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We isolated phage Ec_Makalu_002, which infects uropathogenic strains of Escherichia coli. Here, we report its complete genome sequence, annotated features, and relatedness to other phages.

How genomes are organized within cells and how the 3D architecture of a genome influences cellular functions are significant questions in biology. A bacterial genomic DNA resides inside cells in a highly condensed and functionally organized form called nucleoid (nucleus-like structure without a nuclear membrane). The Escherichia coli chromosome or nucleoid is composed of the genomic DNA, RNA, and protein. The nucleoid forms by condensation and functional arrangement of a single chromosomal DNA with the help of chromosomal architectural proteins and RNA molecules as well as DNA supercoiling. Although a high-resolution structure of a bacterial nucleoid is yet to come, five decades of research has established the following salient features of the E. coli nucleoid elaborated below: 1) The chromosomal DNA is on the average a negatively supercoiled molecule that is folded as plectonemic loops, which are confined into many independent topological domains due to supercoiling diffusion barriers; 2) The loops spatially organize into megabase size regions called macrodomains, which are defined by more frequent physical interactions among DNA sites within the same macrodomain than between different macrodomains; 3) The condensed and spatially organized DNA takes the form of a helical ellipsoid radially confined in the cell; and 4) The DNA in the chromosome appears to have a condition-dependent 3-D structure that is linked to gene expression so that the nucleoid architecture and gene transcription are tightly interdependent, influencing each other reciprocally. Current advents of high-resolution microscopy, single-molecule analysis and molecular structure determination of the components are expected to reveal the total structure and function of the bacterial nucleoid.

Bacteriophage λ of Escherichia coli has two alternative life cycles after infection-host survival with lysogen formation, or host lysis and phage production. In a lysogen, CI represses the two lytic promoters, pR and pL, and activates its own transcription from the auto-regulated pRM promoter. During induction from the lysogenic to lytic state, CI is inactivated, and the two lytic promoters are de-repressed allowing for expression of Cro from pR. Cro is known to repress transcription of CI from pRM to prevent lysogeny. We show here that when Cro and CI are both present but at low levels, the low level of Cro initially stimulates the lytic promoters while CI repressor is still present, stimulating the level of Cro to a concentration required for pRM repression. Cro has no stimulatory effect without the presence of CI. We propose that this early auto-activating role of Cro at lower concentrations is essential in the developmental switch to lytic growth, whereas pRM repression by Cro at relatively higher concentrations avoids restoring lysogeny.

We sequenced a naturally competent bacterial isolate, WY10, cultured from a Wyoming soil sample. Sequence analysis revealed that WY10 is a novel strain of Bacillus simplex . To our knowledge, WY10 is the first B. simplex strain to be characterized as naturally competent for DNA uptake by transformation.

Repeated extragenic palindromes (REPs) in the enterobacterial genomes are usually composed of individual palindromic units separated by linker sequences. A total of 355 annotated REPs are distributed along the Escherichia coli genome. RNA sequence (RNAseq) analysis showed that almost 80% of the REPs in E. coli are transcribed. The DNA sequence of REP325 showed that it is a cluster of six repeats, each with two palindromic units capable of forming cruciform structures in supercoiled DNA. Here, we report that components of the REP325 element and at least one of its RNA products play a role in bacterial nucleoid DNA condensation. These RNA not only are present in the purified nucleoid but bind to the bacterial nucleoid-associated HU protein as revealed by RNA IP followed by microarray analysis (RIP-Chip) assays. Deletion of REP325 resulted in a dramatic increase of the nucleoid size as observed using transmission electron microscopy (TEM), and expression of one of the REP325 RNAs, nucleoid-associated noncoding RNA 4 (naRNA4), from a plasmid restored the wild-type condensed structure. Independently, chromosome conformation capture (3C) analysis demonstrated physical connections among various REP elements around the chromosome. These connections are dependent in some way upon the presence of HU and the REP325 element; deletion of HU genes and/or the REP325 element removed the connections. Finally, naRNA4 together with HU condensed DNA in vitro by connecting REP325 or other DNA sequences that contain cruciform structures in a pairwise manner as observed by atomic force microscopy (AFM). On the basis of our results, we propose molecular models to explain connections of remote cruciform structures mediated by HU and naRNA4., IMPORTANCE Nucleoid organization in bacteria is being studied extensively, and several models have been proposed. However, the molecular nature of the structural organization is not well understood. Here we characterized the role of a novel nucleoid-associated noncoding RNA, naRNA4, in nucleoid structures both in vivo and in vitro. We propose models to explain how naRNA4 together with nucleoid-associated protein HU connects remote DNA elements for nucleoid condensation. We present the first evidence of a noncoding RNA together with a nucleoid-associated protein directly condensing nucleoid DNA.

