Free access
Spotlight Selection
7 October 2016

Pseudomonas aeruginosa Condensins Support Opposite Differentiation States


Condensins play a key role in global chromosome packing. Pseudomonas aeruginosa encodes two condensins, SMC-ScpAB and MksBEF. We report here that the two proteins are involved in the differentiation of the bacterium and impose opposite physiological states. The inactivation of SMC induced a state characterized by increased adhesion to surfaces as well as defects in competitive growth and colony formation. In contrast, MksB-deficient cells were impaired in biofilm formation with no obvious defects during planktonic growth. The phenotype of the double mutant was dominated by the absence of MksB, indicating that the observed growth defects are regulatory in their nature rather than structural. ATPase mutations recapitulated many of the phenotypes of the condensins, indicating their requirement for a functional protein. Additionally, inactivation of condensins dramatically reduced the virulence of the bacterium in a murine model of lung infection. These data demonstrate that condensins are involved in the differentiation of P. aeruginosa and reveal their importance for pathogenicity.
IMPORTANCE Adaptation and differentiation play key roles in bacterial pathogenicity. In Pseudomonas aeruginosa, an opportunistic human pathogen, these processes are mediated by the activity of an intricate regulatory network. We describe here novel members of this network, condensins. We show that the two P. aeruginosa condensins specialize in the establishment of the sessile and planktonic states of the bacterium. Whereas condensins have well-established roles in global chromosome organization, their roles in regulating bacterial physiology have remained unknown. Our data indicate that the two programs may be linked. We further show that condensins are essential for the pathogenicity of P. aeruginosa.


Pseudomonas aeruginosa is an opportunistic human pathogen that presents serious problems to patients with impaired immunity, wound infections, secondary lung infections, or who are in intensive care units (1, 2). Its virulence is largely attributed to its high intrinsic antibiotic resistance and adaptability, which help it occupy diverse niches. Strikingly, the bacterium can undergo clonal diversification and produce colonies with distinct morphologies, such as small-colony variants (SCVs) and biofilms. In particular, SCVs are often found in clinical isolates of P. aeruginosa and can be collected off biofilms or emerge in vitro following antibiotic treatment. They are characterized by the formation of small colonies with clearly defined edges, delays in planktonic growth, increased clumpiness, and sometimes increased antibiotic resistance (35). SCVs retain their phenotypic traits over many generations even in the absence of the initial signal but—at least in some cases—eventually revert to their dominant cell morphology (6). These features bear resemblance to bacterial differentiation. The mechanism that leads to clonal diversification and the subsequent stabilization of the resulting cell lines appears to involve the activity of the signaling network (7), transcriptional positive feedback loops (8), and mutations (3, 9) but remains poorly understood on a causative level. Here, we describe a novel factor that affects P. aeruginosa differentiation, its condensins.
Condensins play a unique role in global chromosome organization (reviewed in references 10, 11, and 12). These multisubunit ABC-type cellular ATPases (Fig. 1A) act as macromolecular clamps that bridge distant DNA segments (13) and have an intrinsic ability to self-organize into chromosome scaffolds (14). Condensins of the structural chromosome maintenance (SMC) superfamily have been implicated in segregation of the origins of DNA replication (1517). The DNA reshaping activities of condensins reside in their core SMC subunits, whereas non-SMC subunits are critical for the regulation and subcellular recruitment of the proteins (1820). In several bacteria, inactivation of condensins leads to dramatic chromosome segregation defects and a reduction in cell viability (2123).
FIG 1 Colony formation by condensin-deficient P. aeruginosa. (A) Architecture of condensins. The cores of the complexes comprise dimeric SMC proteins, which can undergo ATP-dependent cyclization. (B) Operon organization of condensins in P. aeruginosa strain PAO1. (C) Colony formation by PAO1 (WT), Δsmc, Δmks, and ΔmksB Δsmc (ΔΔ) cells. Exponential cells (OD600 of 0.6) were serially diluted (10-fold), spotted onto LB plates, and further incubated at 37°C. (D) Individual colonies observed at ×4 magnification. Size bar, 1 mm. (E) Colony formation (±standard deviation [SD]; n = 6) by the indicated condensin-deficient cells at 23°C and 37°C. When indicated, cells were transformed with pUCP22 or pUCP_SMC plasmids. (F) Distribution of cell sizes for condensin variants at 23°C and 37°C. The data were fit to a bimodal Gaussian distribution.
ATP modulates the activity of the complex by altering its architecture and interaction with DNA (18, 2426). Several distinct intermediates of the ATPase cycle can be trapped by specific mutations. The Walker B EQ mutation stabilizes the dimeric form of the SMC head domain (18, 26). The C motif SR mutation traps the ATP-bound monomeric form of the head (26, 27). The Walker B DA mutant and the Walker A KI and KD mutants are deficient in binding to ATP or DNA (13, 27).
P. aeruginosa encodes two condensins from two distinct superfamilies, SMC-ScpAB and MksBEF (28). MksBEF (PA4684 to PA4686) is encoded as a single operon (Fig. 1B). SMC (PA1527) is encoded downstream of a GntR family transcriptional regulator PA1526 and is separate from ScpA (PA3198) and ScpB (PA3197). Mutational inactivation of SMC results in low-frequency anucleate cell formation and chromosome packing defects (28, 29). We report here that both condensins are expendable under laboratory conditions in P. aeruginosa but are needed for the differentiation of the bacterium. MksB-deficient cells were impaired in biofilm formation, whereas Δsmc cells displayed defects during planktonic growth and bore many traits typical for other small-colony variants. Notably, inactivation of MksB suppressed the growth defects of Δsmc cells, indicating that the observed phenotypes are related to regulation rather than structural deficiencies. Finally, we report a dramatically reduced virulence of condensin-deficient P. aeruginosa in a mouse model of lung infection.


