Lac Repressor Research Articles

How do proteins locate specific targets in DNA?

Many aspects of biology depend on the ability of DNA-binding proteins to locate specific binding sites within the genome. Interest in this target search problem has been reinvigorated through the recent development of microscopy-based technologies capable of tracking individual proteins in real-time as they search for binding sites. In this review we discuss how two different proteins, lac repressor and RNA polymerase, have solved the target search problem through seemingly different mechanisms, with an emphasis on how recent in vitro single-molecule studies have influenced our understanding of these reactions.

Structural insights into the role of architectural proteins in DNA looping deduced from computer simulations.

Bacterial gene expression is regulated by DNA elements that often lie far apart along the genomic sequence, but come close together during genetic processing. The intervening residues form loops, which are organized by the binding of various proteins. For example, the Escherichia coli Lac repressor protein binds DNA operators, separated by 92 or 401bp, and suppresses the formation of gene products involved in the metabolism of lactose. The system also includes several highly abundant architectural proteins, such as the histone-like (heat-unstable) HU protein, which severely deform the double helix upon binding. In order to gain a better understanding of how the naturally stiff DNA double helix forms the short loops detected invivo, we have developed new computational methods to study the effects of various non-specific binding proteins on the three-dimensional configurational properties of DNA sequences. The present article surveys the approach that we use to generate ensembles of spatially constrained protein-decorated DNA structures (minicircles and Lac repressor-mediated loops) and presents some of the insights gained from the correspondence between computation and experiment about the potential contributions of architectural and regulatory proteins to DNA looping and gene expression.

Kinetic approaches to lactose operon induction and bimodality

The quasi-equilibrium approximation is acceptable when molecular interactions are fast enough compared to circuit dynamics, but is no longer allowed when cellular activities are governed by rare events. A typical example is the lactose operon (lac), one of the most famous paradigms of transcription regulation, for which several theories still coexist to describe its behaviors. The lac system is generally analyzed by using equilibrium constants, contradicting single-event hypotheses long suggested by Novick and Weiner (1957). Enzyme induction as an all-or-none phenomenon. Proc. Natl. Acad. Sci. USA 43, 553–566) and recently refined in the study of (Choi et al., 2008. A stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science 322, 442–446). In the present report, a lac repressor (LacI)-mediated DNA immunoprecipitation experiment reveals that the natural LacI-lac DNA complex built in vivo is extremely tight and long-lived compared to the time scale of lac expression dynamics, which could functionally disconnect the abortive expression bursts and forbid using the standard modes of lac bistability. As alternatives, purely kinetic mechanisms are examined for their capacity to restrict induction through: (i) widely scattered derepression related to the arrival time variance of a predominantly backward asymmetric random walk and (ii) an induction threshold arising in a single window of derepression without recourse to nonlinear multimeric binding and Hill functions. Considering the complete disengagement of the lac repressor from the lac promoter as the probabilistic consequence of a transient stepwise mechanism, is sufficient to explain the sigmoidal lac responses as functions of time and of inducer concentration. This sigmoidal shape can be misleadingly interpreted as a phenomenon of equilibrium cooperativity classically used to explain bistability, but which has been reported to be weak in this system.

DNA-recognition process described by MD simulations of the lactose repressor protein on a specific and a non-specific DNA sequence

The lactose repressor protein may bind DNA in two possible configurations: a specific one, if the DNA sequence corresponds to a binding site, and a non-specific one otherwise. To find its target sequences, the lactose repressor first binds non-specifically to DNA, and subsequently, it rapidly searches for a binding site. Atomic structures of non-specific and specific complexes are available from crystallographic and nuclear magnetic resonance experiments. However, what remains unknown is a detailed description of the steps that transform the non-specific complex into the specific one. Here, how the protein first recognizes its binding site has been studied using molecular dynamics simulations. The picture that emerges is that of a protein that is as mobile when interacting with non-specific DNA sequences as when free in solution. This high degree of mobility allows the protein to rapidly sample different DNA sequences. In contrast, when the protein encounters a binding site, the configuration ensemble collapses, and the protein sliding movements along the DNA sequence become scarce. The binding energies in the specific and non-specific complexes were analysed using the Molecular Mechanics Poisson Boltzmann Surface Area approach. These results represent a first step towards a throughout characterization of the DNA-recognition process.