Bacterial antibiotic resistance is one of the foremost public health challenges of our time. Even as efforts to identify novel antibiotics and conserve existing drugs have gained new urgency, so too has the need to investigate less-conventional antibacterial strategies. One such approach is phage therapy, or the use of viruses that infect bacteria (bacteriophages) to kill bacterial pathogens. Phage Therapy: Current Research and Applications presents a timely and comprehensive account of phage therapy's biological and historical underpinnings, empirical support, and biomedical potential.

Sequence alignments of promoters in prokaryotes postulated that the frequency of occurrence of a base pair at a given position of promoter elements reflects its contribution to intrinsic promoter strength. We directly assessed the contribution of the four base pairs in each position in the intrinsic promoter strength by keeping the context constant in Escherichia coli cAMP-CRP (cAMP receptor protein) regulated gal promoters by in vitro transcription assays. First, we show that base pair frequency within known consensus elements correlates well with promoter strength. Second, we observe some substitutions upstream of the ex-10 TG motif that are important for promoter function. Although the galP1 and P2 promoters overlap, only three positions where substitutions inactivated both promoters were found. We propose that RNA polymerase binds to the -12T base pair as part of double-stranded DNA while opening base pairs from -11A to +3 to form the single-stranded transcription bubble DNA during isomerization. The cAMP-CRP complex rescued some deleterious substitutions in the promoter region. The base pair roles and their flexibilities reported here for E. coli gal promoters may help construction of synthetic promoters in gene circuitry experiments in which overlapping promoters with differential controls may be warranted.

Molecular mechanisms of bacterial chromosome packaging are still unclear, as bacteria lack nucleosomes or other apparent basic elements of DNA compaction. Among the factors facilitating DNA condensation may be a propensity of the DNA molecule for folding due to its intrinsic curvature. As suggested previously, the sequence correlations in genome reflect such a propensity [Trifonov and Sussman (1980) Proc. Natl Acad. Sci. USA, 77, 3816-3820]. To further elaborate this concept, we analyzed positioning of A-tracts (the sequence motifs introducing the most pronounced DNA curvature) in the Escherichia coli genome. First, we observed that the A-tracts are over-represented and distributed 'quasi-regularly' throughout the genome, including both the coding and intergenic sequences. Second, there is a 10-12 bp periodicity in the A-tract positioning indicating that the A-tracts are phased with respect to the DNA helical repeat. Third, the phased A-tracts are organized in approximately 100 bp long clusters. The latter feature was revealed with the help of a novel approach based on the Fourier series expansion of the A-tract distance autocorrelation function. Since the A-tracts introduce local bends of the DNA duplex and these bends accumulate when properly phased, the observed clusters would facilitate DNA looping. Also, such clusters may serve as binding sites for the nucleoid-associated proteins that have affinities for curved DNA (such as HU, H-NS, Hfq and CbpA). Therefore, we suggest that the approximately 100 bp long clusters of the phased A-tracts constitute the 'structural code' for DNA compaction by providing the long-range intrinsic curvature and increasing stability of the DNA complexes with architectural proteins.

Small molecules generally activate or inhibit gene transcription as externally added substrates or as internally accumulated end-products, respectively. Rarely has a connection been made that links an intracellular intermediary metabolite as a signal of gene expression. We report that a perturbation in the critical step of a metabolic pathway—the D-galactose amphibolic pathway—changes the dynamics of the pathways leading to accumulation of the intermediary metabolite UDP-galactose. This accumulation causes cell stress and transduces signals that alter gene expression so as to cope with the stress by restoring balance in the metabolite pool. This underscores the importance of studying the global effects of alterations in the level of intermediary metabolites in causing stress and coping with it by transducing signals to genes to reach a stable state of equilibrium (homeostasis). Such studies are an essential component in the integration of metabolomics, proteomics, and transcriptomics.

