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Laboratory-generated hybrids between phage λ and related phages played a seminal role in establishment of the λ model system, which, in turn, served to develop many of the foundational concepts of molecular biology, including gene structure and control. Important λ hybrids with phages 21 and 434 were the earliest of such phages. To understand the biology of these hybrids in full detail, we determined the complete genome sequences of phages 21 and 434. Although both genomes are canonical members of the λ-like phage family, they both carry unsuspected bacterial virulence gene types not previously described in this group of phages. In addition, we determined the sequences of the hybrid phages λ

Recombinational hybrids between phage λ and its relatives were instrumental in the beginnings of molecular biology. Here, we report the complete genome sequences of lambdoid phages 21 and 434 and three of their λ hybrids. In addition, we describe 434B, where the entire lysis gene region was replaced by cryptic prophage sequences.

SUMMARYConserved in bacteria, the histone-like protein HU is crucial for genome organization and expression of many genes. It binds DNA regardless of the sequence and exhibits two binding affinities in vitro, low-affinity to any B-DNA (non-specific) and high-affinity to DNA with distortions like kinks and cruciforms (structure-specific), but the physiological relevance of the two binding modes needed further investigation. We validated and defined the three conserved lysine residues, K3, K18, and K83, in Escherichia coli HU as critical amino acid residues for both non-specific and structure-specific binding and the conserved proline residue P63 additionally for only the structure-specific binding. By mutating these residues in vivo, we showed that two DNA binding modes of HU play separate physiological roles. The DNA structure-specific binding, occurring at specific sites in the E. coli genome, promotes higher-order DNA structure formation, regulating the expression of many genes, including those involved in chromosome maintenance and segregation. The non-specific binding participates in numerous associations of HU with the chromosomal DNA, dictating chromosome structure and organization. Our findings underscore the importance of DNA structure in transcription regulation and promiscuous DNA-protein interactions in a dynamic organization of a bacterial genome.

Burgeoning problems of antimicrobial resistance dictate that new solutions be developed to combat old foes. Use of lytic bacteriophages (phages) for the treatment of drug-resistant bacterial infections is one approach that has gained significant traction in recent years. Fueled by reports of experimental phage therapy cases with very positive patient outcomes, several early-stage clinical trials of therapeutic phage products have been launched in the United States. Eventual licensure enabling widespread access to phages is the goal; however, new paths to regulatory approval and mass-market distribution, distinct from those of small-molecule antibiotics, must be forged first. This review highlights unique aspects related to the clinical use of phages, including advantages to be reaped as well as challenges to be overcome.

Severe infections caused by multidrug-resistant Klebsiella pneumoniae sequence type 258 (ST258) highlight the need for new therapeutics with activity against this pathogen. Bacteriophage (phage) therapy is an alternative treatment approach for multidrug-resistant bacterial infections that has shown efficacy in experimental animal models and promise in clinical case reports. In this study, we assessed microbiologic, histopathologic, and survival outcomes following systemic administration of phage in ST258-infected mice. We found that prompt treatment with two phages, either individually or in combination, rescued mice with K. pneumoniae ST258 bacteremia. Among the three treatment groups, mice that received combination phage therapy demonstrated the greatest increase in survival and the lowest frequency of phage resistance among bacteria recovered from mouse blood and tissue. Our findings support the utility of phage therapy as an approach for refractory ST258 infections and underscore the potential of this treatment modality to be enhanced through strategic phage selection.IMPORTANCE Infections caused by multidrug-resistant K. pneumoniae pose a serious threat to at-risk patients and present a therapeutic challenge for clinicians. Bacteriophage (phage) therapy is an alternative treatment approach that has been associated with positive clinical outcomes when administered experimentally to patients with refractory bacterial infections. Inasmuch as these experimental treatments are prepared for individual patients and authorized for compassionate use only, they lack the rigor of a clinical trial and therefore cannot provide proof of efficacy. Here, we demonstrate that administration of viable phage provides effective treatment for multidrug-resistant K. pneumoniae (sequence type 258 [ST258]) bacteremia in a murine infection model. Moreover, we compare outcomes among three distinct phage treatment groups and identify potential correlates of therapeutic phage efficacy. These findings constitute an important first step toward optimizing and assessing phage therapy's potential for the treatment of severe ST258 infection in humans.

