A method for targeting a specified segment of DNA to a bacterial microorganelle.

Encapsulation of a selected DNA molecule in a cell has important implications for bionanotechnology. Non-viral proteins that can be used as nucleic acid containers include proteinaceous subcellular bacterial microcompartments (MCPs) that self-assemble into a selectively permeable protein shell containing an enzymatic core. Here, we adapted a propanediol utilization (Pdu) MCP into a synthetic protein cage to package a specified DNA segment in vivo, thereby enabling subsequent affinity purification. To this end, we engineered the LacI transcription repressor to be routed, together with target DNA, into the lumen of a Strep-tagged Pdu shell. Sequencing of extracted DNA from the affinity-isolated MCPs shows that our strategy results in packaging of a DNA segment carrying multiple LacI binding sites, but not the flanking regions. Furthermore, we used LacI to drive the encapsulation of a DNA segment containing operators for LacI and for a second transcription factor.

The DNA Damage Inducible SOS Response Is a Key Player in the Generation of Bacterial Persister Cells and Population Wide Tolerance.

Population-wide tolerance and persisters enable susceptible bacterial cells to endure hostile environments, including antimicrobial exposure. The SOS response can play a significant role in the generation of persister cells, population-wide tolerance, and shielding. The SOS pathway is an inducible DNA damage repair system that is also pivotal for bacterial adaptation, pathogenesis, and diversification. In addition to the two key SOS regulators, LexA and RecA, some other stressors and stress responses can control SOS factors. Bacteria are exposed to DNA-damaging agents and other environmental and intracellular factors, including cigarette smoke, that trigger the SOS response at a number of sites within the host. The Escherichia coli TisB/IstR module is as yet the only known SOS-regulated toxin-antitoxin module involved in persister formation. Nevertheless, the SOS response plays a key role in the formation of biofilms that are highly recalcitrant to antimicrobials and can be abundant in persisters. Furthermore, the dynamic biofilm environment generates DNA-damaging factors that trigger the SOS response within the biofilm, fueling bacterial adaptation and diversification. This review highlights the SOS response in relation to antimicrobial recalcitrance to antimicrobials in four clinically significant species, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium tuberculosis.

The Uropathogenic Specific Protein Gene usp from Escherichia coli and Salmonella bongori is a Novel Member of the TyrR and H-NS Regulons.

The Escherichia coli PAIusp is a small pathogenicity island encoding usp, for the uropathogenic specific protein (Usp), a genotoxin and three associated downstream imu1-3 genes that protect the producer against its own toxin. Bioinformatic analysis revealed the presence of the PAIusp also in publically available Salmonella bongori and Salmonella enterica subps. salamae genome sequences. PAIusp is in all examined sequences integrated within the aroP-pdhR chromosomal intergenic region. The focus of this work was identification of the usp promoter and regulatory elements controlling its activity. We show that, in both E. coli and S. bongori, the divergent TyrR regulated P3 promoter of the aroP gene, encoding an aromatic amino acid membrane transporter, drives usp transcription while H-NS acts antagonistically repressing expression. Our results show that the horizontally acquired PAIusp has integrated into the TyrR regulatory network and that environmental factors such as aromatic amino acids, temperature and urea induce usp expression.

The Escherichia coli colibactin resistance protein ClbS is a novel DNA binding protein that protects DNA from nucleolytic degradation.

Cells employ specific and nonspecific mechanisms to protect their genome integrity against exogenous and endogenous factors. The clbS gene is part of the polyketide synthase machinery (pks genomic island) encoding colibactin, a genotoxin implicated in promoting colorectal cancer. The pks is found among the Enterobacteriaceae, in particular Escherichia coli strains of the B2 phylogenetic group. Several resistance mechanisms protect toxin producers against toxicity of their products. ClbS, a cyclopropane hydrolase, was shown to confer colibactin resistance by opening its electrophilic cyclopropane ring. Here we report that ClbS sustained viability and enabled growth also of E. coli expressing another genotoxin, the Usp nuclease. The recA::gfp reporter system showed that ClbS protects against Usp induced DNA damage. To elucidate the mechanism of ClbS mediated protection, we studied the DNA binding ability of the ClbS protein. We show that ClbS directly interacts with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), whereas ssDNA seems to be the preferred substrate. Thus, the ClbS DNA-binding characteristics may serve bacteria to protect their genomes against DNA degradation.

Lytic gene expression in the temperate bacteriophage GIL01 is activated by a phage-encoded LexA homologue.