Interplay of Protein and DNA Structure Revealed in Simulations of the lac Operon

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

DNA looping increases the range of bistability in a stochastic model of the lac genetic switch

Conditions and parameters affecting the range of bistability of the lac genetic switch in Escherichia coli are examined for a model which includes DNA looping interactions with the lac repressor and a lactose analogue. This stochastic gene–mRNA–protein model of the lac switch describes DNA looping using a third transcriptional state. We exploit the fast bursting dynamics of mRNA by combining a novel geometric burst extension with the finite state projection method. This limits the number of protein/mRNA states, allowing for an accelerated search of the model's parameter space. We evaluate how the addition of the third state changes the bistability properties of the model and find a critical region of parameter space where the phenotypic switching occurs in a range seen in single molecule fluorescence studies. Stochastic simulations show induction in the looping model is preceded by a rare complete dissociation of the loop followed by an immediate burst of mRNA rather than a slower build up of mRNA as in the two-state model. The overall effect of the looped state is to allow for faster switching times while at the same time further differentiating the uninduced and induced phenotypes. Furthermore, the kinetic parameters are consistent with free energies derived from thermodynamic studies suggesting that this minimal model of DNA looping could have a broader range of application.

LacI-DNA-IPTG Loops: Equilibria among Conformations by Single-Molecule FRET

The E. coli Lac repressor (LacI) tetramer binds simultaneously to a promoter-proximal DNA binding site (operator) and an auxiliary operator, resulting in a DNA loop, which increases repression efficiency. Induction of the lac operon by allolactose reduces the affinity of LacI for DNA, but induction does not completely prevent looping in vivo. Our previous work on the conformations of LacI loops used a hyperstable model DNA construct, 9C14, that contains a sequence directed bend flanked by operators. Single-molecule fluorescence resonance energy transfer (SM-FRET) on a dual fluorophore-labeled LacI-9C14 loop showed that it adopts a single, stable, high-FRET V-shaped LacI conformation. Ligand-induced changes in loop geometry can affect loop stability, and the current work assesses loop population distributions for LacI-9C14 complexes containing the synthetic inducer IPTG. SM-FRET confirms that the high-FRET LacI-9C14 loop is only partially destabilized by saturating IPTG. LacI titration experiments and FRET fluctuation analysis suggest that the addition of IPTG induces loop conformational dynamics and re-equilibration between loop population distributions that include a mixture of looped states that do not exhibit high-efficiency FRET. The results show that repression by looping even at saturating IPTG should be considered in models for regulation of the operon. We propose that persistent DNA loops near the operator function biologically to accelerate rerepression upon exhaustion of inducer.

In Vivo Facilitated Diffusion Model

Under dilute in vitro conditions transcription factors rapidly locate their target sequence on DNA by using the facilitated diffusion mechanism. However, whether this strategy of alternating between three-dimensional bulk diffusion and one-dimensional sliding along the DNA contour is still beneficial in the crowded interior of cells is highly disputed. Here we use a simple model for the bacterial genome inside the cell and present a semi-analytical model for the in vivo target search of transcription factors within the facilitated diffusion framework. Without having to resort to extensive simulations we determine the mean search time of a lac repressor in a living E. coli cell by including parameters deduced from experimental measurements. The results agree very well with experimental findings, and thus the facilitated diffusion picture emerges as a quantitative approach to gene regulation in living bacteria cells. Furthermore we see that the search time is not very sensitive to the parameters characterizing the DNA configuration and that the cell seems to operate very close to optimal conditions for target localization. Local searches as implied by the colocalization mechanism are only found to mildly accelerate the mean search time within our model.