Bacteriophage T7 and T7-like bacteriophages are valuable genetic models for lytic phage biology that have heretofore been intractable with in vivo genetic engineering methods. This manuscript describes that the presence of &lambda, Red recombination proteins makes in vivo recombineering of T7 possible, so that single base changes and whole gene replacements on the T7 genome can be made. Red recombination functions also increase the efficiency of T7 genome DNA transfection of cells by ~100-fold. Likewise, Red function enables two other T7-like bacteriophages that do not normally propagate in E. coli to be recovered following genome transfection. These results constitute major technical advances in the speed and efficiency of bacteriophage T7 engineering and will aid in the rapid development of new phage variants for a variety of applications.

HU (Histone-like protein from Escherichia coli strain U93) is the most conserved nucleoid-associated protein in eubacteria, but how it impacts global chromosome organization is poorly understood. Using single-molecule tracking, we demonstrate that HU exhibits nonspecific, weak, and transitory interactions with the chromosomal DNA. These interactions are largely mediated by three conserved, surface-exposed lysine residues (triK), which were previously shown to be responsible for nonspecific binding to DNA. The loss of these weak, transitory interactions in a HUα(triKA) mutant results in an over-condensed and mis-segregated nucleoid. Mutating a conserved proline residue (P63A) in the HUα subunit, deleting the HUβ subunit, or deleting nucleoid-associated naRNAs, each previously implicated in HU's high-affinity binding to kinked or cruciform DNA, leads to less dramatically altered interacting dynamics of HU compared to the HUα(triKA) mutant, but highly expanded nucleoids. Our results suggest HU plays a dual role in maintaining proper nucleoid volume through its differential interactions with chromosomal DNA. On the one hand, HU compacts the nucleoid through specific DNA structure-binding interactions. On the other hand, it decondenses the nucleoid through many nonspecific, weak, and transitory interactions with the bulk chromosome. Such dynamic interactions may contribute to the viscoelastic properties and fluidity of the bacterial nucleoid to facilitate proper chromosome functions.

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.

Significance This study focuses on the molecular mechanisms of a noncoding RNA-mediated chromosome organization in Escherichia coli . We show for the first time that noncoding RNA, naRNA4, a critical element at least in part of chromosome structural organization (DNA condensation), may form DNA–RNA complexes in an uncommon way. Surprisingly, we found that HU protein catalyzes the DNA–RNA interactions without being part of the “final” complexes. There are several important examples of noncoding RNA playing critical roles in eukaryotic chromatin functioning, e.g., heterochromatin assembly, gene silencing, and X-inactivation, although nothing is known about the nature of DNA–RNA interaction(s) in these processes. Our findings will be of great interest in the chromatin field, providing mechanistic insights into the large-scale organization of DNA.

Bacillus phages use a communication system, termed “arbitrium,” to coordinate lysis-lysogeny decisions. Arbitrium communication is mediated by the production and secretion of a hexapeptide (AimP) during lytic cycle. Once internalized, AimP reduces the expression of the negative regulator of lysogeny, AimX, by binding to the transcription factor, AimR, promoting lysogeny. We have elucidated the crystal structures of AimR from the Bacillus subtilis SPbeta phage in its apo form, bound to its DNA operator and in complex with AimP. AimR presents intrinsic plasticity, sharing structural features with the RRNPP quorum-sensing family. Remarkably, AimR binds to an unusual operator with a long spacer that interacts nonspecifically with the receptor TPR domain, while the HTH domain canonically recognizes two inverted repeats. AimP stabilizes a compact conformation of AimR that approximates the DNA-recognition helices, preventing AimR binding to the aimX promoter region. Our results establish the molecular basis of the arbitrium communication system.