The GIL01 bacteriophage is a temperate phage that infects the insect pathogen Bacillus thuringiensis. During the lytic cycle, phage gene transcription is initiated from three promoters: P1 and P2, which control the expression of the early phage genes involved in genome replication and P3, which controls the expression of the late genes responsible for virion maturation and host lysis. Unlike most temperate phages, GIL01 lysogeny is not maintained by a dedicated phage repressor but rather by the host's regulator of the SOS response, LexA. Previously we showed that the lytic cycle was induced by DNA damage and that LexA, in conjunction with phage-encoded protein gp7, repressed P1. Here we examine the lytic/lysogenic switch in more detail and show that P3 is also repressed by a LexA-gp7 complex, binding to tandem LexA boxes within the promoter. We also demonstrate that expression from P3 is considerably delayed after DNA damage, requiring the phage-encoded DNA binding protein, gp6. Surprisingly, gp6 is homologous to LexA itself and, thus, is a rare example of a LexA homologue directly activating transcription. We propose that the interplay between these two LexA family members, with opposing functions, ensures the timely expression of GIL01 phage late genes.

A Cytolethal Distending Toxin Variant from Aggregatibacter actinomycetemcomitans with an Aberrant CdtB That Lacks the Conserved Catalytic Histidine 160.

The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtBΔ116-188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate secretes CdtBΔ116-188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained.

Antibiotic induced bacterial lysis provides a reservoir of persisters.

In a genetically uniform bacterial population a small subset of antibiotic-susceptible cells enter an antibiotic tolerant state and are hence referred to as persisters. These have been proposed to be rare phenotypic variants with several stochastically activated independent parallel processes. Here we show an overlooked phenomenon, bacterial tolerance of extraordinary high levels of ampicillin due to encasement of viable cells by an antibiotic induced network of cell debris. This matrix shields the entrapped cells from contact with the bacteriolytic ß-lactam antibiotic ampicillin and may be an underlying cause of notable variations in the level of ampicillin tolerant persisters as well as of considerable medical significance. Disruption of the matrix leads to the rapid elimination of hidden survivors, revealing their metabolically active state.

Recombinant expression and predicted structure of parborlysin, a cytolytic protein from the Antarctic heteronemertine Parborlasia corrugatus.

The heteronemertine Parborlasia corrugatus contains a cytolytic protein, parborlysin, which after extensive purification was found by Edman sequencing to be a mixture of several homologues. To investigate this microheterogeneity and enable the analysis of single toxins, we have obtained seven parborlysin isoform genes from P. corrugatus collected in Antarctica. Total RNA was isolated from the homogenized head region and parborlysin genes were identified from a cDNA library using degenerate primers. The translated sequences reveal that the isoforms are ∼ 10 kDa basic (pI ∼ 10) proteins of which all but one harbour six cysteine residues. We generated a model of the three dimensional structure of parborlysins, which suggests that they are composed of five alpha-helical segments that include large, exposed hydrophobic surfaces. Finally, we constructed plasmids and inserted them into Escherichia coli to obtain overexpressed amino- or carboxy-terminal polyhistidine-tagged parborlysin isoforms fused to the third domain of the E. coli periplasmic-protein TolA to facilitate toxin isolation. One of the isoforms adversely affected growth in the E. coli expressing it. Although we succeeded in isolating one of the recombinant parborlysin constructs, it lacked haemolytic activity.

Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8), after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has "chosen" highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.

The Escherichia coli uropathogenic-specific-protein-associated immunity protein 3 (Imu3) has nucleic acid -binding activity.

BACKGROUND: The Escherichia coli uropathogenic-specific protein (Usp) is a bacteriocin-like genotoxin, active against mammalian cells and associated with E. coli strains that provoke pyelonephritis, prostatitis and bacteraemia. Usp is encoded by a small pathogenicity island with three downstream small open reading frames (Imu1-3) that are believed to provide immunity to the producer. To prevent host suicide, colicins, bacteriocins of E. coli, form tight complexes with their cognate immunity proteins. Colicin - immunity protein complexes are among the strongest protein complexes known. Here, the Usp associated immunity protein 3 (Imu3) was partially characterized to gain insight into its role and mechanism of activity. RESULTS: Isolation and partial characterisation of the Usp-associated immunity protein-3 (Imu3) revealed that, while Usp and Imu3 do not form a high affinity complex, Imu3 exhibits DNA and RNA binding activity. Imu3 was also shown to protect DNA against degradation by colicin E7. CONCLUSIONS: Our data infer that nonspecific DNA binding of the Imu3 immunity protein, prevents suicide of E. coli producing the genotoxin Usp.

Escherichia coli uropathogenic-specific protein, Usp, is a bacteriocin-like genotoxin.