Ethics statement.

All experiments were done in accordance with the protocols approved by the Oklahoma State University Institutional Animal Care and Use Committee (VM-14-1). These protocols are in compliance with the U.S. Animal Welfare Act and the guidelines of the National Research Council.

Plasmids and strains.

P. aeruginosa strains used in this study are summarized in Table 1. PAO1-Lac (ATCC 47085) was used as the wild-type (WT) strain unless otherwise noted. Mutations were introduced using allele replacement as described earlier for OP103 (28), followed by the excision, when indicated, of the gentamicin resistance marker (Gm) with the help of the pFLP2 plasmid (30). Deletions of sspB were constructed using the pEXG2-based plasmids as described elsewhere (31). All strains were verified by PCR to confirm correct replacement.
TABLE 1 Strains and plasmids used in this study
Strain or plasmidCharacteristic(s) or comment(s)aReference or source
P. aeruginosa strains  
    PAO1-LaclacIq+Δ(lacZ)M15+ tetA+ tetR+ATCC 47085
    OP103PAO1-Lac Δsmc::Gm28
    OP105PAO1-Lac lacIq PT7-mksFEB28
    OP107PAO1-Lac Δsmc ΔGmThis study
    OP108PAO1-Lac ΔmksB::GmThis study
    OP109PAO1-Lac ΔmksB::ΔGmThis study
    OP110PAO1-Lac lacIq-PT7-smc GmThis study
    OP111PAO1-Lac lacIq-PT7-smc Gm mksBThis study
    OP112PAO1-Lac ΔmksB Δsmc::GmThis study
    OP113PAO1-Lac ΔmksB Δsmc::ΔGmThis study
    OP114PAO1-Lac lacIq-PT7-smcThis study
    OP115PAO1-Lac lacIq-PT7-PA1526 GmThis study
    OP116smc::smc* ΔGmThis study
    OP117mksB::mksB* GmThis study
    OP130PAO1-Lac ΔsspBThis study
    OP131PAO1-Lac ΔsspB mksB::mksB-DAS4 GmThis study
    OP132PAO1-Lac ΔsspB mksB::mksB-DAS4 ΔGmThis study
    pNPA_SMCAp, pBADN with smc-His8, used for SMC purificationThis study
    pEX_18APAp oriT+ sacB+, gene replacement vector30
    pEXG2Gm, deletion plasmid31
    pEXG2_SspBGm, sspB deletion plasmid31
    pUCP22-SspBAp, PBAD-SspB expression vectorThis study
    pUCP_SMCConstitutive expression of SMC28
lacIq+, lacIq positive.
OP107 (Δsmc) was constructed by the excision of the gentamicin resistance gene from OP103. OP108 and OP109 contain deletions of mksB (PA4686) between nucleotides 33 and 2331. To construct OP113 (Δsmc::ΔGm ΔmksB::ΔGm), the Δsmc::Gm trait was transferred into OP109 and was followed by the removal of Gm. OP110 and OP114 each encode an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible SMC (PA1527). In these strains, 18 nucleotides upstream of PA1527 (7 nucleotides downstream from the stop codon of PA1526) were replaced with the lacIq-PT7 cassette (32). OP115 harbors the lacIq-PT7 cassette upstream of PA1526. In OP111, lacIq-PT7-smc was introduced into OP109. OP116 strains harbor point mutations in the ATPase site of the endogenous smc as indicated. Tested point mutations were K37D (KD), D1092A (DA), E1093Q (EQ), and S1064R (SR). OP130 was constructed using plasmid pEXG2-SspB, which was a gift from S. L. Dove (31). In OP131 and OP132, the endogenous mksB was replaced with its DAS4-tagged version (31). The ATPase mutations of MksB (strains OP117) included D864A (DA), E865Q (EQ), and S829R (SR). These mutations were constructed using overlap extension PCR followed by the insertion of the fragment into plasmid pEX_ΔmksB (28) between the KpnI and BlpI sites. This was followed by the integration of the resulting mutants onto the endogenous mksB locus of the chromosome of PAO1.
pNPA_SMC is a pBAD-based plasmid that encodes SMC-HisGlyHis8. pNPA_SMC* carries point mutations in the ATPase site of SMC, which were introduced using the QuikChange site-directed mutagenesis kit, and was used for purification of mutant SMC. pUCP-SspB encodes arabinose-inducible SspB, which was derived from pUCP-MksB (28) by replacing mksB with sspB.

Bacterial growth and microscopy.

