNA μM of the consensus sequence and 1 μM ParBpSM = ± after a of dissociation rate (see for (Figure This was or higher compared to ParBpSM or nsDNA Similarly, 4R-parSpSM DNA supported release by ParBpSM, but 3R-parSpSM DNA not (Figure The results those shown in Figure the that a functional parSpSM four copies of ParBpSM sequence repeats. The dependence of ParApSM dissociation on the heptad repeat number of the parSpSM fragment combined with 1 μM ParBpSM in the solution when the was with ParApSM bound to the nsDNA-carpet at a saturating was However, the dissociation were significantly (Figure supplement reflecting the approximately higher on the for which the concentration of ParBpSM used was release of ParApSM from only after of ParBpSM on the in the presence of or 4R-parSpSM The release from the nsDNA-carpet when with or parSpSM complexes after an rate of ParApSM release (Figure This to the binding of ParBpSM to the nsDNA-carpet at different concentrations of ParBpSM. the flow to the solution without parSpSM, ParBpSM bound to the nsDNA-carpet to a of and decreased to a of (Figure The of ParBpSM binding appeared to roughly with the dissociation of the slow dissociation of ParApSM, ParBpSM on the nsDNA-carpet relatively with the protein ratio after several of with 1 μM ParBpSM (Figure Figure 3 Download asset Open asset ParBpSM-parSpSM concentration of ATP ParApSM disassembly from the (A) with 1 ATP was into the nsDNA-carpeted flow cell at and when the on the nsDNA-carpet of the saturation the flow the was = to solution containing ParBpSM or nM) and the concentration of parSpSM fragments indicated in the in on the of each of was to protein and and Each represents mean and standard errors of mean (SEM) of N repeated indicate the ParBpSM on the (B) The of the ratio for the four in the in (A) with ParBpSM concentration with different parSpSM in the (C) ATP hydrolysis is required for ParApSM release from the The experiments shown in (A) were repeated using bound to the nsDNA-carpet and ParBpSM nM) plus parSpSM fragment, 3R, or without parSpSM nM heptad in the was to protein and Each represents mean and of N (D) The of the ratio for the four of (C) Each represents the mean with to the of N repeated Figure data 1

Active partition systems and postsegregational killing (PSK) systems are present together in naturally occurring low-copy-number plasmids. Theory suggests that PSK may act as the ultimate determinant of plasmid retention, whereas the partition system may minimize the growth penalty to the host, resulting in a near-ideal symbiosis when the systems combine. Here, we prove the validity of this principle for a specific case involving the P1par system and the mvp PSK system.