BACKGROUND: Bacterial genotoxins provoke DNA damage and carcinogenesis. The Escherichia coli uropathogenic-specific protein gene, usp, and its linked genes, imu1-3, are associated with strains from pyelonephritis, prostatitis, and bacteremia of urinary tract origin. While the Usp C-terminal domain exhibits similarity with DNase-like colicins and pyocins, its role and mechanisms of action, as well as those of the 3 associated proteins, is unknown. METHODS: We isolated Usp and Imu1-3 and examined their activity on plasmid DNA, human umbilical vein endothelial cells, and human embryonic kidney cells (cell line HEK293). The affect of Usp and Imu1-3 was assessed by MTT and Comet assays, infection assays, caspase 3/7 activity, fluorescently labeled actin staining, and Western blotting. RESULTS: Usp possesses DNase activity and, particularly when coapplied with Imu2, exhibits genotoxic activity in mammalian cells. Infection assays demonstrated that E. coli usp(+) imu1-3(+) affects the viability of mammalian cells, induces increased caspase 3/7 activity, and perturbs cell cytoskeleton structure. CONCLUSIONS: Usp is a novel E. coli genotoxin active against mammalian cells. Optimal in vivo activity of Usp requires Imu2. Infection with E. coli usp(+) imu1-3(+) induces a response characteristic of apoptosis.

Structural insight into LexA-RecA* interaction.

RecA protein is a hallmark for the bacterial response to insults inflicted on DNA. It catalyzes the strand exchange step of homologous recombination and stimulates self-inactivation of the LexA transcriptional repressor. Importantly, by these activities, RecA contributes to the antibiotic resistance of bacteria. An original way to decrease the acquisition of antibiotic resistance would be to block RecA association with LexA. To engineer inhibitors of LexA-RecA complex formation, we have mapped the interaction area between LexA and active RecA-ssDNA filament (RecA*) and generated a three-dimensional model of the complex. The model revealed that one subunit of the LexA dimer wedges into a deep helical groove of RecA*, forming multiple interaction sites along seven consecutive RecA protomers. Based on the model, we predicted that LexA in its DNA-binding conformation also forms a complex with RecA* and that the operator DNA sterically precludes interaction with RecA*, which guides the induction of SOS gene expression. Moreover, the model shows that besides the catalytic C-terminal domain of LexA, its N-terminal DNA-binding domain also interacts with RecA*. Because all the model-based predictions have been confirmed experimentally, the presented model offers a validated insight into the critical step of the bacterial DNA damage response.

Interactions of archaeal chromatin proteins Alba1 and Alba2 with nucleic acids.

BACKGROUND: Architectural proteins have important roles in compacting and organising chromosomal DNA. There are two potential histone counterpart peptide sequences (Alba1 and Alba2) in the Aeropyrum pernix genome (APE1832.1 and APE1823). METHODOLOGY/PRINCIPAL FINDINGS: THESE TWO PEPTIDES WERE EXPRESSED AND THEIR INTERACTIONS WITH VARIOUS DNAS WERE STUDIED USING A COMBINATION OF VARIOUS EXPERIMENTAL TECHNIQUES: surface plasmon resonance, UV spectrophotometry, circular dichroism-spectropolarimetry, gel-shift assays, and isothermal titration calorimetry. CONCLUSIONS/SIGNIFICANCE: Our data indicate that there are significant differences in the properties of the Alba1 and Alba2 proteins. Both of these Alba proteins can thermally stabilise DNA polynucleotides, as seen from UV melting curves. Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT). Surface plasmon resonance sensorgrams for binding of Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 to DNA oligonucleotides show different binding patterns. Circular dichroism indicates that Alba2 has a less-ordered secondary structure than Alba1. The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures. Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs.

Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response.

The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations.

Genes regulated by the Escherichia coli SOS repressor LexA exhibit heterogeneous expression.

BACKGROUND: Phenotypic heterogeneity may ensure that a small fraction of a population survives environmental perturbations or may result in lysis in a subpopulation, to increase the survival of siblings. Genes involved in DNA repair and population dynamics play key roles in rapid responses to environmental conditions. In Escherichia coli the transcriptional repressor LexA controls a coordinated cellular response to DNA damage designated the SOS response. Expression of LexA regulated genes, e.g. colicin encoding genes, recA, lexA and umuDC, was examined utilizing transcription fusions with the promoterless gfp at the single cell level. RESULTS: The investigated LexA regulated genes exhibited heterogeneity, as only in a small fraction of the population more intense fluorescence was observed. Unlike recA and lexA, the pore forming and nuclease colicin activity genes as well as umuDC, exhibited no basal level activity. However, in a lexA defective strain high level expression of the gene fusions was observed in the large majority of the cells. All of the investigated genes were expressed in a recA defective strain, albeit at lower levels, revealing expression in the absence of a spontaneous SOS response. In addition, the simultaneous expression of cka, encoding the pore forming colicin K, and lexA, investigated at the single cell level revealed high level expression of only cka in rare individual cells. CONCLUSION: LexA regulated genes exhibit phenotypic heterogeneity as high level expression is observed in only a small subpopulation of cells. Heterogeneous expression is established primarily by stochastic factors and the binding affinity of LexA to SOS boxes.

