An Operon of Three Transcriptional Regulators Controls Horizontal Gene Transfer of the Integrative and Conjugative Element ICE in B13

Summary

Integrative and conjugative elements (ICEs) are a relatively newly recognized class of mobile elements in bacteria, which integrate at one or more positions in a host chromosome, can be excised, circularized, and transfer by conjugation to a new recipient cell. Genome sequencing indicated that ICEs often carry genes with potential adaptive functions for the host. Various ICE-types have been described and ICEclc is a useful model for a wide class of elements found in Beta- and Gammaproteobacteria. Because ICEs normally remain “silent” in the host chromosome and often lack selectable markers, their lifestyle is difficult to study. One of the characteristics of ICEclc is that transfer is initiated in only 3-5% of donor cells in a population during stationary phase. Here, we describe an operon of three regulatory genes, two of which control the transfer initiation of ICEclc. Our findings suggest that the low transfer rate results from the repression of an activator and that this is essential to minimize the deleterious effect of hyper-activation of transfer initiation. While the individual regulatory genes are quite common on ICEs, they rarely occur in this configuration.

Introduction

Comparisons between ever-increasing numbers of sequenced genomes reveal the large extent to which prokaryotic genomes have undergone horizontal gene transfer (HGT) [1]-[5]. HGT has traditionally been viewed as the consequence of natural transformation, or of the action of mobile elements such as conjugative plasmids and phages [6], [7]. During the last decade, however, other types of mobile genetic elements such as integrative and conjugative elements (ICEs) have been recognized, which are widespread and thus may significantly contribute to HGT [8]-[13]. In contrast to phages and plasmids, however, we still know little about the life styles of the diverse ICE types, their modes of self-transfer and regulatory pathways controlling self-transfer. Like temperate phages, ICEs mostly exist in an integrated form at one or more specific sites in the host's chromosome (often in genes for tRNA), and are vertically transmitted to daughter cells by chromosome replication and segregation [8], [10], [14]. In order to transfer horizontally, ICEs excise themselves by site-specific recombination (attL and attR, Figure 1). This produces a circular double-stranded DNA molecule, which can transfer by conjugation to a new recipient cell, where it can reintegrate [14]. Autonomous plasmid-like replication of the excised form may occur [15]-[17], but is not required for the transfer itself.

The regulatory mechanisms that control the switch from integrated to excised state vary widely among different ICE types insofar as this has been studied. In several ICEs, this switch is the consequence of a cascade of a variety of regulatory factors, such as PhrI/RapI and ImmR/ImmA in ICEBs1 [18], SetR/SetCD in ICESXT [19], [20], KorSA/Pra in pSAM2 [21]-[23] or QseM/TraR in ICEMlSym^R7 [24], [25]. Most wild-type ICEs transfer at low frequencies (i.e., less than 1 per 10³ donors), suggesting that the regulatory cascades keep extremely tight control and allow only a small subset of cells in a population to follow the path of ICE excision and transfer, but the need for such tight control is a priori unclear. This bistability is most pronounced and well-studied for a model ICE named ICEclc in Pseudomonas [26], [27], which is evolutionary very distinct from the afore-mentioned ICEs [8], [28]. ICEclc is originally found in two copies in Pseudomonas knackmussii B13 and is member of a family of ICEclc-like elements widely distributed among proteobacterial species [29]. ICEclc is integrated at the 3′-end of tRNA^Gly genes but can excise itself by the action of the IntB13 integrase encoded on the element (Figure 1A). Expression of intB13 in the integrated form is under control of the promoter P_int, which by single cell reporter gene analysis was shown to become active only in 3-5% of a bacterial population during stationary phase [26]. Direct single cell visualization further confirmed that only cells which express reporter gene fused to P_int above a threshold are capable of transferring ICEclc to new recipients, a bistable state which we recently named “transfer competence” (tc) [30]. Irrespective of the success of ICEclc transfer, tc cells can only divide a few times once they re-enter exponential phase before they arrest growth. We recently showed that this is due to the expression of the ICEclc genes shi and parA [30]. Expression of intB13 is dependent on a variety of factors, most notably a gene named inrR (Figure 1A), which itself is also bistably expressed [26]. Both inrR and intB13 expression are dependent on the abundance of the stationary phase sigma factor RpoS, with cells having highest RpoS levels being more likely to activate P_int and P_inR [29]. RpoS and InrR are important for activating ICEclc excision and transfer, but are not sufficient. Therefore, we hypothesized that additional factors are necessary for the tc state to develop [29].

In this study, we report a locus of three consecutive regulatory genes on ICEclc, which is essential for controlling its transfer. The locus was uncovered by random transposon mutagenesis, and further studied by creation of deletion mutants and complementation. The effect of mutations was studied at the level of ICEclc expression through microarray hybridizations, RT-PCR and reporter gene-based single cell fluorescence microscopy, and further in ICEclc transfer assays. Fitness of mutants compared to wild-type was examined in growth assays and individual cell fates were followed by microscopy. Bioinformatics was used to analyze the configuration of the ICEclc regulatory locus within this ICE family, and to possibly reconstruct the steps that may have led to selection of the specific regulatory control mechanism of ICEclc. The results of our study help to explain why a careful balance has to be maintained between ICE transfer frequency and fitness loss.