Single Molecule Tracking of Lac Repressor Diffusing on Stretched DNA

Lac Repressor (LacI) is a DNA-binding protein that regulates genes expression by specific binding to its target sequence on DNA. This sequence is found rapidly among millions of base pairs in the genome, presumably via a combination of 3D and 1D-diffusion (following the interaction with non-specific DNA). Here we report diffusion analysis through the combination of single molecule localization and manipulation. For single molecule localization, Atto532 dye has been covalently linked to a single-cysteine mutant of LacI, LacIQ231C. A double optical tweezers system is used for trapping and stretching a single DNA molecule. All components of the experiment (protein, DNA, beads and buffer) are assembled (and/or rapidly exchanged) with a flow-system. This integrated configuration has several advantages: biomolecules are not in the proximity of the glass surface thus preventing possible electrostatic effects; controlled forces can be measured or applied. We characterized LacI 1D-diffusion under different forces applied to the DNA. The figure shows an example of the data obtained in the form of a kymogram.Acknowledgments:The research leading to the results has received funding from Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAXView Large Image | View Hi-Res Image | Download PowerPoint Slide

Lactose Repressor Functions as a DNA Topological Barrier in Escherichia Coli Lactose Operon

Escherichia coli lac repressor (LacI) is a paradigm transcriptional factor that controls the expression of three genes in the lac operon. It is a tetrameric protein, specifically binds to lac operators, such as O1, O2 and O3, and forms a DNA loop to negatively control transcription initiation. Previously, we found that LacI upon binding to multiple lacO1 operators is capable of acting as a DNA topological barrier to block DNA supercoil diffusion and dividing a supercoiled DNA molecule into two independent topological domains (Leng et al. (2011) Proc Natl Acad Sci USA 108: 19973-78). In this study, we showed that LacI is able to function as a topological barrier and block supercoil diffusion upon binding to O1, O2, and O3 operators in the lac operon. Specifically, we demonstrated that LacI kept two supercoils within the 401 bp DNA-loop between O1 and O2 operators. Additionally, We carried out time course studies to determine the stability of the topological barriers that are produced by the different LacI-operator complexes. Our results showed that the stability of the topological barriers correlates with the DNA-binding affinity of LacI to the different operators i.e., O1, O2, O3, and Os operators. Furthermore, we confirmed our previous observation in which LacI is able to “keep” certain superhelical energy to stabilize LacI-lacO1 complexes. Our results can be explained by a model in which LacI behaves as a topological barrier in the lac operon to regulate the expression of lacZYA genes in Escherichia coli cells.

A Binary Molecular Gate Made of DNA and Protein

Many proteins have evolved the ability to recognize and bind to specific sequences of DNA. This specificity is quite high and often is the basis for genetic switches which can repress or induce transcription in cells. One of the best known DNA-binding proteins is the lac repressor protein which binds tightly to the lac operator but loses its affinity for this site upon the binding of lactose. We have used this lac repressor protein and the lac operator sequence to construct a binary molecular gate which can be controlled by the effector molecule lactose. The device is constructed of three DNA strands which form an elongated hairpin structure of two dsDNA arms with a short ssDNA hinge at one end, and two ssDNA regions on the opposite end. A fourth ssDNA molecule (the 'locking' strand), complementary to the two ssDNA regions, binds by complementary base pairing to seal the gate in the closed configuration. Addition of lac repressor protein to the device causes a displacement of the locking strand by binding to two lac operators embedded in the double-stranded hairpin structure. Binding of the proteins is rapid and sufficiently strong to cause a displacement of the locking strand, switching the gate into the open conformation. Addition of lactose quickly causes a release of the lac repressor protein from the operators allowing the gate to be closed by the binding of the locking strand. The opening and closing of this binary gate was detected by gel shift assays and by fluorescence resonance energy transfer spectroscopy of two dyes located near the opening of this gate. The gate could be cycled repeatedly, responded uniquely to lactose, and may be useful as a device for moving or holding structures on a molecular scale.