DNA-binding proteins play a central role in all aspects of genetic activity within an organism, such as transcription, DNA rearrangement, replication and repair. It is therefore extremely important to analyse the nature of the complexes that are formed between proteins and DNA, as they form the basis of our knowledge of how these processes occur. Partitioning of low copy-number plasmids at cell division is quite similar to mitosis in that, before cell division, paired plasmid molecules are separated from each other and actively moved apart. There are three components that are essential for partitioning to occur, two plasmid encoded proteins and a cis-acting centromere-like site on the plasmid DNA. The first gene of the partition operon involves either an ATPase with Walker-type ATP binding motifs (ParA) or an evolutionary unrelated actin-type-ATPase (ParM). The second partition protein (ParB or ParR) binds to the cis-acting DNA partition site and recruits the ATPase into the nucleoprotein complex. Genes for homologues of ParA and ParB exist on many bacterial chromosomes and plasmids. The IncPα plasmids, exemplified by the low copy-number plasmid RP4, encode for an active partitioning system with homologues of ParA and ParB partitioning proteins called IncC, a putative ATPase, and KorB, a specific DNA-binding protein, respectively. Although KorB has a net negative charge (pI = 4.6), it recognizes and binds specifically to palindromic operator OB (consensus sequence 5´ TTTAGCG/CGCTAAA 3´) occurring at 12 different sites on the plasmid (opera­tors OB1 to OB12), 6 of which are involved in transcriptional regulation. The KorB protein acts as a multifunctional regulator in control circuits of plasmid housekeeping genes involved in replication, maintenance and conjugation. Additionally, KorB functions as the ParB homologue of the plasmid's partitioning system. The major part of the work presented in this thesis concerns the crystal structure of the DNA-binding domain of a global regulator, KorB. This work reveals for the first time the structural basis of DNA binding of a family member of the ParB DNA partitioning factors to its recognition sequence. In this thesis, the X-ray crystal structure of the complex comprising the DNA-binding domain of KorB (KorB-O) and its operator sequence OB, determined to 2.2-Å resolution, is presented. The KorB-O�OB complex crystallized in spacegroup P3221 with cell parameters a = 110.44 Å, c= 160.53 Å, with two copies of the complex in the asymmetric unit. Each half-site of the palindromic operator DNA binds one copy of the protein into the major groove. The KorB-O bound operator DNA adopts a standard B-DNA conform­a­tion with a straight helix axis. The protein structure consists of eight helices two of which belong to a canonical helix- turn-helix DNA-binding motif. The α3-turn-α4 segment of KorB-O has a sequence signature and conformation typical for a helix-turn-helix motif generally involved in specific DNA major groove interactions. However, residues from these two helices do not form direct base contacts in the OB region. The structure reveals mainly two residues, Thr211 and Arg240, which recognize the OB sequence through direct hydrogen bonding. This was further confirmed by mutagenesis, where mutant KorB proteins Thr211Ala and Arg240Ala do not recognize OB specifically suggesting that the mode of operator binding by the KorB-O fragment observed in the crystal structure reflects the specific DNA binding of the wildtype protein. The outer surface of the DNA-bound KorB-O mirrors the overall acidity of KorB, whereas DNA binding occurs via a basic interaction surface. A model of KorB including the structure of its dimerization domain is presented that considers the inter­action with the highly basic ParA homologue IncC. The last chapter of the thesis describes work done to characterize and crystallize the DNA-binding domain of the transposase "Sleeping Beauty" of vertebrate origin. Efforts were made to purify and crystallize different constructs of the N-terminal DNA binding domain. In particular, a protocol is presented for the purification of an active form of the full-length transposase.

Pseudomonas putida KT2440 is an ideal soil bacterium for expanding the range of degradable compounds via the recruitment of various catabolic plasmids. In the course of our investigation of the host range of IncP-7 catabolic plasmids pCAR1, pDK1 and pWW53, we found that the IncP-7 miniplasmids composed of replication and partition loci were exceptionally unstable in KT2440, which is the authentic host of the archetypal IncP-9 plasmid pWW0. This study identified ParI, a homologue of ParA family of plasmid partitioning proteins encoded on the KT2440-specific cryptic genomic island, as a negative host factor for the maintenance of IncP-7 plasmids. The miniplasmids were destabilized by ectopic expression of ParI, and the loss rate correlated with the copy number of ParB binding sites in the centromeric parS region. Mutations in the conserved ATPase domains of ParI abolished destabilization of miniplasmids. Furthermore, ParI destabilized miniplasmid derivatives carrying the partition-deficient parA mutations but failed to impact the stability of miniplasmid derivatives with parB mutations in the putative arginine finger. Altogether, these results indicate that ParI interferes with the IncP-7 plasmid partition system. This study extends canonical partition-mediated incompatibility of plasmids beyond heterogeneous mobile genetic elements, namely incompatibility between plasmid and genomic island.

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.