Results

Discovery of an ICEclc transfer control locus by transposon mutagenesis

In order to discover ICEclc-located factors involved in its self-transfer, a library of P. knackmussii B13 mutants was generated by using random Tn5 mutagenesis [31]. Next, we recovered ICEclc elements with Km-insertions by conjugating the pool of B13 mutants en masse to Pseudomonas putida UWC1 and selecting for Km-resistant P. putida (Figure S1). We hypothesized that mutant ICEclc with insertions in genes implicated in self-transfer could still be transferred to UWC1, when the second copy of ICEclc in the same B13 donor cell is intact and complements transfer of the mutant copy. A total of 1920 Km-resistant P. putida transconjugants was recovered and subsequently conjugated each individually with a second P. putida recipient, resistant to nalidixic acid (Figure S1). For those conjugations in which no Km- and nalidixic acid-resistant transconjugant growth was detected, the corresponding P. putida donor was recovered and the location of the Km^R-gene insertion on ICEclc was mapped. A total of 18 clones was recovered, which had insertions in an ICEclc open reading frame numbered orf18502, that we renamed mfsR (Figure 1B). Surprisingly, apart from one donor with an insertion in intB13, no other mutants with impaired ICEclc transfer were found in this screening. The Km^R-gene had been inserted in four different positions in mfsR, at ICEclc nucleotide positions 19033, 18758, 18730 and 18618 (Figure 1B, accession number: AJ617440.2). This suggests that transposon insertions in strain B13 were sufficiently frequent to cover all genes, but that the selection procedure was biased for the recovery of the mfsR insertion, which may have been due to the function of mfsR as regulator in ICEclc transfer (see below). Alternatively, it is possible that insertions in ICEclc genes needed for transfer might not be efficiently complemented by the second ICEclc copy and would thus be underrepresented in the P. putida library. Frequencies of ICEclc transfer of the strains P. putida UWC1-2961 (Km^R-gene insertion at 19033) and UWC1-2962 (insertion at 18618) in a filter-based conjugation assay were 10³-fold and 10²-fold lower than of a P. putida with one integrated wild-type ICEclc copy, respectively (Figure 2A).

mfsR is part of an operon formed by three consecutive transcriptional regulators

Closer inspection indicated mfsR to be the first open reading frame in a series of three consecutive transcriptional regulators, previously designated as orf18502, orf17984 and orf17162 (Figure 1B). mfsR encodes a TetR-like regulator harboring helix-turn-helix motifs TetR_N and TetR_C_7 (pfam0040 and pfam14246, respectively, see Figure 1D). The orf17984 gene overlaps with the end of the mfsR open reading frame by 4 bp and encodes a putative regulator of the MarR family (smart00347 HTH_MARR motif). The last gene of this cluster starts 24 bp downstream of the stop codon of orf17984 and is predicted to code for a LysR-type transcriptional regulator, harboring an N-terminal HTH_1 motif (pfam00126) and a C-terminal substrate-binding domain (PBP2_LTTR_aromatics_like; cd08414). The gene orf17162 was renamed tciR (transfer competence inducer regulator) in anticipation of the results described further below. Reverse transcription of P. putida UWC1 (ICEclc) RNA isolated from exponential phase-grown cells, followed by specific PCR amplification confirmed that the three genes are transcribed on the same mRNA, which ends downstream of tciR (Figure S2). This implies that mfsR-marR-tciR form a single polycistronic unit.

Effects of tciR, marR, and mfsR deletions on ICEclc transfer

In order to more precisely investigate the role of the three regulators on ICEclc transfer, their open reading frames were each individually and partially deleted in separate strains, namely P. putida UWC1 (ICEclc-ΔmfsR, strain 4322), UWC1 (ICEclc-ΔmarR, strain 4372), UWC1 (ICEclc-ΔmfsR-Δ'marR, strain 3453) and P. putida UWC1 (ICEclc-ΔtciR, strain 4321) (Figure 1B, Table 1). ICEclc transfer frequencies in plate-mating assays with a gentamicin-resistant P. putida UWC1 as recipient were 2·10³-fold lower for UWC1 donors with ICEclc having an internal deletion in tciR compared to intact ICEclc (Figure 2B). Complementation of the ICEclc-ΔtciR mutation with a single copy mini-Tn7 transposed fragment containing the tciR gene under the P_mfsR-promoter (strain 4649, Figure 1C) restored transfer, even to much higher levels than wild-type ICEclc (Figure 2B).

ICEclc transfer frequencies were 27-fold lower for UWC1 donors with ICEclc having an internal deletion in marR compared to intact ICEclc (Figure 2B). Complementation of the ICEclc-ΔmarR mutation with a similar single copy mini-Tn7-marR insertion did not change transfer rates (strain 4804, Figure 2B). This suggests that the effect of the marR deletion on ICEclc transfer is rather due to polar disturbance of the expression of the downstream-located tciR.