Transcription of many genes is regulated by combinations of multiple signals. In Escherichia coli, combinatorial control is typical in the case of operons related to utilization of different sugars in the absence of glucose. To understand regulation of the transport and metabolic pathways in the galactose system, we measured activities of the six gal regulon promoters simultaneously, using an in vitro transcription system containing purified components. Input functions were computed on the basis of the experimental measurements. We observed four different shapes of input functions. From the results, we can conclude that the structure of the regulatory network is insufficient for the determination of signal integration. It is the actual structure of the promoter and regulatory region, the mechanism of transcription regulation, and the interplay between transcription factors that shape the input function to be suitable for adaptation.

Many transcription factors repress transcription of their own genes. Negative autoregulation has been shown to reduce cell-cell variation in regulatory protein levels and speed up the response time in gene networks. In this work we examined transcription regulation of the galS gene and the function of its product, the GalS protein. We observed a unique operator preference of the GalS protein characterized by dominant negative autoregulation. We show that this pattern of regulation limits the repression level of the target genes in steady states. We suggest that transcription factors with dominant negative autoregulation are designed for regulating gene expression during environmental transitions.

Bending is not only required to accommodate DNA within the cell but also is a mechanism used by proteins to initiate DNA replication, transcription, and recombination. Determining the angles by which regulatory DNA segments deviate from linearity upon binding of proteins is a necessary step toward a better understanding of a large number of essential biological functions. The pBend plasmids contain duplicate sets of restriction sites and, when combined with "gel shift" experiments, allow the straightforward determination of the bending angle in a DNA molecule. The steps for successfully carrying out a binding/bending experiment are described. They include the cloning of the protein-binding site into the chosen pBend vector, the isolation of a series of DNA fragments with identical in length but variable placing of the protein-binding site, and the gel electrophoretic analysis of the free and protein-bound fragments.

The virulent bacteriophage EPS7 active against a number of Salmonella serovar and Escherichia coli strains, isolated from the local sewage in Korea, belongs to the family Siphoviridae. The ESP7 genome constitutes a linear double-stranded DNA of 111 382 bp. DNA sequencing and genomic analysis of EPS7 showed that it belongs to the phage T5 family. We identified the EPS7 genes involved in DNA repair, replication, viral structure and bacterial lysis by comparing the EPS7 genome with that of T5. In contrast, the tail genes encoding for putative host receptor-binding protein and the putative receptor-blocking lipoprotein precursor of EPS7 exhibit high homologies with the corresponding gene products of BF23, another member of the T5-family. BF23 binds to BtuB, a surface receptor in the host and involved in vitamin B(12) uptake, but its infection is independent of TonB. By constructing a series of deletion mutants in Salmonella and in E. coli and studying phage infection in the mutant hosts, we showed that BtuB is also the host receptor of the phage EPS7. Whether EPS7 infection depends on TonB needs to be further studied.

With current concerns of antibiotic-resistant bacteria and biodefense, it has become important to rapidly identify infectious bacteria. Traditional technologies involving isolation and amplification of the pathogenic bacteria are time-consuming. We report a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides specific detection of as few as 10 bacterial cells per milliliter in experimental samples, with an ≈100-fold amplification of the signal over background in 1 h. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium , or are highly infectious, e.g., Bacillus anthracis . The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are discussed.

Escherichia coli strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cell's invasiveness, allowing the bacteria to avoid phagocytosis and inactivation by complement. It is also recognized as a receptor by some phages, such as K1F and K1-5, which have virion-associated enzymes that degrade the polysaccharide. In this report we show that expression of the K1 capsule in E. coli physically blocks infection by T7, a phage that recognizes lipopolysaccharide as the primary receptor. Enzymatic removal of the K1 antigen from the cell allows T7 to adsorb and replicate. This observation suggests that the capsule plays an important role as a defense against some phages that recognize structures beneath it and that the K1-specific phages evolved to counter this physical barrier.

In Gal repressosome assembly, a DNA loop is formed by the interaction of two GalR, bound to two distal operators, and the binding of the histone-like protein, HU, to an architecturally critical position on DNA to facilitate the GalR–GalR interaction. We show that GalR piggybacks HU to the critical position on the DNA through a specific GalR–HU interaction. This is the first example of HU making a specific contact with another protein. The GalR–HU contact that results in cooperative binding of the two proteins to DNA may be transient and absent in the final repressosome structure. A sequence-independent DNA-binding protein being recruited to an architectural site on DNA through a specific association with a regulatory protein may be a common mode for assembly of complex nucleoprotein structures.