Revealing fosfomycin primary effect on Staphylococcus aureus transcriptome: modulation of cell envelope biosynthesis and phosphoenolpyruvate induced starvation.

BACKGROUND: Staphylococcus aureus is a highly adaptable human pathogen and there is a constant search for effective antibiotics. Fosfomycin is a potent irreversible inhibitor of MurA, an enolpyruvyl transferase that uses phosphoenolpyruvate as substrate. The goal of this study was to identify the pathways and processes primarily affected by fosfomycin at the genome-wide transcriptome level to aid development of new drugs. RESULTS: S. aureus ATCC 29213 cells were treated with sub-MIC concentrations of fosfomycin and harvested at 10, 20 and 40 minutes after treatment. S. aureus GeneChip statistical data analysis was complemented by gene set enrichment analysis. A visualization tool for mapping gene expression data into biological pathways was developed in order to identify the metabolic processes affected by fosfomycin. We have shown that the number of significantly differentially expressed genes in treated cultures increased with time and with increasing fosfomycin concentration. The target pathway - peptidoglycan biosynthesis - was upregulated following fosfomycin treatment. Modulation of transport processes, cofactor biosynthesis, energy metabolism and nucleic acid biosynthesis was also observed. CONCLUSIONS: Several pathways and genes downregulated by fosfomycin have been identified, in contrast to previously described cell wall active antibiotics, and was explained by starvation response induced by phosphoenolpyruvate accumulation. Transcriptomic profiling, in combination with meta-analysis, has been shown to be a valuable tool in determining bacterial response to a specific antibiotic.

Human perforin permeabilizing activity, but not binding to lipid membranes, is affected by pH.

The various steps that perforin (PFN), a critical mediator of innate immune response, undertakes to form a transmembrane pore remains poorly understood. We have used surface plasmon resonance (SPR) to dissect mechanism of pore formation. The membrane association of PFN was calcium dependent irrespective of pH. However, PFN does not permeabilize large or giant unilamellar vesicles (GUV) at pH 5.5 even though the monomers bind to the membranes in the presence of calcium. It was possible to activate adsorbed PFN and to induce membrane permeabilization by simply raising pH to a physiological level (pH 7.4). These results were independently confirmed on GUV and Jurkat cells. The conformational state of PFN at either pH was further assessed with monoclonal antibodies Pf-80 and Pf-344. Pf-344 maps to a linear epitope within region 373-388 of epidermal growth factor (EGF)-like domain while the Pf-80 appears to recognize a conformational epitope. Pf-344 interacts with the EGF-like domain after PFN monomers undergo pore formation, the site recognized by Pf-80 is only accessible at acidic but not neutral pH. Thus, the Pf-80 mAb likely interacts with a region of the monomer that participates in oligomerization prior to insertion of the monomer into the lipid bilayer and thus may have therapeutic utility against PFN-mediated immunopathology.

The SOS response affects thermoregulation of colicin K synthesis.

Temperature is one of the key environmental parameters affecting bacterial gene expression. This study investigated the effect of temperature on synthesis of Escherichia coli colicins E1, K, N and E7 as well as the molecular basis underlying thermoregulation of the colicin K activity gene cka. The results of our study show that synthesis of the investigated colicins is higher at 37 degrees C than at 22 degrees C and that temperature regulates cka expression at the level of transcription. We propose that the SOS response indirectly regulates thermoregulation of colicin K (and possibly of the other examined colicins). Two LexA dimers bind cooperatively with high affinity to the two overlapping LexA boxes in a temperature-independent manner. At 22 degrees C the relative degree of repression is higher as a result of less LexA cleavage due to a slower growth rate, while at 37 degrees C the extent of LexA cleavage is higher due to a higher growth rate. Thermoregulation of colicin synthesis is an additional example of the connection between the SOS regulon and cell physiology.

Molecular determinants of sphingomyelin specificity of a eukaryotic pore-forming toxin.

Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp(112) and Tyr(113) exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins.

Intradomain LexA rotation is a prerequisite for DNA binding specificity.

In the absence of DNA damage the LexA protein represses the bacterial SOS system. We performed molecular dynamic simulations of two LexA dimers bound to operators. Our model predicted that rotation of the LexA DNA binding domain, with respect to the dimerised C-terminal domain, is required for selective DNA binding. To confirm the model, double and quadruple cysteine LexA mutants were engineered. Electrophoretic mobility-shift assay and surface plasmon resonance showed that disulfide bond formation between the introduced cysteine residues precluded LexA specific DNA binding due to blocked domain reorientation. Our model could provide the basis for novel drug design.