In contrast, ICEclc elements with mfsR deletions [i.e., P. putida UWC1 (ICEclc-ΔmfsR) and UWC1 (ICEclc-ΔmfsR-Δ'marR)] transferred with 25- and 15-fold higher frequencies than wild type ICEclc, respectively (Figure 2A, B). Complementation of the ICEclc-ΔmfsR mutation with a single copy mini-transposed mfsR gene under control of its own promoter reduced ICEclc-ΔmfsR transfer frequencies by 10⁴-fold, also here much stronger than predicted from wild-type ICEclc itself (Figure 2B). These results suggested that tciR is the actual regulator of ICEclc transfer, and further that mfsR is regulating expression of the mfsR-marR-tciR operon. Since MfsR is expected to be a repressor, its deletion would lead to higher expression of the downstream genes marR and tciR, which results in increased ICEclc transfer. The effect of the transposon insertions in mfsR (i.e., lower ICEclc transfer rates, Figure 2A) seems therefore due to a polar effect on marR-tciR expression.

tciR encodes a global activator of the genes in the ICEclc core region

Next, we examined the effect of regulatory gene deletions on gene expression of ICEclc as a whole, using semi-tiling microarray analyses (Figure 3). When P. putida UWC1 (ICEclc) wild-type cells are growing exponentially on 3-chlorobenzoate (3CBA), expression from the genes in the ICEclc core region (roughly the second half of ICEclc) plus the integrase intB13 is silent, whereas they are highly transcribed when cells are in stationary phase (Figure 3A). Among others, the core region encodes genes implicated in ICEclc conjugative transfer [32], [33]. P. putida with mutant ICEclc lacking either marR or tciR strongly diminished expression in the core region and of the integrase gene in stationary phase when compared to wild type (Figure 3C, Figure S3). Lower core and integrase gene expression explains the lower ICEclc transfer rates from these mutants (Figure 2B). In contrast, mfsR deletion resulted in much higher expression from the ICEclc core genes in exponentially growing cells (Figure 3B), and even slightly higher expression in stationary phase than in wild-type ICEclc (Figure S4), which explains the 10- to 100-fold ICEclc higher transfer rates (Figure 2B). Expression of the mfsR-marR-tciR cluster itself was the same in the tciR and marR deletion mutants, and no different to the wild-type (Figure 4A, C, E). In contrast, expression of the mfsR-marR-tciR cluster was higher in the mfsR deletion mutants than in wild-type, both in exponential and stationary phase cells (Figure 4B, D). Since gene expression from ICEclc is similar in mutants lacking mfsR alone or mfsR plus the first 117 bp of marR (Figure S4), we conclude that it is the LysR-type regulator encoded by tciR, which is the main activator for ICEclc core gene expression.

Microarray analysis also helped to understand the behaviour of the mfsR Km-insertion mutant (Figure 3D). As for the tciR deletion mutant, expression of the ICEclc core region and of the integrase was dramatically lower than wild-type in stationary phase cells (Figure 3D, Figure S4). On the other hand, both mfsR deletion and mfsR Km-insertion mutants showed increased expression of a group of genes on ICEclc coding for a putative efflux system (Figure 3B, D). Detailed inspection of mfsR-marR-tciR expression in the Km-insertion mutant revealed that the first 160 bp of mfsR, upstream of the Km^R-gene insertion were higher expressed than in wild-type cells (Figure 4F). In contrast, the downstream genes marR and tciR were lower expressed compared to wild-type and to the mfsR deletion mutant (Figure 4B, D, F). This confirmed, therefore, that insertion of the Km^R-gene had caused a polar effect on expression of marR and tciR, which explains the strongly diminished expression of the ICEclc core genes in stationary phase in the mfsR Km-insertion mutant, and decreased ICEclc transfer.

Inserting the presumed mfsR promoter region upstream of a promoterless mcherry gene in single copy on the chromosome of P. putida UWC1 without ICEclc produced strong and homogenous mCherry expression among all cells (Figure 5A, strain 3482). Inserting into this strain a single copy mfsR gene expressed from its own promoter abolished mCherry expression (Figure 5A, strain 4302). Expression of mCherry from P_mfsR in P. putida UWC1 (ICEclc) was very low, whereas disruption of mfsR on ICEclc again resulted in high mCherry expression (Figure 5A, strain 3606). Complementation of this strain by a single copy mfsR gene under its own promoter caused repression of mCherry expression (Figure 5A). All these data are consistent with the hypothesis that MfsR is repressing expression of itself and the downstream located marR and tciR genes.

mfsR deletion leads to an increase in the number of cells activating ICEclc

We then tested whether changed ICEclc transfer rates and core gene expression were in fact due to changes in the proportion of cells activating ICEclc. Hereto, a double promoter-reporter construct, carrying P_int-gfp and P_inR-echerry was inserted in single copy on the chromosomes of P. putida UWC1 (ICEclc), P. putida UWC1 (ICEclc-ΔmfsR), P. putida UWC1 (ICEclc-Δ'marR) and P. putida UWC1 (ICEclc-ΔtciR) (Table 1). P_int and P_inR are the respective promoters for the integrase gene intB13 (integrated form) and the integrase activator gene inrR. Previous studies showed that both promoters are active only in a small subpopulation of cells, which are representative for transfer competent cells [26], [29], [30]. Consistent with previous data, the subpopulation of P. putida UWC1 (ICEclc) wild-type cells expressing P_int and P_inR in stationary phase suspended cultures represented a few percent (Figure 5B). In contrast, deletion in mfsR resulted in 80-100% of cells expressing P_int- and P_inR-promoters (Figure 5B). Expression of both promoters in P. putida UWC1 (ICEclc-ΔmfsR) occurred in early stationary phase whereas in wild-type cells their expression is maximal in late stationary phase (Figure 5B). Conversely, P. putida UWC1 (ICEclc-ΔtciR) and P. putida UWC1 (ICEclc-ΔmarR) did not produce any detectable P_int- or P_inR-expressing cells, neither in exponential nor in stationary phase (Figure 5B). Considering a detection limit by microscopy of ∼1 fluorescent cell among 1000-10,000 non fluorescent cells, the absence of detectable P_int- or P_inR-expressing cells in those mutants would be in accordance with absence of ICEclc core gene activation on microarrays (Figure 3D) and lower transfer frequencies (Figure 2B).