Transcription repression of the galactose operon of Escherichia coli requires (1) the binding of the GalR repressor to tandem operators flanking the promoters, (2) the binding of histone-like protein, HU, to a site between the GalR-binding sites, and (3) negatively supercoiled DNA. Under these conditions, protein-protein interactions mediate the formation of a nucleoprotein complex in the form of a DNA loop, which we have termed a repressosome. To analyze the structure of the repressosome, we have screened and isolated galR mutants in which single amino acid substitutions in GalR lead to defects in loop formation while the protein's operator-binding activity is retained. The mutant proteins were purified and their properties confirmed in vitro. We verified that in the case of the two stronger mutations, the proteins had secondary structures that were identical to that of wild-type GalR as reflected by circular dichroism spectroscopy. Homology-based modeling of GalR by use of the crystal structures of PurR and LacI has enabled us to place the three sites of mutation in a structural context. They occur in the carboxy-terminal subdomain of the GalR core, are surface exposed, and, therefore, may be involved in protein-protein interactions. On the basis of our model of GalR and its structural alignment with LacI and PurR, we have identified additional residues, the substitution of which leads to a specific defect in repression by looping. The effects of the mutations are the same in the presence of HMG-17, a eukaryotic protein unrelated to HU, which can also mediate GalR-dependent repression of the gal promoter. This observation suggests that the mutations define sites of GalR-GalR interaction rather than HU-GalR interaction in the repressosome.

Regulation of gene transcription in both prokaryotes and eukaryotes involves formation of various DNA-multiprotein complexes of higher order structure through communication between distant regions of DNA. The communication between distant DNA sites occurs by interaction between proteins bound to the sites by looping out the intervening DNA segments. The repression of transcription of two overlapping promoters of the gal operon in Escherichia coli requires Gal repressor (GalR) and the histone-like protein HU. Both in vivo and in vitro data support a proposed HU containing complex responsive to induction in which GalR molecules bound to two distant operator sites interact by looping out DNA. We successfully applied atomic force microscope (AFM) imaging to visualize galDNA complexes with proteins. We report GalR mediated DNA looping in which HU plays an obligatory role by helping GalR tetramerization. Supercoiling of DNA, which is also critical for GalR action, may stabilize the DNA loops by providing an energetically favorable geometry of the DNA.

Gal or Lac repressor binding to an upstream DNA segment, in the absence of DNA looping, represses the P1 promoter located on the same face and activates the P2 promoter situated on the opposite face of the DNA helix in the gal operon. Both inhibition and stimulation of transcription requires the physical presence of the C-terminal domain of the alpha subunit of RNA polymerase although the latter is not required for transcription itself. We propose that Gal and Lac repressors inhibit or stimulate transcription initiation by disabling or stimulating RNA polymerase activity at a post-binding step by directly or indirectly altering the C-terminal alpha domain to an unfavorable state at P1 or a more favorable state at P2, respectively.

The gal operon of Escherichia coli is negatively regulated by repressor binding to bipartite operators separated by 11 helical turns of DNA. Synergistic binding of repressor to separate sites on DNA results in looping, with the intervening DNA as a topologically closed domain containing the two promoters. A closed DNA loop of 11 helical turns, which is in-flexible to torsional changes, disables the promoters either by resisting DNA unwinding needed for open complex formation or by impeding the processive DNA contacts by an RNA polymerase in flux during transcription initiation. Interaction between two proteins bound to different sites on DNA modulating the activity of the intervening segment toward other proteins by allostery may be a common mechanism of regulation in DNA-multiprotein complexes.

The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit. It requires binding of cAMP for a conformational change in order to function as a site-specific DNA-binding protein that regulates gene activity. The hinge region connecting the cAMP-binding domain to the DNA-binding domain is involved in the cAMP-induced allosteric change. We studied the structural changes in CRP that are required for gene regulation by making a large number of single and double amino acid substitutions at four different positions in or near the hinge. To achieve cAMP-independent transcription by CRP, amino acid residues 138 (located within the hinge region) and 141 (located in the D alpha-helix adjacent to the hinge) must be polar. This need for polar residues at positions 138 and 141 suggests an interaction that causes the C and D alpha-helices to come together. As a consequence, the F alpha-helix is released from the D alpha-helix and can interact with DNA. At position 144 in the D alpha-helix and within interacting distances of the F alpha-helix, replacement of alanine by an amino acid with a larger side chain, regardless of its nature, allows cAMP independence. This result indicates that pushing against the F alpha-helix may be a way of making the helix available for DNA binding. We believe that the cAMP-induced allosteric change involves similar hinge reorientation to adjust the C and D alpha-helices, allowing outward movement of the F alpha-helix.