Bacteria were grown in LB or M9 medium supplemented with 0.4% Casamino Acids (M9CA) and 0.4% glycerol as indicated. Antibiotics were used at the following concentrations: 30 μg/ml gentamicin and 200 μg/ml carbenicillin for P. aeruginosa and 10 μg/ml gentamicin and 100 μg/ml ampicillin for Escherichia coli.
To evaluate competitive growth, overnight cultures of the indicated gentamicin-resistant mutant and gentamicin-sensitive PAO1 cells were mixed in equal ratios, where 2 × 104 cells were inoculated into fresh LB medium and further incubated at 37°C. The cell mixture was then reinoculated every 8 h (optical density at 600 nm [OD600] of 0.5). Aliquots of the mixture were removed at the indicated times and were plated on LB and LB plus gentamicin plates to quantify the mutant and total cell numbers. The strain loss rate was determined by fitting the data to single exponential decay.
Biofilm formation on polyvinyl chloride (PVC) was evaluated as previously described (33). Stationary cells were diluted 1:100 in M9CA medium containing 0.4% glycerol, dispensed at 100 μl into each microplate well, and incubated at 37°C for the indicated times. The cells were stained with 20 μl of 0.1% crystal violet for 10 min and then removed. The wells were washed three times with phosphate-buffered saline (PBS) and air dried for 15 min. The remaining dye was resuspended in 100 μl of 95% ethanol and quantified by measuring the absorbance at 600 nm. Cell adhesion to PVC was quantified in essentially the same way, except that the dispensed cells were at an OD600 of 0.6 and their incubation was for 5 min or 1 h as indicated in the text.
Exopolysaccharide production was measured using the Congo red assay (34) with minor modifications. Briefly, cells were washed and suspended in PBS at an OD600 of 3, supplemented with Congo red at 0.002% (mass/vol) for exponential cells or 0.005% for stationary cells, incubated for 30 min, and then pelleted by centrifugation. The amount of the remaining Congo red was determined by measuring the OD490 of the supernatant. These values were then normalized by comparing the absorbance values to those of a serially diluted Congo red stock.
For controlled degradation of MksB, we employed the ClpXP-dependent degron system (31). OP132 cells encode only the DAS4-tagged version of MksB and lack the endogenous adaptor protein SspB, a protein required for ClpXP-dependent proteolysis (35) (Table 1). Transformation of OP132 cells with the pUCP-SspB plasmid (degron on) but not the vector alone (degron off) resulted in nearly complete degradation of MksB-DAS4. Due to the leaky expression of SspB, MksB-DAS4 degradation was nearly complete in the presence and absence of the arabinose inducer. Similar results were obtained using a pPSPK-SspB plasmid (31), which encodes an IPTG-controlled SspB (data not shown).
Microscopy was performed as previously described (28). Cell sizes were quantified using the program Nucleus (36).


MksB and MksEF were purified as previously described (28). SMC-His8 was cloned into the pBADN plasmid, overproduced. and purified using nickel chelate and heparin chromatography as described earlier for MksB (28). The main modification of this procedure was the use of Tris-Cl, pH 8.5, as a buffer at all steps and an additional gel filtration step on a Sephacryl S-300 column. The protein was dialyzed against 20 mM Tris-Cl (pH 8.5), 300 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol (DTT), and 50% glycerol for storage. Rabbit polyclonal antibodies against SMC, MksB, and MksE were raised at Covance and affinity purified as described previously (28). ATPase rates were measured using the Molecular Probes EnzChek phosphate assay kit in 20 mM HEPES (pH 7.7), 40 mM NaCl, 2 mM MgCl2, 1 mM DTT, 5% glycerol, and 1 mM MgATP.

Mouse lung infection.

The mouse model of P. aeruginosa lung infection was adapted from previously published protocols (37). Bacteria were grown at room temperature up to an OD600 of 0.5 and were then collected by centrifugation, washed once in PBS, and resuspended again in PBS. Anesthetized (isoflurane) 10- to 12-week-old female mice of the C57BL/6 strain (Charles River) were intranasally inoculated with 3 × 106 CFU of wild-type or mutant P. aeruginosa and observed at least twice daily. One mouse in each group was intranasally inoculated with PBS only and treated as a control. No signs of infection were detected in the controls. Euthanized mice were immediately necropsied, and lung tissue was collected for histology and bacterial load. For histological analysis, 2-mm sections of lungs were fixed in formalin, paraffin-embedded, sectioned, and stained with hematoxylin-eosin. The tissues were microscopically evaluated by a board-certified veterinary pathologist who was blinded to the experimental groups. The remainder of the lung tissue was homogenized in PBS and plated on LB to determine the colony count.


SMC and MksB are expendable and antagonistic to each other.