Mutants with mfsR deletion in ICEclc face a strong fitness cost

Given that P. putida UWC1 carrying ICEclc-ΔmfsR transferred at a much higher rate than wild-type ICEclc, and also expressed both P_int- and P_inR- promoters in almost all cells, we wondered why such mutants did not become selected spontaneously. Both P. putida UWC1 (ICEclc) wild-type and (ICEclc-ΔmfsR) displayed statistically indistinguishable generation times during exponential growth on minimal medium with either 3CBA or succinate as carbon source (Table 2), although P. putida UWC1 (ICEclc-ΔmfsR) went through a longer lag phase (Figure S5A). In contrast, the proportion of colony forming units (CFU) in samples taken from stationary phase cultures on 3CBA or succinate and plated on 3CBA solid medium was dramatically reduced for P. putida UWC1 (ICEclc-ΔmfsR) (Figure 6). Whereas the number of colonies formed on 3CBA plates was the same as that on succinate for P. putida UWC1 (ICEclc), only 0.3-5.6% appeared on 3CBA plates for UWC1 (ICEclc-ΔmfsR) (Table 2). Also in absolute terms, the number of CFU/ml for UWC1 (ICEclc-ΔmfsR) cells taken from stationary phase cultures both on 3CBA and succinate was lower than that for UWC1 (ICEclc) (Figure 6). Moreover, 8 of 10 tested colonies of UWC1 (ICEclc-ΔmfsR) grown on MM plates with succinate did no longer amplify the clcA gene of ICEclc (not shown), the remaining two still being able to grow on 3CBA. Furthermore, half or more of microcolonies formed from UWC1 (ICEclc-ΔmfsR) with a single copy P_int-egfp insertion showed incidence of malformations and cell lysis, similar to what was reported previously for nutrient-reactivated tc cells [30] (Figure 6B), but cells in the other microcolonies divided with generation times even slightly faster (1.49±0.15 h) than those in microcolonies of P. putida UWC1 (ICEclc, 1.79±0.09 h). This indicates that the mfsR deletion in ICEclc imposes a strong fitness cost on P. putida UWC1. Survival of UWC1 was restored to wild-type level when the ICEclc-ΔmfsR was complemented by the mini-Tn7 inserted mfsR gene (Figure S6). In contrast, neither P. putida UWC1 (ICEclc) with marR or tciR deletion, nor the mini-Tn7 complemented strains of P. putida UWC1 (ICEclc-ΔtciR) and (ICEclc-ΔmarR) were impaired in survival compared to UWC1 (ICEclc) (Figure S6).

tciR is a very widespread ortholog among ICE closely related to ICEclc

Using bioinformatic queries, we retrieved orthologs to tciR and mfsR from sequenced bacterial genomes, and examined manually whether they occur in chromosomal regions qualifying as ICE (e.g., presence of an integrase gene nearby, see Materials and Methods). Interestingly, orthologs to the individual components of the mfsR operon are widespread but rarely occur in the same configuration (Figure 7). So far, the ICEclc mfsR-marR-tciR configuration is only found in the ICEclc variant of Burkholderia xenovorans LB400, whereas the Tn4371-element of Acidovorax sp. strain JS42 and Aeromonas hydropohila SSU (accession number AGWR01000022.1) both carry an mfsR homolog and a MarR-type regulator immediately downstream, but no tciR equivalent nearby. There is a tciR ortholog in Acidovorax sp. strain JS42, but not on the same chromosomal region as mfsR (Figure 7).

On the other hand, tciR seems much more widespread among ICEs, as homologs can be found in ICEclc-like elements GI1 and GI6 of Bordetella petrii DSM12804, in PAGI-2 of P. aeruginosa strain C, in diverse ICEs of X. campestris pv. vesicatoria str. 85-106, suspected ICEs in Herminiimonas arsenicoxydans, Cupriavidus metallidurans CH34, and Tolumonas auensis DSM 9187, among several dozens of others (Figure 7). Given that TciR is such a common regulator found in ICEs of the ICEclc family, it might be similarly implicated in their transfer control. This regulation is likely different in detail from ICEclc, given the absence of an mfsR and marR.