Tn10 insertion in the galS (ultrainduction factor) gene of Escherichia coli allows the gal operon to be constitutively expressed at a very high level, equal to that seen in a delta galR strain in the presence of an inducer. The insertion has been mapped by criss-cross Hfr matings and by marker rescue into Kohara phages at 46 min on the E. coli chromosome.

Two operators, spatially separated from each other and from the promoters, repress the gal operon when bound to Gal repressor. Conversion of either gal operator to a lac operator results in derepression, although both Gal and Lac repressors are present, suggesting that mere occupation of operator sites is not sufficient to cause repression. Conversion of both operators to lac operators restores normal repression in the presence of Lac repressor protein. We propose that normal repression requires interaction between operator-bound like repressor molecules; this generates a DNA loop, which is part of a higher order structure. RNA polymerase and cyclic AMP receptor protein are present in this complex but unable to initiate transcription because of the higher order structure. Such higher order DNA-multiprotein complexes could occur in a variety of genetic regulatory systems that are controlled from distal sites by regulatory proteins.

When the gal operator region is present in a multicopy plasmid it binds to all ("titrates") the gal repressor and "induces" the chromosomal gal operon. To make operator mutations (Oa) with reduced affinity toward the repressor, plasmid DNA was irradiated with UV light and mutant derivatives were isolated that were unable to release the chromosomal gal genes from repression. Then with such an Oa plasmid operator revertants were isolated that had reacquired the ability to release repression. Both sets of mutations have been localized by DNA sequence analysis. When the Oa mutations were transferred from the plasmid to the chromosome by recombination these mutant operators were found to make gal expression constitutive (independent of repressor) but still dependent on cAMP, whereas the previously reported gal operator mutants (Oc) are constitutive both in the presence and in the absence of cAMP. The titration method of isolating mutants enables the isolation of strains with operator mutations that also affect normal promoter activity, and it provides an easy way to isolate revertants of operator mutations.

Bacteria with strongly depressed phosphoglucomutase (EC 2.7.5.1) activity are found among the mutants of Escherichia coli which, when grown on maltose, accumulate sufficient amylose to be detectable by iodine staining. These pgm mutants grow poorly on galactose but also accumulate amylose on this carbon source. Growth on lactose does not produce high amylose but, instead, results in the induction of the enzymes of maltose metabolism, presumably by accumulation of maltose. These facts suggest that the catabolism of glucose-1-phosphate is strongly depressed in pgm mutants, although not completely abolished. Anabolism of glucose-1-phosphate is also strongly depressed, since amino acid- or glucose-grown pgm mutants are sensitive to phage C21, indicating a deficiency in the biosynthesis of uridine diphosphoglucose or uridine diphosphogalactose, or both. All pgm mutations isolated map at about 16 min on the genetic map, between pur E and the gal operon.

It has been reported that dimerized GalR could bind to 7 sites along the E.coli chromosome to regulate gene expression. Here, we label GalR at the C terminus with Venus fluorescence protein, for in-vivo localization in E.coli. In cells growing in stationary phase, 2 or 3 fluorescence spots per cell (55% and 31%) were detected regardless of the presence or absence of galactose. The spots indicate GalR oligomerization since tagged GalR monomers are below our detection limit. In log phase, however, we could not observe any similar spots. Furthermore, GalR mutations inhibiting tetramer formation abolished the spots also in stationary phase. We propose that GalR that binds to operator sites could oligomerize and facilitate long distance looping.To examine this, we employed Atomic Force Microscopy (AFM) and Chromosome Conformation Capture (3C) analysis. By AFM we visualize GalR binding and consequent DNA looping. Analysis of the loop sizes identifies operator pairs bound via GalR oligomers and quantifies the cooperativity that brings distant operators together.3C analysis suggests that DNA targets of GalR distributed around the chromosome are connected with one another mostly in stationary phase cells and that connection is lost in the log phase. Furthermore, we identified five additional potential binding sites of GalR along the investigated region of the E-coli chromosome.