One of the best understood systems in genetic regulatory biology is the so-called "genetic switch" that determines the choice the phage-encoded CI repressor binds cooperatively to tripartite operators, OL and OR, in a defined pattern, thus blocking the transcription at two lytic promoters, PL and PR, and auto-regulating the promoter, PRM, which directs CI synthesis by the prophage. Fine-tuning of the maintenance of lysogeny is facilitated by interactions between CI dimers bound to OR and OL through the formation of a loop by the intervening DNA segment. By using a purified in vitro transcription system, we have genetically dissected the roles of individual operator sites in the formation of the DNA loop and thus have gained several new and unexpected insights into the system. First, although both OR and OL are tripartite, the presence of only a single active CI binding site in one of the two operators is sufficient for DNA loop formation. Second, in PL, unlike in PR, the promoter distal operator site, OL3, is sufficient to directly repress PL. Third, DNA looping mediated by the formation of CI octamers arising through the interaction of pairs of dimers bound to adjacent operator sites in OR and OL does not require OR and OL to be aligned "in register", that is, CI bound to "out-of-register" sub-operators, for example, OL1~Ol2 and OR2~OR3, can also mediate loop formation. Finally, based on an examination of the mechanism of activation of PRM when only OR1 or OR2 are wild type, we hypothesize that RNA polymerase bound at PR interferes with DNA loop formation. Thus, the formation of DNA loops involves potential interactions between proteins bound at numerous cis-acting sites, which therefore very subtly contribute to the regulation of the "switch".

[This corrects the article DOI: 10.1371/journal.pgen.1008456.].

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.

Bacteriophages infect an estimated 1023 to 1025 bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub “superspreaders,” releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications., IMPORTANCE Bacteriophages (phages), viruses that infect bacteria, are the planet’s most numerous biological entities and kill vast numbers of bacteria in natural environments. Many of these bacteria carry plasmids, extrachromosomal DNA elements that frequently encode antibiotic resistance. However, it is largely unknown whether plasmids are destroyed during phage infection or released intact upon phage lysis, whereupon their encoded resistance could be acquired and manifested by other bacteria (transformation). Because phages are being developed to combat antibiotic-resistant bacteria and because transformation is a principal form of horizontal gene transfer, this question has important implications for biomedicine and microbial evolution alike. Here we report the isolation and characterization of two novel Escherichia coli phages, dubbed “superspreaders,” that promote extensive plasmid transformation and efficiently disperse antibiotic resistance genes. Our work suggests that phage superspreaders are not suitable for use in medicine but may help drive bacterial evolution in natural environments.

Background Bacteriophage (phage) therapy is being considered as a treatment option for patients with multi-drug-resistant bacterial infections. However, there is a dearth of controlled clinical data to support therapeutic phage efficacy. As a first step toward addressing this deficiency, we tested the ability of two well-characterized phages, alone and in combination, to kill carbapenem-resistant Klebsiella pneumoniae (ST258) in blood in vitro and rescue mice from lethal ST258 infection. Methods Wild-type C57BL/6J mice were infected with a lethal inoculum of ST258 by intra-peritoneal (IP) injection followed 1 hour later by IP administration of lytic phage P1, P2, or P1+P2 at a multiplicity of infection (MOI) estimated at 1. Survival of each group of mice was tracked for 10 days. In separate experiments, mice were sacrificed at 1 hour, 24 hours, and 48 hours post-phage treatment. Mouse blood and tissues were collected at each timepoint for enumeration of bacteria and phage, screening for phage resistance, and histopathology. Results ST258 survival in mouse blood in vitro was significantly less after 1 hour of incubation with P1 or P1+P2 (MOI 1) compared with the control group (no phage). Consistent with the in vitro data, none of the mice (0/15) in the control group (no phage) survived to 10 days post-infection, whereas 12/15, 14/15, and 15/15 mice survived in the P2, P1, and P1+P2-treated groups, respectively (P < 0.0001). Conclusion Prompt, systemic administration of lytic bacteriophages rescued mice from lethal ST258 infection. These data support the potential of phage therapy to effectively treat infections caused by ST258. It will be important to assess whether, for other phage-bacteria combinations, in vitro lysis in blood correlates with in vivo treatment efficacy and therefore may have predictive utility. Disclosures All Authors: No reported Disclosures.

Although discovered decades ago, the molecular identification, the diversity and versatility of functions, and the evolutionary origin of repeat DNA sequences (REPs) containing palindromic units in prokaryotes are now bringing attention to a wide range of biological scientists. A brief account of the current state of the repeat DNA sequences is presented here.