Flanking DNA Matters in DNA Loop Formation and Breakdown Mediated by Lac Repressor Protein

The lac Repressor protein (LacI) is a paradigm for the study of protein-mediated DNA loops in bacteria. When it binds to two distant operator sites on substrate DNA, it causes the formation of DNA loops. Although past in vitro and in vivo experiments have shown that changes in the inter-loop DNA can affect loop formation and breakdown, here I will present in vitro experimental evidence that the DNA outside the loop that is flanking the operators can also affect looping kinetics. Observing loop formation and breakdown in single molecules with Tethered Particle Motion (TPM) we found fluctuations in how much DNA is bound to the protein, and these fluctuations depend on the AT- or GC-content of the flanking regions outside the loop. This suggests an interaction between the looped complex and the flanking region which has not been previously observed. The studies complement previous experiments which found that DNA between the operator sites interacts with LacI while in the looped state. These results suggest that the presence or absence of flanking DNA interactions with the protein complex could give rise to additional states in TPM experiments that are not caused by conformational changes of the protein complex or topologic variations of the loop.

Gene Location and DNA Density Determine Transcription Factor Distributions in E. Coli

The diffusion coefficient of the canonical transcription factor Lac Repressor, LacI, within living Escherichia coli has been measured directly by in vivo tracking to be D = 0.4 μm∧2/s. At this rate, simple models of diffusion lead to the expectation that LacI and other proteins will rapidly homogenize throughout the cell.We have tested this expectation of spatial homogeneity by high-throughput single molecule visualization of LacI molecules non-specifically bound to DNA in fixed cells to generate an ensemble average of the steady-state distribution of protein in the cell. Contrary to expectation, we find that the distribution of LacI depends on the spatial location of its encoding gene. We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid. We then show that LacI inhomogeneity increases the strength with which targets located proximally to the LacI gene are regulated. Finally, we propose a model for intranucleoid diffusion which can reconcile these results with previous measurements of LacI diffusion.

DNA Competition Experiments Reveal the Importance of Operator Binding Strength and Inter-Operator Sequence in Protein-Mediated DNA Looping

DNA looping mediated by the lac Repressor protein (LacI) is a paradigm of study for protein-DNA interaction as well as DNA mechanics on the nanoscale. Loop formation in this system occurs by the spontaneous binding of LacI to two distant operators on its DNA substrate. We employ single-molecule Tethered Particle Motion (TPM) to observe loop formation and breakdown in various DNA substrates. We are discussing and aiming to explain substantial differences between loop lifetime measurements in bulk and single-molecule experiments, namely an unexpected difficulty in competing bound protein off in the presence of excess DNA even though loops continued to form and break down rapidly. For this aim, we conduct single-molecule DNA competition experiments using DNAs with different operator strengths and intra-operator sequences giving rise to intrinsic bends. We find that we are able to compete off LacI that is bound to unbent DNA constructs with non-ideal operators in a matter of minutes, in line with typical loop breakdown rates. In contrast, competing off LacI bound to unbent or intrinsically curved DNA substrates with ideal operators took at least hours up to days in both single-molecule and bulk experiments, even though loops continue to break down and form repeatedly within minutes. To explain this resistance to competition of the LacI-DNA complex in DNA substrates with ideal operators while loops continue to break down, we posit a weak binding of LacI to non-operator DNA in the unlooped state; this hypothesis is supported by recent complementary experiments that reveal interactions between LacI and non-operator DNA outside the looping region.

Determining the Elasticity of Short DNA Fragments using Optical Tweezers and Protein-Mediated DNA Loop Formation Assays

In the light of the recent controversy over whether DNA on short length scales becomes more elastic or not, we contribute two additional measurements that take sequence effects into account: We have directly stretched short DNA molecules of varying AT / CG content with optical tweezers to determine their persistence length. Scaling the result for known end-point entropic effects, we find a persistence length of 44±2nm and 59±3nm for the AT and CG rich constructs, respectively, but no appreciable overall softening, in line with our previous measurements that found no softening in DNA molecules as short as 250 bp. Secondly, we have used tethered particle motion (TPM) microscopy to observe protein-mediated DNA loop formation in the lactose repressor system with the same DNA constructs between the operators. While an absolute determination of the persistence length of the DNA from such kinetic measurements is notoriously difficult, as ongoing discussions of ring cyclization experiments by various groups suggest, we can unequivocally state that any sequence effect in these measurements is much smaller than what we found in the stretching experiments with the optical tweezers. This suggests two distinct response regimes of DNA to force: one for low curvatures commensurate with thermal forces, in which elastic-rod descriptions for the DNA hold and sequence effects are observed, and a second non-linear high curvature region in which elasticity is not sequence-dependent and likely dominated by the conformation of the phosphate backbone.