After more than 150 years of research in microbiology, new technologies and new insights into the microbial world have sparked a revolution in the field. This is a much needed development, not only to renew interest in prokaryote research, but also to meet many emerging challenges in medicine, agriculture and industrial processes. Although many microbiologists—such as Emil von Behring, Robert Koch, Jacques Monod, Francois Jacob, Andre Lwoff, Alexander Fleming, Selman A. Waksman and Joshua Lederberg—grace the list of Nobel laureates, attention moved away from microbiology as biologists focused their interest on eukaryotic cells and higher organisms in the 1970s and 1980s. Furthermore, from the beginning, research on prokaryotes has suffered from an anthropocentric view, regarding as interesting only those organisms that cause disease or that can be exploited for industrial or agricultural use. But the advent of new technologies, some of which have been driven by a need to understand eukaryotes, may change this. We are increasingly realizing how little we know about microbes in general, their diversity, the mechanisms of their evolution and adaptation and their modes of existence within, and communication with, their environment and higher organisms. As bacteria have succeeded in occupying virtually all ecological niches on this planet, ranging from arctic regions to oceanic hot springs, they hold an immense wealth of genetic information that we have barely started to explore and that may provide many useful applications for humans. The new technologies that allow us to sequence and annotate whole genomes more rapidly and to analyse the expression of thousands of genes in a single experiment are likely to speed up this change, particularly as microbes are well suited for high‐throughput analysis. Any microbial genome can now be sequenced within a few hours and, in the near future, new bioinformatics tools will enable scientists not …

Most eukaryotic extra-chromosomal elements are found in mitochondria or chloroplasts, or are transient viral visitors. There are just a handful of stably maintained nuclear plasmids, and of these, only the 2μm circle plasmid of S. cerevisiae has been investigated in detail. Although this plasmid is a compact 6 kbp in size, and has only four genes, it nevertheless has a site specific recombination system, a partition system, a copy number amplification system and a copy number control system. These inter-dependent systems provide the plasmid with a distinctively eukaryotic solution to the problem of maintenance in a population.

The highly conserved ParA family of partitioning systems is responsible for positioning DNA and protein complexes in bacteria. In Escherichia coli, plasmids that rely upon these systems are positioned at mid-cell and are repositioned at the quarter-cell positions after replication. How they remain fixed at these positions throughout the cell cycle is unknown. We use fluorescence recovery after photobleaching and time-lapse microscopy to measure plasmid mobility in living E. coli cells. We find that a minimalized version of plasmid RK2 that lacks its Par system is highly mobile, that the intact RK2 plasmid is relatively immobile, and that the addition of a Par system to the minimalized RK2 plasmid limits its mobility to that of the intact RK2. Mobility is thus the default state, and Par systems are required not only to position plasmids, but also to hold them at these positions. The intervention of Par systems is required continuously throughout the cell cycle to restrict plasmid movement that would, if unrestricted, subvert the segregation process. Our results reveal an important function for Par systems in plasmid DNA segregation that is likely to be conserved in bacteria.

On plasmid incompatibility

Background Antimicrobial resistance is a critical global concern. Pathogens may become resistant through acquisition of mobile genetic elements harboring antimicrobial resistance genes (ARGs). Environmental biofilms provide an excellent venue for ARG transfer via mobile elements. Mobile ARG dissemination pathways in the environment require a better understanding. Here we use proximity-ligation (Hi-C) and metagenomic sequencing to pair plasmids and ARGs to their bacterial hosts and present evidence of plasmid sharing between bacterial species. Methods Eight environmental biofilm swabs collected from water sources in Sindh Province, Pakistan were equally aliquoted for preparation by 1) DNA cross-linking for proximity ligation and Hi-C metagenomic sequencing, and 2) traditional shotgun metagenomic sequencing. Metagenomic Hi-C was used to analyze the proximity-ligation and shotgun sequencing data to construct metagenomic-assembled genomes, determine if ARGs were chromosomally- or plasmid-located, and associate mobile genetic elements and ARGs with their host cells. Results Actinomycetia, Gammaproteobacteria, and Alphaproteobacteria were the most frequently detected taxa in most samples, with Bacteroidia also being at higher numbers than other taxa in most samples. Multiple classes of ARGs in the sample were identified as being located on plasmids, including aminoglycoside ARGs (e.g. aph(3'), aph(6)), beta-lactam RGs (blaCTX-M-15, blaEC, blaTEM-1, blaOKP-A), macrolide RGs (ere(A), erm(X), mph(A), mef(C), mph(G)), quinolone RG qnrS1, sulfonamide RG sul1, and several tetracycline and metal ARGs. TetC, blaA2, and aac(3)I, were only found on bacterial chromosomes. Analysis of unique assembled ARG-bearing plasmids and taxonomic clusters revealed plasmid sharing between bacterial species, some of which were not closely related taxa. Conclusion Mobile ARGs are common in environmental biofilms. These ARGs have the potential to spread within biofilms between species, as evidenced by shared plasmid between different bacterial species. The environment should be considered an important source for mobile ARG dissemination between bacterial species. Disclosures All Authors: No reported disclosures