Discussion

ICEclc has two distinctive modes of existence: the integrated form, which is transmitted vertically, and the circular form, which can be horizontally transferred. Previous work in our laboratory has shown that the transition between these two states occurs in only a few percent of cells in a population under stationary phase conditions [26], [30], [34]. We have recently suggested to name cells in which the molecular decision occurs to activate the ICEclc horizontal transfer mode transfer competent (tc) cells [30]. Single cell time-lapse experiments indicated that ICEclc transfer - at least insofar as detectable by microscopy, only occurs from tc cells, which can be distinguished through simultaneous expression of fluorescent proteins from single copy transcriptional fusions to the P_int and P_inR promoters of ICEclc [30]. Activation of those two promoters is the likely outcome of a multi-step regulatory cascade that orchestrates expression of some fifty genes [32], but the key factors that determine the onset of this cascade and control the extent of bistability are still obscure. Previous work provided evidence for the role of the stationary phase sigma factor RpoS in activation of ICEclc promoters, and we could show that tc cells on average have higher levels of RpoS [34]. In the present study, we report the discovery of a cluster of three regulatory genes, two of which globally control ICEclc activation and transfer, and additionally maintain a cap on fitness loss induced by the ICE (Figure 8).

The three regulatory genes occur in a unique configuration of a TetR-type repressor (encoded by mfsR), followed by a MarR-type (orf17984) and a LysR-type regulator (tciR). Transcript and microarray analysis of the locus in wild-type and mutant ICEclc, plus analysis of reporter gene expression from the mfsR promoter in a variety of host backgrounds, showed that the three genes are expressed as a polycistronic unit and are under autoregulatory control by MfsR (Figure 5A).

Precise gene deletions and complementations indicated that tciR is likely the main global regulator of ICEclc transfer activation. Deletion of tciR caused a 2·10³-fold lower frequency of ICEclc transfer compared to wild type (Figure 2B), silenced expression of the ICEclc core region (Figure 3B) and reduced the proportion of cells expressing P_int and P_inR in stationary phase (Figure 5B). Complementation of the tciR deletion on ICEclc with a single copy mini-Tn7 inserted mfsR^fs-marR^fs-tciR fragment (Figure 1C) fused to the mfsR promoter region restored the expected phenotype (Figure 2B). TciR may act either directly as a regulator on a variety of individual ICEclc core promoters, or as a “master” regulator in a hierarchical activation cascade (Figure 8).

The role of marR is less clear and as yet unsolved. Deletion in marR resulted in essentially the same ICEclc transcriptome profile as deletion in tciR (Figure 3D). It also resulted in a lower transfer frequency than wild-type but not as low as the deletion in tciR (Figure 2B), and produced no detectable reporter gene expression from P_int or P_inR (Figure 5). In contrast, complementation of the marR deletion on ICEclc by a single copy marR gene through mini-Tn7 delivery (Figure 1C) did not restore ICEclc transfer (Figure 2B). Furthermore, mutants with deletions in mfsR or mfsR plus the first 116 bp of marR (Figure 1B), behaved quite similar in transfer frequency (Figure 2) and showed similar ICEclc transcriptomes (Figure S4). The marR gene therefore seems to have no clear role in ICEclc core gene expression.

The most surprising effect of deletions in mfsR was a complete deregulation of the ICEclc core gene expression. This became obvious from frequencies of ICEclc transfer being 10-100 fold higher than wild type, approximating 1 transfer per donor cell (Figure 2B). The deregulation was also obvious in microarray data showing the ICEclc core region in the mfsR deletion mutants being already transcribed in exponential phase (Figure 3B, Figure S3). Finally, 80-100% of individual cells in stationary phase expressed the reporter genes from P_int and P_inR in the mfsR deletion strain compared to 3-5% in wild-type (Figure 5B). This can be explained by the fact that deletion of mfsR would abolish autorepression, which would lead to constant high expression of marR and tciR. This overinitiates ICEclc core gene expression, leads to more cells entering the tc state and to higher transfer rates. The balance of mfsR control appears to be extremely delicate, since even complementation with a single gene copy under control of the original promoter results in a stronger effect than the wild-type, both for tciR and mfsR (Figure 2B). The delicate balance became also obvious from the polar effects of insertion of the Km-resistance gene within mfsR, leading to decreased transcription of marR and tciR, diminished core gene expression (Figure 3D) and reduced transfer rates (Figure 2A).

The finding that deletion and complementation of mfsR or tciR drastically changes the proportion of cells activating ICEclc, could imply that the bistability seen in wild-type situation (i.e., 3-5% of cells in stationary phase becoming transfer competent) is a result of feedback at this locus. Cells activating ICEclc in the wild-type situation could arise as a consequence of “sloppy” control by MfsR, incidentally causing a few cells to escape its control and transcribing marR and tciR. We think this is an unlikely scenario, because mCherry expression from the mfsR promoter is homogenous among cells (Figure 5A). Alternatively, there might be a chemical ligand that specifically binds to MfsR in a small subset of cells, upon which its repression is relieved in those cells. The resulting TciR would then be the necessary activator to trigger ICEclc core expression in cells with on average highest RpoS levels [29] (Figure 8). On the other hand, even though mfsR may be the first level of control, bistability may also originate at later checkpoints in the regulatory cascade, which depend on the presence of sufficient TciR.