HU networks control chromatin-like DNA compaction to synchronize bacterial responses for pathogenesis and changing environments., Molecular mechanisms controlling functional bacterial chromosome (nucleoid) compaction and organization are surprisingly enigmatic but partly depend on conserved, histone-like proteins HUαα and HUαβ and their interactions that span the nanoscale and mesoscale from protein-DNA complexes to the bacterial chromosome and nucleoid structure. We determined the crystal structures of these chromosome-associated proteins in complex with native duplex DNA. Distinct DNA binding modes of HUαα and HUαβ elucidate fundamental features of bacterial chromosome packing that regulate gene transcription. By combining crystal structures with solution x-ray scattering results, we determined architectures of HU-DNA nucleoproteins in solution under near-physiological conditions. These macromolecular conformations and interactions result in contraction at the cellular level based on in vivo imaging of native unlabeled nucleoid by soft x-ray tomography upon HUβ and ectopic HUα38 expression. Structural characterization of charge-altered HUαα-DNA complexes reveals an HU molecular switch that is suitable for condensing nucleoid and reprogramming noninvasive Escherichia coli into an invasive form. Collective findings suggest that shifts between networking and cooperative and noncooperative DNA-dependent HU multimerization control DNA compaction and supercoiling independently of cellular topoisomerase activity. By integrating x-ray crystal structures, x-ray scattering, mutational tests, and x-ray imaging that span from protein-DNA complexes to the bacterial chromosome and nucleoid structure, we show that defined dynamic HU interaction networks can promote nucleoid reorganization and transcriptional regulation as efficient general microbial mechanisms to help synchronize genetic responses to cell cycle, changing environments, and pathogenesis.

DNA in bacterial cells primarily exists in a negatively supercoiled state. The extent of supercoiling differs between regions of the chromosome, changes in response to external conditions and regulates gene expression. Here we report the use of trimethylpsoralen intercalation to map the extent of supercoiling across the Escherichia coli chromosome during exponential and stationary growth phases. We find that stationary phase E. coli cells display a gradient of negative supercoiling, with the terminus being more negatively supercoiled than the origin of replication, and that such a gradient is absent in exponentially growing cells. This stationary phase pattern is correlated with the binding of the nucleoid-associated protein HU, and we show that it is lost in an HU deletion strain. We suggest that HU establishes higher supercoiling near the terminus of the chromosome during stationary phase, whereas during exponential growth DNA gyrase and/or transcription equalizes supercoiling across the chromosome., Bacterial DNA primarily exists in a negatively supercoiled or under-wound state. Here, by mapping the supercoiling state, the authors show that there is a gradient of supercoiling across the bacterial chromosome with the terminus being more negatively supercoiled than the origin.

Background It is unclear how DNA is packaged in a bacterial cell in the absence of nucleosomes. To investigate the initial level of DNA condensation in bacterial nucleoid we used in vivo DNA digestion coupled with high-throughput sequencing of the digestion-resistant fragments. To this end, we transformed E. coli cells with a plasmid expressing micrococcal nuclease. The nuclease expression was under the control of AraC repressor, which enabled us to perform an inducible digestion of bacterial nucleoid inside a living cell. Results Analysis of the genomic localization of the digestion-resistant fragments revealed their non-random distribution. The patterns observed in the distribution of the sequenced fragments indicate the presence of short DNA segments protected from the enzyme digestion, possibly because of interaction with DNA-binding proteins. The average length of such digestion-resistant segments is about 50 bp and the characteristic repeat in their distribution is about 90 bp. The gene starts are depleted of the digestion-resistant fragments, suggesting that these genomic regions are more exposed than genomic sequences on average. Sequence analysis of the digestion-resistant segments showed that while the GC-content of such sequences is close to the genome-wide value, they are depleted of A-tracts as compared to the bulk genomic DNA or to the randomized sequence of the same nucleotide composition. Conclusions Our results suggest that DNA is packaged in the bacterial nucleoid in a non-random way that facilitates interaction of the DNA binding factors with regulatory regions of the genome. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0637-3) contains supplementary material, which is available to authorized users.