To delineate the roles of condensins in the physiology of P. aeruginosa, we generated in-frame deletions of SMC and MksB (see Materials and Methods). Surprisingly, these deletions failed to produce dramatic defects in bacterial viability. When tested for colony formation, ΔmksB and Δsmc ΔmksB cells were virtually identical to the parental cells. The Δsmc strain, however, deviated by producing smaller and fewer colonies (Fig. 1C), which differed from the other strains in their structure. They had well-defined edges devoid of the large translucent zone typical for the wild-type P. aeruginosa (Fig. 1D). This appearance is similar to other small-colony variants of P. aeruginosa obtained by other means (46). In contrast, ΔmksB cells appeared to be identical to the wild type in colony formation as did ΔmksB Δsmc cells. The latter result is notable and indicates that SMC and MksB play opposite roles in regulating P. aeruginosa physiology.
The frequency of colony formation by Δsmc cells was reduced 2.6-fold compared to the parental strain (Fig. 1E). Curiously, it was observed at 23°C but not at 37°C. This reduction could be complemented by constitutive expression of the smc gene from a pUCP22 plasmid but not the plasmid alone. When visualized by microscopy, Δsmc cells showed a propensity to form short chains of up to four cells at 23°C but not at 37°C (Fig. 1F). Thus, the reduction in colony formation was likely caused by a delay in the separation of cells following division.
Immunoblot analysis revealed that SMC and MksBEF are constitutively expressed in growing and stationary-phase cells at approximately 100 copies of SMC and MksE and 3,000 copies of MksB (Fig. 2A and B). Although somewhat larger, the excess of MksB over MksE is not unlike that observed for the E. coli MukBEF during stationary phase and is consistent with the dynamic organization of the complex (19). The copy numbers of the condensins were not affected by the deletion of their counterparts (Fig. 2C), indicating that the proteins do not directly affect each other's expression.
FIG 2 MksB, MksE, and SMC are constitutively expressed in P. aeruginosa. (A) Immunoblot analysis of condensin expression. PAO1 cells were collected at the indicated optical densities (also shown with arrows on the growth curve on the right) and boiled in the sample buffer, and 0.1 OD600 or 0.01 OD600 unit was analyzed by quantitative immunoblotting alongside serially diluted purified proteins. (B) Copy numbers of MksB, MksE, and SMC (±SD; n = 3). (C) Immunoblot verification of the lack of cross talk in condensin expression.

Inactivation of SMC leads to defects during planktonic growth.

Mutations in smc and mksB had different effects on bacterial fitness. Δsmc cells but not ΔmksB cells performed poorly during competitive growth (Fig. 3A). When cultured together with the parental strain, Δsmc cells were lost within several reinoculations. No such loss occurred when the mutants were cultured alone. The strain loss rate was markedly reduced when reinoculations were carried out with reduced frequency (Fig. 3C) and was completely absent during the stationary phase. Therefore, the reduced competitiveness of the mutant develops during cell growth.
FIG 3 Fitness defects of SMC-deficient cells. (A) Competitive growth defects. PAO1 cells were mixed with either Δsmc or ΔmksB cells and grown in LB at 37°C with regular dilution into fresh medium. Aliquots were removed at the indicated times, and the ratio (±SD; n = 6) between mutant and wild-type cells was measured by plating. (B) Immunoblot analysis of IPTG-dependent expression in lacIq-PT7-smc cells. WT, PAO1; Δ, Δsmc strain. (C) Strain loss rate during competitive growth for OP103 (Δsmc), OP103 harboring SMC producing the pUCP_SMC plasmid (pSMC), conditional mutant OP110 (lacIq-PT7-smc) grown with or without IPTG (PSMC or P+SMC), OP111 (lacIq-PT7-smc DmksB), or OP115 (lacIq-PT7-PA1526) grown with or without IPTG. Unless indicated otherwise (24 h), cells were diluted every 8 h. (D) A lag in growth is observed for Δsmc cells but not the other condensin variants.
Competitive growth defects were partially complemented by the inducible expression of SMC. To this end, we examined several constructs. First, we transformed the Δsmc cells with a pUCP22-based plasmid pUCP_SMC, which encodes SMC under the control of an IPTG-inducible PT7 promoter at approximately 4× its endogenous level (Fig. 3B). A significant reduction in the strain loss rate was observed with the addition of a plasmid harboring the smc gene; however, no rate change occurred due to the vector alone (Fig. 3C). We next introduced a PT7 promoter into the intergenic region between PA1526 and SMC. Depending on the amount of IPTG, the resulting cells produced SMC below or above its endogenous level (Fig. 3B). In all cases, we observed partial complementation of the competitive growth defects in the presence, but not in the absence, of IPTG whether or not mksB was present inside the cell (Fig. 3C). Thus, the residual fitness defects in Δsmc cells were likely due to some cis consequences of the deletion rather than the amount of the expressed protein. Accordingly, no loss of fitness was detected in OP115 (lacIq-PT7-PA1526) cells, which produce SMC from the PT7 promoter placed upstream to PA1526 (Fig. 3C). Under noninducing conditions, however, these cells produced notably lower competitive growth defects, which is perhaps due to an interaction between SMC and PA1526 or the leaky expression of smc.
Only SMC-deficient cells, not other condensin mutants, displayed anomalies during planktonic growth. They remained clumpy for several hours after transfer into fresh medium, resulting in a characteristic lag in the growth curves (Fig. 3D). This lag may potentially be the reason why the strain performed poorly during competitive growth.

Inactivation of MksB impairs biofilm formation.