Quasi-global appearance of transfer competence across all cells in the mfsR deletion mutant resulted in massive fitness loss (Figure 6), which became evident at two levels. First of all, time-lapse observations indicated lysis and aberrant cell growth in more than 50% of microcolonies (Figure 6B). This lysis and growth arrest are similar to what we previously described as being a side consequence of becoming transfer competent in wild-type cells [30], and is caused by the parA-shi gene products on ICEclc [30]. Secondly, there was a strong loss of the capacity to grow on 3CBA among cells sampled from stationary phase cultures of the mfsR mutant compared to wild-type (Figure 6A, Figure S6), indicative for loss of ICEclc and counterselection against maintaining ICEclc-ΔmfsR. However, those cells that maintained ICEclc-ΔmfsR could still grow on 3CBA and showed indistinguishable exponential growth rate (Table 2). This paradox can be understood when modeling the number of tc cells in batch culture populations for ICEclc wild-type (probability of tc arisal, P_tc, in stationary phase of 0.025) and for the ICEclc-ΔmfsR mutant (P_tc = 0.5). This model (Figure S5B) shows that whereas a large proportion of tc cells appear in ICEclc-ΔmfsR mutant cultures in stationary phase, these can only divide 2-3 times upon reinoculation into fresh medium before lysing. This causes an apparent prolongation of a lag phase visible as stagnant culture turbidity, but does not influence the overall predicted population exponential growth rate in batch culture (Figure S5B).

As expected from the postulated role of TciR, its complementation in trans also leads to increased ICEclc transfer, but interestingly, only the mfsR deletion caused strongly decreased cell survival (Figure S6). We therefore hypothesize that ICEclc activation may follow two separate processes: transfer and tc cell growth arrest [30], that may both be initiated at the mfsR locus. Deleting mfsR would then deregulate both processes, whereas expressing tciR in trans would only increase activation through the transfer branch (Figure 8).

The configuration of the mfsR-marR-tciR operon of ICEclc is unique, but tciR alone is a very common part of ICE similar to ICEclc (Figure 7). We therefore speculate that mfsR-marR are a more recent acquisition in ICEclc, which drastically changed the expression of the tciR gene. Unfortunately, expression of tciR analogs in other ICEs has not been studied and very little has been reported on the transferability of ICEs related to ICEclc. The exceptions being GI3 of B. petrii that transfers at extremely low frequencies (∼10^-7) [35], and the P. aeruginosa PAGI-2 element for which transfer has not been detected at all [16]. In comparison, wild-type ICEclc transfers at rates of 10^-2 to 10^-3 per donor (Figure 2), suggesting that it was perhaps the acquisition of the mfsR regulatory control that led to expression of transfer activity in a larger proportion of cells in the population. As we show here, the downside of increasing the proportion of ICEclc tc cells is an increase of the proportion of cells displaying growth arrest through the shi-parA pathway [30]. Likely, the MfsR autoregulation evolved to a stage of permitting efficient transfer but avoiding too much fitness loss to the population. Even though the mechanistic details are different for ICEclc, double control layers are more common for various ICEs and typically involve a variety of regulators acting on each other and/or in response to specific chemical ligands [18], [19], [23]-[25]. It will be highly interesting to further study the mechanistic details of the control systems that maintain very low ICE transfer rates, and to understand whether and how such control can evolve to allow hyperefficient transfer.

Materials and Methods

Strains and culture conditions

Table 1 lists the strains used in this study. Escherichia coli DH5α (Gibco Life Technologies, Gaithersburg, Md.), E. coli DH5α λpir, E. coli BW20767/pRL27 were cultured at 37°C on Luria-Bertani (LB) medium [36]. Pseudomonas species were cultured at 30°C on LB or 21C minimal medium (MM) [37] complemented with one of the following carbon sources: 0.5, 5, or 10 mM 3-chlorobenzoate (3CBA), 15 mM succinate or 10 mM fructose. Antibiotics were supplemented to the growth medium to select for maintenance of genetic constructions at the following concentrations: kanamycin (Km) 25 µg/ml, chloramphenicol (Cm) 20 µg/ml, rifampicin (Rif) 50 µg/ml, nalidixic acid (Nal) 50 µg/ml, gentamicin (Gm) 20 µg/ml, and ampicillin (Ap) 100 µg/ml.

Strain constructions and DNA techniques

DNA purification, PCR, restriction enzyme digestions, DNA ligations and electro-transformations were performed according to standard procedures [36]. Deletions in ICEclc genes were created by double recombination techniques as described elsewhere [29], [38]. Nucleotide positions are given according to AJ617740 (ICEclc). Primers used for strain constructions are listed in table S1.