Plasmids are major drivers of increasing antibiotic resistance, necessitating an urgent need to understand their biology. Here we describe a detailed dissection of the molecular components controlling the genetics of I-complex plasmids, a group of antibiotic resistance plasmids found frequently in pathogenic Escherichia coli and other Enterobacteriaceae that cause significant human disease. We show these plasmids cluster into four distinct subgroups, with the prototype IncI1 plasmid R64 subgroup displaying low nucleotide sequence conservation to other I-complex plasmids. Using pMS7163B, an I-complex plasmid distantly related to R64, we performed a high-resolution transposon-based genetic screen and defined genes involved in replication, stability, and conjugative transfer. We identified the replicon and a partitioning system as essential for replication/stability. Genes required for conjugation included the type IV secretion system, relaxosome, and several uncharacterised genes located in the pMS7163B leading transfer region that exhibited an upstream strand-specific transposon insertion bias. The overexpression of these genes severely impacted host cell growth or reduced fitness during mixed competitive growth, demonstrating that their expression must be controlled to avoid deleterious impacts. These genes were present in >80% of all I-complex plasmids and broadly conserved across multiple plasmid incompatibility groups, implicating an important role in plasmid dissemination.

The partitioning locus (par) of plasmid pRA2 belongs to a recently discovered subgroup of plasmid partitioning systems that are evolutionarily distinct from the P1, F and R1/NR1 prototypes. The pRA2 par region was effective in stabilizing both pRA2 and F mini-replicons. Analysis of the nucleotide sequence revealed three potential coding regions that were designated parA, parB and parC. Through mutagenesis, parA and parB were found to be essential for partitioning function, whereas parC did not appear to be required. Using transcriptional reporter systems, it was demonstrated in vivo that ParB repressed par promoter activity by 60-fold and that ParA had little effect on transcriptional activity. Primer extension analysis revealed that the par transcriptional start point was located 47 nucleotides upstream of the parA translational start codon. Based on this information, putative -10 and -35 transcriptional signals were identified, and their subsequent deletion resulted in a dramatic reduction in promoter activity. The par promoter region was also demonstrated to exert incompatibility towards a plasmid with an active pRA2 par system. Nested deletions in this region allowed the incompatibility determinant, designated parS, to be localized. Recombinant ParA and ParB proteins were overexpressed and purified by affinity chromatography. Through in vitro binding experiments, purified ParB was shown to interact specifically with the par promoter region. DNase I footprinting revealed that ParB not only binds to the conserved sequence 5'-TCA AA(T/C) (G/C)CT CAA (A/T)A, which is present in three copies in the par promoter region, but also binds to the pRA2 partitioning site, parS. It appears that ParB has a dual role in pRA2 partitioning, being responsible for both the regulation of par transcription and the formation of a partition nucleoprotein complex at parS.

Inoviruses

Agrobacterium tumefaciens pTAR parA promoter region involved in autoregulation, incompatibility and plasmid partitioning

Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as blaTEM−1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical importance. Such phenomenon is bothersome when the plasmids are transmissible, facilitating the spread of virulence and resistance plasmids among pathogenic bacteria. Notably, certain TA systems are more commonly found in particular ExPEC plasmid types, indicating the possible relationships between certain TA systems and ExPEC pathogenesis.