Although fully functional during competitive growth, MksB-deficient cells displayed a markedly reduced ability to form biofilms both on glass and on polyvinyl chloride (PVC) surfaces (Fig. 4A). In contrast, Δsmc cells were, if anything, more proficient in producing biofilms than the parental strain (Fig. 4B). This was mostly due to the high adherence of Δsmc mutants to the surface. Indeed, even 5 min of incubation of Δsmc but not PAO1 cells resulted in significant cell attachment to the PVC surface (Fig. 4C). Similar to the case with colony formation (Fig. 1), the phenotype of the double mutant was dominated by the absence of mksB.
FIG 4 Biofilm formation defects. (A) Biofilm formation by condensin mutants on glass (top) and PVC (bottom). (B) Cell adhesion to PVC quantified by crystal violet staining (±SD; n = 3). Overnight cells were diluted 100-fold into M9CA medium, grown for indicated times, and stained with crystal violet. (C) Adhesion of cells at an OD600 of 0.6 to PVC after incubation for 5 min or 60 min. (D) Depletion of MksB using the degron system. DAS4-tagged but not wild-type MksB is depleted in cells that express plasmid-encoded SspB (pSspB) as determined using immunoblotting. (E) Inducible depletion of MksB impairs biofilm formation. Biofilm formation (±SD; n = 3) was measured for PAO1 (WT), ΔmksB (none), or OP132 (ΔsspB mksB::mksB-DAS4; DAS4) cells that were transformed with the pUCP22 (OFF) or pSspB (ON) plasmid. (F and G) Congo red depletion by pelleted exponential-phase cells (OD600 of 0.6; exp) and stationary-phase (stat) cells.
Depletion of MksB using the degron system (31) also resulted in impaired biofilm formation, demonstrating that this effect was indeed due to the absence of the protein (Fig. 4D and E). To this end, we removed sspB, which encodes the protease adapter protein, from PAO1 and replaced the endogenous mksB with another version that encodes a C-terminal DAS4 tag (31). The resulting strain produced a tagged MksB at its endogenous level until it was transformed with a plasmid that harbors sspB (Fig. 4D). As expected, biofilm formation was impaired in all strains where MksB production was depleted (Fig. 4E). Because the cell physiology changes in response to the production of the plasmid-borne SspB protein, we conclude that the phenotype is associated with the presence of MksB.
The stickiness of P. aeruginosa can often be attributed to the production of exopolysaccharides by the Pel/Psl system (38). To determine whether or not the same holds true for our mutants, we employed the Congo red assay (34, 39). The assay exploits the high affinity of the dye to polysaccharides. The cells were supplemented with Congo red for 30 min and pelleted by centrifugation, and the remaining Congo red was spectrophotometrically quantified (Fig. 4F). We found that Δsmc cells produce Pel/Psl on par with the wild type in the stationary phase, but unlike the wild type, they fail to switch it off upon the resumption of growth (Fig. 4G). In contrast, ΔmksB and ΔmksB Δsmc cells downregulate the synthesis of polysaccharides both during exponential growth and in the stationary phase. Thus, the adhesion behaviors of the condensin mutants are mediated by the Pel/Psl system.

ATPase activity is required for fitness and biofilm formation.

ATP is central to the operation of condensins. We generated point mutations in the ATPase site of SMC as a means of modulating its activity and exploring correlations between its phenotypes. We tested the following three types of mutations (Fig. 5A): (i) the Walker B DA and Walker A KD mutations, which preclude ATP binding and dimerization of the SMC heads (13, 27); (ii) the Walker B EQ mutation, which precludes ATP hydrolysis but not binding and stabilizes the dimeric SMC heads (18, 26); and (iii) the C motif SR mutation, which interferes with head dimerization but not ATP binding (26, 27). All tested mutants were expressed in P. aeruginosa at their normal level (Fig. 5B) and, for the case of SMC, confirmed for their deficiency in ATP hydrolysis (Fig. 5C).
FIG 5 Phenotypes of ATPase mutants of SMC and MksB. (A) Effects of ATPase mutations on the conformation of condensins. (B) Immunoblot analysis of expression levels for the tested mutants. (C) ATPase activity of the purified variants of SMC. (D to F) OP116 (smc::smc* Gm) cells, which harbor the indicated mutations in smc in its endogenous location, were assayed for anucleate cell formation (±SD; n > 800) (D), colony formation (±SD; n = 3) (E), and competitive growth (±SD; n = 3) (F). (G) Biofilm formation by the indicated ATPase or deletion MksB mutants (±SD; n = 6). Note formation of oligomers by MksBEQ, which is consistent with head disengagement defects expected for this mutant.
The Walker B mutant SMCEQ was indistinguishable from the wild-type protein in all tested assays, including anucleate cell formation (Fig. 5D), colony formation (Fig. 5E), and competitive growth (Fig. 5F). All other mutants were deficient in one or another aspect of SMC activity. In particular, DA, SR, and KD mutations prompted the formation of anucleate cells at the level found in the deletion mutant (Fig. 5D). However, defects in colony formation and competitive growth were observed only for SMCKD (Fig. 5E and F). All of these mutations interfere with the dimerization of the SMC heads, whereas the charge inversion KD mutant is deficient in DNA bridging (13, 26, 27).
These results reveal that the phenotypes of SMC mutants are only partially related. For example, the DA and SR mutants of SMC produced anucleate cells but did not show any fitness defects. It appears, therefore, that at least some of the regulatory functions of SMC are unrelated to their role in chromosome packing.
ATPase activity was similarly essential for the function of MksB. Two out of three tested ATPase mutants, EQ and SR, were deficient in biofilm formation (Fig. 5G). However, one of the variants, MksBDA, failed to produce the said phenotype. This is not unlike the EQ mutant of SMC, which behaved similarly to the wild type in all fitness assays (Fig. 5D to F). It appears, therefore, that the regulatory functions of condensins can be performed by one of their intermediates in the ATPase cycle.