For complementation of P. putida UWC1 (ICEclc-ΔmfsR, strain 4322) we first amplified the mfsR gene plus the 429 bp upstream region containing the mfsR promoter using PCR. This fragment was cloned into pGEM-T-easy and verified for correctness by DNA sequencing. The fragment containing the correct mfsR region was then recovered by restriction enzyme digestion with PstI and BamHI, and ligated into the mini-Tn7 vector pUC-miniTn7-Gm [39]. After transformation and verification in E. coli, the mini-Tn7 construct was introduced into P. putida UWC1 (ICEclc-ΔmfsR) by using the pUX-BF13 helper plasmid [40]. Clones resistant to Gm were selected and verified by PCR for correct insertion of the mfsR DNA in the attTn7 locus. To complement P. putida UWC1 (ICEclc-ΔtciR, strain 4321) and (ICEclc-ΔmarR, strain 4372) we amplified the complete mfsR-marR-tciR locus including the 429-bp upstream region. This fragment was cloned into pGEM-T-easy and again verified for correctness by DNA sequencing. The fragment was recovered by digestion with BamHI and StuI, and ligated with the mini-Tn7 vector. A frameshift was then introduced in the mfsR coding region by digestion at the unique NcoI-site, filling in using Klenow and religation. This will cause premature ending of the mfsR gene product (mfsR^fs). A second frameshift was subsequently introduced to inactivate the marR gene product, using the unique BsgI-site (marR^fs). After transformation and verification in E. coli, the construct was introduced in P. putida UWC1 (ICEclc-ΔtciR) as outlined above. This procedure was repeated to create a fragment with frameshifts in mfsR and in tciR (using the unique KpnI site), but maintaining an intact marR. This construct was introduced into P. putida UWC1 (ICEclc-ΔmarR). Gm-resistant clones were verified by PCR for the correct insertion at the attTn7-site, and for the presence of ICEclc.

A 656-bp region upstream of mfsR was amplified by PCR and fused to a promoterless mcherry gene. This fragment was introduced in single copy on the chromosome of P. putida UWC1, P. putida UWC1 (ICEclc) or P. putida UWC1 (ICEclc-ΔmfsR) using mini-Tn5 delivery. Three independent Km-resistant colonies were verified by PCR for the correct insertion and stored individually. P. putida UWC1 mini-Tn5-P_mfsR-mcherry and P. putida UWC1 (ICEclc-ΔmfsR) mini-Tn5-P_mfsR-mcherry were then further used as recipient to introduce the mini-Tn7-mfsR construct.

Random mutagenesis and screening

Random mini-transposon insertions in P. knackmussii B13 were generated by mobilization of the pRL27 suicide plasmid from E. coli BW20767 in a biparental mating. Hereto both strains were each cultured overnight in 3 ml LB, pelleted down, resuspended in 50 µl sterile saline solution (0.9% NaCl), mixed in a 1∶1 (v/v) ratio and incubated on the surface of an LB agar plate for 24 hours at 30°C. The mixture was then resuspended with 1 ml saline solution, which was inoculated in 100 ml MM with 0.5 mM 3CBA plus Km to select for the mini-transposon insertion and Cm to counterselect against E. coli, and incubated at 30°C for 16 h with orbital shaking (180 rpm). An aliquot of 3 ml of this pool of enriched Km^R B13 mutants was used en masse as donor in a subsequent mating procedure. Hereto, cells from the 3 ml suspension were pelleted by centrifugation, washed with 3 ml sterile saline and mixed with 3 ml of suspension of P. putida UWC1 recipient, that had been grown for 16 h on LB, was pelleted by centrifugation and resuspended in sterile saline. The mating mixture was again centrifuged, the cell pellet was resuspended in 50 µl sterile saline solution and spotted on the surface of a MM agar plate containing 0.5 mM 3CBA. The mixture was incubated for 72 hours at 30°C, after which the cells were washed from the plate with 1 ml sterile saline, which was further serially diluted and plated on MM agar plates with 5 mM 3CBA plus Km and Rif to select for transconjugants carrying mutant ICEclc. Individual colonies were purified, recultured in organized 96-well format and stored at -80°C after addition of and mixing with glycerol to 15% (v/v). Libraries were replicated and regrown in 100 µl LB plus Rif for 16 h in 96-well microtiter plates, mixed with 100 µl P. putida UWC1 Nal^R recipient suspension, and incubated at 30°C for 48 h. Then 50 µl of each well was reinoculated into 170 µl of MM containing 5 mM 3CBA plus Km, Rif and Nal, and growth was measured by continuous OD-measurements in a multiplate reader (FluoStar Omega, BMG labtech). Absence of growth was taken as indication for absence of ICEclc transfer, in which case the donor culture was recovered for mapping of the transposon insertion.

Insertion mappings

DIG-labeled primers 070934 or 070935, annealing to one of the ends of the Km^R insert but facing outward, were used (separately) in single-primer PCR with DNA from mutant UWC1 donors as templates. The reactions produced oligonucleotide probes with the 5′-DIG label, the sequence of the end of the Km^R gene and the adjacent sequence of the ICEclc insert position. Such products were used for rough localization of the insertion position by hybridizing to macroblot membranes (Eurogentec, UK), whose set of 55-mer oligonucleotides covers most of the ICEclc genes. Hybridization and detection of the DIG-marker were carried out according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany). Once the insertion was roughly mapped on ICEclc, PCR-based sequencing was used to determine exact position of the Km^R-gene insertion.

ICEclc transfer assays

The frequency of ICEclc transfer was determined in experimental conditions described previously [33]. P. putida UWC1 ICEclc wild-type or mutant derivatives were used as donors, whereas P. putida UWCGC (constitutively fluorescent, Gm^R) or P. putida UWC1 Km^R were used as the recipient (Table 1). Briefly, donors and recipient were each cultivated on 5 mM 3CBA MM and 10 mM fructose MM, respectively, and combined on 0.5 mM 3CBA agar plates as a single concentrated pellet. After 48 hours incubation at 30°C, mating mixes were resuspended, diluted and plated on 5 mM 3CBA MM agar (counting of donor CFU) or 5 mM 3CBA Gm or Km agar (counting of transconjugant CFU). Transconjugants were checked by PCR and frequencies were expressed as the number of transconjugant CFU per donor CFU. Donor survival was used for the data shown in Figure S6.