Some unidentified RNA molecules, together with the nucleoid protein HU, were suggested to be involved in the nucleoid structure of Escherichia coli . HU is a conserved protein known for its role in binding to DNA and maintaining negative supercoils in the latter. HU also binds to a few RNAs, but the full spectrum of its binding targets in the cell is not known. To understand any interaction of HU with RNA in the nucleoid structure, we immunoprecipitated potential HU-RNA complexes from cells and examined bound RNAs by hybridization to whole-genome tiling arrays. We identified associations between HU and 10 new intragenic and intergenic noncoding RNAs (ncRNAs), 2 of which are homologous to the annotated bacterial interspersed mosaic elements (BIMEs) and boxC DNA repeat elements. We confirmed direct binding of HU to BIME RNA in vitro . We also studied the nucleoid shape of HU and two of the ncRNA mutants (nc1 and nc5) by transmission electron microscopy and showed that both HU and the two ncRNAs play a role in nucleoid morphology. We propose that at least two of the ncRNA species complex with HU and help the formation or maintenance of the architecture of the E. coli chromosome. We also observed binding of HU with rRNA and tRNA segments, a few small RNAs, and a distinct small set of mRNAs, although the significance, if any, of these associations is not known.

By microscopic analysis of fluorescent-labeled GalR, a regulon-specific transcription factor in Escherichia coli , we observed that GalR is present in the cell as aggregates (one to three fluorescent foci per cell) in nongrowing cells. To investigate whether these foci represent GalR-mediated association of some of the GalR specific DNA binding sites ( gal operators), we used the chromosome conformation capture (3C) method in vivo. Our 3C data demonstrate that, in stationary phase cells, many of the operators distributed around the chromosome are interacted. By the use of atomic force microscopy, we showed that the observed remote chromosomal interconnections occur by direct interactions between DNA-bound GalR not involving any other factors. Mini plasmid DNA circles with three or five operators positioned at defined loci showed GalR-dependent loops of expected sizes of the intervening DNA segments. Our findings provide unique evidence that a transcription factor participates in organizing the chromosome in a three-dimensional structure. We believe that these chromosomal connections increase local concentration of GalR for coordinating the regulation of widely separated target genes, and organize the chromosome structure in space, thereby likely contributing to chromosome compaction.

The DNA loop that represses transcription from galactose (gal) promoters is infrequently formed in stationary-phase cells because the concentration of the loop architectural protein HU is significantly low at that state, resulting in expression of the operon in the absence of the gal inducer D-galactose. Unexpectedly, transcription from the gal promoters under these conditions overrides physical block because of the presence of the Gal repressor bound to an internal operator (O(I)) located downstream of the promoters. We have shown here that although a stretch of pyrimidine residues (UUCU) in the RNA:DNA hybrid located immediately upstream of O(I) weakens the RNA:DNA hybrid and favors RNA polymerase (RNAP) pausing and backtracking, a stretch of purines (GAGAG) in the RNA present immediately upstream of the pause sequence in the hybrid acts as an antipause element by stabilizing the RNA:DNA duplex and preventing backtracking. This facilitates forward translocation of RNAP, including overriding of the DNA-bound Gal repressor barrier at O(I). When the GAGAG sequence is separated from the pyrimidine sequence by a 5-bp DNA insertion, RNAP backtracking is favored from a weak hybrid to a more stable hybrid. RNAP backtracking is sensitive to Gre factors, D-galactose, and antisense oligonucleotides. The ability of a native DNA sequence to override transcription elongation blocks in the gal operon uncovers a previously unknown way of regulating gal metabolism in Escherichia coli. It also explains the synthesis of gal enzymes in the absence of inducer for biosynthetic reactions.

The poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsA-GFP. ManY, an E. coli protein that is required for phage lambda DNA injection, was found to localize to the bacterial poles as well. Furthermore, labelling of lambda DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to lambda's developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation.

Inactivation of bacteriophage lambda CI repressor leads almost exclusively to lytic development. Prophage induction can be initiated either by DNA damage or by heat treatment of a temperature-sensitive repressor. These two treatments also cause a concurrent activation of either the host SOS or heat-shock stress responses respectively. We studied the effects of these two methods of induction on the lytic pathway by monitoring the activation of different lambda promoters, and found that the lambda genetic network co-ordinates information from the host stress response networks. Our results show that the function of the CII transcriptional activator, which facilitates the lysogenic developmental pathway, is not observed following either method of induction. Mutations in the cro gene restore the CII function irrespective of the induction method. Deletion of the heat-shock protease gene ftsH can also restore CII function following heat induction but not following SOS induction. Our findings highlight the importance of the elimination of CII function during induction as a way to ensure an efficient lytic outcome. We also show that, despite the common inhibitory effect on CII function, there are significant differences in the heat- and SOS-induced pathways leading to the lytic cascade.