Reduced virulence of condensin-deficient P. aeruginosa.

Even though the phenotype of condensin mutations appeared rather mild, their effect on epigenetic states was quite dramatic. To determine whether or not these changes alter the virulence of P. aeruginosa, we evaluated Δsmc and Δsmc ΔmksB strains in a mouse model of lung infection (37).
Eight C57BL/6 mice were each intranasally infected with 3 × 106 CFU of PAO1 (WT), Δsmc, or Δsmc ΔmksB cells, and their survival followed (Fig. 6A). All of the mice infected with PAO1 and Δsmc ΔmksB cells initially appeared clinically ill, which was primarily characterized by depression and increased respiratory rate. However, the Δsmc ΔmksB mice quickly stabilized, whereas the PAO1 mice continued to deteriorate and most had to be euthanized by day 1 postinfection. No clinical signs of illness were detected in the Δsmc mice. Thus, both condensin mutants were clinically less virulent than the parental strain based on survival data.
FIG 6 Virulence analysis of condensin mutants. (A) Survival curves for mice infected with PAO1 (WT), OP107 (Δsmc), and OP113 (Δsmc ΔmksB) cells. (B) Histopathological analysis of lung sections from mice infected with the indicated strains. Mice infected with WT cells exhibited myriad, conspicuous colonies of bacteria within airways and alveolar spaces, which are seen as large purple masses (arrows). Septal capillaries were markedly congested. Although there were accompanying edema and mild neutrophilic infiltrates, significant inflammation was absent. Both mutant strains exhibited similar lesions of pneumonia characterized by airway and alveolar infiltrates by primarily neutrophils and macrophages. Conspicuous bacterial colonies (as present for the WT) were not seen. Size bars, 100 μm. (C) Bacterial loads in lungs of infected mice. Horizontal lines indicate the geometric averages of the loads. Note that the Δsmc strain-infected mice did not yield any colonies (plotted at the detection limit of 100 CFU/g).
All PAO1 mice euthanized on day 1 had severe bacterial colonization of the lungs along with pulmonary hemorrhage and mild neutrophilic infiltrates (Fig. 6B). The inflammatory reaction appeared mild most likely because the mice were killed before significant infiltrates (neutrophils and macrophages) had time to accumulate. It seems like the lesions and death in the mice inoculated with the WT were more from hemodynamic events (hemorrhage, fluid leakage, and shock) related to the pathogen rather than any accompanying component of severe host inflammatory response.
The Δsmc mice all had pneumonias; however, their pneumonias were characterized by heavy cellular infiltrates (primarily neutrophils and macrophages) within the airways and alveolar spaces. The myriad bacterial colonies that were so conspicuous in the lungs of PAO1 mice were not seen in the Δsmc-infected animals (Fig. 6B). The Δsmc ΔmksB mice that lived until the end of the study had histological lesions that were indistinguishable from the single smc mutants, whereas the lone Δsmc ΔmksB mouse that had to be killed on day 2 had histological lesions that resembled the WT.
The bacterial load analysis was consistent with the histological results. The PAO1 mice had significantly higher bacterial loads than the Δsmc ΔmksB mice (1.8 × 107 versus 5.3 × 106 CFU/g), and the number of bacteria in the Δsmc lungs was below detection (less than 100 CFU/g) (Fig. 6C). According to the Student test analysis, the difference between the PAO1 and Δsmc ΔmksB mice is significant (P = 0.02) just as it is between the PAO1 and Δsmc mice (P < 2 × 10−4). The CFU count also formed distinct distributions for mice that fell to the sickness and those that survived it (P = 4 × 10−5), which is consistent with mortality being caused by infection.
Based on these results, we conclude that condensin-deficient P. aeruginosa are significantly less virulent than the parent. We further conclude that Δsmc P. aeruginosa is even less virulent than the double mutant Δsmc ΔmksB. This result, while somewhat counterintuitive, is fully consistent with the phenotypic profiles of the two strains, which revealed the enhanced fitness of Δsmc ΔmksB and ΔmksB cells.