ICEclc transcriptome analysis by microarrays

The ICEclc transcriptomes of P. putida UWC1 (ICEclc), P. putida UWC1 (ICEclc), P. putida UWC1 (ICEclc-Km^R₁₉₀₃₃), P. putida UWC1 (ICEclc-Δ'marR), P. putida UWC1 (ICEclc-Δ'tciR), P. putida UWC1 (ICEclc-ΔmfsR-Δ'marR), P. putida UWC1 (ICEclc-ΔmfsR) were investigated by microarray analysis, as described previously [32]. Total RNA was extracted from cells grown on 10 mM 3CBA MM, and harvested at mid exponential phase (OD₆₀₀ = 0.6) and 48 h after entrance in stationary phase. Reverse transcription using cyanine-dCTP among the dNTPs produced labeled cDNA that was further purified and hybridized on 8×15 K microarray slides (Agilent, Santa Clara, CA, USA). Slides were washed and scanned according to manufacturer's instructions (Agilent). Data were recovered and analyzed using GeneSpring GX. Microarray data can be accessed from the GEO database (accession number: GSE51391).

Time-lapse microscopy

P. putida UWC1 strains were precultured for 16 h at 30°C in LB medium, after which 100 µl were transferred to 20 ml fresh MM 4 mM 3CBA medium in presence of the appropriate antibiotics. This culture was incubated for 96 hours at 30°C and 200 rpm shaking, after which the cells were 100-fold diluted in MM without C-source and inoculated on agarose surfaces (gel patches) for time-lapse microscopy [41]. Medium for gel patches consisted of 1% agarose dissolved by heating into MM with 0.1 mM 3CBA. Gel patches were created by pipetting 130 µl of the agarose-MM-3CBA solution kept at 55 °C on the surface of a circular cover glass (42 mm ø and 0.17-mm thick), placed in an autoclaved perfusion chamber (POC, H. Saur, Reutlingen, Germany), separated with a 0.5 mm thick silicon spacer ring and covering them with a second cover glass. After solidification of the agarose, the upper cover slip was removed and 6 µl of the diluted cell suspension was placed onto the agarose gel patch. As soon as the drops were dried on the surface, the patches were turned upside down and placed bacteria-facing-down on a new round cover glass [41]. A second silicon spacer ring was added to allow air circulation within the closed chamber and the glass sandwich was fixed into the metal cast POC chamber with a metal ring. Up to four patches could be placed simultaneously within a single glass sandwich in a POC chamber.

Microcolony development was followed directly on a Nikon Inverted Microscope Eclipse Ti-E, equipped with a Perfect Focus System (PFS), pE-100 CoolLED and a Plan Apo λ 100×1.45 Oil objective (Nikon), installed in a controlled temperature room (22°C). Ten random regions of every patch were imaged automatically during 48 hours with intervals of 1 h, in Phase Contrast mode (10 ms exposure), eGFP (500 ms) and eCherry (500 ms). Images were recorded using Micro-Manager 1.4 (http://www.micro-manager.org/) and fluorescence values were extracted using MetaMorph (Series 7.5, MDS, Analytical Technologies).

P. putida UWC1 ICEclc and mutant fitness tests

Triplicates of strains UWC1 (ICEclc) and UWC1 (ICEclc-ΔmfsR) were grown for 16 h in LB medium at 30°C. Both strains were then 500-fold diluted (starting OD₆₀₀ 0.001) in MM with 5 mM 3CBA or 10 mM succinate. Upon reaching early stationary phase, strains were again diluted into fresh MM (starting OD 0.001) with the same carbon source, and growth was followed by frequent culture turbidity measurements (OD₆₀₀). 24 h after reaching stationary phase, each replicate culture was serially diluted in MM and plated onto MM agar plates with 5 mM 3CBA or with 10 mM succinate. The number of CFU/ml was scored and the ratio was calculated between the number of CFU/ml on MM agar with 5 mM 3CBA and the number of CFU/ml on MM with 10 mM succinate.

Ten randomly chosen colonies of UWC1 (ICEclc-ΔmfsR) cultivated in MM with succinate and grown on MM-succinate agar plates were retested for growth on MM agar with 5 mM 3CBA. The presence of ICEclc was determined by colony PCR on the same colonies by amplifying the clcA gene, which is carried by ICEclc and the gene product of which is essential for 3CBA metabolism.

Bioinformatic screening for ICE related to ICEclc

Homologues to tciR of ICEclc were detected by BLASTN to the nr/nt database at E-value <1·10^-15. The corresponding whole or draft genome sequences were retrieved and compaired by aligning to ICEclc (Accession number AJ617740.2) using Megablast. Detected regions were manually recovered and searched for the tciR homologue and an intB13 homologue within a 1-100 kb window. If annotated, the presence of a gene for tRNA-Gly nearby the intB13 homologue was scored. Regions covering all criteria (i.e., homology to ICEclc core region, tciR homologue and presence of integrase gene) were retained as containing putative ICE. Selected regions were further individually pair-wise compared by using the Artemis Comparison Tool within the WebACT service [42].

Supporting Information

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