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Cell envelope plays a dual role in the life of bacteria by simultaneously protecting it from a hostile environment and facilitating access to beneficial molecules. At the heart of this ability lie the restrictive properties of the cellular membrane augmented by efflux transporters, which preclude intracellular penetration of most molecules except with the help of specialized uptake mediators. Recently, kinetic properties of the cell envelope came into focus driven on one hand by the urgent need in new antibiotics and, on the other hand, by experimental and theoretical advances in studies of transmembrane transport. A notable result from these studies is the development of a kinetic formalism that integrates the Michaelis–Menten behavior of individual transporters with transmembrane diffusion and offers a quantitative basis for the analysis of intracellular penetration of bioactive compounds. This review surveys key experimental and computational approaches to the investigation of transport by individual translocators and in whole cells, summarizes key findings from these studies and outlines implications for antibiotic discovery. Special emphasis is placed on Gram-negative bacteria, whose envelope contains two separate membranes. This feature sets these organisms apart from Gram-positive bacteria and eukaryotic cells by providing them with full benefits of the synergy between slow transmembrane diffusion and active efflux. [ABSTRACT FROM AUTHOR]

Most clinical antibiotics do not have efficacy against Gram‐negative pathogens, mainly because these cells are protected by the permeability barrier comprising the two membranes with active efflux. The emergence of multidrug‐resistant Gram‐negative strains threatens the utility even of last resort therapeutic treatments. Significant efforts at different levels of resolution are currently focused on finding a solution to this nonpermeation problem and developing new approaches to the optimization of drug activities against multidrug‐resistant pathogens. The exceptional efficiency of the Gram‐negative permeability barrier is the result of a complex interplay between the two opposing fluxes of drugs across the two membranes. In this review, we describe the current state of understanding of the problem and the recent advances in theoretical and empirical approaches to characterization of drug permeation and active efflux in Gram‐negative bacteria. [ABSTRACT FROM AUTHOR]

Abstract Antibiotic resistance is a serious threat to public health. Significant efforts are currently directed toward containment of the spread of resistance, finding new therapeutic options concerning resistant human and animal pathogens, and addressing the gaps in the fundamental understanding of mechanisms of resistance. Experimental data and kinetic modeling revealed a major factor in resistance, the synergy between active efflux and the low permeability barrier of the outer membrane, which dramatically reduces the intracellular accumulation of many antibiotics. The structural and mechanistic particularities of trans-envelope efflux pumps amplify the effectiveness of cell envelopes as permeability barriers. An important feature of this synergism is that efflux pumps and the outer membrane barriers are mechanistically independent and select antibiotics based on different physicochemical properties. The synergism amplifies even weak polyspecificity of multidrug efflux pumps and creates a major hurdle in the discovery and development of new therapeutics against Gram-negative pathogens. [ABSTRACT FROM AUTHOR]

Global folding of bacterial chromosome requires the activity of condensins. These highly conserved proteins are involved in various aspects of higher-order chromatin dynamics in a diverse range of organisms. Two distinct superfamilies of condensins have been identified in bacteria. The SMC-ScpAB proteins bear significant homology to eukaryotic condensins and cohesins and are found in most of the presently sequenced bacteria. This review focuses on the MukBEF/MksBEF superfamily, which is broadly distributed across diverse bacteria and is characterized by low sequence conservation. The prototypical member of this superfamily, the Escherichia coli condensin MukBEF, continues to provide critical insights into the mechanism of the proteins. MukBEF acts as a complex molecular machine that assists in chromosome segregation and global organization. The review focuses on the mechanistic analysis of DNA organization by MukBEF with emphasis on its involvement in the formation of chromatin scaffold and plausible other roles in chromosome segregation. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Replication and segregation of genetic information are the activities central to the well-being of all living cells. Concerted mechanisms have evolved that ensure that each cellular chromosome is replicated once and only once per cell cycle and then faithfully segregated into daughter cells. Despite remarkable taxonomic diversity, these mechanisms are largely conserved across eubacteria, although species-specific distinctions can often be noted. Here, we provide an overview of the current state of knowledge about maintenance of the chromosome structure in Pseudomonas aeruginosa. We focus on global chromosome organization and its dynamics during DNA replication and cell division. Special emphasis is made on contrasting these activities in P. aeruginosa and other bacteria. Among unique P. aeruginosa, features are the presence of two distinct autonomously replicating sequences and multiple condensins, which suggests existence of novel regulatory mechanisms. [ABSTRACT FROM AUTHOR]

Reports on findings which imply that type II topoisomerases use the energy of adenosine triphosphate hydrolysis to preferentially remove the topological links that provide barriers to DNA segregation. Details of previous studies; Plausible explanation of results; Propositions.

The ability Gram-negative pathogens have at adapting and protecting themselves against antibiotics has increasingly become a public health threat. Data-driven models identifying molecular properties that correlate with outer membrane (OM) permeation and growth inhibition while avoiding efflux could guide the discovery of novel classes of antibiotics. Here we evaluate 174 molecular descriptors in 1260 antimicrobial compounds and study their correlations with antibacterial activity in Gram-negative Pseudomonas aeruginosa. The descriptors are derived from traditional approaches quantifying the compounds' intrinsic physicochemical properties, together with, bacterium-specific from ensemble docking of compounds targeting specific MexB binding pockets, and all-atom molecular dynamics simulations in different subregions of the OM model. Using these descriptors and the measured inhibitory concentrations, we design a statistical protocol to identify predictors of OM permeation/inhibition. We find consistent rules across most of our data highlighting the role of the interaction between the compounds and the OM. An implementation of the rules uncovered in our study is shown, and it demonstrates the accuracy of our approach in a set of previously unseen compounds. Our analysis sheds new light on the key properties drug candidates need to effectively permeate/inhibit P. aeruginosa, and opens the gate to similar data-driven studies in other Gram-negative pathogens. Identifying molecular properties of compounds that best correlate with outer membrane permeation and growth inhibition could guide the discovery of new antibiotics. Here, the authors evaluate 174 molecular descriptors in 1260 antimicrobial compounds and study their correlations with antibacterial activity in Gram-negative Pseudomonas aeruginosa to derive a statistical protocol to identify mechanistic predictors of outer membrane permeation. [ABSTRACT FROM AUTHOR]

MukBEF, the bacterial prototype of eukaryotic condensins and cohesins, has a key role in the global chromosomal organization of Escherichia coli and its close relatives. The recent report of the crystal structure of the MukB head domain in complex with its accessory subunits MukEF clearly demonstrates that MukBEF functions as a macromolecular assembly rather than a set of individual molecules and offers clues on how ATP and MukEF regulate the architecture of this complex. [ABSTRACT FROM AUTHOR]

Antibiotics effective against Gram‐negative ESKAPE pathogens are a critical area of unmet need. Infections caused by these pathogens are not only difficult to treat but finding new therapies to overcome Gram‐negative resistance is also a challenge. There are not enough antibiotics in development that target the most dangerous pathogens and there are not enough novel drugs in the pipeline. The major obstacle in the antibiotic discovery pipeline is the lack of understanding of how to breach antibiotic permeability barriers of Gram‐negative pathogens. These barriers are created by active efflux pumps acting across both the inner and the outer membranes. Overproduction of efflux pumps alone or together with either modification of the outer membrane or antibiotic‐inactivating enzymes and target mutations contribute to clinical levels of antibiotics resistance. Recent efforts have generated significant advances in the rationalization of compound efflux and permeation across the cell envelopes of Gram‐negative pathogens. Combined with earlier studies and novel mathematical models, these efforts have led to a multilevel understanding of how antibiotics permeate these barriers and how multidrug efflux and permeation contribute to the development of antibiotic resistance and heteroresistance. Here, we discuss the new developments in this area. [ABSTRACT FROM AUTHOR]

Two membrane cell envelopes act as selective permeability barriers in Gram-negative bacteria, protecting cells against antibiotics and other small molecules. Significant efforts are being directed toward understanding how small molecules permeate these barriers. In this study, we developed an approach to analyze the permeation of compounds into Gram-negative bacteria and applied it to Pseudomonas aeruginosa, an important human pathogen notorious for resistance to multiple antibiotics. The approach uses mass spectrometric measurements of accumulation of a library of structurally diverse compounds in four isogenic strains of P. aeruginosa with varied permeability barriers. We further developed a machine learning algorithm that generates a deterministic classification model with minimal synonymity between the descriptors. This model predicted good permeators into P. aeruginosa with an accuracy of 89% and precision above 58%. The good permeators are broadly distributed in the property space and can be mapped to six distinct regions representing diverse chemical scaffolds. We posit that this approach can be used for more detailed mapping of the property space and for rational design of compounds with high Gram-negative permeability. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a significant opportunistic pathogen responsible for numerous human infections. Its high pathogenicity resides in a diverse array of virulence factors and an ability to adapt to hostile environments. We report that these factors are tied to the activity of condensins, SMC and MksBEF, which primarily function in structural chromosome maintenance. This study revealed that both proteins are required for P. aeruginosa virulence during corneal infection. The reduction in virulence was traced to broad changes in gene expression. Transcriptional signatures of smc and mksB mutants were largely dissimilar and non‐additive, with the double mutant displaying a distinct gene expression profile. Affected regulons included those responsible for lifestyle control, primary metabolism, surface adhesion and biofilm growth, iron and sulfur assimilation, and numerous virulence factors, including type 3 and type 6 secretion systems. The in vitro phenotypes of condensin mutants mirrored their transcriptional profiles and included impaired production and secretion of multiple virulence factors, growth deficiencies under nutrient limiting conditions, and altered c‐di‐GMP signaling. Notably, c‐di‐GMP mediated some but not all transcriptional responses of the mutants. Thus, condensins are integrated into the control of multiple genetic programs related to epigenetic and virulent behavior of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Summary Condensins play a central role in global chromatin organization. In bacteria, two families of condensins have been identified, the MukBEF and SMC-ScpAB complexes. Only one of the two complexes is usually found in a given species, giving rise to a paradigm that a single condensin organizes bacterial chromosomes. Using sequence analysis, we identified a third family of condensins, MksBEF (MukBEF-like SMC proteins), which is broadly present in diverse bacteria. The proteins appear distantly related to MukBEF, have a similar operon organization and similar predicted secondary structures albeit with notably shorter coiled-coils. All three subunits of MksBEF exhibit significant sequence variation and can be divided into a series of overlapping subfamilies. MksBEF often coexists with the SMC-ScpAB, MukBEF and, sometimes, other MksBEFs. In Pseudomonas aeruginosa, both SMC and MksB contribute to faithful chromosome partitioning, with their inactivation leading to increased frequencies of anucleate cells. Moreover, MksBEF can complement anucleate cell formation in SMC-deficient cells. Purified PaMksB showed activities typical for condensins including ATP-modulated DNA binding and condensation. Notably, DNA binding by MksB is negatively regulated by ATP, which sets it apart from other known SMC proteins. Thus, several specialized condensins might be involved in organization of bacterial chromosomes. [ABSTRACT FROM AUTHOR]

In Gram-negative pathogens, multidrug efflux pumps that provide clinically significant levels of antibiotic resistance function as threecomponent complexes. They are composed of the inner membrane transporters belonging to one of three superfamilies of proteins, RND, ABC, or MF; periplasmic proteins belonging to the membrane fusion protein (MFP) family; and outer membrane channels exemplified by the Escherichia coli ToIC. The three-component complexes span the entire two-membrane envelope of Gram-negative bacteria and expel toxic molecules from the cytoplasmic membrane to the medium. The architecture of these complexes is expected to vary significantly because of the structural diversity of the inner membrane transporters. How the three-component pumps are assembled, their architecture, and their dynamics remain unclear. In this study, we reconstituted interactions and compared binding kinetics of the E. coli ToIC with AcrA, MacA, and EmrA, the periplasmic MFP5 that function in multidrug efflux with transporters from the RND, ABC, and MF superfamilies, respectively. By using surface plasmon resonance, we demonstrate that ToIC interactions with MFP5 are highly dynamic and sensitive to pH. The affinity of ToIC to MFP5 decreases in the order MacA > EmrA > AcrA. We further show that MFP5 are prone to oligomerization, but differ dramatically from each other in oligomerization kinetics and stability of oligomers. The propensity of MFP5 to oligomerize correlates with the stability of MFP-ToIC complexes and structural features of inner membrane transporters. We propose that recruitment of ToIC by various MFP5 is determined not only by kinetics of MFP-TolC interactions but also by oligomerization kinetics of MFPs and pH. [ABSTRACT FROM AUTHOR]

Correct folding of the chromosome into its highly ordered structure requires the action of condensins. The multisubunit condensins are highly conserved from bacteria to humans, and at their core they contain the characteristic V-shaped dimer of structural maintenance of chromosome proteins. The mechanism of DNA rearrangements by condensins remains unclear. Using magnetic tweezers, we show that bacterial condensin MukB acts as an ATP-modulated macromolecular assemblage in DNA condensation. Condensation occurs in a highly cooperative manner, resulting in the formation of force-resilient clusters. ATP regulates nucleation but not propagation of the clusters and seems to play a structural role. MukB clusters can further interact with each other, thereby bringing distant DNA segments together. The resulting activity has not previously been described among DNA-remodeling machines and may explain how the protein can organize the global structure of the chromosome. [ABSTRACT FROM AUTHOR]

MukBEF is a bacterial SMC (structural maintenance of chromosome) complex required for faithful chromosome segregation in Escherichia coli. The SMC subunit of the complex, MukB, promotes DNA condensation in vitro and in vivo; however, all three subunits are required for the function of MukBEF. We report here that MukEF disrupts MukB·DNA complex. Pre-assembled MukBEF was inert in DNA binding or reshaping. Similarly, the association of MukEF with DNA-bound MukB served to displace MukB from DNA. When purified from cells, MukBEF existed as a mixture of MukEF-saturated and unsaturated complexes. The holoenzyme was unstable and could only bind DNA upon dissociation of MukEF. The DNA reshaping properties of unsaturated MukBEF were identical to those of MukB. Furthermore, the unsaturated MukBEF was stable and proficient in DNA binding. These results support the view that kleisins are not directly involved in DNA binding but rather bridge distant DNA-bound MukBs. [ABSTRACT FROM AUTHOR]

Proposes a model for the mechanism of topology simplification by type II DNA topoisomerases. Qualitative analysis of the model by computer simulations; Description of the mechanism used by type II topoisomerases to actively disentangle DNA.

The original article can be found online at https://doi.org/10.1038/s41598-022-12376-1. Correction to: I Scientific Reports i https://doi.org/10.1038/s41598-022-12376-1, published online 17 May 2022 The original version of this Article contained an error in the spelling of the author Dinesh Nath which was incorrectly given as Dinesh Nash. [Extracted from the article]

Cell envelopes of many bacteria consist of two membranes studded with efflux transporters. Such organization protects bacteria from the environment and gives rise to multidrug resistance. We report a kinetic model that accurately describes the permeation properties of this system. The model predicts complex non-linear patterns of drug uptake complete with a bifurcation, which recapitulate the known experimental anomalies. We introduce two kinetic parameters, the efflux and barrier constants, which replace those of Michaelis and Menten for trans-envelope transport. Both compound permeation and efflux display transitions, which delineate regimes of efficient and inefficient efflux. The first transition is related to saturation of the transporter by the compound and the second one behaves as a bifurcation and involves saturation of the outer membrane barrier. The bifurcation was experimentally observed in live bacteria. We further found that active efflux of a drug can be orders of magnitude faster than its diffusion into a cell and that the efficacy of a drug depends both on its transport properties and therapeutic potency. This analysis reveals novel physical principles in the behavior of the cellular envelope, creates a framework for quantification of small molecule permeation into bacteria, and should invigorate structure-activity studies of novel antibiotics. [ABSTRACT FROM AUTHOR]

In Gram-negative bacteria, efflux pumps are able to prevent effective cellular concentrations from being achieved for a number of antibiotics. Small molecule adjuvants that act as efflux pump inhibitors (EPIs) have the potential to reinvigorate existing antibiotics that are currently ineffective due to efflux mechanisms. Through a combination of rigorous experimental screening and in silico virtual screening, we recently identified novel classes of EPIs that interact with the membrane fusion protein AcrA, a critical component of the AcrAB-TolC efflux pump in Escherichia coli. Herein, we present initial optimization efforts and structure-activity relationships around one of those previously described hits, NSC 60339 (1). From these efforts we identified two compounds, SLUPP-225 (17h) and SLUPP-417 (17o), which demonstrate favorable properties as potential EPIs in E. coli cells including the ability to penetrate the outer membrane, improved inhibition of efflux relative to 1, and potentiation of the activity of novobiocin and erythromycin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

MukB is a bacterial SMC (structural maintenance of chromosome) protein that regulates the global folding of the Escherichia coli chromosome by bringing distant DNA segments together. We report that moderate overproduction of MukB may lead, depending on strain and growth conditions, to transient growth arrest. In DH5α cells, overproduction of MukB or MukBEF using pBAD expression system triggered growth arrest 2.5 h after induction. The exit from growth arrest was accompanied by the loss of the overproducing plasmid and a decline in the abundance of MukBEF. The arrested cells showed a compound gene expression profile which can be characterized by the following features: (i) a broad and deep downregulation of ribosomal proteins (up to 80-fold); (ii) downregulation of groups of genes encoding enzymes involved in nucleotide metabolism, respiration, and central metabolism; (iii) upregulation of some of the genes responsive to general stress; and (iv) degradation of the patterns of spatial correlations in the transcriptional activity of the chromosome. The transcriptional state of the MukB induced arrest is most similar to stationary cells and cells recovered from stationary phase into a nutrient deprived medium, to amino acid starved cells and to the cells shifting from glucose to acetate. The mukB++ state is dissimilar from all examined transcriptional states generated by protein overexpression with the possible exception of RpoE and RpoH overexpression. Thus, the transcription profile of MukB-arrested cells can be described as a combination of responses typical for other growth-arrested cells and those for overproducers of DNA binding proteins with a particularly deep down-regulation of ribosomal genes. [ABSTRACT FROM AUTHOR]

Bacterial condensin MukBEF is essential for global folding of the Escherichia coli chromosome. MukB, a SMC (structural maintenance of chromosome) protein, comprises the core of this complex and is responsible for its ATP-modulated DNA binding and reshaping activities. MukF serves as a kleisin that modulates MukB- DNA interactions and links MukBs into macromolecular assemblies. Little is known about the function of MukE. Using random mutagenesis, we generated six loss-of-function point mutations in MukE. The surface mutations clustered in two places. One of them was at or close to the interface with MukF while the other was away from the known interactions of the protein. All loss-of-function mutations affected focal localization of MukBEF in live cells. In vitro, however, only some of them interfered with the assembly of MukBEF into a complex or the ability of MukEF to disrupt MukB- DNA interactions. Moreover, some MukE mutants were able to join intracellular foci formed by endogenous MukBEF and most of the mutants were efficiently incorporated into MukBEF even in the presence of endogenous MukE. These data reveal that focal localization of MukBEF involves other activities besides DNA binding and that MukE plays a central role in them. [ABSTRACT FROM AUTHOR]

Lipid-coated DNA nanoparticles (lipoplexes) are a powerfulgenedelivery tool with promising therapeutic applications. The mechanismof lipoplex assembly remains poorly understood. We explored DNA packingby a cationic lipid DSTAP (distearoyl trimethylammonium-propane) usingmagnetic tweezers. DSTAP-induced DNA condensation occurred as a seriesof bursts with the mean step size of 60–80 nm. The pause timepreceding the steps could be approximated as a bimodal distribution,which reveals at least two distinct condensation pathways. The rapidlycondensed DNA was more resilient to force-induced decondensation.The proportion of the stable, fast-formed complexes decreased at highsalt concentrations. A similar trend was observed in bulk experiments.Lipoplexes assembled at low salt concentration more efficiently shieldedDNA from fluorescent dyes and DNase even after transfer to the highsalt conditions. These data reveal that lipoplex folding occurs viatwo parallel pathways even at the single molecule level. The progressthrough the two pathways can be monitored in real time using singleDNA manipulations. The relative efficiency of the two pathways canbe varied by external conditions. [ABSTRACT FROM AUTHOR]

Structural maintenance of chromosome (SMC) proteins comprise the core of several specialized complexes that stabilize the global architecture of the chromosomes by dynamically linking distant DNA fragments. This reaction however remains poorly understood giving rise to numerous proposed mechanisms of the proteins. Using two novel assays, we investigated real-time formation of DNA bridges by bacterial condensin MukBEF. We report that MukBEF can efficiently bridge two DNAs and that this reaction involves multiple steps. The reaction begins with the formation of a stable MukB–DNA complex, which can further capture another protein-free DNA fragment. The initial tether is unstable but is quickly strengthened by additional MukBs. DNA bridging is modulated but is not strictly dependent on ATP and MukEF. The reaction revealed high preference for right-handed DNA crossings indicating that bridging involves physical association of MukB with both DNAs. Our data establish a comprehensive view of DNA bridging by MukBEF, which could explain how SMCs establish both intra- and interchromosomal links inside the cell and indicate that DNA binding and bridging could be separately regulated. [ABSTRACT FROM AUTHOR]

MukB is a bacterial SMC (structural maintenance of chromosome) protein required for faithful chromosome segregation in Escherichia coli. We report here that purified MukB introduces right-handed knots into DNA in the presence of type-2 topoisomerase, indicating that the protein promotes intramolecular DNA condensation. The pattern of generated knots suggests that MukB, similar to eukaryotic condensins, stabilizes large right-handed DNA loops. In contrast to eukaryotic condensins, however, the net super-coiling stabilized by MukB was negative. Furthermore, DNA reshaping by MukB did not require ATP. These data establish that bacterial condensins alter the shape of double-stranded DNA in vitro and lend support to the notions that the right-handed knotting is the most conserved biochemical property of condensins. Finally, we found that MukB can be eluted from a heparin column in two distinct forms, one of which is inert in DNA binding or reshaping. Furthermore, we find that the activity of MukB is reversibly attenuated during chromatographic separation. Thus, MukB has a unique set of topological properties, compared with other SMC proteins, and is likely to exist in two different conformations. [ABSTRACT FROM AUTHOR]

MukB is a bacterial SMC(structural maintenance of chromosome) protein required for correct folding of the Escherichia coli chromosome. MukB acts in complex with the two non-SMC proteins, MukE and MukF. The role of MukEF is unclear. MukEF disrupts MukB-DNA interactions in vitro. In vivo, however, MukEF stimulates MukB-induced DNA condensation and is required for the assembly of MukB clusters at the quarter positions of the cell length. We report here that MukEF is essential for stable association of MukB with the chromosome. We found that MukBEF forms a stable complex with the chromosome that copurifies with nucleoids following gentle cell lysis. Little MukB could be found with the nucleoids in the absence or upon overproduction of MukEF. Similarly, overproduced MukEF recruited MukB-green fluorescent protein (GFP) from its quarter positions, indicating that formation of MukB-GFP clusters and stable association with the chromosome could be mechanistically related. Finally, we report that MukE-GFP forms foci at the quarter positions of the cell length but not in cells that lack MukB or overproduce MukEF, suggesting that the clusters are formed by MukBEF and not by its individual subunits. These data support the view that MukBEF acts as a macromolecular assembly, a scaffold, in chromosome organization and that MukEF is essential for the assembly of this scaffold. [ABSTRACT FROM AUTHOR]

MukBEF is a bacterial SMC (structural maintenance of chromosome) complex required for chromosome partitioning in Escherichia coli. We report that overproduction of MukBEF results in marked chromosome condensation. This condensation is rapid and precedes the effects of overproduction on macromolecular synthesis. Condensed nucleoids are often mispositioned; however, cell viability is only mildly affected. The overproduction of MukB leads to a similar chromosome condensation, even in the absence of MukE and MukF. Thus, the non-SMC subunits of MukBEF play only an auxiliary role in chromosome condensation. MukBEF, however, was often a better condensin than MukB. Furthermore, the chromosome condensation by MukB did not rescue the temperature sensitivity of MukEF-deficient cells, nor did it suppress the high frequency of anucleate cell formation. We infer that the role of MukBEF in stabilizing chromatin architecture is more versatile than its role in controlling chromosome size. We further propose that MukBEF could be directly involved in chromosome segregation. [ABSTRACT FROM AUTHOR]