P. aeruginosa is notorious for its ability to thrive in a hostile environment. To achieve this, it encodes numerous biosynthetic and regulatory enzymes that help it adapt to adverse conditions. Befittingly, P. aeruginosa harbors two condensins. One of them is a conventional SMC condensin while the other, MksBEF, belongs to a recently discovered and poorly characterized family that is distantly related to E. coli MukBEF. We show here that the two proteins are involved in the differentiation of the bacterium. This is a novel activity of condensins, which may or may not be related to their function in chromosome maintenance.
Inactivation of SMC and MksB had opposite effects on the physiology of P. aeruginosa. The ΔmksB cells were deficient in their adhesion to surfaces and the formation of biofilms (Fig. 4) but did not display defects during planktonic growth (Fig. 3). If anything, ΔmksB cells appeared to be better fit for planktonic growth since they reached high densities sooner than the parental cells (Fig. 3D). In contrast, Δsmc cells had marked fitness defects (Fig. 3) but were well adapted for sessile growth (Fig. 4). These effects appear to be regulatory rather than structural in nature since the phenotype of the double mutant was dominated by the absence of MksB. Additive effects would instead be expected if the inactivation of condensins resulted in the inactivation of an essential pathway. In this sense, MksB and SMC appear to be integrated into opposite developmental programs in P. aeruginosa (Fig. 7).
FIG 7 P. aeruginosa condensins are integrated into opposite epigenetic programs. Inactivation of SMC renders P. aeruginosa poorly fit for planktonic growth but well adapted for adhesion to surfaces. Hence, the activity of the protein appears essential for planktonic growth. Conversely, MksB-deficient cells grow well in bulk medium but are impaired in surface adhesion, which points to their role in sessile growth.
The phenotype of Δsmc cells was multifaceted and included such features as a decrease in competitive index (Fig. 3A), a lag in planktonic growth (Fig. 3D), increased adhesion to surfaces (Fig. 4B and C), and the formation of small colonies with sharp edges (Fig. 1D). All of these phenotypes have been previously reported for other SCVs of P. aeruginosa, which were obtained by antibiotic treatment or isolated in clinics (35). The commonality of these phenotypes, as well as the ability of SCVs and “normal” cells to slowly covert into each other, suggests that they represent a distinct differentiation state in P. aeruginosa, which can be induced by a variety of means. Curiously, many of these phenotypes can be attributed to a single trait, the expression of the exopolysaccharides by the Pel/Psl system (Fig. 4F and G). Given the epigenetic nature of this transition and because it helps the bacterium to colonize new niches, it might be prudent to refer to this as differentiation. Our finding that this transition can be triggered by condensins, a low-copy-number motor protein, suggests new ways to induce differentiation programs.
Despite their relatively mild phenotype, condensin mutants had dramatically reduced virulence in a murine model of lung infection. Δsmc cells were completely cleared by the immune system without producing visible clinical signs (Fig. 6). Even the Δsmc ΔmksB mutant, which has fewer growth defects in planktonic bacteria, was much less virulent than the parental strain. These data demonstrate that condensins are essential for virulence in P. aeruginosa.
The finding that condensins participate in epigenetic control was unexpected. Indeed, these proteins contribute to an essential cellular function, chromosome replication and segregation. Their participation in an additional cellular program increases the costs of mitigating mutational pressure. Clearly, the benefits of maintaining such coordination must outweigh the costs. A plausible benefit from such coordination may involve adjusting chromosome structure to a particular differentiated form. Sporulating bacteria, for example, condense the chromosome in the spore but not the mother (40). Intriguingly, our data suggest that this link may also work in the opposite direction where information on chromosome packing can influence epigenetic switching.


We are indebted to Marie Montelongo and Pulavendron Sivasamiv for mouse inoculations and to Herbert Schweizer and Simon Dove for sharing strains and plasmids.