Lac Repressor-DNA Interactions assessed by Ultrafast Force-Clamp Spectroscopy

We recently developed an ultrafast force-clamp laser trap technique [Capitanio et al., Nature Methods 9,1013-1019(2012)] that allows probing, under controlled force, both long- and short-lived biomolecular interactions (100μs to tens/hundreds of seconds), as well as sub-nanometer conformational changes occurring upon bond formation. Here, we show the application of our method to the study of lactose repressor (LacI). Our results show two kinetically well-distinct populations of interactions, which clearly represent strong interactions (targeting the two operators located 100nm apart from each other: long events in the figure) and fast scanning of LacI along non-cognate DNA (during target-search: short events in the figure). Our results demonstrate the effectiveness of the method to study the sequence-dependent affinity of DNA-binding proteins along the DNA molecule and the effects of force on a wide range of interaction durations, including μs time scales not accessible to other methods. This improvement in time resolution provides also important means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational changes occurring upon target recognition.This research is funded by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies (1-3). These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres (4-8). Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell. Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest (9). The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed. This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase(10). The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes(11). In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

Dependence of transcription-coupled DNA supercoiling on promoter strength in Escherichia coli topoisomerase I deficient strains

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA in vitro and in vivo. This phenomenon has been nicely explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In Escherichia coli topA strains, DNA gyrase selectively converts the positively supercoiled domain into negative supercoils to produce hypernegatively supercoiled DNA. In this article, in order to examine whether promoter strength affects transcription-coupled DNA supercoiling (TCDS), we developed a two-plasmid system in which a linear, non-supercoiled plasmid was used to express lac repressor constitutively while a circular plasmid was used to gage TCDS in E. coli cells. Using this two-plasmid system, we found that TCDS in topA strains is dependent on promoter strength. We also demonstrated that transcription-coupled hypernegative supercoiling of plasmid DNA did not need the expression of a membrane-insertion protein for strong promoters; however, it might require co-transcriptional synthesis of a polypeptide. Furthermore, we found that for weak promoters the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. Our results can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA.

Mechanism of promoter repression by Lac repressor–DNA loops

The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repressor concentration come from free repressor and repressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap.

Mutation Frequency and Spectrum of Mutations Vary at Different Chromosomal Positions of Pseudomonas putida

It is still an open question whether mutation rate can vary across the bacterial chromosome. In this study, the occurrence of mutations within the same mutational target sequences at different chromosomal locations of Pseudomonas putida was monitored. For that purpose we constructed two mutation detection systems, one for monitoring the occurrence of a broad spectrum of mutations and transposition of IS element IS1411 inactivating LacI repressor, and another for detecting 1-bp deletions. Our results revealed that both the mutation frequency and the spectrum of mutations vary at different chromosomal positions. We observed higher mutation frequencies when the direction of transcription of the mutational target gene was opposite to the direction of replisome movement in the chromosome and vice versa, lower mutation frequency was accompanied with co-directional transcription and replication. Additionally, asymmetry of frameshift mutagenesis at homopolymeric and repetitive sequences during the leading and lagging-strand replication was found. The transposition frequency of IS1411 was also affected by the chromosomal location of the target site, which implies that regional differences in chromosomal topology may influence transposition of this mobile element. The occurrence of mutations in the P. putida chromosome was investigated both in growing and in stationary-phase bacteria. We found that the appearance of certain mutational hot spots is strongly affected by the chromosomal location of the mutational target sequence especially in growing bacteria. Also, artificial increasing transcription of the mutational target gene elevated the frequency of mutations in growing bacteria.