Analysing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2lambda) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface of phage lambda that are marked with different selectable drug-resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug-resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)(4) and (Gly-Arg)(4) peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library. The unique usefulness to 2lambda is also described.

Summary The gal regulon of Escherichia coli contains genes involved in galactose transport and metabolism. Transcription of the gal regulon genes is regulated in different ways by two iso-regulatory proteins, Gal repressor (GalR) and Gal isorepressor (GalS), which recognize the same binding sites in the absence of d-galactose. DNA binding by both GalR and GalS is inhibited in the presence of d-galactose. Many of the gal regulon genes are activated in the presence of the adenosine cyclic-3′,5′-monophosphate (cAMP)–cAMP receptor protein (CRP) complex. We studied transcriptional regulation of the gal regulon promoters simultaneously in a purified system and attempted to integrate the two small molecule signals, d-galactose and cAMP, that modulate the isoregulators and CRP respectively, at each promoter, using Boolean logic. Results show that similarly organized promoters can have different input functions. We also found that in some cases the activity of the promoter and the cognate gene can be described by different logic gates. We combined the transcriptional network of the galactose regulon, obtained from our experiments, with literature data to construct an integrated map of the galactose network. Structural analysis of the network shows that at the interface of the genetic and metabolic network, feedback loops are by far the most common motif.

Bacteriophage λ is a temperate bacteriophage, meaning that it can reproduce and develop either in a lytic or lysogenic state. When λ infects its bacterial host Escherichia coli , the phage may develop lytically, causing cell lysis with the release of hundreds of progeny virus, or it may abort

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.

In bacteria, the contribution of global nucleoid organization in determining cellular transcription programs is unclear. Using a mutant form of the most abundant nucleoid-associated protein HU, HUαE38K,V42L, we previously showed that nucleoid remodeling by the mutant protein re-organizes the global transcription pattern. Here, we demonstrate that, unlike the dimeric wild-type HU, HUαE38K,V42L is an octamer and wraps DNA around its surface. The formation of wrapped nucleoprotein complexes by HUαE38K,V42L leads to a high degree of DNA condensation. The DNA wrapping is right-handed, which restrains positive supercoils. In vivo, HUαE38K,V42L shows altered association and distribution patterns with the genetic loci whose transcription are differentially affected in the mutant strain.

S100B is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effect by binding and affecting various target proteins. A consensus sequence for S100B target proteins was published as (K/R)(L/I)xWxxIL and matches a region in the actin capping protein CapZ (V.V. Ivanenkov, G.A. Jamieson, Jr., E. Gruenstein, R.V. Dimlich, Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ, J. Biol. Chem. 270 (1995) 14651–14658). Several additional S100B targets are known including p53, a nuclear Dbf2 related (NDR) kinase, the RAGE receptor, neuromodulin, protein kinase C, and others. Examining the binding sites of such targets and new protein sequence searches provided additional potential target proteins for S100B including Hdm2 and Hdm4, which were both found to bind S100B in a calcium-dependent manner. The interaction between S100B and the Hdm2 and/or the Hdm4 proteins may be important physiologically in light of evidence that like Hdm2, S100B also contributes to lowering protein levels of the tumor suppressor protein, p53. For the S100B–p53 interaction, it was found that phosphorylation of specific serine and/or threonine residues reduces the affinity of the S100B–p53 interaction by as much as an order of magnitude, and is important for protecting p53 from S100B-dependent down-regulation, a scenario that is similar to what is found for the Hdm2–p53 complex.

The tethered particle motion (TPM) technique has attracted great interest because of its simplicity and the wealth of information that it can provide on protein-induced conformational changes in nucleic acids. Here we present an approach to TPM methodology and analysis that increases the efficiency of data acquisition and facilitates interpretation of TPM assays. In particular, the statistical analysis that we propose allows fast data processing, minimal data selection and visual display of the distribution of molecular behaviour. The methodology proved useful in verifying CI protein-mediated DNA looping in bacteriophage λ and in differentiating between two different types of loops, stable and dynamic, whose relative occurrence seems to be a function of the distance between the operators as well as their relative angular orientation. Furthermore, the statistical analysis indicates that CI binding per se slightly shortens the DNA.