Lister PD, Wolter DJ, Hanson ND. 2009. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev 22:582–610.
Wagner VE, Iglewski BH. 2008. P. aeruginosa biofilms in CF infection. Clin Rev Allergy Immunol 35:124–134.
Bragonzi A, Paroni M, Nonis A, Cramer N, Montanari S, Rejman J, Di Serio C, Doring G, Tummler B. 2009. Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence. Am J Respir Crit Care Med 180:138–145.
Drenkard E, Ausubel FM. 2002. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416:740–743.
Wei Q, Tarighi S, Dotsch A, Haussler S, Musken M, Wright VJ, Camara M, Williams P, Haenen S, Boerjan B, Bogaerts A, Vierstraete E, Verleyen P, Schoofs L, Willaert R, De Groote VN, Michiels J, Vercammen K, Crabbe A, Cornelis P. 2011. Phenotypic and genome-wide analysis of an antibiotic-resistant small colony variant (SCV) of Pseudomonas aeruginosa. PLoS One 6:e29276.
Deziel E, Comeau Y, Villemur R. 2001. Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities. J Bacteriol 183:1195–1204.
Balasubramanian D, Schneper L, Kumari H, Mathee K. 2013. A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res 41:1–20.
Turner KH, Vallet-Gely I, Dove SL. 2009. Epigenetic control of virulence gene expression in Pseudomonas aeruginosa by a LysR-type transcription regulator. PLoS Genet 5:e1000779.
Hoboth C, Hoffmann R, Eichner A, Henke C, Schmoldt S, Imhof A, Heesemann J, Hogardt M. 2009. Dynamics of adaptive microevolution of hypermutable Pseudomonas aeruginosa during chronic pulmonary infection in patients with cystic fibrosis. J Infect Dis 200:118–130.
Kleine Borgmann LA, Graumann PL. 2014. Structural maintenance of chromosome complex in bacteria. J Mol Microbiol Biotechnol 24:384–395.
Reyes-Lamothe R, Nicolas E, Sherratt DJ. 2012. Chromosome replication and segregation in bacteria. Annu Rev Genet 46:121–143.
Rybenkov VV, Herrera V, Petrushenko ZM, Zhao H. 2014. MukBEF, a chromosomal organizer. J Mol Microbiol Biotechnol 24:371–383.
Petrushenko ZM, Cui Y, She W, Rybenkov VV. 2010. Mechanics of DNA bridging by bacterial condensin MukBEF in vitro and in singulo. EMBO J 29:1126–1135.
Cui Y, Petrushenko ZM, Rybenkov VV. 2008. MukB acts as a macromolecular clamp in DNA condensation. Nat Struct Mol Biol 15:411–418.
Gruber S, Errington J. 2009. Recruitment of condensin to replication origin regions by ParB/SpoOJ promotes chromosome segregation in B. subtilis. Cell 137:685–696.
Minnen A, Attaiech L, Thon M, Gruber S, Veening JW. 2011. SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae. Mol Microbiol 81:676–688.
Sullivan NL, Marquis KA, Rudner DZ. 2009. Recruitment of SMC by ParB-parS organizes the origin region and promotes efficient chromosome segregation. Cell 137:697–707.
Hirano M, Hirano T. 2004. Positive and negative regulation of SMC-DNA interactions by ATP and accessory proteins. EMBO J 23:2664–2673.
Petrushenko ZM, Lai CH, Rybenkov VV. 2006. Antagonistic interactions of kleisins and DNA with bacterial condensin MukB. J Biol Chem 281:34208–34217.
She W, Mordukhova E, Zhao H, Petrushenko ZM, Rybenkov VV. 2013. Mutational analysis of MukE reveals its role in focal subcellular localization of MukBEF. Mol Microbiol 87:539–552.
Britton RA, Lin DC, Grossman AD. 1998. Characterization of a prokaryotic SMC protein involved in chromosome partitioning. Genes Dev 12:1254–1259.
Jensen RB, Shapiro L. 1999. The Caulobacter crescentus smc gene is required for cell cycle progression and chromosome segregation. Proc Natl Acad Sci U S A 96:10661–10666.
Niki H, Jaffe A, Imamura R, Ogura T, Hiraga S. 1991. The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli. EMBO J 10:183–193.
Kleine Borgmann LA, Hummel H, Ulbrich MH, Graumann PL. 2013. SMC condensation centers in Bacillus subtilis are dynamic structures. J Bacteriol 195:2136–2145.
Schwartz MA, Shapiro L. 2011. An SMC ATPase mutant disrupts chromosome segregation in Caulobacter. Mol Microbiol 82:1359–1374.
Woo JS, Lim JH, Shin HC, Suh MK, Ku B, Lee KH, Joo K, Robinson H, Lee J, Park SY, Ha NC, Oh BH. 2009. Structural studies of a bacterial condensin complex reveal ATP-dependent disruption of intersubunit interactions. Cell 136:85–96.
Hirano M, Anderson DE, Erickson HP, Hirano T. 2001. Bimodal activation of SMC ATPase by intra- and inter-molecular interactions. EMBO J 20:3238–3250.
Petrushenko ZM, She W, Rybenkov VV. 2011. A new family of bacterial condensins. Mol Microbiol 81:881–896.
Vallet-Gely I, Boccard F. 2013. Chromosomal organization and segregation in Pseudomonas aeruginosa. PLoS Genet 9:e1003492.
Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP. 1998. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86.
Castang S, McManus HR, Turner KH, Dove SL. 2008. H-NS family members function coordinately in an opportunistic pathogen. Proc Natl Acad Sci U S A 105:18947–18952.
Morita Y, Narita S, Tomida J, Tokuda H, Kawamura Y. 2010. Application of an inducible system to engineer unmarked conditional mutants of essential genes of Pseudomonas aeruginosa. J Microbiol Methods 82:205–213.
O'Toole GA, Kolter R. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28:449–461.
Spiers AJ, Bohannon J, Gehrig SM, Rainey PB. 2003. Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. Mol Microbiol 50:15–27.
McGinness KE, Baker TA, Sauer RT. 2006. Engineering controllable protein degradation. Mol Cell 22:701–707.
Wang Q, Mordukhova EA, Edwards AL, Rybenkov VV. 2006. Chromosome condensation in the absence of the non-SMC subunits of MukBEF. J Bacteriol 188:4431–4441.
Peluso L, de Luca C, Bozza S, Leonardi A, Giovannini G, Lavorgna A, De Rosa G, Mascolo M, Ortega De Luna L, Catania MR, Romani L, Rossano F. 2010. Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization. BMC Microbiol 10:9.
Colvin KM, Irie Y, Tart CS, Urbano R, Whitney JC, Ryder C, Howell PL, Wozniak DJ, Parsek MR. 2012. The Pel and Psl polysaccharides provide Pseudomonas aeruginosa structural redundancy within the biofilm matrix. Environ Microbiol 14:1913–1928.
Merritt JH, Brothers KM, Kuchma SL, O'Toole GA. 2007. SadC reciprocally influences biofilm formation and swarming motility via modulation of exopolysaccharide production and flagellar function. J Bacteriol 189:8154–8164.
Higgins D, Dworkin J. 2012. Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev 36:131–148.

Information & Contributors


Published In

cover image Journal of Bacteriology
Journal of Bacteriology
Volume 198Number 211 November 2016
Pages: 2936 - 2944
Editor: G. A. O'Toole, Geisel School of Medicine at Dartmouth
PubMed: 27528506


Received: 5 June 2016
Accepted: 7 August 2016
Published online: 7 October 2016


Request permissions for this article.



Hang Zhao
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA
April L. Clevenger
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA
Jerry W. Ritchey
Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, Oklahoma, USA
Helen I. Zgurskaya
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA
Valentin V. Rybenkov
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA


G. A. O'Toole
Geisel School of Medicine at Dartmouth


Address correspondence to Valentin V. Rybenkov, [email protected].

Metrics & Citations


Note: There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.

Citation counts come from the Crossref Cited by service.


If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.

View Options

Figures and Media






Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy